Accuracy of point-of-care HIV and CD4 field testing by lay healthcare workers in the Botswana Combination Prevention Project.

Accurate HIV and CD4 testing are critical in program implementation, with HIV misdiagnosis having serious consequences at both the client and/or community level. We implemented a comprehensive training and Quality Assurance (QA) program to ensure accuracy of point-of-care HIV and CD4 count testing by lay counsellors during the Botswana Combination Prevention Project (BCPP). We compared the performance of field testing by lay counsellors to results from an accredited laboratory to ascertain accuracy of testing. All trained lay counsellors passed competency assessments and performed satisfactorily in proficiency testing panel evaluations in 2013, 2014, and 2015. There was excellent agreement (99.6 %) between field and laboratory-based HIV test results; of the 3002 samples tested, 960 and 2030 were concordantly positive and negative respectively, with 12 misclassifications (kappa score 0.99, p < 0.0001). Of the 149 HIV-positive samples enumerated for CD4 count in the field using PIMA at a threshold of ≤ 350 cells/µl; there was 86 % agreement with laboratory testing, with only 21 misclassified. The mean difference between field and lab CD4 testing was - 16.16 cells/µl (95 % CI -5.4 to 26.9). Overall, there was excellent agreement between field and laboratory results for both HIV rapid test and PIMA CD4 results. A standard training package to train lay counsellors to accurately perform HIV and CD4 point-of-care testing in field settings was feasible, with point-of-care results obtained by lay counsellors comparable to laboratory-based testing.

Doravirine-associated resistance mutations in antiretroviral therapy naïve and experienced adults with HIV-1 subtype C infection in Botswana.

OBJECTIVES: There are limited data on the prevalence of doravirine (DOR)-associated drug resistance mutations in people with HIV (PWH) in Botswana. This cross-sectional, retrospective study aimed to explore the prevalence of DOR-associated resistance mutations among ART-naïve and -experienced PWH in Botswana enrolled in the population-based Botswana Combination Prevention Project (BCPP). METHODS: A total of 6078 HIV-1C pol sequences were analysed for DOR-associated resistance mutations using the Stanford HIV drug resistance database, and their levels were predicted according to the Stanford DRM penalty scores and resistance interpretation. Virologic failure was defined as HIV-1 RNA load (VL) >400 copies/mL. RESULTS: Among 6078 PWH, 5999 (99%) had known ART status, and 4529/5999 (79%) were on ART at time of sampling. The suppression rate among ART-experienced was 4517/4729 (96%). The overall prevalence of any DOR-associated resistance mutations was 181/1473 (12.3% [95% confidence interval {CI}: 10.7-14.1]); by ART status: 42/212 (19.8% [95% CI: 14.7-25.4]) among ART-failing individuals (VL ≥400 copies/mL) and 139/1261 (11.0% [95% CI: 9.3-12.9]) among ART-naïve individuals (P < 0.01). Intermediate DOR-associated resistance mutations were observed in 106/1261 (7.8% [95% CI: 6.9-10.1]) in ART-naïve individuals and 29/212 (13.7% [95% CI: 9.4-8.5]) among ART-experienced participants (P < 0.01). High-level DOR-associated resistance mutations were observed in 33/1261 (2.6% [95% CI: 1.8-3.7]) among ART-naïve and 13/212 (6.1% [95% CI: 3.6-10.8]) among ART-failing PWH (P < 0.01). PWH failing ART with at least one EFV/NVP-associated resistance mutation had high prevalence 13/67 (19.4%) of high-level DOR-associated resistance mutations. CONCLUSION: DOR-associated mutations were rare (11.0%) among ART-naive PWH but present in 62.7% of Botswana individuals who failed NNRTI-based ART with at least one EFV/NVP-associated resistance mutation. Testing for HIV drug resistance should underpin the use of DOR in PWH who have taken first-generation NNRTIs.

Leveraging Partnerships to Maximize Global Health Security Improvements in Guinea, 2015-2019.

In response to the 2014-2016 West Africa Ebola virus disease (EVD) outbreak, a US congressional appropriation provided funds to the US Centers for Disease Control and Prevention (CDC) to support global health security capacity building in 17 partner countries, including Guinea. The 2014 funding enabled CDC to provide more than 300 deployments of personnel to Guinea during the Ebola response, establish a country office, and fund 11 implementing partners through cooperative agreements to support global health security engagement efforts in 4 core technical areas: workforce development, surveillance systems, laboratory systems, and emergency management. This article reflects on almost 4 years of collaboration between CDC and its implementing partners in Guinea during the Ebola outbreak response and the recovery period. We highlight examples of collaborative synergies between cooperative agreement partners and local Guinean partners and discuss the impact of these collaborations in strengthening the above 4 core capacities. Finally, we identify the key elements of the successful collaborations, including communication and information sharing as a core cooperative agreement activity, a flexible funding mechanism, and willingness to adapt to local needs. We hope these observations can serve as guidance for future endeavors seeking to establish strong and effective partnerships between government and nongovernment organizations providing technical and operational assistance.

Increasing knowledge of HIV status in a country with high HIV testing coverage: Results from the Botswana Combination Prevention Project.

INTRODUCTION: Achieving widespread knowledge of HIV-positive status is a crucial step to reaching universal ART coverage, population level viral suppression, and ultimately epidemic control. We implemented a multi-modality HIV testing approach to identify 90% or greater of HIV-positive persons in the Botswana Combination Prevention Project (BCPP) intervention communities. METHODS: BCPP is a cluster-randomized trial designed to evaluate the impact of combination prevention interventions on HIV incidence in 30 communities in Botswana. Community case finding and HIV testing that included home and targeted mobile testing were implemented in the 15 intervention communities. We described processes for identifying HIV-positive persons, uptake of HIV testing by age, gender and venue, characteristics of persons newly diagnosed through BCPP, and coverage of knowledge of status reached at the end of study. RESULTS: Of the 61,655 eligible adults assessed in home or mobile settings, 13,328 HIV-positive individuals, or 93% of the estimated 14,270 positive people in the communities were identified through BCPP. Knowledge of status increased by 25% over the course of the study with the greatest increases seen among men (37%) as compared to women (19%) and among youth aged 16-24 (77%) as compared to older age groups (21%). Although more men were tested through mobile than through home-based testing, higher rates of newly diagnosed HIV-positive men were found through home than mobile testing. CONCLUSIONS: Even when HIV testing coverage is high, additional gains can be made using a multi-modality HIV testing strategy to reach different sub-populations who are being missed by non-targeted program activities. Men and youth can be reached and will engage in community testing when services are brought to places they access routinely.

Creating a National Specimen Referral System in Guinea: Lessons From Initial Development and Implementation.

In the wake of the 2014-2016, West Africa Ebola virus disease (EVD) outbreak, the Government of Guinea recognized an opportunity to strengthen its national laboratory system, incorporating capacity and investments developed during the response. The Ministry of Health (MOH) identified creation of a holistic, safe, secure, and timely national specimen referral system as a priority for improved detection and confirmation of priority diseases, in line with national Integrated Disease Surveillance and Response guidelines. The project consisted of two parts, each led by different implementing partners working collaboratively together and with the Ministry of Health: the development and approval of a national specimen referral policy, and pilot implementation of a specimen referral system, modeled on the policy, in three prefectures. This paper describes the successful execution of the project, highlighting the opportunities and challenges of building sustainable health systems capacity during and after public health emergencies, and provides lessons learned for strengthening national capabilities for surveillance and disease diagnosis.

Assessing health systems in Guinea for prevention and control of priority zoonotic diseases: A One Health approach.

To guide One Health capacity building efforts in the Republic of Guinea in the wake of the 2014-2016 Ebola virus disease (EVD) outbreak, we sought to identify and assess the existing systems and structures for zoonotic disease detection and control. We partnered with the government ministries responsible for human, animal, and environmental health to identify a list of zoonotic diseases - rabies, anthrax, brucellosis, viral hemorrhagic fevers, trypanosomiasis and highly pathogenic avian influenza - as the country's top priorities. We used each priority disease as a case study to identify existing processes for prevention, surveillance, diagnosis, laboratory confirmation, reporting and response across the three ministries. Results were used to produce disease-specific systems "maps" emphasizing linkages across the systems, as well as opportunities for improvement. We identified brucellosis as a particularly neglected condition. Past efforts to build avian influenza capabilities, which had degraded substantially in less than a decade, highlighted the challenge of sustainability. We observed a keen interest across sectors to reinvigorate national rabies control, and given the regional and global support for One Health approaches to rabies elimination, rabies could serve as an ideal disease to test incipient One Health coordination mechanisms and procedures. Overall, we identified five major categories of gaps and challenges: (1) Coordination; (2) Training; (3) Infrastructure; (4) Public Awareness; and (5) Research. We developed and prioritized recommendations to address the gaps, estimated the level of resource investment needed, and estimated a timeline for implementation. These prioritized recommendations can be used by the Government of Guinea to plan strategically for future One Health efforts, ideally under the auspices of the national One Health Platform. This work demonstrates an effective methodology for mapping systems and structures for zoonotic diseases, and the benefit of conducting a baseline review of systemic capabilities prior to embarking on capacity building efforts.

Evaluation of dried blood spots collected on filter papers from three manufacturers stored at ambient temperature for application in HIV-1 drug resistance monitoring.

As more HIV-infected people gain access to antiretroviral therapy (ART), monitoring HIV drug resistance (HIVDR) becomes essential to combat both acquired and transmitted HIVDR. Studies have demonstrated dried blood spots (DBS) are a suitable alternative in HIVDR monitoring using DBS collected on Whatman 903 (W-903). In this study, we sought to evaluate two other commercially available filter papers, Ahlstrom 226 (A-226) and Munktell TFN (M-TFN), for HIVDR genotyping following ambient temperature storage. DBS were prepared from remnant blood specimens collected from 334 ART patients and stored at ambient temperature for a median time of 30 days. HIV-1 viral load was determined using NucliSENS EasyQ® HIV-1 v2.0 RUO test kits prior to genotyping of the protease and reverse transcriptase regions of the HIV-1 pol gene using an in-house assay. Among the DBS tested, 26 specimens had a viral load ≥ 1000 copies/mL in all three types of filter paper and were included in the genotyping analysis. Genotyping efficiencies were similar between DBS collected on W-903 (92.3%), A-226 (88.5%), and M-TFN (92.3%) filter papers (P = 1.00). We identified 50 DR-associated mutations in DBS collected on W-903, 33 in DBS collected on A-226, and 48 in DBS collected on M-TFN, resulting in mutation detection sensitivities of 66.0% for A-226 and 88.0% for M-TFN when compared to W-903. Our data indicate that differences among filter papers may exist at this storage condition and warrant further studies evaluating filter paper type for HIVDR monitoring.

Molecular characterization of ambiguous mutations in HIV-1 polymerase gene: implications for monitoring HIV infection status and drug resistance.

Detection of recent HIV infections is a prerequisite for reliable estimations of transmitted HIV drug resistance (t-HIVDR) and incidence. However, accurately identifying recent HIV infection is challenging due partially to the limitations of current serological tests. Ambiguous nucleotides are newly emerged mutations in quasispecies, and accumulate by time of viral infection. We utilized ambiguous mutations to establish a measurement for detecting recent HIV infection and monitoring early HIVDR development. Ambiguous nucleotides were extracted from HIV-1 pol-gene sequences in the datasets of recent (HIVDR threshold surveys [HIVDR-TS] in 7 countries; n=416) and established infections (1 HIVDR monitoring survey at baseline; n=271). An ambiguous mutation index of 2.04×10(-3) nts/site was detected in HIV-1 recent infections which is equivalent to the HIV-1 substitution rate (2×10(-3) nts/site/year) reported before. However, significantly higher index (14.41×10(-3) nts/site) was revealed with established infections. Using this substitution rate, 75.2% subjects in HIVDR-TS with the exception of the Vietnam dataset and 3.3% those in HIVDR-baseline were classified as recent infection within one year. We also calculated mutation scores at amino acid level at HIVDR sites based on ambiguous or fitted mutations. The overall mutation scores caused by ambiguous mutations increased (0.54×10(-2)3.48×10(-2)/DR-site) whereas those caused by fitted mutations remained stable (7.50-7.89×10(-2)/DR-site) in both recent and established infections, indicating that t-HIVDR exists in drug-naïve populations regardless of infection status in which new HIVDR continues to emerge. Our findings suggest that characterization of ambiguous mutations in HIV may serve as an additional tool to differentiate recent from established infections and to monitor HIVDR emergence.

Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis.

Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = -0.034 ± 0.246 log(10) copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = -0.028 ± 0.186 log(10) copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥ 3.00 log(10) copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.

Evaluation of blood collection filter papers for HIV-1 DNA PCR.

BACKGROUND: The collection of dried blood spots (DBS) on Whatman 903 cards has facilitated for years the detection of HIV-1 in infants by DNA PCR as early as 4-6 weeks after birth in resource-limited settings (RLS), but alternate blood collection devices are proving to be necessary. OBJECTIVES: The qualitative detection of HIV-1 DNA by PCR from DBS prepared on three commercially available blood collection cards was evaluated at the Centers for Disease Control and Prevention (CDC) and in four laboratories in Africa. STUDY DESIGN: DBS were prepared on Ahlstrom grade 226, Munktell TFN and Whatman 903, and stored under a variety of conditions. DBS were stored at ambient temperature (RT), 37°C with high humidity, and -20°C for varying lengths of time. The presence of HIV-1 DNA was tested using Roche Amplicor HIV-1 DNA (v 1.5) weekly for 4 weeks and at weeks 8 and 12 (RT and 37°C), at weeks 4, 8, and 18 (-20°C) of storage. DBS specimens were also tested after international shipment at RT. In addition, after nearly 3 years storage at -20°C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. RESULTS: HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. CONCLUSIONS: Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for HIV-1 DNA detection and may be considered as optional blood collection devices in resource-limited countries.

Prevalence of transmitted HIV drug resistance in Botswana: lessons learned from the HIVDR-Threshold Survey conducted among women presenting for routine antenatal care as part of the 2007 national sentinel survey.

The emergence and spread of transmitted drug resistance (TDR) poses a major threat to the success of the rapidly expanding antiretroviral treatment (ART) programs in resource-limited countries. The World Health Organization recommends the use of the HIV Drug Resistance Threshold Survey (HIVDR-TS) as an affordable means to monitor the presence of TDR in these settings. We report our experiences and results of the 2007 HIVDR-TS in Botswana, a country with one of the longest-existing national public ART programs in Africa. The HIVDR-TS and HIV-1 incidence testing were performed in the two largest national sites as part of the 2007 antenatal Botswana Sentinel Survey. The HIVDR-TS showed no significant drug resistance mutations (TDR less than 5%) in one site. TDR prevalence, however, could not be ascertained at the second site due to low sample size. The agreement between HIVDR-TS eligibility criteria and laboratory-based methodologies (i.e., BED-CEIA and LS-EIA) in identifying recently HIV-1 infected adults was poor. Five years following the establishment of Botswana's public ART program, the prevalence of TDR remains low. The HIVDR-TS methodology has limitations for low-density populations as in Botswana, where the majority of antenatal sites are too small to recruit sufficient numbers of patients. In addition, the eligibility criteria (age <25 years and parity (first pregnancy)) of the HIVDR-TS performed poorly in identifying recent HIV-1 infections in Botswana. An alternative sampling strategy should be considered for the surveillance of HIVDR in Botswana and similar geographic settings.

Ensuring the quality of HIV rapid testing in resource-poor countries using a systematic approach to training.

HIV rapid testing is a key tool in the fight against the HIV/AIDS epidemic; it enables the rapid expansion of prevention and treatment programs in resource-limited countries. Meeting the goals of these programs means that millions of people will need testing annually. Accuracy and reliability of these tests are critical to the success of these programs. Given the enormous number of rapid tests that are performed each year, even a low error rate of 0.5% applied to 100 million people will result in 500,000 erroneous results. Ensuring the quality of HIV rapid testing presents unique challenges in that testing is often performed in various settings by personnel without formal laboratory training. This article describes the development and implementation of a generic HIV rapid test training package using a systems approach in an effort to standardize training and ensure the quality of rapid tests. It also highlights achievements from Uganda, Haiti, and Botswana.

HIV type 1 drug resistance in adults receiving highly active antiretroviral therapy in Abidjan, Côte d'Ivoire.

As antiretroviral therapy continues to scale-up in developing countries, there is concern that high levels of HIV drug resistance to antiretroviral drugs will occur. Here we describe rates of emergence of HIV-1 drug resistance and factors associated with their occurrence among adults who received antiretroviral therapy (ART) for >1 year through the Côte d'Ivoire national drug access program from 1998 to 2003. To detect genotypic drug resistance, we sequenced all 1- and 2-year specimens with detectable HIV RNA viral load. To assess factors associated with emerging drug resistance, we used log normal regression with interval censoring, including covariates in the model for self-reported drug adherence, CD4 cell count, and HIV viral load at therapy initiation, and observed changes in these measures, type of prescribed ART drugs, diagnoses of opportunistic illness, and demographic characteristics. An estimated 14.2% [95% confidence limits (CL) 11.7, 16.9] and 26.6% (95% CL 22.7, 30.8) of patients developed primary drug-resistant mutations within 1 year and 2 years after initiation of therapy, respectively. Factors associated with drug resistance included drug nonadherence, partial or lack of viral suppression, higher viral load or lower CD4 at initiation of therapy, and initiation of ART with what is now considered substandard dual combination therapy. Our results demonstrate the need to strengthen adherence and continuity in treatment programs in order to avoid interruption of ART drugs. Treatment programs should pay attention to indicators of emerging drug resistance: incomplete or lesser decreases in viral load or increases in CD4 cell counts following initiation of therapy, and the occurrence of AIDS opportunistic illnesses.

Early diagnosis of human immunodeficiency virus in infants using polymerase chain reaction on dried blood spots in Botswana's national program for prevention of mother-to-child transmission.

BACKGROUND: Botswana has high antenatal human immunodeficiency virus (HIV) prevalence (33.4%). The public health system provides free services for prevention of mother to child transmission of HIV (PMTCT) and antiretroviral therapy, which can reduce vertical HIV transmission from 35% to <5%. Infant HIV diagnosis is challenging in resource-limited settings, and HIV prevalence among HIV-exposed infants in Botswana is unknown. Dried blood spot (DBS) polymerase chain reaction (PCR) provides a feasible method to assess PMTCT programs and identify HIV-infected children. METHODS: We trained staff in 15 clinics and a hospital to obtain DBS on HIV-exposed infants age 6 weeks to 17 months receiving routine care. Samples were sent to the national HIV reference laboratory. Roche Amplicor 1.5 DNA PCR testing was performed. RESULTS: Between June-December 2005, 1931 HIV-exposed infants age 6 weeks to 17 months were tested for HIV, of whom 136 (7.0%) were HIV infected. Among infants

Performance of drug-resistance genotypic assays among HIV-1 infected patients with predominantly CRF02_AG strains of HIV-1 in Abidjan, Cote d'Ivoire.

OBJECTIVE: We evaluated the performance of three genotypic assays to detect resistant mutations among HIV-1 infected patients with known antiretroviral drug resistance profile in Abidjan, Cote d'Ivoire, most of whom had the circulating recombinant form (CRF02_A/G) of HIV-1. METHODS: The 56 patients analyzed in this study were enrolled in a pilot program to make available antiretroviral therapy (ART) to HIV-infected patients in Abidjan through the UNAIDS Drug Access Initiative (DAI). These patients had failed ART, as demonstrated by rebound in RNA viral load. Their plasma samples had been previously analyzed for ART genotypic drug-resistance by VircoGEN (Mechelen, Belgium) and were known to have primary and secondary resistance mutations, and also had phenotypic drug-resistance by a recombinant virus assay technology (Mechelen, Belgium). The two assays we evaluated were: VircoGEN, TruGene HIV-1, and ViroSEQ HIV-1 assays. RESULTS: For the reverse transcriptase gene, all 27 samples that had the T215Y/F mutation were detected by VircoGEN , ViroSEQ, and TrueGene. All 19 (100%) samples that had the K70R/E mutation detected by VircoGEN were detected by ViroSEQ, and 18 (94.7%) by TrueGene. All ten samples with the M184V mutation, three with the K65R, two with the G190A mutation, one with the K103N mutation, and one with the V75T mutation were detected similarly by all three assays. For the protease gene, all three assays detected the I84V (n = 1), M46I (n = 1), and L90M (n = 1) mutations. CONCLUSION: These results suggest that any of these assays should be considered for monitoring the occurrence of drug resistance among HIV-infected patients receiving antiretroviral therapy in West Africa.