Low levels of HIV-1 drug resistance mutations in patients who achieved viral re-suppression without regimen switch: a retrospective study.

BACKGROUND: We identified a HIV-positive cohort in virologic failure (VF) who re-suppressed without drug switch. We characterized their drug resistance mutations (DRM) and adherence profiles to learn how to better manage HIV drug resistance. A retrospective cohort study utilizing clinical data and stored samples. Patients received ART at three Nigerian treatment centres. Plasma samples stored when they were in VF were genotyped. RESULT: Of 126 patients with samples available, 57 were successfully genotyped. From ART initiation, the proportion of patients with adherence ≥90% increased steadily from 54% at first high viral load (VL) to 67% at confirmed VF, and 81% at time of re-suppressed VL. Sixteen (28%) patients had at least one DRM. Forty-six (81%) patients had full susceptibility to the three drugs in their first-line (1 L) regimen. Thirteen (23%) were resistant to at least one antiretroviral drug but three were resistant to drugs not used in Nigeria. Ten patients had resistance to their 1 L drug(s) and six were fully susceptible to the three drugs in the recommended second-line regimen. CONCLUSION: This cohort had little drug resistance mutations. We conclude that if adherence is not assured, patients could exhibit virologic failure without having developed mutations associated with drug resistance.

High prevalence of resistance to third-generation cephalosporins detected among clinical isolates from sentinel healthcare facilities in Lagos, Nigeria.

BACKGROUND: Antimicrobial resistance (AMR) in bacterial pathogens is a worldwide concern that demands immediate attention. Most information on AMR originates from high-income countries and little is known about the burden in Africa, particularly Nigeria. Using four sentinel sites (General hospitals) in Lagos State, this study sought to estimate the burden of AMR. METHODS: This is a hospital-based surveillance using secondary health care centres. Four sites were randomly selected and included in the study. Clinical isolates were collected over a period of 6 months for each site from August 2020 to March 2021. All isolates were characterised and analysed for resistance to 15 antibiotics using the Kirby-Baur method. Multiplex PCR assay was used for the detection of Extended spectrum beta lactamase genes. Data analysis was done using SPSS version 27.0. RESULTS: Four hundred and ninety-nine (499) patients consented and participated in this study, consisting of 412 (82.6%) females and 87 (17.4%) males. The mean age ± SD of the participants was 33.9 ± 13.8 with a range of 1-89 years. The majority (90.8%) of the participants were outpatients. Two hundred and thirty-two (232) isolates were obtained from 219 samples, comprising of 120 (51.7%) Gram positive and 112 (48.3%) Gram negative organisms. Key bacterial pathogens isolated from this study included Staphylococcus aureus (22.8%), Escherichia coli (16.4%), Staphylococcus spp. (15.9%), Enterococcus spp. (7.3%) and Klebsiella pneumoniae (6.5%). There was high prevalence of multi-drug resistance (79.3%) among the isolates with 73.6% of Staphylococcus aureus phenotypically resistant to methicillin and 70% possessed the MecA gene. 76.5% of Enterococcus spp. isolated were Vancomycin resistant. Overall, resistance to Cephalosporins was most frequently/commonly observed (Cefotaxime 87.5%). CONCLUSION: A high incidence of AMR was identified in clinical bacteria isolates from selected general hospitals in Lagos State, highlighting the necessity for the implementation of national action plans to limit the prevalence of AMR. Surveillance via collection of isolates has a lot of promise, especially in resource-limited environments.

Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria.

In an outbreak, effective detection of the aetiological agent(s) involved using molecular techniques is key to efficient diagnosis, early prevention and management of the spread. However, sequencing is necessary for mutation monitoring and tracking of clusters of transmission, development of diagnostics and for vaccines and drug development. Many sequencing methods are fast evolving to reduce test turn-around-time and to increase through-put compared to Sanger sequencing method; however, Sanger sequencing remains the gold standard for clinical research sequencing with its 99.99% accuracy This study sought to generate sequence data of SARS-CoV-2 using Sanger sequencing method and to characterize them for possible site(s) of mutations. About 30 pairs of primers were designed, synthesized, and optimized using endpoint PCR to generate amplicons for the full length of the virus. Cycle sequencing using BigDye Terminator v.3.1 and capillary gel electrophoresis on ABI 3130xl genetic analyser were performed according to the manufacturers' instructions. The sequence data generated were assembled and analysed for variations using DNASTAR Lasergene 17 SeqMan Ultra. Total length of 29,760bp of SARS-CoV-2 was assembled from the sample analysed and deposited in GenBank with accession number: MT576584. Blast result of the sequence assembly shows a 99.97% identity with the reference sequence. Variations were noticed at positions: nt201, nt2997, nt14368, nt16535, nt20334, and nt28841-28843, which caused amino acid alterations at the S (aa614) and N (aa203-204) regions. The mutations observed at S and N-gene in this study may be indicative of a gradual changes in the genetic coding of the virus hence, the need for active surveillance of the viral genome.

Laboratory Optimization Tweaks for Sanger Sequencing in a Resource-Limited Setting.

Despite various challenges that hinder the implementation of high-tech molecular methods in resource-limited settings, we have been able to implement and achieve International Organization for Standardization 15189:2012 accreditation for genotypic HIV drug resistance testing in our facility. At the Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Nigeria has recorded a high sequencing success rate and good quality sequence data. This was achieved by optimizing laboratory processes from 2008 to the current date. We have optimized sample preparation, RT-PCR, several post-PCR processes, and the cycle sequencing to improve the sensitivity of amplification even with limited plasma samples and low viral copy numbers. The optimized workflow maximizes output, minimizes reagent wastage, and achieves substantial cost savings without compromising the quality of the sequence data. Our performance at our last external quality assurance program is a testimonial to the efficiency of the workflow. For the 5-sample panel, each with 67-68 mutation points evaluated, we scored 100% for all 5 specimens. Our optimized laboratory workflow is thus documented to support laboratories and to help researchers achieve excellent results the first time and eliminate contamination while minimizing the wastage of costly sequencing reagents.

Implication of First-Line Antiretroviral Therapy Choice on Second-Line Options.

BACKGROUND: Although there are a number of studies comparing the currently recommended preferred and alternative first-line (1L) antiretroviral therapy (ART) regimens on clinical outcomes, there are limited data examining the impact of 1L regimen choice and duration of virologic failure (VF) on accumulation of drug resistance mutations (DRM). The patterns of DRM from patients failing zidovudine (AZT)-containing versus tenofovir (TDF)-containing ART were assessed to evaluate the predicted susceptibility to second-line (2L) nucleoside reverse-transcriptase inhibitor (NRTI) backbone options in the context of an ongoing programmatic setting that uses viral load (VL) monitoring. METHODS: Paired samples from Nigerian ART patients who experienced VF and switched to 2L ART were retrospectively identified. For each sample, the human immunodeficiency virus (HIV)-1 polymerase gene was sequenced at 2 time points, and DRM was analyzed using Stanford University's HIVdb program. RESULTS: Sequences were generated for 191 patients. At time of 2L switch, 28.2% of patients on AZT-containing regimens developed resistance to TDF, whereas only 6.8% of patients on TDF-containing 1L had mutations compromising susceptibility to AZT. In a stratified evaluation, patients with 0-6 months between tested VL samples had no difference in proportion compromised to 2L, whereas those with >6 months between samples had a statistically significant difference in proportion with compromised 2L NRTI. In multivariate analyses, patients on 1L AZT had 9.90 times higher odds of having a compromised 2L NRTI option than patients on 1L TDF. CONCLUSIONS: In the context of constrained resources, where VL monitoring is limited, we present further evidence to support use of TDF as the preferred 1L NRTI because it allows for preservation of the recommended 2L NRTI option.

Potential for false-positive HIV test results using rapid HIV testing algorithms.

Background: In order to scale up access to HIV counselling and testing in Nigeria, an HIV diagnostic algorithm based on rapid testing was adopted. However, there was the need to further evaluate the testing strategy in order to better assess its performance, because of the potential for false positivity. Objectives: The objective of this study was to compare positive HIV test results obtained from the approved rapid testing algorithm with results from western blot tests performed on samples from the same patient. Methodology: A retrospective review was conducted of HIV screening and confirmatory results for patients seen between 2007 and 2008. Rapid test and western blot results were extracted and compared for concordance. Discordant results were further reviewed using a combination of HIV-1 RNA viral load and CD4+ cell count test results and clinical presentation from medical records. Results: Analysis of 2228 western blot results showed that 98.3% (n = 2191) were positive for HIV-1, 0.4% (n = 8) were positive for HIV-2 and 0.3% (n = 7) were dual infections (positive for both HIV-1 and HIV-2); 0.6% (n = 13) were indeterminate and 0.4% (n = 9) were negative. Further investigation of the 13 indeterminate results showed nine to be HIV-1 positive and four to be HIV-negative, for a total of 13 negative results. The positive predictive value of the HIV counselling and testing algorithm was 99.4%. Conclusion: Using the rapid testing algorithm alone, false positives were detected. Therefore, effective measures such as training and retraining of staff should be prioritised in order to minimise false-positive diagnoses and the associated potential for long-term psychological and financial impact on the patients.

Foetal loss and enhanced fertility observed in mice treated with Zidovudine or Nevirapine.

BACKGROUND: Health concerns for HIV-infected persons on antiretroviral therapy (ART) have moved from morbidity to the challenges of long-term ART. We investigated the effect of Zidovudine or Nevirapine on reproductive capacity across two mouse generations. METHODS: A prospective mouse study with drugs administered through one spermatogenic cycle. Mouse groups (16 males and 10 females) were given Zidovudine or Nevirapine for 56 days. Males were mated to untreated virgin females to determine dominant lethal effects. Twenty females (10 treated and 10 untreated) mated with the treated males per dose and gave birth to the F1 generation. Parental mice were withdrawn from drugs for one spermatogenic cycle and mated to the same dams to ascertain if effects are reversible. The F1 generation were exposed for another 56 days and mated to produce the F2 generation. RESULTS: Foetal loss was indicated in the dominant lethal assay as early as four weeks into drug administration to the males. At the first mating of the parental generation to produce the F1 generation, births from 10 dams/dose when the 'father-only' was exposed to Zidovudine (10, 100 and 250 mg/kg) was 3, 2 and 1 while it was 7, 1 and 4 respectively when 'both-parents' were exposed. Similarly births from the parental generation first mating when the 'father-only' was exposed to Nevirapine (5, 50 and 150 mg/kg) was 2, 2 and 0 while it was 6, 5 and 9 respectively when 'both-parents' were exposed. However, fertility was not significantly different neither by dose nor by the parental exposure. The F1 mice mated to produce the F2 generation recorded only one birth. CONCLUSION: The dominant lethal analysis showed foetal loss occurred when the "fathers-only" were treated while fertility was enhanced when "both-parents" were on therapy at the time of mating.

Exposure of Allium cepa root cells to zidovudine or nevirapine induces cytogenotoxic changes.

Antiretroviral drugs have proved useful in the clinical management of HIV-infected persons, though there are concerns about the effects of exposure to these DNA-reactive drugs. We investigated the potential of the plant model Allium cepa root tip assay to demonstrate the cytogenotoxicity of zidovudine and nevirapine and as a replace-reduce-refine programme amenable to resource-poor research settings. Cells mitotic index were determined in squashed root cells from Allium cepa bulbs exposed to zidovudine or nevirapine for 48 hr. The concentration of zidovudine and nevirapine inhibiting 50% root growth after 96 hr exposure was 65.0 µM and 92.5 µM respectively. Root length of all antiretroviral-exposed roots after 96 hr exposure was significantly shorter than the unexposed roots while additional root growth during a subsequent 48 hr recovery period in the absence of drug was not significantly different. By ANOVA, there was a significant association between percentage of cells in mitosis and zidovudine dose (p=0.004), but not nevirapine dose (p=0.68). Chromosomal aberrations such as sticky chromosomes, chromatin bridges, multipolar mitoses and binucleated cells were observed in root cells exposed to zidovudine and nevirapine for 48 hr. The most notable chromosomal aberration was drug-related increases in sticky chromosomes. Overall, the study showed inhibition in root length growth, changes in the mitotic index, and the induction of chromosomal aberrations in Allium bulbs treated for 96 hr or 48 hr with zidovudine and nevirapine. The study reveals generalized cytogenotoxic damage induced by exposure to zidovudine and nevirapine, and further show that the two compounds differ in their effects on mitosis and the types of chromosomal aberrations induced.

Proficiency testing for HIV, tuberculosis and malaria diagnosis in clinical laboratories in Nigeria.

BACKGROUND: Proficiency testing (PT) is a means of verifying the reliability of laboratory results, but such programmes are not readily available to laboratories in developing countries. This project provided PT to laboratories in Nigeria. OBJECTIVES: To assess the proficiency of laboratories in the diagnosis of HIV, tuberculosis and malaria. METHODS: This was a prospective study carried out between 2009 and 2011. A structured questionnaire was administered to 106 randomly-selected laboratories. Forty-four indicated their interest in participation and were enrolled. Four rounds of pre-characterised plasma panels for HIV, sputum films for tuberculosis and blood films for malaria were distributed quarterly by courier over the course of one year. The results were returned within two weeks and scores of ≥ 80% were reported as satisfactory. Mentoring was offered after the first and second PT rounds. RESULTS: Average HIV PT scores increased from 74% to 95% from the first round to the third round, but decreased in the fourth round. For diagnosis of tuberculosis, average scores increased from 42% in the first round to 78% in the second round; but a decrease to 34% was observed in the fourth round. Malaria PT performance was 2% at first, but average scores increased between the second and fourth rounds, culminating in a fourth-round score of 39%. Many participants requested training and mentoring. CONCLUSIONS: There were gross deficiencies in the quality of laboratory services rendered across Nigeria. In-country PT programmes, implemented in conjunction with mentoring, will improve coverage and diagnosis of HIV, tuberculosis and malaria.

Comparison of the COBAS/Ampliprep Taqman and Amplicor HIV-1 monitor tests in Lagos, Nigeria.

BACKGROUND: The use of real-time Polymerase chain reaction (PCR) technology options is increasing in resource-limited settings because they are faster, improve assay sensitivity, have higher throughput, larger dynamic ranges and reduced rates of contamination. In 2010, UNAIDS ranked Nigeria as the second highest population of people living with HIV and AIDS (2.98 million people) in the world. OBJECTIVE: The objective of this study was to compare the analytical performances of the Amplicor HIV-1 Monitor (version 1.5) and the COBAS Ampliprep/Taqman (version 2.0) used in monitoring HIV disease progression in HIV-infected individuals. METHOD: In a cross-sectional study, HIV-1 RNA values obtained with the Amplicor HIV-1 monitor version 1.5 were compared with those of the COBAS/Ampliprep TaqMan HIV-1 version 2.0 in a routine clinical setting. Between May and November 2011, 176 plasma samples collected were analysed in parallel using both techniques. Data analysis was done using statgraphics Centurion XVI and Medcalc version 12.0. RESULT: The correlation coefficient for the two assays was 0.83 and the level of agreement using a Bland-Altman plot was 94.2%. CONCLUSION: These findings suggest that the results from the two methods were comparable, hence the COBAS/Ampliprep Taqman version 2.0 is recommended for high-volume laboratories.

Accumulation of protease mutations among patients failing second-line antiretroviral therapy and response to salvage therapy in Nigeria.

BACKGROUND: To date, antiretroviral therapy (ART) guidelines and programs in resource-limited settings (RLS) have focused on 1(st)- and 2(nd)-line (2 L) therapy. As programs approach a decade of implementation, policy regarding access to 3(rd)-line (3 L) ART is needed. We aimed to examine the impact of maintaining patients on failing 2 L ART on the accumulation of protease (PR) mutations. METHODS AND FINDINGS: From 2004-2011, the Harvard/APIN PEPFAR Program provided ART to >100,000 people in Nigeria. Genotypic resistance testing was performed on a subset of patients experiencing 2 L failure, defined as 2 consecutive viral loads (VL)>1000 copies/mL after ≥6 months on 2 L. Of 6714 patients who received protease inhibitor (PI)-based ART, 673 (10.0%) met virologic failure criteria. Genotypes were performed on 61 samples. Patients on non-suppressive 2 L therapy for <12 months prior to genotyping had a median of 2 (IQR: 0-5) International AIDS Society (IAS) PR mutations compared with 5 (IQR: 0-6) among patients failing for >24 months. Patients developed a median of 0.6 (IQR: 0-1.4) IAS PR mutations per 6 months on failing 2 L therapy. In 38% of failing patients no PR mutations were present. For patients failing >24 months, high- or intermediate-level resistance to lopinavir and atazanavir was present in 63%, with 5% to darunavir. CONCLUSIONS: This is the first report assessing the impact of duration of non-suppressive 2 L therapy on the accumulation of PR resistance in a RLS. This information provides insight into the resistance cost of failing to switch non-suppressive 2 L regimens and highlights the issue of 3 L access.

Experience of quality management system in a clinical laboratory in Nigeria.

ISSUES: Quality-management systems (QMS) are uncommon in clinical laboratories in Nigeria, and until recently, none of the nation's 5 349 clinical laboratories have been able to attain the certifications necessary to begin the process of attaining international accreditation. Nigeria's Human Virology Laboratory (HVL), however, began implementation of a QMS in 2006, and in 2008 it was determined that the laboratory conformed to the requirements of ISO 9001:2000 (now 2008), making it the first diagnostic laboratory to be certified in Nigeria. The HVL has now applied for the World Health Organization (WHO) accreditation preparedness scheme. The experience of the QMS implementation process and the lessons learned therein are shared here. DESCRIPTION: In 2005, two personnel from the HVL spent time studying quality systems in a certified clinical laboratory in Dakar, Senegal. Following this peer-to-peer technical assistance, several training sessions were undertaken by HVL staff, a baseline assessment was conducted, and processes were established. The HVL has monitored its quality indicators and conducted internal and external audits; these analyses (from 2007 to 2009) are presented herein. LESSONS LEARNED: Although there was improvement in the pre-analytical and analytical indicators analysed and although data-entry errors decreased in the post-analytical process, the delay in returning laboratory test results increased significantly. There were several factors identified as causes for this delay and all of these have now been addressed except for an identified need for automation of some high-volume assays (currently being negotiated). Internal and external audits showed a trend of increasing non-conformities which could be the result of personnel simply becoming lax over time. Application for laboratory accreditation, however, could provide the renewed vigour needed to correct these non-conformities. RECOMMENDATION: This experience shows that sustainability of the QMS at present is a cause for concern. However, the tiered system of accreditation being developed by WHO-Afro may act as a driving force to preserve the spirit of continual improvement.