Simon M. Agwale

HIV point-of-care diagnostics: meeting the special needs of sub-Saharan Africa

Sub-Saharan Africa, accounting for 70% of the 35 million people living with HIV worldwide, obviously carries the heaviest burden of the HIV epidemic. Moreover, the regions poor health system occasioned by limited resources and inadequate skilled clinical personnel usually makes decentralization of HIV care difficult. Therefore, quality diagnostics that are easy to use, inexpensive, and amenable for use at point of care (POC) are a dire necessity. Clearly, such diagnostics will significantly lessen the pressure on the existing over-stretched centralized HIV laboratory services. Thankfully, some POC diagnostics are already being validated, while others are in the pipeline. As POC test kits emerge, implementation hurdles should be envisaged and planned for. This review examines emerging HIV diagnostic platforms, HIV POC product pipelines, gaps, perceived POC implementation challenges, and general recommendations for quality care.

Molecular Characterization of Hepatitis A Virus Isolates from Nigeria

Objective: Despite the endemicity of hepatitis A virus (HAV) in Nigeria, genetic information on the HAV genotypes/subgenotypes circulating in the country remains unknown. The objective of this study was to investigate HAV strains using molecular epidemiological and genetic analyses among apparently healthy adult Nigerian subjects. Methods: Testing for HAV-RNA was performed on 114 serum samples by the reverse transcription-polymerase chain reaction and sequenced with primers encompassing the VP1/P2A junction. Results: Twelve serum samples tested were found to be HAV-RNA positive. Phylogenetic analysis revealed that all 12 HAV isolates were classified as subgenotype IA exhibiting 98.3% nucleotide identity. Interestingly, the Nigerian HAV/IA subgenotype consisted of two distinct genomic sublineages with a unique majority (n = 11) corresponding to strains endemic in Cameroon and the other (n = 1) shows a probable link with European sequences. Predicted conserved amino acid sequences and the few deduced substitution in the VP1/P2A junction might play a role in the development of a novel Nigerian-Cameroon sublineage within the HAV/IA subgenotype and might explain the stability of HAV/IA in this subregion. Conclusion: This study reveals the development of a new HAV/IA sublineage in the Nigerian-Cameroon subregion. The presence of a single subgenotype indicates that this HAV strain has been predominantly circulating in Nigeria.

Genetic Analysis of Hepatitis A Virus Variants Circulating Among Children Presenting With Acute Diarrhea in Cameroon

Molecular investigation was undertaken of circulating hepatitis A virus (HAV) associated with cases of acute diarrhea among children under 5 years of age in Kumba–Cameroon. Reverse transcription PCR, sequencing, and phylogenetic analysis of a 371 bp segment of the VP1/P2A junction of six isolates obtained from stool samples showed the exclusive emergence of genetically related HAV subgenotype IA. All the isolates clustered within a unique lineage exhibiting a 99.5% nucleotide identity suggesting infection from a common source. The Cameroonian HAV isolates did not intermix or cluster with those from other regions of Africa and the rest of the world. Tajimas neutralization tests using the six sequences suggested HAV/IA population expansion (D = −1.37; P = 0.016). This is the first description of indigenous HAV genotypes circulating in Cameroon revealing a community‐wide spread and predominance of HAV/1A infection in the Kumba area. These findings stress the need for routine molecular tracking of HAV infection as a contributory cause of acute diarrhea in Cameroonian children. J. Med. Virol. 84:728–732, 2012.

Absence of routine molecular testing and prevalence of HIV-2 infection in regions hardest-hit by HIV infection

INTRODUCTION Investigating the incidence and dynamics of HIV-2 and false-negative HIV test results in a highly sexually active population where frequent opportunities exist for acquiring and transmitting infections provides additional understanding of the epidemiology of the virus in Africa. METHODOLOGY The HIV status of 900 active female sex workers (FSWs) was determined using two lateral flow rapid assays in series. The second rapid test device incorporates type-specific recombinant peptides that discriminate between HIV-1 and HIV-2 infection. HIV sero-negative samples were re-tested for HIV infection and their viral loads determined using the NucliSENS real-time nucleic acid sequence-based amplification (NASBA) platform. RESULTS In total, 335 FSWs were determined to be HIV positive, the majority (227; 67.8%) of whom were between the ages of 20 and 30 years. Eighteen (5.4%) were found to have evidence of HIV-2 infection, 17 of whom were co-infected with HIV-1. Only one HIV-2 mono-infection was observed. Out of 565 HIV-negative individuals determined by serology, 11(1.9%; p > 0.05) were found to be HIV-1 positive when tested via the NASBA platform. CONCLUSION False negative test results, HIV-2 infection, and complex transmission networks among FSWs may aid in fueling the HIV epidemic in the Nigerian population. These findings demonstrate the need to reevaluate the quality of HIV serological diagnostics, control services, and stress the need for widespread introduction of molecular testing among high-risk populations in the country.

Estimates of human immunodeficiency virus incidence among female sex workers in north central Nigeria: implications for HIV clinical trials.

We estimated the HIV incidence among commercial female sex workers (FSWs) in north central Nigeria using bimodal methodology. Using a cross-sectional study design, a total of 900 active FSWs between the ages of 18 and 35 years were recruited from 52 brothels within Nasarawa State, Nigeria. A rapid test algorithm was used to determine their HIV status. The BED IgG-Capture enzyme immunoassay (CEIA) was applied on the HIV-seropositive samples to detect recent HIV-1 infection for the estimation of incidence among those with HIV infection. Of the 900 FSWs tested, 335 (37.2%) were found to be positive for HIV. Of these, 63 showed evidence of recent infection. Using two third-generation BED analysis approaches that account for false-recent rate, an annualized adjusted HIV incidence of 11.97% (95% CI: 8.51-15.43%) and 12.36% (95% CI: 8.18-16.34%) was observed; difference P>0.05. In addition, 875 (97.2%) of the FSWs readily agreed to participate in HIV clinical trials. We have developed the expertise and infrastructure necessary for clinical trial investigations in sub-Saharan Africa. We have recorded a high proportion of recent HIV infections among FSWs in Nigeria and also provided an enabling environment for future studies of HIV prevention methods.

Broad Antibody Mediated Cross-Neutralization and Preclinical Immunogenicity of New Codon-Optimized HIV-1 Clade CRF02_AG and G Primary Isolates

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary “street strain” isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

CD4+ T lymphocyte reference values of immunocompetent subjects in an African university

CD4+ T cells play critical roles in the immune system and, being primary targets of HIV infection, they are used to measure disease progression and response to combination antiretroviral therapy (cART), alongside other parameters, in HIV/AIDS patients. The aim of this study was to determine the reference values of CD4+ T cells in a student population that was HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) negative. After ethical clearance and informed consent, 500 subjects (mean age = 26 years) were recruited, of whom 56 (11.2%) had HIV, HBV or HCV and were excluded. Blood samples were collected from the remaining 444 subjects into vacutainer tubes and analysed using the BD FACScount cytometer according to the manufacturers instructions. Of the 444 subjects, 266 (59.9%) were male and 178 (40.1%) were female. The mean (± standard deviation) CD4+ T cell count was 987 cells/µL (± 336). The mean counts among males and females were 957 cells/µL (± 306) and 991 cells/µL (± 340), respectively. Values of CD4+ T cells ranged from 651 cells/µL to 1705 cells/µL. Subjects with higher CD4+ T Cells were more likely to be female than male. There was no direct correlation between CD4+ T cell values and age of the participants. Our findings offer the first insight into the CD4+ T cell reference values of a Nigerian student population and provide useful data that will guide future cART decisions and other immune-based therapies.