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.

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.

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.

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.

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.

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.

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.

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.

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.

Prevalence and antimicrobial resistance profiles of Neisseria gonorrhea and Chlamydia trachomatis isolated from individuals attending STD clinics in Kampala, Uganda.

Background: Sexually transmitted diseases (STDs) management in sub-Saharan Africa is syndromic but molecular diagnostics provide quicker, sensitive diagnosis and treatment. Effective STD control hinges on identification and treatment of infected persons and sexual partner contact tracing. Objectives: This study assessed feasibility of using the Xpert CT/NG test to identify prevalent Chlamydia trachomatis (CT) and Neisseria gonorrhea (NG) infections among STD clinic attendees and their sexual partners and tested for antimicrobial resistance for N. gonorrhea. Methods: A cross-sectional study was conducted at 4 outpatient STD clinics in Kampala, Uganda from February 2019 to October 2019. Participants received a syndromic diagnosis, were tested for NG and CT, as well as their sexual partners. Urine (men) and high vaginal swabs (women) were collected, examined using Xpert CT/NG assay. A total of 79 participants were enrolled at baseline of whom 25 had CT/NG. 21 partners of infected baseline participants and 7 partners of the 21 primary partners were enrolled. Results: The mean age of the reported sexual partners was 26 (18-43) years. The prevalence of NG was 25% at baseline and 18 % for CT. Nine (11.4%) people were dually infected. Men were more likely to have NG (p<0.001) at multivariable level. Two participants tested HIV-1 positive. On microbiological culture, 8 samples (2.5%) grew NG and all were resistant to penicillin, ciprofloxacin. For CT, we found a preponderance of the F-serovar in this population. Conclusion: The most prevalent organism was Neisseria gonorrhea. Generally, the prevalence of CT and NG was high. Infection proportions increased among primary partners, particularly women. Etiologic testing without partner tracing and treatment may underestimate burden of CT/NG in this population and contribute to re-infection.

Prevalence and antimicrobial resistance profiles of Neisseria gonorrhea and Chlamydia trachomatis isolated from individuals attending STD clinics in Kampala, Uganda

Background: Sexually transmitted diseases (STDs) management in sub-Saharan Africa is syndromic but molecular diagnostics provide quicker, sensitive diagnosis and treatment. Effective STD control hinges on identification and treatment of infected persons and sexual partner contact tracing. Objectives: This study assessed feasibility of using the Xpert CT/NG test to identify prevalent Chlamydia trachomatis (CT) and Neisseria gonorrhea (NG) infections among STD clinic attendees and their sexual partners and tested for antimicrobial resistance for N. gonorrhea. Methods: A cross-sectional study was conducted at 4 outpatient STD clinics in Kampala, Uganda from February 2019 to October 2019. Participants received a syndromic diagnosis, were tested for NG and CT, as well as their sexual partners. Urine (men) and high vaginal swabs (women) were collected, examined using Xpert CT/NG assay. A total of 79 participants were enrolled at baseline of whom 25 had CT/NG. 21 partners of infected baseline participants and 7 partners of the 21 primary partners were enrolled. Results: The mean age of the reported sexual partners was 26 (18-43) years. The prevalence of NG was 25% at baseline and 18 % for CT. Nine (11.4%) people were dually infected. Men were more likely to have NG (p<0.001) at multivariable level. Two participants tested HIV-1 positive. On microbiological culture, 8 samples (2.5%) grew NG, and all were resistant to penicillin, ciprofloxacin. For CT, we found a preponderance of the F-serovar in this population. Conclusion: The most prevalent organism was Neisseria gonorrhea. Generally, the prevalence of CT and NG was high. Infection proportions increased among primary partners, particularly women. Etiologic testing without partner tracing and treatment may underestimate burden of CT/NG in this population and contribute to re-infection

Microbiological Contamination of Mobile Phones and Mobile Phone Hygiene of Final-Year Medical Students in Uganda: A Need for Educational Intervention.

INTRODUCTION: Contaminated mobile phones act as reservoirs for organisms causing hospital-acquired infections (HAI). Little is known about medical school students' awareness of infection prevention and control (IPC) regarding mobile phone use among medical students. We demonstrated the presence of organisms on mobile phones of final-year medical students at Makerere University College of Health Sciences and evaluated their awareness of IPC regarding mobile phone hygiene and use in a hospital setting. METHODS: In this cross-sectional study, organisms from swabs obtained from 79 medical students' mobile phones were identified and antimicrobial susceptibility test carried out using standard biochemical tests and the automated BD Phoenix instrument. Data were collected using a self-administered questionnaire to assess the students' awareness. The analysis was carried out using STATA software version 16. RESULTS: Seventy (88.6%) mobile phones were contaminated with at least one organism. One hundred forty-eight bacteria were isolated, of which 123 (83.1%) were Gram-positive, 24 (16.2%) were Gram-negative, and 1 (0.7%) was yeast (Candida spp). Coagulase negative staphylococci were the most frequently isolated among Gram-positive bacteria. Acinetobacter baumannii were the most frequently isolated among Gram-negative bacteria. The average IPC practical score regarding mobile phone hygiene (34%) was significantly lower than the average IPC awareness score (77%) (p ≤ 0.0001). Seventy-four (93.7%) students use their phones while rotating in the various wards. Forty (50.6%) of the students cleaned their phones with alcohol-based sanitizer after rotations in the ward. Thirty-five (44.3%) students were aware of IPC programs in the hospital they rotated in. CONCLUSION: There is a high prevalence of bacterial contamination from mobile phones of medical students. The students had lower IPC practical scores compared to IPC awareness scores regarding mobile phone hygiene irrespective of the ward of rotation. Curriculum of final-year medical students should include IPC-related topics, which incorporate practical skills.

Performance and cost-effectiveness of a pooled testing strategy for SARS-CoV-2 using real-time polymerase chain reaction in Uganda.

Real-time polymerase chain reaction (RT-PCR) remains the gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This study tested the performance of a pooled testing strategy for RT-PCR and its cost-effectiveness. In total, 1280 leftover respiratory samples collected between 19 April and 6 May 2021 were tested in 128 pools of 10 samples each, out of which 16 pools were positive. The positivity rate of the unpooled samples was 1.9% (24/1280). After parallel testing using the individual and pooled testing strategies, positive agreement was 100% and negative agreement was 99.8%. The overall median cycle threshold (Ct) value of the unpooled samples was 29.8 (interquartile range 22.3-34.3). Pools that remained positive when compared with the results of individual samples had lower median Ct values compared with those that turned out to be negative (28.8 versus 34.8; P=0.0.035). Pooled testing reduced the cost >4-fold. Pooled testing may be a more cost-effective approach to diagnose SARS-CoV-2 in resource-limited settings without compromising diagnostic performance.

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.

Unmapped exome reads implicate a role for Anelloviridae in childhood HIV-1 long-term non-progression.

Human immunodeficiency virus (HIV) infection remains a significant public health burden globally. The role of viral co-infection in the rate of progression of HIV infection has been suggested but not empirically tested, particularly among children. We extracted and classified 42 viral species from whole-exome sequencing (WES) data of 813 HIV-infected children in Botswana and Uganda categorised as either long-term non-progressors (LTNPs) or rapid progressors (RPs). The Ugandan participants had a higher viral community diversity index compared to Batswana (p = 4.6 × 10-13), and viral sequences were more frequently detected among LTNPs than RPs (24% vs 16%; p = 0.008; OR, 1.9; 95% CI, 1.6-2.3), with Anelloviridae showing strong association with LTNP status (p = 3 × 10-4; q = 0.004, OR, 3.99; 95% CI, 1.74-10.25). This trend was still evident when stratified by country, sex, and sequencing platform, and after a logistic regression analysis adjusting for age, sex, country, and the sequencing platform (p = 0.02; q = 0.03; OR, 7.3; 95% CI, 1.6-40.5). Torque teno virus (TTV), which made up 95% of the Anelloviridae reads, has been associated with reduced immune activation. We identify an association between viral co-infection and prolonged AIDs-free survival status that may have utility as a biomarker of LTNP and could provide mechanistic insights to HIV progression in children, demonstrating the added value of interrogating off-target WES reads in cohort studies.

Plasmodium interspecies interactions during a period of increasing prevalence of Plasmodium ovale in symptomatic individuals seeking treatment: an observational study.

BACKGROUND: The epidemiology and severity of non-falciparum malaria in endemic settings has garnered little attention. We aimed to characterise the prevalence, interaction, clinical risk factors, and temporal trends of non-falciparum Plasmodium species among symptomatic individuals presenting at health-care facilities in endemic settings of Kenya. METHODS: We diagnosed and analysed infecting malaria species (Plasmodium falciparum, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and Plasmodium malariae) via PCR in clinical samples collected between March 1, 2008, and Dec 31, 2016, from six hospitals located in different regions of Kenya. We recruited patients aged 6 months or older who presented at outpatient departments with symptoms of malaria or tested positive for uncomplicated malaria by malaria rapid diagnostic test. Descriptive statistics were used to describe the prevalence and distribution of Plasmodium species. A statistical model was designed and used for estimating the frequency of Plasmodium species and assessing interspecies interactions. Mixed-effect linear regression models with random slopes for each location were used to test for change in prevalence over time. FINDINGS: Samples from 2027 symptomatic participants presenting at care facilities were successfully analysed for all Plasmodium species. 1469 (72·5%) of the samples were P falciparum single-species infections, 523 (25·8%) were mixed infections, and only 35 (1·7%) were single non-falciparum species infections. 452 (22·3%) were mixed infections containing P ovale spp. A likelihood-based model calculation of the population frequency of each species estimated a significant within-host interference between P falciparum and P ovale curtisi. Mixed-effect logistic regression models identified a significant increase in P ovale wallikeri (2·1% per year; p=0·043) and P ovale curtisi (0·7% per year; p=0·0002) species over time, with a reciprocal decrease in P falciparum single-species infections (2·5% per year; p=0·0065). The frequency of P malariae infections did not significantly change over time. Risk of P falciparum infections presenting with fever was lower if co-infected with P malariae (adjusted odds ratio 0·43, 95% CI 0·25-0·74; p=0·0023). INTERPRETATION: Our results show a prevalence of non-falciparum species infections of 27·5% among symptomatic individuals presenting at care facilities, which is higher than expected from previous cross-sectional surveys. The proportion of infections with P ovale wallikeri and P ovale curtisi was observed to significantly increase over the period of study, which could be due to attenuated responsiveness of these species to malaria drug treatment. The increase in frequency of P ovale spp could threaten the malaria control efforts in Kenya and pose increased risk of malaria to travellers. FUNDING: Armed Forces Health Surveillance Branch and its Global Emerging Infections Surveillance Section.

High Levels of Acquired HIV Drug Resistance Following Virological Nonsuppression in HIV-Infected Women from a High-Risk Cohort in Uganda.

HIV drug resistance (HIVDR) is of increasing health concern, especially among key populations. We investigated the prevalence of virological suppression (VS), prevalence and correlates of HIVDR in HIV-infected women, enrolled in a high-risk cohort. We enrolled 267 women initiated on first-line antiretroviral therapy (ART) between 2015 and 2018. Participants' plasma samples were analyzed for HIV RNA viral load (VL) and genotypic resistance testing was performed on those with VL nonsuppression (defined as VL ≥1,000 copies/mL). We used the Stanford HIVDR database-algorithm to assess HIVDR mutations and logistic regression to assess risk factors for VL nonsuppression and HIVDR. We observed an overall VS prevalence of 76.0% (203/267) and detected respective acquired drug resistance prevalence to non-nucleoside reverse transcriptase inhibitors (NNRTIs) and nucleoside reverse transcriptase inhibitors (NRTIs) of 81.3% [confidence interval (CI) 67.4-91.1] and 45.8% (CI 31.4-60.8) among the 48 successfully genotyped VL nonsuppressors. NNRTI mutations were observed in 81.3% (39/48) of the genotyped participants and 45.8% (22/48) had both NRTI and NNRTI mutations. The mutation K103N was detected in 62.5% (30/48) of participants, 41.7% (20/48) had M184V/I, 14.6% had K65R, and 12.5% (6/48) had thymidine analog mutations (TAMs). None of the analyzed potential risk factors, including age and duration on ART, was significantly correlated with VL nonsuppression or HIVDR. Although high levels of NNRTI mutations support the transition to dolutegravir, the presence of NRTI mutations, especially TAMs, may compromise dolutegravir-based regimens or other second-line ART options. The moderate VS prevalence and high HIVDR prevalence therefore call for timely ART switching and intensive adherence counseling.

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.

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.