Correction: Youth and healthcare workers' perspectives on the feasibility and acceptability of self-testing for HIV, Hepatitis and Syphilis among young people: Qualitative findings from a pilot study in Gaborone, Botswana.

[This corrects the article DOI: 10.1371/journal.pone.0288971.].

Sub-Saharan Africa preparedness and response to the COVID-19 pandemic: A perspective of early career African scientists.

Emerging highly transmissible viral infections such as SARS-CoV-2 pose a significant global threat to human health and the economy. Since its first appearance in December 2019 in the city of Wuhan, Hubei province, China, SARS-CoV-2 infection has quickly spread across the globe, with the first case reported on the African continent, in Egypt on February 14 th, 2020. Although the global number of COVID-19 infections has increased exponentially since the beginning of the pandemic, the number of new infections and deaths recorded in African countries have been relatively modest, suggesting slower transmission dynamics of the virus on the continent, a lower case fatality rate, or simply a lack of testing or reliable data. Notably, there is no significant increase in unexplained pneumonias or deaths on the continent which could possibly indicate the effectiveness of interventions introduced by several African governments. However, there has not yet been a comprehensive assessment of sub-Saharan Africa's (SSA) preparedness and response to the COVID-19 pandemic that may have contributed to prevent an uncontrolled outbreak so far. As a group of early career scientists and the next generation of African scientific leaders with experience of working in medical and diverse health research fields in both SSA and resource-rich countries, we present a unique perspective on the current public health interventions to fight COVID-19 in Africa. Our perspective is based on extensive review of the available scientific publications, official technical reports and announcements released by governmental and non-governmental health organizations as well as from our personal experiences as workers on the COVID-19 battlefield in SSA. We documented public health interventions implemented in seven SSA countries including Uganda, Kenya, Rwanda, Cameroon, Zambia, South Africa and Botswana, the existing gaps and the important components of disease control that may strengthen SSA response to future outbreaks.

Increased Prevalence of Liver Fibrosis and HIV Viremia among Patients with HIV, HBV, and Tuberculosis in Botswana.

People with concomitant human immunodeficiency virus (HIV) and tuberculosis (TB) have an increased risk of hepatotoxic reactions due to antiretroviral therapy (ART) and anti-TB therapy (ATT). Concomitant hepatitis B virus (HBV) in these patients may lead to poorer health outcomes. To assess liver enzyme levels and immune response in adults with HIV, HBV, and TB, data from 300 antiretroviral-naïve people living with HIV (PLWHIV) were analyzed. The prevalence of HIV/HBV (cHIV/HBV) and HIV/TB (cHIV/TB) was 28% (95% CI: 23.0-33.4) and 10% (95% CI: 6.8-14.0), respectively. HIV/HBV/TB (cHIV/HBV/TB) prevalence was 5.3% (95% CI: 3.1-8.5). There was a statistically significant difference between the groups of participants in HIV viral load (p = 0.004), hemoglobin levels (p = 0.025), and body mass index (p = 0.011). A larger proportion of cHIV/HBV/TB participants (37.5%) had an aspartate aminotransferase to platelet ratio index (APRI) score ≥0.5 (p = 0.013), a lower cutoff for significant liver fibrosis. Immunological non-responders (CD4+ T-cell count <20% gain and HIV viral load <400 copies/mL at 6 months) were observed in all groups except those with cHIV/TB. Our findings support the need to screen for infections that could cause excessive liver damage prior to ATT or ART initiation, such as HBV.

Immune Phenotype and Functionality of Mtb-Specific T-Cells in HIV/TB Co-Infected Patients on Antiretroviral Treatment.

The performance of host blood-based biomarkers for tuberculosis (TB) in HIV-infected patients on antiretroviral therapy (ART) has not been fully assessed. We evaluated the immune phenotype and functionality of antigen-specific T-cell responses in HIV positive (+) participants with TB (n = 12) compared to HIV negative (-) participants with either TB (n = 9) or latent TB infection (LTBI) (n = 9). We show that the cytokine profile of Mtb-specific CD4+ T-cells in participants with TB, regardless of HIV status, was predominantly single IFN-γ or dual IFN-γ/ TNFα. Whilst ESAT-6/CFP-10 responding T-cells were predominantly of an effector memory (CD27-CD45RA-CCR7-) profile, HIV-specific T-cells were mainly of a central (CD27+CD45RA-CCR7+) and transitional memory (CD27+CD45RA+/-CCR7-) phenotype on both CD4+ and CD8+ T-cells. Using receiving operating characteristic (ROC) curve analysis, co-expression of CD38 and HLA-DR on ESAT-6/CFP-10 responding total cytokine-producing CD4+ T-cells had a high sensitivity for discriminating HIV+TB (100%, 95% CI 70-100) and HIV-TB (100%, 95% CI 70-100) from latent TB with high specificity (100%, 95% CI 68-100 for HIV-TB) at a cut-off value of 5% and 13%, respectively. TB treatment reduced the proportion of Mtb-specific total cytokine+CD38+HLA-DR+ CD4+ T-cells only in HIV-TB (p = 0.001). Our results suggest that co-expression of CD38 and HLA-DR on Mtb-specific CD4+ T-cells could serve as a TB diagnosis tool regardless of HIV status.

High incidence of tuberculosis in the first year of antiretroviral therapy in the Botswana National antiretroviral therapy programme between 2011 and 2015.

OBJECTIVE: Tuberculosis (TB) remains one of the leading causes of mortality and morbidity among people living with HIV. We sought to estimate the incidence of TB in a national database of HIV-infected patients receiving antiretroviral therapy (ART) in Botswana. DESIGN: A retrospective analysis of HIV-infected adult patients (≥18years) who initiated ART between 2011 and 2015 in the Botswana ART program. METHODS: Multivariable analysis using Cox regression included sex, age, viral load and CD4 T-cell counts. RESULTS: Of 45 729 patients, with a median follow-up of 1.7 years Q1 : Q3, 0.5, 3.1), 1791 patients developed TB over a median of 1.5 years (Q1 : Q3, 0.3, 3.1) of follow-up (incidence rate 1.9 per 100 person-years; 95% CI 1.8-2.0). At baseline, the median CD4 T-cell count was 272 cells/µl (Q1, Q3 146, 403). The risk of TB was greatest within the first year of ART (incidence rate 2.9 per 100 person-years; 95% CI 2.7-3.1) and in patients with CD4 T-cell counts below 50 cells/µl (incidence rate 8.3/100 person-years; 95% CI 7.1-9.7). Patients with viral loads above 10 000 copies/ml at 3 months post-ART initiation had two times higher risk of TB, hazard ratio 2.5 (95% CI 1.8-2.3). CONCLUSION: We report a high incidence of TB within the first year of ART and in patients with advanced immunodeficiency. Improved screening strategies and virologic monitoring during this early period on ART, coupled with TB preventive treatment, will reduce the burden of TB.

Comparison of an in-house 'home-brew' and commercial ViroSeq integrase genotyping assays on HIV-1 subtype C samples.

BACKGROUND: Roll-out of Integrase Strand Transfer Inhibitors (INSTIs) such as dolutegravir for HIV combination antiretroviral therapy (cART) in sub-Saharan Africa necessitates the development of affordable HIV drug resistance (HIVDR) assays targeting the Integrase gene. We optimised and evaluated an in-house integrase HIV-1 drug resistance assay (IH-Int) and compared it to a commercially available assay, ViroSeq™ Integrase Genotyping kit (VS-Int) amongst HIV-1 clade C infected individuals. METHODS: We used 54 plasma samples from treatment naïve participants and one plasma sample from a patient failing INSTI based cART. Specimens were genotyped using both the VS-Int and IH-Int assays. Stanford HIV drug resistance database were used for integrase resistance interpretation. We compared the major and minor resistance mutations, pairwise nucleotide and amino-acid identity, costs and assay time. RESULTS: Among 55 specimens tested with IH-Int, 53 (96.4%) successfully amplified compared to 45/55 (81.8%) for the VS-Int assay. The mean nucleotide and amino acid similarity from 33 paired sequences was 99.8% (SD ± 0.30) and 99.8% (SD ± 0.39) for the IH-Int and VS-Int assay respectively. The reagent cost/sample were 32 USD and 147 USD for IH-Int and VS-Int assay, respectively. All sequenced samples were confirmed as HIV-1 subtype C. CONCLUSIONS: The IH-Int assay had a high amplification success rate and high concordance with the commercial assay. It is significantly cheaper compared to the commercial assay. Our assay has the needed specifications for routine monitoring of participants on Dolutegravir based regimens in Botswana.

Detection of Second Line Drug Resistance among Drug Resistant Mycobacterium Tuberculosis Isolates in Botswana.

The emergence and transmission of multidrug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (M.tb) strains is a threat to global tuberculosis (TB) control. The early detection of drug resistance is critical for patient management. The aim of this study was to determine the proportion of isolates with additional second-line resistance among rifampicin and isoniazid resistant and MDR-TB isolates. A total of 66 M.tb isolates received at the National Tuberculosis Reference Laboratory between March 2012 and October 2013 with resistance to isoniazid, rifampicin or both were analyzed in this study. The genotypes of the M.tb isolates were determined by spoligotyping and second-line drug susceptibility testing was done using the Hain Genotype MTBDRsl line probe assay version 2.0. The treatment outcomes were defined according to the Botswana national and World Health Organization (WHO) guidelines. Of the 57 isolates analyzed, 33 (58%) were MDR-TB, 4 (7%) were additionally resistant to flouroquinolones and 3 (5%) were resistant to both fluoroquinolones and second-line injectable drugs. The most common fluoroquinolone resistance-conferring mutation detected was gyrA A90V. All XDR-TB cases remained smear or culture positive throughout the treatment. Our study findings indicate the importance of monitoring drug resistant TB cases to ensure rapid detection of second-line drug resistance.

Brief Report: High Rates of Adverse Birth Outcomes in HIV and Syphilis Coinfected Women in Botswana.

BACKGROUND: Little is known about the combined impact of HIV/syphilis coinfection on birth outcomes. METHODS: Antenatal HIV and syphilis test results, obstetric history, and infant birth outcomes were collected from obstetric records in maternity wards in Botswana between 2008 and 2011 (5 sites) and 2014 and 2016 (8 sites). We used logistic regression to compare adverse birth outcomes by HIV and syphilis status. Outcomes included stillbirth, preterm delivery, low birth weight, and in-hospital neonatal death. RESULTS: Of 76,466 women, 75,770 (99.1%) had HIV test results, and 20,520 (27.1%) were HIV positive. Syphilis test results were available for 67,290 (88.0%), and 697 (1.0%) had reactive rapid plasma reagin. Among 692 women with syphilis and an HIV test result, 261 (37.7%) were coinfected. HIV-infected women were more likely to be infected with syphilis than HIV-uninfected women [odds ratio (OR) = 1.68; 95% confidence interval (CI): 1.44 to 1.96]. From 2008-2011 to 2014-2016, the proportion of women with syphilis remained constant (1.1% vs. 1.0%, P = 0.41), but HIV/syphilis coinfection declined from 45% to 27% (P < 0.0001). Stillbirth occurred in 5.8% of coinfected women, compared with 1.9% with no HIV/syphilis (OR = 3.09; 95% CI: 1.83 to 5.23); 3.4% with HIV alone (OR = 1.75; 95% CI: 1.03 to 2.97), or 3.7% with syphilis alone (OR = 1.58; 95% CI: 0.77 to 3.25). Low birth weight occurred in 24.1% of coinfected women, compared with 12.1% with no HIV/syphilis (OR 2.31; 95% CI: 1.74 to 3.08; 20% with HIV alone (OR = 1.27; 95% CI: 0.96 to 1.69); or 14.6% with syphilis alone (OR = 1.85; 95% CI: 1.26 to 2.74). CONCLUSIONS: Although HIV/syphilis coinfection in pregnancy has declined in the past decade, coinfection was associated with adverse birth outcomes.

Genetic diversity of Mycobacterium tuberculosis strains circulating in Botswana.

BACKGROUND: Molecular typing of Mycobacterium tuberculosis (M.tb) isolates can inform Tuberculosis (TB) control programs on the relative proportion of transmission driving the TB epidemic. There is limited data on the M. tb genotypes that are circulating in Botswana. The aim of this study was to generate baseline data on the genetic diversity of M.tb isolates circulating in the country. METHODS: A total of 461 M.tb isolates received at the Botswana National Tuberculosis Reference Laboratory between March 2012 and October 2013 were included in this study. Drug susceptibility testing was conducted using the BD BACTEC MGIT 960 System. M.tb strains were genotyped using spoligotyping and spoligotype patterns were compared with existing patterns in the SITVIT Web database. A subset of drug resistant isolates which formed spoligo clusters (n = 65) was additionally genotyped with 12-loci MIRU. Factors associated with drug resistance and clustering were evaluated using logistic regression. RESULTS: Of the 461 isolates genotyped, 458 showed 108 distinct spoligotype patterns. The predominant M.tb lineages were Lineage 4 (81.9%), Lineage 2 (9%) and Lineage 1 (7.2%). The predominant spoligotype families within Lineage 4 were LAM (33%), S (14%), T (16%), X (16%). Three hundred and ninety-two (86%) isolates could be grouped into 44 clusters (2-46 isolates per cluster); giving a clustering rate of 76%. We identified 173 (37.8%) drug resistant isolates, 48 (10.5%) of these were multi-drug resistant. MIRU typing of the drug resistant isolates allowed grouping of 46 isolates into 14 clusters, giving a clustering rate of 49.2%. There was no association between age, sex, treatment category, region and clustering. CONCLUSIONS: This study highlights the complexity of the TB epidemic in Botswana with multiple strains contributing to disease and provides baseline data on the population structure of M.tb strains in Botswana.

Effect of Xpert MTB/RIF on clinical outcomes in routine care settings: individual patient data meta-analysis.

BACKGROUND: Xpert MTB/RIF, the most widely used automated nucleic acid amplification test for tuberculosis, is available in more than 130 countries. Although diagnostic accuracy is well documented, anticipated improvements in patient outcomes have not been clearly identified. We performed an individual patient data meta-analysis to examine improvements in patient outcomes associated with Xpert MTB/RIF. METHODS: We searched PubMed, Embase, ClinicalTrials.gov, and the Pan African Clinical Trials Registry from inception to Feb 1, 2018, for randomised controlled trials (RCTs) comparing the use of Xpert MTB/RIF with sputum smear microscopy as tests for tuberculosis diagnosis in adults (aged 18 years or older). We excluded studies of patients with extrapulmonary tuberculosis, and studies in which mortality was not assessed. We used a two-stage approach for our primary analysis and a one-stage approach for the sensitivity analysis. To assess the primary outcome of cumulative 6-month all-cause mortality, we first performed logistic regression models (random effects for cluster randomised trials, with robust SEs for multicentre studies) for each trial, and then pooled the odds ratio (OR) estimates by a fixed-effects (inverse variance) or random-effects (Der Simonian Laird) meta-analysis. We adjusted for age and gender, and stratified by HIV status and previous tuberculosis-treatment history. The study protocol has been registered with PROSPERO, number CRD42014013394. FINDINGS: Our search identified 387 studies, of which five RCTs were eligible for analysis. 8567 adult clinic attendees (4490 [63·5%] of 7074 participants for whom data were available were HIV-positive) were tested for tuberculosis with Xpert MTB/RIF (Xpert group) versus sputum smear microscopy (sputum smear group), across five low-income and middle-income countries (South Africa, Brazil, Zimbabwe, Zambia, and Tanzania). The primary outcome (reported in three studies) occurred in 182 (4·5%) of 4050 patients in the Xpert group and 217 (5·3%) of 4093 patients in the smear group (pooled adjusted OR 0·88, 95% CI 0·68-1·14 [p=0·34]; for HIV-positive individuals OR 0·83, 0·65-1·05 [p=0·12]). Kaplan-Meier estimates showed a lower rate of death (12·73 per 100 person-years in the Xpert group vs 16·38 per 100 person-years in the sputum smear group) for HIV-positive patients (hazard ratio 0·76, 95% CI 0·60-0·97; p=0·03). The risk of bias was assessed as reasonable and the statistical heterogeneity across studies was low (I2<20% for the primary outcome). INTERPRETATION: Despite individual patient data analysis from five RCTs, we were unable to confidently rule in nor rule out an Xpert MTB/RIF-associated reduction in mortality among outpatients tested for tuberculosis. Reduction in mortality among HIV-positive patients in a secondary analysis suggests the possibility of population-level impact. FUNDING: US National Institutes of Health.

Cross-sectional estimates revealed high HIV incidence in Botswana rural communities in the era of successful ART scale-up in 2013-2015.

BACKGROUND: Botswana is close to reaching the UNAIDS "90-90-90" HIV testing, antiretroviral treatment (ART), and viral suppression goals. We sought to determine HIV incidence in this setting with both high HIV prevalence and high ART coverage. METHODS: We used a cross-sectional approach to assessing HIV incidence. A random, population-based sample of adults age 16-64 years was enrolled in 30 rural and peri-urban communities as part of the Botswana Combination Prevention Project (BCPP), from October 2013 -November 2015. Data and samples from the baseline household survey were used to estimate cross-sectional HIV incidence, following an algorithm that combined Limiting-Antigen Avidity Assay (LAg-Avidity EIA), ART status (documented or by testing ARV drugs in plasma) and HIV-1 RNA load. The LAg-Avidity EIA cut-off normalized optical density (ODn) was set at 1.5. The HIV-1 RNA cut-off was set at 400 copies/mL. For estimation purposes, the Mean Duration of Recent Infection was 130 days and the False Recent Rate (FRR) was evaluated at values of either 0 or 0.39%. RESULTS: Among 12,610 individuals participating in the baseline household survey, HIV status was available for 12,570 participants and 3,596 of them were HIV positive. LAg-Avidity EIA data was generated for 3,581 (99.6%) of HIV-positive participants. Of 326 participants with ODn ≤1.5, 278 individuals were receiving ART verified through documentation and were considered to represent longstanding HIV infections. Among the remaining 48 participants who reported no use of ART, 14 had an HIV-1 RNA load ≤400 copies/mL (including 3 participants with ARVs in plasma) and were excluded, as potential elite/viremic controllers or undisclosed ART. Thus, 34 LAg-Avidity-EIA-recent, ARV-naïve individuals with detectable HIV-1 RNA (>400 copies/mL) were classified as individuals with recent HIV infections. The annualized HIV incidence among 16-64 year old adults was estimated at 1.06% (95% CI 0.68-1.45%) with zero FRR, and at 0.64% (95% CI 0.24-1.04%) using a previously defined FRR of 0.39%. Within a subset of younger individuals 16-49 years old, the annualized HIV incidence was estimated at 1.29% (95% CI 0.82-1.77%) with zero FRR, and at 0.90% (95% CI 0.42-1.38%) with FRR set to 0.39%. CONCLUSIONS: Using a cross-sectional estimate of HIV incidence from 2013-2015, we found that at the time of near achievement of the UNAIDS 90-90-90 targets, ~1% of adults (age 16-64 years) in Botswana's rural and peri-urban communities became HIV infected annually.

Correction: Immunological non-response and low hemoglobin levels are predictors of incident tuberculosis among HIV-infected individuals on Truvada-based therapy in Botswana.

[This corrects the article DOI: 10.1371/journal.pone.0192030.].

HIV disease is associated with increased biomarkers of endothelial dysfunction despite viral suppression on long-term antiretroviral therapy in Botswana.

BACKGROUND: Untreated HIV infection is associated with increased biomarkers of endothelial dysfunction. However, the predictors and degree of endothelial dysfunction among virally suppressed HIV-infected adults on long-term antiretroviral therapy (ART) have not been well studied in sub-Saharan Africa (SSA). METHODS: We enrolled 112 HIV-infected adults with virological suppression on long-term ART and 84 HIV-uninfected controls in Botswana. We measured plasma levels of markers of endothelial injury [soluble vascular adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and E-selectin] and plasma levels of biomarkers of inflammation [interleukin 6 (IL-6)] and monocyte activation (sCD163). Baseline traditional cardiovascular disease (CVD) risk factors and bilateral common carotid intima-media thickness (cIMT) were also available for all participants. We assessed whether HIV status (despite virological suppression on ART) was associated with biomarkers of endothelial dysfunction after controlling for traditional CVD risk factors in linear regression models. We additionally assessed the association between IL-6, sCD163 and cIMT with endothelial dysfunction in separate multivariate linear regression models, controlling for cIMT, among virally suppressed HIV-infected participants only. RESULTS: In multivariate analysis, HIV infection was significantly associated with increased VCAM-1 (p < 0.01) and ICAM-1 (p = 0.03) but not E-selectin (p = 0.74) levels. Within the HIV-positive group, higher sCD163 levels were associated with decreased ICAM-1 and E-selectin (p < 0.01 and p = 0.01, respectively) but not VCAM-1 (p = 0.13) levels. IL-6 was not associated with any of the biomarkers of endothelial dysfunction. CONCLUSION: HIV disease was associated with biomarkers of endothelial dysfunction among virally suppressed adults in Botswana on long-term ART after controlling for traditional CVD risk factors. Future work should explore the clinical impact of persistent endothelial dysfunction following long-term HIV viral suppression on the risk of CVD clinical endpoints among HIV-infected patients in this setting.

Immunological non-response and low hemoglobin levels are predictors of incident tuberculosis among HIV-infected individuals on Truvada-based therapy in Botswana.

BACKGROUND: There is a high burden of tuberculosis (TB) in HIV antiretroviral programmes in Africa. However, few studies have looked at predictors of incident TB while on Truvada-based combination antiretroviral therapy (cART) regimens. METHODS: We estimated TB incidence among individuals enrolled into an observational cohort evaluating the efficacy and tolerability of Truvada-based cART in Gaborone, Botswana between 2008 and 2011. We used Cox proportional hazards regressions to determine predictors of incident TB. RESULTS: Of 300 participants enrolled, 45 (15%) had a diagnosis of TB at baseline. During 428 person-years (py) of follow-up, the incidence rate of TB was 3.04/100py (95% CI, 1.69-5.06), with 60% of the cases occurring within 3 months of ART initiation. Incident cases had low baseline CD4+ T cell counts (153cells/mm3 [Q1, Q3: 82, 242]; p = 0.69) and hemoglobin levels (9.2g/dl [Q1, Q3: 8.5,10.1]; p<0.01). In univariate analysis, low BMI (HR = 0.73; 95% CI 0.58-0.91; p = 0.01) and hemoglobin levels <8 g/dl (HR = 10.84; 95%CI: 2.99-40.06; p<0.01) were risk factors for TB. Time to incident TB diagnosis was significantly reduced in patients with poor immunological recovery (p = 0.04). There was no association between baseline viral load and risk of TB (HR = 1.75; 95%CI: 0.70-4.37). CONCLUSION: Low hemoglobin levels prior to initiation of ART are significant predictors of incident tuberculosis. Therefore, there is potential utility of iron biomarkers to identify patients at risk of TB prior to initiation on ART. Furthermore, additional strategies are required for patients with poor immunological recovery to reduce excess risk of TB while on ART.

Mycobacterium tuberculosis Next-Generation Whole Genome Sequencing: Opportunities and Challenges.

Mycobacterium tuberculosis drug resistance is a threat to global tuberculosis (TB) control. Comprehensive and timely drug susceptibility determination is critical to inform appropriate treatment of drug-resistant tuberculosis (DR-TB). Phenotypic drug susceptibility testing (DST) is the gold standard for M. tuberculosis drug resistance determination. M. tuberculosis whole genome sequencing (WGS) has the potential to be a one-stop method for both comprehensive DST and epidemiological investigations. We discuss in this review the tremendous opportunities that next-generation WGS presents in terms of understanding the molecular epidemiology of tuberculosis and mechanisms of drug resistance. The potential clinical value and public health impact in the areas of DST for patient management and tracing of transmission chains for timely public health intervention are also discussed. We present the current challenges for the implementation of WGS in low and middle-income settings. WGS analysis has already been adapted routinely in laboratories to inform patient management and public health interventions in low burden high-income settings such as the United Kingdom. We predict that the technology will be adapted similarly in high burden settings where the impact on the epidemic will be greatest.

Brief Report: High Sensitivity and Specificity of the Cepheid Xpert HIV-1 Qualitative Point-of-Care Test Among Newborns in Botswana.

BACKGROUND: HIV point-of-care (POC) testing allows for early infant HIV diagnosis and treatment, but POC accuracy at birth and in the setting of antiretroviral prophylaxis for the prevention of mother-to-child HIV transmission is unknown. METHODS: We evaluated the Cepheid Xpert HIV-1 Qual POC test against the Roche Taqman HIV-1 DNA polymerase chain reaction (PCR) platform using dried blood spots from 15 HIV-infected and 75 HIV-exposed uninfected newborns. These infants were screened for HIV at <96 hours of life at 5 hospital maternity wards in Botswana; all infants received postexposure antiretroviral prophylaxis with single-dose nevirapine and zidovudine, and most mothers received 3-drug antiretroviral therapy in pregnancy and at delivery. RESULTS: Fourteen of the 15 PCR positive samples tested positive by Cepheid POC, yielding a sensitivity of 93.3% (95% confidence interval: 68.1 to 99.8). Baseline viral load among positive infants ranged from <40 to >10,000,000 copies/mL, with a median of 2403 copies/mL. The HIV RNA for the infant with false-negative POC testing was 1661 copies/mL. Of note, 2 infants with low HIV RNA (<40 and 272 copies/mL) were correctly identified as HIV positive by Cepheid POC. All the 75 PCR-negative samples tested negative by Cepheid POC, yielding a specificity of 100% (95% confidence interval: 96.1 to 100). DISCUSSION: Our study demonstrates high sensitivity and specificity for the Cepheid POC assay in the first week of life despite early infection and antiretroviral prophylaxis. This platform may be a useful approach for adding early infant HIV diagnosis to current testing programs.

Short Communication: Low False Recent Rate of Limiting Antigen-Avidity Assay Combined with HIV-1 RNA Data in Botswana.

Cross-sectional estimation of HIV incidence could misclassify some established or chronic HIV infections as recent. Usually long-term nonprogressors, elite and viremic controllers, and individuals on ART contribute to misclassification. Local data on the false recent rate (FRR) could minimize misclassification during estimation of HIV incidence. To improve monitoring of HIV incidence, we estimated local FRR in Botswana. A total of 1,036 specimens from individuals infected for at least 1.5-2 years were sampled between 2004 and 2009 and tested using the limiting antigen (LAg)-avidity assay using a cutoff of 1.5 normalized optical density units. The FRR was 0.97% (10/1,036; 95% confidence interval [CI] 0.46-1.77). Four samples had HIV-1 RNA >1,000 cps/ml, giving an adjusted FRR of 0.39% (4/1,036; 95% CI 0.11-0.99). A combination of LAg and HIV-1 RNA load data resulted in FRR below 1% in the Botswana population.

Point-of-Care Cepheid Xpert HIV-1 Viral Load Test in Rural African Communities Is Feasible and Reliable.

Routine monitoring of HIV-1 RNA or viral load (VL) in patients on antiretroviral therapy (ART) is important, but there are multiple impediments to VL testing in resource-constrained settings. An accurate point-of-care (POC) HIV-1 VL test could alleviate many of these challenges. We compared the performance of the Cepheid Xpert HIV-1 VL assay against the laboratory-based Abbott m2000sp/m2000rt assay (Abbott assay). ART-naive individuals participating in the Botswana Combination Prevention Project in 20 communities provided EDTA-blood specimens during household surveys. Both the POC Xpert HIV-1 VL and Abbott assays were performed on specimens sampled from 277 individuals. We found a high correlation between the Xpert HIV-1 VL and Abbott assay results (r2 = 0.92; P < 0.001). The overall mean difference in the HIV-1 RNA values obtained by Xpert HIV-1 VL assay and Abbott assay was 0.34 log10 copies/ml (95% confidence interval [CI], 0.26 to 0.40 log10 copies/ml) (P < 0.001). Using a clinically relevant level of 1,000 copies/ml as a threshold, agreement was 90.6% (95% CI, 87.9 to 93.1%), with a sensitivity of 98.6% (95% CI, 97.2 to 100%). The two methods agreed on their detectability of HIV-1 RNA (>40 copies/ml) at 97.1% (95% CI, 95.5 to 98.7%), with a sensitivity of 99.6% (95% CI, 97.2 to 100%). The POC Cepheid Xpert HIV-1 VL assay showed high agreement and accuracy with a laboratory-based method of HIV-1 RNA testing. The POC Xpert HIV-1 VL assay tended to overestimate HIV-1 VL, although the difference was below a clinically relevant threshold of 0.5 log10 copies/ml. The POC Cepheid Xpert HIV-1 VL assay is a promising tool for monitoring patients on ART in southern Africa.

Prevalence of Extensively Drug Resistant Tuberculosis among Archived Multidrug Resistant Tuberculosis Isolates in Zimbabwe.

We conducted a cross-sectional study of second line drug resistance patterns and genetic diversity of MDR-TB isolates archived at the BRTI-TB Laboratory, Harare, between January 2007 and December 2011. DSTs were performed for second line antituberculosis drugs. XDR-TB strains were defined as MDR-TB strains with resistance to either kanamycin and ofloxacin or capreomycin and ofloxacin. Strain types were identified by spoligotyping. No resistance to any second line drugs was shown in 73% of the isolates, with 23% resistant to one or two drugs but not meeting the definition of XDR-TB. A total of 26 shared types were identified, and 18 (69%) matched preexisting shared types in the current published spoligotype databases. Of the 11 out of 18 clustered SITs, 4 predominant (>6 isolates per shared type) were identified. The most and least abundant types were SIT 1468 (LAM 11-ZWE) with 12 (18%) isolates and SIT 53 (T1) with 6 (9%) isolates, respectively. XDR-TB strains are rare in Zimbabwe, but the high proportion of "pre-XDR-TB" strains and treatment failure cases is of concern. The genetic diversity of the MDR-TB strains showed no significant association between SITs and drug resistance.

Microscopic-observation drug-susceptibility assay for the diagnosis of drug-resistant tuberculosis in Harare, Zimbabwe.

INTRODUCTION: Limited data exist on use of the microscopic-observation drug-susceptibility (MODS) assay among persons suspected of MDR-TB living in high HIV-prevalence settings. METHODS: We retrospectively reviewed available clinical and drug susceptibility data for drug-resistant TB suspects referred for culture and drug-susceptibility testing between April 1, 2011 and March 1, 2012. The diagnostic accuracy of MODS was estimated against a reference standard including Löwenstein-Jensen (LJ) media and manual liquid (BACTEC MGIT) culture. The accuracy of MODS drug-susceptibility testing (DST) was assessed against a reference standard absolute concentration method. RESULTS: One hundred thirty-eight sputum samples were collected from 99 drug-resistant TB suspects; in addition, six previously cultured MDR isolates were included for assessment of DST accuracy. Among persons with known HIV infection status, 39/59 (66%) were HIV-infected. Eighty-six percent of patients had a history of prior TB treatment, and 80% of individuals were on antituberculous treatment at the time of sample collection. M. tuberculosis was identified by reference standard culture among 34/98 (35%) MDR-TB suspects. Overall MODS sensitivity for M. tuberculosis detection was 85% (95% CI, 69-95%) and specificity was 93% (95% CI, 84-98%); diagnostic accuracy did not significantly differ by HIV infection status. Median time to positivity was significantly shorter for MODS (7 days; IQR 7-15 days) than MGIT (12 days; IQR 6-16 days) or LJ (28 days; IQR 21-35 days; p<0.001). Of 33 specimens with concurrent DST results, sensitivity of the MODS assay for detection of resistance to isoniazid, rifampin, and MDR-TB was 88% (95% CI, 68-97%), 96% (95% CI, 79-100%), and 91% (95% CI, 72-99%), respectively; specificity was 89% (95% CI, 52-100%), 89% (95% CI, 52-100%), and 90% (95% CI, 56-100%), respectively. CONCLUSION: In a high HIV-prevalence setting, MODS diagnosed TB and drug-resistant TB with high sensitivity and shorter turnaround time compared with standard culture and DST methods.