Retrospective longitudinal analysis of low-level viremia among HIV-1 infected adults on antiretroviral therapy in Kenya.

Background: HIV low-level viremia (LLV) (51-999 copies/mL) can progress to treatment failure and increase potential for drug resistance. We analyzed retrospective longitudinal data from people living with HIV (PLHIV) on antiretroviral therapy (ART) in Kenya to understand LLV prevalence and virologic outcomes. Methods: We calculated rates of virologic suppression (≤50 copies/mL), LLV (51-999 copies/mL), virologic non-suppression (≥1000 copies/mL), and virologic failure (≥2 consecutive virologic non-suppression results) among PLHIV aged 15 years and older who received at least 24 weeks of ART during 2015-2021. We analyzed risk for virologic non-suppression and virologic failure using time-dependent models (each viral load (VL) <1000 copies/mL used to predict the next VL). Findings: Of 793,902 patients with at least one VL, 18.5% had LLV (51-199 cp/mL 11.1%; 200-399 cp/mL 4.0%; and 400-999 cp/mL 3.4%) and 9.2% had virologic non-suppression at initial result. Among all VLs performed, 26.4% were LLV. Among patients with initial LLV, 13.3% and 2.4% progressed to virologic non-suppression and virologic failure, respectively. Compared to virologic suppression (≤50 copies/mL), LLV was associated with increased risk of virologic non-suppression (adjusted relative risk [aRR] 2.43) and virologic failure (aRR 3.86). Risk of virologic failure increased with LLV range (aRR 2.17 with 51-199 copies/mL, aRR 3.98 with 200-399 copies/mL and aRR 7.99 with 400-999 copies/mL). Compared to patients who never received dolutegravir (DTG), patients who initiated DTG had lower risk of virologic non-suppression (aRR 0.60) and virologic failure (aRR 0.51); similarly, patients who transitioned to DTG had lower risk of virologic non-suppression (aRR 0.58) and virologic failure (aRR 0.35) for the same LLV range. Interpretation: Approximately a quarter of patients experienced LLV and had increased risk of virologic non-suppression and failure. Lowering the threshold to define virologic suppression from <1000 to <50 copies/mL to allow for earlier interventions along with universal uptake of DTG may improve individual and program outcomes and progress towards achieving HIV epidemic control. Funding: No specific funding was received for the analysis. HIV program support was provided by the President's Emergency Plan for AIDS Relief (PEPFAR) through the United States Centers for Disease Control and Prevention (CDC).

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.

Enhancing accreditation outcomes for medical laboratories on the Strengthening Laboratory Management Toward Accreditation programme in Kenya via a rapid results initiative.

Background: Since 2010, Kenya has used SLIPTA to prepare and improve quality management systems in medical laboratories to achieve ISO 15189 accreditation. However, less than 10% of enrolled laboratories had done so in the initial seven years of SLMTA implementation. Objective: We described Kenya's experience in accelerating medical laboratories on SLMTA to attain ISO 15189 accreditation. Methods: From March 2017 to July 2017, an aggressive top-down approach through high-level management stakeholder engagement for buy-in, needs-based expedited SLIPTA mentorship and on-site support as a rapid results initiative (RRI) was implemented in 39 laboratories whose quality improvement process had stagnated for 2-7 years. In July 2017, SLIPTA baseline and exit audit average scores on quality essential elements were compared to assess performance. Results: After RRI, laboratories achieving greater than a 2-star SLMTA rating increased significantly from 15 (38%) at baseline to 33 (85%) (p < 0.001). Overall, 34/39 (87%) laboratories received ISO 15189 accreditation within two years of RRI, leading to a 330% increase in the number of accredited laboratories in Kenya. The most improved of the 12 quality system essentials were Equipment Management (mean increase 95% CI: 5.31 ± 1.89) and Facilities and Biosafety (mean increase [95% CI: 4.05 ± 1.78]) (both: p < 0.0001). Information Management and Corrective Action Management remained the most challenging to improve, despite RRI interventions. Conclusion: High-level advocacy and targeted mentorship through RRI dramatically improved laboratory accreditation in Kenya. Similar approaches of strengthening SLIPTA implementation could improve SLMTA outcomes in other countries with similar challenges.

HIV Viral Load Monitoring Among Patients Receiving Antiretroviral Therapy - Eight Sub-Saharan Africa Countries, 2013-2018.

One component of the Joint United Nations Programme on HIV/AIDS (UNAIDS) goal to end the HIV/AIDS epidemic by 2030, is that 95% of all persons receiving antiretroviral therapy (ART) achieve viral suppression.† Thus, testing all HIV-positive persons for viral load (number of copies of viral RNA per mL) is a global health priority (1). CDC and other U.S. government agencies, as part of the U.S. President's Emergency Plan for AIDS Relief (PEPFAR), together with other stakeholders, have provided technical assistance and supported the cost for multiple countries in sub-Saharan Africa to expand viral load testing as the preferred monitoring strategy for clinical response to ART. The individual and population-level benefits of ART are well understood (2). Persons receiving ART who achieve and sustain an undetectable viral load do not transmit HIV to their sex partners, thereby disrupting onward transmission (2,3). Viral load testing is a cost-effective and sustainable programmatic approach for monitoring treatment success, allowing reduced frequency of health care visits for patients who are virally suppressed (4). Viral load monitoring enables early and accurate detection of treatment failure before immunologic decline. This report describes progress on the scale-up of viral load testing in eight sub-Saharan African countries from 2013 to 2018 and examines the trajectory of improvement with viral load testing scale-up that has paralleled government commitments, sustained technical assistance, and financial resources from international donors. Viral load testing in low- and middle-income countries enables monitoring of viral load suppression at the individual and population level, which is necessary to achieve global epidemic control. Although there has been substantial achievement in improving viral load coverage for all patients receiving ART, continued engagement is needed to reach global targets.

Field evaluation of Abbott Real Time HIV-1 Qualitative test for early infant diagnosis using dried blood spots samples in comparison to Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Qual test in Kenya.

Timely diagnosis and treatment of infants infected with HIV are critical for reducing infant mortality. High-throughput automated diagnostic tests like Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Qual Test (Roche CAPCTM Qual) and the Abbott Real Time HIV-1 Qualitative (Abbott Qualitative) can be used to rapidly expand early infant diagnosis testing services. In this study, the performance characteristics of the Abbott Qualitative were evaluated using two hundred dried blood spots (DBS) samples (100 HIV-1 positive and 100 HIV-1 negative) collected from infants attending the antenatal facilities in Kisumu, Kenya. The Abbott Qualitative results were compared to the diagnostic testing completed using the Roche CAPCTM Qual in Kenya. The sensitivity and specificity of the Abbott Qualitative were 99.0% (95% CI: 95.0-100.0) and 100.0% (95% CI: 96.0-100.0), respectively, and the overall reproducibility was 98.0% (95% CI: 86.0-100.0). The limits of detection for the Abbott Qualitative and Roche CAPCTM Qual were 56.5 and 6.9copies/mL at 95% CIs (p=0.005), respectively. The study findings demonstrate that the Abbott Qualitative test is a practical option for timely diagnosis of HIV in infants.

Using standard and institutional mentorship models to implement SLMTA in Kenya.

BACKGROUND: Kenya is home to several high-performing internationally-accredited research laboratories, whilst most public sector laboratories have historically lacked functioning quality management systems. In 2010, Kenya enrolled an initial eight regional and four national laboratories into the Strengthening Laboratory Management Toward Accreditation (SLMTA) programme. To address the challenge of a lack of mentors for the regional laboratories, three were paired, or 'twinned', with nearby accredited research laboratories to provide institutional mentorship, whilst the other five received standard mentorship. OBJECTIVES: This study examines results from the eight regional laboratories in the initial SLMTA group, with a focus on mentorship models. METHODS: Three SLMTA workshops were interspersed with three-month periods of improvement project implementation and mentorship. Progress was evaluated at baseline, mid-term, and exit using the Stepwise Laboratory Quality Improvement Process Towards Accreditation (SLIPTA) audit checklist and scores were converted into a zero- to five-star scale. RESULTS: At baseline, the mean score for the eight laboratories was 32%; all laboratories were below the one-star level. At mid-term, all laboratories had measured improvements. However, the three twinned laboratories had increased an average of 32 percentage points and reached one to three stars; whilst the five non-twinned laboratories increased an average of 10 percentage points and remained at zero stars. At exit, twinned laboratories had increased an average 12 additional percentage points (44 total), reaching two to four stars; non-twinned laboratories increased an average of 28 additional percentage points (38 total), reaching one to three stars. CONCLUSION: The partnership used by the twinning model holds promise for future collaborations between ministries of health and state-of-the-art research laboratories in their regions for laboratory quality improvement. Where they exist, such laboratories may be valuable resources to be used judiciously so as to accelerate sustainable quality improvement initiated through SLMTA.

Comparison of short-term and long-term protocols for stabilization and preservation of RNA and DNA of Leishmania, Trypanosoma, and Plasmodium.

Molecular tools continue to be important in the prevention and control of parasitic diseases. However, using these techniques directly in the field remains a major challenge. Therefore, the preservation of clinical samples collected from endemic field areas for later analysis remains an important preanalytical process. This study aimed at identifying a suitable protocol for stabilization and preservation of RNA and DNA in bioclinical specimens for Trypanosoma, Leishmania, and Plasmodium research. Both spiked and unspiked blood samples were preserved in 7 protocols (different media; storage temperatures). Samples were evaluated for possible degradation of DNA and RNA along the storage duration up to the 10th week. Nucleic acid targets were assessed as follows: (i) Trypanosoma and Plasmodium RNA analysis was done using real-time nucleic acid sequence-based amplification (RT-NASBA) for 18S rRNA and for stage-specific Pfs25 mRNA, respectively; (ii) Trypanosoma DNA assessment analysis was conducted by using a conventional PCR for 18S rDNA; (iii) Leishmania RNA analysis was performed with a quantitative NASBA for 18S rRNA and Leishmania DNA assessment with an RT-PCR for 18S rDNA. Findings suggested that a newly developed L3™ buffer proved to be reliable and suitable for both short- and long-term preservation of parasite nucleic acid material. This buffer is envisaged to be suitable for utilization in field situations where resources are limited.

Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania.

OBJECTIVE: To evaluate the repeatability and reproducibility of four simplified molecular assays for the diagnosis of Trypanosoma brucei spp. or Leishmania ssp. in a multicentre ring trial with seven participating laboratories. METHODS: The tests are based on PCR or NASBA amplification of the parasites nucleic acids followed by rapid read-out by oligochromatographic dipstick (PCR-OC and NASBA-OC). RESULTS: On purified nucleic acid specimens, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 91.7% and 95.5%; Tryp-NASBA-OC, 95.8% and 100%; Leish-PCR-OC, 95.9% and 98.1%; Leish-NASBA-OC, 92.3% and 98.2%. On blood specimens spiked with parasites, the repeatability and reproducibility of the tests were Tryp-PRC-OC, 78.4% and 86.6%; Tryp-NASBA-OC, 81.5% and 89.0%; Leish-PCR-OC, 87.1% and 91.7%; Leish-NASBA-OC, 74.8% and 86.2%. CONCLUSION: As repeatability and reproducibility of the tests were satisfactory, further phase II and III evaluations in clinical and population specimens from disease endemic countries are justified.

Sensitivity and specificity of the Leishmania OligoC-TesT and NASBA-oligochromatography for diagnosis of visceral leishmaniasis in Kenya.

OBJECTIVE: To estimate the sensitivity and specificity of the OligoC-TesT and nucleic acid sequence-based amplification coupled to oligochromatography (NASBA-OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya. METHODS: Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays. RESULTS: The Leishmania OligoC-TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90-98.8%) and a specificity of 88.8% (95% CI: 81-93.6%), while the sensitivity and specificity of the NASBA-OC were 79.8% (95% CI: 67-87%) and 100% (95% CI: 96.3-100%), respectively. CONCLUSION: Our findings indicate high sensitivity of the Leishmania OligoC-TesT on blood while the NASBA-OC is a better marker for active disease.

Simplified molecular detection of Leishmania parasites in various clinical samples from patients with leishmaniasis.

BACKGROUND: Molecular methods to detect Leishmania parasites are considered specific and sensitive, but often not applied in endemic areas of developing countries due to technical complexity. In the present study isothermal, nucleic acid sequence based amplification (NASBA) was coupled to oligochromatography (OC) to develop a simplified detection method for the diagnosis of leishmaniasis. NASBA-OC, detecting Leishmania RNA, was evaluated using clinical samples from visceral leishmaniasis patients from East Africa (n = 30) and cutaneous leishmaniasis from South America (n = 70) and appropriate control samples. RESULTS: Analytical sensitivity was 10 parasites/ml of spiked blood, and 1 parasite/ml of culture. Diagnostic sensitivity of NASBA-OC was 93.3% (95% CI: 76.5%-98.8%) and specificity was 100% (95% CI: 91.1%-100%) on blood samples, while sensitivity and specificity on skin biopsy samples was 98.6% (95% CI: 91.2%-99.9%) and 100% (95% CI: 46.3%-100%), respectively. CONCLUSION: The NASBA-OC format brings implementation of molecular diagnosis of leishmaniasis in resource poor countries one step closer.