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Consultant Psychiatrist, AMRI Hospitals, Kolkata, West Bengal, IndiaClick here for where can i buy viagra over the counter usa correspondence address and email Date of Submission11-Jun-2021Date of Decision11-Jun-2021Date of http://vicstyles.com/can-u-buy-viagra-over-the-counter/ Acceptance11-Jun-2021Date of Web Publication17-Jun-2021 How to cite this article:Singh OP. Grief management in erectile dysfunction treatment. Indian context.

Indian J Psychiatry 2021;63:211Grief is where can i buy viagra over the counter usa a normal response to loss and bereavement. Human beings are aware of the concept of death and permanence of loss leading to grief and bereavement. It may be seen in some other species also.

While there has been where can i buy viagra over the counter usa a neurobiological mechanism explaining grief, it primarily remains a sociocultural phenomenon affecting the brain and the body. The perception of death followed by the gradual “sinking in” of its consequences leads to psychobiological reaction. Grief which is unmanaged can lead to serious health reactions like increased cardiovascular mortality (broken heart) and psychiatric disorders like depression and suicide.erectile dysfunction treatment as an epidemic has brought grief and bereavement to the doorstep of each and every person.

Constantly hearing, seeing about death, and losing where can i buy viagra over the counter usa friends and family has brought enormous strain to people's lives. Death rituals have a therapeutic function wherein they allow a family and a group to mourn in a ritualistic way. This allows people to share grief and keep the deceased as focus of attention for a fixed time and then to move on with life.

Sometimes, this process is hampered by what Kenneth Doka called “disenfranchised grief” in 1989 and defined it “as a process in which loss is felt as not being openly acknowledged, socially validated or publicly mourned.”[1] Externally imposed disenfranchised grief leads to grief remaining unresolved and unaddressed, and the person where can i buy viagra over the counter usa feels that his right to grieve has been denied.erectile dysfunction treatment has unexpectedly disturbed the process of death rituals as it leads to:Unexpected or sudden lossDepletion of emotional and coping resourcesLimitation in visiting and end of care supportNot able to perform last ritualsLack of social support due to erectile dysfunction treatment restrictions.[2]The mechanical and impersonal process has led to severe psychological trauma in the survivors, particularly in the early phase of the disease when the knowledge was less and health-care workers were burdened and under cover of personal protective equipment, communication was difficult. Realizing this, the Indian Council of Medical Research has come out with guidelines for health-care workers to deal with death and guide family members. However, persistence of grief reaction remains a problem, and due to lack of social support due to erectile dysfunction treatment, people are increasingly relying on professionals to take care of their grief reactions.In India, the sharing of grief is very important.

People try to reach the grieving where can i buy viagra over the counter usa family. So, what should be the model of care for these people?. We should try to increase the sharing of grief and the handling of the person should be allowed to take placeThe physical support and the economical support have to be arranged, particularly where both parents have diedThere are some common modes like “condolence meetings” or “smaran sabha” which should be attended by both family members and colleagues.erectile dysfunction treatment has brought an unprecedented amount of grief, and it is our duty to manage grief with innovative solutions to prevent the emergence of prolonged grief reaction, depression, and suicide.

References 1.Doka KJ, editor where can i buy viagra over the counter usa. Disenfranchised Grief. New Directions, Challenges, and Strategies for Practice.

Champaign, IL where can i buy viagra over the counter usa. Research Press. 2002.

2.Albuquerque where can i buy viagra over the counter usa S, Teixeira AM, Rocha JC. erectile dysfunction treatment and Disenfranchised Grief. Front Psychiatry 2021;12:638874.

Correspondence Address:Om where can i buy viagra over the counter usa Prakash SinghDepartment of Psychiatry, WBMES, Kolkata, West Bengal. AMRI Hospitals, Kolkata, West Bengal IndiaSource of Support. None, Conflict of Interest.

NoneDOI. 10.4103/indianjpsychiatry.indianjpsychiatry_489_21How to cite this article:Parthasarathy R, Channaveerachari NK, Manjunatha N, Sadh K, Kalaivanan RC, Gowda GS, Basvaraju V, Harihara SN, Rao GN, Math SB, Thirthalli J. Mental health care in Karnataka.

Moving beyond the Bellary model of District Mental Health Program. Indian J Psychiatry 2021;63:212-4How to cite this URL:Parthasarathy R, Channaveerachari NK, Manjunatha N, Sadh K, Kalaivanan RC, Gowda GS, Basvaraju V, Harihara SN, Rao GN, Math SB, Thirthalli J. Mental health care in Karnataka.

Moving beyond the Bellary model of District Mental Health Program. Indian J Psychiatry [serial online] 2021 [cited 2021 Jul 25];63:212-4. Available from.

Https://www.indianjpsychiatry.org/text.asp?. 2021/63/3/212/318719Karnataka state has taken many strides forward with regard to the District Mental Health Program (DMHP) and is one of the few states to have dedicated DMHP psychiatrists as team leaders in all the districts. Moreover, some of the recent developments have moved beyond the Bellary model and augur well for the nation.

This article attempts to provide a summary of such developments in the state and discusses the future directions. Core Services DMHP in Karnataka offers (a) clinical services, including the outreach services (on a rotation basis), covering the primary health centers (PHCs), community health centers, and taluk hospitals. (b) training of all the medical officers and other health professionals such as nurses and pharmacists of the district.

(c) information, education, and communication (IEC) activities – posters, wall paintings in PHCs, IEC activities for schools, colleges, police personnel, judicial departments, elected representatives, faith healers, bus branding, radio talks, etc., In addition, sensitization of Anganwadi workers, accredited social health activists, auxiliary nurse midwives, police/prison staff, agriculture department/horticulture department/primary land development bank staff, village rehabilitation workers, staff of noncommunicable disease/revised National Tuberculosis Control Program, etc.. And (d) targeted interventions are being focused on life skills education and counseling in schools, college counseling services, workplace stress management, and suicide prevention services. These initiatives have led to a phenomenal increase in patient footfalls to clinics [Figure 1] and >100,000 stakeholders are trained in various aspects of mental health (in the past 3 years).Figure 1.

Chart showing the phenomenal increase in the number of footfalls covered over the past 3 yearsClick here to view Seamless Medication Availability The procurement has been streamlined. The state-level purchase is done by the Karnataka Drugs and Logistics Society, based on the indents collated from each of the districts, and then, sent to their respective district warehouses. Individual indenters (taluk hospitals, community health centers, and primary health centers) then need to procure them from the district warehouses.

The amount spent for the purpose has gone up drastically to INR 3 crores (30 million rupees) in the past financial year (2017–2018). However, further streamlining is possible in the sense that the delays can be further curtailed. The Collaboration with the Karnataka State Wakf Board The WAKF board of Karnataka runs a “Darga” in south interior Karnataka.

Thousands of persons with mental illnesses do come over here for religious cure. On a day of every week, the attendance crosses 10,000 footfalls. Recently, the authorities have agreed to come up with an allopathic PHC inside the campus of the Darga.

The idea is to have integrated and comprehensive care for patients without hurting their religious sentiments. Although such collaborative initiatives are spread across the country, this one is occurring at a larger scale with involvement of governmental agencies [Table 1].Table 1. Details of the key developments and innovations in mental health care in IndiaClick here to view Research Initiatives Although excellent evidence-based studies have come out in community settings, actual involvement of government machinery in these kinds of initiatives is few and far.

Their involvement is imperative for the evidence to become pragmatic and generalizable. Of course, by doing so, the methodological rigor compromises a bit. NIMHANS and Government of Karnataka have been collaborating for such service-driven research initiatives for over a decade and a half.

Community-based interventions are going on in three taluks – Thirthahalli, Turuvekere, and Jagaluru, wherein cohorts of severe mental disorders are being cared for. In addition, several research questions (of public health significance) are being answered.[6],[7] Exciting new initiatives are also underway. Examining the magnitude of reduction of treatment gap by these community interventions, impact of care at doorsteps (CAD) services from the DMHP machinery, impact of technology-based mentoring program for DMHP staff, evaluation of the impact of tele-OCT, etc.

Discussion and Future Directions All the above-mentioned activities in Karnataka take it beyond the Bellary model of DMHP. For example, the Memorandum of understanding (MOU) between NIMHANS and the state gives the flexibility and easy maneuverability for active collaboration. Odisha is another state which has taken this path of MOU.

This collaborative activity can be expanded pan India as there are several Centers of Excellence spread throughout India. Another aspect of the Karnataka story is collaborative research activity. As described above, many activities going on across the state have the potential to inform public health policies.

Karnataka has also been able to counter long-standing and well-known criticisms of DMHP/NMHP. For example, issues related to human resources, availability of medications, funding, mentoring and monitoring, and sustenance, etc., at least to an extent. Of course, the state needs to do much more for mental health care.

For example, compliance with Mental Health Care Act-2017. Handling unequal distribution of mental health human resources. Rigorous involvement of local administration to tackle micro-level issues.

Refining DMHP to suit special populations such as geriatric, children, and adolescents. And perinatal and upscaling urban DMHP, in areas such as Bengaluru Metropolitan City. Another area for improvement is that the DMHP evaluation strategies should move beyond head counting and consider meaningful patient-related outcomes, including cost-effective analysis.

Digital technology should further be exploited. The upcoming Karnataka Mental Healthcare Management System is a step in the right direction.[8] Finally, the DMHP should involve health and wellness centers to cater to the mental health needs, particularly for follow-up services, case detection, providing basic counseling, stress management, advocating lifestyle changes, relapse prevention strategies, and other preventive and promotive strategies. References 1.Manjunatha N, Kumar CN, Chander KR, Sadh K, Gowda GS, Vinay B, et al.

Taluk Mental Health Program. The new kid on the block?. Indian J Psychiatry 2019;61:635-9.

[PUBMED] [Full text] 2.Manjunatha N, Kumar CN, Math SB, Thirthalli J. Designing and implementing an innovative digitally driven primary care psychiatry program in India. Indian J Psychiatry 2018;60:236-44.

[PUBMED] [Full text] 3.Pahuja E, Santhosh KT, Fareeduzzafar, Manjunatha N, Kumar CK, Gupta R, et al. An impact of digitally-driven Primary Care Psychiatry Pr. Indian J Psychiatry 2020;62 Suppl 1:S17.

4.Manjunatha N, Singh G. Manochaitanya. Integrating mental health into primary health care.

Lancet 2016;387:647-8. 5.Manjunatha N, Singh G, Chaturvedi SK. Manochaitanya programme for better utilization of primary health centres.

Indian J Med Res 2017;145:163-5. [PUBMED] [Full text] 6.Agarwal PP, Manjunatha N, Parthasarathy R, Kumar CN, Kelkar R, Math SB, et al. A performance audit of first 30 months of Manochaitanya programme at secondary care level of Karnataka, India.

Indian J Community Med 2019;44:222-4. [PUBMED] [Full text] 7.Kumar CN, Thirthalli J, Suresha KK, Arunachala U, Gangadhar BN. Alcohol use disorders in patients with schizophrenia.

Comparative study with general population controls. Addict Behav 2015;45:22-5. 8.

Correspondence Address:Naveen Kumar ChannaveerachariDepartment of Psychiatry, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka IndiaSource of Support. None, Conflict of Interest.

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Blood transcriptomics generic viagra sildenafil have revealed http://www.em-gliesberg-strasbourg.ac-strasbourg.fr/?page_id=44 major characteristics of the immune response in active TB, but the signature early after is unknown. In a unique clinically and temporally well-defined cohort of household contacts of active TB patients that progressed to TB, we define minimal changes in gene expression in incipient TB increasing in subclinical and clinical TB. While increasing with time, changes in gene expression were highest at 30 d before diagnosis, with heterogeneity in the response in household TB contacts and in a published cohort of TB progressors as they progressed generic viagra sildenafil to TB, at a bulk cohort level and in individual progressors. Blood signatures from patients before and during anti-TB treatment robustly monitored the treatment response distinguishing early and late responders.

Blood transcriptomics thus reveal the evolution and resolution of the immune response in TB, which may help in clinical management of the disease. Tuberculosis (TB) resulted in 1.5 million deaths in generic viagra sildenafil 2018. Although a quarter of the world’s population is estimated to have been infected by Mycobacterium tuberculosis (WHO, 2019), most infected individuals remain asymptomatic (latently infected [LTBI]. Richeldi, 2006) and are suggested to have a 5–15% lifetime risk of developing TB (Vynnycky and Fine, 2000).

However, recent epidemiological studies suggest that most cases occur within 2 yr generic viagra sildenafil after (Behr et al., 2018. Behr et al., 2019. Behr et al., 2021) with the median time to TB disease during occurring in the first year or earlier (Emery et al., 2021. Menzies et al., 2021), implicating early immune events as key determinants of outcome (Cadena et al., 2016) generic viagra sildenafil.

Heterogeneity of LTBI in HIV-coinfected humans and nonhuman primates has been reported (Barry et al., 2009. Esmail et al., 2016. Lin et al., 2016), but current assays cannot characterize the underlying heterogeneity of generic viagra sildenafil immune responses to M. Tuberculosis determining TB risk or those that accompany disease progression.

Clinically, the progressor LTBI state has been categorized into two phenotypes. (1) incipient TB, no clinical symptoms, radiological abnormalities or microbiological evidence of active TB generic viagra sildenafil disease. And (2) subclinical TB, no clinical symptoms, but either radiological changes or microbiological evidence of active TB disease (Davies and Pai, 2008. Drain et al., 2018.

Kendall et al., generic viagra sildenafil 2021. Pfyffer et al., 1997. Richeldi, 2006. WHO, 2019) generic viagra sildenafil.

Clinical TB patients display radiological features and microbiological evidence of active TB disease (Davies and Pai, 2008. Drain et al., 2018 generic viagra sildenafil. Kendall et al., 2021. Pfyffer et al., 1997.

Richeldi, 2006 generic viagra sildenafil. WHO, 2019). Thus, a proportion of patients presumed as LTBI may either be incipient or already have subclinical disease, contributing to onward transmission of (Dowdy et al., 2013. Drain et al., generic viagra sildenafil 2018.

Kendall et al., 2021). Reported reduced blood transcriptional signatures of TB risk were not related to subclinical TB or incipient disease or to the blood signature of active TB (Gupta et al., 2020. Penn-Nicholson et al., generic viagra sildenafil 2020. Scriba et al., 2017.

Singhania et al., 2018a. Singhania et al., generic viagra sildenafil 2018b. Suliman et al., 2018. Zak et al., 2016).

Earlier detection could generic viagra sildenafil inform treatment and limit transmission. Diagnosis of active pulmonary TB requires microbiological samples for evidence of , which can be difficult to obtain (Davies and Pai, 2008. Richeldi, 2006). A blood transcriptional signature has generic viagra sildenafil been reported in patients with active TB (Berry et al., 2010.

Blankley et al., 2016. Bloom et al., 2013. Joosten et al., generic viagra sildenafil 2013. Maertzdorf et al., 2011.

Ottenhoff et al., 2012. Roe et al., generic viagra sildenafil 2016. Scriba et al., 2017), which is dominated by type I IFN signaling, reflects the extent of radiographical lung disease (Berry et al., 2010. Moreira-Teixeira et al., 2020), and is generic viagra sildenafil diminished upon treatment (Berry et al., 2010.

Bloom et al., 2012. Cliff et al., 2013. Thompson et al., 2017) generic viagra sildenafil. Biomarkers to monitor TB treatment success are needed to accelerate assessment of treatment responses and determine the required treatment duration to adapt drug treatment regimens.

The accepted biomarker is sputum conversion to negative culture after 2 mo, which has low sensitivity and modest specificity for prediction of treatment failure (Horne et al., 2010. Mitchison, 1993) generic viagra sildenafil. Chest x rays (CRXs) and inflammatory markers commonly used to assess the response to treatment are not universally available and difficult to standardize (Walzl et al., 2011). How the host response evolves after of humans with M.

Tuberculosis toward the peak response in active TB is as yet unclear generic viagra sildenafil. Sequential immune responses were reported during TB progression but not linked to the clinical disease status, with adolescents evaluated at enrollment and then only sampled every 6 mo with follow-up over 2 yr, or evaluated at baseline and at the end of 2 yr (Scriba et al., 2017). Since this study was not on household contacts, knowledge of when each individual was exposed to M. Tuberculosis could not generic viagra sildenafil be estimated, limiting the scope for detailed temporal evaluation of changes in the immune response during progressive .

Without detailed clinical characterization of patients upon serial sampling before TB diagnosis, differential gene expression during different phenotypic stages of disease ranging from incipient TB to subclinical TB to clinical TB cannot be assessed. Moreover, confounding interpretations due to re in high TB burden settings during the prospective period of observation and sampling cannot be ruled out (Charalambous et al., 2008. Van Helden et generic viagra sildenafil al., 2008. Van Rie et al., 2005.

Van Rie et al., 1999. Verver et al., 2005 generic viagra sildenafil. Warren et al., 2004). Although blood transcriptional signatures have been shown to reflect the response to TB treatment (Berry et al., 2010.

Bloom et generic viagra sildenafil al., 2012. Cliff et al., 2013. Penn-Nicholson et al., 2020. Thompson et al., 2017), the patterns of resolution with treatment in different patient groups using detailed kinetic generic viagra sildenafil analysis at multiple time points has not been evaluated.

To address these questions, we undertook a prospective cohort study comprising participants with microbiologically confirmed pulmonary TB and household contacts of pulmonary TB at Leicester, UK, a high-income, moderate TB setting (TB incidence circa 40 per 100,000 population). An integrated clinical-research platform enabled recruitment, regular follow-up, and detailed characterization of participants at serial time points of prospective observation (Materials and methods), with a low probability of new generic viagra sildenafil community-acquired during prospective follow-up of TB contacts. In total, 356 household contacts of pulmonary TB and 74 participants with incident TB were recruited between 2015 and 2018 and prospectively followed for 24 mo. TB contacts were reviewed every 3–6 mo with RNA sequencing (RNA-Seq) samples collected, whole-genome sequencing of the M.

Tuberculosis strain to trace back contacts to the index case, and CRX performed to screen for subclinical TB at each visit, with detailed radiological characterization and clinical investigation, including invasive sampling (bronchoscopy), if x-ray abnormalities were suspected generic viagra sildenafil or symptoms reported. This allowed reliable characterization of participants with incipient, subclinical, or clinically active TB, and linking blood transcriptional signatures to the clinical phenotype as disease progressed. Active TB patients were sampled, and clinical characterization was undertaken before starting TB treatment, and prospectively at scheduled visits during treatment, with microbiological investigation, radiological surveillance with CRX, and computed tomography scan as clinically indicated. Changes in blood gene expression in different clinical subgroups of generic viagra sildenafil active TB patients were related to the time of diagnosis and to detailed time points during treatment.

Bioinformatics analysis of blood RNA-Seq data of contacts revealed minimal changes in gene expression in incipient TB, increasing as patients progressed to subclinical and clinical TB, with similar expression profiles in these clinical phenotypes for published reduced risk signatures of TB. Moreover, gene expression changes in the blood of Leicester TB progressors, and a published cohort of TB progressors from a high burden TB setting, were most pronounced at 30 d before diagnosis, although heterogeneity was observed over time before diagnosis. The signature of TB progression in the Leicester cohort was compared with active TB disease, before and during treatment, to understand the immune events underlying the evolution and resolution of generic viagra sildenafil TB disease (Fig. 1.

Study design). Our study provides information of the underlying host immune response at the different stages of disease and generic viagra sildenafil a roadmap to describe the temporality of gene expression changes that occur during progression and treatment of active TB, which may help in clinical management of TB patients. To determine how global changes in differential gene expression develop as individuals progress from incipient TB to subclinical TB and then to to clinical TB and whether these clinical phenotypes show a graded increase in the immune response, we performed detailed analysis of changes in gene expression over time in blood of clinically defined Leicester household TB contacts who then progressed to TB (Fig. 2).

Contacts who progressed to TB generic viagra sildenafil were subdivided according to their clinical phenotype at the time point of sampling (Table S1). In the 14 household contacts, incipient TB was concurrent with samples (n = 10) collected earlier than 40 d before diagnosis. Subclinical TB spread between earlier than day 40 (n = 1), 21–40 d (n = 3), and <20 d (n = 6) before diagnosis. Clinical TB spread between 21–40 d (n = 4) or <20 d (n = 14) generic viagra sildenafil before diagnosis (Table S1).

Numbers of up- and down-regulated genes were minimal in incipient TB (94 up-regulated and 48 down-regulated genes), increasing in subclinical TB (483 up-regulated genes and 81 down-regulated genes) and in clinical TB (572 up-regulated and 136 down-regulated genes. Fig. 2 A) generic viagra sildenafil. Fewer down-regulated genes were detected in each of the different clinical phenotypes of the TB contacts as they progressed to TB (Data S1).

Of the up-regulated genes, Metacore pathway analysis showed a dominance of the IFN-α/β signaling pathways in subclinical TB and clinical TB with an increase in the ratio in the number of genes per pathway, 14/62 and 16/62, respectively, with much lower representation in incipient TB, 5/62 (Fig. 2 B) generic viagra sildenafil. Incipient TB showed IFN-γ activation of macrophages and the classical complement as the top represented pathways. However, only 5/50 and 5/53 genes were represented in each pathway, although with generic viagra sildenafil a much lower ratio of genes per pathway overall (Fig.

2 B and Data S1). The P values for the different clinical subgroups showed a corresponding increasing statistical significance for the IFN-α/β signaling pathways, progressing from the incipient TB (8.65 × 10−5), subclinical TB (4.75 × 10−13), and clinical TB (2.44 × 10−14) respectively (Fig. 2 B) generic viagra sildenafil. Similarly, the type I IFN modules increased in subclinical TB and clinical TB, although clinical TB progressors showed additional changes resembling the signature of active TB, including an increase in the innate/hemopoeitic mediators module (Fig.

2 C). A decrease in the natural killer (NK) and T cell module in incipient, subclinical, and clinical TB was observed, with incipient TB showing no other detectable changes generic viagra sildenafil at this stringent level of statistical analysis (Fig. 2 C). The top 30 differentially expressed coding genes (false discovery rate [FDR] P <.

0.05, log2 fold change >1) ranked by fold change, selected from a total of 47 genes in incipient TB, 233 genes in subclinical TB, and 311 genes in clinical generic viagra sildenafil TB, showed that many genes were differentially expressed across all three clinical phenotypes, albeit to different levels (Table 1 and Data S1). These included the genes C1QC***, SERPING1***, ETV7***, and BATF2*** expressed in all three clinical phenotypes. C1QA**, C1QB**, C2**, and EXOC3L1** were expressed in two of the clinical phenotypes, and ANKRD22** and GBP6** were significantly expressed in subclinical TB and clinical TB and were barely elevated above controls in the incipient TB (Data S1, full incipient TB versus control tab). Although certain generic viagra sildenafil genes appeared to be uniquely expressed within each clinical phenotype, most could be detected across the three clinical phenotypes, albeit to differing levels.

Seemingly unique genes within the top 30 gene set of the incipient TB subgroup, such as CCL2, HESX1, PCGF2, LCN8, and SIGLEC1, were only elevated to a very low level against controls in the full set of differentially expressed genes, potentially suggesting that they may come up early in the immune response to M. Tuberculosis, although they were also expressed at a low level in clinical TB (Data S1. Full incipient TB, full clinical generic viagra sildenafil TB versus control tabs). The expression of the complement fixing genes C1QC and C1QB in the top 30 genes of the incipient TB versus control group is in keeping with the Metacore pathway analysis in Fig.

2 B. However, these genes were also differentially generic viagra sildenafil expressed within the top 30 genes of subclinical TB and clinical TB (Data S1. Full subclinical TB, full clinical TB versus control tabs). BATF2 expression increased significantly with increasing disease.

1.4 log2 fold change, P value generic viagra sildenafil 0.0011 in incipient TB. 2.9 log2 fold change, P value 7.6 × 10−12 in subclinical TB. And 3.48 log2 fold change, P value 6.07 × 10−24 in clinical TB. Expression of SERPING1 and generic viagra sildenafil ETV7 showed a similar increase in expression as individuals who progressed to TB (Table 1 and Data S1).

We next analyzed blood transcriptional changes that occurred over time in Leicester TB household contacts as they progressed to TB, together with patients sampled before they were diagnosed with TB (progressors) in view of our findings that high levels of differential gene expression are mainly seen in progressors with subclinical TB and clinical TB, rather than in incipient TB. RNA-Seq data were analyzed in blood from Leicester household contacts of active TB patients generic viagra sildenafil at different time points after recruitment as they progressed to clinical TB (Fig. 4 A. Table S2, top, n = 12 TB contacts.

Total of 21 samples) together generic viagra sildenafil with Leicester patients sampled before they were diagnosed with active TB by culture/microbiological/clinical positivity (Fig. 4 A. Table S2, bottom, n = 11 progressors, total of 12 samples), all before treatment. Active TB patients at the time generic viagra sildenafil of diagnosis (Fig.

4 A, far right. Table S2, top, n = 49 TB patients), all as compared with healthy controls (Table S2, top, n = 38 healthy controls). The biggest changes in gene expression (log2 fold, FDR P generic viagra sildenafil value of 0.05 cutoff) were observed at 0–20 d before TB diagnosis in the contacts (n = 11) and progressors (n = 9. 765 up-regulated and 125 down-regulated genes.

Fig. 4 A) generic viagra sildenafil. Although the change in the number of genes just before diagnosis appeared similar to that observed in active TB patients at the time of diagnosis (1,231 up-regulated and 511 down-regulated genes. Fig.

4 A, far generic viagra sildenafil right), the extent of differential expression in the blood of active TB patients at the time of diagnosis was higher (Fig. 4 A, far right, scale on y axis 0–100. ˆ’log10 P adjusted) as compared with the contacts and progressors sampled before diagnosis (Fig. 4 A generic viagra sildenafil.

0–20 d before diagnosis, scale on y axis 0–15. ˆ’log10 P adjusted). Changes in gene generic viagra sildenafil expression were substantially lower between 21–40 d before diagnosis with low level up-regulation of 185 and down-regulation of 80 genes (Fig. 4 A.

Representative of four TB contacts that progressed to TB and three TB progressors sampled before diagnosis). At 41–832 d before TB diagnosis, when six samples were all from TB contacts subsequently progressing to clinical TB, this change in gene expression was further reduced, with very generic viagra sildenafil low levels of 109 up-regulated and 34 down-regulated genes (Fig. 4 A, far left). We next performed generic viagra sildenafil more in-depth analyses on Leicester TB household contacts alone as they progressed to TB, recruited and sampled from 2015 to 2018 and followed up to date, by pooling our more recently recruited dataset with our previously published dataset (Singhania et al., 2018a.

Total 38 samples from 14 TB household contacts, sampled as they progressed to TB against matched controls. Fig. 4 B and Table generic viagra sildenafil S3). Lower levels of gene expression were now observed between 0 and 31 d before diagnosis with only 23 up-regulated and 1 down-regulated gene (Fig.

4 B. Log2 fold, generic viagra sildenafil FDR P value of 0.05 cutoff. Scale on y axis 0–4. ˆ’log10 P adjusted), including up-regulation of GBP5, SEPTIN4, ETV7, C1QC, BATF2, C1QB, FCGR1A, GBP6, and SERPING1.

Gene expression changes at earlier time points fluctuated over time, with generic viagra sildenafil 15 up-regulated and 8 down-regulated genes observed between days 86 and 150 but not at 32–58 before diagnosis (Fig. 4 B). Up-regulation of these genes was still detected, albeit to a much lower level, at all the earliest time points before diagnosis (Data S2). Some exceptions included C1QB, C1QC, and C1QA, which were in the top seven DEGs at the time points 86–150 before diagnosis, while in the 0–31 d before diagnosis, only C1QB and C1QC were in the top eight expressed genes at the level of fold change generic viagra sildenafil over controls, suggesting heterogeneity of gene expression over time.

We next analyzed a bigger dataset of individuals from South Africa reported to have subsequently progressed to TB, reported to have been sampled at 6 monthly intervals for blood RNA-Seq analysis before diagnosis, although without serial clinical follow-up (Scriba et al., 2017. Zak et al., 2016). We further generic viagra sildenafil subdivided the sampling into tighter time points and examined differential gene expression levels as compared with LTBI nonprogressors recruited in parallel. Again the highest levels of gene expression changes were observed between 0 and 31 d before diagnosis, with 1,155 up-regulated and 473 down-regulated genes (Fig.

4 C. Log2 fold, FDR P value of 0.05 cutoff generic viagra sildenafil. Scale on y axis 0–15. ˆ’log10 P adjusted), including up-regulation of SEPTIN4, SERPING1, BATF2, GBP6, ETV7, and FCGR1A, similar to those detected in our Leicester contacts 0–31 d before diagnosis (Fig.

4 B) generic viagra sildenafil. Expression of C1QA, C1QB, and C1QC was only detectable 0–31 d before diagnosis, perhaps reflecting the level of detectability over controls. Changes in differential gene expression at most other time points before diagnosis were very low (Fig. 4 C and Data S3), although fluctuations in differential gene expression were observed over time, for example with marked changes at 181–250 (88 up-regulated and 46 down-regulated genes) and 251–360 d (51 up-regulated and 3 down-regulated genes) before generic viagra sildenafil diagnosis, as compared with other time points showing minimal changes.

Among the top 30 genes found to be up-regulated between 181 and 250 d before diagnosis were SEPTIN4, GBP6, BATF2, ETV7, SERPING1, and FCGR1A, although these genes were also among the top up-regulated 30 genes at 0–31 d before diagnosis, albeit then at a more significant level (Data S3), suggesting a graded increase in the expression of these genes as progressors approached TB diagnosis, with some heterogeneity of gene expression over time. The blood modular signature of TB contacts and TB patients sampled prediagnosis as generic viagra sildenafil in Fig. 4 A showed a reduction in the NK and T cell module (dominated by IFNG and effector T and NK cell genes) at >40 d before diagnosis, followed by an increase in the innate/hemopoeitic mediator module from 40 d. Increased type I IFN–inducible and inflammasome/granulocyte modules together with a reduction in the NK and T cell, and T and B cell modules were detected at 0–20 d before diagnosis (Fig.

4 D), similar to the reported generic viagra sildenafil TB blood signature (Moreira-Teixeira et al., 2020. Singhania et al., 2018a). The decrease in the NK and T cell module fluctuated over time before diagnosis in progressors, which could reflect fluctuation in the response or heterogeneity in the progressors. Changes in the type I IFN/C’/myeloid and inflammasome/granulocyte modules together with a reduction in the NK and T cell module were observed in Leicester TB contacts alone, generic viagra sildenafil but to a lesser extent only detectable from 30 d before diagnosis using a nominal P value of 0.05 rather than FDR (Fig.

4 E). The initial change at 200–850 d before diagnosis again consisted of a reduction in the NK and T cell module, although this was not consistent, again reflecting heterogeneity over time. Changes in the Zak modular signature over time were also mainly detectable over time using a nominal P generic viagra sildenafil value of 0.05 rather than FDR (Fig. 4 F).

At 0–31 d before diagnosis, the modular signature for the Zak progressors (Fig. 4 F) was almost identical to that of generic viagra sildenafil active TB (Fig. 4 D, far right. Moreira-Teixeira et al., 2020.

Singhania et al., 2018a), although with less enrichment generic viagra sildenafil as at a nominal P value of 0.05 (Fig. 4 F), including enrichment of inflammasome/granulocytes, innate/hemopoetic mediators, innate immunity PRR/C’/granulocytes, IFN/PRR, and IFN/C’/myeloid modules and decreased enrichment of T cell, B cell, and NK and T cell modules. The modular signature was barely detectable at other time points before diagnosis, with the IFN/PRR and IFN/C’/myeloid modules missing at 32–78 and 79–104 d but then present at 181–250 and 251–360 d before diagnosis, again suggesting temporal heterogeneity of gene expression or potential re as reported in high-burden TB settings (Charalambous et al., 2008. Uys et al., 2015 generic viagra sildenafil.

Van Helden et al., 2008. Van Rie et al., 2005. Van Rie et al., 1999 generic viagra sildenafil. Verver et al., 2005).

To investigate the heterogeneity among Leicester TB household contacts and the Zak progressors, the average gene expression value of the 30-gene signatures (from Table 1) derived from incipient TB (blue), subclinical TB (orange), and clinical TB (red) was assessed at different time points before diagnosis in individual Leicester TB contacts (n = 9) and individual Zak progressors (SupTab1. SupTab6_RNA-Seq-Metadata from Zak et al., generic viagra sildenafil 2016. Training set n = 18. GEO accession generic viagra sildenafil no.

GSE79362) where two or more sampling time points were evident. The average 30-gene incipient TB, subclinical TB, and clinical TB signatures were shown to be marginally elevated over the baseline (dotted line for each) in four of the Leicester TB household contacts at all time points analyzed before TB diagnosis, 0–30 d, with the subclinical TB and clinical TB signatures showing slightly better performance (Fig. 5 A, shorter time generic viagra sildenafil points, n = 4). Three out of four of these contacts who progressed rapidly to TB disease had been infected with an outbreak strain of M.

Tuberculosis identified by whole genome sequencing. TB contacts who progressed over 100–200 d showed generic viagra sildenafil a greater elevation against baseline, similar for the incipient, subclinical, and clinical TB 30-gene signatures, increasing at times close to TB diagnosis (Fig. 5 A, longer time points). One TB contact (Patient ID 493) showed some fluctuation, although always above baseline for all three signatures (Fig.

5 A, longer time points, n = 5) generic viagra sildenafil. The average 30-gene incipient TB, subclinical TB, and clinical TB signatures showed an increase over the baseline (dotted line, LTBI controls) in the Zak progressor patients between 4–600 d before diagnosis (Fig. 5 B), although a sharp increase in the signatures over time was only observed in around five of the progressors from just >200 d to a maximum before diagnosis. Other patients showed heterogeneity generic viagra sildenafil in expression of these 30-gene signatures over time, many showing elevated signatures maintained at the same level over time, with others actually decreasing (Fig.

5 B). The published Zak 16-gene signature showed almost superimposable curves with very similar increases above the baseline controls over time in the individual Leicester TB household contacts (Fig. S1 A, shorter time points and longer time points), and in the Zak progressors, with identical increases in the five individuals and the same heterogeneity as observed generic viagra sildenafil with the 30-gene signatures (Fig. S1 B).

There is currently a need for early biomarkers to monitor TB treatment success earlier and to evaluate robustly the duration of treatment required in TB patients to adapt drug treatment regimens. To establish treatment response signatures, RNA-Seq was performed on blood from 74 TB patients at diagnosis (treatment-naive), and longitudinally, at generic viagra sildenafil carefully planned time points during TB treatment. We first monitored the transcriptional response to treatment across the whole cohort, and second monitored the transcriptional response of individual patients to identify distinct profiles of their transcriptional response that might help to stratify clinical treatment phenotypes. Blood was collected and subjected to RNA-Seq from the 74 TB patients at diagnosis before treatment and thereafter, at 1 and 2 wk, at 1, 2, 4, 5, 6, 7/8, 9/10, and 11/12 mo, and at >1-yr after treatment (Fig.

S2, A generic viagra sildenafil and B) from clinically defined patients. Pulmonary TB, difficult TB cases, TB drug–resistant, outbreak TB strain, and other TB progressors (Table S4). TB patients received either standard anti-TB treatment (ATT. 200 d or less) or generic viagra sildenafil extended ATT (>200 d.

Table S4), according to their clinical assessment through treatment, with smear-positive patients mostly falling within the extended ATT patient group (Fig. S2 C) generic viagra sildenafil. The sample-to-sample correlation heatmap and principal component analysis (PCA) of all the active TB patients at diagnosis before treatment and at the different time points during the treatment course showed samples to mainly cluster according to time points, with some heterogeneity (Fig. S2, D and E).

The top 1,000 most variable gene expression heatmap distinguished patients according to time of treatment, and generic viagra sildenafil according to smear positivity and negativity at treatment initiation (T0. Fig. S2 D). The innate/hemopoietic, IFN/PRR, and IFN/C’/myeloid modules were found to be over-abundant as compared with controls before treatment and decrease in abundance to different degrees within all the subgroups after T0, except for in the TB drug resistant generic viagra sildenafil subgroup (Fig.

6 A). These modules decreased in abundance after 1 wk of treatment and were completely abrogated after 4 mo of treatment in the standard ATT subgroup. Although the extended generic viagra sildenafil ATT and difficult TB cases subgroups showed a similar pattern to the standard ATT subgroup, a stronger modular signature before treatment and an incomplete diminishment after 6 mo were observed, when a standard treatment course would be completed. The outbreak TB strains subgroup showed a similar but weaker global modular signature to the standard ATT subgroup, also resolving within 4 mo of treatment.

However, a small subgroup of four patients, the TB drug–resistant subgroup, showed a distinct modular signature that for the most part was not diminished, in accordance with these patients requiring altered drug treatment regimens for a longer period (Fig. 6 A) generic viagra sildenafil. The standard and extended ATT subgroups contained a large number of patients such that the modular signature was more robust than in the other three subgroups, which contained lower numbers of patients (Fig. 6 A).

We therefore examined the members of each of these three subgroups individually and show that at the level of the individual, these modular responses to treatment are heterogeneous and so should be validated in larger cohorts in generic viagra sildenafil future studies (Fig. S3). The number of DEGs compared with controls was also reduced upon treatment (Fig. 6 B) generic viagra sildenafil.

Smear-positive and smear-negative TB patients showed a similar modular and gene expression decrease during treatment with complete diminishment by 4–5 mo, although the smear-positive patients had a stronger modular signature before treatment (data not shown). We then identified a 212-gene signature (TREAT-TB212) that showed the response to treatment across the whole Leicester cohort, mainly showing decreased gene expression as compared with controls over the treatment course, which reverted to the expression profile of healthy controls by 4 mo of treatment in most but not all of the patients (Fig. 7 A) and in an independent treatment response cohort dataset (Fig generic viagra sildenafil. 7 B.

Thompson et al., 2017). The TREAT-TB212 signature in not-cured patients from the Thompson cohort was sustained at all time points up to 24 wk at similar levels to that of the generic viagra sildenafil pre- and very early treatment response signatures (Fig. 7 B), at a comparable level to the Leicester cohort of active TB patients, TB progressors recruited as TB household contacts after diagnosis, and most different clinical treatment response subgroups (Fig. 7 A) generic viagra sildenafil.

A higher TREAT-TB212 signature was observed in patients receiving treatment for >200 d but this was observed only early after T0, as compared with those receiving standard treatment of up to 200 d (Fig. 7 A). In keeping with the modular and differential gene expression analyses, the TREAT-TB212 signature was only different in the smear-positive and -negative patients at T0 but generic viagra sildenafil not between 1 wk to 1 yr after T0, indicating that the patients were responding similarly to treatment (Fig. 7 A).

The log2 fold-change of all TREAT-TB212 genes against controls verified changes in gene expression upon treatment (Fig. 7 C, generic viagra sildenafil Leicester cohort) with a similar profile in the Thompson cohort (Fig. 7 D). Again, most of the gene expression profiles reverted to that of healthy controls by 4 mo of treatment (Fig.

7 C, Leicester cohort), although this could not be evaluated in the Thompson cohort due to fewer sampling visits generic viagra sildenafil that did not include this time point (Fig. 7 D). The TREAT-TB212 signature was further reduced to a 27-gene signature (TREAT-TB27), which selected genes with the greatest changes in expression over the treatment course in the whole cohort (Fig. 7 E), and generic viagra sildenafil its validity was confirmed also in the Thompson cohort (Fig.

7 F). Although TB patients had been subgrouped according to their clinical phenotype in response to treatment, as standard ATT, extended ATT, difficult TB cases, TB drug–resistant, and outbreak TB strains, the TREAT-TB212 signature did not show a clear transcriptional response trend according to their clinical definition except for most of the drug-resistant group (Fig. 7 G, generic viagra sildenafil compared with T0). However, by monitoring the transcriptional response of individual patients according to their TREAT-TB212 signature profile, regardless of their clinical subgroups but where samples at all time points had been obtained, four distinct transcriptional profiles were revealed.

Expected, resembling standard ATT. Weaker, as generic viagra sildenafil compared with standard ATT. Or stronger initial or stronger delayed, as compared with standard ATT (Fig. 7 H, compared with T0).

Strikingly, stronger initial generic viagra sildenafil or stronger delayed transcriptional response patient groups showed differences in the transcriptional response already at 1 and 2 wk after T0, although at week 1 after treatment, C-reactive protein (CRP) levels in both groups were comparable (46.00 mg/l, stronger initial. 34.00 mg/l, stronger delayed). The stronger delayed patient group displayed elevated levels of CRP even after 1 mo of treatment as compared to stronger initial (8 mg/l, stronger initial. 38 mg/l, stronger generic viagra sildenafil delayed group), also correlating with minimal changes in radiographical signs of disease (data not shown), suggesting continued inflammation and potentially in the stronger delayed.

Thus the treatment response could not be predicted clinically by CRP levels early but could be predicted by the different kinetics of the transcriptional response observed as early as 1 wk after T0 in the stronger delayed as compared with the stronger initial group, supporting the role of transcriptional biomarkers as more sensitive measures of the treatment response than existing clinical markers. To develop a reduced transcriptional signature that may enable early identification of poorer treatment responders, based on generic viagra sildenafil the stronger initial and stronger delayed groups, the differential expression of TREAT-TB212 between two consecutive time points from T0 to 1 wk, 1 to 2 wk, and 2 wk to 1 mo was computed leading to a reduced signature (EarlyRESP-TB25. Fig. 7 H and Fig.

S4). EarlyRESP-TB25 showed differences in the stronger initial and stronger delayed patient groups by their different transcriptomic profiles at 1–2 wk after T0 (Fig. 7 J), with similar but not optimal results observed for TREAT-TB27 (Fig. 7 I.

Derived gene lists TREAT-TB27, EarlyRESP-TB25. Fig. S4, A and B). Reported reduced blood signatures of TB diagnosis or risk show little to no overlap with each other, and most have been tested for distinguishing active TB from LTBI but not active TB from other diseases (ODs.

Kaforou et al., 2013. Maertzdorf et al., 2016. Roe et al., 2016. Singhania et al., 2018a.

Suliman et al., 2018. Sweeney et al., 2016. Zak et al., 2016. Reviewed in Singhania et al., 2018b).

We set out to develop an optimized reduced signature that would distinguish active TB from ODs as well as LTBI. Combined reduced blood signatures of TB diagnosis or risk comprising 101 distinct genes (Kaforou et al., 2013. Maertzdorf et al., 2016. Roe et al., 2016.

Singhania et al., 2018a. Suliman et al., 2018. Sweeney et al., 2016. Zak et al., 2016.

Unpublished data) were analyzed in 10 published datasets from multiple clinical disease cohorts including active TB, LTBI, and ODs and healthy controls (Bloom et al., 2013. Parnell et al., 2012. Suarez et al., 2015. Zhai et al., 2015), and the pooled dataset was batch-corrected (Fig.

S5, A and B. And Materials and methods). 12 genes were identified in the reduced signature that were shared between the top 30 genes distinguishing active TB from LTBI, and active TB from ODs, ranked by decreasing importance (mean decrease accuracy. Fig.

8, A and B), further reduced to 10 genes (TB10) based on performance (area under the curve [AUC] and accuracy) on pooled cohort datasets with independent validation (Fig. S5 C and Table S5). This TB10 signature originating from the different reduced reported signatures (Fig. 8 C) comprised genes that were either up- or down-regulated in active TB as compared with controls, LTBI, and ODs (Fig.

8 D) and was shown to be significantly different in TB versus ODs and LTBI and controls using ANOVA (Data S4). Removal of the GBP5 gene (12th in rank) reported to discriminate active TB and LTBI did not improve the performances for discrimination of active TB from ODs. Individual gene expression was heterogeneous across patients with active TB and the ODs (Fig. 8 D, likely reflecting the different extents of morbidity.

Berry et al., 2010. Bloom et al., 2013). The performances of TB10 were tested and compared with the signatures that we describe in this study, including the 30-gene incipient TB, the 30-gene subclinical TB, the 30-gene clinical TB, the TREAT-TB27, and the EarlyRESP-TB25 (Fig. S5 D), and previously published reduced signatures (Fig.

8 E and Fig. S5 E. Kaforou et al., 2013. Maertzdorf et al., 2016.

Roe et al., 2016. Singhania et al., 2018a. Suliman et al., 2018. Sweeney et al., 2016.

Zak et al., 2016. Described in Materials and methods). TB10 showed the best performance for TB versus ODs (Fig. 8 E and Fig.

S5 D. AUC, 0.999. Accuracy, 0.976. And 95% confidence interval [CI], 0.9959–1), and TB versus LTBI (Fig.

S5 E. AUC, 0.971. Accuracy, 0.943. 95% CI, 0.9343–1).

Although the Suliman reduced signature was comparable to TB10 for distinguishing TB from LTBI (Fig. S5 E), it showed poorer performance for distinguishing TB from ODs (Fig. 8 E). Blood transcriptomics have revealed major characteristics of the immune response in TB, show promise to support TB diagnosis, and would be of great use to identify individuals with asymptomatic incipient TB or subclinical TB before they progress to clinical TB to facilitate targeted early treatment and reduce onward transmission.

Moreover, new tools for effective TB treatment monitoring are needed to determine when and if patients are responding to treatment to provide a personalized approach to treatment and accelerate screening of new anti-TB drugs. To achieve this, a detailed knowledge of how the host immune response develops over time and relates to the state of M. Tuberculosis is needed. We now show, in a unique clinically and temporally well-defined cohort of household contacts of active TB patients, that minimal changes in blood gene expression are detectable in incipient TB, increasing as patients progress to subclinical TB, and maximal at the time of presentation with clinical TB, with similar results for published reduced risk signatures of TB.

Although the transcriptional signatures increased with time and were most highly expressed around 30 d before diagnosis, there was heterogeneity over time in the response in the TB contacts as they progressed to TB and a published cohort of TB progressors from a high-burden TB setting. Blood signatures at detailed time points during ATT additionally allowed us to define signatures that can distinguish early and late responders. Finally, we demonstrate comparable performance of immune signatures developed for TB diagnosis and detection of early stages of M. Tuberculosis and their reduction upon TB treatment monitoring, with subtle differences for different signatures at different stages of development and diminishment of TB disease.

The temporality of gene expression changes during progression, and resolution of active TB may provide mechanistic insights toward the development of host therapies and supports a framework for future development of biomarkers to improve the clinical management of LTBI and active TB. TB contacts that progress to TB showed a decrease in the NK and T cell effector modular signature, which was detectable from the earliest stages of progression, in keeping with findings that cytotoxic effector molecules and NK cells are important for protection against M. Tuberculosis in human TB (Roy Chowdhury et al., 2018). We observed an increase in the inflammatory and IFN modular signatures in subclinical TB and clinical TB at time points closest to TB diagnosis in both Leicester contacts and Zak TB progressors, but not in incipient TB.

That the IFN modular signature fluctuated with time in Zak progressors potentially explains differing reports that type I/II IFN signaling and the complement cascade were elevated 18 mo before TB disease diagnosis in this cohort (Scriba et al., 2017), with others suggesting increased expression of complement genes in subclinical TB closer to diagnosis (Esmail et al., 2018). Our analysis at more detailed time points of the Zak cohort suggests elevated type I IFN signaling, and complement genes at 18 mo before diagnosis may indicate individual patient heterogeneity, as we discuss below. A marked decrease in the B and T cell modular signatures and increase in other modules found in active TB including myeloid inflammation, lymphoid, and monocyte and neutrophil gene modules occurred in progressors more proximally to TB disease, in keeping with Scriba et al. (2017).

A reciprocal reduction in the inflammation and IFN modules was observed after a week of successful treatment and was restored to that of healthy controls by 4 mo, together with the B and T cell modular signatures. Our findings that the evolution of the immune response on progression to TB shows the reverse upon treatment is suggestive that reduced signatures optimized to support early diagnosis of TB may also reflect the changes that occur in response to treatment. Reduced blood signatures have been proposed to determine the risk of exposed individuals to their subsequently developing TB (Penn-Nicholson et al., 2020. Singhania et al., 2018a.

Singhania et al., 2018b. Suliman et al., 2018. Zak et al., 2016). However, it was unclear at the time whether any of these reported blood signatures of TB risk predicted progressors at stages of incipient or subclinical TB.

We now show that while these published gene risk signatures are expressed during subclinical TB and clinical TB, only SERPING1, ETV7, and BATF2 from the 16-gene Zak signature were up-regulated, albeit at a very low level, in incipient TB, similarly to the global transcriptional expression signature in these Leicester contact clinical phenotypes. 7 of the 16-gene signature reported by Zak et al. (2016) were among the 30 most highly expressed genes across the Leicester contact clinical phenotypes of TB progression, including FCRGR1A, SEPT4, GBP5, ANKRD22, SERPING1, ETV7, and BATF2. Although C1QC, SERPING1, ETV7, and BATF2 were among the top 30 DEGs in all the clinical phenotypes, increasing statistical significance of differential gene expression was observed with progression from incipient to subclinical to clinical TB, suggesting that they may be early indicators of M.

Tuberculosis and reflect the evolution of the immune response with time after . In support of this, IFN-α/β signaling pathways by modular and Metacore analysis were observed in subclinical TB and more so in clinical TB, but were not apparent in incipient TB. Collectively, our findings suggest that the levels of gene expression increase with progressive from incipient to subclinical to active TB, potentially explaining why distinct published signatures of TB risk show little overlap with each other (Penn-Nicholson et al., 2020. Singhania et al., 2018a.

Suliman et al., 2018. Zak et al., 2016. Reviewed in Singhania et al., 2018b) due to differential levels of detectable gene expression and/or uncertainty in the stage of at which sampling was performed. The unique gene set expressed in incipient TB is characterized by low-level gene expression changes across multiple pathways, which may limit their value for predicting which incipient TB patients will progress to clinical TB.

However, genes that were differentially expressed in subclinical TB and clinical TB at a high and significant level were detectable in incipient TB, supporting suggestions that serial testing among carefully selected clinical target groups might be required for optimal implementation of biomarkers for TB risk (Esmail et al., 2020. Gupta et al., 2020). Indeed, inclusion of clinical details of TB progressors over detailed sampling times after exposure/ with M. Tuberculosis allowed us to define progressors as incipient TB and subclinical TB (Drain et al., 2018) and to assign changes in blood gene expression at an early stage of to each clinical phenotype, providing a framework for improved biomarker selection to target early TB treatment and block onward transmission.

Our findings of temporal heterogeneity in the blood transcriptional response of Leicester TB progressors and more so in the Zak TB progressors (Zak et al., 2016), both at bulk cohort levels as well as in individual progressors, likely reflect the dynamic nature of the host–pathogen interaction over time, and are consistent with observations in positron emission tomography scan and computed tomography scan studies of progressive in humans with LTBI coinfected with HIV/TB and in nonhuman primates (Barry et al., 2009. Esmail et al., 2016. Lin et al., 2016). The majority of Leicester contacts progressed over 100–200 d, displaying a greater elevation in their blood signatures against the baseline as they progressed to TB.

However, a subset showed rapid progression with an increased signature close to diagnosis, mostly explained by with an outbreak strain of M. Tuberculosis, supporting previous reports of differing virulence of M. Tuberculosis strains (Coscolla and Gagneux, 2014), suggesting that the time at which protective immune responses were overwhelmed leading to TB progression could differ according to the infecting strain of M. Tuberculosis.

Detailed analysis of individuals in the Zak et al. (2016) cohort, sampled for >600 d, showed a sharp increase in the signatures over time in only five progressors from 200 d before diagnosis, progressing sharply to the highest signature before diagnosis, again suggesting that immune responses had been overwhelmed. However, in other Zak progressors, the fate of appeared to hang in the balance for a prolonged period until diagnosis, possibly reflecting subclinical disease, although in-depth clinical analysis was not performed at these earlier sampling time points in the study. Moreover, since it was not a study of recent TB contacts, there was no knowledge of time of exposure or the infecting M.

Tuberculosis strain. Our findings are consistent with a recent randomized controlled clinical trial for biomarker-guided tuberculosis preventive therapy (termed CORTIS. NCT02735590), which concluded that a reduced signature RISK11 derived from the Zak 16-gene signature (Zak et al., 2016) was better suited to screening of symptomatic individuals with possible early clinical TB than for mass community-based screening for incipient TB (Scriba et al., 2021). It is unclear whether this signature can distinguish subclinical or active TB from other s, particularly viral s, which may present with symptoms similar to clinical TB and are also dominated by type I IFN signaling (Singhania et al., 2018a.

Singhania et al., 2018b). Improved biomarkers to monitor TB treatment success are needed to reliably evaluate the duration of treatment required in individual TB patients and deliver optimal drug treatment regimens. We adopted both clinical and bioinformatics approaches to develop blood signatures of the treatment response. Using the clinical approach, individual patients of the Leicester TB cohort were stratified on the basis of treatment response (standard ATT, extended ATT, and difficult TB) and infecting strain characteristics (drug-resistant TB and outbreak TB strains).

The treatment response signature corresponded with their clinical treatment response, discriminating both the subgroup of drug resistant patients in Leicester responding more slowly to treatment, and the subgroup labeled as not cured in the African Thompson cohort (Thompson et al., 2017), supporting identification of patients not responding to treatment. Using the bioinformatics approach, we monitored the transcriptional response of individual patients, independent of their clinically defined treatment phenotype, and defined four types of TB patients. The expected group responding standardly to treatment. The weaker group defining a subgroup with a lower grade of (low CRP, and longer time to M.

Tuberculosis culture positivity). And the stronger initial and stronger delayed groups, showing differences in their transcriptional response at 1 wk after T0. Differences between these latter two subgroups were not discernible by clinical measures of their treatment response, such as CRP and x ray, at these early time points, supporting the utility of transcriptional biomarkers as more sensitive measures of the treatment response than existing clinical markers, to inform clinical management of TB patients and to support drug development platforms and future drug treatment trials. Existing TB diagnostic tools are limited in their scope and dependent on sputum availability for rapid identification of TB.

Diagnostic blood transcriptomic signatures of TB may provide a pathway to support early diagnosis for a broader spectrum of disease phenotypes, although there is little consensus between reported reduced signatures for TB risk and those that distinguish active TB from LTBI (Kaforou et al., 2013. Maertzdorf et al., 2016. Penn-Nicholson et al., 2020. Roe et al., 2016.

Scriba et al., 2021. Singhania et al., 2018a. Singhania et al., 2018b. Suliman et al., 2018.

Sweeney et al., 2016. Zak et al., 2016). Moreover, there is a need to distinguish TB from other confounding diseases (Kaforou et al., 2013. Singhania et al., 2018a.

Singhania et al., 2018b). Our blood signature, TB10, derived from published reduced signatures tested on multiple disease cohorts, optimally distinguished patients with TB from those with LTBI, and TB from those with ODs. Although some of the published signatures had similar performance in distinguishing TB from LTBI, they had poorer performance than TB10 in distinguishing TB from ODs. All reduced signatures derived in this study, including the 30-gene signatures of incipient TB, subclinical TB, and clinical TB progression and the new treatment-monitoring signatures TREAT-TB27 and EarlyRESP-TB25, showed poorer performances than TB10 in distinguishing TB from LTBI and in distinguishing TB from ODs, suggesting that the top gene expression changes that occur temporally upon progression to TB may not exactly match the top gene set that is temporally diminished after TB treatment.

All signatures increased in subclinical TB and maximally in clinical TB, also decreasing at week 1 after treatment, with a further decrease at month 2 and complete disappearance of gene expression changes by month 6. However, all signatures barely showed a significant increase in expression in incipient TB above controls, with the exception of the 30-gene incipient TB signature, which potentially could be further reduced to the most highly expressed genes and optimized to give the highest sensitivity of gene expression to detect what is likely to be early M. Tuberculosis in asymptomatic individuals with incipient TB and subclinical TB, at risk of progression to TB. The global aim of our study, however, was not to develop optimized signatures of risk and progression to TB, but to use signatures developed at different stages of disease progression and treatment to determine how and to what extent they are perturbed in the different clinical phenotypes of progression to TB, during active TB, and upon treatment.

In conclusion, using in-depth temporal analysis of gene expression changes over time in a cohort of clinically well-characterized household contacts of TB patients from a moderate-burden TB setting with minimal risk of re, together with reanalysis of gene expression at more detailed time points in a published cohort of TB progressors from a high TB burden setting, we demonstrate significant heterogeneity in changes of gene expression, at both the bulk cohort level and in individual patients, as they progress to TB. This has major implications for assessing TB risk in individuals with LTBI. Our characterization of the immune response underlying the evolution and resolution of TB provides a framework for biomarker development to improve clinical management of this disease. Between September 2015 and September 2018, longitudinal cohorts of active TB and TB contacts were recruited from the clinical TB service at Glenfield Hospital, University Hospitals of Leicester National Health Service Trust, Leicester, UK (Table S1, top).

All participants were prospectively enrolled and sampled before the initiation of any TB treatment. The Research Ethics Committee for East Midlands–Nottingham 1, Nottingham, UK (REC 15/EM/0109) approved the study. All participants gave written informed consent. All active TB patients had microbiologically confirmed disease with whole-genome sequencing of culture isolates performed for case linkage with contacts.

All participants were prospectively followed with visits at scheduled time points from the time of diagnosis (pretreatment) until 12 mo after completing treatment. Household TB contacts were identified through routine contact tracing and underwent systematic baseline investigation with routine CXR, IFN-γ release assay for M. Tuberculosis reactivity (IGRA) testing, and a symptom questionnaire. Sputum was collected if participants were spontaneously expectorating, and bronchoscopy performed in those not expectorating with clinical or radiological suspicion of TB.

On this basis, participants were classified with LTBI, subclinical TB, or active TB. Participants with LTBI were prospectively followed up for a minimum period of 2 yr with scheduled follow-up visits at 3–6 monthly intervals. At each visit, a symptom screen and CXR was performed and a blood RNA sample collected. In total, 356 TB contacts were recruited (150 IGRA-positive and 206 IGRA-negative [IGRA-ve]).

To date, 20 participants from this cohort have been diagnosed with TB and classified as TB progressors, although 6 were excluded from the gene expression study due to either cDNA library failure (n = 1) or failure to secure microbiological confirmation (n = 5) essential for case linkage with the index case. In these cases, the diagnosis of active TB was based on clinical symptoms, typical radiological features, and supporting histology from the site of . At each visit, a symptom screen and CXR were performed and a blood RNA sample collected. At the time of our publication in 2018 (Singhania et al., 2018a), we reported a modified disease risk score using a TB-specific 20-gene signature in 9 TB contacts who had developed TB during the study, with 99 contacts remaining healthy for 2 yr or more, but performed no detailed analysis on changes in gene expression and the immune response.

Since then, not considering the excluded progressors detailed above, an additional five TB contacts (n = 5) developed TB and were included for in-depth temporal analysis of the blood signature of TB progressors at different time points before diagnosis. In total, blood samples from 14 contacts who progressed to TB were subjected to RNA sequencing out of the 20 contacts who progressed to TB. Six were excluded from the RNA-Seq gene expression study due to either cDNA library failure (n = 1) or failure to secure microbiological confirmation (n = 5). This was important as essential for case linkage with the index case, although they were confirmed as progressing to TB by positive histology showing caseating lungs.

The contacts had the following characteristics. Gender, 35.7% male and 64.3% female. Ethnicity, 28.6% South Asian, 14.3% East African, 42.9% British Caucasian (5/6 outbreak strain), and 14.3% European. The controls had the following characteristics.

Gender, 47.1% male and 52.9% female. Ethnicity, 64.7% South Asian, 11.8% East African, 5.9% European, and 17.6% British Indian. Since these were small numbers and skewed somewhat by the British Caucasian and gender, we applied COMBAT batch correction as described later in the Materials and methods. We collected blood at detailed time points to examine in detail changes in gene expression in the different clinical phenotypes, incipient TB, subclinical TB, and clinical TB (Table S1), and also to examine changes in gene expression occurring over time in Leicester cohorts of contacts of active TB patients (Table S2, top.

N = 12 TB contacts. 25 samples. Singhania et al., 2018a) and from noncontact patients with symptoms before they were diagnosed with active TB by culture/microbiological/clinical positivity (Table S2, bottom. N = 10 TB progressors.

14 samples), all before treatment, and from active TB patients at the time of diagnosis (Table S2, top, n = 49 TB patients), all as compared with healthy controls (Table S2, top, n = 38 healthy controls). Blood from TB contacts who progressed to TB and from TB progressors was subjected to RNA-Seq and analysis at the time points indicated (Table S2), together with that from 49 newly recruited active TB patients at the time of diagnosis, before initiation of treatment. To investigate this further in Leicester TB contacts who progressed to TB only, datasets representing all time points, sampled before diagnosis throughout 2015 to 2018, were analyzed in TB contacts only that progressed to TB, by pooling our previously published TB contact dataset (Singhania et al., 2018a), which had not been investigated in depth with respect to kinetic changes in the immune responses, with our more recently recruited TB contact dataset (Table S3). This pooled dataset, now consisting of 38 samples from 14 TB contacts as they progressed to TB, was batch corrected and analyzed against matched controls, as described later in the Materials and methods.

The treatment response cohort had the following characteristics. The Leicester active TB cohort composed of 74 patients with pulmonary TB was simultaneously recruited between September 2015 and September 2018, at the Glenfield Hospital, University Hospitals of Leicester National Health Service Trust, Leicester, UK, at the time of diagnosis (treatment-naive. Fig. S2 A and Table S4).

A cohort of 38 healthy IGRA-ve controls was recruited in parallel. To follow the transcriptional response after treatment, whole blood samples were collected and were subjected to RNA-Seq at diagnosis before initiation of any ATT (T0), and thereafter, at 1 and 2 wk. 1, 2, 4, 5, 6, 7/8, 9/10, and 11/12 mo. And >1 yr after T0 with clinical assessment including CXR, CRP, and symptom assessment.

All RNA isolation and processing were performed on all blood samples simultaneously (Fig. S2 and Table S4). Patients who had previous TB, had previous treatment for LTBI, were pregnant, were under 16 yr age, or were immunosuppressed were excluded from this study. All participants had routine HIV testing, and patients with a positive result were excluded.

Patients with active TB were all confirmed by laboratory isolation of M. Tuberculosis on the culture of a respiratory specimen (sputum or bronchoalveolar wash/lavage) with sensitivity testing performed by the Public Health Laboratory Birmingham, Heart of England National Health Service Foundation Trust, Birmingham, UK. All participants were prospectively enrolled and sampled before the initiation of any TB treatment. The Research Ethics Committee for East Midlands–Nottingham 1, Nottingham, UK (REC 15/EM/0109), approved the study.

All participants gave written informed consent. We used 10 published blood RNA-Seq or microarray datasets (Berry et al., 2010. Bloom et al., 2013. Leong et al., 2018.

Parnell et al., 2012. Singhania et al., 2018a. Suarez et al., 2015. Thompson et al., 2017.

Zak et al., 2016. Zhai et al., 2015) from multiple clinical disease cohorts including active TB (225 patients) and LTBI (217 individuals) from Berry, London and South Africa (GEO accession nos. GSE107991 and GSE107992). Bloom (GEO accession no.

GSE42834). Singhania, Leicester (GEO accession no. GSE107993). Zak (GEO accession no.

GSE79362). Thompson (GEO accession no. GSE89403). Leong (GEO accession no.

GSE101705). And ODs influenza (Parnell [GEO accession no. GSE40012] and Zhai [GEO accession no. GSE68310]), Bloom lung cancer (GEO accession no.

GSE42834), Bloom pneumonia (GEO accession no. GSE42834), Bloom sarcoidosis (GEO accession no. GSE42834), and Suarez bacterial/viral s (GEO accession no. GSE60244.

Total 186 patients). And healthy controls from each respective dataset (223 individuals). We downloaded from GEO the filtered and normalized datasets, which have been normalized with different methods, according to type of data (RNA-Seq or Illumina microarray) or laboratory practices. We then pooled the 10 datasets together, matching the targets by gene names.

When a gene was absent in a least one dataset of origin, we completely removed a gene from the pooled dataset, so that we had a robust and stringent pooled dataset. A gene could be absent from any dataset for multiple reasons. One of the platforms did not target this gene, the gene annotation databases used were different for each dataset (different version of the genomes) and there was no correspondence of gene name, or the gene was filtered out due to low expression in the filtered dataset of origin. Using these stringent criteria, we had a pooled dataset containing 11,912 genes in total, regrouping 851 individual whole blood samples.

We then batch-corrected the pooled dataset, the batch being the origin of dataset, with the reference COMBAT algorithm (Johnson et al., 2007) from the sva library in R. We checked the impact of batch correction on a mix of RNA-Seq and microarray datasets by drawing PCA plots (Fig. S5 A). We also verified high correlations before/after batch correction per group of patients (Fig.

S5 B) and expression on gene of interest (data not shown). From the 11,912 batch-corrected genes, we then selected the 101 genes that were contained in at least one of the nine published reduced gene signatures (Kaforou et al., 2013. Maertzdorf et al., 2016. Roe et al., 2016.

Singhania et al., 2018a. Suliman et al., 2018. Sweeney et al., 2016. Zak et al., 2016.

And reviewed in Singhania et al., 2018b. And unpublished data). Trang Tran’s 20-gene signature was independently derived from both Berry London (Berry et al., 2010. GEO accession no.

GSE107991) and Leicester RNA-Seq datasets (GEO accession no. GSE107993). Differential gene expression analyses were performed on active TB patients compared with controls, LTBI, or controls plus LTBI individuals, using Wald tests (DESeq2 library. Love et al., 2014) by fitting generalized linear models.

A gradient-boosting machine algorithm (gbm v2.1.7 library) was applied on the lists of DEGs to determine the high order ranking of genes predicting the active TB status. For signature reduction, we performed a random forest algorithm (randomForest library v4.6-14) based on cumulative sensitivity of genes in their importance order. Finally, the meta-data with cross-validation analysis combining two optimal signatures from microarray datasets from Berry et al. (2010) and six optimal signatures from the Berry London and Leicester RNA-Seq datasets (TB versus controls, TB versus LTBI, TB versus controls plus LTBI for each cohort) yielded 20 gene signatures (FCGR1A, GBP5, SEPT4, ANKRD22, BATF2, FCGR1B, GBP1, GBP6, LHFPL2, SERPING1, C1QB, CD274, GBP4, AIM2, FBXO6, PSTPIP2, ASPHD2, FCMR, RTP4,and APOL6).

Genes ranked by DESeq2 Wald statistic for TB progressor patients at different time points or with different clinical symptoms compared with controls were used to look for enrichment of either the hallmark gene set using Broad’s gene set enrichment analysis preranked analysis and default settings. The normalized enrichment score and the FDR were plotted using ggplot2. The different genes lists of DEG in the different clinical phenotypes, incipient TB, subclinical TB, and clinical TB (Fig. 2) were functionally annotated using Metacore (Thomson Reuters v 19.4).

R libraries used. All analyses have been made with R 3.5.1, using multiple libraries, and Bioconductor v3.8 (Anders et al., 2015). The libraries are. Arules v1.6-4, sva v3.30.1 (Leek et al., 2012), Boruta v6.0.0 (Kursa and Rudnicki, 2010), ranger v0.12.1, ImpulseDE2 v1.6.1 (Fischer, 2019), VennDiagram v1.6.20, caret v6.0-85, lattice v0.20-38, RColorBrewer v1.1-2, tibble v2.1.3, tidyr v0.8.3, dplyr v0.8.1, DESeq2 v1.22.2 (Love et al., 2014), ComplexHeatmap v2.3.1 (Gu et al., 2016), ROCR v1.0-7, randomForest v4.6-14, ggbiplot v0.55, ggplot2 v3.2.0, and qusage v2.16.1 (Yaari et al., 2013).

We acknowledge the Francis Crick Advanced Sequencing Facility, Bioinformatics and Biostatistics Science Technology Platforms, for their contribution to our sequencing processing, L. Moreira-Teixeira for reviewing the manuscript, and Marisol Alvarez Martinez for helpful advice on bioinformatic approaches used. A. O’Garra, O.

Tabone, C.M. Graham, and A. Singhania and part of the project were funded by the Francis Crick Institute (Crick 10126. Crick 10468), which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.

The project, part of O. Tabone's salary, and salaries for J. Lee and R. Verma were funded by the BIOASTER Microbiology Technology Institute, Lyon, France (with funding from the French Government Investissement d’Avenir program ANR-10-AIRT-03) and the Medical Diagnostic Discovery Department, bioMérieux SA, France.

P. Haldar and R. Verma were supported by the National Institute for Health Research Leicester Biomedical Research Centre and the University of Leicester. W.J.

Branchett is funded by a Wellcome Investigator Award to A. O’Garra (FC11028). Author contributions. A.

O’Garra and P. Haldar co-led the study. A. O’Garra, P.

Haldar, R. Verma, G. Woltmann, M. Rodrigue, and P.

Leissner designed the study and discussed the findings throughout the project with K. Kaiser, O. Tabone, P. Chakravarty, A.

Singhania, and W.J. Branchett. R. Verma and J.

Lee recruited TB, LTBI, and contacts for the Leicester cohort. C.M. Graham performed RNA-Seq sample processing and helped to organize receipt of samples at The Crick and identifiable storage. O.

Tabone and P. Chakravarty performed bioinformatics analysis with major input from A. Singhania, and some input from T. Trang and F.

Reynier, all overseen by A. O’Garra. A. O’Garra wrote the manuscript with input from P.

Haldar, W.J. Branchett, O. Tabone, and A. Singhania.

All co-authors read, reviewed, and approved the paper..

Blood transcriptomics have revealed major characteristics of the immune response in active TB, but the signature where can i buy viagra over the counter usa early after is unknown. In a unique clinically and temporally well-defined cohort of household contacts of active TB patients that progressed to TB, we define minimal changes in gene expression in incipient TB increasing in subclinical and clinical TB. While increasing with time, changes in gene expression were highest at 30 d where can i buy viagra over the counter usa before diagnosis, with heterogeneity in the response in household TB contacts and in a published cohort of TB progressors as they progressed to TB, at a bulk cohort level and in individual progressors.

Blood signatures from patients before and during anti-TB treatment robustly monitored the treatment response distinguishing early and late responders. Blood transcriptomics thus reveal the evolution and resolution of the immune response in TB, which may help in clinical management of the disease. Tuberculosis (TB) resulted in where can i buy viagra over the counter usa 1.5 million deaths in 2018.

Although a quarter of the world’s population is estimated to have been infected by Mycobacterium tuberculosis (WHO, 2019), most infected individuals remain asymptomatic (latently infected [LTBI]. Richeldi, 2006) and are suggested to have a 5–15% lifetime risk of developing TB (Vynnycky and Fine, 2000). However, recent epidemiological studies suggest that most cases occur within 2 where can i buy viagra over the counter usa yr after (Behr et al., 2018.

Behr et al., 2019. Behr et al., 2021) with the median time to TB disease during occurring in the first year or earlier (Emery et al., 2021. Menzies et al., 2021), implicating early immune events where can i buy viagra over the counter usa as key determinants of outcome (Cadena et al., 2016).

Heterogeneity of LTBI in HIV-coinfected humans and nonhuman primates has been reported (Barry et al., 2009. Esmail et al., 2016. Lin et where can i buy viagra over the counter usa al., 2016), but current assays cannot characterize the underlying heterogeneity of immune responses to M.

Tuberculosis determining TB risk or those that accompany disease progression. Clinically, the progressor LTBI state has been categorized into two phenotypes. (1) incipient TB, no clinical symptoms, radiological abnormalities or microbiological evidence of where can i buy viagra over the counter usa active TB disease.

And (2) subclinical TB, no clinical symptoms, but either radiological changes or microbiological evidence of active TB disease (Davies and Pai, 2008. Drain et al., 2018. Kendall et al., 2021 where can i buy viagra over the counter usa.

Pfyffer et al., 1997. Richeldi, 2006. WHO, 2019) where can i buy viagra over the counter usa.

Clinical TB patients display radiological features and microbiological evidence of active TB disease (Davies and Pai, 2008. Drain et where can i buy viagra over the counter usa al., 2018. Kendall et al., 2021.

Pfyffer et al., 1997. Richeldi, 2006 where can i buy viagra over the counter usa. WHO, 2019).

Thus, a proportion of patients presumed as LTBI may either be incipient or already have subclinical disease, contributing to onward transmission of (Dowdy et al., 2013. Drain et where can i buy viagra over the counter usa al., 2018. Kendall et al., 2021).

Reported reduced blood transcriptional signatures of TB risk were not related to subclinical TB or incipient disease or to the blood signature of active TB (Gupta et al., 2020. Penn-Nicholson et where can i buy viagra over the counter usa al., 2020. Scriba et al., 2017.

Singhania et al., 2018a. Singhania et where can i buy viagra over the counter usa al., 2018b. Suliman et al., 2018.

Zak et al., 2016). Earlier detection could inform treatment and limit where can i buy viagra over the counter usa transmission. Diagnosis of active pulmonary TB requires microbiological samples for evidence of , which can be difficult to obtain (Davies and Pai, 2008.

Richeldi, 2006). A blood transcriptional signature has been reported in patients with active TB (Berry et where can i buy viagra over the counter usa al., 2010. Blankley et al., 2016.

Bloom et al., 2013. Joosten et where can i buy viagra over the counter usa al., 2013. Maertzdorf et al., 2011.

Ottenhoff et al., 2012. Roe et al., where can i buy viagra over the counter usa 2016. Scriba et al., 2017), which is dominated by type I IFN signaling, reflects the extent of radiographical lung disease (Berry et al., 2010.

Moreira-Teixeira et where can i buy viagra over the counter usa al., 2020), and is diminished upon treatment (Berry et al., 2010. Bloom et al., 2012. Cliff et al., 2013.

Thompson et where can i buy viagra over the counter usa al., 2017). Biomarkers to monitor TB treatment success are needed to accelerate assessment of treatment responses and determine the required treatment duration to adapt drug treatment regimens. The accepted biomarker is sputum conversion to negative culture after 2 mo, which has low sensitivity and modest specificity for prediction of treatment failure (Horne et al., 2010.

Mitchison, 1993) where can i buy viagra over the counter usa. Chest x rays (CRXs) and inflammatory markers commonly used to assess the response to treatment are not universally available and difficult to standardize (Walzl et al., 2011). How the host response evolves after of humans with M.

Tuberculosis toward the peak response in active TB is as yet where can i buy viagra over the counter usa unclear. Sequential immune responses were reported during TB progression but not linked to the clinical disease status, with adolescents evaluated at enrollment and then only sampled every 6 mo with follow-up over 2 yr, or evaluated at baseline and at the end of 2 yr (Scriba et al., 2017). Since this study was not on household contacts, knowledge of when each individual was exposed to M.

Tuberculosis could not be estimated, limiting the scope for detailed temporal evaluation of changes in the immune response during where can i buy viagra over the counter usa progressive . Without detailed clinical characterization of patients upon serial sampling before TB diagnosis, differential gene expression during different phenotypic stages of disease ranging from incipient TB to subclinical TB to clinical TB cannot be assessed. Moreover, confounding interpretations due to re in high TB burden settings during the prospective period of observation and sampling cannot be ruled out (Charalambous et al., 2008.

Van Helden where can i buy viagra over the counter usa et al., 2008. Van Rie et al., 2005. Van Rie et al., 1999.

Verver et where can i buy viagra over the counter usa al., 2005. Warren et al., 2004). Although blood transcriptional signatures have been shown to reflect the response to TB treatment (Berry et al., 2010.

Bloom et where can i buy viagra over the counter usa al., 2012. Cliff et al., 2013. Penn-Nicholson et al., 2020.

Thompson et al., 2017), the patterns of resolution where can i buy viagra over the counter usa with treatment in different patient groups using detailed kinetic analysis at multiple time points has not been evaluated. To address these questions, we undertook a prospective cohort study comprising participants with microbiologically confirmed pulmonary TB and household contacts of pulmonary TB at Leicester, UK, a high-income, moderate TB setting (TB incidence circa 40 per 100,000 population). An integrated clinical-research where can i buy viagra over the counter usa platform enabled recruitment, regular follow-up, and detailed characterization of participants at serial time points of prospective observation (Materials and methods), with a low probability of new community-acquired during prospective follow-up of TB contacts.

In total, 356 household contacts of pulmonary TB and 74 participants with incident TB were recruited between 2015 and 2018 and prospectively followed for 24 mo. TB contacts were reviewed every 3–6 mo with RNA sequencing (RNA-Seq) samples collected, whole-genome sequencing of the M. Tuberculosis strain to trace back contacts to the index case, and CRX performed to screen for subclinical TB at each visit, with detailed radiological characterization and clinical investigation, including invasive sampling (bronchoscopy), if x-ray abnormalities were suspected or symptoms reported where can i buy viagra over the counter usa.

This allowed reliable characterization of participants with incipient, subclinical, or clinically active TB, and linking blood transcriptional signatures to the clinical phenotype as disease progressed. Active TB patients were sampled, and clinical characterization was undertaken before starting TB treatment, and prospectively at scheduled visits during treatment, with microbiological investigation, radiological surveillance with CRX, and computed tomography scan as clinically indicated. Changes in blood gene where can i buy viagra over the counter usa expression in different clinical subgroups of active TB patients were related to the time of diagnosis and to detailed time points during treatment.

Bioinformatics analysis of blood RNA-Seq data of contacts revealed minimal changes in gene expression in incipient TB, increasing as patients progressed to subclinical and clinical TB, with similar expression profiles in these clinical phenotypes for published reduced risk signatures of TB. Moreover, gene expression changes in the blood of Leicester TB progressors, and a published cohort of TB progressors from a high burden TB setting, were most pronounced at 30 d before diagnosis, although heterogeneity was observed over time before diagnosis. The signature of TB progression in the Leicester cohort was compared with active TB disease, before and during treatment, to understand the immune events underlying the where can i buy viagra over the counter usa evolution and resolution of TB disease (Fig.

1. Study design). Our study provides information of the underlying host immune response at the different stages of disease and a roadmap to describe the temporality of gene expression changes that occur during progression and treatment of active TB, which where can i buy viagra over the counter usa may help in clinical management of TB patients.

To determine how global changes in differential gene expression develop as individuals progress from incipient TB to subclinical TB and then to to clinical TB and whether these clinical phenotypes show a graded increase in the immune response, we performed detailed analysis of changes in gene expression over time in blood of clinically defined Leicester household TB contacts who then progressed to TB (Fig. 2). Contacts who progressed to TB were subdivided according to their clinical where can i buy viagra over the counter usa phenotype at the time point of sampling (Table S1).

In the 14 household contacts, incipient TB was concurrent with samples (n = 10) collected earlier than 40 d before diagnosis. Subclinical TB spread between earlier than day 40 (n = 1), 21–40 d (n = 3), and <20 d (n = 6) before diagnosis. Clinical TB spread where can i buy viagra over the counter usa between 21–40 d (n = 4) or <20 d (n = 14) before diagnosis (Table S1).

Numbers of up- and down-regulated genes were minimal in incipient TB (94 up-regulated and 48 down-regulated genes), increasing in subclinical TB (483 up-regulated genes and 81 down-regulated genes) and in clinical TB (572 up-regulated and 136 down-regulated genes. Fig. 2 A) where can i buy viagra over the counter usa.

Fewer down-regulated genes were detected in each of the different clinical phenotypes of the TB contacts as they progressed to TB (Data S1). Of the up-regulated genes, Metacore pathway analysis showed a dominance of the IFN-α/β signaling pathways in subclinical TB and clinical TB with an increase in the ratio in the number of genes per pathway, 14/62 and 16/62, respectively, with much lower representation in incipient TB, 5/62 (Fig. 2 B) where can i buy viagra over the counter usa.

Incipient TB showed IFN-γ activation of macrophages and the classical complement as the top represented pathways. However, only 5/50 and 5/53 genes were represented in each pathway, although with a much lower ratio of genes per where can i buy viagra over the counter usa pathway overall (Fig. 2 B and Data S1).

The P values for the different clinical subgroups showed a corresponding increasing statistical significance for the IFN-α/β signaling pathways, progressing from the incipient TB (8.65 × 10−5), subclinical TB (4.75 × 10−13), and clinical TB (2.44 × 10−14) respectively (Fig. 2 B) where can i buy viagra over the counter usa. Similarly, the type I IFN modules increased in subclinical TB and clinical TB, although clinical TB progressors showed additional changes resembling the signature of active TB, including an increase in the innate/hemopoeitic mediators module (Fig.

2 C). A decrease in the natural killer (NK) and T cell module in incipient, subclinical, and where can i buy viagra over the counter usa clinical TB was observed, with incipient TB showing no other detectable changes at this stringent level of statistical analysis (Fig. 2 C).

The top 30 differentially expressed coding genes (false discovery rate [FDR] P <. 0.05, log2 fold change >1) ranked by fold change, selected from a total of 47 genes in incipient TB, 233 genes in subclinical TB, and 311 genes in clinical TB, showed that many genes were differentially expressed across all three where can i buy viagra over the counter usa clinical phenotypes, albeit to different levels (Table 1 and Data S1). These included the genes C1QC***, SERPING1***, ETV7***, and BATF2*** expressed in all three clinical phenotypes.

C1QA**, C1QB**, C2**, and EXOC3L1** were expressed in two of the clinical phenotypes, and ANKRD22** and GBP6** were significantly expressed in subclinical TB and clinical TB and were barely elevated above controls in the incipient TB (Data S1, full incipient TB versus control tab). Although certain genes appeared to be uniquely expressed within each clinical phenotype, most where can i buy viagra over the counter usa could be detected across the three clinical phenotypes, albeit to differing levels. Seemingly unique genes within the top 30 gene set of the incipient TB subgroup, such as CCL2, HESX1, PCGF2, LCN8, and SIGLEC1, were only elevated to a very low level against controls in the full set of differentially expressed genes, potentially suggesting that they may come up early in the immune response to M.

Tuberculosis, although they were also expressed at a low level in clinical TB (Data S1. Full incipient TB, full clinical TB versus where can i buy viagra over the counter usa control tabs). The expression of the complement fixing genes C1QC and C1QB in the top 30 genes of the incipient TB versus control group is in keeping with the Metacore pathway analysis in Fig.

2 B. However, these genes were also differentially where can i buy viagra over the counter usa expressed within the top 30 genes of subclinical TB and clinical TB (Data S1. Full subclinical TB, full clinical TB versus control tabs).

BATF2 expression increased significantly with increasing disease. 1.4 log2 fold where can i buy viagra over the counter usa change, P value 0.0011 in incipient TB. 2.9 log2 fold change, P value 7.6 × 10−12 in subclinical TB.

And 3.48 log2 fold change, P value 6.07 × 10−24 in clinical TB. Expression of SERPING1 and ETV7 showed a similar increase where can i buy viagra over the counter usa in expression as individuals who progressed to TB (Table 1 and Data S1). We next analyzed blood transcriptional changes that occurred over time in Leicester TB household contacts as they progressed to TB, together with patients sampled before they were diagnosed with TB (progressors) in view of our findings that high levels of differential gene expression are mainly seen in progressors with subclinical TB and clinical TB, rather than in incipient TB.

RNA-Seq data where can i buy viagra over the counter usa were analyzed in blood from Leicester household contacts of active TB patients at different time points after recruitment as they progressed to clinical TB (Fig. 4 A. Table S2, top, n = 12 TB contacts.

Total of 21 samples) together with Leicester where can i buy viagra over the counter usa patients sampled before they were diagnosed with active TB by culture/microbiological/clinical positivity (Fig. 4 A. Table S2, bottom, n = 11 progressors, total of 12 samples), all before treatment.

Active TB patients at the time of where can i buy viagra over the counter usa diagnosis (Fig. 4 A, far right. Table S2, top, n = 49 TB patients), all as compared with healthy controls (Table S2, top, n = 38 healthy controls).

The biggest changes in gene expression (log2 fold, FDR P value of 0.05 cutoff) were observed at 0–20 d before TB diagnosis in the contacts (n where can i buy viagra over the counter usa = 11) and progressors (n = 9. 765 up-regulated and 125 down-regulated genes. Fig.

4 A) where can i buy viagra over the counter usa. Although the change in the number of genes just before diagnosis appeared similar to that observed in active TB patients at the time of diagnosis (1,231 up-regulated and 511 down-regulated genes. Fig.

4 A, far right), the where can i buy viagra over the counter usa extent of differential expression in the blood of active TB patients at the time of diagnosis was higher (Fig. 4 A, far right, scale on y axis 0–100. ˆ’log10 P adjusted) as compared with the contacts and progressors sampled before diagnosis (Fig.

4 A where can i buy viagra over the counter usa. 0–20 d before diagnosis, scale on y axis 0–15. ˆ’log10 P adjusted).

Changes in gene expression were substantially lower between 21–40 where can i buy viagra over the counter usa d before diagnosis with low level up-regulation of 185 and down-regulation of 80 genes (Fig. 4 A. Representative of four TB contacts that progressed to TB and three TB progressors sampled before diagnosis).

At 41–832 d before TB diagnosis, when six samples were all from TB contacts subsequently progressing to clinical TB, this change in gene expression where can i buy viagra over the counter usa was further reduced, with very low levels of 109 up-regulated and 34 down-regulated genes (Fig. 4 A, far left). We next performed more in-depth analyses on Leicester TB household contacts alone as they progressed to where can i buy viagra over the counter usa TB, recruited and sampled from 2015 to 2018 and followed up to date, by pooling our more recently recruited dataset with our previously published dataset (Singhania et al., 2018a.

Total 38 samples from 14 TB household contacts, sampled as they progressed to TB against matched controls. Fig. 4 B where can i buy viagra over the counter usa and Table S3).

Lower levels of gene expression were now observed between 0 and 31 d before diagnosis with only 23 up-regulated and 1 down-regulated gene (Fig. 4 B. Log2 fold, FDR where can i buy viagra over the counter usa P value of 0.05 cutoff.

Scale on y axis 0–4. ˆ’log10 P adjusted), including up-regulation of GBP5, SEPTIN4, ETV7, C1QC, BATF2, C1QB, FCGR1A, GBP6, and SERPING1. Gene expression changes at earlier time points fluctuated over time, with 15 up-regulated and 8 down-regulated genes observed between days 86 and where can i buy viagra over the counter usa 150 but not at 32–58 before diagnosis (Fig.

4 B). Up-regulation of these genes was still detected, albeit to a much lower level, at all the earliest time points before diagnosis (Data S2). Some exceptions included C1QB, C1QC, and C1QA, which were in the top seven DEGs at the time points 86–150 before diagnosis, while in the 0–31 d before diagnosis, only C1QB and C1QC were in the top eight expressed genes where can i buy viagra over the counter usa at the level of fold change over controls, suggesting heterogeneity of gene expression over time.

We next analyzed a bigger dataset of individuals from South Africa reported to have subsequently progressed to TB, reported to have been sampled at 6 monthly intervals for blood RNA-Seq analysis before diagnosis, although without serial clinical follow-up (Scriba et al., 2017. Zak et al., 2016). We further subdivided the sampling where can i buy viagra over the counter usa into tighter time points and examined differential gene expression levels as compared with LTBI nonprogressors recruited in parallel.

Again the highest levels of gene expression changes were observed between 0 and 31 d before diagnosis, with 1,155 up-regulated and 473 down-regulated genes (Fig. 4 C. Log2 fold, FDR P value of 0.05 cutoff where can i buy viagra over the counter usa.

Scale on y axis 0–15. ˆ’log10 P adjusted), including up-regulation of SEPTIN4, SERPING1, BATF2, GBP6, ETV7, and FCGR1A, similar to those detected in our Leicester contacts 0–31 d before diagnosis (Fig. 4 B) where can i buy viagra over the counter usa.

Expression of C1QA, C1QB, and C1QC was only detectable 0–31 d before diagnosis, perhaps reflecting the level of detectability over controls. Changes in differential gene expression at most other time points before diagnosis were very low (Fig. 4 C and Data S3), although fluctuations in differential gene expression were observed over time, for example where can i buy viagra over the counter usa with marked changes at 181–250 (88 up-regulated and 46 down-regulated genes) and 251–360 d (51 up-regulated and 3 down-regulated genes) before diagnosis, as compared with other time points showing minimal changes.

Among the top 30 genes found to be up-regulated between 181 and 250 d before diagnosis were SEPTIN4, GBP6, BATF2, ETV7, SERPING1, and FCGR1A, although these genes were also among the top up-regulated 30 genes at 0–31 d before diagnosis, albeit then at a more significant level (Data S3), suggesting a graded increase in the expression of these genes as progressors approached TB diagnosis, with some heterogeneity of gene expression over time. The blood modular signature of where can i buy viagra over the counter usa TB contacts and TB patients sampled prediagnosis as in Fig. 4 A showed a reduction in the NK and T cell module (dominated by IFNG and effector T and NK cell genes) at >40 d before diagnosis, followed by an increase in the innate/hemopoeitic mediator module from 40 d.

Increased type I IFN–inducible and inflammasome/granulocyte modules together with a reduction in the NK and T cell, and T and B cell modules were detected at 0–20 d before diagnosis (Fig. 4 D), similar to the reported TB where can i buy viagra over the counter usa blood signature (Moreira-Teixeira et al., 2020. Singhania et al., 2018a).

The decrease in the NK and T cell module fluctuated over time before diagnosis in progressors, which could reflect fluctuation in the response or heterogeneity in the progressors. Changes in the type I IFN/C’/myeloid and inflammasome/granulocyte modules together with a reduction in the NK and T cell module were observed in Leicester TB contacts alone, but to a lesser extent only detectable from 30 d before diagnosis using a where can i buy viagra over the counter usa nominal P value of 0.05 rather than FDR (Fig. 4 E).

The initial change at 200–850 d before diagnosis again consisted of a reduction in the NK and T cell module, although this was not consistent, again reflecting heterogeneity over time. Changes in the Zak modular signature over time were also mainly detectable over time using a nominal P value of where can i buy viagra over the counter usa 0.05 rather than FDR (Fig. 4 F).

At 0–31 d before diagnosis, the modular signature for the Zak progressors (Fig. 4 F) where can i buy viagra over the counter usa was almost identical to that of active TB (Fig. 4 D, far right.

Moreira-Teixeira et al., 2020. Singhania et al., 2018a), although with less enrichment as at a nominal P value of 0.05 where can i buy viagra over the counter usa (Fig. 4 F), including enrichment of inflammasome/granulocytes, innate/hemopoetic mediators, innate immunity PRR/C’/granulocytes, IFN/PRR, and IFN/C’/myeloid modules and decreased enrichment of T cell, B cell, and NK and T cell modules.

The modular signature was barely detectable at other time points before diagnosis, with the IFN/PRR and IFN/C’/myeloid modules missing at 32–78 and 79–104 d but then present at 181–250 and 251–360 d before diagnosis, again suggesting temporal heterogeneity of gene expression or potential re as reported in high-burden TB settings (Charalambous et al., 2008. Uys et where can i buy viagra over the counter usa al., 2015. Van Helden et al., 2008.

Van Rie et al., 2005. Van Rie et where can i buy viagra over the counter usa al., 1999. Verver et al., 2005).

To investigate the heterogeneity among Leicester TB household contacts and the Zak progressors, the average gene expression value of the 30-gene signatures (from Table 1) derived from incipient TB (blue), subclinical TB (orange), and clinical TB (red) was assessed at different time points before diagnosis in individual Leicester TB contacts (n = 9) and individual Zak progressors (SupTab1. SupTab6_RNA-Seq-Metadata from Zak et where can i buy viagra over the counter usa al., 2016. Training set n = 18.

GEO accession where can i buy viagra over the counter usa no. GSE79362) where two or more sampling time points were evident. The average 30-gene incipient TB, subclinical TB, and clinical TB signatures were shown to be marginally elevated over the baseline (dotted line for each) in four of the Leicester TB household contacts at all time points analyzed before TB diagnosis, 0–30 d, with the subclinical TB and clinical TB signatures showing slightly better performance (Fig.

5 A, where can i buy viagra over the counter usa shorter time points, n = 4). Three out of four of these contacts who progressed rapidly to TB disease had been infected with an outbreak strain of M. Tuberculosis identified by whole genome sequencing.

TB contacts who progressed over 100–200 d showed a greater elevation against baseline, similar for the incipient, subclinical, and clinical TB 30-gene signatures, increasing at times close to TB where can i buy viagra over the counter usa diagnosis (Fig. 5 A, longer time points). One TB contact (Patient ID 493) showed some fluctuation, although always above baseline for all three signatures (Fig.

5 A, where can i buy viagra over the counter usa longer time points, n = 5). The average 30-gene incipient TB, subclinical TB, and clinical TB signatures showed an increase over the baseline (dotted line, LTBI controls) in the Zak progressor patients between 4–600 d before diagnosis (Fig. 5 B), although a sharp increase in the signatures over time was only observed in around five of the progressors from just >200 d to a maximum before diagnosis.

Other patients showed heterogeneity in expression where can i buy viagra over the counter usa of these 30-gene signatures over time, many showing elevated signatures maintained at the same level over time, with others actually decreasing (Fig. 5 B). The published Zak 16-gene signature showed almost superimposable curves with very similar increases above the baseline controls over time in the individual Leicester TB household contacts (Fig.

S1 A, shorter time points where can i buy viagra over the counter usa and longer time points), and in the Zak progressors, with identical increases in the five individuals and the same heterogeneity as observed with the 30-gene signatures (Fig. S1 B). There is currently a need for early biomarkers to monitor TB treatment success earlier and to evaluate robustly the duration of treatment required in TB patients to adapt drug treatment regimens.

To establish treatment response signatures, RNA-Seq was performed on blood from 74 TB patients at diagnosis where can i buy viagra over the counter usa (treatment-naive), and longitudinally, at carefully planned time points during TB treatment. We first monitored the transcriptional response to treatment across the whole cohort, and second monitored the transcriptional response of individual patients to identify distinct profiles of their transcriptional response that might help to stratify clinical treatment phenotypes. Blood was collected and subjected to RNA-Seq from the 74 TB patients at diagnosis before treatment and thereafter, at 1 and 2 wk, at 1, 2, 4, 5, 6, 7/8, 9/10, and 11/12 mo, and at >1-yr after treatment (Fig.

S2, A and where can i buy viagra over the counter usa B) from clinically defined patients. Pulmonary TB, difficult TB cases, TB drug–resistant, outbreak TB strain, and other TB progressors (Table S4). TB patients received either standard anti-TB treatment (ATT.

200 d where can i buy viagra over the counter usa or less) or extended ATT (>200 d. Table S4), according to their clinical assessment through treatment, with smear-positive patients mostly falling within the extended ATT patient group (Fig. S2 C) where can i buy viagra over the counter usa.

The sample-to-sample correlation heatmap and principal component analysis (PCA) of all the active TB patients at diagnosis before treatment and at the different time points during the treatment course showed samples to mainly cluster according to time points, with some heterogeneity (Fig. S2, D and E). The top 1,000 most variable gene expression heatmap distinguished patients according to time of treatment, and according to smear positivity and negativity at treatment initiation (T0 where can i buy viagra over the counter usa.

Fig. S2 D). The innate/hemopoietic, IFN/PRR, and IFN/C’/myeloid modules were found to be over-abundant as compared with controls before treatment and decrease in abundance to different degrees within all the subgroups after T0, except for in where can i buy viagra over the counter usa the TB drug resistant subgroup (Fig.

6 A). These modules decreased in abundance after 1 wk of treatment and were completely abrogated after 4 mo of treatment in the standard ATT subgroup. Although the extended ATT where can i buy viagra over the counter usa and difficult TB cases subgroups showed a similar pattern to the standard ATT subgroup, a stronger modular signature before treatment and an incomplete diminishment after 6 mo were observed, when a standard treatment course would be completed.

The outbreak TB strains subgroup showed a similar but weaker global modular signature to the standard ATT subgroup, also resolving within 4 mo of treatment. However, a small subgroup of four patients, the TB drug–resistant subgroup, showed a distinct modular signature that for the most part was not diminished, in accordance with these patients requiring altered drug treatment regimens for a longer period (Fig. 6 A) where can i buy viagra over the counter usa.

The standard and extended ATT subgroups contained a large number of patients such that the modular signature was more robust than in the other three subgroups, which contained lower numbers of patients (Fig. 6 A). We therefore examined the members of each of these three subgroups individually and show that at the level of the individual, these modular responses to treatment are heterogeneous and so should be validated in larger where can i buy viagra over the counter usa cohorts in future studies (Fig.

S3). The number of DEGs compared with controls was also reduced upon treatment (Fig. 6 B) where can i buy viagra over the counter usa.

Smear-positive and smear-negative TB patients showed a similar modular and gene expression decrease during treatment with complete diminishment by 4–5 mo, although the smear-positive patients had a stronger modular signature before treatment (data not shown). We then identified a 212-gene signature (TREAT-TB212) that showed the response to treatment across the whole Leicester cohort, mainly showing decreased gene expression as compared with controls over the treatment course, which reverted to the expression profile of healthy controls by 4 mo of treatment in most but not all of the patients (Fig. 7 A) where can i buy viagra over the counter usa and in an independent treatment response cohort dataset (Fig.

7 B. Thompson et al., 2017). The TREAT-TB212 signature in not-cured patients from the Thompson cohort was sustained at where can i buy viagra over the counter usa all time points up to 24 wk at similar levels to that of the pre- and very early treatment response signatures (Fig.

7 B), at a comparable level to the Leicester cohort of active TB patients, TB progressors recruited as TB household contacts after diagnosis, and most different clinical treatment response subgroups (Fig. 7 A) where can i buy viagra over the counter usa. A higher TREAT-TB212 signature was observed in patients receiving treatment for >200 d but this was observed only early after T0, as compared with those receiving standard treatment of up to 200 d (Fig.

7 A). In keeping with the modular and differential gene expression analyses, the TREAT-TB212 signature was only different in the smear-positive and -negative patients at T0 but not between 1 wk to 1 yr after T0, indicating that the patients were where can i buy viagra over the counter usa responding similarly to treatment (Fig. 7 A).

The log2 fold-change of all TREAT-TB212 genes against controls verified changes in gene expression upon treatment (Fig. 7 C, Leicester cohort) with a similar profile in where can i buy viagra over the counter usa the Thompson cohort (Fig. 7 D).

Again, most of the gene expression profiles reverted to that of healthy controls by 4 mo of treatment (Fig. 7 C, Leicester cohort), although this could not be evaluated in the where can i buy viagra over the counter usa Thompson cohort due to fewer sampling visits that did not include this time point (Fig. 7 D).

The TREAT-TB212 signature was further reduced to a 27-gene signature (TREAT-TB27), which selected genes with the greatest changes in expression over the treatment course in the whole cohort (Fig. 7 E), and its where can i buy viagra over the counter usa validity was confirmed also in the Thompson cohort (Fig. 7 F).

Although TB patients had been subgrouped according to their clinical phenotype in response to treatment, as standard ATT, extended ATT, difficult TB cases, TB drug–resistant, and outbreak TB strains, the TREAT-TB212 signature did not show a clear transcriptional response trend according to their clinical definition except for most of the drug-resistant group (Fig. 7 G, where can i buy viagra over the counter usa compared with T0). However, by monitoring the transcriptional response of individual patients according to their TREAT-TB212 signature profile, regardless of their clinical subgroups but where samples at all time points had been obtained, four distinct transcriptional profiles were revealed.

Expected, resembling standard ATT. Weaker, as where can i buy viagra over the counter usa compared with standard ATT. Or stronger initial or stronger delayed, as compared with standard ATT (Fig.

7 H, compared with T0). Strikingly, stronger initial where can i buy viagra over the counter usa or stronger delayed transcriptional response patient groups showed differences in the transcriptional response already at 1 and 2 wk after T0, although at week 1 after treatment, C-reactive protein (CRP) levels in both groups were comparable (46.00 mg/l, stronger initial. 34.00 mg/l, stronger delayed).

The stronger delayed patient group displayed elevated levels of CRP even after 1 mo of treatment as compared to stronger initial (8 mg/l, stronger initial. 38 mg/l, stronger delayed group), also correlating with where can i buy viagra over the counter usa minimal changes in radiographical signs of disease (data not shown), suggesting continued inflammation and potentially in the stronger delayed. Thus the treatment response could not be predicted clinically by CRP levels early but could be predicted by the different kinetics of the transcriptional response observed as early as 1 wk after T0 in the stronger delayed as compared with the stronger initial group, supporting the role of transcriptional biomarkers as more sensitive measures of the treatment response than existing clinical markers.

To develop a reduced transcriptional signature that may enable early where can i buy viagra over the counter usa identification of poorer treatment responders, based on the stronger initial and stronger delayed groups, the differential expression of TREAT-TB212 between two consecutive time points from T0 to 1 wk, 1 to 2 wk, and 2 wk to 1 mo was computed leading to a reduced signature (EarlyRESP-TB25. Fig. 7 H and Fig.

S4). EarlyRESP-TB25 showed differences in the stronger initial and stronger delayed patient groups by their different transcriptomic profiles at 1–2 wk after T0 (Fig. 7 J), with similar but not optimal results observed for TREAT-TB27 (Fig.

7 I. Derived gene lists TREAT-TB27, EarlyRESP-TB25. Fig.

S4, A and B). Reported reduced blood signatures of TB diagnosis or risk show little to no overlap with each other, and most have been tested for distinguishing active TB from LTBI but not active TB from other diseases (ODs. Kaforou et al., 2013.

Maertzdorf et al., 2016. Roe et al., 2016. Singhania et al., 2018a.

Suliman et al., 2018. Sweeney et al., 2016. Zak et al., 2016.

Reviewed in Singhania et al., 2018b). We set out to develop an optimized reduced signature that would distinguish active TB from ODs as well as LTBI. Combined reduced blood signatures of TB diagnosis or risk comprising 101 distinct genes (Kaforou et al., 2013.

Maertzdorf et al., 2016. Roe et al., 2016. Singhania et al., 2018a.

Suliman et al., 2018. Sweeney et al., 2016. Zak et al., 2016.

Unpublished data) were analyzed in 10 published datasets from multiple clinical disease cohorts including active TB, LTBI, and ODs and healthy controls (Bloom et al., 2013. Parnell et al., 2012. Suarez et al., 2015.

Zhai et al., 2015), and the pooled dataset was batch-corrected (Fig. S5, A and B. And Materials and methods).

12 genes were identified in the reduced signature that were shared between the top 30 genes distinguishing active TB from LTBI, and active TB from ODs, ranked by decreasing importance (mean decrease accuracy. Fig. 8, A and B), further reduced to 10 genes (TB10) based on performance (area under the curve [AUC] and accuracy) on pooled cohort datasets with independent validation (Fig.

S5 C and Table S5). This TB10 signature originating from the different reduced reported signatures (Fig. 8 C) comprised genes that were either up- or down-regulated in active TB as compared with controls, LTBI, and ODs (Fig.

8 D) and was shown to be significantly different in TB versus ODs and LTBI and controls using ANOVA (Data S4). Removal of the GBP5 gene (12th in rank) reported to discriminate active TB and LTBI did not improve the performances for discrimination of active TB from ODs. Individual gene expression was heterogeneous across patients with active TB and the ODs (Fig.

8 D, likely reflecting the different extents of morbidity. Berry et al., 2010. Bloom et al., 2013).

The performances of TB10 were tested and compared with the signatures that we describe in this study, including the 30-gene incipient TB, the 30-gene subclinical TB, the 30-gene clinical TB, the TREAT-TB27, and the EarlyRESP-TB25 (Fig. S5 D), and previously published reduced signatures (Fig. 8 E and Fig.

S5 E. Kaforou et al., 2013. Maertzdorf et al., 2016.

Roe et al., 2016. Singhania et al., 2018a. Suliman et al., 2018.

Sweeney et al., 2016. Zak et al., 2016. Described in Materials and methods).

TB10 showed the best performance for TB versus ODs (Fig. 8 E and Fig. S5 D.

AUC, 0.999. Accuracy, 0.976. And 95% confidence interval [CI], 0.9959–1), and TB versus LTBI (Fig.

95% CI, 0.9343–1). Although the Suliman reduced signature was comparable to TB10 for distinguishing TB from LTBI (Fig. S5 E), it showed poorer performance for distinguishing TB from ODs (Fig.

8 E). Blood transcriptomics have revealed major characteristics of the immune response in TB, show promise to support TB diagnosis, and would be of great use to identify individuals with asymptomatic incipient TB or subclinical TB before they progress to clinical TB to facilitate targeted early treatment and reduce onward transmission. Moreover, new tools for effective TB treatment monitoring are needed to determine when and if patients are responding to treatment to provide a personalized approach to treatment and accelerate screening of new anti-TB drugs.

To achieve this, a detailed knowledge of how the host immune response develops over time and relates to the state of M. Tuberculosis is needed. We now show, in a unique clinically and temporally well-defined cohort of household contacts of active TB patients, that minimal changes in blood gene expression are detectable in incipient TB, increasing as patients progress to subclinical TB, and maximal at the time of presentation with clinical TB, with similar results for published reduced risk signatures of TB.

Although the transcriptional signatures increased with time and were most highly expressed around 30 d before diagnosis, there was heterogeneity over time in the response in the TB contacts as they progressed to TB and a published cohort of TB progressors from a high-burden TB setting. Blood signatures at detailed time points during ATT additionally allowed us to define signatures that can distinguish early and late responders. Finally, we demonstrate comparable performance of immune signatures developed for TB diagnosis and detection of early stages of M.

Tuberculosis and their reduction upon TB treatment monitoring, with subtle differences for different signatures at different stages of development and diminishment of TB disease. The temporality of gene expression changes during progression, and resolution of active TB may provide mechanistic insights toward the development of host therapies and supports a framework for future development of biomarkers to improve the clinical management of LTBI and active TB. TB contacts that progress to TB showed a decrease in the NK and T cell effector modular signature, which was detectable from the earliest stages of progression, in keeping with findings that cytotoxic effector molecules and NK cells are important for protection against M.

Tuberculosis in human TB (Roy Chowdhury et al., 2018). We observed an increase in the inflammatory and IFN modular signatures in subclinical TB and clinical TB at time points closest to TB diagnosis in both Leicester contacts and Zak TB progressors, but not in incipient TB. That the IFN modular signature fluctuated with time in Zak progressors potentially explains differing reports that type I/II IFN signaling and the complement cascade were elevated 18 mo before TB disease diagnosis in this cohort (Scriba et al., 2017), with others suggesting increased expression of complement genes in subclinical TB closer to diagnosis (Esmail et al., 2018).

Our analysis at more detailed time points of the Zak cohort suggests elevated type I IFN signaling, and complement genes at 18 mo before diagnosis may indicate individual patient heterogeneity, as we discuss below. A marked decrease in the B and T cell modular signatures and increase in other modules found in active TB including myeloid inflammation, lymphoid, and monocyte and neutrophil gene modules occurred in progressors more proximally to TB disease, in keeping with Scriba et al. (2017).

A reciprocal reduction in the inflammation and IFN modules was observed after a week of successful treatment and was restored to that of healthy controls by 4 mo, together with the B and T cell modular signatures. Our findings that the evolution of the immune response on progression to TB shows the reverse upon treatment is suggestive that reduced signatures optimized to support early diagnosis of TB may also reflect the changes that occur in response to treatment. Reduced blood signatures have been proposed to determine the risk of exposed individuals to their subsequently developing TB (Penn-Nicholson et al., 2020.

Singhania et al., 2018a. Singhania et al., 2018b. Suliman et al., 2018.

Zak et al., 2016). However, it was unclear at the time whether any of these reported blood signatures of TB risk predicted progressors at stages of incipient or subclinical TB. We now show that while these published gene risk signatures are expressed during subclinical TB and clinical TB, only SERPING1, ETV7, and BATF2 from the 16-gene Zak signature were up-regulated, albeit at a very low level, in incipient TB, similarly to the global transcriptional expression signature in these Leicester contact clinical phenotypes.

7 of the 16-gene signature reported by Zak et al. (2016) were among the 30 most highly expressed genes across the Leicester contact clinical phenotypes of TB progression, including FCRGR1A, SEPT4, GBP5, ANKRD22, SERPING1, ETV7, and BATF2. Although C1QC, SERPING1, ETV7, and BATF2 were among the top 30 DEGs in all the clinical phenotypes, increasing statistical significance of differential gene expression was observed with progression from incipient to subclinical to clinical TB, suggesting that they may be early indicators of M.

Tuberculosis and reflect the evolution of the immune response with time after . In support of this, IFN-α/β signaling pathways by modular and Metacore analysis were observed in subclinical TB and more so in clinical TB, but were not apparent in incipient TB. Collectively, our findings suggest that the levels of gene expression increase with progressive from incipient to subclinical to active TB, potentially explaining why distinct published signatures of TB risk show little overlap with each other (Penn-Nicholson et al., 2020.

Singhania et al., 2018a. Suliman et al., 2018. Zak et al., 2016.

Reviewed in Singhania et al., 2018b) due to differential levels of detectable gene expression and/or uncertainty in the stage of at which sampling was performed. The unique gene set expressed in incipient TB is characterized by low-level gene expression changes across multiple pathways, which may limit their value for predicting which incipient TB patients will progress to clinical TB. However, genes that were differentially expressed in subclinical TB and clinical TB at a high and significant level were detectable in incipient TB, supporting suggestions that serial testing among carefully selected clinical target groups might be required for optimal implementation of biomarkers for TB risk (Esmail et al., 2020.

Gupta et al., 2020). Indeed, inclusion of clinical details of TB progressors over detailed sampling times after exposure/ with M. Tuberculosis allowed us to define progressors as incipient TB and subclinical TB (Drain et al., 2018) and to assign changes in blood gene expression at an early stage of to each clinical phenotype, providing a framework for improved biomarker selection to target early TB treatment and block onward transmission.

Our findings of temporal heterogeneity in the blood transcriptional response of Leicester TB progressors and more so in the Zak TB progressors (Zak et al., 2016), both at bulk cohort levels as well as in individual progressors, likely reflect the dynamic nature of the host–pathogen interaction over time, and are consistent with observations in positron emission tomography scan and computed tomography scan studies of progressive in humans with LTBI coinfected with HIV/TB and in nonhuman primates (Barry et al., 2009. Esmail et al., 2016. Lin et al., 2016).

The majority of Leicester contacts progressed over 100–200 d, displaying a greater elevation in their blood signatures against the baseline as they progressed to TB. However, a subset showed rapid progression with an increased signature close to diagnosis, mostly explained by with an outbreak strain of M. Tuberculosis, supporting previous reports of differing virulence of M.

Tuberculosis strains (Coscolla and Gagneux, 2014), suggesting that the time at which protective immune responses were overwhelmed leading to TB progression could differ according to the infecting strain of M. Tuberculosis. Detailed analysis of individuals in the Zak et al.

(2016) cohort, sampled for >600 d, showed a sharp increase in the signatures over time in only five progressors from 200 d before diagnosis, progressing sharply to the highest signature before diagnosis, again suggesting that immune responses had been overwhelmed. However, in other Zak progressors, the fate of appeared to hang in the balance for a prolonged period until diagnosis, possibly reflecting subclinical disease, although in-depth clinical analysis was not performed at these earlier sampling time points in the study. Moreover, since it was not a study of recent TB contacts, there was no knowledge of time of exposure or the infecting M.

Tuberculosis strain. Our findings are consistent with a recent randomized controlled clinical trial for biomarker-guided tuberculosis preventive therapy (termed CORTIS. NCT02735590), which concluded that a reduced signature RISK11 derived from the Zak 16-gene signature (Zak et al., 2016) was better suited to screening of symptomatic individuals with possible early clinical TB than for mass community-based screening for incipient TB (Scriba et al., 2021).

It is unclear whether this signature can distinguish subclinical or active TB from other s, particularly viral s, which may present with symptoms similar to clinical TB and are also dominated by type I IFN signaling (Singhania et al., 2018a. Singhania et al., 2018b). Improved biomarkers to monitor TB treatment success are needed to reliably evaluate the duration of treatment required in individual TB patients and deliver optimal drug treatment regimens.

We adopted both clinical and bioinformatics approaches to develop blood signatures of the treatment response. Using the clinical approach, individual patients of the Leicester TB cohort were stratified on the basis of treatment response (standard ATT, extended ATT, and difficult TB) and infecting strain characteristics (drug-resistant TB and outbreak TB strains). The treatment response signature corresponded with their clinical treatment response, discriminating both the subgroup of drug resistant patients in Leicester responding more slowly to treatment, and the subgroup labeled as not cured in the African Thompson cohort (Thompson et al., 2017), supporting identification of patients not responding to treatment.

Using the bioinformatics approach, we monitored the transcriptional response of individual patients, independent of their clinically defined treatment phenotype, and defined four types of TB patients. The expected group responding standardly to treatment. The weaker group defining a subgroup with a lower grade of (low CRP, and longer time to M.

Tuberculosis culture positivity). And the stronger initial and stronger delayed groups, showing differences in their transcriptional response at 1 wk after T0. Differences between these latter two subgroups were not discernible by clinical measures of their treatment response, such as CRP and x ray, at these early time points, supporting the utility of transcriptional biomarkers as more sensitive measures of the treatment response than existing clinical markers, to inform clinical management of TB patients and to support drug development platforms and future drug treatment trials.

Existing TB diagnostic tools are limited in their scope and dependent on sputum availability for rapid identification of TB. Diagnostic blood transcriptomic signatures of TB may provide a pathway to support early diagnosis for a broader spectrum of disease phenotypes, although there is little consensus between reported reduced signatures for TB risk and those that distinguish active TB from LTBI (Kaforou et al., 2013. Maertzdorf et al., 2016.

Penn-Nicholson et al., 2020. Roe et al., 2016. Scriba et al., 2021.

Singhania et al., 2018a. Singhania et al., 2018b. Suliman et al., 2018.

Sweeney et al., 2016. Zak et al., 2016). Moreover, there is a need to distinguish TB from other confounding diseases (Kaforou et al., 2013.

Singhania et al., 2018a. Singhania et al., 2018b). Our blood signature, TB10, derived from published reduced signatures tested on multiple disease cohorts, optimally distinguished patients with TB from those with LTBI, and TB from those with ODs.

Although some of the published signatures had similar performance in distinguishing TB from LTBI, they had poorer performance than TB10 in distinguishing TB from ODs. All reduced signatures derived in this study, including the 30-gene signatures of incipient TB, subclinical TB, and clinical TB progression and the new treatment-monitoring signatures TREAT-TB27 and EarlyRESP-TB25, showed poorer performances than TB10 in distinguishing TB from LTBI and in distinguishing TB from ODs, suggesting that the top gene expression changes that occur temporally upon progression to TB may not exactly match the top gene set that is temporally diminished after TB treatment. All signatures increased in subclinical TB and maximally in clinical TB, also decreasing at week 1 after treatment, with a further decrease at month 2 and complete disappearance of gene expression changes by month 6.

However, all signatures barely showed a significant increase in expression in incipient TB above controls, with the exception of the 30-gene incipient TB signature, which potentially could be further reduced to the most highly expressed genes and optimized to give the highest sensitivity of gene expression to detect what is likely to be early M. Tuberculosis in asymptomatic individuals with incipient TB and subclinical TB, at risk of progression to TB. The global aim of our study, however, was not to develop optimized signatures of risk and progression to TB, but to use signatures developed at different stages of disease progression and treatment to determine how and to what extent they are perturbed in the different clinical phenotypes of progression to TB, during active TB, and upon treatment.

In conclusion, using in-depth temporal analysis of gene expression changes over time in a cohort of clinically well-characterized household contacts of TB patients from a moderate-burden TB setting with minimal risk of re, together with reanalysis of gene expression at more detailed time points in a published cohort of TB progressors from a high TB burden setting, we demonstrate significant heterogeneity in changes of gene expression, at both the bulk cohort level and in individual patients, as they progress to TB. This has major implications for assessing TB risk in individuals with LTBI. Our characterization of the immune response underlying the evolution and resolution of TB provides a framework for biomarker development to improve clinical management of this disease.

Between September 2015 and September 2018, longitudinal cohorts of active TB and TB contacts were recruited from the clinical TB service at Glenfield Hospital, University Hospitals of Leicester National Health Service Trust, Leicester, UK (Table S1, top). All participants were prospectively enrolled and sampled before the initiation of any TB treatment. The Research Ethics Committee for East Midlands–Nottingham 1, Nottingham, UK (REC 15/EM/0109) approved the study.

All participants gave written informed consent. All active TB patients had microbiologically confirmed disease with whole-genome sequencing of culture isolates performed for case linkage with contacts. All participants were prospectively followed with visits at scheduled time points from the time of diagnosis (pretreatment) until 12 mo after completing treatment.

Household TB contacts were identified through routine contact tracing and underwent systematic baseline investigation with routine CXR, IFN-γ release assay for M. Tuberculosis reactivity (IGRA) testing, and a symptom questionnaire. Sputum was collected if participants were spontaneously expectorating, and bronchoscopy performed in those not expectorating with clinical or radiological suspicion of TB.

On this basis, participants were classified with LTBI, subclinical TB, or active TB. Participants with LTBI were prospectively followed up for a minimum period of 2 yr with scheduled follow-up visits at 3–6 monthly intervals. At each visit, a symptom screen and CXR was performed and a blood RNA sample collected.

In total, 356 TB contacts were recruited (150 IGRA-positive and 206 IGRA-negative [IGRA-ve]). To date, 20 participants from this cohort have been diagnosed with TB and classified as TB progressors, although 6 were excluded from the gene expression study due to either cDNA library failure (n = 1) or failure to secure microbiological confirmation (n = 5) essential for case linkage with the index case. In these cases, the diagnosis of active TB was based on clinical symptoms, typical radiological features, and supporting histology from the site of .

At each visit, a symptom screen and CXR were performed and a blood RNA sample collected. At the time of our publication in 2018 (Singhania et al., 2018a), we reported a modified disease risk score using a TB-specific 20-gene signature in 9 TB contacts who had developed TB during the study, with 99 contacts remaining healthy for 2 yr or more, but performed no detailed analysis on changes in gene expression and the immune response. Since then, not considering the excluded progressors detailed above, an additional five TB contacts (n = 5) developed TB and were included for in-depth temporal analysis of the blood signature of TB progressors at different time points before diagnosis.

In total, blood samples from 14 contacts who progressed to TB were subjected to RNA sequencing out of the 20 contacts who progressed to TB. Six were excluded from the RNA-Seq gene expression study due to either cDNA library failure (n = 1) or failure to secure microbiological confirmation (n = 5). This was important as essential for case linkage with the index case, although they were confirmed as progressing to TB by positive histology showing caseating lungs.

The contacts had the following characteristics. Gender, 35.7% male and 64.3% female. Ethnicity, 28.6% South Asian, 14.3% East African, 42.9% British Caucasian (5/6 outbreak strain), and 14.3% European.

The controls had the following characteristics. Gender, 47.1% male and 52.9% female. Ethnicity, 64.7% South Asian, 11.8% East African, 5.9% European, and 17.6% British Indian.

Since these were small numbers and skewed somewhat by the British Caucasian and gender, we applied COMBAT batch correction as described later in the Materials and methods. We collected blood at detailed time points to examine in detail changes in gene expression in the different clinical phenotypes, incipient TB, subclinical TB, and clinical TB (Table S1), and also to examine changes in gene expression occurring over time in Leicester cohorts of contacts of active TB patients (Table S2, top. N = 12 TB contacts.

25 samples. Singhania et al., 2018a) and from noncontact patients with symptoms before they were diagnosed with active TB by culture/microbiological/clinical positivity (Table S2, bottom. N = 10 TB progressors.

14 samples), all before treatment, and from active TB patients at the time of diagnosis (Table S2, top, n = 49 TB patients), all as compared with healthy controls (Table S2, top, n = 38 healthy controls). Blood from TB contacts who progressed to TB and from TB progressors was subjected to RNA-Seq and analysis at the time points indicated (Table S2), together with that from 49 newly recruited active TB patients at the time of diagnosis, before initiation of treatment. To investigate this further in Leicester TB contacts who progressed to TB only, datasets representing all time points, sampled before diagnosis throughout 2015 to 2018, were analyzed in TB contacts only that progressed to TB, by pooling our previously published TB contact dataset (Singhania et al., 2018a), which had not been investigated in depth with respect to kinetic changes in the immune responses, with our more recently recruited TB contact dataset (Table S3).

This pooled dataset, now consisting of 38 samples from 14 TB contacts as they progressed to TB, was batch corrected and analyzed against matched controls, as described later in the Materials and methods. The treatment response cohort had the following characteristics. The Leicester active TB cohort composed of 74 patients with pulmonary TB was simultaneously recruited between September 2015 and September 2018, at the Glenfield Hospital, University Hospitals of Leicester National Health Service Trust, Leicester, UK, at the time of diagnosis (treatment-naive.

Fig. S2 A and Table S4). A cohort of 38 healthy IGRA-ve controls was recruited in parallel.

To follow the transcriptional response after treatment, whole blood samples were collected and were subjected to RNA-Seq at diagnosis before initiation of any ATT (T0), and thereafter, at 1 and 2 wk. 1, 2, 4, 5, 6, 7/8, 9/10, and 11/12 mo. And >1 yr after T0 with clinical assessment including CXR, CRP, and symptom assessment.

All RNA isolation and processing were performed on all blood samples simultaneously (Fig. S2 and Table S4). Patients who had previous TB, had previous treatment for LTBI, were pregnant, were under 16 yr age, or were immunosuppressed were excluded from this study.

All participants had routine HIV testing, and patients with a positive result were excluded. Patients with active TB were all confirmed by laboratory isolation of M. Tuberculosis on the culture of a respiratory specimen (sputum or bronchoalveolar wash/lavage) with sensitivity testing performed by the Public Health Laboratory Birmingham, Heart of England National Health Service Foundation Trust, Birmingham, UK.

All participants were prospectively enrolled and sampled before the initiation of any TB treatment. The Research Ethics Committee for East Midlands–Nottingham 1, Nottingham, UK (REC 15/EM/0109), approved the study. All participants gave written informed consent.

We used 10 published blood RNA-Seq or microarray datasets (Berry et al., 2010. Bloom et al., 2013. Leong et al., 2018.

Parnell et al., 2012. Singhania et al., 2018a. Suarez et al., 2015.

Thompson et al., 2017. Zak et al., 2016. Zhai et al., 2015) from multiple clinical disease cohorts including active TB (225 patients) and LTBI (217 individuals) from Berry, London and South Africa (GEO accession nos.

GSE107991 and GSE107992). Bloom (GEO accession no. GSE42834).

Singhania, Leicester (GEO accession no. GSE107993). Zak (GEO accession no.

GSE79362). Thompson (GEO accession no. GSE89403).

Leong (GEO accession no. GSE101705). And ODs influenza (Parnell [GEO accession no.

GSE40012] and Zhai [GEO accession no. GSE68310]), Bloom lung cancer (GEO accession no. GSE42834), Bloom pneumonia (GEO accession no.

GSE42834), Bloom sarcoidosis (GEO accession no. GSE42834), and Suarez bacterial/viral s (GEO accession no. GSE60244.

Total 186 patients). And healthy controls from each respective dataset (223 individuals). We downloaded from GEO the filtered and normalized datasets, which have been normalized with different methods, according to type of data (RNA-Seq or Illumina microarray) or laboratory practices.

We then pooled the 10 datasets together, matching the targets by gene names. When a gene was absent in a least one dataset of origin, we completely removed a gene from the pooled dataset, so that we had a robust and stringent pooled dataset. A gene could be absent from any dataset for multiple reasons.

One of the platforms did not target this gene, the gene annotation databases used were different for each dataset (different version of the genomes) and there was no correspondence of gene name, or the gene was filtered out due to low expression in the filtered dataset of origin. Using these stringent criteria, we had a pooled dataset containing 11,912 genes in total, regrouping 851 individual whole blood samples. We then batch-corrected the pooled dataset, the batch being the origin of dataset, with the reference COMBAT algorithm (Johnson et al., 2007) from the sva library in R.

We checked the impact of batch correction on a mix of RNA-Seq and microarray datasets by drawing PCA plots (Fig. S5 A). We also verified high correlations before/after batch correction per group of patients (Fig.

S5 B) and expression on gene of interest (data not shown). From the 11,912 batch-corrected genes, we then selected the 101 genes that were contained in at least one of the nine published reduced gene signatures (Kaforou et al., 2013. Maertzdorf et al., 2016.

Roe et al., 2016. Singhania et al., 2018a. Suliman et al., 2018.

Sweeney et al., 2016. Zak et al., 2016. And reviewed in Singhania et al., 2018b.

And unpublished data). Trang Tran’s 20-gene signature was independently derived from both Berry London (Berry et al., 2010. GEO accession no.

GSE107991) and Leicester RNA-Seq datasets (GEO accession no. GSE107993). Differential gene expression analyses were performed on active TB patients compared with controls, LTBI, or controls plus LTBI individuals, using Wald tests (DESeq2 library.

Love et al., 2014) by fitting generalized linear models. A gradient-boosting machine algorithm (gbm v2.1.7 library) was applied on the lists of DEGs to determine the high order ranking of genes predicting the active TB status. For signature reduction, we performed a random forest algorithm (randomForest library v4.6-14) based on cumulative sensitivity of genes in their importance order.

Finally, the meta-data with cross-validation analysis combining two optimal signatures from microarray datasets from Berry et al. (2010) and six optimal signatures from the Berry London and Leicester RNA-Seq datasets (TB versus controls, TB versus LTBI, TB versus controls plus LTBI for each cohort) yielded 20 gene signatures (FCGR1A, GBP5, SEPT4, ANKRD22, BATF2, FCGR1B, GBP1, GBP6, LHFPL2, SERPING1, C1QB, CD274, GBP4, AIM2, FBXO6, PSTPIP2, ASPHD2, FCMR, RTP4,and APOL6). Genes ranked by DESeq2 Wald statistic for TB progressor patients at different time points or with different clinical symptoms compared with controls were used to look for enrichment of either the hallmark gene set using Broad’s gene set enrichment analysis preranked analysis and default settings.

The normalized enrichment score and the FDR were plotted using ggplot2. The different genes lists of DEG in the different clinical phenotypes, incipient TB, subclinical TB, and clinical TB (Fig. 2) were functionally annotated using Metacore (Thomson Reuters v 19.4).

R libraries used. All analyses have been made with R 3.5.1, using multiple libraries, and Bioconductor v3.8 (Anders et al., 2015). The libraries are.

Arules v1.6-4, sva v3.30.1 (Leek et al., 2012), Boruta v6.0.0 (Kursa and Rudnicki, 2010), ranger v0.12.1, ImpulseDE2 v1.6.1 (Fischer, 2019), VennDiagram v1.6.20, caret v6.0-85, lattice v0.20-38, RColorBrewer v1.1-2, tibble v2.1.3, tidyr v0.8.3, dplyr v0.8.1, DESeq2 v1.22.2 (Love et al., 2014), ComplexHeatmap v2.3.1 (Gu et al., 2016), ROCR v1.0-7, randomForest v4.6-14, ggbiplot v0.55, ggplot2 v3.2.0, and qusage v2.16.1 (Yaari et al., 2013). We acknowledge the Francis Crick Advanced Sequencing Facility, Bioinformatics and Biostatistics Science Technology Platforms, for their contribution to our sequencing processing, L. Moreira-Teixeira for reviewing the manuscript, and Marisol Alvarez Martinez for helpful advice on bioinformatic approaches used.

Graham, and A. Singhania and part of the project were funded by the Francis Crick Institute (Crick 10126. Crick 10468), which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.

The project, part of O. Tabone's salary, and salaries for J. Lee and R.

Verma were funded by the BIOASTER Microbiology Technology Institute, Lyon, France (with funding from the French Government Investissement d’Avenir program ANR-10-AIRT-03) and the Medical Diagnostic Discovery Department, bioMérieux SA, France. P. Haldar and R.

Verma were supported by the National Institute for Health Research Leicester Biomedical Research Centre and the University of Leicester. W.J. Branchett is funded by a Wellcome Investigator Award to A.

O’Garra (FC11028). Author contributions. A.

O’Garra and P. Haldar co-led the study. A.

Woltmann, M. Rodrigue, and P. Leissner designed the study and discussed the findings throughout the project with K.

Verma and J. Lee recruited TB, LTBI, and contacts for the Leicester cohort. C.M.

Graham performed RNA-Seq sample processing and helped to organize receipt of samples at The Crick and identifiable storage. O. Tabone and P.

Chakravarty performed bioinformatics analysis with major input from A. Singhania, and some input from T. Trang and F.

Reynier, all overseen by A. O’Garra. A.

O’Garra wrote the manuscript with input from P. Haldar, W.J. Branchett, O.

Tabone, and A. Singhania. All co-authors read, reviewed, and approved the paper..

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Yr awr gyflwyno ynghyd ag awr ar gyfer y dyletswyddau sy'n gysylltiedig â'r awr honno o gyflawni gan gynnwys paratoi ac asesu.Byddwch yn weithiwr proffesiynol brwdfrydig a chyfeillgar gyda sgiliau cyfathrebu a rhyngbersonol rhagorol ynghyd ag ymrwymiad i ddarparu profiad myfyriwr rhagorol.Beth fyddwch chi'n ei wneud - dyletswyddau allweddol Cael a bod yn gyfarwydd â dogfennau cwrs perthnasol sy'n rhoi gwybodaeth sy'n ymwneud â nodau a gweithrediad y cwrs, gan gynnwys y maes llafur, dulliau asesu, y dulliau addysgu a ffefrir, gwybodaeth sydd ei hangen ar fyfyrwyr, dyddiadau ac amseroedd y cwrs, ac ati.Cysylltu ag aelodau o dîm y rhaglen, fel y bo'n briodol, er mwyn deall gofynion, cydlynu cynlluniau a chamau gweinyddol ac i drafod newidiadau.Paratoi deunydd cwrs effeithiol a fydd yn cyflawni nodau'r cwrs, gan gynnwys gwella'r profiad dysgu trwy ddull myfyriwr-ganolog.Mynychu a darparu sesiynau fel y cytunwyd, darlithio, arwain trafodaethau a darparu arweiniad a chyngor fel arall a hyrwyddo dysgu, yn ôl yr angen. Paratoi a marcio, yn ôl yr angen, unrhyw waith ysgrifenedig cysylltiedig, gwaith stiwdio, gwaith dosbarth a phapurau arholiad, o fewn y gweithdrefnau asesu priodol.Rhoi adborth adeiladol ac amserol i fyfyrwyr ar eu cynnydd. Cadw cofnodion o'r fath o asesu a phresenoldeb ag sydd eu hangen ar y Brifysgol.Arsylwi amseroedd agor a chau dosbarthiadau yn llym.Sicrhau bod Cyfarwyddwr/Arweinydd y Rhaglen a'r dosbarth yn cael gwybod am eu habsenoldeb, lle nad oes modd osgoi hyn, cyn belled â phosibl. I ddarparu, lle bo hynny'n briodol, cwrs/cynllun gwaith, set o amcanion a rhestrau darllen i fyfyrwyr.Beth ddewch chi i’r rôl - profiad a chryfderau hanfodolY gallu i gyflwyno, yn ôl yr angen, ddarlithoedd ar-lein.Lefel dda o wybodaeth sy'n berthnasol i'r pwnc a'r maes proffesiynol. Tystiolaeth o ymgymryd â datblygiad proffesiynol parhaus (DPP).Dealltwriaeth gadarn o addysgeg.Y gallu i ddylunio deunydd addysgu a dysgu.Y gallu i ddefnyddio dulliau asesu priodol.Y gallu i gyflawni dyletswyddau gweinyddol mewn modd cywir ac amserol.Y gallu i gynllunio llwythi gwaith.Y gallu i nodi meysydd i'w gwella a defnyddio sgiliau menter a datrys problemau i wella perfformiad.Y gallu i gyfathrebu a lledaenu syniadau cymhleth a chysyniadol mewn amryw o ffyrdd - cyflwyniadau, adroddiadau, deunyddiau dysgu.Y gallu i ddatblygu perthnasoedd gwaith cynhyrchiol fel rhan o dîm proffesiynol.Y gallu i ddangos ymrwymiad i Gydraddoldeb ac Amrywiaeth, Iechyd a Diogelwch, Safonau Ansawdd.Beth fydd gennych chi - cymwysterau hanfodol Gradd anrhydedd dda a/neu gymhwyster proffesiynol cyfatebol.Statws Cymrawd fel rhan o gynllun Cydnabyddiaeth Proffesiynol yr Academi Addysg Uwch.Aelodaeth o gorff proffesiynol.Cyfanswm ein pecyn gwobrwyo Mae gennym ystod o fuddion gwych i weithwyr gan gynnwys:Aelodaeth o'r Cynllun Pensiwn Llywodraeth Leol gyda chyfraniadau haelCyfleoedd gweithio hyblyg ac anghysbellCyfleusterau chwaraeon a ffitrwydd arobryn gydag aelodaeth â chymhorthdal, ynghyd â ffisiotherapi / therapi cyflenwol / tylino chwaraeon â chymhorthdalMynediad i holl gyfleusterau'r llyfrgellCynlluniau aberthu cyflog gan gynnwys beicio i'r gwaithSut i wneud cais Os hoffech siarad â ni am y cyfle hwn, cysylltwch â Professor Sarah Curran, Athro Meddygaeth Podiatrig ac Adsefydlu, I gychwyn eich cais ewch i www.cardiffmet.ac.uk/jobsDyddiad cau.

Dydd Iau 25 Chwefror 2021, 4:30 pm Pe bai nifer uchel o geisiadau yn cael eu derbyn, efallai y bydd angen i ni gau'r swydd wag hon yn gynnar. Felly rydym yn eich annog i wneud cais mor gynnar â phosibl.Rydym wedi ymrwymo i gefnogi a hyrwyddo cydraddoldeb ac amrywiaeth ac i greu amgylchedd gwaith cynhwysol. Mae ein Cynllun Strategol yn sail i'n hymrwymiad i recriwtio a chadw'r dalent orau ac rydym yn croesawu ceisiadau gan bobl o gefndiroedd amrywiol. Rydym yn penodi yn ôl teilyngdod.Oherwydd y pandemig coronafeirws mae ein holl recriwtio bron yn cael ei gynnal fwy neu lai. Ein nod yw ymateb i'ch cais cyn gynted â phosibl.Gwybodaeth Bellach Disgrifiad Swydd Llawn a Manyleb Person Ein Pecyn YmgeisyddEwch i'n gwefan i gael gwybodaeth am weithio i ni, ein diwylliant, buddion, cydbwysedd rhwng bywyd a gwaith a'n cyfleoedd datblygu a datblygu gyrfa..

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£49.32 per hour (includes 18% annual leave payment)Tenure. Casual (as and when required)Welsh University of the Year 2021 by The Times where can i buy viagra over the counter usa and The Sunday Times Good University Guide. Our performance in this year’s Guide is where can i buy viagra over the counter usa exceptional. Our University was the biggest riser in Wales, where can i buy viagra over the counter usa and the third biggest in the UK, placing us in the UK top 40 for student satisfaction with teaching quality and the wider student experience.

We are very pleased that our strong performance has resulted in the University attracting high numbers of well qualified students and we are now seeking additional staff in both our academic schools and professional services departments.The opportunity Cardiff School of Sport and Health Sciences is a recognised centre of excellence in the UK and has established a national and international reputation for the quality of its academic and research work in the areas of sport and health sciences. Based across the Cyncoed and where can i buy viagra over the counter usa Llandaff campuses, we provide a broad range of vocationally focused academic programmes and pride ourselves on good student feedback and excellent graduate employability. Many of our programmes are professionally accredited to meet the requirements of the sport and healthcare professions and we where can i buy viagra over the counter usa engage closely with industry to provide consultancy services and professional development programmes. Our School also delivers a thriving research culture, with over 100 PhD students where can i buy viagra over the counter usa across a variety of disciplines.

The wellbeing of our staff, students and our local community underpins everything we do as we continue to add value through our teaching, innovation and research.You will work as part where can i buy viagra over the counter usa of a multi-disciplinary team to guide and supervise students in a learning environment on the HCPC approved BSc (Hons) Podiatry and MSc Muskuloskeletal Studies programmes in on-site clinical facilities,working in collaboration with NHS Staff. You will be expected to provide support to student and facilitate their development in accordance with the PACE competency framework and assist them in their development as clinical leaders.Please note that the session fee is inclusive of both teaching and associated duties, i.e. The hour of delivery plus one hour for the duties associated with that hour of delivery including preparation and assessment.You will be an enthusiastic and friendly professional with excellent communication and interpersonal skills coupled with a commitment to providing an excellent student experience.What you’ll do – key duties To obtain and be familiar with relevant course documents which give information related to the aims and operation of the course, including the syllabus, methods of assessment, preferred teaching methods, information needed by students, course dates and times, etc.To liaise with members of the programme team, as appropriate, in order to understand requirements, coordinate where can i buy viagra over the counter usa plans and administrative actions and to discuss changes.To prepare effective course material which will fulfil the aims of the course, including enhancement of the learning experience through a student-centred approach.To attend and deliver sessions as agreed, lecturing, leading discussions and otherwise providing guidance and advice and promoting learning, as required.To prepare and mark, as required, any related written work, studio work, class work and examination papers, within the appropriate assessment procedures.To provide constructive and timely feedback to students on their progress.To keep such records of assessment and attendance as the University requires.To observe class opening and closing times strictly.To ensure that the Programme Director/Leader and the class are informed of their absence, where this is unavoidable, as far in advance as possible.To provide, where appropriate, a course/work plan, set of objectives and reading lists to students.What you’ll bring – essential experience &. StrengthsAbility to deliver, as and where required online lectures.A good level of knowledge relevant to the subject and professional area.Evidence of undertaking continuous professional development (CPD).A sound understanding of pedagogy.Ability to design teaching and learning material.Ability to employ appropriate assessment methods.Ability to where can i buy viagra over the counter usa undertake administrative duties in an accurate and timely fashion.Ability to plan workloads.Ability to identify areas for improvement and to use initiative and problem solving skills to improve performance.Ability to communicate and disseminate complex and conceptual ideas in a variety of ways – presentations, reports, learning materials.Ability to develop productive working relationships as part of a professional team.Ability to demonstrate a commitment to Equality and Diversity, Health and Safety, Quality Standards.

What you’ll have - essential qualifications A good honours degree and/or equivalent professional qualification.Ability to achieve Fellow Status as part of the Higher Education Academy’s Professional Recognition scheme.Membership of a professional body.Our total reward package We have a range of great benefits for employees including:Membership of the Local Government Pension Scheme with generous contributionsFlexible and remote working opportunitiesAward winning sports and fitness facilities with subsidised membership, plus subsidised physiotherapy/complementary therapy/sports massageAccess to where can i buy viagra over the counter usa all library facilitiesSalary sacrifice schemes including cycle to work. How to apply If you would like to talk with us about this opportunity please contact Professor Sarah Curran, Professor of Podiatric Medicine and Rehabilitation at scurran@cardiffmet.ac.ukTo start your application please visit www.cardiffmet.ac.uk/jobsClosing date. Thursday 25 February 2021, 4:30 pm Should a high volume of applications be received, we may need where can i buy viagra over the counter usa to close this vacancy early. We therefore encourage you to where can i buy viagra over the counter usa apply as early as possible.We are committed to supporting and promoting equality and diversity and to creating an inclusive working environment.

Our Strategic Plan underpins our commitment to where can i buy viagra over the counter usa recruit and retain the best talent and we welcome applications from people from diverse backgrounds. We appoint on merit.Due to the erectile dysfunction viagra all of our recruitment is where can i buy viagra over the counter usa being conducted virtually. We aim to respond to your application as soon as possible.Further Information Full Job Description and Person Specification Our Candidate PackPlease visit our website for information about working for us, our culture, benefits, work-life balance and our career progression and development opportunities.Teitl y Swydd. Ysgol where can i buy viagra over the counter usa / Uned.

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Mae ein where can i buy viagra over the counter usa perfformiad yn y Canllaw eleni yn eithriadol, y dyrchafiad uchaf yng Nghymru, a'r trydydd mwyaf yn y DU, gan ein gosod yn 40 uchaf y DU ar gyfer boddhad myfyrwyr ag ansawdd addysgu a phrofiad ehangach myfyrwyr. Rydym yn where can i buy viagra over the counter usa falch iawn bod ein perfformiad cryf wedi arwain at y Brifysgol yn denu niferoedd uchel o fyfyrwyr â chymwysterau da ac rydym nawr yn chwilio am staff ychwanegol yn ein hysgolion academaidd a'n hadrannau gwasanaethau proffesiynol.Y cyfle Mae Ysgol Chwaraeon a Gwyddorau Iechyd Caerdydd yn ganolfan ragoriaeth gydnabyddedig yn y DU ac mae wedi sefydlu enw da cenedlaethol a rhyngwladol am ansawdd ei gwaith academaidd ac ymchwil ym meysydd chwaraeon a gwyddorau iechyd. Wedi'i leoli where can i buy viagra over the counter usa ar draws campysau Cyncoed a Llandaf, rydym yn darparu ystod eang o raglenni academaidd â ffocws galwedigaethol ac yn ymfalchïo mewn adborth da gan fyfyrwyr a chyflogadwyedd graddedig rhagorol. Mae llawer o'n rhaglenni wedi'u hachredu'n broffesiynol i fodloni gofynion y proffesiynau chwaraeon a gofal iechyd ac rydym yn ymgysylltu'n agos â diwydiant i ddarparu gwasanaethau ymgynghori a rhaglenni datblygiad proffesiynol.

Mae ein Hysgol hefyd yn cyflwyno diwylliant ymchwil ffyniannus, gyda dros 100 o where can i buy viagra over the counter usa fyfyrwyr PhD ar draws amrywiaeth o ddisgyblaethau. Mae lles ein staff, ein myfyrwyr a'n cymuned where can i buy viagra over the counter usa leol yn sail i bopeth a wnawn wrth i ni barhau i ychwanegu gwerth trwy ein haddysgu, ein harloesedd a'n hymchwil.Yn benodol, bydd deiliad y swydd yn gweithio fel rhan o dîm amlddisgyblaethol i arwain a goruchwylio myfyrwyr mewn amgylchedd dysgu ar y rhaglenni BSc (Anrh) Podiatreg ac MSc Astudiaethau Cyhyrysgerbydol MSc mewn cyfleusterau clinigol ar y safle, gan weithio ar y cyd â Staff y GIG. Disgwylir i diwtoriaid ddarparu cefnogaeth i fyfyrwyr a hwyluso eu datblygiad yn unol â fframwaith cymhwysedd PACE a'u cynorthwyo yn eu datblygiad fel arweinwyr clinigol.Sylwch fod y where can i buy viagra over the counter usa ffi sesiwn yn cynnwys dyletswyddau addysgu a dyletswyddau cysylltiedig, h.y. Yr awr gyflwyno ynghyd ag awr ar gyfer y dyletswyddau sy'n gysylltiedig â'r awr honno o gyflawni gan gynnwys paratoi ac asesu.Byddwch yn weithiwr proffesiynol brwdfrydig a chyfeillgar gyda sgiliau cyfathrebu a rhyngbersonol rhagorol ynghyd ag ymrwymiad i ddarparu profiad myfyriwr rhagorol.Beth fyddwch chi'n ei wneud - dyletswyddau allweddol Cael a bod yn gyfarwydd where can i buy viagra over the counter usa â dogfennau cwrs perthnasol sy'n rhoi gwybodaeth sy'n ymwneud â nodau a gweithrediad y cwrs, gan gynnwys y maes llafur, dulliau asesu, y dulliau addysgu a ffefrir, gwybodaeth sydd ei hangen ar fyfyrwyr, dyddiadau ac amseroedd y cwrs, ac ati.Cysylltu ag aelodau o dîm y rhaglen, fel y bo'n briodol, er mwyn deall gofynion, cydlynu cynlluniau a chamau gweinyddol ac i drafod newidiadau.Paratoi deunydd cwrs effeithiol a fydd yn cyflawni nodau'r cwrs, gan gynnwys gwella'r profiad dysgu trwy ddull myfyriwr-ganolog.Mynychu a darparu sesiynau fel y cytunwyd, darlithio, arwain trafodaethau a darparu arweiniad a chyngor fel arall a hyrwyddo dysgu, yn ôl yr angen.

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Dydd Iau 25 Chwefror 2021, 4:30 pm Pe bai nifer uchel o geisiadau yn cael eu derbyn, efallai y bydd angen i ni gau'r swydd wag hon yn gynnar. Felly rydym yn eich annog i wneud cais mor gynnar â phosibl.Rydym wedi ymrwymo i where can i buy viagra over the counter usa gefnogi a hyrwyddo cydraddoldeb ac amrywiaeth ac i greu amgylchedd gwaith cynhwysol. Mae ein Cynllun Strategol yn sail i'n hymrwymiad i recriwtio a chadw'r dalent orau ac rydym yn croesawu ceisiadau gan bobl o gefndiroedd amrywiol where can i buy viagra over the counter usa. Rydym yn penodi yn ôl teilyngdod.Oherwydd y pandemig coronafeirws mae ein holl recriwtio bron yn cael ei gynnal fwy neu lai.

Ein nod yw ymateb i'ch cais cyn gynted â phosibl.Gwybodaeth Bellach Disgrifiad Swydd Llawn a Manyleb Person Ein Pecyn YmgeisyddEwch i'n gwefan i gael gwybodaeth am weithio i ni, ein diwylliant, buddion, cydbwysedd rhwng bywyd a gwaith a'n cyfleoedd datblygu a datblygu gyrfa..

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IntroductionEarly life is regarded as a can you take viagra with alcohol crucial period of neurobiological, emotional, social and physical development in all animal species and may have long-term implications for health across the life course. The first studies examining the can you take viagra with alcohol preadult origins of chronic disease were probably published more than 50 years ago and based on rodent models.1 By briefly administering a suboptimal diet to newborn mice, Dubos and others1 demonstrated a marked impact on subsequent growth and resistance to . In the 1970s, Forsdahl,2 using infant mortality rates as a proxy for living conditions at birth, arguably provided the first evidence in humans for an association with heart disease in later life. In the last two decades, findings from longitudinal can you take viagra with alcohol studies with extended mortality and morbidity surveillance have implicated a host of preadult characteristics as potential risk factors for several chronic disease outcomes, including perinatal and postnatal growth,3 coordination,4 intelligence,5 6 mental health,7 overweight,8 9 physical stature,10 raised blood pressure,11 12 cigarette smoking,13 physical strength14 and diet15 among many others.16An array of prospective studies has also demonstrated associations of childhood socioeconomic disadvantage–indexed by paternal social class or education, the presence of household amenities and domestic overcrowding—with somatic health outcomes in adulthood, chiefly premature mortality and cardiovascular disease.17 18 Parallel work has been undertaken by psychologists and psychiatrists exploring the consequences of childhood maeatment for later psychopathologies—perhaps the most well examined health endpoint in this context.19 20 Collectively, these early life circumstances have been more widely defined to comprise the separate themes of material deprivation (eg, economic hardship and long-term unemployment). Stressful family dynamics (eg, physical and can you take viagra with alcohol emotional abuse, psychiatric illness or substance abuse by a family member).

Loss or threat of loss (eg, death or serious illness …INTRODUCTIONSevere acute respiratory syndrome erectile dysfunction 2 (erectile dysfunction), causative agent of erectile dysfunction disease (erectile dysfunction treatment), emerged in Wuhan, China, in late 2019. On 11 March 2020, the World Health Organization (WHO) declared erectile dysfunction treatment a viagra, with over 10 million confirmed cases as of can you take viagra with alcohol the beginning of July 2020.1 2 The first patient in the Netherlands was confirmed on 27 February 2020.3 Cases primarily clustered in the southeastern part of the country, but were reported in other regions quickly hereafter. Multi-pronged interventions to suppress the spread of the viagra, including social distancing, school and bar/restaurant closure, and stringent advice to home quarantine when feeling ill and work from home, were implemented on 16 March 2020—and were relaxed gradually since 1 June 2020. By 1 July 2020, 50 273 cases, 11 877 hospitalisations, and 6113 related deaths were reported in the Netherlands.3Supplemental materialReported erectile dysfunction treatment cases worldwide can you take viagra with alcohol are an underestimation of the true magnitude of the viagra. The scope of undetected cases remains largely unknown due to difference in restrictive testing policy and registration across countries, and occurrence of can you take viagra with alcohol asymptomatic s.4 5 Large-scale nationwide serosurveillance studies measuring erectile dysfunction-specific serum antibodies could help to better assess the number of s, viral spread, and groups at risk of in the general population by incorporating extensive questionnaire data, for example, on lifestyle, behaviour and profession.

This might yield different factors than those identified for (severely-ill) clinical cases investigated more frequently up until now.6 7 Unfortunately, such nationwide studies (eg, in Spain8 and Iceland,9) also referred to as Unity Studies by the WHO,10 are scarce and mainly set up through convenience sampling.Therefore, a nationwide serosurveillance study (PIENTER-Corona, PICO) was initiated quickly after the lockdown was in effect. This cohort is unique as it comprises data available from a previous serosurvey established in 2016/17 (PIENTER-3) of a randomised nationwide sample of Dutch citizens, across all ages and a separate sample enriched for Orthodox-Reformed Protestants, whom might can you take viagra with alcohol have been exposed to erectile dysfunction more frequently due to their socio-geographical-clustered lifestyle.11 12 The presented serological framework and findings of our first round of inclusion can support public health policy in the Netherlands as well as internationally.METHODSStudy designIn 2016/17, the National Institute for Public Health and the Environment of the Netherlands (RIVM) initiated a large-scale nationwide serosurveillance study (PIENTER-3) (n=7600. Age-range 0–89 years). The primary aim was to obtain insights into the can you take viagra with alcohol protection against treatment-preventable diseases offered by the National Immunisation Programme in the Netherlands. A comprehensive description of PIENTER-3 has been published previously.13 Briefly, participants were selected via a two-stage cluster design, comprising 40 municipalities in five regions nationwide can you take viagra with alcohol (henceforth ‘national sample’, NS), and nine municipalities in the low vaccination coverage municipalities (LVC), inhabited by a relative large proportion of Orthodox-Reformed Protestants (figure 1).

Among other materials, sera and questionnaire data had been collected from all participants. Hence, the PIENTER-3 study acted as baseline sample of can you take viagra with alcohol the Dutch population for the present cross-sectional PICO-study since 6102 participants (80%) consented to be approached for follow-up (after updating addresses and screening of possible deaths). The study was powered to estimate an overall seroprevalence with a precision of at least 2.5%.13 The PICO-study protocol was approved by the Medical Ethics Committee MEC-U, the Netherlands (Clinical Trial Registration NTR8473), and conformed to the principles embodied in the Declaration of Helsinki.Geographical representation of number of participants in the PICO-study, the Netherlands, first round of inclusion, per municipality. The size can you take viagra with alcohol of the dots reflect the absolute number of participants. Thicker grey and smaller light grey boundaries represent provinces and municipalities, respectively, and orange and blue boundaries characterise municipalities from the national and low vaccination coverage sample, respectively." data-icon-position data-hide-link-title="0">Figure 1 Geographical representation can you take viagra with alcohol of number of participants in the PICO-study, the Netherlands, first round of inclusion, per municipality.

The size of the dots reflect the absolute number of participants. Thicker grey and smaller light grey boundaries represent provinces and municipalities, respectively, and orange and blue boundaries characterise municipalities from the national and low vaccination coverage sample, respectively.Study population and materialsOn 25 March 2020, can you take viagra with alcohol an invitation letter was sent. Invitees (age-range 2–92 years) willing to participate registered online. After enrolment, participants received an instruction letter on how can you take viagra with alcohol to self-collect a fingerstick blood sample in a microtainer (maximum of 0.3 mL). Blood samples were returned to can you take viagra with alcohol the RIVM-laboratory in safety envelopes.

Serum samples were stored at −20°C awaiting analyses. Materials were collected between March 31 and May 11, with the majority (80%) in the first week can you take viagra with alcohol of April 2020 (median collection date April 3). Simultaneous with the blood collection, participants were asked to complete an (online) questionnaire, including questions regarding sociodemographic characteristics, erectile dysfunction treatment-related symptoms, and potential other determinants for erectile dysfunction seropositivity, such as comorbidities, medication use and behavioural factors. All participants can you take viagra with alcohol provided written informed consent.Laboratory methodsSerum samples (diluted 1:200) were tested for the presence of erectile dysfunction spike S1-specific IgG antibodies using a validated fluorescent bead-based multiplex-immunoassay as described.14 A cut-off concentration for seropositivity (2.37 AU/mL. With specificity of 99% and sensitivity of 84.4%) was determined by ROC-analysis of 400 pre-viagra control samples (including a nationwide random cross-sectional sample (n=108)) as well as patients with confirmed influenza-like illnesses caused by erectile dysfunctiones and other viagraes, and a can you take viagra with alcohol selection of sera from 115 PCR-confirmed erectile dysfunction treatment cases with mild, or severe disease symptoms.

Seropositive PICO-samples and those with a concentration 25% below the cut-off were retested (n=138), and the geometric mean concentration (GMC) was calculated. Paired pre-viagra PIENTER-3-samples of these retested can you take viagra with alcohol PICO-samples (available from 129/138) were tested correspondingly as described above to correct for false-positive results (online supplemental figure S1A).Statistical analysesStudy population, erectile dysfunction treatment-related symptoms and antibody responsesData management and analyses were conducted in SAS v.9.4 (SAS Institute Inc., USA) and R v.3.6. P values <0.05 were considered statistically significant. Sociodemographic characteristics and erectile dysfunction treatment-related symptoms (general, respiratory, and gastrointestinal) developed since the start can you take viagra with alcohol of the epidemic were stratified by sample (NS vs LVC), or sex, respectively, and described for seropositive and seronegative participants. Differences were can you take viagra with alcohol tested via Pearson’s χ², or Fisher’s exact test if appropriate.

Differences in GMC between reported symptoms in seropositive participants were determined by calculating the difference in log-transformed concentrations of those who developed symptoms at least 4 weeks prior to the sampling—ensuring a plateaued response—and tested by means of a Mann-Whitney U-test.Seroprevalence estimatesSeroprevalence estimates (with 95% Wilson CIs (CI)) for erectile dysfunction-specific antibodies were calculated taking into account the survey design (ie, controlling for region and municipality) and weighted by sex, age, ethnic background and degree of urbanisation to match the distribution of the general Dutch population in both the NS and LVC sample. Estimates were corrected for test performance via the Rogan can you take viagra with alcohol &. Gladen bias correction (with sensitivity of 84.4% and assuming a specificity of 100% after cross-validation with pre-sera).15 Smooth age-specific seroprevalence estimates were obtained with a logistic regression in a Generalised Additive Model using penalised splines.16Risk factors for erectile dysfunction seropositivityA random-effects logistic regression model was used to identify risk factors for erectile dysfunction seropositivity, applying a full case analysis (n=3100. Values were missing for <5% of can you take viagra with alcohol the participants). Potential risk factors included sociodemographic characteristics (sex, age group, region, ethnic background, Orthodox-Reformed Protestants, educational can you take viagra with alcohol level, household size, (parent with a) contact profession, healthcare worker), and erectile dysfunction treatment-related factors (contact with a erectile dysfunction treatment confirmed case, number of persons contacted yesterday, working from home (normally and in the last week), comorbidities (combining diabetes, history of malignancy, immunodeficiency, cardio-vascular, kidney and chronic lung disease (note.

As a sensitivity analysis, comorbidities were also included separately)), and use of blood pressure medication, immunosuppressants, statins and antivirals/antibiotics in the last month). Models included can you take viagra with alcohol a random intercept, potential clustering by municipality and region was accounted for, and odds ratios (OR) in univariable analyses were a priori adjusted for sex and age. Variables with p<0.10 were entered in the multivariable analysis, and backward selection was performed—manually dropping variables one-by-one based on p≥0.05—to identify significant risk factors. Adjusted ORs and corresponding can you take viagra with alcohol 95% CIs were provided.RESULTSStudy populationOf 6102 invitees, 3207 (53%) donated a serum sample and filled-out the questionnaire, of which 2637 persons from the NS and 570 from the LVC. Participants from across the country participated (figure 1), with can you take viagra with alcohol age ranging from 2 to 90 years (table 1).

In the NS, slightly more women (55%) participated, most (88%) were of Dutch descent, nearly half had a high educational level, and 45% was religious. 20 percent of persons between age 25–66 can you take viagra with alcohol years were healthcare workers and 56% of the (parents of) participants reported to have had daily contact with patients, clients and/or children in their profession/volunteer work normally. Over half of the participants lived in a ≥2-person household, and 78% reported to have had physical contact with <5 people outside their own household yesterday (during lockdown), of which more than half with nobody. Comorbidities most can you take viagra with alcohol frequently reported included chronic lung and cardiovascular disease (both 13%), and a history of malignancy (5%). In line with the population distribution, the LVC sample can you take viagra with alcohol was characterised by a relative high proportion of Orthodox-Reformed Protestants from Dutch descent (table 1).

Sociodemographic characteristics between responders and non-responders are provided in online supplemental table S1.View this table:Table 1 Sociodemographic characteristics of participants in the PICO-study and weighted seroprevalence in the general population of the Netherlands, first round of inclusion, by national sample and low vaccination coverage sampleSupplemental materialerectile dysfunction treatment-related symptoms and antibody responsesIn total, 63% of participants reported to have had ≥1 erectile dysfunction treatment-related symptom(s) since the start of the epidemic, with runny nose (37%), headache (33%), and cough (30%) being most common (table 2). All reported symptoms were significantly higher in seropositive compared to seronegative persons, except for can you take viagra with alcohol stomach ache. The majority of those seropositive (93%) reported to have had can you take viagra with alcohol symptoms (90% of men vs 95% of women), of whom three already in mid-February, 2 weeks prior to the official first notification. Median duration of illness in the seropositive participants was 8.5 days (IQR. 4.0–12.5), 16% (n=12) visited can you take viagra with alcohol ageneral practitioner and one was admitted to the hospital.

Among seropositive persons, most reported to have had ≥1 respiratory symptom(s) (86%), with runny nose and cough (both 61%) most regularly, and ≥1 general (84%) symptom(s), of which anosmia/ageusia (53%) was most discriminative as compared to the seronegative participants (4%, p<0.0001) (table 2). Symptoms were more common in women, except for anosmia/ageusia, can you take viagra with alcohol cough and irritable/confusion. Almost 75% of the seropositive participants met the erectile dysfunction treatment case definition of fever and/or cough and/or dyspnoea, which improved to can you take viagra with alcohol 80% when anosmia/ageusia was included—while remaining 36% in those seronegative. GMC was significantly higher among seropositive persons with fever vs without (48.2 vs 11.6 AU/mL, p=0.01), and with dyspnoea vs without (78.6 vs 13.5 AU/mL, p=0.04).View this table:Table 2 erectile dysfunction treatment-related symptoms since the start of the epidemic among all participants in the PICO-study reporting symptoms (n=3147), first round of inclusionSeroprevalence estimatesOverall weighted seroprevalence in the NS was 2.8% (95% CI 2.1 to 3.7), did not differ between sexes or ethnic backgrounds (table 1), and was not higher among healthcare workers (2.7% vs non-healthcare workers 2.5%). Seroprevalence was lowest in the northern region (1.3%) and can you take viagra with alcohol highest in the mid-west (4.0%).

Estimates were lowest in children—gradually increasing from below 1% at age 2 years to 3% at 17 years—was highest in age group 18–39 years (4.9%) and ranged between 2 and 4% up to 90 years of age (figure 2). In both samples, seroprevalence was can you take viagra with alcohol highest in Orthodox-Reformed Protestants (>7%) (table 1). Online supplement figure S1B displays the distribution of IgG concentrations can you take viagra with alcohol for all participants by age, and online supplemental figure S2 ⇓shows the seroprevalence smoothed by age in the LVC.Smooth age-specific erectile dysfunction seroprevalence in the general population of the Netherlands, beginning of April 2020." data-icon-position data-hide-link-title="0">Figure 2 Smooth age-specific erectile dysfunction seroprevalence in the general population of the Netherlands, beginning of April 2020.Risk factors for erectile dysfunction seropositivityVariables that were associated with erectile dysfunction seropositivity in univariable analyses included age group, Orthodox-Reformed Protestant, had been in contact with a erectile dysfunction treatment case, use of immunosuppressants, and antibiotic/antiviral medication in the last month (table 3). In multivariable analysis, substantial higher odds were observed for those who took immunosuppressants the last month, were Orthodox-Reformed Protestant, had been in contact with a erectile dysfunction treatment confirmed case, and from age groups 18–24 and 25–39 years (compared to 2–12 years).View this table:Table 3 Risk factor analysis for erectile dysfunction seropositivity among all participants (n=3100. Full case analysis) in the PICO-study, first round of inclusionDISCUSSIONHere, we can you take viagra with alcohol have estimated the seroprevalence of erectile dysfunction-specific antibodies and identified risk factors for seropositivity in the general population of the Netherlands during the first epidemic wave in April 2020.

Although overall seroprevalence was still low at this phase, important risk factors for seropositivity could be identified, including adults aged 18–39 years, persons using immunosuppressants, and Orthodox-Reformed Protestants. These data can you take viagra with alcohol can guide future interventions, including strategies for vaccination, believed to be a realistic solution to overcome this viagra.This PICO-study revealed that 2.8% (95% CI 2.1 to 3.7) of the Dutch population had detectable erectile dysfunction-specific serum IgG antibodies, suggesting that almost half a million inhabitants (of in total 17 423 98117) were infected (487 871 (95% CI 365 904 to 644 687)) in mid-March, 2020 (taking into account the median time to seroconvert18). Several seropositive can you take viagra with alcohol participants reported to have had erectile dysfunction treatment-related symptoms back in mid-February, suggesting the viagra circulated in our country at the beginning of February already. Our overall estimate is in line with preliminary results from another study conducted in the Netherlands in the beginning of April which found 2.7% to be seropositive, although this study was performed in healthy blood donors aged 18–79 years.19 Worldwide, various seroprevalence studies are ongoing. A large nationwide study in Spain showed that around 5% (ranging between can you take viagra with alcohol 3.7% and 6.2%) was seropositive, indicating that only a small proportion of the population had been infected in one of the hardest hit countries in Europe.

Current studies in literature mostly cover erectile dysfunction treatment hotspots or specific regions—with possibly bias in selection of participants and/or smaller age-ranges—with rates ranging between 1–7% in April (eg, in Los Angeles County (CA, USA)20 or ten other sites in the USA,21 Geneva (Switzerland),22 and Luxembourg23). Estimates also very can you take viagra with alcohol much depend on test performances. Particularly, when seroprevalence is relatively low, can you take viagra with alcohol specificity of the assay should approach near 100% to diminish false-positive results and minimise overestimation. Although we cannot rule-out false-positive samples completely, our assay was validated using a broad range of positive and negative erectile dysfunction samples. PICO-samples were can you take viagra with alcohol cross-linked to pre-viagra concentration.

And bias correction for test performance was applied to represent most accurate estimates. In addition, future studies should establish whether epidemiologically dominant genetic changes in the spike protein of erectile dysfunction influence binding to spike S1 used in our and other assays.Seroprevalence was highest in adults aged 18–39 years, which is in line with the serosurvey among blood donors in the Netherlands, but contrary to the low incidence rate as reported can you take viagra with alcohol in Dutch surveillance, caused by restrictive testing of risk groups and healthcare workers at the beginning of the epidemic, primarily identifying severe cases.3 19 The elevation in these younger adults may be explained by increased social contacts typical for this age group, in addition to specific social activities in February, such as skiing holidays in the Alps (from where the viagra disseminated quickly across Europe), or carnival festivities in the Netherlands (ie, multiple superspreading events primarily in the mid and Southern part, explaining local elevation in seroprevalence). In correspondence with other nationwide studies8 9 and can you take viagra with alcohol reports from the Dutch government,3 24 seroprevalence was lowest in children. Although some rare events of paediatric inflammatory multisystem syndrome have been reported, this group seems to be at decreased risk for developing (severe) erectile dysfunction treatment in general, which may be explained by less severe possibly resulting in a limited humoral response.25 26 Further, significantly higher odds for seropositivity were seen in Orthodox-Reformed Protestants. This community can you take viagra with alcohol lives socio-geographically clustered in the Netherlands, that is, work, school, leisure and church are intertwined heavily.

As observed in other countries, particularly frequent attendance of church with close distance to others, including singing activities, might have fuelled the spread of erectile dysfunction within this community in the beginning of the epidemic.11 12 Whereas the comorbidities with possible increased risk of severe erectile dysfunction treatment were not associated with seropositivity in this study, immunosuppressants use did display higher odds (note. We did not have information of specific can you take viagra with alcohol drugs). Recent data indicate that immunosuppressive treatment is not associated with worse erectile dysfunction treatment outcomes,27 28 yet continued surveillance is warranted as these patients might be more prone to can you take viagra with alcohol (future) , for instance due to a possible attenuated humoral immune response.29The majority of seropositive participants exhibited ≥1 symptom(s), mostly general and respiratory. A recent meta-analysis found a pooled asymptomatic proportion of 16%,5 hence the observed overall fraction in the present study (7%) might be a conservative estimate as the self-reported symptoms could have been due to other reasons or circulating pathogens along the recalled period (ie, 62% of the seronegative participants reported symptoms too). The asymptomatic proportion might be different across ages5 and should be explored further can you take viagra with alcohol along with elucidating the overall contribution of asymptomatic transmission via well-designed contact-tracing studies.

Interestingly, clinical studies have observed anosmia/ageusia to be associated with erectile dysfunction , and this notion is supported here at a population-based level.30 In the viagra context, sudden onset of anosmia/ageusia seems to be a useful surveillance tool, which can contribute to early disease recognition and minimise transmission by rapid self-isolation.This study has some limitations. First, although half of the total municipalities in the Netherlands were included, can you take viagra with alcohol some erectile dysfunction treatment hotspots might be missed due to the study design. Second, our study population consisted of more Dutch (88%) than non-Dutch persons and relative more healthcare workers (20%) when compared to the general population (76% and 14%, respectively).17 Healthcare workers in the Netherlands do not can you take viagra with alcohol seem to have had a higher likelihood of , and transmission seems to have taken place mostly in household settings.3 31 Although selectivity in response was minimised by weighting our study sample on a set of sociodemographic characters to match the Dutch population, seroprevalence might still be slightly influenced. Third, some potential determinants for seropositivity could have been missed as we might have been underpowered to detect small differences given the low prevalence in this phase, or because these questions had not been included in the questionnaire (as it was designed in the very beginning of the epidemic). Finally, at this stage the proportion of infected individuals that fail to show can you take viagra with alcohol detectable seroconversion is unknown, potentially leading to underestimation of the percentage of infected persons.To conclude, we estimated that 2.8% of the Dutch inhabitants, that is, nearly half a million, were infected with erectile dysfunction amidst the first epidemic wave in the beginning of April 2020.

This is in striking contrast with the 30-fold lower number of reported cases (of approximately 15 000)3, and underlines the importance of seroepidemiological studies to estimate the true viagra size. The proportion of persons still susceptible to erectile dysfunction is high and IFR is substantial.4 Globally, nationwide seroepidemiological studies are urgently needed for better understanding of related risk factors, viral spread, and measures applied to mitigate dissemination.7 The prospective nature of our study will enable us to gain key insights on the duration and quality of antibody responses in infected persons, and hence possible protection of disease by antibodies.6 Serosurveys will thus play a major role in guiding future interventions, such as strategies for vaccination (of risk groups), since even when treatments become available, initial treatment availability will be limited.What is already known on this topicReported erectile dysfunction treatment cases worldwide are an underestimation of the true magnitude of the viagra as the scope of undetected cases remains largely unknown.Various symptoms and risk factors have been identified in patients seeking medical advice, however, these may not be representative for s in the general population.Seroepidemiological studies in outbreak settings have been performed, however, studies on a nationwide level covering all ages remain limited.What this study addsThis nationwide seroepidemiological study covering all ages reveals that 2.8% of the Dutch population had been infected with erectile dysfunction at the beginning of April 2020, that is, 30 times higher than the official cases reported, leaving a large proportion of the population still susceptible for .The highest seroprevalence was observed in young adults can you take viagra with alcohol from 18 to 39 years of age and lowest in children aged 2 to 17 years, indicating marginal erectile dysfunction s among children in general.Persons taking immunosuppressants as well as those from the Orthodox-Reformed Protestant community had over four times higher odds of being seropositive compared to others.The extend of the spread of erectile dysfunction and the risk groups identified here, can inform monitoring strategies and guide future interventions internationally.AcknowledgmentsFirst of all, we gratefully acknowledge the participants of the PICO-study. Secondly, this study would not have been possible without the instrumental contribution of colleagues from the National Institute of Public Health and Environment (RIVM), Bilthoven, the Netherlands, more specially the department of Immunology of Infectious Diseases and treatments, regarding logistics and/or laboratory analyses (Marjan Bogaard-van Maurik, Annemarie Buisman, Pieter van Gageldonk, Hinke ten Hulscher-van Overbeek, Petra Jochemsen, Deborah Kleijne, Jessica Loch, Marjan Kuijer, Milou Ohm, Hella Pasmans, Lia de Rond, Debbie van Rooijen, Liza Tymchenko, Esther van Woudenbergh, can you take viagra with alcohol and Mary-lene de Zeeuw-Brouwer), the Epidemiology and Surveillance department concerning logistics (Francoise van Heiningen, Alies van Lier, Jeanet Kemmeren, Joske Hoes, Maarten Immink, Marit Middeldorp, Christiaan Oostdijk, Ilse Schinkel-Gordijn, Yolanda van Weert, and Anneke Westerhof), methodological insights (Hendriek Boshuizen, Susan Hahné, Scott McDonald, Rianne van Gageldonk-Lafeber, Jan van de Kassteele, and Maarten Schipper) and manuscript reviewing (Susan van den Hof, and Don Klinkenberg), department of IT and Communication for help with the invitations (Luppo de Vries, Daphne Gijselaar, and Maaike Mathu), student interns for additional support (Stijn Andeweg for creating online supplemental figures 1A and 1B. Janine Wolf, Natasha Kaagman, and Demi Wagenaar for logistics. And Lisette van Cooten for data entry of can you take viagra with alcohol paper questionnaires), and Sidekick-IT, Breda, the Netherlands, regarding data flow (Tim de Hoog).

This study was funded by the ministry of Health, Welfare and Sports (VWS), the Netherlands..

IntroductionEarly life is regarded as a crucial where can i buy viagra over the counter usa period of neurobiological, emotional, social and physical development in all animal species and may have long-term implications for health across the http://gavran-hausmeister.de/impressum-datenschutz/ life course. The first studies examining the preadult origins where can i buy viagra over the counter usa of chronic disease were probably published more than 50 years ago and based on rodent models.1 By briefly administering a suboptimal diet to newborn mice, Dubos and others1 demonstrated a marked impact on subsequent growth and resistance to . In the 1970s, Forsdahl,2 using infant mortality rates as a proxy for living conditions at birth, arguably provided the first evidence in humans for an association with heart disease in later life. In the last two decades, findings from longitudinal studies with extended mortality and morbidity surveillance have implicated a host of preadult characteristics as potential risk factors for several chronic disease outcomes, including perinatal and postnatal growth,3 coordination,4 intelligence,5 6 mental health,7 overweight,8 9 physical stature,10 raised blood pressure,11 12 cigarette smoking,13 physical strength14 and diet15 among many others.16An array of prospective studies has also demonstrated associations of childhood socioeconomic disadvantage–indexed by paternal social class or education, the presence of household amenities and domestic overcrowding—with somatic health outcomes in adulthood, chiefly premature mortality and cardiovascular disease.17 18 Parallel work has been undertaken by psychologists and psychiatrists exploring the consequences of childhood maeatment for later psychopathologies—perhaps the most well where can i buy viagra over the counter usa examined health endpoint in this context.19 20 Collectively, these early life circumstances have been more widely defined to comprise the separate themes of material deprivation (eg, economic hardship and long-term unemployment).

Stressful family dynamics (eg, physical and emotional where can i buy viagra over the counter usa abuse, psychiatric illness or substance abuse by a family member). Loss or threat of loss (eg, death or serious illness …INTRODUCTIONSevere acute respiratory syndrome erectile dysfunction 2 (erectile dysfunction), causative agent of erectile dysfunction disease (erectile dysfunction treatment), emerged in Wuhan, China, in late 2019. On 11 March 2020, the World Health Organization (WHO) declared erectile dysfunction treatment a viagra, with over 10 million confirmed cases as of the beginning of July 2020.1 2 The first patient in the Netherlands was confirmed on 27 February 2020.3 Cases primarily clustered in the southeastern part of the country, but were reported in other where can i buy viagra over the counter usa regions quickly hereafter. Multi-pronged interventions to suppress the spread of the viagra, including social distancing, school and bar/restaurant closure, and stringent advice to home quarantine when feeling ill and work from home, were implemented on 16 March 2020—and were relaxed gradually since 1 June 2020.

By 1 July 2020, 50 273 cases, 11 877 hospitalisations, and 6113 related deaths were reported in the Netherlands.3Supplemental materialReported erectile dysfunction treatment cases worldwide are an where can i buy viagra over the counter usa underestimation of the true magnitude of the viagra. The scope of undetected cases remains largely unknown due to difference in restrictive testing policy and registration across countries, and occurrence of asymptomatic s.4 5 Large-scale where can i buy viagra over the counter usa nationwide serosurveillance studies measuring erectile dysfunction-specific serum antibodies could help to better assess the number of s, viral spread, and groups at risk of in the general population by incorporating extensive questionnaire data, for example, on lifestyle, behaviour and profession. This might yield different factors than those identified for (severely-ill) clinical cases investigated more frequently up until now.6 7 Unfortunately, such nationwide studies (eg, in Spain8 and Iceland,9) also referred to as Unity Studies by the WHO,10 are scarce and mainly set up through convenience sampling.Therefore, a nationwide serosurveillance study (PIENTER-Corona, PICO) was initiated quickly after the lockdown was in effect. This cohort is unique as it comprises data available from a previous serosurvey established in 2016/17 (PIENTER-3) of a randomised nationwide sample of Dutch citizens, across all ages and a separate sample enriched for Orthodox-Reformed Protestants, whom might have been exposed to erectile dysfunction more frequently due to their socio-geographical-clustered lifestyle.11 12 The presented serological framework and findings of our first round of inclusion can support public health policy in the Netherlands as well as internationally.METHODSStudy designIn 2016/17, the National Institute for Public Health and the Environment of the Netherlands (RIVM) initiated a large-scale nationwide serosurveillance study where can i buy viagra over the counter usa (PIENTER-3) (n=7600.

Age-range 0–89 years). The primary where can i buy viagra over the counter usa aim was to obtain insights into the protection against treatment-preventable diseases offered by the National Immunisation Programme in the Netherlands. A comprehensive description of PIENTER-3 has been published previously.13 Briefly, participants were selected via a two-stage cluster design, comprising 40 municipalities in five regions nationwide (henceforth ‘national sample’, NS), and nine municipalities in the low vaccination coverage municipalities (LVC), inhabited by a relative large where can i buy viagra over the counter usa proportion of Orthodox-Reformed Protestants (figure 1). Among other materials, sera and questionnaire data had been collected from all participants.

Hence, the PIENTER-3 study acted as baseline sample of the Dutch population for the present cross-sectional PICO-study since 6102 participants (80%) consented to be approached for follow-up (after updating addresses and screening of possible deaths) where can i buy viagra over the counter usa. The study was powered to estimate an overall seroprevalence with a precision of at least 2.5%.13 The PICO-study protocol was approved by the Medical Ethics Committee MEC-U, the Netherlands (Clinical Trial Registration NTR8473), and conformed to the principles embodied in the Declaration of Helsinki.Geographical representation of number of participants in the PICO-study, the Netherlands, first round of inclusion, per municipality. The size of the where can i buy viagra over the counter usa dots reflect the absolute number of participants. Thicker grey and smaller light grey boundaries represent provinces and municipalities, respectively, and orange and blue boundaries characterise municipalities from the national and low vaccination coverage sample, respectively." data-icon-position data-hide-link-title="0">Figure 1 Geographical representation of number of participants in the PICO-study, where can i buy viagra over the counter usa the Netherlands, first round of inclusion, per municipality.

The size of the dots reflect the absolute number of participants. Thicker grey and smaller light grey boundaries represent provinces and where can i buy viagra over the counter usa municipalities, respectively, and orange and blue boundaries characterise municipalities from the national and low vaccination coverage sample, respectively.Study population and materialsOn 25 March 2020, an invitation letter was sent. Invitees (age-range 2–92 years) willing to participate registered online. After enrolment, participants received an instruction letter on how where can i buy viagra over the counter usa to self-collect a fingerstick blood sample in a microtainer (maximum of 0.3 mL).

Blood samples were returned to the RIVM-laboratory in where can i buy viagra over the counter usa safety envelopes. Serum samples were stored at −20°C awaiting analyses. Materials were collected between March 31 and May 11, with the majority (80%) in the first where can i buy viagra over the counter usa week of April 2020 (median collection date April 3). Simultaneous with the blood collection, participants were asked to complete an (online) questionnaire, including questions regarding sociodemographic characteristics, erectile dysfunction treatment-related symptoms, and potential other determinants for erectile dysfunction seropositivity, such as comorbidities, medication use and behavioural factors.

All participants provided written informed consent.Laboratory methodsSerum where can i buy viagra over the counter usa samples (diluted 1:200) were tested for the presence of erectile dysfunction spike S1-specific IgG antibodies using a validated fluorescent bead-based multiplex-immunoassay as described.14 A cut-off concentration for seropositivity (2.37 AU/mL. With specificity of 99% and sensitivity of 84.4%) was determined by ROC-analysis of 400 pre-viagra control where can i buy viagra over the counter usa samples (including a nationwide random cross-sectional sample (n=108)) as well as patients with confirmed influenza-like illnesses caused by erectile dysfunctiones and other viagraes, and a selection of sera from 115 PCR-confirmed erectile dysfunction treatment cases with mild, or severe disease symptoms. Seropositive PICO-samples and those with a concentration 25% below the cut-off were retested (n=138), and the geometric mean concentration (GMC) was calculated. Paired pre-viagra PIENTER-3-samples of these retested PICO-samples (available from 129/138) were tested correspondingly as described above to correct for false-positive results (online supplemental figure S1A).Statistical analysesStudy population, erectile dysfunction treatment-related symptoms and antibody responsesData management and analyses were where can i buy viagra over the counter usa conducted in SAS v.9.4 (SAS Institute Inc., USA) and R v.3.6.

P values <0.05 were considered statistically significant. Sociodemographic characteristics and erectile dysfunction treatment-related symptoms (general, respiratory, and gastrointestinal) developed since the start of the epidemic were stratified by sample where can i buy viagra over the counter usa (NS vs LVC), or sex, respectively, and described for seropositive and seronegative participants. Differences were tested via Pearson’s χ², or Fisher’s exact where can i buy viagra over the counter usa test if appropriate. Differences in GMC between reported symptoms in seropositive participants were determined by calculating the difference in log-transformed concentrations of those who developed symptoms at least 4 weeks prior to the sampling—ensuring a plateaued response—and tested by means of a Mann-Whitney U-test.Seroprevalence estimatesSeroprevalence estimates (with 95% Wilson CIs (CI)) for erectile dysfunction-specific antibodies were calculated taking into account the survey design (ie, controlling for region and municipality) and weighted by sex, age, ethnic background and degree of urbanisation to match the distribution of the general Dutch population in both the NS and LVC sample.

Estimates were corrected for where can i buy viagra over the counter usa test performance via the Rogan &. Gladen bias correction (with sensitivity of 84.4% and assuming a specificity of 100% after cross-validation with pre-sera).15 Smooth age-specific seroprevalence estimates were obtained with a logistic regression in a Generalised Additive Model using penalised splines.16Risk factors for erectile dysfunction seropositivityA random-effects logistic regression model was used to identify risk factors for erectile dysfunction seropositivity, applying a full case analysis (n=3100. Values were missing for <5% where can i buy viagra over the counter usa of the participants). Potential risk factors included sociodemographic characteristics (sex, age group, region, ethnic background, Orthodox-Reformed Protestants, educational level, household size, (parent with a) contact profession, healthcare worker), and erectile dysfunction treatment-related factors (contact with where can i buy viagra over the counter usa a erectile dysfunction treatment confirmed case, number of persons contacted yesterday, working from home (normally and in the last week), comorbidities (combining diabetes, history of malignancy, immunodeficiency, cardio-vascular, kidney and chronic lung disease (note.

As a sensitivity analysis, comorbidities were also included separately)), and use of blood pressure medication, immunosuppressants, statins and antivirals/antibiotics in the last month). Models included a random intercept, potential clustering where can i buy viagra over the counter usa by municipality and region was accounted for, and odds ratios (OR) in univariable analyses were a priori adjusted for sex and age. Variables with p<0.10 were entered in the multivariable analysis, and backward selection was performed—manually dropping variables one-by-one based on p≥0.05—to identify significant risk factors. Adjusted ORs where can i buy viagra over the counter usa and corresponding 95% CIs were provided.RESULTSStudy populationOf 6102 invitees, 3207 (53%) donated a serum sample and filled-out the questionnaire, of which 2637 persons from the NS and 570 from the LVC.

Participants from across the country participated (figure 1), with age ranging from 2 to 90 years where can i buy viagra over the counter usa (table 1). In the NS, slightly more women (55%) participated, most (88%) were of Dutch descent, nearly half had a high educational level, and 45% was religious. 20 percent of persons between age 25–66 years were healthcare workers and 56% of the (parents of) participants reported to have had daily where can i buy viagra over the counter usa contact with patients, clients and/or children in their profession/volunteer work normally. Over half of the participants lived in a ≥2-person household, and 78% reported to have had physical contact with <5 people outside their own household yesterday (during lockdown), of which more than half with nobody.

Comorbidities most frequently reported included chronic lung and cardiovascular disease (both 13%), and a history of malignancy where can i buy viagra over the counter usa (5%). In line with the population distribution, the LVC sample was characterised by a relative high proportion of Orthodox-Reformed Protestants from where can i buy viagra over the counter usa Dutch descent (table 1). Sociodemographic characteristics between responders and non-responders are provided in online supplemental table S1.View this table:Table 1 Sociodemographic characteristics of participants in the PICO-study and weighted seroprevalence in the general population of the Netherlands, first round of inclusion, by national sample and low vaccination coverage sampleSupplemental materialerectile dysfunction treatment-related symptoms and antibody responsesIn total, 63% of participants reported to have had ≥1 erectile dysfunction treatment-related symptom(s) since the start of the epidemic, with runny nose (37%), headache (33%), and cough (30%) being most common (table 2). All reported symptoms were significantly higher in seropositive compared to seronegative persons, where can i buy viagra over the counter usa except for stomach ache.

The majority of those seropositive (93%) reported to have had symptoms (90% of men vs 95% of women), of whom three already in mid-February, 2 weeks prior to the official first where can i buy viagra over the counter usa notification. Median duration of illness in the seropositive participants was 8.5 days (IQR. 4.0–12.5), 16% (n=12) visited ageneral practitioner and where can i buy viagra over the counter usa one was admitted to the hospital. Among seropositive persons, most reported to have had ≥1 respiratory symptom(s) (86%), with runny nose and cough (both 61%) most regularly, and ≥1 general (84%) symptom(s), of which anosmia/ageusia (53%) was most discriminative as compared to the seronegative participants (4%, p<0.0001) (table 2).

Symptoms were more common in women, except for where can i buy viagra over the counter usa anosmia/ageusia, cough and irritable/confusion. Almost 75% of the seropositive participants met the erectile dysfunction treatment case definition of fever and/or cough and/or dyspnoea, which improved to 80% when anosmia/ageusia was included—while remaining 36% in those where can i buy viagra over the counter usa seronegative. GMC was significantly higher among seropositive persons with fever vs without (48.2 vs 11.6 AU/mL, p=0.01), and with dyspnoea vs without (78.6 vs 13.5 AU/mL, p=0.04).View this table:Table 2 erectile dysfunction treatment-related symptoms since the start of the epidemic among all participants in the PICO-study reporting symptoms (n=3147), first round of inclusionSeroprevalence estimatesOverall weighted seroprevalence in the NS was 2.8% (95% CI 2.1 to 3.7), did not differ between sexes or ethnic backgrounds (table 1), and was not higher among healthcare workers (2.7% vs non-healthcare workers 2.5%). Seroprevalence was lowest in the where can i buy viagra over the counter usa northern region (1.3%) and highest in the mid-west (4.0%).

Estimates were lowest in children—gradually increasing from below 1% at age 2 years to 3% at 17 years—was highest in age group 18–39 years (4.9%) and ranged between 2 and 4% up to 90 years of age (figure 2). In both samples, seroprevalence was highest in Orthodox-Reformed Protestants (>7%) where can i buy viagra over the counter usa (table 1). Online supplement figure S1B displays the distribution of IgG concentrations for all participants by age, and online supplemental figure S2 ⇓shows the seroprevalence smoothed by age where can i buy viagra over the counter usa in the LVC.Smooth age-specific erectile dysfunction seroprevalence in the general population of the Netherlands, beginning of April 2020." data-icon-position data-hide-link-title="0">Figure 2 Smooth age-specific erectile dysfunction seroprevalence in the general population of the Netherlands, beginning of April 2020.Risk factors for erectile dysfunction seropositivityVariables that were associated with erectile dysfunction seropositivity in univariable analyses included age group, Orthodox-Reformed Protestant, had been in contact with a erectile dysfunction treatment case, use of immunosuppressants, and antibiotic/antiviral medication in the last month (table 3). In multivariable analysis, substantial higher odds were observed for those who took immunosuppressants the last month, were Orthodox-Reformed Protestant, had been in contact with a erectile dysfunction treatment confirmed case, and from age groups 18–24 and 25–39 years (compared to 2–12 years).View this table:Table 3 Risk factor analysis for erectile dysfunction seropositivity among all participants (n=3100.

Full case analysis) in the PICO-study, first round of inclusionDISCUSSIONHere, we have estimated the seroprevalence where can i buy viagra over the counter usa of erectile dysfunction-specific antibodies and identified risk factors for seropositivity in the general population of the Netherlands during the first epidemic wave in April 2020. Although overall seroprevalence was still low at this phase, important risk factors for seropositivity could be identified, including adults aged 18–39 years, persons using immunosuppressants, and Orthodox-Reformed Protestants. These data can guide future interventions, including strategies for vaccination, believed to be a realistic solution to overcome this viagra.This PICO-study revealed that 2.8% (95% CI 2.1 to 3.7) of the Dutch population had detectable erectile dysfunction-specific serum IgG antibodies, suggesting that almost half a million inhabitants (of in total 17 423 98117) were infected (487 871 (95% CI 365 904 to 644 687)) in mid-March, 2020 where can i buy viagra over the counter usa (taking into account the median time to seroconvert18). Several seropositive where can i buy viagra over the counter usa participants reported to have had erectile dysfunction treatment-related symptoms back in mid-February, suggesting the viagra circulated in our country at the beginning of February already.

Our overall estimate is in line with preliminary results from another study conducted in the Netherlands in the beginning of April which found 2.7% to be seropositive, although this study was performed in healthy blood donors aged 18–79 years.19 Worldwide, various seroprevalence studies are ongoing. A large nationwide study in Spain showed that around 5% (ranging between 3.7% and 6.2%) was seropositive, indicating that only a small proportion of where can i buy viagra over the counter usa the population had been infected in one of the hardest hit countries in Europe. Current studies in literature mostly cover erectile dysfunction treatment hotspots or specific regions—with possibly bias in selection of participants and/or smaller age-ranges—with rates ranging between 1–7% in April (eg, in Los Angeles County (CA, USA)20 or ten other sites in the USA,21 Geneva (Switzerland),22 and Luxembourg23). Estimates also where can i buy viagra over the counter usa very much depend on test performances.

Particularly, when seroprevalence where can i buy viagra over the counter usa is relatively low, specificity of the assay should approach near 100% to diminish false-positive results and minimise overestimation. Although we cannot rule-out false-positive samples completely, our assay was validated using a broad range of positive and negative erectile dysfunction samples. PICO-samples were cross-linked to pre-viagra concentration where can i buy viagra over the counter usa. And bias correction for test performance was applied to represent most accurate estimates.

In addition, future studies should establish whether epidemiologically dominant genetic changes in the spike protein of erectile dysfunction influence binding to spike S1 used in our and other assays.Seroprevalence was highest in adults aged 18–39 years, which is in line with the serosurvey among blood donors in the Netherlands, but contrary to the low incidence rate as reported in Dutch surveillance, caused by restrictive testing of risk groups and healthcare workers at the beginning of the epidemic, primarily identifying severe cases.3 19 The elevation in these younger adults may be explained by increased social contacts typical for this age group, in addition to specific social activities in February, such as skiing holidays in the Alps (from where the viagra disseminated quickly across Europe), where can i buy viagra over the counter usa or carnival festivities in the Netherlands (ie, multiple superspreading events primarily in the mid and Southern part, explaining local elevation in seroprevalence). In correspondence with other nationwide studies8 9 and reports from the Dutch government,3 24 where can i buy viagra over the counter usa seroprevalence was lowest in children. Although some rare events of paediatric inflammatory multisystem syndrome have been reported, this group seems to be at decreased risk for developing (severe) erectile dysfunction treatment in general, which may be explained by less severe possibly resulting in a limited humoral response.25 26 Further, significantly higher odds for seropositivity were seen in Orthodox-Reformed Protestants. This community lives where can i buy viagra over the counter usa socio-geographically clustered in the Netherlands, that is, work, school, leisure and church are intertwined heavily.

As observed in other countries, particularly frequent attendance of church with close distance to others, including singing activities, might have fuelled the spread of erectile dysfunction within this community in the beginning of the epidemic.11 12 Whereas the comorbidities with possible increased risk of severe erectile dysfunction treatment were not associated with seropositivity in this study, immunosuppressants use did display higher odds (note. We did where can i buy viagra over the counter usa not have information of specific drugs). Recent data indicate that immunosuppressive treatment is not associated with worse erectile dysfunction treatment outcomes,27 28 yet continued surveillance is warranted as these patients might where can i buy viagra over the counter usa be more prone to (future) , for instance due to a possible attenuated humoral immune response.29The majority of seropositive participants exhibited ≥1 symptom(s), mostly general and respiratory. A recent meta-analysis found a pooled asymptomatic proportion of 16%,5 hence the observed overall fraction in the present study (7%) might be a conservative estimate as the self-reported symptoms could have been due to other reasons or circulating pathogens along the recalled period (ie, 62% of the seronegative participants reported symptoms too).

The asymptomatic proportion might be different across ages5 and should be explored further along with elucidating the overall contribution of where can i buy viagra over the counter usa asymptomatic transmission via well-designed contact-tracing studies. Interestingly, clinical studies have observed anosmia/ageusia to be associated with erectile dysfunction , and this notion is supported here at a population-based level.30 In the viagra context, sudden onset of anosmia/ageusia seems to be a useful surveillance tool, which can contribute to early disease recognition and minimise transmission by rapid self-isolation.This study has some limitations. First, although half of the total municipalities in the Netherlands were included, some erectile dysfunction treatment hotspots might be missed due to where can i buy viagra over the counter usa the study design. Second, our study population consisted of more Dutch (88%) than non-Dutch persons and relative more healthcare workers (20%) when compared to the general population (76% and 14%, respectively).17 Healthcare workers in the Netherlands do not seem to have had a higher likelihood of , and transmission seems to have taken place mostly in household settings.3 31 Although selectivity in response was minimised by weighting our study sample on where can i buy viagra over the counter usa a set of sociodemographic characters to match the Dutch population, seroprevalence might still be slightly influenced.

Third, some potential determinants for seropositivity could have been missed as we might have been underpowered to detect small differences given the low prevalence in this phase, or because these questions had not been included in the questionnaire (as it was designed in the very beginning of the epidemic). Finally, at this stage the proportion of infected individuals that fail to show detectable seroconversion where can i buy viagra over the counter usa is unknown, potentially leading to underestimation of the percentage of infected persons.To conclude, we estimated that 2.8% of the Dutch inhabitants, that is, nearly half a million, were infected with erectile dysfunction amidst the first epidemic wave in the beginning of April 2020. This is in striking contrast with the 30-fold lower number of reported cases (of approximately 15 000)3, and underlines the importance of seroepidemiological studies to estimate the true viagra size. The proportion of persons still susceptible to erectile dysfunction is high and IFR is substantial.4 Globally, nationwide seroepidemiological studies are urgently needed for better understanding of related risk factors, viral spread, and measures applied to mitigate dissemination.7 The prospective nature of our study will enable us to gain key insights on the duration and quality of antibody responses in infected persons, and hence possible protection of disease by antibodies.6 Serosurveys will thus play a major role in guiding future interventions, such as strategies for vaccination (of risk groups), since even when treatments become available, initial treatment availability will be limited.What is where can i buy viagra over the counter usa already known on this topicReported erectile dysfunction treatment cases worldwide are an underestimation of the true magnitude of the viagra as the scope of undetected cases remains largely unknown.Various symptoms and risk factors have been identified in patients seeking medical advice, however, these may not be representative for s in the general population.Seroepidemiological studies in outbreak settings have been performed, however, studies on a nationwide level covering all ages remain limited.What this study addsThis nationwide seroepidemiological study covering all ages reveals that 2.8% of the Dutch population had been infected with erectile dysfunction at the beginning of April 2020, that is, 30 times higher than the official cases reported, leaving a large proportion of the population still susceptible for .The highest seroprevalence was observed in young adults from 18 to 39 years of age and lowest in children aged 2 to 17 years, indicating marginal erectile dysfunction s among children in general.Persons taking immunosuppressants as well as those from the Orthodox-Reformed Protestant community had over four times higher odds of being seropositive compared to others.The extend of the spread of erectile dysfunction and the risk groups identified here, can inform monitoring strategies and guide future interventions internationally.AcknowledgmentsFirst of all, we gratefully acknowledge the participants of the PICO-study.

Secondly, this where can i buy viagra over the counter usa study would not have been possible without the instrumental contribution of colleagues from the National Institute of Public Health and Environment (RIVM), Bilthoven, the Netherlands, more specially the department of Immunology of Infectious Diseases and treatments, regarding logistics and/or laboratory analyses (Marjan Bogaard-van Maurik, Annemarie Buisman, Pieter van Gageldonk, Hinke ten Hulscher-van Overbeek, Petra Jochemsen, Deborah Kleijne, Jessica Loch, Marjan Kuijer, Milou Ohm, Hella Pasmans, Lia de Rond, Debbie van Rooijen, Liza Tymchenko, Esther van Woudenbergh, and Mary-lene de Zeeuw-Brouwer), the Epidemiology and Surveillance department concerning logistics (Francoise van Heiningen, Alies van Lier, Jeanet Kemmeren, Joske Hoes, Maarten Immink, Marit Middeldorp, Christiaan Oostdijk, Ilse Schinkel-Gordijn, Yolanda van Weert, and Anneke Westerhof), methodological insights (Hendriek Boshuizen, Susan Hahné, Scott McDonald, Rianne van Gageldonk-Lafeber, Jan van de Kassteele, and Maarten Schipper) and manuscript reviewing (Susan van den Hof, and Don Klinkenberg), department of IT and Communication for help with the invitations (Luppo de Vries, Daphne Gijselaar, and Maaike Mathu), student interns for additional support (Stijn Andeweg for creating online supplemental figures 1A and 1B. Janine Wolf, Natasha Kaagman, and Demi Wagenaar for logistics. And Lisette van Cooten for data entry where can i buy viagra over the counter usa of paper questionnaires), and Sidekick-IT, Breda, the Netherlands, regarding data flow (Tim de Hoog). This study was funded by the ministry of Health, Welfare and Sports (VWS), the Netherlands..

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Justice, one of the four Beauchamp and daily viagra dosage Childress prima facie basic principles of biomedical ethics, is explored in two excellent papers in the current issue of the journal. The papers stem from a British Medical Association (BMA) essay competition on justice and fairness in medical practice and policy. Although the competition was open to (almost) all comers, of the 235 entries both the winning paper by Alistair Wardrope1 and the highly commended runner-up by Zoe Fritz and Caitríona Cox2 daily viagra dosage were written by practising doctors—a welcome indication of the growing importance being accorded to philosophical reflection about medical practice and practices within medicine itself. Both papers are thoroughly thought provoking and represent two very different approaches to the topic. Each deserves a careful read.The competition was a component of a BMA 2019/2020 ‘Presidential project’ on fairness and justice and asked candidates to ‘use ethical reasoning and theory to tackle challenging, practical, contemporary, problems in health care and help provide a solution based on an explained and defended sense of fairness/justice’.In this guest editorial I’d like to explain why, in 2018 on becoming president-elect of the BMA, I chose the theme of justice and fairness in medical ethics for my 2019–2020 Presidential project—and why in a world of massive and ever-increasing and remediable health inequalities biomedical ethics requires greater international and interdisciplinary efforts to try to reach agreement on the need to achieve greater ‘health justice’ and to reach agreement on what that commitment actually means and on what in practice it requires.First, some background.

As president I was offered the wonderful opportunity to pursue, daily viagra dosage with the organisation’s formidable assistance, a ‘project’ consistent with the BMA’s interests and values. As a hybrid of general medical practitioner and philosopher/medical ethicist, and as a firm defender of the Beauchamp and Childress four principles approach to medical ethics,3 I chose to try to raise the ethical profile of justice and fairness within medical ethics.My first objective was to ask the BMA to ask the World Medical Association (WMA) to add an explicit commitment ‘to strive to practise fairly and justly throughout my professional life’ to its contemporary version of the Hippocratic Oath—the Declaration of Geneva4—and to the companion document the International Code of Medical Ethics.5 The stimulus for this proposal was the WMA’s addition in 2017 of the principle of respect for patients’ autonomy. Important as daily viagra dosage that addition is, it is widely perceived (though in my own view mistakenly) as being too much focused on individual patients and not enough on communities, groups and populations. The simple addition of a commitment to fairness and justice would provide a ‘balancing’ moral commitment.Adding the fourth principleIt would also explicitly add the fourth of those four prima facie moral commitments, increasingly widely accepted by doctors internationally. Two of them—benefiting our patients (beneficence) and doing so with as little harm as possible (non-maleficence)—have been an integral part of medical ethics since Hippocratic times.

Respect for autonomy and justice daily viagra dosage are very much more recent additions to medical ethics. The WMA, having added respect for autonomy to the Declaration of Geneva, should, I proposed, complete the quartet by adding the ‘balancing’ principle of fairness and justice.Since the Declaration is unlikely to be revised for several years, it seems likely that the proposal to add to it an explicit commitment to practise fairly and justly will have to wait. However, an explicit commitment to justice daily viagra dosage and fairness has, at the BMA’s request, been added to the draft of the International Code of Medical Ethics and it seems reasonable to hope and expect that it will remain in the final document.Adding a commitment to fairness and justice is the easy part!. Few doctors would on reflection deny that they ought to try to practise fairly and justly. It is far more difficult to say what is actually meant by this.

Two additional components of my Presidential project—the essay competition and a conference (which with luck will have been held, virtually, shortly before publication of this editorial)—sought to help elucidate just what is meant by practising fairly and justly.One of the most striking features of the essay competition was the readiness of many writers to point to injustices in the context of medical practice and policy and describe ways of remedying them, but without giving a specific account of justice and fairness on daily viagra dosage the basis of which the diagnosis of injustice was made and the remedy offered.Wardrope’s winning essay comes close to such an approach by challenging the implied premise that an account of justice and fairness must provide some such formal theory. In preference, he points to the evident injustice and unsustainability of humans’ degradation of ‘the Land’ and its atmosphere and its inhabitants and then challenges some assumptions of contemporary philosophy and ethics, especially what he sees as their anthropocentric and individualistic focus. Instead, he invokes Leopold Aldo’s ‘Land Ethic’ (as well daily viagra dosage as drawing in aid Isabelle Stenger’s focus on ‘the intrusion of Gaia’). In his thoughtful and challenging paper, he seeks to refocus our ethics—including our medical ethics and our sense of justice and fairness—on mankind’s exploitative threat, during this contemporary ‘anthropocene’ stage of evolution, to the continuing existence of humans and of all forms of life in our ‘biotic community’. As remedy, the author, allying his approach to those of contemporary virtue ethics, recommends the beneficial outcomes that would be brought about by a sense of fairness and justice—a developed and sensitive ‘ecological conscience’ as he calls it—that embraces the interests of the entire biotic community of which we humans are but a part.Fritz and Cox pursue a very different and philosophically more conventional approach to the essay competition’s question and offer a combination and development of two established philosophical theories, those of John Rawls and Thomas Scanlon, to provide a philosophically robust and practically beneficial methodology for justice and fairness in medical practice and policy.

Briefly summarised, they recommend a two-stage approach daily viagra dosage for healthcare justice. First, those faced with a problem of fairness or justice in healthcare or policy should use Thomas Scanlon’s proposed contractualist approach whereby reasonable people seek solutions that they and others could not ‘reasonably reject’. This stage would involve committees of decision-makers and representatives of relevant stakeholders looking at the immediate and longer term impact on existing stakeholders of proposed solutions. They would then check those solutions against substantive criteria of justice derived from Rawls’ theory (which, via his theoretical device of the ‘veil of ignorance’, Rawls daily viagra dosage and the authors argue that all reasonable people can be expected to accept!. ).

The Rawlsian criteria relied on by Fritz and Cox are daily viagra dosage equity of access to healthcare. The ‘difference principle’ whereby avoidable inequalities of primary goods can only be justified if they benefit the most disadvantaged. The just savings principle, of particular importance for ensuring intergenerational justice and sustainability. And a criterion of increased openness, transparency and accountability.It would of course be naïve to expect a single universalisable solution to the question ‘what daily viagra dosage do we mean by fairness and justice in health care?. €™ As the papers by Wardrope1 and Fritz and Cox2 demonstrate, there can be very wide differences of approach in well-defended accounts.

My own hope for my project is to emphasise the importance first of committing ourselves within medicine to practising fairly and justly in whatever branch daily viagra dosage we practise. And then to think carefully about what we do mean by that and act accordingly.Following AristotleFor my own part, over 40 years of looking, I have not yet found a single substantive theory of justice that is plausibly universalisable and have had to content myself with Aristotle’s formal, almost content-free but probably universalisable theory, according to which equals should be treated equally and unequals unequally in proportion to the relevant inequalities—what some health economists refer to as horizontal and vertical justice or equity.6Beauchamp and Childress in their recent eighth and ‘perhaps final’ edition of their foundational ‘Principles of biomedical ethics’1 acknowledge that ‘[t]he construction of a unified theory of justice that captures our diverse conceptions and principles of justice in biomedical ethics continues to be controversial and difficult to pin down’.They still cite Aristotle’s formal principle (though with less explanation than in their first edition back in 1979) and they still believe that this formal principle requires substantive or ‘material’ content if it is to be useful in practice. They then describe six different theories of justice—four ‘traditional’ (utilitarian, libertarian, communitarian and egalitarian) and two newer theories, which they suggest may be more helpful in the context of health justice, one based on capabilities and the other on actual well-being.They again end their discussion of justice with their reminder that ‘Policies of just access to health care, strategies of efficiencies in health care institutions, and global needs for the reduction of health-impairing conditions dwarf in social importance every other issue considered in this book’ ……. €˜every society daily viagra dosage must ration its resources but many societies can close gaps in fair rationing more conscientiously than they have to date’ [emphasis added]. And they go on to stress their own support for ‘recognition of global rights to health and enforceable rights to health care in nation-states’.For my own part I recommend, perhaps less ambitiously, that across the globe we extract from Aristotle’s formal theory of justice a starting point that ethically requires us to focus on equality and always to treat others as equals and treat them equally unless there are moral justifications for not doing so.

Where such justifications exist we should say what they are, explain the moral assumptions that justify them and, to the extent possible, seek the agreement of those affected.IntroductionIt did not occur to the Governor that there might be more than one definition of what is good … It daily viagra dosage did not occur to him that while the courts were writing one definition of goodness in the law books, fires were writing quite another one on the face of the land. (Leopold, ‘Good Oak’1, pp 10–11)As I wrote the abstract that would become this essay, wildfires were spreading across Australia’s east coast. By the time I was invited to write the essay, back-to-back winter storms were flooding communities all around my home. The essay has been written in moments of respite between shifts daily viagra dosage during the erectile dysfunction treatment viagra. Every one of these events was described as ‘unprecedented’.

Yet each is becoming increasingly likely, and that due to our interactions with our environment.Public discourse surrounding these events is dominated by questions of justice and fairness. How to daily viagra dosage balance competing imperatives of protecting individual lives against risk of spreading contagion. How best to allocate scarce resources like intensive care beds or mechanical ventilators. The conceptual tools daily viagra dosage of clinical ethics are well tailored to these sorts of questions. The rights of the individual versus the community, issues of distributive justice—these are familiar to anyone with even a passing acquaintance with its canonical debates.What biomedical ethics has remained largely silent on is how we have been left to confront these decisions.

How human activity has eroded Earth’s life support systems to make the ‘unprecedented’ the new normal. A medical ethic fit for the Anthropocene—our (still tentative) geological epoch defined by human influence on natural systems—must be able not just to react to the consequences of our exploitation of the natural world, but reimagine our relationship with it.Those reimaginations already exist, daily viagra dosage if we know where to look for them. The ‘Land Ethic’ of the US conservationist Aldo Leopold offers one such vision.i Developed over decades of experience working in and teaching land management, the Land Ethic is most famously formulated in an essay of the same name published shortly before Leopold’s death fighting a wildfire on a neighbour’s farm. It begins with a reinterpretation of the ethical relationship between humanity and the ‘land community’, daily viagra dosage the ecosystems we live within and depend upon. Moving us from ‘conqueror’ to ‘plain member and citizen’ of that community1 (p 204).

Land ceases to be a resource to be exploited for human need once we view ourselves as part of, and only existing within, the land community. Our moral evaluations shift consonantly:A thing is right when it tends to daily viagra dosage preserve the integrity, stability, and beauty of the biotic community. It is wrong when it tends otherwise.1 (pp 224–225)The justice of the Land Ethic questions many presuppositions of biomedical ethics. By valuing the community in itself—in a way irreducible to the welfare of its members—it steps away from the individualism axiomatic in contemporary bioethics.2 Viewing ourselves as citizens of the land community also extends the moral horizons of healthcare from a solely human focus, taking seriously the interests of the non-human members of that daily viagra dosage community. Taking into account the ‘stability’ of the community requires intergenerational justice—that we consider those affected by our actions now, and their implications for future generations.3 The resulting vision of justice in healthcare—one that takes climate and environmental justice seriously—could offer health workers an ethic fit for the future, demonstrating ways in which practice must change to do justice to patients, public and planet—now and in years to come.Healthcare in the AnthropoceneSeemeth it a small thing unto you to have fed upon good pasture, but ye must tread down with your feet the residue of your pasture?.

And to have drunk of the clear waters, but ye must foul the residue with your feet?. (Ezekiel 34:18, quoted in Leopold, ‘Conservation in the Southwest’4, p 94)The majority of the development of human societies worldwide—including all of recorded human history—has taken place within a single daily viagra dosage geological epoch, a roughly 11 600 yearlong period of relative warmth and climatic stability known as the Holocene. That stability, however, can no longer be taken for granted. The epoch that has sustained most of human development is giving way to one shaped by the planetary consequences of that development—the Anthropocene.The Anthropocene is marked by accelerating degradation of the ecosystems that have sustained human societies. Human activity is already estimated to have raised global temperatures 1°C above preindustrial levels, and if emissions continue at current levels we are likely to reach 1.5°C between 2030 and 2052.5 The global rate of species extinction is orders of magnitude higher than the average over the past 10 million years.6 Ocean acidification, deforestation and disruption of nitrogen and phosphorus flows are likely at or beyond sustainable planetary boundaries.7Yet this period has also seen rapid (if uneven) improvements in human health, with improved life expectancy, falling child mortality and falling numbers of daily viagra dosage people living in extreme poverty.

The 2015 report of the Rockefeller Foundation-Lancet Commission on planetary health explained this dissonance in stark terms. €˜we have been mortgaging the health of future generations to realise economic and development gains daily viagra dosage in the present.’7In the instrumental rationality of modernity, nature has featured only as inexhaustible resource and infinite sink to fuel social and economic ends. But this disenchanted worldview can no longer hide from the implausibility of these assumptions. It cannot resist what the philosopher Isabelle Stengers has called ‘the intrusion of Gaia’.8 The present viagra—made more likely by deforestation, land use change and biodiversity loss9—is just the most immediately salient of these intrusions. Anthropogenic environmental changes are increasing undernutrition, increasing range and transmissibility of many vectorborne and waterborne diseases like dengue fever and cholera, increasing frequency and severity of extreme weather events like heatwaves and wildfires, and driving population exposure to air pollution—which already accounts for over 7 million deaths annually.10These daily viagra dosage intrusions will shape healthcare in the Anthropocene.

This is because health workers will have to deal with their consequences, and because modern industrialised healthcare as practised in most high-income countries—and considered aspirational elsewhere—was borne of the same worldview that has mortgaged the health of future generations. The health sector in the USA is estimated to account for 8% of the country’s greenhouse gas footprint.11 Pharmaceutical production and waste causes more local environmental degradation, accumulating in water supplies with damaging effects for local flora and fauna.12 Public health has similarly embraced daily viagra dosage short-term gains with neglect of long-term consequences. Health messaging was instrumental to the development and popularisation of many disposable and single-use products, while a 1947 report funded by the Rockefeller Foundation (who would later fund the landmark 2015 Lancet report on planetary health) popularised the high-meat, high-dairy ‘American’ diet—dependent on fossil fuel-driven intensive agricultural practices—as the healthy ideal.13Healthcare fit for the Anthropocene requires a shift in perspectives that allows us to see and work with the intrusion of Gaia. But can dominant approaches in bioethics incorporate that shift?. A perfect moral daily viagra dosage stormWe have built a beautiful piece of social machinery … which is coughing along on two cylinders because we have been too timid, and too anxious for quick success, to tell the farmer the true magnitude of his obligations.

(Leopold, ‘The Ecological Conscience’4, p 341)At local, national and international scales, the lifestyles of the wealthiest pose an existential threat to the poorest and most marginalised in society. Our actions now are depriving future generations of the daily viagra dosage environmental prerequisites of good health and social flourishing. If justice means, as Ranaan Gillon parses it, ‘the moral obligation to act on the basis of fair adjudication between competing claims’,14 then this state of affairs certainly seems unjust. However, the tools available for grappling with questions of justice in bioethics seem ill equipped to deal with these sorts of injustice.To illustrate this problem, consider how Gillon further fleshes out his description of justice. In terms of fair distribution of daily viagra dosage scarce resources, respect for people’s rights, and respect for morally acceptable laws.

The first of these—labelled distributive justice—concerns how fairly to allot finite resources among potential beneficiaries. Classic problems of distributive justice in healthcare concern a group of people at a particular time (usually patients), who could each benefit from a particular resource (historically, discussions have often focused on transplant organs. More recently, intensive care beds and ventilators have come to the fore) daily viagra dosage. But there are fewer of these resources than there are people with a need for them. Such discussions are not daily viagra dosage easy, but they are at least familiar—we know where to begin with them.

We can consider each party’s need, their potential to benefit from the resource, any special rights or other claims they may have to it, and so forth. The distribution of benefits and harms in the Anthropocene, however, does not comfortably fit this formalism. It is daily viagra dosage one thing to say that there is but one intensive care bed, from which Smith has a good chance of gaining another year of life, Jones a poor chance, and so offer it to Smith. Another entirely to say that production of the materials consumed in Smith’s care has contributed to the degradation of scarce water supplies on the other side of the globe, or that the unsustainable pattern of energy use will affect innumerable other future persons in poorly quantifiable ways through fuelling climate change. The calculations of distributive justice are well suited daily viagra dosage to problems where there are a set pool of potential beneficiaries, and the use of the scarce resources available affects only those within that pool.

But global environmental problems do not fit this pattern—the effects of our actions are spatially and temporally dispersed, so that large numbers of present and future people are affected in different ways.Nor can this problem be readily addressed by turning to Gillon’s second category of obligations of justice, those grounded in human rights. For while it might be plausible (if not entirely uncontroversial) to say that those communities whose water supplies are degraded by pharmaceutical production have a right to clean water, it is another thing entirely to say that Smith’s healthcare is directly violating that right. It would not be true to say that, were it not for the resources used in caring for Smith, that the daily viagra dosage communities in question would face no threat to water security—indeed, they would likely make no appreciable difference. Similarly for the effects of Smith’s care on future generations facing accelerating environmental change.iiThe issue here is of fragmentation of agency. While it is not the case that Smith’s care is directly responsible for these environmental harms, the cumulative consequences of many such acts—and the ways in which these acts are embedded in particular systems of energy generation, waste management, international trade, and so on—are daily viagra dosage reliably producing these harms.

The injustice is structural, in Iris Marion Young’s terminology—arising from the ways in which social structures constrain individuals from pursuing certain courses of action, and enable them to follow others, with side effects that cumulatively produce devastating impacts.15Gillon describes the third component of justice as respect for morally acceptable laws. But there is little reason to believe that existing legal frameworks provide sufficient guidance to address these structural injustices. While the intricacies of global governance are well beyond what I can hope to address here, the stark fact remains that, despite the international commitment of the 2015 Paris Agreement daily viagra dosage to attempt to keep global temperature rise to 1.5°C above preindustrial levels, the Intergovernmental Panel on Climate Change estimates that present national commitments—even if these are substantially increased in coming years—will take us well beyond that target.5 Confronted by such institutional inadequacy, respect for the rule of law is inadequate to remedy injustice.The confluence of these particular features—dispersion of causes and effects, fragmentation of agency and institutional inadequacy—makes it difficult for us to reason ethically about the choices we have to make. Stephen Gardiner calls this a ‘perfect moral storm’.16 Each of these factors individually would be difficult to address using the resources of contemporary biomedical ethics. Their convergence makes it seem insurmountable.This perfect storm was not, however, unpredictable.

Van Rensselaer Potter, a professor of Oncology responsible for introducing the term ‘bioethics’ into Anglophone discourse, daily viagra dosage observed that since he coined the phrase, the study of bioethics had diverged from his original usage (governing all issues at the intersection of ethics and the biological sciences) to a narrow focus on the moral dilemmas arising in interactions between individuals in biomedical contexts. Potter predicted that the short-term, individualistic and medicalised focus of this approach would result in a neglect of population-level and ecological-level issues affecting human and planetary health, with catastrophic consequences.17 His proposed solution was a new ‘global bioethics’, grounded in a new understanding of humanity’s position within planetary systems—one articulated by the Land Ethic.The Land EthicA land ethic changes the role of Homo sapiens from conqueror of the land-community to plain member and citizen of it. It implies respect daily viagra dosage for his fellow-members, and also respect for the community as such.iii (Leopold, ‘The Land Ethic’1, p 204)Developed throughout a career in forestry, conservation and wildlife management, the Land Ethic is less an attempt to provide a set of maxims for moral action, than to shift our perspectives of the moral landscape. In his working life, Aldo Leopold witnessed how actions intended to optimise short-term economic outcomes eroded the environments on which we depend—whether soil degradation arising from intensive farming and deforestation, or disruption of freshwater ecosystems by industrial dairy farming. He also saw that contemporary morality remained silent on such actions, even when their consequences were to the collective detriment of all.Leopold argued that a series of ‘historical accidents’ left our morality particularly ill suited to handle these intrusions of Gaia—with a worldview that considered them ‘intrusions’, rather than the predictable response of our biotic community.

These ‘accidents’ were daily viagra dosage. The unusual resilience of European ecological communities to anthropogenic interference (England survived an almost wholesale deforestation without consequent loss of ecosystem resilience, while similar changes elsewhere resulted in permanent environmental degradation). And the legacy of European settler colonialism, meaning that an ethic arising daily viagra dosage in these particular conditions came to dominate global social arrangements4 (p 311). The first of these supported a worldview in which ‘Land … is … something to be tamed rather than something to be understood, loved, and lived with. Resources are still regarded as separate entities, indeed, as commodities, rather than as our cohabitants in the land community’4 (p 311).

The second enabled the marginalisation of other daily viagra dosage views. In this genealogy, Leopold anticipated the perfect moral storm discussed above. His intent with the Land Ethic was to navigate it.There are three key components of the Land Ethic that daily viagra dosage comprise the first three sections of Leopold’s final essay on the subject. (1) the ‘community concept’ that allows communities as wholes to have intrinsic value. (2) the ‘ethical sequence’ that situates the value of such communities as extending, not replacing, values assigned to individuals.

And (3) the ‘ecological conscience’ that views ethical action not in terms of following a particular code, but in developing appropriate moral perception.The community conceptThe most widely quoted passage of Leopold’s opus—already cited daily viagra dosage above, and frequently (mis)taken as a summary maxim of the ethic—states that:A thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community. It is wrong when it tends otherwise.1 (pp 224–225)This passage makes the primary object of our moral responsibilities ‘the biotic community’, a term Leopold uses interchangeably with the ‘land community’. Leopold’s community concept is notable in at least three respects. Its holism—an embrace of the moral significance of communities in a way that is not simply reducible to the significance daily viagra dosage of its individual members. Its understanding of communities as temporally extended, placing importance on their ‘integrity’ and ‘stability’.

And its rejection of anthropocentrism, affording humanity a place as ‘plain member and citizen’ of a broader daily viagra dosage land community.Individualism is so prevalent in biomedical ethics that it is scarcely argued for, instead forming part of the ‘background constellation of values’2 tacitly assumed within the field. We are used to evaluating the well-being of a community as a function of the well-being of its individual members—this is the rationale underlying quality-adjusted life year calculations endemic within health economics, and most discussions of distributive justice adopt some variation of this approach. Holism instead proposes that this makes no more sense than evaluating a person’s well-being as an aggregate of the well-being of their individual organs. While we can sensibly talk about people’s hearts, livers or kidneys, their health is defined in terms of and constitutively dependent on daily viagra dosage the health of the person as a whole. Similarly, holism proposes, while individuals can be identified separately, it only makes sense to talk about them and their well-being in the context of the larger biotic community which supports and defines us.Holism helps us to negotiate the issues that confront individualistic accounts of collective well-being in Anthropocene health injustices.

In the previous section, we found in the environmental consequences of industrialised healthcare that it is difficult to identify which parties in particular are daily viagra dosage harmed, and how much each individual action contributes to those harms. But our intuition that the overall result is unfair or unjust is itself a holistic assessment of the overall outcome, not dependent on our calculation of the welfare of every party involved. Holism respects the intuition that says—no matter the individuals involved—a world where people now exploit ecological resources in a fashion that deprives people in the future of the prerequisites of survival, is worse than one where communities now and in the future live in a sustainable relationship with their environment.The second aspect of Leopold’s community concept is that the community is something that does not exist at a single time and place—it is defined in terms of its development through time. Promoting the ‘integrity’ and ‘stability’ of the community requires that we not just daily viagra dosage consider its immediate interests, but how that will affect its long-term sustainability or resilience. We saw earlier the difficulties in trying to say just who is harmed and how when we approach harm to future generations individualistically.

But from daily viagra dosage the perspective of the Land Ethic, when we exploit environmental resources in ways that will have predictable damaging results for future generations, the object of our harm is not just some purely notional future person. It is a presently existing, temporally extended entity—the community of which they will be part.Lastly, Leopold’s community is quite consciously a biotic—not merely human—community. Leopold defines the land community as the open network of energy and mineral exchange that sustains all aspects of that network:Land… is not merely soil. It is a fountain of daily viagra dosage energy flowing through a circuit of soils, plants, and animals. Food chains are the living channels which conduct energy upward.

Death and decay return it to the soil. The circuit is not closed daily viagra dosage. Some energy is dissipated in decay, some is added by absorption, some is stored in soils, peats, and forests, but it is a sustained circuit, like a slowly augmented revolving fund of life.4 (pp 268–269)While the components within this network may change, the land community as a whole remains stable when the overall complexity of the network is not disrupted—other components are able to adjust to these changes, or new ones arise to take their place.ivThe normative inference Leopold makes from his understanding of the land community is this. It makes no sense to single out individual entities within the community as being especially valuable or useful, without taking into account the whole community upon which they mutually depend daily viagra dosage. To do so is self-defeating.

By privileging the interests of a few members of the community, we ultimately undermine the prerequisites of their existence.The ethical sequenceThe Land Ethic’s holism is in fact its most frequently critiqued feature. Its emphasis on the value of the biotic community leads some to allege a subjugation of individual interests to the needs of daily viagra dosage the environment. This critique neglects how Leopold positions the Land Ethic in what he calls the ‘ethical sequence’. This is the gradual extension of scope of ethical considerations, both in terms of the complexity of social interactions they cover (from interactions between two people, to the structure of progressively larger social groups), and in the kinds of person they acknowledge as worthy of moral consideration (as we resist, for example, classist, sexist or racist exclusions from personhood).This sequence serves less as a description of the history of morality, than a prescription for how we should understand the Land Ethic as adding daily viagra dosage to, rather than supplanting, our responsibilities to others. We do not argue that taking seriously health workers’ responsibilities for public health and health promotion supplants their duties to the patients they work with on a daily basis.

Similarly, the Land Ethic implies ‘respect for [our] fellow members, and also respect for the community as such’1 (p 204). At times, our responsibilities towards daily viagra dosage these different parties may come into tension. But balancing these responsibilities has always been part of the work of clinical ethics.The ecological conscienceIf the community concept gives a definition of the good, and the ethical sequence situates this definition within the existing moral landscape, neither offers an explicit decision procedure to guide right action. In arguing for the ‘ecological conscience’, Leopold explains his rationale for not attempting to articulate daily viagra dosage such a procedure. In his career as conservationist, Leopold witnessed time and again laws nominally introduced in the name of environmental protection that did little to achieve their long-term goals, while exacerbating other environmental threats.v This is not surprising, given the ‘perfect moral storm’ of Anthropocene global health and environmental threats discussed above.

The cumulative results of apparently innocent actions can be widespread and damaging.Leopold’s response to this problem is to advocate the cultivation of an ‘ecological conscience’. What is needed to promote a healthy human relationship with the land community is not for us to be told exactly how and how not to act in the face of environmental health threats, but rather to shift our view of the land from daily viagra dosage ‘a commodity belonging to us’ towards ‘a community to which we belong’1 (p viii). To understand what the Land Ethic requires of us, therefore, we should learn more about the land community and our relationship with it, to develop our moral perception and extend its scope to embrace the non-human members of our community.Seen in this light, the Land Ethic shares much in common with virtue ethics, where right action is defined in terms of what the moral agent would do, rather than vice versa. But rather than the Eudaimonia of individual human flourishing proposed by Aristotle, the phronimos of the Land Ethic sees their telos coming from their position within the land community. While clinical virtue ethicists have traditionally taken the virtues of medical practice to be grounded in the interaction with individual patients, the realities of healthcare in the Anthropocene mean that limiting our moral perceptions in daily viagra dosage this way would ultimately be self-defeating—hurting those very patients we mean to serve (and many more besides).18 The virtuous clinician must adopt a view of the moral world that can focus on a person both as an individual, and simultaneously as member of the land community.

I will close by exploring how adopting that perspective might change our practice.Justice in the AnthropoceneFailing this, it seems to me we fail in the ultimate test of our vaunted superiority—the self-control of environment. We fall back into the biological daily viagra dosage category of the potato bug which exterminated the potato, and thereby exterminated itself. (Leopold, ‘The River of the Mother of God’4, p 127)I have articulated some of the challenges healthcare faces in the Anthropocene. I have suggested that the tools presently available to clinical ethics may be inadequate to meet them. The Land daily viagra dosage Ethic invites us to reimagine our position in and relationship with the land community.

I want to close by suggesting how the development of an ecological conscience might support a transition to more just healthcare. I will not endeavour to give detailed prescriptions daily viagra dosage for action, given Leopold’s warnings about the limitations of such codifications. Rather, I will attempt to show how the cultivation of an ecological conscience might change our perception of what justice demands. Following the tradition of virtue ethics with which the Land Ethic holds much in common, this is best achieved by looking at models of virtuous action, and exploring what makes it virtuous.19Industrialised healthcare developed within a paradigm that saw the environment as inert resource and held that the scope of clinical ethics ranged only over the clinician’s interaction with their patients. When we begin to see clinician and patient not as standing apart from the environment, but as ‘member daily viagra dosage and citizen of the land community’, their relationship with one another and with the world around them changes consonantly.

The present viagra has only begun to make commonplace the idea that health workers do not simply treat infectious diseases, but interact with them in a range of ways, including as vector—and as a result our moral obligations in confronting them may extend beyond the immediate clinical encounter, to cover all the other ways we may contract or spread disease. But we daily viagra dosage may be responsible for disease outbreaks with conditions other than erectile dysfunction treatment, and in ways beyond simply becoming infected. The development of an ecological conscience would show how our practices of consumption may fuel deforestation that accelerates the emergence of novel pathogens, or support intensive animal rearing that drives antibiotic resistance.18The Land Ethic also challenges us not to abstract our work away from the places in which it takes place. General practitioner surgeries and hospitals are situated within social and land communities alike, shaping and shaped by them. These spaces can be used in ways that daily viagra dosage support or undermine those communities.

Surgeries can work to empower their communities to pursue more sustainable and healthy diets by doubling as food cooperatives, or providing resources and ‘social prescriptions’ for increased walking and cycling. Hospitals can use their extensive real estate to provide publicly accessible green and wild spaces within urban environments, and use their role as major nodes in transport infrastructure to change that infrastructure to support active travel alternatives.ivThe Land Ethic reminds us that a community (human or land) is not healthy if its flourishing cannot be sustainably maintained. An essential daily viagra dosage component of Anthropocene health justice is intergenerational justice. Contemporary industrialised healthcare has an unsustainable ecological footprint. Continuing with such a model of care would serve only to mortgage the health of future generations for the sake of those living now daily viagra dosage.

Ecologically conscious practice must take seriously the sorts of downstream, distributed consequences of activity that produce anthropogenic global health threats, and evaluate to what extent our most intensive healthcare practices truly serve to promote public and planetary health. It is not enough for the clinician to assume that our resource usage is a necessary evil in the pursuit of best clinical outcomes, for it is already apparent that much of our environmental exploitation is of minimal or even negative long-term value. The work of the National Health Service (NHS) Sustainable Development Unit has seen a 10% reduction in greenhouse gas emissions in the NHS from 2007 to 2015 despite an 18% increase in clinical activity,20 while different models of care used in less industrialised nations manage to provide high-quality health outcomes in less resource-intensive daily viagra dosage fashion.21ConclusionOur present problem is one of attitudes and implements. We are remodelling the Alhambra with a steam-shovel. We shall hardly relinquish the steam-shovel, which after daily viagra dosage all has many good points, but we are in need of gentler and more objective criteria for its successful use.

(Leopold, ‘The Land Ethic’1, p 226)The moral challenges of the Anthropocene do not solely confront health workers. But the potentially catastrophic health effects of anthropogenic global environmental change, and the contribution of healthcare activity to driving these changes provide a specific and unique imperative for action from health workers.Yet it is hard to articulate this imperative in the language of contemporary clinical ethics, ill equipped for this intrusion of Gaia. Justice in daily viagra dosage the Anthropocene requires us to be able to adopt a perspective from which these changes no longer appear as unexpected intrusions, but that acknowledges the land community as part of our moral community. The Land Ethic articulates an understanding of justice that is holistic, structural, intergenerational, and rejects anthropocentrism. This understanding seeks not to supplant, but to augment, daily viagra dosage our existing one.

It aims to do so by helping us to develop an ‘ecological conscience’, seeing ourselves as ‘plain member and citizen’ of the land community. The Land Ethic does not provide a step-by-step guide to just action. Nor does it definitively adjudicate on how to balance the interests of our patients, other populations now and in daily viagra dosage the future, and the planet. It could, however, help us on the first step towards that change—showing how to cultivate the ‘internal change in our intellectual emphasis, loyalties, affections, and convictions’1 (pp 209–210) necessary to realise the virtues of just healthcare in the Anthropocene.AcknowledgmentsThis essay was written as a submission for the BMA Presidential Essay Prize. I am grateful to the organisers and judging panel for the opportunity..

Justice, one where can i buy viagra over the counter usa of the four Beauchamp and Childress prima facie where to get female viagra basic principles of biomedical ethics, is explored in two excellent papers in the current issue of the journal. The papers stem from a British Medical Association (BMA) essay competition on justice and fairness in medical practice and policy. Although the competition was open to (almost) all comers, of the 235 entries both the winning paper by Alistair Wardrope1 and the highly commended runner-up by Zoe Fritz and Caitríona Cox2 were written by practising where can i buy viagra over the counter usa doctors—a welcome indication of the growing importance being accorded to philosophical reflection about medical practice and practices within medicine itself.

Both papers are thoroughly thought provoking and represent two very different approaches to the topic. Each deserves a careful read.The competition was a component of a BMA 2019/2020 ‘Presidential project’ on fairness and justice and asked candidates to ‘use ethical reasoning and theory to tackle challenging, practical, contemporary, problems in health care and help provide a solution based on an explained and defended sense of fairness/justice’.In this guest editorial I’d like to explain why, in 2018 on becoming president-elect of the BMA, I chose the theme of justice and fairness in medical ethics for my 2019–2020 Presidential project—and why in a world of massive and ever-increasing and remediable health inequalities biomedical ethics requires greater international and interdisciplinary efforts to try to reach agreement on the need to achieve greater ‘health justice’ and to reach agreement on what that commitment actually means and on what in practice it requires.First, some background. As president where can i buy viagra over the counter usa I was offered the wonderful opportunity to pursue, with the organisation’s formidable assistance, a ‘project’ consistent with the BMA’s interests and values.

As a hybrid of general medical practitioner and philosopher/medical ethicist, and as a firm defender of the Beauchamp and Childress four principles approach to medical ethics,3 I chose to try to raise the ethical profile of justice and fairness within medical ethics.My first objective was to ask the BMA to ask the World Medical Association (WMA) to add an explicit commitment ‘to strive to practise fairly and justly throughout my professional life’ to its contemporary version of the Hippocratic Oath—the Declaration of Geneva4—and to the companion document the International Code of Medical Ethics.5 The stimulus for this proposal was the WMA’s addition in 2017 of the principle of respect for patients’ autonomy. Important as that addition is, it is widely perceived (though in my own view mistakenly) as being too much focused on individual patients and not where can i buy viagra over the counter usa enough on communities, groups and populations. The simple addition of a commitment to fairness and justice would provide a ‘balancing’ moral commitment.Adding the fourth principleIt would also explicitly add the fourth of those four prima facie moral commitments, increasingly widely accepted by doctors internationally.

Two of them—benefiting our patients (beneficence) and doing so with as little harm as possible (non-maleficence)—have been an integral part of medical ethics since Hippocratic times. Respect for autonomy and justice are very much more recent additions where can i buy viagra over the counter usa to medical ethics. The WMA, having added respect for autonomy to the Declaration of Geneva, should, I proposed, complete the quartet by adding the ‘balancing’ principle of fairness and justice.Since the Declaration is unlikely to be revised for several years, it seems likely that the proposal to add to it an explicit commitment to practise fairly and justly will have to wait.

However, an explicit commitment to justice and fairness has, at the BMA’s request, been where can i buy viagra over the counter usa added to the draft of the International Code of Medical Ethics and it seems reasonable to hope and expect that it will remain in the final document.Adding a commitment to fairness and justice is the easy part!. Few doctors would on reflection deny that they ought to try to practise fairly and justly. It is far more difficult to say what is actually meant by this.

Two additional components of my Presidential project—the essay competition and a conference (which with luck will have been held, virtually, shortly before publication of this editorial)—sought to help elucidate just what is meant by where can i buy viagra over the counter usa practising fairly and justly.One of the most striking features of the essay competition was the readiness of many writers to point to injustices in the context of medical practice and policy and describe ways of remedying them, but without giving a specific account of justice and fairness on the basis of which the diagnosis of injustice was made and the remedy offered.Wardrope’s winning essay comes close to such an approach by challenging the implied premise that an account of justice and fairness must provide some such formal theory. In preference, he points to the evident injustice and unsustainability of humans’ degradation of ‘the Land’ and its atmosphere and its inhabitants and then challenges some assumptions of contemporary philosophy and ethics, especially what he sees as their anthropocentric and individualistic focus. Instead, he invokes Leopold Aldo’s ‘Land Ethic’ (as well as drawing in aid Isabelle Stenger’s where can i buy viagra over the counter usa focus on ‘the intrusion of Gaia’).

In his thoughtful and challenging paper, he seeks to refocus our ethics—including our medical ethics and our sense of justice and fairness—on mankind’s exploitative threat, during this contemporary ‘anthropocene’ stage of evolution, to the continuing existence of humans and of all forms of life in our ‘biotic community’. As remedy, the author, allying his approach to those of contemporary virtue ethics, recommends the beneficial outcomes that would be brought about by a sense of fairness and justice—a developed and sensitive ‘ecological conscience’ as he calls it—that embraces the interests of the entire biotic community of which we humans are but a part.Fritz and Cox pursue a very different and philosophically more conventional approach to the essay competition’s question and offer a combination and development of two established philosophical theories, those of John Rawls and Thomas Scanlon, to provide a philosophically robust and practically beneficial methodology for justice and fairness in medical practice and policy. Briefly summarised, they recommend a two-stage approach for healthcare where can i buy viagra over the counter usa justice.

First, those faced with a problem of fairness or justice in healthcare or policy should use Thomas Scanlon’s proposed contractualist approach whereby reasonable people seek solutions that they and others could not ‘reasonably reject’. This stage would involve committees of decision-makers and representatives of relevant stakeholders looking at the immediate and longer term impact on existing stakeholders of proposed solutions. They would then check those solutions against substantive criteria of justice derived from Rawls’ theory (which, via his theoretical device of the ‘veil of ignorance’, Rawls and the authors argue that all reasonable people can be expected where can i buy viagra over the counter usa to accept!.

). The Rawlsian criteria relied where can i buy viagra over the counter usa on by Fritz and Cox are equity of access to healthcare. The ‘difference principle’ whereby avoidable inequalities of primary goods can only be justified if they benefit the most disadvantaged.

The just savings principle, of particular importance for ensuring intergenerational justice and sustainability. And a criterion of increased openness, transparency and accountability.It would of course be naïve to expect a single universalisable solution to the question ‘what do we mean by where can i buy viagra over the counter usa fairness and justice in health care?. €™ As the papers by Wardrope1 and Fritz and Cox2 demonstrate, there can be very wide differences of approach in well-defended accounts.

My own hope for my project is to emphasise the importance first of committing where can i buy viagra over the counter usa ourselves within medicine to practising fairly and justly in whatever branch we practise. And then to think carefully about what we do mean by that and act accordingly.Following AristotleFor my own part, over 40 years of looking, I have not yet found a single substantive theory of justice that is plausibly universalisable and have had to content myself with Aristotle’s formal, almost content-free but probably universalisable theory, according to which equals should be treated equally and unequals unequally in proportion to the relevant inequalities—what some health economists refer to as horizontal and vertical justice or equity.6Beauchamp and Childress in their recent eighth and ‘perhaps final’ edition of their foundational ‘Principles of biomedical ethics’1 acknowledge that ‘[t]he construction of a unified theory of justice that captures our diverse conceptions and principles of justice in biomedical ethics continues to be controversial and difficult to pin down’.They still cite Aristotle’s formal principle (though with less explanation than in their first edition back in 1979) and they still believe that this formal principle requires substantive or ‘material’ content if it is to be useful in practice. They then describe six different theories of justice—four ‘traditional’ (utilitarian, libertarian, communitarian and egalitarian) and two newer theories, which they suggest may be more helpful in the context of health justice, one based on capabilities and the other on actual well-being.They again end their discussion of justice with their reminder that ‘Policies of just access to health care, strategies of efficiencies in health care institutions, and global needs for the reduction of health-impairing conditions dwarf in social importance every other issue considered in this book’ …….

€˜every society must ration its resources but many societies can close gaps where can i buy viagra over the counter usa in fair rationing more conscientiously than they have to date’ [emphasis added]. And they go on to stress their own support for ‘recognition of global rights to health and enforceable rights to health care in nation-states’.For my own part I recommend, perhaps less ambitiously, that across the globe we extract from Aristotle’s formal theory of justice a starting point that ethically requires us to focus on equality and always to treat others as equals and treat them equally unless there are moral justifications for not doing so. Where such justifications exist we should say what they are, explain the moral assumptions that justify them and, to the extent possible, seek the agreement of those affected.IntroductionIt did not occur to the Governor that there might be more than one definition of what is good … It did not occur where can i buy viagra over the counter usa to him that while the courts were writing one definition of goodness in the law books, fires were writing quite another one on the face of the land.

(Leopold, ‘Good Oak’1, pp 10–11)As I wrote the abstract that would become this essay, wildfires were spreading across Australia’s east coast. By the time I was invited to write the essay, back-to-back winter storms were flooding communities all around my home. The essay has been written in moments of respite between where can i buy viagra over the counter usa shifts during the erectile dysfunction treatment viagra.

Every one of these events was described as ‘unprecedented’. Yet each is becoming increasingly likely, and that due to our interactions with our environment.Public discourse surrounding these events is dominated by questions of justice and fairness. How to balance competing imperatives of protecting individual lives against where can i buy viagra over the counter usa risk of spreading contagion.

How best to allocate scarce resources like intensive care beds or mechanical ventilators. The conceptual tools of clinical ethics are where can i buy viagra over the counter usa well tailored to these sorts of questions. The rights of the individual versus the community, issues of distributive justice—these are familiar to anyone with even a passing acquaintance with its canonical debates.What biomedical ethics has remained largely silent on is how we have been left to confront these decisions.

How human activity has eroded Earth’s life support systems to make the ‘unprecedented’ the new normal. A medical ethic fit for the Anthropocene—our (still where can i buy viagra over the counter usa tentative) geological epoch defined by human influence on natural systems—must be able not just to react to the consequences of our exploitation of the natural world, but reimagine our relationship with it.Those reimaginations already exist, if we know where to look for them. The ‘Land Ethic’ of the US conservationist Aldo Leopold offers one such vision.i Developed over decades of experience working in and teaching land management, the Land Ethic is most famously formulated in an essay of the same name published shortly before Leopold’s death fighting a wildfire on a neighbour’s farm.

It begins with a reinterpretation of the ethical relationship between where can i buy viagra over the counter usa humanity and the ‘land community’, the ecosystems we live within and depend upon. Moving us from ‘conqueror’ to ‘plain member and citizen’ of that community1 (p 204). Land ceases to be a resource to be exploited for human need once we view ourselves as part of, and only existing within, the land community.

Our moral evaluations shift consonantly:A thing is right when it tends to preserve the integrity, stability, and beauty where can i buy viagra over the counter usa of the biotic community. It is wrong when it tends otherwise.1 (pp 224–225)The justice of the Land Ethic questions many presuppositions of biomedical ethics. By valuing where can i buy viagra over the counter usa the community in itself—in a way irreducible to the welfare of its members—it steps away from the individualism axiomatic in contemporary bioethics.2 Viewing ourselves as citizens of the land community also extends the moral horizons of healthcare from a solely human focus, taking seriously the interests of the non-human members of that community.

Taking into account the ‘stability’ of the community requires intergenerational justice—that we consider those affected by our actions now, and their implications for future generations.3 The resulting vision of justice in healthcare—one that takes climate and environmental justice seriously—could offer health workers an ethic fit for the future, demonstrating ways in which practice must change to do justice to patients, public and planet—now and in years to come.Healthcare in the AnthropoceneSeemeth it a small thing unto you to have fed upon good pasture, but ye must tread down with your feet the residue of your pasture?. And to have drunk of the clear waters, but ye must foul the residue with your feet?. (Ezekiel 34:18, quoted in Leopold, ‘Conservation in the Southwest’4, p 94)The majority of the development of human societies worldwide—including all of recorded human history—has taken place within a single geological epoch, a roughly 11 600 yearlong period of relative warmth and climatic stability known as where can i buy viagra over the counter usa the Holocene.

That stability, however, can no longer be taken for granted. The epoch that has sustained most of human development is giving way to one shaped by the planetary consequences of that development—the Anthropocene.The Anthropocene is marked by accelerating degradation of the ecosystems that have sustained human societies. Human activity is already estimated to have raised global temperatures 1°C above preindustrial levels, and if emissions continue at current levels we are likely to reach 1.5°C between 2030 where can i buy viagra over the counter usa and 2052.5 The global rate of species extinction is orders of magnitude higher than the average over the past 10 million years.6 Ocean acidification, deforestation and disruption of nitrogen and phosphorus flows are likely at or beyond sustainable planetary boundaries.7Yet this period has also seen rapid (if uneven) improvements in human health, with improved life expectancy, falling child mortality and falling numbers of people living in extreme poverty.

The 2015 report of the Rockefeller Foundation-Lancet Commission on planetary health explained this dissonance in stark terms. €˜we have been mortgaging the health of future generations to realise economic and development gains in the present.’7In the instrumental rationality of modernity, nature has featured only as inexhaustible resource and infinite sink to fuel social and economic ends where can i buy viagra over the counter usa. But this disenchanted worldview can no longer hide from the implausibility of these assumptions.

It cannot resist what the philosopher Isabelle Stengers has called ‘the intrusion of Gaia’.8 The present viagra—made more likely by deforestation, land use change and biodiversity loss9—is just the most immediately salient of these intrusions. Anthropogenic environmental changes are increasing undernutrition, increasing range and transmissibility of many where can i buy viagra over the counter usa vectorborne and waterborne diseases like dengue fever and cholera, increasing frequency and severity of extreme weather events like heatwaves and wildfires, and driving population exposure to air pollution—which already accounts for over 7 million deaths annually.10These intrusions will shape healthcare in the Anthropocene. This is because health workers will have to deal with their consequences, and because modern industrialised healthcare as practised in most high-income countries—and considered aspirational elsewhere—was borne of the same worldview that has mortgaged the health of future generations.

The health sector in the USA where can i buy viagra over the counter usa is estimated to account for 8% of the country’s greenhouse gas footprint.11 Pharmaceutical production and waste causes more local environmental degradation, accumulating in water supplies with damaging effects for local flora and fauna.12 Public health has similarly embraced short-term gains with neglect of long-term consequences. Health messaging was instrumental to the development and popularisation of many disposable and single-use products, while a 1947 report funded by the Rockefeller Foundation (who would later fund the landmark 2015 Lancet report on planetary health) popularised the high-meat, high-dairy ‘American’ diet—dependent on fossil fuel-driven intensive agricultural practices—as the healthy ideal.13Healthcare fit for the Anthropocene requires a shift in perspectives that allows us to see and work with the intrusion of Gaia. But can dominant approaches in bioethics incorporate that shift?.

A perfect moral stormWe have built a beautiful piece of social machinery … which is coughing along on two cylinders because we have been too where can i buy viagra over the counter usa timid, and too anxious for quick success, to tell the farmer the true magnitude of his obligations. (Leopold, ‘The Ecological Conscience’4, p 341)At local, national and international scales, the lifestyles of the wealthiest pose an existential threat to the poorest and most marginalised in society. Our actions now are depriving future generations of the environmental prerequisites of good where can i buy viagra over the counter usa health and social flourishing.

If justice means, as Ranaan Gillon parses it, ‘the moral obligation to act on the basis of fair adjudication between competing claims’,14 then this state of affairs certainly seems unjust. However, the tools available for grappling with questions of justice in bioethics seem ill equipped to deal with these sorts of injustice.To illustrate this problem, consider how Gillon further fleshes out his description of justice. In terms of fair distribution of scarce resources, respect for people’s where can i buy viagra over the counter usa rights, and respect for morally acceptable laws.

The first of these—labelled distributive justice—concerns how fairly to allot finite resources among potential beneficiaries. Classic problems of distributive justice in healthcare concern a group of people at a particular time (usually patients), who could each benefit from a particular resource (historically, discussions have often focused on transplant organs. More recently, intensive care where can i buy viagra over the counter usa beds and ventilators have come to the fore).

But there are fewer of these resources than there are people with a need for them. Such discussions are not easy, but they are at least familiar—we know where to begin with them where can i buy viagra over the counter usa. We can consider each party’s need, their potential to benefit from the resource, any special rights or other claims they may have to it, and so forth.

The distribution of benefits and harms in the Anthropocene, however, does not comfortably fit this formalism. It is one thing where can i buy viagra over the counter usa to say that there is but one intensive care bed, from which Smith has a good chance of gaining another year of life, Jones a poor chance, and so offer it to Smith. Another entirely to say that production of the materials consumed in Smith’s care has contributed to the degradation of scarce water supplies on the other side of the globe, or that the unsustainable pattern of energy use will affect innumerable other future persons in poorly quantifiable ways through fuelling climate change.

The calculations of distributive justice are well where can i buy viagra over the counter usa suited to problems where there are a set pool of potential beneficiaries, and the use of the scarce resources available affects only those within that pool. But global environmental problems do not fit this pattern—the effects of our actions are spatially and temporally dispersed, so that large numbers of present and future people are affected in different ways.Nor can this problem be readily addressed by turning to Gillon’s second category of obligations of justice, those grounded in human rights. For while it might be plausible (if not entirely uncontroversial) to say that those communities whose water supplies are degraded by pharmaceutical production have a right to clean water, it is another thing entirely to say that Smith’s healthcare is directly violating that right.

It would not be true to say that, were where can i buy viagra over the counter usa it not for the resources used in caring for Smith, that the communities in question would face no threat to water security—indeed, they would likely make no appreciable difference. Similarly for the effects of Smith’s care on future generations facing accelerating environmental change.iiThe issue here is of fragmentation of agency. While it is not the case that Smith’s care is directly responsible for these environmental harms, the cumulative consequences of many such acts—and the ways in which these acts are embedded in particular systems of energy where can i buy viagra over the counter usa generation, waste management, international trade, and so on—are reliably producing these harms.

The injustice is structural, in Iris Marion Young’s terminology—arising from the ways in which social structures constrain individuals from pursuing certain courses of action, and enable them to follow others, with side effects that cumulatively produce devastating impacts.15Gillon describes the third component of justice as respect for morally acceptable laws. But there is little reason to believe that existing legal frameworks provide sufficient guidance to address these structural injustices. While the intricacies of global governance are well beyond what I can hope to address here, the stark fact remains that, despite the international commitment of the 2015 Paris Agreement to attempt to keep global temperature rise to 1.5°C above preindustrial levels, the Intergovernmental Panel on Climate Change estimates that present national commitments—even if these are substantially increased in coming years—will take us well beyond that target.5 Confronted by where can i buy viagra over the counter usa such institutional inadequacy, respect for the rule of law is inadequate to remedy injustice.The confluence of these particular features—dispersion of causes and effects, fragmentation of agency and institutional inadequacy—makes it difficult for us to reason ethically about the choices we have to make.

Stephen Gardiner calls this a ‘perfect moral storm’.16 Each of these factors individually would be difficult to address using the resources of contemporary biomedical ethics. Their convergence makes it seem insurmountable.This perfect storm was not, however, unpredictable. Van Rensselaer Potter, a professor of Oncology where can i buy viagra over the counter usa responsible for introducing the term ‘bioethics’ into Anglophone discourse, observed that since he coined the phrase, the study of bioethics had diverged from his original usage (governing all issues at the intersection of ethics and the biological sciences) to a narrow focus on the moral dilemmas arising in interactions between individuals in biomedical contexts.

Potter predicted that the short-term, individualistic and medicalised focus of this approach would result in a neglect of population-level and ecological-level issues affecting human and planetary health, with catastrophic consequences.17 His proposed solution was a new ‘global bioethics’, grounded in a new understanding of humanity’s position within planetary systems—one articulated by the Land Ethic.The Land EthicA land ethic changes the role of Homo sapiens from conqueror of the land-community to plain member and citizen of it. It implies respect for his fellow-members, and also respect for the community as such.iii (Leopold, ‘The Land Ethic’1, p 204)Developed throughout where can i buy viagra over the counter usa a career in forestry, conservation and wildlife management, the Land Ethic is less an attempt to provide a set of maxims for moral action, than to shift our perspectives of the moral landscape. In his working life, Aldo Leopold witnessed how actions intended to optimise short-term economic outcomes eroded the environments on which we depend—whether soil degradation arising from intensive farming and deforestation, or disruption of freshwater ecosystems by industrial dairy farming.

He also saw that contemporary morality remained silent on such actions, even when their consequences were to the collective detriment of all.Leopold argued that a series of ‘historical accidents’ left our morality particularly ill suited to handle these intrusions of Gaia—with a worldview that considered them ‘intrusions’, rather than the predictable response of our biotic community. These ‘accidents’ where can i buy viagra over the counter usa were. The unusual resilience of European ecological communities to anthropogenic interference (England survived an almost wholesale deforestation without consequent loss of ecosystem resilience, while similar changes elsewhere resulted in permanent environmental degradation).

And the legacy of European settler colonialism, meaning that an ethic arising in these where can i buy viagra over the counter usa particular conditions came to dominate global social arrangements4 (p 311). The first of these supported a worldview in which ‘Land … is … something to be tamed rather than something to be understood, loved, and lived with. Resources are still regarded as separate entities, indeed, as commodities, rather than as our cohabitants in the land community’4 (p 311).

The second enabled the marginalisation of other where can i buy viagra over the counter usa views. In this genealogy, Leopold anticipated the perfect moral storm discussed above. His intent with the Land Ethic was to navigate it.There are three key components of the Land Ethic that comprise the first three sections where can i buy viagra over the counter usa of Leopold’s final essay on the subject.

(1) the ‘community concept’ that allows communities as wholes to have intrinsic value. (2) the ‘ethical sequence’ that situates the value of such communities as extending, not replacing, values assigned to individuals. And (3) the ‘ecological conscience’ that views ethical action not in terms of following a particular code, but in developing appropriate moral perception.The community conceptThe most widely quoted passage of Leopold’s opus—already cited above, and frequently (mis)taken as a summary maxim of the ethic—states that:A where can i buy viagra over the counter usa thing is right when it tends to preserve the integrity, stability, and beauty of the biotic community.

It is wrong when it tends otherwise.1 (pp 224–225)This passage makes the primary object of our moral responsibilities ‘the biotic community’, a term Leopold uses interchangeably with the ‘land community’. Leopold’s community concept is notable in at least three respects. Its holism—an embrace of the moral significance of communities in a way that is not simply reducible to the significance of where can i buy viagra over the counter usa its individual members.

Its understanding of communities as temporally extended, placing importance on their ‘integrity’ and ‘stability’. And its rejection of anthropocentrism, affording humanity a place as ‘plain member and citizen’ of a broader land community.Individualism is so prevalent where can i buy viagra over the counter usa in biomedical ethics that it is scarcely argued for, instead forming part of the ‘background constellation of values’2 tacitly assumed within the field. We are used to evaluating the well-being of a community as a function of the well-being of its individual members—this is the rationale underlying quality-adjusted life year calculations endemic within health economics, and most discussions of distributive justice adopt some variation of this approach.

Holism instead proposes that this makes no more sense than evaluating a person’s well-being as an aggregate of the well-being of their individual organs. While we can sensibly talk where can i buy viagra over the counter usa about people’s hearts, livers or kidneys, their health is defined in terms of and constitutively dependent on the health of the person as a whole. Similarly, holism proposes, while individuals can be identified separately, it only makes sense to talk about them and their well-being in the context of the larger biotic community which supports and defines us.Holism helps us to negotiate the issues that confront individualistic accounts of collective well-being in Anthropocene health injustices.

In the previous section, we found in the environmental consequences of industrialised healthcare that it is difficult to identify which parties in particular where can i buy viagra over the counter usa are harmed, and how much each individual action contributes to those harms. But our intuition that the overall result is unfair or unjust is itself a holistic assessment of the overall outcome, not dependent on our calculation of the welfare of every party involved. Holism respects the intuition that says—no matter the individuals involved—a world where people now exploit ecological resources in a fashion that deprives people in the future of the prerequisites of survival, is worse than one where communities now and in the future live in a sustainable relationship with their environment.The second aspect of Leopold’s community concept is that the community is something that does not exist at a single time and place—it is defined in terms of its development through time.

Promoting the ‘integrity’ and ‘stability’ of the community requires that we not just where can i buy viagra over the counter usa consider its immediate interests, but how that will affect its long-term sustainability or resilience. We saw earlier the difficulties in trying to say just who is harmed and how when we approach harm to future generations individualistically. But from the perspective of the Land Ethic, when where can i buy viagra over the counter usa we exploit environmental resources in ways that will have predictable damaging results for future generations, the object of our harm is not just some purely notional future person.

It is a presently existing, temporally extended entity—the community of which they will be part.Lastly, Leopold’s community is quite consciously a biotic—not merely human—community. Leopold defines the land community as the open network of energy and mineral exchange that sustains all aspects of that network:Land… is not merely soil. It is a fountain of energy flowing through a circuit of where can i buy viagra over the counter usa soils, plants, and animals.

Food chains are the living channels which conduct energy upward. Death and decay return it to the soil. The circuit is where can i buy viagra over the counter usa not closed.

Some energy is dissipated in decay, some is added by absorption, some is stored in soils, peats, and forests, but it is a sustained circuit, like a slowly augmented revolving fund of life.4 (pp 268–269)While the components within this network may change, the land community as a whole remains stable when the overall complexity of the network is not disrupted—other components are able to adjust to these changes, or new ones arise to take their place.ivThe normative inference Leopold makes from his understanding of the land community is this. It makes no sense to where can i buy viagra over the counter usa single out individual entities within the community as being especially valuable or useful, without taking into account the whole community upon which they mutually depend. To do so is self-defeating.

By privileging the interests of a few members of the community, we ultimately undermine the prerequisites of their existence.The ethical sequenceThe Land Ethic’s holism is in fact its most frequently critiqued feature. Its emphasis on the value of the biotic community leads some to allege a subjugation where can i buy viagra over the counter usa of individual interests to the needs of the environment. This critique neglects how Leopold positions the Land Ethic in what he calls the ‘ethical sequence’.

This is the gradual extension of scope of ethical considerations, both in terms of the complexity of social interactions they cover (from interactions between two people, to the structure of progressively larger social groups), and in the kinds of person they acknowledge as worthy of moral consideration (as we resist, for example, classist, sexist or racist exclusions from personhood).This sequence serves less as a description of the history of morality, than where can i buy viagra over the counter usa a prescription for how we should understand the Land Ethic as adding to, rather than supplanting, our responsibilities to others. We do not argue that taking seriously health workers’ responsibilities for public health and health promotion supplants their duties to the patients they work with on a daily basis. Similarly, the Land Ethic implies ‘respect for [our] fellow members, and also respect for the community as such’1 (p 204).

At times, where can i buy viagra over the counter usa our responsibilities towards these different parties may come into tension. But balancing these responsibilities has always been part of the work of clinical ethics.The ecological conscienceIf the community concept gives a definition of the good, and the ethical sequence situates this definition within the existing moral landscape, neither offers an explicit decision procedure to guide right action. In arguing for the ‘ecological conscience’, Leopold explains his rationale for not attempting to articulate such where can i buy viagra over the counter usa a procedure.

In his career as conservationist, Leopold witnessed time and again laws nominally introduced in the name of environmental protection that did little to achieve their long-term goals, while exacerbating other environmental threats.v This is not surprising, given the ‘perfect moral storm’ of Anthropocene global health and environmental threats discussed above. The cumulative results of apparently innocent actions can be widespread and damaging.Leopold’s response to this problem is to advocate the cultivation of an ‘ecological conscience’. What is needed to promote a healthy human relationship with the land community is not for us to be told exactly how and how not to act in the face of environmental health threats, but rather to shift our view of the land from ‘a commodity belonging to us’ towards ‘a community where can i buy viagra over the counter usa to which we belong’1 (p viii).

To understand what the Land Ethic requires of us, therefore, we should learn more about the land community and our relationship with it, to develop our moral perception and extend its scope to embrace the non-human members of our community.Seen in this light, the Land Ethic shares much in common with virtue ethics, where right action is defined in terms of what the moral agent would do, rather than vice versa. But rather than the Eudaimonia of individual human flourishing proposed by Aristotle, the phronimos of the Land Ethic sees their telos coming from their position within the land community. While clinical virtue ethicists have traditionally taken the virtues of medical practice to be grounded in the interaction with individual patients, the realities of healthcare in the Anthropocene mean that limiting our moral perceptions in this way would ultimately be self-defeating—hurting those very patients we mean to serve (and many more besides).18 The virtuous clinician must adopt a view of where can i buy viagra over the counter usa the moral world that can focus on a person both as an individual, and simultaneously as member of the land community.

I will close by exploring how adopting that perspective might change our practice.Justice in the AnthropoceneFailing this, it seems to me we fail in the ultimate test of our vaunted superiority—the self-control of environment. We fall back into the biological category of the potato bug where can i buy viagra over the counter usa which exterminated the potato, and thereby exterminated itself. (Leopold, ‘The River of the Mother of God’4, p 127)I have articulated some of the challenges healthcare faces in the Anthropocene.

I have suggested that the tools presently available to clinical ethics may be inadequate to meet them. The Land Ethic invites us to reimagine our where can i buy viagra over the counter usa position in and relationship with the land community. I want to close by suggesting how the development of an ecological conscience might support a transition to more just healthcare.

I will where can i buy viagra over the counter usa not endeavour to give detailed prescriptions for action, given Leopold’s warnings about the limitations of such codifications. Rather, I will attempt to show how the cultivation of an ecological conscience might change our perception of what justice demands. Following the tradition of virtue ethics with which the Land Ethic holds much in common, this is best achieved by looking at models of virtuous action, and exploring what makes it virtuous.19Industrialised healthcare developed within a paradigm that saw the environment as inert resource and held that the scope of clinical ethics ranged only over the clinician’s interaction with their patients.

When we begin to see clinician and patient not as standing apart from the environment, but as ‘member and where can i buy viagra over the counter usa citizen of the land community’, their relationship with one another and with the world around them changes consonantly. The present viagra has only begun to make commonplace the idea that health workers do not simply treat infectious diseases, but interact with them in a range of ways, including as vector—and as a result our moral obligations in confronting them may extend beyond the immediate clinical encounter, to cover all the other ways we may contract or spread disease. But we may be responsible for disease outbreaks with conditions other than where can i buy viagra over the counter usa erectile dysfunction treatment, and in ways beyond simply becoming infected.

The development of an ecological conscience would show how our practices of consumption may fuel deforestation that accelerates the emergence of novel pathogens, or support intensive animal rearing that drives antibiotic resistance.18The Land Ethic also challenges us not to abstract our work away from the places in which it takes place. General practitioner surgeries and hospitals are situated within social and land communities alike, shaping and shaped by them. These spaces can be where can i buy viagra over the counter usa used in ways that support or undermine those communities.

Surgeries can work to empower their communities to pursue more sustainable and healthy diets by doubling as food cooperatives, or providing resources and ‘social prescriptions’ for increased walking and cycling. Hospitals can use their extensive real estate to provide publicly accessible green and wild spaces within urban environments, and use their role as major nodes in transport infrastructure to change that infrastructure to support active travel alternatives.ivThe Land Ethic reminds us that a community (human or land) is not healthy if its flourishing cannot be sustainably maintained. An essential component of Anthropocene health justice is where can i buy viagra over the counter usa intergenerational justice.

Contemporary industrialised healthcare has an unsustainable ecological footprint. Continuing with such a model of care would where can i buy viagra over the counter usa serve only to mortgage the health of future generations for the sake of those living now. Ecologically conscious practice must take seriously the sorts of downstream, distributed consequences of activity that produce anthropogenic global health threats, and evaluate to what extent our most intensive healthcare practices truly serve to promote public and planetary health.

It is not enough for the clinician to assume that our resource usage is a necessary evil in the pursuit of best clinical outcomes, for it is already apparent that much of our environmental exploitation is of minimal or even negative long-term value. The work of the National Health Service (NHS) Sustainable Development Unit has seen a 10% reduction in greenhouse gas emissions in the NHS from 2007 to 2015 despite an 18% increase in clinical activity,20 while different models of care used in less industrialised nations manage to provide high-quality health outcomes in less resource-intensive fashion.21ConclusionOur where can i buy viagra over the counter usa present problem is one of attitudes and implements. We are remodelling the Alhambra with a steam-shovel.

We shall hardly relinquish the steam-shovel, which after all has many good points, but we are in need of gentler and more objective where can i buy viagra over the counter usa criteria for its successful use. (Leopold, ‘The Land Ethic’1, p 226)The moral challenges of the Anthropocene do not solely confront health workers. But the potentially catastrophic health effects of anthropogenic global environmental change, and the contribution of healthcare activity to driving these changes provide a specific and unique imperative for action from health workers.Yet it is hard to articulate this imperative in the language of contemporary clinical ethics, ill equipped for this intrusion of Gaia.

Justice in the Anthropocene requires us to be able to adopt a perspective from which these changes no longer appear as unexpected intrusions, but that where can i buy viagra over the counter usa acknowledges the land community as part of our moral community. The Land Ethic articulates an understanding of justice that is holistic, structural, intergenerational, and rejects anthropocentrism. This understanding where can i buy viagra over the counter usa seeks not to supplant, but to augment, our existing one.

It aims to do so by helping us to develop an ‘ecological conscience’, seeing ourselves as ‘plain member and citizen’ of the land community. The Land Ethic does not provide a step-by-step guide to just action. Nor does it definitively adjudicate on how to balance the where can i buy viagra over the counter usa interests of our patients, other populations now and in the future, and the planet.

It could, however, help us on the first step towards that change—showing how to cultivate the ‘internal change in our intellectual emphasis, loyalties, affections, and convictions’1 (pp 209–210) necessary to realise the virtues of just healthcare in the Anthropocene.AcknowledgmentsThis essay was written as a submission for the BMA Presidential Essay Prize. I am grateful to the organisers and judging panel for the opportunity..

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