Whole-genome sequencing illuminates the evolution and spread of multidrug-resistant tuberculosis in Southwest Nigeria.

Nigeria has an emerging problem with multidrug-resistant tuberculosis (MDR-TB). Whole-genome sequencing was used to understand the epidemiology of tuberculosis and genetics of multi-drug resistance among patients from two tertiary referral centers in Southwest Nigeria. In line with previous molecular epidemiology studies, most isolates of Mycobacterium tuberculosis from this dataset belonged to the Cameroon clade within the Euro-American lineage. Phylogenetic analysis showed this clade was undergoing clonal expansion in this region, and suggests that it was involved in community transmission of sensitive and multidrug-resistant tuberculosis. Five patients enrolled for retreatment were infected with pre-extensively drug resistant (pre-XDR) due to fluoroquinolone resistance in isolates from the Cameroon clade. In all five cases resistance was conferred through a mutation in the gyrA gene. In some patients, genomic changes occurred in bacterial isolates during the course of treatment that potentially led to decreased drug susceptibility. We conclude that inter-patient transmission of resistant isolates, principally from the Cameroon clade, contributes to the spread of MDR-TB in this setting, underscoring the urgent need to curb the spread of multi-drug resistance in this region

A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

Background Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. Methodology/Principal Findings DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria

J Gen Virol

Hepatitis C virus (HCV) is classified into seven genotypes based on genetic diversity, and most genotypes have been found in Africa. Infections with HCV genotype 2 (HCV2) are most prevalent in West Africa and it was suggested that HCV2 originated in West Africa. To better understand the evolutionary epidemiology of HCV2 in Africa, we examined new NS5B sequences of HCV2 strains obtained from C\uf4te d'Ivoire, Ghana and Nigeria sequenced at the Centers for Disease Control and Prevention with those available from West, North and Central Africa. Bayesian phylogeographic analysis using a discrete trait model showed that Ghana was the most likely geographical region for the origin of HCV2. Spread of HCV2 from Ghana did not appear to be through diffusion to adjacent countries along the coast. Rather, it was transmitted from Ghana to many distant countries in Africa, suggesting that certain routes of geographical dissemination were historically more efficient than mere proximity and that the HCV2 epidemic history in West Africa is extremely complex.20152016-08-01T00:00:00ZCC999999/Intramural CDC HHS/United States25888623PMC4681065664

Mycobacterium tuberculosis ecology in Venezuela: epidemiologic correlates of common spoligotypes and a large clonal cluster defined by MIRU-VNTR-24

<p>Abstract</p> <p>Background</p> <p>Tuberculosis remains an endemic public health problem, but the ecology of the TB strains prevalent, and their transmission, can vary by country and by region. We sought to investigate the prevalence of <it>Mycobacterium tuberculosis </it>strains in different regions of Venezuela. A previous study identified the most prevalent strains in Venezuela but did not show geographical distribution nor identify clonal genotypes. To better understand local strain ecology, we used spoligotyping to analyze 1298 <it>M. tuberculosis </it>strains isolated in Venezuela from 1997 to 2006, predominantly from two large urban centers and two geographically distinct indigenous areas, and then studied a subgroup with MIRU-VNTR 24 loci.</p> <p>Results</p> <p>The distribution of spoligotype families is similar to that previously reported for Venezuela and other South American countries: LAM 53%, T 10%, Haarlem 5%, S 1.9%, X 1.2%, Beijing 0.4%, and EAI 0.2%. The six most common shared types (SIT's 17, 93, 605, 42, 53, 20) accounted for 49% of the isolates and were the most common in almost all regions, but only a minority were clustered by MIRU-VNTR 24. One exception was the third most frequent overall, SIT 605, which is the most common spoligotype in the state of Carabobo but infrequent in other regions. MIRU-VNTR homogeneity suggests it is a clonal group of strains and was named the "Carabobo" genotype. Epidemiologic comparisons showed that patients with SIT 17 were younger and more likely to have had specimens positive for Acid Fast Bacilli on microscopy, and patients with SIT 53 were older and more commonly smear negative. Female TB patients tended to be younger than male patients. Patients from the high incidence, indigenous population in Delta Amacuro state were younger and had a nearly equal male:female distribution.</p> <p>Conclusion</p> <p>Six SIT's cause nearly half of the cases of tuberculosis in Venezuela and dominate in nearly all regions. Strains with SIT 17, the most common pattern overall may be more actively transmitted and SIT 53 strains may be less virulent and associated with reactivation of past infections in older patients. In contrast to other common spoligotypes, strains with SIT 605 form a clonal group centered in the state of Carabobo.</p

Comparative genomics shows differences in the electron transport and carbon metabolic pathways of Mycobacterium africanum relative to Mycobacterium tuberculosis and suggests an adaptation to low oxygen tension

YesThe geographically restricted Mycobacterium africanum lineages (MAF) are primarily found in West Africa, where they account for a significant proportion of tuberculosis. Despite this phenomenon, little is known about the co-evolution of these ancient lineages with West Africans. MAF and M. tuberculosis sensu stricto lineages (MTB) differ in their clinical, in vitro and in vivo characteristics for reasons not fully understood. Therefore, we compared genomes of 289 MAF and 205 MTB clinical isolates from the 6 main human-adapted M. tuberculosis complex lineages, for mutations in their Electron Transport Chain and Central Carbon Metabolic pathway in order to explain these metabolic differences. Furthermore, we determined, in silico, whether each mutation could affect the function of genes encoding enzymes in these pathways. We found more mutations with the potential to affect enzymes in these pathways in MAF lineages compared to MTB lineages. We also found that similar mutations occurred in these pathways between MAF and some MTB lineages. Generally, our findings show further differences between MAF and MTB lineages that may have contributed to the MAF clinical and growth phenotype and indicate potential adaptation of MAF lineages to a distinct ecological niche, which we suggest includes areas characterized by low oxygen tension.European Research CouncilINTERRUPTB starting grant nr. 311725 (to BdJ, FG, CM, LR, BO, MA) and The UK Medical Research Council and the European & Developing Countries Clinical Trials Partnership (EDCTP) Grant No. CB. 2007. 41700.007.Research Development Fund Publication Prize Award winner, January 2020

Biodiversity and strain differentiation of cassava-infecting geminiviruses and Bemisa tabaci in southern Africa

Thesis (M.Sc.)--University of the Witwatersrand, Faculty of Science, School of Molecular and Cell Biology, 2001.Cassava mosaic disease is prevalent in Africa and significantly affects the growth and yield of cassava. This disease is caused by a number of whiteflytransmitted begomoviruses. The aims of this study were to establish the identity of cassava begomoviruses in southern Africa and to develop assays for their differentiation. Using primers that target the highly conserved core region of the coat protein gene it was possible to identify and establish the geographical distribution and relatedness of cassava begomoviruses in 6 countries within southern Africa. It was found that African cassava mosaic virus occurred in five countries (except Angola), East African cassava mosaic virus was present in all countries (except Zambia) and South African cassava mosaic virus was present in South Africa and Swaziland. In addition, this study reports for the first time in southern Africa, the Ugandan variant virus (UgV) which occurs frequently in mixed infections with other cassava-infecting begomoviruses. Bemisia tabaci (Gennadius) is the vector of begomoviruses that cause cassava mosaic disease (CMD) in Africa and India. The taxonomy of the B. tabaci complex is problematic, making it unclear whether more than one variant or 'type' of the vector is involved in the transmission of cassavainfecting begomoviruses. Phylogenetic analysis of mitochondrial COl gene sequences revealed that B. tabaci colonising cassava in Africa form 3 distinct clades (clade 1: Mozambique, South Africa, Swaziland, Zambia; clade 2: Cameroon; clade 3 : Zimbabwe). These results indicate that topotypes within B. tabaci vector populations from cassava exist and suggest a geographic basis for the separation of cassava-colonising B. tabaci in Africa. New cassava viruses and viral strains continue to be discovered and simple, rapid and sensitive techniques are needed for screening of cassava plantations. Here we report on the development of a heteroduplex mobility assay (HMA) for differentiating cassava-infecting begomoviruses. The HMA profiles were able to differentiate four different viral species and eleven different virus strains, and showed a good correlation with sequencing results and phylogenetic comparisons with other sequenced cassava viruses. This technique was found to be sensitive and rapid and had the added advantage of being able to detect mixtures of viruses in field-grown cassava. Current serological methods and antibodies are limited in their usefulness and specificity and new antibodies need to be developed to detect all the possible viral species. The viability of using phage antibodies to detect begomoviruses proved promising as a number of phage clones were isolated and characterised. These clones, when used in combination, were able to differentiate between several cassava-infecting begomoviruses. However a number of improvements on this technique would need to be implemented before it became an acceptable method for producing antibodies to identify and distinguish between cassava begomovirus species and strains

Evaluation of HIV testing strategies and monitoring of immune responses in HIV-vaccinated individuals in Tanzania

This thesis describes studies on the evaluation of human immunodeficiency virus (HIV) enzyme-linked immunosorbent assays (ELISAs) and simple rapid HIV assays for use in HIV testing strategies in resource-limited settings and studies of HIV vaccine-induced immune responses. Peripheral blood mononuclear cell (PBMC) preparation techniques were also studied in preparation for use in the HIV vaccine trials. The performance of two antibody ELISAs (Vironostika Uni-Form II plus O and Enzygnost anti-HIV-1/2 Plus) and two new diagnostic HIV antigen/antibody combination ELISAs (Murex and Vironostika HIV Uni-Form II antigen/antibody) was evaluated using 1380 serum samples from Tanzanian individuals (paper I). The sensitivity at initial testing was 100% for all assays except Vironostika Uni-Form II plus O which showed one false negative sample at initial testing but 100% sensitivity after repeat testing. The initial specificity was 99.8% for Enzygnost, 98.9% for each of the antigen/antibody ELISAs and 97.0% for Vironostika Plus O ELISA. An alternative confirmatory HIV testing strategy based on initial testing on any of the two antigen/antibody assays followed by testing of reactive samples on the Enzygnost anti-HIV-1/2 Plus assay gave 100% specificity (95% CI; 99.7-100%). The performance of five simple rapid HIV antibody assays was evaluated using 1433 whole blood samples (paper II). The sensitivity at initial testing of Determine, SD Bioline and Uni-Gold was 100% while First Response and Stat-Pak had a sensitivity of 99.5% and 97.7%, respectively, which increased to 100% on repeat testing. The initial specificity of the Uni-Gold assay was 100% while the specificities were 99.6%, 99.4%, 99.6% and 99.8% for Determine, SD Bioline, First Response and Stat-Pak assays, respectively. An alternative confirmatory HIV testing strategy based on initial testing on SD Bioline followed by testing of reactive samples on the Determine gave 100% sensitivity (95% CI; 99.1-100) and 100% specificity (95% CI; 96-99.1) with Uni-Gold as tiebreaker for discordant results and was adopted as a national algorithm in Tanzania. Standard Ficoll-Paque gradient (FIP) centrifugation, BD vacutainer cell preparation tube (CPT) and Greiner Bio-One LeucoSep tube techniques for PBMC preparation were evaluated (paper III). No differences in mean recovery or mean viability of fresh PBMCs were observed between FIP centrifugation and CPT techniques used in Stockholm. In Dar es Salaam, recovery and viability of PBMCs isolated by FIP technique was higher compared to CPT purified cells. LeucoSep cell separation gave a higher yield and viability than FIP cell separation. The cells purified by the different techniques at the two sites performed equally well in interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assays. In a phase 1 HIV-1 DNA prime MVA boost vaccine trial in Sweden (HIVIS01/02), HIV-specific lymphoproliferative responses were tested by a [3H]-thymidine uptake assay and a flow-cytometric assay using whole blood (FASCIA-WB) (paper IV). A FASCIA using PBMC (FASCIA-PBMC) was also employed (n=14).Two weeks after the HIV-MVA boost 35 of 38 (92%) vaccinees were reactive by the thymidine uptake assay. Thirty-two of 38 (84%) vaccinees were reactive by the CD4+ T-cell FASCIA-WB, and 7 of 38 (18%) also exhibited CD8+ T-cell responses. There was strong correlation between the proliferative responses measured by the thymidine uptake assay and CD4+ T-cell FASCIA-WB (r=0.68; P < 0.01). Fourteen vaccinees were analyzed using all three assays. Ten of 14 (71%) and 11/14 (79%) demonstrated CD4+ T-cell responses in FASCIA-WB and FASCIA-PBMC, respectively. CD8+ T-cell reactivity was observed in 3/14 (21%) and 7/14 (50%) using the FASCIA-WB and FASCIA-PBMC, respectively. All 14 were reactive by the thymidine uptake assay. A FASCIA-PBMC, which allows simultaneous phenotyping, may be an option to the [3H] thymidine uptake assay for assessment of vaccine-induced T-cell proliferation, especially in isotope-restricted settings. In the HIVIS03 phase I/II HIV vaccine trial in Tanzania, sixty HIV-uninfected volunteers randomised to three groups of 20, received DNA plasmid vaccine 1 mg intradermally (id) or 3.8 mg intramuscularly (im) or placebo using a needle-free injection device (paper V). DNA plasmids vectoring HIV-1 genes gp160 subtypes A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (108 pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. The vaccines were well tolerated. Two weeks after the first HIV-MVA boost 35/35 (100%) vaccinees had IFN-γ ELISpot responses; 35 (100%) to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ responses. The id -primed recipients had significantly higher responses to Env than im recipients after HIV-MVA boost. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T-cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. A high neutralizing antibody response rate (31-83% depending on the clade B or AE virus tested) was demonstrated using a PBMC assay. In conclusion, this vaccine approach was safe and highly immunogenic

A Novel Methodology for Isolating Broadly Neutralizing HIV-1 Human Monoclonal Antibodies

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-53. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

Elicitation of broadly neutralizing HIV-1 antibodies by guiding the immune responses using primary and secondary immunogens

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-44. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

On reconstructing Proto-Bantu grammar

This book is about reconstructing the grammar of Proto-Bantu, the ancestral language at the origin of current-day Bantu languages. While Bantu is a low-level branch of Niger-Congo, the world’s biggest phylum, it is still Africa’s biggest language family. This edited volume attempts to retrieve the phonology, morphology and syntax used by the earliest Bantu speakers to communicate with each other, discusses methods to do so, and looks at issues raised by these academic endeavours. It is a collective effort involving a fine mix of junior and senior scholars representing several generations of expert historical-comparative Bantu research. It is the first systematic approach to Proto-Bantu grammar since Meeussen’s Bantu Grammatical Reconstructions (1967). Based on new bodies of evidence from the last five decades, most notably from northwestern Bantu languages, this book considerably transforms our understanding of Proto-Bantu grammar and offers new methodological approaches to Bantu grammatical reconstruction