Contribution of PEPFAR-Supported HIV and TB Molecular Diagnostic Networks to COVID-19 Testing Preparedness in 16 Countries.

The US President's Emergency Plan for AIDS Relief (PEPFAR) supports molecular HIV and tuberculosis diagnostic networks and information management systems in low- and middle-income countries. We describe how national programs leveraged these PEPFAR-supported laboratory resources for SARS-CoV-2 testing during the COVID-19 pandemic. We sent a spreadsheet template consisting of 46 indicators for assessing the use of PEPFAR-supported diagnostic networks for COVID-19 pandemic response activities during April 1, 2020, to March 31, 2021, to 27 PEPFAR-supported countries or regions. A total of 109 PEPFAR-supported centralized HIV viral load and early infant diagnosis laboratories and 138 decentralized HIV and TB sites reported performing SARS-CoV-2 testing in 16 countries. Together, these sites contributed to >3.4 million SARS-CoV-2 tests during the 1-year period. Our findings illustrate that PEPFAR-supported diagnostic networks provided a wide range of resources to respond to emergency COVID-19 diagnostic testing in 16 low- and middle-income countries.

Laboratory and field evaluation of the STANDARD Q and Panbio™ SARS-CoV-2 antigen rapid test in Namibia using nasopharyngeal samples.

BACKGROUND: As new SARS-CoV-2 variants of concern emerge, there is a need to scale up testing to minimize transmission of the Coronavirus disease 2019 (COVID-19). Many countries especially those in the developing world continue to struggle with scaling up reverse transcriptase polymerase reaction (RT-PCR) to detect SARS-CoV-2 due to scarcity of resources. Alternatives such as antigen rapid diagnostics tests (Ag-RDTs) may provide a solution to enable countries scale up testing. METHODS: In this study, we evaluated the Panbio™ and STANDARD Q Ag-RDTs in the laboratory using 80 COVID-19 RT-PCR confirmed and 80 negative nasopharyngeal swabs. The STANDARD Q was further evaluated in the field on 112 symptomatic and 61 asymptomatic participants. RESULTS: For the laboratory evaluation, both tests had a sensitivity above 80% (Panbio™ = 86% vs STANDARD Q = 88%). The specificity of the Panbio™ was 100%, while that of the STANDARD Q was 99%. When evaluated in the field, the STANDARD Q maintained a high specificity of 99%, however the sensitivity was reduced to 56%. CONCLUSION: Using Ag-RDTs in low resource settings will be helpful in scaling-up SARS-CoV-2 testing, however, negative results should be confirmed by RT-PCR where possible to rule out COVID-19 infection.

Pretreatment Human Immunodeficiency Virus (HIV) Drug Resistance Among Treatment-Naive Infants Newly Diagnosed With HIV in 2016 in Namibia: Results of a Nationally Representative Study.

Background: The World Health Organization (WHO) recommends routine surveillance of pretreatment human immunodeficiency virus (HIV) drug resistance (HIVDR) in children <18 months of age diagnosed with HIV through early infant diagnosis (EID). In 2016, 262 children <18 months of age were diagnosed with HIV in Namibia through EID. Levels of HIVDR in this population are unknown. Methods: In 2016, Namibia surveyed pretreatment HIVDR among children aged <18 months following WHO guidance. Reverse transcriptase, protease, and integrase regions of HIV-1 were genotyped from remnant dried blood spot specimens from all infants diagnosed with HIV in Namibia in 2016. HIVDR was predicted using the Stanford HIVdb algorithm. Results: Of 262 specimens genotyped, 198 HIV-1 protease and reverse transcriptase sequences and 118 HIV-1 integrase sequences were successfully amplified and analyzed. The prevalence of efavirenz/nevirapine (EFV/NVP), abacavir (ABC), zidovudine, lamivudine/emtricitabine (3TC/FTC), and tenofovir (TDF) resistance was 62.6%, 17.7%, 5.6%, 15.7%, and 10.1%, respectively. No integrase inhibitor resistance was detected. Conclusions: The high level of EFV/NVP resistance is unsurprising; however, levels of ABC and TDF resistance are among the highest observed to date in infants in sub-Saharan Africa. The absence of resistance to dolutegravir (DTG) is reassuring but underscores the need to further study the impact of ABC and 3TC/FTC resistance on pediatric protease inhibitor- and DTG-based regimens and accelerate access to other antiretroviral drugs. Results underscore the need for antiretroviral therapy optimization and prompt management of high viral loads in infants and pregnant and breastfeeding women.

Persistence of Positive RT-PCR Results for Over 70 Days in Two Travelers with COVID-19.

The relation of continuing to test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription real-time polymerase chain reaction (RT-PCR) to infectivity remains unclear, with numerous consequences. This report describes 2 patients with persistent viral detection by RT-PCR for 77 and 72 days, respectively, longer than other reported case-patients who were otherwise healthy.

High levels of HIV drug resistance among adults failing second-line antiretroviral therapy in Namibia.

To support optimal third-line antiretroviral therapy (ART) selection in Namibia, we investigated the prevalence of HIV drug resistance (HIVDR) at time of failure of second-line ART. A cross-sectional study was conducted between August 2016 and February 2017. HIV-infected people ≥15 years of age with confirmed virological failure while receiving ritonavir-boosted protease inhibitor (PI/r)-based second-line ART were identified at 15 high-volume ART clinics representing over >70% of the total population receiving second-line ART. HIVDR genotyping of dried blood spots obtained from these individuals was performed using standard population sequencing methods. The Stanford HIVDR algorithm was used to identify sequences with predicted resistance; genotypic susceptibility scores for potential third-line regimens were calculated. Two hundred thirty-eight individuals were enrolled; 57.6% were female. The median age and duration on PI/r-based ART at time of enrolment were 37 years and 3.46 years, respectively. 97.5% received lopinavir/ritonavir-based regimens. The prevalence of nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), and PI/r resistance was 50.6%, 63.1%, and 13.1%, respectively. No significant association was observed between HIVDR prevalence and age or sex. This study demonstrates high levels of NRTI and NNRTI resistance and moderate levels of PI resistance in people receiving PI/r-based second-line ART in Namibia. Findings underscore the need for objective and inexpensive measures of adherence to identify those in need of intensive adherence counselling, routine viral load monitoring to promptly detect virological failure, and HIVDR genotyping to optimize selection of third-line drugs in Namibia.

Human Immunodeficiency Virus-1 Drug Resistance Patterns Among Adult Patients Failing Second-Line Protease Inhibitor-Containing Regimens in Namibia, 2010-2015.

Three hundred sixty-six adult patients in Namibia with second-line virologic failures were evaluated for human immunodeficiency virus drug-resistant (HIVDR) mutations. Less than half (41.5%) harbored ≥1 HIVDR mutations to standardized second-line antiretroviral therapy (ART) regimen. Optimizing adherence, viral load monitoring, and genotyping are critical to prevent emergence of resistance, as well as unnecessary switching to costly third-line ART regimens.

Population-based surveillance of HIV drug resistance emerging on treatment and associated factors at sentinel antiretroviral therapy sites in Namibia.

OBJECTIVE: The World Health Organization (WHO) prospective surveys of acquired HIV drug resistance (HIVDR) evaluate HIVDR emerging after the first year of antiretroviral therapy (ART) and associated factors. METHODS: Consecutive ART starters in 2009 were enrolled at 3 sentinel sites in Namibia. Genotyping was performed at start and after 12 months in patients with HIV viral load (VL) >1000 copies per mL. HIVDR outcomes were: HIVDR prevention (VL ≤1000 copies/mL), possible HIVDR (VL >1000 copies/mL without detectable HIVDR or loss to follow-up or ART stop), and HIVDR (VL >1000 copies/mL with detectable HIVDR). Adherence was assessed using medication possession ratio (MPR). RESULTS: Of 394 starters, at 12 months, 80% were on first-line ART, 1% died, 4% transferred out, 1% stopped ART, <1% switched to second-line, and 15% were lost to follow-up. Among patients on first-line, 77% had VL testing, and 94% achieved VL ≤1000 copies per mL. At baseline, 7% had HIVDR. After 12 months, among patients with VL testing, 5% had HIVDR. A majority of patients failing therapy had high-level resistance to nonnucleoside reverse transcriptase inhibitors but none to protease inhibitors. All sites achieved the WHO target of ≥70% HIVDR prevention. Factors associated with not achieving HIVDR prevention were: baseline resistance to nonnucleoside reverse transcriptase inhibitors [odds ratio (OR) 3.0, P = 0.023], WHO stage 3 or 4 at baseline (OR 2.0, P = 0.012), and MPR <75% (OR 4.9, P = 0.021). CONCLUSIONS: Earlier ART initiation and removal of barriers to on-time drug pickups may help to prevent HIVDR. These data inform decisions at national and global levels on the effectiveness of first- and second-line regimens.

Limited utility of dried-blood- and plasma spot-based screening for antiretroviral treatment failure with Cobas Ampliprep/TaqMan HIV-1 version 2.0.

The 2013 WHO antiretroviral therapy (ART) guidelines recommend dried blood spots (DBS) as an alternative specimen type for viral load (VL) monitoring. We assessed the programmatic utility of screening for antiretroviral (ARV) treatment failure (TF) at 5,000 and 1,000 copies/ml using DBS and dried plasma spots (DPS) with a commonly used VL assay, the Roche Cobas Ampliprep/Cobas TaqMan V.2.0 (CAP/CTM). Plasma, DBS, and DPS were prepared from 839 whole-blood specimens collected from patients on ART for ≥ 6 months at three public facilities in Namibia. Using the CAP/CTM test, VL were measured in plasma, DBS, and DPS, and the results were compared using the plasma VL as the reference standard. The clinical sensitivities, specificities, and positive (PPV) and negative predictive values (NPV) of DBS at ARV TF diagnostic thresholds of 5,000 copies/ml and 1,000 copies/ml were 0.99, 0.55, 0.33, and 0.99 and 0.99, 0.26, 0.29, and 0.99, respectively, and for DPS at TF diagnostic thresholds of 5,000 copies/ml and 1,000 copies/ml, they were 0.88, 0.98, 0.92, and 0.97 and 0.91, 0.96, 0.89, and 0.97, respectively. The prevalences of TF were overestimated in DBS by 33% and 57% at these two thresholds, respectively. A high rate of false-positive results would occur if the CAP/CTM with DBS were to be used to screen for ARV TF. WHO recommendations for DBS-based VL monitoring should be specific to the VL assay version and type. Despite the better performance of DPS, the programmatic utility for TF screening may be limited by requirements for processing the whole blood at the collection site.

Population-based monitoring of HIV drug resistance in Namibia with early warning indicators.

INTRODUCTION: HIV drug resistance (HIVDR) testing is not routinely available in many resource-limited settings, therefore, antiretroviral therapy (ART) program and site factors known to be associated with HIVDR should be monitored to optimize the quality of patient care and minimize the emergence of preventable HIVDR. METHODS: In 2009, Namibia selected 5 World Health Organization Early Warning Indicators (EWIs) and piloted abstraction at 9 ART sites: "ART prescribing practices, patients lost to follow-up at 12 months, patient retention on first-line ART at 12 months, on-time antiretroviral drug pick-up, and antiretroviral drug-supply continuity". RESULTS: Records supported monitoring of 3 of 5 selected EWIs. Nine of 9 (100%) sites met the target of 100% initiated on appropriate first-line regimens. Eight of 9 (89%) sites met the target of ≤20% lost to follow-up, although 20.8% of ART starters (range: 4.6%-44.6%) had a period of absence without documented ART coverage of 2.3 months (range: 1.5-3.9 months). Six of 9 (67%) sites met the target of 0% switched to a second-line regimen. CONCLUSIONS: EWI monitoring directly resulted in public health action which will optimize the quality of care, specifically the strengthening of ART record systems permitting monitoring of 5 EWIs in future years and protocols for improved ART patient defaulter tracing.

Heterologous expression of the benzoate para-hydroxylase encoding gene (CYP53B1) from Rhodotorula minuta by Yarrowia lipolytica.

There is currently an increasing number of cytochrome P450 (CYP450) monooxygenase encoding genes becoming available from various genome-sequencing projects. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. In this study, the CYP53B1 gene, which encodes a benzoate para-hydroxylase, was successfully cloned from Rhodotorula minuta and overexpressed in Yarrowia lipolytica E150. Multiple copies of the CYP53B1 cDNA were cloned under the POX2 promoter, while the Y. lipolytica CPR was cloned under the isocitrate lyase promoter. Whole cell biotransformation of benzoic acid to para-hydroxybenzoic acid (pHBA) was used to analyse the hydroxylase activity of the recombinant Y. lipolytica UOFS Y-2366. Different induction conditions were tested in shake flask cultures. The highest concentration of pHBA produced by UOFS Y-2366 was 1.6 g l(-1) after 200 h when stearic acid was repeatedly added to the media. R. minuta accumulated up to 1.8 g l(-1) of pHBA within only 24 h. Thus, the specific hydroxylase activity of Y. lipolytica UOFS Y-2366 [approximately 0.07 U (g dry wt.)(-1)] was about 30 times lower than the specific hydroxylase activity of R. minuta [2.62 U (g dry wt.)(-1)]. However, the hydroxylation activity obtained with Y. lipolytica was one of the highest hydroxylation activities thus reported for whole cell biotransformation studies carried out with yeasts expressing foreign CYP450s.