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Le Dantec virus (LDV), assigned to the species Ledantevirus ledantec, genus Ledantevirus, family Rhabdoviridae has been associated with human disease but has gone undetected since the 1970s. We describe the detection of LDV in a human case of undifferentiated fever in Uganda by metagenomic sequencing and demonstrate a serological response using ELISA and pseudotype neutralisation. By screening 997 individuals sampled in 2016, we show frequent exposure to ledanteviruses with 76% of individuals seropositive in Western Uganda, but lower seroprevalence in other areas. Serological cross-reactivity as measured by pseudotype-based neutralisation was confined to ledanteviruses, indicating population seropositivity may represent either exposure to LDV or related ledanteviruses. We also describe the discovery of a closely related ledantevirus in blood from the synanthropic rodent Mastomys erythroleucus. Ledantevirus infection is common in Uganda but is geographically heterogenous. Further surveys of patients presenting with acute fever are required to determine the contribution of these emerging viruses to febrile illness in Uganda.

Understanding SARS-CoV-2 vaccine-induced antibody responses in varied antigenic and serological prior exposures can guide optimal vaccination strategies for enhanced immunogenicity. We evaluated spike (S)-directed IgG, IgM, and IgA antibody optical densities (ODs) and concentrations to the two-dose ChAdOx1-S Oxford-AstraZeneca (ChAdOx1-S, Covishield) SARS-CoV-2 vaccine in 67 Ugandans, categorised by prior infection and baseline S-IgG histories: uninfected and S-IgG-negative (n = 12); previously infected yet S-IgG-negative (n = 17); and previously infected with S-IgG-positive status (n = 38). Antibody dynamics were compared across eight timepoints from baseline till nine months. S-IgG antibodies remained consistently potent across all groups. Individuals with prior infections maintained robust S-IgG levels, underscoring the endurance of hybrid immunity. In contrast, those without prior exposure experienced an initial surge in S-IgG after the primary dose but no subsequent significant increase post-boost. However, they reached levels parallel to the previously exposed groups. S-IgM levels remained moderate, while S-IgA persisted in individuals with prior antigen exposure. ChAdOx1-S, Covishield vaccine elicited robust and sustained antibody responses in recipients, irrespective of their initial immune profiles. Hybrid immunity showed higher responses, aligning with global observations. Early post-vaccination antibody levels could predict long-term immunity, particularly in individuals without virus exposure. These findings can inform vaccine strategies and pandemic management.

BackgroundSignificant milestones have been made in the development of COVID19 diagnostics Technologies. Government of the republic of Uganda and the line Ministry of Health mandated Uganda Virus Research Institute to ensure quality of COVID19 diagnostics. Re-testing was one of the methods initiated by the UVRI to implement External Quality assessment of COVID19 molecular diagnostics.Methodparticipating laboratories were required by UVRI to submit their already tested and archived nasopharyngeal samples and corresponding meta data. These were then re-tested at UVRI using the WHO Berlin protocol, the UVRI results were compared to those of the primary testing laboratories in order to ascertain performance agreement for the qualitative & quantitative results obtained. Ms Excel window 12 and GraphPad prism ver 15 was used in the analysis. Bar graphs, pie charts and line graphs were used to compare performance agreement between the reference Laboratory and primary testing Laboratories.ResultsEleven (11) Ministry of Health/Uganda Virus Research Institute COVID19 accredited laboratories participated in the re-testing of quality control samples. 5/11 (45%) of the primary testing laboratories had 100% performance agreement with that of the National Reference Laboratory for the final test result. Even where there was concordance in the final test outcome (negative or positive) between UVRI and primary testing laboratories, there were still differences in CT values. The differences in the Cycle Threshold (CT) values were insignificant except for Tenna & Pharma Laboratory and the UVRI(p = 0.0296). The difference in the CT values were not skewed to either the National reference Laboratory(UVRI) or the primary testing laboratory but varied from one laboratory to another. In the remaining 6/11 (55%) laboratories where there were discrepancies in the aggregate test results, only samples initially tested and reported as positive by the primary laboratories were tested and found to be false positives by the UVRI COVID19 National Reference Laboratory.ConclusionFalse positives were detected from public, private not for profit and private testing laboratories in almost equal proportion. There is need for standardization of molecular testing platforms in Uganda. There is also urgent need to improve on the Laboratory quality management systems of the molecular testing laboratories in order to minimize such discrepancies.

• Human immunodeficiency virus (HIV) drug resistance has implications for antiretroviral treatment strategies and for containing the HIV pandemic because the development of HIV drug resistance leads to the requirement for antiretroviral drugs that may be less effective, less well-tolerated, and more expensive than those used in first-line regimens. • HIV drug resistance studies are designed to determine which HIV mutations are selected by antiretroviral drugs and, in turn, how these mutations affect antiretroviral drug susceptibility and response to future antiretroviral treatment regimens. • Such studies collectively form a vital knowledge base essential for monitoring global HIV drug resistance trends, interpreting HIV genotypic tests, and updating HIV treatment guidelines. • Although HIV drug resistance data are collected in many studies, such data are often not publicly shared, prompting the need to recommend best practices to encourage and standardize HIV drug resistance data sharing. • In contrast to other viruses, sharing HIV sequences from phylogenetic studies of transmission dynamics requires additional precautions as HIV transmission is criminalized in many countries and regions. • Our recommendations are designed to ensure that the data that contribute to HIV drug resistance knowledge will be available without undue hardship to those publishing HIV drug resistance studies and without risk to people living with HIV.

Schistosomiasis is a disease caused by parasitic flatworms of the Schistosoma spp., and is increasingly recognized to alter the immune system, and the potential to respond to vaccines. The impact of endemic infections on protective immunity is critical to inform vaccination strategies globally. We assessed the influence of Schistosoma mansoni worm burden on multiple host vaccine-related immune parameters in a Ugandan fishing cohort (n = 75) given three doses of a Hepatitis B (HepB) vaccine at baseline and multiple timepoints post-vaccination. We observed distinct differences in immune responses in instances of higher worm burden, compared to low worm burden or non-infected. Concentrations of pre-vaccination serum schistosome-specific circulating anodic antigen (CAA), linked to worm burden, showed a significant bimodal distribution associated with HepB titers, which was lower in individuals with higher CAA values at month 7 post-vaccination (M7). Comparative chemokine/cytokine responses revealed significant upregulation of CCL19, CXCL9 and CCL17 known to be involved in T cell activation and recruitment, in higher CAA individuals, and CCL17 correlated negatively with HepB titers at month 12 post-vaccination. We show that HepB-specific CD4+ T cell memory responses correlated positively with HepB titers at M7. We further established that those participants with high CAA had significantly lower frequencies of circulating T follicular helper (cTfh) subpopulations pre- and post-vaccination, but higher regulatory T cells (Tregs) post-vaccination, suggesting changes in the immune microenvironment in high CAA could favor Treg recruitment and activation. Additionally, we found that changes in the levels of innate-related cytokines/chemokines CXCL10, IL-1β, and CCL26, involved in driving T helper responses, were associated with increasing CAA concentration. This study provides further insight on pre-vaccination host responses to Schistosoma worm burden which will support our understanding of vaccine responses altered by pathogenic host immune mechanisms and memory function and explain abrogated vaccine responses in communities with endemic infections.

PrEPVacc is an international, multi-centre, double-blind vaccine study comparing experimental combination vaccine regimens including DNA/AIDSVAX BE and DNA/CN54gp140 with placebo control. Simultaneously, daily oral PrEP is compared for efficacy against daily Truvada in the context of the current PrEP availability situation at the study sites. An important clinical trial outcome is the accurate measurement of in vivo antibody titer induced through vaccination. Here we report the validation of two ELISAs for CN54gp140 and AIDSVAX BE at Uganda Virus Research Institute that demonstrates precision, specificity, and robustness for assessing the reciprocal antibody end point titer in human serum. This is a critical endpoint for determining whether vaccination can provide any protection against HIV in populations at risk of acquiring HIV.

Objective The objective of this study was to evaluate the performance of seven antigen rapid diagnostic tests (Ag RDTs) in a clinical setting to identify those that could be recommended for use in the diagnosis of SARS-CoV-2 infection in Uganda. Methods This was a cross-sectional prospective study. Nasopharyngeal swabs were collected consecutively from COVID-19 PCR positive and COVID-19 PCR negative participants at isolation centers and points of entry, and tested with the SARS-CoV-2 Ag RDTs. Test sensitivity and specificity were generated by comparing results against qRT-PCR results (Berlin Protocol) at a cycle threshold (Ct) cut-off of ≤39. Sensitivity was also calculated at Ct cut-offs ≤29 and ≤33. Results None of the Ag RDTs had a sensitivity of ≥80% at Ct cut-off values ≤33 and ≤39. Two kits, Panbio™ COVID-19 Ag and VivaDiag™ SARS-CoV-2 Ag had a sensitivity of ≥80% at a Ct cut-off value of ≤29. Four kits: BIOCREDIT COVID -19 Ag, COVID-19 Ag Respi-Strip, MEDsan® SARS-CoV-2 Antigen Rapid Test and Panbio™ COVID-19 Ag Rapid Test had a specificity of ≥97%. Conclusions This evaluation identified one Ag RDT, Panbio™ COVID-19 Ag with a performance at high viral load (Ct value ≤29) reaching that recommended by WHO. This kit was recommended for screening of patients with COVID -19-like symptoms presenting at health facilities.

BackgroundCryptococcal meningitis (CCM) remains one of the leading causes of mortality among HIV infected patients. Due to factors such as the severity of CCM pathology, the quality of life (QOL) of patients post-treatment is likely to be poor. Few studies have reported on QOL of CCM patients post treatment completion. We used data collected among patients in the CryptoDex trial (ISRCTN59144167) to determine QOL and associated factors at week 10 and six months from treatment initiation.MethodologyCryptoDex was a double-blind placebo-controlled trial of adjunctive dexamethasone in HIV infected adults with CCM, conducted between 2013 and 2015 in six countries in Asia and Africa. QOL was determined using the descriptive and Visual Analog Scales (VAS) of the EuroQol Five-Dimension-Three-Level (EQ-5D-3L) tool. We derived index scores, and described these and the VAS scores at 10 weeks and 6 months; and used linear regression to determine the relationship between various characteristics and VAS scores at both time points. VAS scores were interpreted as very good (81-100), good (51-80), normal (31-50) and bad/very bad (0-30).ResultsOf 451 patients enrolled in the trial, 238 had QOL evaluations at week 10. At baseline, their mean age (SD) was 35.2(8.5) years. The mean index scores (SD) were 0.785(0.2) and 0.619(0.4) among African and Asian patients respectively at week 10, and 0.879(0.2) and 0.731(0.4) among African and Asian patients respectively at month six. The overall mean VAS score (SD) at 10 weeks was 57.2 (29.7), increasing significantly to 72(27.4) at month six (pConclusionQOL was good among patients that had completed therapy for CCM, but below perfect. Strategies to improve QOL among CCM survivors are required.

There is growing recognition of the burden of depression in people living with HIV/AIDS (PLWHA), associated with negative behavioural and clinical outcomes. Unfortunately, most HIV care providers in sub-Saharan Africa do not routinely provide mental health services to address this problem. This article describes the process of developing a model for integrating the management of depression in HIV care in Uganda. Theory of Change (ToC) methodology was used to guide the process of developing the model. Three successive ToC workshops were held with a multi-disciplinary group of 38 stakeholders within Wakiso district, in the Central region of Uganda. The first 2 workshops were for generating practical ideas for a feasible and acceptable model of integrating the management of depression in HIV care at all levels of care within the district healthcare system; while the third and final workshop was for consensus building. Following meaningful brainstorming and discussions, the stakeholders suggested improved mental wellbeing among PLWHA as the ultimate outcome of the program. This would be preceded by short-term and intermediate outcomes including reduced morbidity among persons with HIV attributable to depression, allocation of more resources towards management of depression, increased help-seeking among depressed PLWHA and more health workers detecting and managing depression. These would be achieved following several interventions undertaken at all levels of care. The participants further identified some indicators of successful implementation such as emphasis of depression management in the district healthcare plans, increased demand for anti-depressants etc; as well as various assumptions underlying the intervention. All these were graphically aligned in a causal pathway, leading to a ToC map, contextualizing and summarizing the intervention model. The ToC was a valuable methodology that brought together stakeholders to identify key strategies for development of a comprehensible contextualized intervention model for managing depression within HIV care in Uganda; allowing greater stakeholder engagement and buy-in.

BackgroundHealthcare workers (HCWs) are at high risk of acquiring SARS-CoV-2 and COVID-19 and may therefore be a suitable population for COVID-19 vaccine trials. We conducted a survey to evaluate willingness-to-participate in COVID-19 vaccine trials in a population of HCWs at three hospitals in Uganda.MethodsThe survey was conducted between September and November 2020. Using a standardised questionnaire, data were collected on socio-demographics, previous participation in health research, COVID-19 information sources, underlying health conditions, and willingness-to-participate in COVID-19 vaccine trials. Data were analysed descriptively and a binomial generalised linear model with a log link function used to investigate factors associated with unwillingness to participate.Results657 HCWs (female, 63%) were enrolled with a mean age of 33 years (Standard Deviation, 10). Overall willingness-to-participate was 70.2%. Key motivating factors for participation were: hope of being protected against COVID-19 (81.1%), altruism (73.3%), and the opportunity to get health care (26.0%). Selected hypothetical trial attributes reduced willingness-to-participate as follows: weekly-quarterly study visits over a 12-month period (70.2%-63.2%, P = 0.026); provision of approximately 50ml of blood at each study visit (70.2%-63.2%, P = 0.026); risk of mild-moderate local adverse reactions (70.2%-60.3%, PConclusionsWillingness-to-participate in COVID-19 vaccine trials among HCWs in Uganda is high but may be affected by vaccine trial requirements and concerns about the safety of candidate vaccines.

Globally, early initiation of antiretroviral therapy for HIV led to a reduction in the estimated mortality from cryptococcal meningitis (CCM) from 624,700 in 2009 to 181,100 in 2014. However, CCM remains one of the leading causes of mortality among HIV infected patients especially in sub-Saharan Africa where 75% of the deaths occur. Most of the studies evaluating mortality have reported short-term mortality (at or before 10 weeks of therapy). We determined mortality and associated factors among patients treated for CCM in the CryptoDex trial (ISRCTN59144167) in Uganda, and the effect of dexamethasone adjunctive therapy on mortality at two years. We conducted a retrospective cohort study between May 2017 and July 2017 to determine the long term survival (up to 2 years post-randomization) of all patients who had been enrolled into the CryptoDex trial in Uganda. The CryptoDex trial recruited between April 2013 and February 2015. We estimated mortality rates and determined factors affecting mortality at two years using Cox regression. The study followed up 211 participants, 127 (60.2%) of whom were male. Sixteen participants (7.58%) were diagnosed with HIV at the same admission when CCM was diagnosed. By two years following randomization 127 (60%) participants had died, a mortality rate of 67 deaths per 100 person-years. Mortality was associated with Glasgow coma score (GCS) below 15 (adjusted Hazard ratio (aHR) 1.77, 95% CI: 1.02-2.44), p = 0.040; weight (aHR 0.97, per 1 Kg increase; 95% CI: 0.94-0.99), p = 0.003; and presence of convulsions (aHR 2.31, 95% CI: 1.32-4.04), p = 0.004, while dexamethasone use and fungal burden had no effect. Long-term mortality in CCM patients remains high even among patients receiving recommended therapy. Strategies to improve long-term survival in CCM patients are urgently needed, especially targeting those with reduced GCS, low weight, and convulsions.

IntroductionWith the scale-up of antiretroviral therapy (ART) there is a need to monitor programme performance to maximize ART efficacy and to prevent emergence of HIV drug resistance (HIVDR). In keeping with the elements of the World Health Organisation (WHO) guidance we carried out a nationally representative assessment of early warning indicators (EWI) at 304 randomly selected ART service outlets in Uganda.MethodsRetrospective patient data was extracted for the six EWIs for HIVDR including; on-time antiretroviral (ARV) drug pick-up, patient retention on ART at 12 months, ART dispensing practices, ARV drug stock-outs, viral load suppression (VLS) and viral load (VL) testing completion. Point prevalence for each clinic and national aggregate prevalence with 95% confidence intervals (CI) for all clinics were estimated and facility performances were computed and association between EWIs and programmatic factors assessed using Fisher's Exact Test.ResultsFacilities meeting the EWI targets: on-time pill pick-up was 9.5%, more facilities in the north met this target (p = 0.040). Retention on ART at 12 months was 24.1%, facilities in Kampala region (pConclusionDispensing practices in all facilities were in line with the national guidelines however, there still remains a challenge to long-term ART programmatic success in monitoring patient response to treatment, and maintaining patients on ART without interruptions arising due to poor patient adherence and as a consequence of ARV supply interruption. It is therefore of high importance that the national ART program ensures intensified follow-up for patients, ensuring uninterrupted supply of ARV drugs and increasing VL monitoring at treatment centres, in order to improve patient outcomes and avert preventable HIVDR.

INTRODUCTION:In October 2017, a blood sample from a resident of Kween District, Eastern Uganda, tested positive for Marburg virus. Within 24 hour of confirmation, a rapid outbreak response was initiated. Here, we present results of epidemiological and laboratory investigations. METHODS:A district task force was activated consisting of specialised teams to conduct case finding, case management and isolation, contact listing and follow up, sample collection and testing, and community engagement. An ecological investigation was also carried out to identify the potential source of infection. Virus isolation and Next Generation sequencing were performed to identify the strain of Marburg virus. RESULTS:Seventy individuals (34 MVD suspected cases and 36 close contacts of confirmed cases) were epidemiologically investigated, with blood samples tested for MVD. Only four cases met the MVD case definition; one was categorized as a probable case while the other three were confirmed cases. A total of 299 contacts were identified; during follow- up, two were confirmed as MVD. Of the four confirmed and probable MVD cases, three died, yielding a case fatality rate of 75%. All four cases belonged to a single family and 50% (2/4) of the MVD cases were female. All confirmed cases had clinical symptoms of fever, vomiting, abdominal pain and bleeding from body orifices. Viral sequences indicated that the Marburg virus strain responsible for this outbreak was closely related to virus strains previously shown to be circulating in Uganda. CONCLUSION:This outbreak of MVD occurred as a family cluster with no additional transmission outside of the four related cases. Rapid case detection, prompt laboratory testing at the Uganda National VHF Reference Laboratory and presence of pre-trained, well-prepared national and district rapid response teams facilitated the containment and control of this outbreak within one month, preventing nationwide and global transmission of the disease.

BackgroundCryptococcal meningitis (CCM) remains a leading cause of mortality amongst HIV infected patients in sub-Saharan Africa. When patients receive recommended therapy, mortality at 10 weeks has been reported to vary between 20 to 36%. However, mortality rate and factors affecting mortality after completing recommended therapy are not well known. We investigated mortality rate, and factors affecting mortality at 2 years among CCM patients following completion of recommended CCM therapy in Uganda.MethodsA retrospective cohort study was conducted among HIV infected patients that had completed 10 weeks of recommended therapy for CCM (2 weeks of intravenous amphotericin B 1mg/kg and 10 weeks of oral Fluconazole 800mg daily) in the CryptoDex trial (ISRCTN59144167) between 2013 and 2015. Survival analysis applying Cox regression was used to determine the mortality rate and factors affecting mortality at 2 years.ResultsThis study followed up 112 participants for 2 years. Mean age (±SD) was 34.9 ± 8, 48 (57.1%) were female and 80 (74.8%) had been on ART for less than 1 year. At 2 years, overall mortality was 30.9% (20 deaths per 100 person-years). Majority of deaths (61.8%) occurred during the first 6 months. In multivariable analysis, mortality was associated with ever being re-admitted since discharge after hospital-based management of CCM (aHR = 13.33, 95% CI: 5.92-30.03), p75% compared to ConclusionThere remains a considerable risk of mortality in the first two years after completion of standard therapy for CCM in resource-limited settings with risk highest during the first 6 months. Maintenance of patient follow up during this period may reduce mortality.

Helminth infection may affect vaccine immunogenicity and efficacy. Adolescents, a target population for tuberculosis booster vaccines, often have a high helminth burden. We investigated effects of Schistosoma mansoni (Sm) on the immunogenicity and safety of MVA85A, a model candidate tuberculosis vaccine, in BCG-vaccinated Ugandan adolescents.In this phase II open label trial we enrolled 36 healthy, previously BCG-vaccinated adolescents, 18 with no helminth infection detected, 18 with Sm only. The primary outcome was immunogenicity measured by Ag85A-specific interferon gamma ELISpot assay. Tuberculosis and schistosome-specific responses were also assessed by whole-blood stimulation and multiplex cytokine assay, and by antibody ELISAs.Ag85A-specific cellular responses increased significantly following immunisation but with no differences between the two groups. Sm infection was associated with higher pre-immunisation Ag85A-specific IgG4 but with no change in antibody levels following immunisation. There were no serious adverse events. Most reactogenicity events were of mild or moderate severity and resolved quickly.The significant Ag85A-specific T cell responses and lack of difference between Sm-infected and uninfected participants is encouraging for tuberculosis vaccine development. The implications of pre-existing Ag85A-specific IgG4 antibodies for protective immunity against tuberculosis among those infected with Sm are not known. MVA85A was safe in this population.ClinicalTrials.gov NCT02178748.

BackgroundFishing communities around Lake Victoria in sub-Saharan Africa have been characterised as a population at high risk of HIV-infection.MethodsUsing data from a cohort of HIV-positive individuals aged 13-49 years, enrolled from 5 fishing communities on Lake Victoria between 2009-2011, we sought to identify factors contributing to the epidemic and to understand the underlying structure of HIV transmission networks. Clinical and socio-demographic data were combined with HIV-1 phylogenetic analyses. HIV-1 gag-p24 and env-gp-41 sub-genomic fragments were amplified and sequenced from 283 HIV-1-infected participants. Phylogenetic clusters with ≥2 highly related sequences were defined as transmission clusters. Logistic regression models were used to determine factors associated with clustering.ResultsAltogether, 24% (n = 67/283) of HIV positive individuals with sequences fell within 34 phylogenetically distinct clusters in at least one gene region (either gag or env). Of these, 83% occurred either within households or within community; 8/34 (24%) occurred within household partnerships, and 20/34 (59%) within community. 7/12 couples (58%) within households clustered together. Individuals in clusters with potential recent transmission (11/34) were more likely to be younger 71% (15/21) versus 46% (21/46) in un-clustered individuals and had recently become resident in the community 67% (14/21) vs 48% (22/46). Four of 11 (36%) potential transmission clusters included incident-incident transmissions. Independently, clustering was less likely in HIV subtype D (adjusted Odds Ratio, aOR = 0.51 [95% CI 0.26-1.00]) than A and more likely in those living with an HIV-infected individual in the household (aOR = 6.30 [95% CI 3.40-11.68]).ConclusionsA large proportion of HIV sexual transmissions occur within house-holds and within communities even in this key mobile population. The findings suggest localized HIV transmissions and hence a potential benefit for the test and treat approach even at a community level, coupled with intensified HIV counselling to identify early infections.

BACKGROUND:Although the association between alcohol consumption and HIV risk is well documented, few studies have examined the magnitude of new HIV infections that could be prevented by controlling alcohol use. We report the population attributable fraction (PAF) of incident HIV infections due to alcohol consumption among the HIV high-risk population of fishing communities along Lake Victoria, Uganda. METHODS:In a community-based cohort study, 1607 HIV sero-negative participants aged 18-49 years were enrolled from eight fishing communities along Lake Victoria, Uganda. At follow up 12 months later, 1288 (80.1%) were seen and interviewed. At baseline and follow-up visits, participants completed interviewer-administered questionnaires on alcohol consumption, demographics, and sexual risk behavior, and were tested for HIV infection. HIV incidence and adjusted incident rate ratios (adjusted IRRs) were estimated using Poisson regression models; the crude and adjusted PAFs of incident HIV infections associated with alcohol consumption were calculated using the Greenland and Drescher method for cohort studies. RESULTS:Among the 1288 participants seen at follow up, 53.5% reported drinking alcohol of whom 24.4% drank occasionally (2 days a week or less) and 29.1% drank regularly (3-7 days a week). Forty eight incident HIV infections occurred giving an incidence rate of 3.39/100 person years at-risk (pyar) (95% CI, 2.55-4.49). Compared to non-drinkers, the adjusted IRR of HIV was 3.09 (1.13-8.46) among occasional drinkers and 5.34 (2.04-13.97) among regular drinkers. The overall adjusted PAF of incident HIV infections due alcohol was 64.1 (95% CI; 23.5-83.1); ranging from 52.3 (11.9-74.2) among Muslims to 71.2 (32.6-87.7) for participants who reported ≥ 2 sexual partners in the past 12 months. CONCLUSION:In fishing communities along Lake Victoria, Uganda, 64% of new HIV infections can be attributed to drinking alcohol. Interventions to reduce alcohol consumption should be integrated in HIV/AIDS prevention activities for populations in whom both HIV and alcohol consumption are highly prevalent.

[This corrects the article DOI: 10.1371/journal.pone.0137834.].

Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes.We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling.Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

BackgroundMajor protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs.MethodsSamples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain.ResultsWe cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08-6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39-7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure.ConclusionsHere we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic determinants of protease inhibitor failure in those who fail without traditional resistance mutations whilst PI use is being scaled up globally.

We examined several measures of self-reported HIV risk behaviour in mutually disclosed sero-discordant couples over time to see if a couples counselling intervention was associated with changes in these behaviors.We analysed data from a prospective cohort study of HIV sero-discordant couples in Jinja, Uganda collected between June 2009 and December 2011. Participants received couples counselling, at 3-monthly intervals. We examined trends in reported condom-use, number of concurrent sexual partners, knowledge of HIV serostatus of concurrent partners and condom use of concurrent partners using Generalized Estimating Equation models, comparing responses at study enrollment with responses at six, 12 18 and 24 months of follow-up.A total of 586 couples were enrolled and the female member was HIV positive in 255 (44%) of them. The median age for female participants was 35 years and 42 years for men. Reported condom use at last sex with spouse increased over time (p

HIV epidemiology informs prevention trial design and program planning. Nine clinical research centers (CRC) in sub-Saharan Africa conducted HIV observational epidemiology studies in populations at risk for HIV infection as part of an HIV prevention and vaccine trial network. Annual HIV incidence ranged from below 2% to above 10% and varied by CRC and risk group, with rates above 5% observed in Zambian men in an HIV-discordant relationship, Ugandan men from Lake Victoria fishing communities, men who have sex with men, and several cohorts of women. HIV incidence tended to fall after the first three months in the study and over calendar time. Among suspected transmission pairs, 28% of HIV infections were not from the reported partner. Volunteers with high incidence were successfully identified and enrolled into large scale cohort studies. Over a quarter of new cases in couples acquired infection from persons other than the suspected transmitting partner.

We examined the real-world effectiveness of ART as an HIV prevention tool among HIV serodiscordant couples in a programmatic setting in a low-income country.We enrolled individuals from HIV serodiscordant couples aged ≥18 years of age in Jinja, Uganda from June 2009 - June 2011. In one group of couples the HIV positive partner was receiving ART as they met clinical eligibility criteria (a CD4 cell count ≤250 cells/ μL or WHO Stage III/IV disease). In the second group the infected partner was not yet ART-eligible. We measured HIV incidence by testing the uninfected partner every three months. We conducted genetic linkage studies to determine the source of new infections in seroconverting participants.A total of 586 couples were enrolled of which 249 (42%) of the HIV positive participants were receiving ART at enrollment, and an additional 99 (17%) initiated ART during the study. The median duration of follow-up was 1.5 years. We found 9 new infections among partners of participants who had been receiving ART for at least three months and 8 new infections in partners of participants who had not received ART or received it for less than three months, for incidence rates of 2.09 per 100 person-years (PYRs) and 2.30 per 100 PYRs, respectively. The incidence rate ratio for ART-use was 0.91 (95% confidence interval 0.31-2.70; p=0.999). The hazard ratio for HIV seroconversion associated with ART-use by the positive partner was 1.07 (95% CI 0.41-2.80). A total of 5/7 (71%) of the transmissions on ART and 6/7 (86%) of those not on ART were genetically linked.Overall HIV incidence was low in comparison to previous studies of serodiscordant couples. However, ART-use was not associated with a reduced risk of HIV transmission in this study.

BACKGROUND:With the scale-up of antiretroviral therapy (ART), monitoring programme performance is needed to maximize ART efficacy and limit HIV drug resistance (HIVDR). METHODS:We implemented a WHO HIVDR prospective survey protocol at three treatment centers between 2012 and 2013. Data were abstracted from patient records at ART start (T1) and after 12 months (T2). Genotyping was performed in the HIV pol region at the two time points. RESULTS:Of the 425 patients enrolled, at T2, 20 (4.7%) had died, 66 (15.5%) were lost to follow-up, 313 (73.6%) were still on first-line, 8 (1.9%) had switched to second-line, 17 (4.0%) had transferred out and 1 (0.2%) had stopped treatment. At T2, 272 out of 321 on first and second line (84.7%) suppressed below 1000 copies/ml and the HIV DR prevention rate was 70.1%, just within the WHO threshold of ≥ 70%. The proportion of participants with potential HIVDR was 20.9%, which is higher than the 18.8% based on pooled analyses from African studies. Of the 35 patients with mutations at T2, 80% had M184V/I, 65.7% Y181C, and 48.6% (54.8% excluding those not on Tenofovir) had K65R mutations. 22.9% had Thymidine Analogue Mutations (TAMs). Factors significantly associated with HIVDR prevention at T2 were: baseline viral load (VL)

Yegor Voronin and colleagues explore how monoclonal antibodies against HIV could provide a new opportunity to further reduce mother-to-child transmission of HIV and propose that new interventions should consider issues related to implementation, feasibility, and access. Please see later in the article for the Editors' Summary.

In contrast to resource-rich countries, most HIV-infected patients in resource-limited countries receive treatment without virological monitoring. There are few long-term data, in this setting, on rates of viral suppression or switch to second-line antiretroviral therapy. The DART trial compared clinically driven monitoring (CDM) versus routine laboratory (CD4/haematology/biochemistry) and clinical monitoring (LCM) in HIV-infected adults initiating therapy. There was no virological monitoring in either study group during follow-up, but viral load was measured in Ugandan participants at trial closure. Two thousand three hundred and seventeen (2317) participants from this country initiated antiretroviral therapy with zidovudine/lamivudine plus tenofovir (n = 1717), abacavir (n = 300), or nevirapine (n = 300). Of 1896 (81.8%) participants who were alive and in follow-up at trial closure (median 5.1 years after therapy initiation), 1507 (79.5%) were on first-line and 389 (20.5%) on second-line antiretroviral therapy. The overall switch rate after the first year was 5.6 per 100 person-years; the rate was substantially higher in participants with low baseline CD4 counts (

High HIV-1 incidence rates were reported among persons in fisherfolk communities (FFC) in Uganda who were selected for high risk behaviour. We assessed the incidence of HIV-1 and associated risk factors in a general population FFC to determine population-wide HIV rates.A community-based cohort study was conducted among a random sample of 2191 participants aged 18-49 years. At baseline and 12 months post-baseline, data were collected on socio-demographic characteristics and risky behaviors (including number of partners, new partners, condom use, use of alcohol and illicit drug use). Venous blood was collected for HIV serological testing. HIV incidence was calculated per 100 person years at-risk (pyar) and adjusted incidence rate ratios (Adj.IRR) were estimated by multivariable Poisson regression.Overall follow up at 12 months was 76.9% (1685/2191) and was significantly higher among HIV uninfected persons and those with at least 1 year duration of stay in community. Overall HIV-1 incidence was 3.39/100 pyar (95% CI: 2.55-4.49). Among the 25-29 years who drank alcohol, HIV incidence was 7.67/100 pyar (95% CI;4.62-12.7) while it was 5.67/100 pyar (95% CI;3.14-10.2) for 18-24 year olds who drank alcohol. The risk of HIV infection was higher among 25-29 years (adj.IRR = 3.36; 95% CI: 1.48-7.65) and 18-24 years (adj.IRR = 2.65; 95% CI: 1.05-6.70) relative to 30+ years. Compared to non-drinkers, HIV incidence increased by frequency of alcohol drinking--occasional drinkers (adj.IRR = 3.18; 95% CI: 1.18-8.57) and regular drinkers (adj.IRR = 4.93; 95% CI: 1.91-12.8).HIV-1 incidence in general fisherfolk population along L. Victoria, Uganda, is high and is mainly associated with young age and alcohol drinking. HIV prevention and control strategies are urgently needed in this population.

OBJECTIVE:We examined the association of HIV-1 subtypes with disease progression based on three viral gene regions. DESIGN:A prospective HIV-1 clinical cohort study in rural Uganda. METHODS:Partial gag, env and pol genes were sequenced. Cox proportional hazard regression modelling was used to estimate adjusted hazard ratios (aHRs) of progression to: CD4≤250, AIDS onset and death, adjusted for sex, age and CD4 count at enrolment. RESULTS:Between 1990 and 2010, 292 incident cases were subtyped: 25% had subtype A, 45% had D, 26% had A/D recombinants, 1% had C and 4% were other recombinant forms. Of the 278 incident cases included in the disease progression analysis, 62% progressed to CD4≤250, 32% to AIDS, and 34% died with a higher proportion being among subtype D cases. The proportions of individuals progressing to the three endpoints were significantly higher among individuals infected with subtype D. Throughout the study period, individuals infected with subtype D progressed faster to CD4≤250, adjusted HR (aHR), (95% CI) = 1.72 (1.16-2.54), but this was mainly due to events in the period before antiretroviral therapy (ART) introduction, when individuals infected with subtype D significantly progressed faster to CD4≤250 than subtype A cases; aHR (95% CI) = 1.78 (1.01-3.14). CONCLUSIONS:In this population, HIV-1 subtype D was the most prevalent and was associated with faster HIV-1 disease progression than subtype A. Further studies are needed to examine the effect of HIV-1 subtypes on disease progression in the ART period and their effect on the virological and immunological ART outcomes.

Pre-eclampsia/eclampsia usually resolves after delivery but sometimes hypertension persists and cardiovascular disease develops later. Our objective was to determine the incidence and maternal socio-demographic and obstetric risk factors for persistence of hypertension in women with pre-eclampsia/eclampsia.This was a prospective cohort study conducted from July 2009 to June 2011 at Mulago Hospital labour ward and postnatal clinics. We followed up 188 women admitted with pre-eclampsia/eclampsia until 3 months after delivery. Data was collected using interviewer-administered questionnaires, examination of participants and review of medical records. Stata (version12) software was used for data analysis. Univariable analysis was used to compute the relative risk of persistent hypertension at the 95% confidence level. This was followed by multivariable logistic regression analysis to determine factors independently associated with persistence of hypertension.64 (34%) out of the 188 women analysed had persistent hypertension three months after delivery. Maternal age, gestational age at delivery and parity were predictors of persistent hypertension.The proportion of women with pre-eclampsia/eclampsia at risk of persistent hypertension at three months after delivery was high, with nearly one of three mothers remaining hypertensive. Follow up of mothers who develop pre-eclampsia is important so that early diagnosis and management of chronic hypertension can be made to avoid long term morbidity and mortality.

OBJECTIVE:A minority of HIV-1 positive individuals treated with antiretroviral therapy (ART) in primary HIV-1 infection (PHI) maintain viral suppression on stopping. Whether this is related to ART duration has not been explored. DESIGN:And Methods: Using SPARTAC trial data from individuals recruited within 6 months of seroconversion, we present an observational analysis investigating whether duration of ART was associated with post-treatment viraemic control. Kaplan-Meier estimates, logistic regression and Cox models were used. RESULTS:165 participants reached plasma viral loads (VL) 12 weeks compared to ≤12 weeks (p=0.061). Cumulative probabilities of remaining 12 weeks. In multivariable regression, ART for >12 weeks was independently associated with a lower probability of being ≥400 copies/ml within 12 weeks of ART stop (OR=0.11 (95%CI=0.03,0.34), p

The CHAVI002 study was designed to characterize immune responses, particularly HIV-specific T-cell responses, amongst 2 cohorts of HIV-exposed seronegative (HESN) individuals. The absence of a clear definition of HESNs has impaired comparison of research within and between such cohorts. This report describes two distinct HESN cohorts and attempts to quantify HIV exposure using a 'HIV risk index' (RI) model.HIV serodiscordant couples (UK; 24, Uganda; 72) and HIV unexposed seronegative (HUSN) controls (UK; 14, Uganda; 26 couples, 3 individuals) completed sexual behavior questionnaires every 3 months over a 9 month period. The two cohorts were heterogeneous, with most HESNs in the UK men who have sex with men (MSM), while all HESNs in Uganda were in heterosexual relationships. Concordance of responses between partners was determined. Each participant's sexual behavior score (SBS) was estimated based on the number and type of unprotected sex acts carried out in defined time periods. Independent HIV acquisition risk factors (partner plasma viral load, STIs, male circumcision, pregnancy) were integrated with the SBS, generating a RI for each HESN.96 HIV serodiscordant couples completed 929 SBQs. SBSs remained relatively stable amongst the UK cohort, whilst decreasing from Visit 1 to 2 in the Ugandan cohort. Compared to the Ugandan cohort, SBSs and RIs in the UK cohort were lower at visit 1, and generally higher at later visits. Differences between the cohorts, with lower rates of ART use in Uganda and higher risk per-act sex in the UK, had major impacts on the SBSs and RIs of each cohort. There was one HIV transmission event in the UK cohort.Employment of a risk quantification model facilitated quantification and comparison of HIV acquisition risk across two disparate HIV serodiscordant couple cohorts.

The use of cellular coreceptors and modulation of cytokine concentrations by HIV to establish a productive infection is well documented. However, it is unknown whether the expression of these proteins affects the course of HIV clade A and D disease, reported to have different progression rates.We investigated whether the number of CD4(+) T-cells expressing CCR5 or CXCR4, the density of these coreceptors and concentrations of specific immune proteins linked to HIV pathogenesis vary between individuals infected with HIV clade A or D. We undertook additional analyses stratifying participants by early (CD4>500 cells/µl) or late (CD4

The Comprehensive T Cell Vaccine Immune Monitoring Consortium (CTC-VIMC) was created to provide standardized immunogenicity monitoring services for HIV vaccine trials. The ex vivo interferon-gamma (IFN-γ) ELISpot is used extensively as a primary immunogenicity assay to assess T cell-based vaccine candidates in trials for infectious diseases and cancer. Two independent, GCLP-accredited central laboratories of CTC-VIMC routinely use their own standard operating procedures (SOPs) for ELISpot within two major networks of HIV vaccine trials. Studies are imperatively needed to assess the comparability of ELISpot measurements across laboratories to benefit optimal advancement of vaccine candidates.We describe an equivalence study of the two independently qualified IFN-g ELISpot SOPs. The study design, data collection and subsequent analysis were managed by independent statisticians to avoid subjectivity. The equivalence of both response rates and positivity calls to a given stimulus was assessed based on pre-specified acceptance criteria derived from a separate pilot study.Detection of positive responses was found to be equivalent between both laboratories. The 95% C.I. on the difference in response rates, for CMV (-1.5%, 1.5%) and CEF (-0.4%, 7.8%) responses, were both contained in the pre-specified equivalence margin of interval [-15%, 15%]. The lower bound of the 95% C.I. on the proportion of concordant positivity calls for CMV (97.2%) and CEF (89.5%) were both greater than the pre-specified margin of 70%. A third CTC-VIMC central laboratory already using one of the two SOPs also showed comparability when tested in a smaller sub-study.The described study procedure provides a prototypical example for the comparison of bioanalytical methods in HIV vaccine and other disease fields. This study also provides valuable and unprecedented information for future vaccine candidate evaluations on the comparison and pooling of ELISpot results generated by the CTC-VIMC central core laboratories.

Clinical laboratory reference intervals have not been established in many African countries, and non-local intervals are commonly used in clinical trials to screen and monitor adverse events (AEs) among African participants. Using laboratory reference intervals derived from other populations excludes potential trial volunteers in Africa and makes AE assessment challenging. The objective of this study was to establish clinical laboratory reference intervals for 25 hematology, immunology and biochemistry values among healthy African adults typical of those who might join a clinical trial.Equal proportions of men and women were invited to participate in a cross sectional study at seven clinical centers (Kigali, Rwanda; Masaka and Entebbe, Uganda; two in Nairobi and one in Kilifi, Kenya; and Lusaka, Zambia). All laboratories used hematology, immunology and biochemistry analyzers validated by an independent clinical laboratory. Clinical and Laboratory Standards Institute guidelines were followed to create study consensus intervals. For comparison, AE grading criteria published by the U.S. National Institute of Allergy and Infectious Diseases Division of AIDS (DAIDS) and other U.S. reference intervals were used. 2,990 potential volunteers were screened, and 2,105 (1,083 men and 1,022 women) were included in the analysis. While some significant gender and regional differences were observed, creating consensus African study intervals from the complete data was possible for 18 of the 25 analytes. Compared to reference intervals from the U.S., we found lower hematocrit and hemoglobin levels, particularly among women, lower white blood cell and neutrophil counts, and lower amylase. Both genders had elevated eosinophil counts, immunoglobulin G, total and direct bilirubin, lactate dehydrogenase and creatine phosphokinase, the latter being more pronounced among women. When graded against U.S. -derived DAIDS AE grading criteria, we observed 774 (35.3%) volunteers with grade one or higher results; 314 (14.9%) had elevated total bilirubin, and 201 (9.6%) had low neutrophil counts. These otherwise healthy volunteers would be excluded or would require special exemption to participate in many clinical trials.To accelerate clinical trials in Africa, and to improve their scientific validity, locally appropriate reference ranges should be used. This study provides ranges that will inform inclusion criteria and evaluation of adverse events for studies in these regions of Africa.

Some HIV infected individuals remain asymptomatic for protracted periods of time in the absence of antiretroviral therapy (ART). Virological control, CD4 T cell loss and HIV-specific responses are some of the key interrelated determinants of HIV-1 disease progression. In this study, possible interactions between viral load, CD4 T cell slopes, host genetics and HIV-specific IFN-gamma responses were evaluated in chronically HIV-1-infected adults.Multilevel regression modeling was used to stratify clade A or D HIV-infected individuals into disease progression groups based on CD4 T cell slopes. ELISpot assays were used to quantify the frequency and magnitude of HIV-induced IFN-gamma responses in 7 defined rapid progressors (RPs) and 14 defined slow progressors (SPs) at a single time point. HLA typing was performed using reference strand conformational analysis (RSCA). Although neither the breadth nor the magnitude of the proteome-wide HIV-specific IFN-gamma response correlated with viral load, slow disease progression was associated with over-representation of host immunogenetic protective HLA B* alleles (10 of 14 SPs compared to 0 of 7; p = 0.004, Fisher's Exact) especially B*57 and B*5801, multiclade Gag T-cell targeting (71%, 10 of 14 SPs compared to 14%, 1 of 7 RPs); p = 0.029, Fisher's Exact test and evident virological control (3.65 compared to 5.46 log10 copies/mL in SPs and RPs respectively); p

An understanding of the health of potential volunteers in Africa is essential for the safe and efficient conduct of clinical trials, particularly for trials of preventive technologies such as vaccines that enroll healthy individuals. Clinical safety laboratory values used for screening, enrolment and follow-up of African clinical trial volunteers have largely been based on values derived from industrialized countries in Europe and North America. This report describes baseline morbidity during recruitment for a multi-center, African laboratory reference intervals study.Asymptomatic persons, aged 18-60 years, were invited to participate in a cross-sectional study at seven sites (Kigali, Rwanda; Masaka and Entebbe, Uganda; Kangemi, Kenyatta National Hospital and Kilifi, Kenya; and Lusaka, Zambia). Gender equivalency was by design. Individuals who were acutely ill, pregnant, menstruating, or had significant clinical findings were not enrolled. Each volunteer provided blood for hematology, immunology, and biochemistry parameters and urine for urinalysis. Enrolled volunteers were excluded if found to be positive for HIV, syphilis or Hepatitis B and C. Laboratory assays were conducted under Good Clinical Laboratory Practices (GCLP).Of the 2990 volunteers who were screened, 2387 (80%) were enrolled, and 2107 (71%) were included in the analysis (52% men, 48% women). Major reasons for screening out volunteers included abnormal findings on physical examination (228/603, 38%), significant medical history (76, 13%) and inability to complete the informed consent process (73, 13%). Once enrolled, principle reasons for exclusion from analysis included detection of Hepatitis B surface antigen (106/280, 38%) and antibodies against Hepatitis C (95, 34%). This is the first large scale, multi-site study conducted to the standards of GCLP to describe African laboratory reference intervals applicable to potential volunteers in clinical trials. Approximately one-third of all potential volunteers screened were not eligible for analysis; the majority were excluded for medical reasons.