Whole-Exome Sequencing Reveals Uncaptured Variation and Distinct Ancestry in the Southern African Population of Botswana.

Large-scale, population-based genomic studies have provided a context for modern medical genetics. Among such studies, however, African populations have remained relatively underrepresented. The breadth of genetic diversity across the African continent argues for an exploration of local genomic context to facilitate burgeoning disease mapping studies in Africa. We sought to characterize genetic variation and to assess population substructure within a cohort of HIV-positive children from Botswana-a Southern African country that is regionally underrepresented in genomic databases. Using whole-exome sequencing data from 164 Batswana and comparisons with 150 similarly sequenced HIV-positive Ugandan children, we found that 13%-25% of variation observed among Batswana was not captured by public databases. Uncaptured variants were significantly enriched (p = 2.2 × 10-16) for coding variants with minor allele frequencies between 1% and 5% and included predicted-damaging non-synonymous variants. Among variants found in public databases, corresponding allele frequencies varied widely, with Botswana having significantly higher allele frequencies among rare (<1%) pathogenic and damaging variants. Batswana clustered with other Southern African populations, but distinctly from 1000 Genomes African populations, and had limited evidence for admixture with extra-continental ancestries. We also observed a surprising lack of genetic substructure in Botswana, despite multiple tribal ethnicities and language groups, alongside a higher degree of relatedness than purported founder populations from the 1000 Genomes project. Our observations reveal a complex, but distinct, ancestral history and genomic architecture among Batswana and suggest that disease mapping within similar Southern African populations will require a deeper repository of genetic variation and allelic dependencies than presently exists.

Heme oxygenase-1 and neopterin plasma/serum levels and their role in diagnosing active and latent TB among HIV/TB co-infected patients: a cross sectional study.

BACKGROUND: Tuberculosis (TB) diagnosis in the context of HIV co-infection remains challenging. Heme oxygenase 1 (HO-1) and neopterin have been validated as potential biomarkers for TB diagnosis. Latent TB infection (LTBI) is diagnosed using tuberculin skin test (TST) and interferon gamma release assays (T-Spot and QuantiFERON TB gold tests, respectively). However, these tests have shown challenges and yet diagnosing LTBI is important for the overall control of TB. This study was conducted to determine the levels of H0-1 and neopterin, and their role in the diagnosis of TB among individuals enrolled in the Community Health and Social Network of Tuberculosis (COHSONET) study and the Kampala TB Drug Resistance Survey (KDRS). METHODS: This was a nested cross-sectional study. Plasma and serum samples collected from 140 patients at Mulago National Referral Hospital, Kampala Uganda were used. M.tb culture was performed on sputum to confirm active TB(ATB) and QuantiFERON TB gold test to confirm latent TB infection (LTBI). ELISAs were performed to determine the levels of HO-1 and neopterin. Data analysis was done using t-test and Receiver Operating Characteristic curves to determine the diagnostic accuracy. RESULTS: HO-1 levels among active tuberculosis (ATB)/HIV-infected patients and LTBI/HIV-infected patients were 10.7 ng/ml (IQR: 7.3-12.7 ng/ml) and 7.5 ng/ml (IQR: 5.4-14.1 ng/ml) respectively. Neopterin levels among ATB/HIV-positive patients and LTBI/HIV-positive patients were 11.7 ng/ml (IQR: 5.2.4 ng/ml) and 8.8 ng/ml (IQR: 2.4-19.8 ng/ml), respectively. HO-1 showed a sensitivity of 58.57% and a specificity of 67.14% with area under the curve (AUC) of 0.57 when used to discriminate between ATB and LTB. Neopterin showed an AUC of 0.62 with a sensitivity of 57.14% and a specificity of 60.0% when used to distinguish ATB from LTB. CONCLUSION: There was no in significant difference in HO-1 concentration levels of ATB individuals compared to LTB individuals. There was a significant difference in neopterin concentrations levels of ATB individuals compared to latently infected individuals. Findings from this study, show that HO-1 and neopterin have poor ability to distinguish between ATB and LTB.

Participatory approaches, local stakeholders and cultural relevance facilitate an impactful community-based project in Uganda.

Sanitation is a major global challenge that is often addressed at national and international levels, while community opinions and beliefs are neglected. To promote water, sanitation and hygiene (WASH) we organized a cross-cultural knowledge exchange workshop to assess participatory methods for engaging local stakeholders. The workshop included 22 participants from all sectors of society. Practical solutions to sanitation challenges were identified and later shared with a local community. Qualitative and quantitative analyses were used to assess impact and showed participatory methods were highly valued to encourage information sharing among widely varied stakeholders, and that video was a particularly successful approach when engaging with local communities. An 8-month follow-up survey of village members revealed excellent information recall, positive behaviour changes and a desire for future visits. Our evidence suggests that community-based participation helped identify solutions to WASH issues affecting rural communities in resource-poor settings. Engaging in a multicultural knowledge-share was particularly valuable as it enabled participants to recognize they have common challenges and allowed them to share low-cost solutions from their different communities. Our use of video was widely viewed as an ideal means of circulating findings, as it communicated information to people with a wide variety of community roles and to all age groups. Its relevance was increased by adopting a culturally appropriate context by involving local communities in workshop activities. We recommend that research in low- and middle-income countries should be mindful of the environmental context in which WASH is implemented, and encourage acceptance by engaging with communities through the use of varied participatory methods.

Anatomic and Cellular Niches for Mycobacterium tuberculosis in Latent Tuberculosis Infection.

Latent tuberculosis has been recognized for over a century, but discovery of new niches, where Mycobacterium tuberculosis resides, continues. We evaluated literature on M.tuberculosis locations during latency, highlighting that mesenchymal and hematopoietic stem cells harbor organisms in sensitized asymptomatic individuals.

Diversity of vaginal microbiota in sub-Saharan Africa and its effects on HIV transmission and prevention.

The vaginal microbial community ("microbiota") is a key component of the reproductive health of women, providing protection against urogenital infections. In sub-Saharan Africa, there is a high prevalence of bacterial vaginosis, a condition defined by bacterial overgrowth and a shift away from a Lactobacillus-dominated profile toward increased percentages of strict anaerobic species. Bacterial vaginosis is associated with an increased risk of HIV acquisition and transmission, as well as an increased risk of acquiring other sexually transmitted infections, preterm births, and pelvic inflammatory disease. Vaginal microbiota, rich in taxa of strict anaerobic species, disrupts the mucosal epithelial barrier through secretion of metabolites and enzymes that mediate inflammation. Advancements in next-generation sequencing technologies such as whole-genome sequencing have led to deeper profiling of the vaginal microbiome and further study of its potential role in HIV pathogenesis and treatment. Until recently data on the composition of the vaginal microbiome in sub-Saharan Africa have been limited; however, a number of studies have been published that highlight the critical role of vaginal microbiota in disease and health in African women. This article reviews these recent findings and identifies gaps in knowledge about variations in female genital commensal bacteria that could provide vital information to improve the effectiveness of interventions to prevent HIV and other sexually transmitted infections. In addition, we review the effects of pregnancy, contraception, and sexual practices on vaginal microbiome and the potential of vaginal microbiota on HIV transmission and prevention. A better understanding of the role of vaginal microbiota in host susceptibility to HIV infection and its prevention among African women could inform the development of novel local and systemic interventions to minimize new HIV infections among high-risk women.

blaVIM- and blaOXA-mediated carbapenem resistance among Acinetobacter baumannii and Pseudomonas aeruginosa isolates from the Mulago hospital intensive care unit in Kampala, Uganda.

BACKGROUND: Between January 2015 and July 2017, we investigated the frequency of carbapenem resistant Acinetobacter baumannii (CRAB) and carbapenem resistant Pseudomonas aeruginosa (CRPA) at the Mulago Hospital intensive care unit (ICU) in Kampala, Uganda. Carbapenemase production and carbapenemase gene carriage among CRAB and CRPA were determined; mobility potential of carbapenemase genes via horizontal gene transfer processes was also studied. METHODS: Clinical specimens from 9269 patients were processed for isolation of CRAB and CRPA. Drug susceptibility testing was performed with the disk diffusion method. Carriage of carbapenemase genes and class 1 integrons was determined by PCR. Conjugation experiments that involved blaVIM positive CRAB/CRPA (donors) and sodium azide resistant Escherichia coli J53 (recipient) were performed. RESULTS: The 9269 specimens processed yielded 1077 and 488 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, respectively. Of these, 2.7% (29/1077) and 7.4% (36/488) were confirmed to be CRAB and CRPA respectively, but 46 were available for analysis (21 CRAB and 25 CRPA). Majority of specimens yielding CRAB and CRPA were from the ICU (78%) while 20 and 2% were from the ENT (Ear Nose & Throat) Department and the Burns Unit, respectively. Carbapenemase assays performed with the MHT assay showed that 40 and 33% of CRPA and CRAB isolates respectively, were carbapenemase producers. Also, 72 and 48% of CRPA and CRAB isolates respectively, were metallo-beta-lactamase producers. All the carbapenemase producing isolates were multidrug resistant but susceptible to colistin. blaVIM was the most prevalent carbapenemase gene, and it was detected in all CRAB and CRPA isolates while blaOXA-23 and blaOXA-24 were detected in 29 and 24% of CRAB isolates, respectively. Co-carriage of blaOXA-23 and blaOXA-24 occurred in 14% of CRAB isolates. Moreover, 63% of the study isolates carried class 1 integrons; of these 31% successfully transferred blaVIM to E. coli J53. CONCLUSIONS: CRAB and CRPA prevalence at the Mulago Hospital ICU is relatively low but carbapenemase genes especially blaVIM and blaOXA-23 are prevalent among them. This requires strengthening of infection control practices to curb selection and transmission of these strains in the hospital.

Species, antibiotic susceptibility profiles and van gene frequencies among enterococci isolated from patients at Mulago National Referral Hospital in Kampala, Uganda.

BACKGROUND: The increase in drug resistance to affordable antibiotics used to treat Gram positive bacterial infections has complicated the management of enterococcal infections. Resistance to vancomycin, one of the most powerful antibiotics, is of utmost concern as both intrinsic and acquired forms of resistance do occur in enterococci. This cross-sectional study aimed to determine the species, antibiotic susceptibility profiles and vanA/vanB gene frequencies among enterococci isolated from patients at Mulago Hospital in Kampala, Uganda. METHODS: Between November 2011 and October 2012, stool, urine, sputum and blood samples, as well as vaginal, endocervical, pus, ear and urethra swabs from 3229 patients were processed for isolation of bacteria, yielding 162 enterococci of which 115 were available for analysis (one isolate per specimen/patient). Species-level confirmation and susceptibility testing were determined with the Phoenix™ AST/ID Automated System, while vanA/vanB gene carriage was determined by PCR. RESULTS: Species-level identification revealed 72 isolates of E. faecalis, 20 E. gallinarum/casseliflavus, 5 E. faecium, 4 E. raffinosus and 2 isolates each for E. hirae and E. durans. Ten isolates could not be identified to species level. Antibiotic resistance was generally low especially to ampicillin, quinolones, nitrofurantoin, glycopeptides and linezolid, but high for erythromycin and tetracycline. Equally, vanA and vanB gene frequencies were low (i.e. 15.8 and 7.9%, respectively) and detected only in E. casseliflavus/gallinarum species that are intrinsically resistant to vancomycin. Vancomycin resistant isolates of E. faecalis and E. faecium were not detected. CONCLUSIONS: Enterococcus species are frequent in clinical specimens at Mulago Hospital but they are highly susceptible to common antibiotics especially ampicillin. While vancomycin resistant enterococcal (VRE) isolates of E. faecium and E. faecalis are rare in the hospital and frequency of multidrug resistance is low, non-faecium and non-faecalis VRE isolates (i.e. E. gallinarum/casseliflavus) are frequent, some with VanA/VanB (high-level) vancomycin resistance. Therefore, species-level identification of enterococci is necessary in resource limited settings to guide infection control and treatment of enterococcal infections.

Mycobacterium tuberculosis thymidylate kinase antigen assays for designating incipient, high-risk latent M.tb infection.

BACKGROUND: Precise designation of high risk forms of latent Mycobacterium tuberculosis-M.tb infections (LTBI) is impossible. Delineation of high-risk LTBI can, however, allow for chemoprophylaxis and curtail majority cases of active tuberculosis (ATB). There is epidemiological evidence to support the view that LTBI in context of HIV-1 co-infection is high-risk for progression to ATB relative to LTBI among HIV-ve persons. We recently showed that assays of M.tb thymidylate kinase (TMKmt) antigen and host specific IgG can differentiate ATB from LTBI and or no TB (NTB, or healthy controls). In this study, we aimed to expose the differential levels of TMKmt Ag among HIV+ve co-infected LTBI relative to HIV-ve LTBI as a strategy to advance these assays for designating incipient LTBI. METHODS: TMKmt host specific IgM and IgG detection Enzyme Immuno-Assays (EIA) were conducted on 40 TB exposed house-hold contacts (22 LTBI vs. 18 no TB (NTB) by QunatiFERON-TB GOLD®); and TMKmt Ag detection EIA done on 82 LTBI (46 HIV+ve vs 36 HIV-ve) and 9 NTB (American donors). Purified recombinant TMKmt protein was used as positive control for the Ag assays. RESULTS: IgM levels were found to be equally low across QuantiFERON-TB GOLD® prequalified NTB and TB exposed house-hold contacts. Higher TMKmt host specific IgG trends were found among TB house-hold contacts relative to NTB controls. TMKmt Ag levels among HIV+ve LTBI were 0.2676 ± 0.0197 (95% CI: 0.2279 to 0.3073) relative to 0.1069 ± 0.01628 (95% CI: 0.07385 to 0.14) for HIV-ve LTBI (supporting incipient nature of LTBI in context of HIV-1 co-infection). NTB had TMKmt Ag levels of 0.1013 ± 0.02505 (5% CI: 0.0421 to 0.1606) (intimating that some were indeed LTBI). CONCLUSIONS: TMKmt Ag levels represent a novel surrogate biomarker for high-risk LTBI, while host-specific IgG can be used to designate NTB from LTBI.

Mycobacterium tuberculosis Uganda II is more susceptible to rifampicin and isoniazid compared to Beijing and Delhi/CAS families.

BACKGROUND: Mycobacterium tuberculosis Uganda family is the predominant cause of tuberculosis in Uganda. Reasons for this are not clear but are likely to be due to the rampant person-to-person transmission or delayed susceptibility of the organism to drugs during treatment, which may lead to continuous shedding of infectious bacilli, among others. The objective of this study was to determine in vitro, the susceptibility patterns of M. tuberculosis Uganda family compared with Beijing and Delhi/CAS, other M. tuberculosis sub-lineages that also circulate in Uganda but are not as prevalent. The comparisons were made after 10 days of exposure of the strains to Rifampicin and Isoniazid, the most important first-line anti-tuberculosis drugs. METHODS: Minimum inhibitory concentrations (MICs) for three Isoniazid- and Rifampicin-susceptible M. tuberculosis strains (Uganda II, Beijing and Delhi/CAS families) were determined by micro-dilution plate assay. Killing curves for each strain were deduced from colony forming units after exposure to Isoniazid and Rifampicin on days 0, 2, 4, 6, 8, and 10 under aerobic and oxygen-depleted conditions. Data were analyzed with GraphPad Prism 5 software. RESULTS: The MIC for Isoniazid was 0.05 µg/ml for M. tuberculosis Uganda II, and 0.03 µg/ml for M. tuberculosis Beijing and Delhi/CAS. Rifampicin MIC was 1 µg/ml for M. tuberculosis Uganda II, and 0.12 µg/ml for Beijing and Delhi/CAS. At low Rifampicin (0.03-2.5 µg/ml) and Isoniazid (0.12-5 µg/ml) concentrations under aerobic conditions, there was no significant difference in susceptibility patterns between M. tuberculosis Uganda II and Beijing or Delhi/CAS. However, at high Rifampicin (5 µg/ml) and Isoniazid (1.25 µg/ml) concentrations under oxygen-depleted conditions, M. tuberculosis Uganda II was more susceptible to the drugs compared with Beijing or Delhi/CAS families. CONCLUSION: The predominance of M. tuberculosis Uganda II family as the main causative agent of tuberculosis in Uganda is not attributed to its susceptibility behavior to Isoniazid and Rifampicin. Probably, its predominance is due to differences in the immune defenses in the general population against the strains, given that Beijing and Delhi/CAS families may have been introduced more recently. Further research beyond susceptibility to anti-tuberculosis drugs is required to fully explore tuberculosis strain predominance in Uganda.

The T2 Mycobacterium tuberculosis genotype, predominant in Kampala, Uganda, shows negative correlation with antituberculosis drug resistance.

Surveillance of the circulating Mycobacterium tuberculosis complex (MTC) strains in a given locality is important for understanding tuberculosis (TB) epidemiology. We performed molecular epidemiological studies on sputum smear-positive isolates that were collected for anti-TB drug resistance surveillance to establish the variability of MTC lineages with anti-TB drug resistance and HIV infection. Spoligotyping was performed to determine MTC phylogenetic lineages. We compared patients' MTC lineages with drug susceptibility testing (DST) patterns and HIV serostatus. Out of the 533 isolates, 497 (93.2%) had complete DST, PCR, and spoligotyping results while 484 (90.1%) participants had results for HIV testing. Overall, the frequency of any resistance was 75/497 (15.1%), highest among the LAM (34.4%; 95% confidence interval [CI], 18.5 to 53.2) and lowest among the T2 (11.5%; 95% CI, 7.6 to 16.3) family members. By multivariate analysis, LAM (adjusted odds ratio [OR(adj)], 5.0; 95% CI, 2.0 to 11.9; P < 0.001) and CAS (OR(adj), 2.9; 95% CI, 1.4.0 to 6.3; P = 0.006) families were more likely to show any resistance than was T2. All other MTC lineages combined were more likely to be resistant to any of the anti-TB drugs than were the T2 strains (OR(adj), 1.7; 95% CI, 1.0 to 2.9; P = 0.040). There were no significant associations between multidrug resistance and MTC lineages, but numbers of multidrug-resistant TB strains were small. No association was established between MTC lineages and HIV status. In conclusion, the T2 MTC lineage negatively correlates with anti-TB drug resistance, which might partly explain the reported low levels of anti-TB drug resistance in Kampala, Uganda. Patients' HIV status plays no role with respect to the MTC lineage distribution.

The Mycobacterium tuberculosis Uganda II family and resistance to first-line anti-tuberculosis drugs in Uganda.

BACKGROUND: The global increase in the burden of multidrug-resistant tuberculosis (MDR-TB) underscores an urgent need for data on factors involved in generation and spread of TB drug resistance. We performed molecular analyses on a representative sample of Mycobacterium tuberculosis (MTB) isolates. Basing on findings of the molecular epidemiological study in Kampala, we hypothesized that the predominant MTB strain lineage in Uganda is negatively associated with anti-TB drug resistance and we set out to test this hypothesis. METHODS: We extracted DNA from mycobacterial isolates collected from smear-positive TB patients in the national TB drug resistance survey and carried out IS6110-PCR. To identify MTB lineages/sub lineages RT-PCR SNP was performed using specific primers and hybridization probes and the 'melting curve' analysis was done to distinguish the Uganda II family from other MTB families. The primary outcome was the distribution of the Uganda II family and its associations with anti-TB drug resistance and HIV infection. RESULTS: Out of the 1537 patients enrolled, MTB isolates for 1001 patients were available for SNP analysis for identification of Uganda II family, of which 973 (97%) had conclusive RT-PCR results. Of these 422 (43.4%) were of the Uganda II family, mostly distributed in the south west zone (55.0%; OR = 4.6 for comparison with other zones; 95% CI 2.83-7.57; p < 0.001) but occurred in each of the other seven geographic zones at varying levels. Compared to the Uganda II family, other genotypes as a group were more likely to be resistant to any anti-TB drug (OR(adj) =2.9; 95% CI 1.63-5.06; p = 0.001) or MDR (OR(adj) 4.9; 95% CI, 1.15-20.60; p = 0.032), even after adjusting for geographic zone, patient category, sex, residence and HIV status. It was commonest in the 25-34 year age group 159/330 (48.2%). No association was observed between Uganda II family and HIV infection. CONCLUSION: The Uganda II family is a major cause of morbidity due to TB in all NTLP zones in Uganda. It is less likely to be resistant to anti-TB drugs than other MTB strain lineages.

CTX-M, TEM, and SHV Genes in Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp Isolated from Hematologic Cancer Patients with Bacteremia in Uganda.

Purpose: We determined the phenotypic resistance to third-generation cephalosporins, phenotypic extended spectrum beta-lactamase (ESBL) prevalence, and genotypic prevalence of ESBL-encoding genes blaCTX-M, blaTEM, and blaSHV in Enterobacteriaceae isolated from hematologic cancer patients with febrile neutropenia and bacteremia at the Uganda Cancer Institute (UCI). Patients and Methods: Blood cultures from hematologic cancer patients with febrile neutropenia were processed in BACTEC 9120. E. coli, K. pneumoniae, and Enterobacter spp. isolates were identified using conventional biochemical methods. Antimicrobial susceptibility tests, phenotypic ESBL characterization, and genotypic characterization of the ESBL-encoding genes blaCTX-M, blaTEM, and blaSHV were determined for pure isolates of E. coli, K. pneumoniae, and Enterobacter spp. Results: Two hundred and two patients were included in the study. Median age of patients was 19 years (IQR: 10-30 years). Majority (N=119, 59%) were male patients. Sixty (30%) of the participants had at least one febrile episode due to Enterobacteriaceae. Eighty-three organisms were isolated with E. coli being predominant (45, 54%). Seventy-nine (95%) Enterobacteriaceae were multidrug resistant. The ESBL phenotype was detected in 54/73 (74%) of Enterobacteriaceae that were resistant to third-generation cephalosporins. A higher proportion of Enterobacteriaceae with ESBL-positive phenotype were resistant to piperacillin-tazobactam (p=0.024), gentamicin (p=0.000), ciprofloxacin (p=0.000), and cotrimoxazole (p=0.000) compared to Enterobacteriaceae, which were sensitive to third-generation cephalosporins. The organisms were more susceptible to carbapenems and chloramphenicol than resistant. ESBL-encoding genes (blaCTX-M, blaTEM, and blaSHV) were detected in 55 (75%) of the 73 Enterobacteriaceae that were resistant to third-generation cephalosporins. BlaCTX-M, was the most common ESBL-encoding gene identified with 50 (91%). Conclusion: ESBL-producing Enterobacteriaceae are a predominant cause of bacteremia in hematologic cancer patients at UCI. The most common ESBL-encoding gene identified in the ESBL-PE was blaCTX-M. Resistance to imipenem and meropenem was low.

HIVseqDB: a portable resource for NGS and sample metadata integration for HIV-1 drug resistance analysis.

Summary: Human immunodeficiency virus (HIV) remains a public health threat, with drug resistance being a major concern in HIV treatment. Next-generation sequencing (NGS) is a powerful tool for identifying low-abundance drug resistance mutations (LA-DRMs) that conventional Sanger sequencing cannot reliably detect. To fully understand the significance of LA-DRMs, it is necessary to integrate NGS data with clinical and demographic data. However, freely available tools for NGS-based HIV-1 drug resistance analysis do not integrate these data. This poses a challenge in interpretation of the impact of LA-DRMs, mainly for resource-limited settings due to the shortage of bioinformatics expertise. To address this challenge, we present HIVseqDB, a portable, secure, and user-friendly resource for integrating NGS data with associated clinical and demographic data for analysis of HIV drug resistance. HIVseqDB currently supports uploading of NGS data and associated sample data, HIV-1 drug resistance data analysis, browsing of uploaded data, and browsing and visualizing of analysis results. Each function of HIVseqDB corresponds to an individual Django application. This ensures efficient incorporation of additional features with minimal effort. HIVseqDB can be deployed on various computing environments, such as on-premises high-performance computing facilities and cloud-based platforms. Availability and implementation: HIVseqDB is available at https://github.com/AlfredUg/HIVseqDB. A deployed instance of HIVseqDB is available at https://hivseqdb.org.

Determinants of QuantiFERON Plus-diagnosed tuberculosis infection in adult Ugandan TB contacts: A cross-sectional study.

BACKGROUND: The tuberculin skin test is commonly used to diagnose latent tuberculosis infection (LTBI) in resource-limited settings, but its specificity is limited by factors including cross-reactivity with BCG vaccine and environmental mycobacteria. Interferon-gamma release assays (IGRA) overcome this problem by detecting M. tuberculosis complex-specific responses, but studies to determine risk factors for IGRA-positivity in high TB burden settings are lacking. METHODS: We conducted a cross-sectional study to determine factors associated with a positive IGRA by employing the QuantiFERON-TB® Gold-plus (QFT Plus) assay in a cohort of asymptomatic adult TB contacts in Kampala, Uganda. Multivariate logistic regression analysis with forward stepwise logit function was employed to identify independent correlates of QFT Plus-positivity. RESULTS: Of the 202 participants enrolled, 129/202 (64%) were female, 173/202 (86%) had a BCG scar, and 67/202 (33%) were HIV-infected. Overall, 105/192 (54%, 95% CI 0.48-0.62) participants had a positive QFT Plus result. Increased risk of QFT-Plus positivity was independently associated with casual employment/unemployment vs. non-casual employment (adjusted odds ratio (aOR) 2.18, 95% CI 1.01-4.72), a family vs. non-family relation to the index patient (aOR 2.87, 95% CI 1.33-6.18), living in the same vs. a different house as the index (aOR 3.05, 95% CI 1.28-7.29), a higher body mass index (BMI) (aOR per additional kg/m2 1.09, 95% CI 1.00-1.18) and tobacco smoking vs. not (aOR 2.94, 95% CI 1.00-8.60). HIV infection was not associated with QFT-Plus positivity (aOR 0.91, 95% CI 0.42-1.96). CONCLUSION: Interferon Gamma Release Assay positivity in this study population was lower than previously estimated. Tobacco smoking and BMI were determinants of IGRA positivity that were previously unappreciated.

Phylogenomic analysis uncovers a 9-year variation of Uganda influenza type-A strains from the WHO-recommended vaccines and other Africa strains.

Genetic characterisation of circulating influenza viruses directs annual vaccine strain selection and mitigation of infection spread. We used next-generation sequencing to locally generate whole genomes from 116 A(H1N1)pdm09 and 118 A(H3N2) positive patient swabs collected across Uganda between 2010 and 2018. We recovered sequences from 92% (215/234) of the swabs, 90% (193/215) of which were whole genomes. The newly-generated sequences were genetically and phylogenetically compared to the WHO-recommended vaccines and other Africa strains sampled since 1994. Uganda strain hemagglutinin (n = 206), neuraminidase (n = 207), and matrix protein (MP, n = 213) sequences had 95.23-99.65%, 95.31-99.79%, and 95.46-100% amino acid similarity to the 2010-2020 season vaccines, respectively, with several mutated hemagglutinin antigenic, receptor binding, and N-linked glycosylation sites. Uganda influenza type-A virus strains sequenced before 2016 clustered uniquely while later strains mixed with other Africa and global strains. We are the first to report novel A(H1N1)pdm09 subclades 6B.1A.3, 6B.1A.5(a,b), and 6B.1A.6 (± T120A) that circulated in Eastern, Western, and Southern Africa in 2017-2019. Africa forms part of the global influenza ecology with high viral genetic diversity, progressive antigenic drift, and local transmissions. For a continent with inadequate health resources and where social distancing is unsustainable, vaccination is the best option. Hence, African stakeholders should prioritise routine genome sequencing and analysis to direct vaccine selection and virus control.

Sputum Microbiome and Chronic Obstructive Pulmonary Disease in a Rural Ugandan Cohort of Well-Controlled HIV Infection.

Sub-Saharan Africa has increased morbidity and mortality related to chronic obstructive pulmonary disease (COPD). COPD among people living with HIV (PLWH) has not been well studied in this region, where HIV/AIDS is endemic. Increasing evidence suggests that respiratory microbial composition plays a role in COPD severity. Therefore, we aimed to investigate microbiome patterns and associations among PLWH with COPD in Sub-Saharan Africa. We conducted a cross-sectional study of 200 adults stratified by HIV and COPD in rural Uganda. Induced sputum samples were collected as an easy-to-obtain proxy for the lower respiratory tract microbiota. We performed 16S rRNA gene sequencing and used PICRUSt2 (version 2.2.3) to infer the functional profiles of the microbial community. We used a statistical tool to detect changes in specific taxa that searches and adjusts for confounding factors such as antiretroviral therapy (ART), age, sex, and other participant characteristics. We could cluster the microbial community into three community types whose distribution was shown to be significantly impacted by HIV. Some genera, e.g., Veillonella, Actinomyces, Atopobium, and Filifactor, were significantly enriched in HIV-infected individuals, while the COPD status was significantly associated with Gammaproteobacteria and Selenomonas abundance. Furthermore, reduced bacterial richness and significant enrichment in Campylobacter were associated with HIV-COPD comorbidity. Functional prediction using PICRUSt2 revealed a significant depletion in glutamate degradation capacity pathways in HIV-positive patients. A comparison of our findings with an HIV cohort from the United Kingdom revealed significant differences in the sputum microbiome composition, irrespective of viral suppression. IMPORTANCE Even with ART available, HIV-infected individuals are at high risk of suffering comorbidities, as shown by the high prevalence of noninfectious lung diseases in the HIV population. Recent studies have suggested a role for the respiratory microbiota in driving chronic lung inflammation. The respiratory microbiota was significantly altered among PLWH, with disease persisting up to 3 years post-ART initiation and HIV suppression. The community structure and diversity of the sputum microbiota in COPD are associated with disease severity and clinical outcomes, both in stable COPD and during exacerbations. Therefore, a better understanding of the sputum microbiome among PLWH could improve COPD prognostic and risk stratification strategies. In this study, we observed that in a virologically suppressed HIV cohort in rural Uganda, we could show differences in sputum microbiota stratified by HIV and COPD, reduced bacterial richness, and significant enrichment in Campylobacter associated with HIV-COPD comorbidity.

Molecular Epidemiology and Evolutionary Dynamics of Human Influenza Type-A Viruses in Africa: A Systematic Review.

Genomic characterization of circulating influenza type-A viruses (IAVs) directs the selection of appropriate vaccine formulations and early detection of potentially pandemic virus strains. However, longitudinal data on the genomic evolution and transmission of IAVs in Africa are scarce, limiting Africa's benefits from potential influenza control strategies. We searched seven databases: African Journals Online, Embase, Global Health, Google Scholar, PubMed, Scopus, and Web of Science according to the PRISMA guidelines for studies that sequenced and/or genomically characterized Africa IAVs. Our review highlights the emergence and diversification of IAVs in Africa since 1993. Circulating strains continuously acquired new amino acid substitutions at the major antigenic and potential N-linked glycosylation sites in their hemagglutinin proteins, which dramatically affected vaccine protectiveness. Africa IAVs phylogenetically mixed with global strains forming strong temporal and geographical evolution structures. Phylogeographic analyses confirmed that viral migration into Africa from abroad, especially South Asia, Europe, and North America, and extensive local viral mixing sustained the genomic diversity, antigenic drift, and persistence of IAVs in Africa. However, the role of reassortment and zoonosis remains unknown. Interestingly, we observed substitutions and clades and persistent viral lineages unique to Africa. Therefore, Africa's contribution to the global influenza ecology may be understated. Our results were geographically biased, with data from 63% (34/54) of African countries. Thus, there is a need to expand influenza surveillance across Africa and prioritize routine whole-genome sequencing and genomic analysis to detect new strains early for effective viral control.

Medical Mycology dissertation topics require prioritisation among Postgraduate Microbiology trainees of Makerere University, Uganda.

Background: As elsewhere worldwide, there is an increasing burden of fungal diseases in Uganda. However, expertise in medical mycology (the study of fungal diseases of medical importance) among clinicians and laboratory personnel remains low. Objective: This study sought to determine the proportion of dissertations on medical mycology among postgraduate medical microbiology trainees at the College of Health Sciences, Makerere University, Uganda. Methods: We retrospectively reviewed the topics of dissertations submitted to the Departments of Medical Microbiology and Immunology & Molecular Biology from 2011 through 2018. The proportion of dissertation topics on medical mycology was analysed using descriptive statistics. Results: A total of 152 dissertations were retrieved. Of these, only 5 (3.3%) were on medical mycology compared to bacteriology (50.7%, n = 77), virology (27.6%, n = 42), parasitology (14.5%, n = 22) and immunology (4.0%, n = 6). Of the 5 dissertations on fungal diseases, the distribution was as follows: cryptococcal meningitis (40%, n = 2), Candidiasis (20%, n = 1), superficial mycoses (20%, n = 1) and other invasive fungal diseases (20%, n = 1). The most common method that was used for studying the fungal diseases was culture 60%, n = 3. Conclusion: There is limited research on medical mycology among the postgraduate medical microbiology trainees of Makerere University, Uganda.

QuasiFlow: a Nextflow pipeline for analysis of NGS-based HIV-1 drug resistance data.

Summary: Next-generation sequencing (NGS) enables reliable detection of resistance mutations in minority variants of human immunodeficiency virus type 1 (HIV-1). There is paucity of evidence for the association of minority resistance to treatment failure, and this requires evaluation. However, the tools for analyzing HIV-1 drug resistance (HIVDR) testing data are mostly web-based which requires uploading data to webservers. This is a challenge for laboratories with internet connectivity issues and instances with restricted data transfer across networks. We present QuasiFlow, a pipeline for reproducible analysis of NGS-based HIVDR testing data across different computing environments. Since QuasiFlow entirely depends on command-line tools and a local copy of the reference database, it eliminates challenges associated with uploading HIV-1 NGS data onto webservers. The pipeline takes raw sequence reads in FASTQ format as input and generates a user-friendly report in PDF/HTML format. The drug resistance scores obtained using QuasiFlow were 100% and 99.12% identical to those obtained using web-based HIVdb program and HyDRA web respectively at a mutation detection threshold of 20%. Availability and implementation: QuasiFlow and corresponding documentation are publicly available at https://github.com/AlfredUg/QuasiFlow. The pipeline is implemented in Nextflow and requires regular updating of the Stanford HIV drug resistance interpretation algorithm. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Microbial characteristics of dental caries in HIV positive individuals.

Background: Dental caries is a multifactorial disease that affects many people. Even though microorganisms play a crucial role in causing dental caries, diagnosis is routinely macroscopic. In order to improve early detection especially in HIV patients who are disproportionately affected, there is need to reconcile the macroscopic and microscopic characteristics of dental caries. Therefore, the aim of this study was to characterize the oral microbiota profile along the decayed, missing, filled teeth (DMFT) index using amplicon sequencing data. Methods: Amplicon sequencing of the V6-V8 region of the 16S rRNA gene was done on DNA recovered from whole unstimulated saliva of 59 HIV positive and 29 HIV negative individuals. The microbial structure, composition and co-occurrence networks were characterized using QIIME-2, Phyloseq, Microbiome-1.9.2 and Metacoder in R. Results: We characterized the oral microbiota into 2,093 operational taxonomic units (OTUs), 21 phyla and 239 genera from 2.6 million high quality sequence reads. While oral microbiota did not cluster participants into distinct groups that track with the DMFT index, we observed the following: (a) The proportion of accessory microbiota was highest in the high DMFT category while the core size (∼50% of richness) remained relatively stable across all categories. (b) The abundance of core genera such as Stomatobaculum, Peptostreptococcus and Campylobacter was high at onset of dental caries, (c) A general difference in oral microbial biomass. (d) The onset of dental caries (low DMFT) was associated with significantly lower oral microbial entropy. Conclusions: Although oral microbial shifts along the DMFT index were not distinct, we demonstrated the potential utility of microbiota dynamics to characterize oral disease. Therefore, we propose a microbial framework using the DMFT index to better understand dental caries among HIV positive people in resource limited settings.