Airway microbiome signature accurately discriminates Mycobacterium tuberculosis infection status.

Mycobacterium tuberculosis remains one of the deadliest infectious agents globally. Amidst efforts to control TB, long treatment duration, drug toxicity, and resistance underscore the need for novel therapeutic strategies. Despite advances in understanding the interplay between microbiome and disease in humans, the specific role of the microbiome in predicting disease susceptibility and discriminating infection status in tuberculosis still needs to be fully investigated. We investigated the impact of M.tb infection and M.tb-specific IFNγ immune responses on airway microbiome diversity by performing TB GeneXpert and QuantiFERON-GOLD assays during the follow-up phase of a longitudinal HIV-Lung Microbiome cohort of individuals recruited from two large independent cohorts in rural Uganda. M.tb rather than IFNγ immune response mainly drove a significant reduction in airway microbiome diversity. A microbiome signature comprising Streptococcus, Neisseria, Fusobacterium, Prevotella, Schaalia, Actinomyces, Cutibacterium, Brevibacillus, Microbacterium, and Beijerinckiacea accurately discriminated active TB from Latent TB and M.tb-uninfected individuals.

The development and implementation of a proficiency testing program for SARS-CoV-2 using dried tube specimens in resource-limited countries.

INTRODUCTION: When COVID-19 hit the world in 2019, an enhanced focus on diagnostic testing for SARS-CoV-2 was essential for a successful pandemic response. Testing laboratories stretched their capabilities for the new coronavirus by adopting different test methods. The necessity of having external quality assurance (EQA) mechanisms was even more critical due to this rapid expansion. However, there was a lack of experience in providing the necessary SARS-CoV-2 EQA materials, especially in locations with constrained resources. OBJECTIVE: We aimed to create a PT (Proficiency testing) programme based on the Dried Tube Specimens (DTS) method that would be a practical option for molecular based SARS-CoV-2 EQA in Low- and Middle-Income Countries. METHODS: Based on previous ISO/IEC 17043:2010 accreditation experiences and with assistance from the US Centers for Disease Control and Prevention, The Supranational Reference Laboratory of Uganda (adapted the DTS sample preparation method and completed a pilot EQA program between 2020 and 2021. Stability and panel validation testing was conducted on the designed materials before shipping to pilot participants in six African countries. Participants received a panel containing five SARS-CoV-2 DTS samples, transported at ambient conditions. Results submitted by participants were compared to validation results. Participants were graded as satisfactory (≥ 80%) or unsatisfactory (< 80%) and performance reports disseminated. RESULTS: Our SARS-CoV-2 stability experiments showed that SARS-CoV-2 RNA was stable (-15 to -25 °C, 4 to 8 °C, (18 to 28 °C) room temperature and 35 to 38 °C) as well as DTS panels (4 to 8 °C, 18 to 28 °C, 35 to 38 °C and 45 °C) for a period of 4 weeks. The SARS-CoV-2 DTS panels were successfully piloted in 35 test sites from Zambia, Malawi, Mozambique, Nigeria, and Seychelles. The pilot results of the participants showed good accuracy, with an average of 86% (30/35) concordance with the original SARS CoV-2 expectations. CONCLUSION: The SARS-CoV-2 DTS PT panel is reliable, stable at ambient temperature, simple to prepare and requires minimal resources.

HIV co-infection is associated with reduced Mycobacterium tuberculosis transmissibility in sub-Saharan Africa.

Persons living with HIV are known to be at increased risk of developing tuberculosis (TB) disease upon infection with Mycobacterium tuberculosis (Mtb). However, it has remained unclear how HIV co-infection affects subsequent Mtb transmission from these patients. Here, we customized a Bayesian phylodynamic framework to estimate the effects of HIV co-infection on the Mtb transmission dynamics from sequence data. We applied our model to four Mtb genomic datasets collected in sub-Saharan African countries with a generalized HIV epidemic. Our results confirm that HIV co-infection is a strong risk factor for developing active TB. Additionally, we demonstrate that HIV co-infection is associated with a reduced effective reproductive number for TB. Stratifying the population by CD4+ T-cell count yielded similar results, suggesting that, in this context, CD4+ T-cell count is not a better predictor of Mtb transmissibility than HIV infection status alone. Together, our genome-based analyses complement observational household contact studies, and more firmly establish the negative association between HIV co-infection and Mtb transmissibility.

Xpert MTB/RIF Ultra versus mycobacterial growth indicator tube liquid culture for detection of Mycobacterium tuberculosis in symptomatic adults: a diagnostic accuracy study.

BACKGROUND: Xpert MTB/RIF Ultra (Ultra) is an automated molecular test for the detection of Mycobacterium tuberculosis in sputum. We compared the sensitivity of Ultra to that of mycobacterial growth indicator tube (MGIT) liquid culture, considered the most sensitive assay in routine clinical use. METHODS: In this prospective, multicentre, cross-sectional diagnostic accuracy study, we used a non-inferiority design to assess whether the sensitivity of a single Ultra test was non-inferior to that of a single liquid culture for detection of M tuberculosis in sputum. We enrolled adults (age ≥18 years) with pulmonary tuberculosis symptoms in 11 countries and each adult provided three sputum specimens with a minimum volume of 2 mL over 2 days. Ultra was done directly on sputum 1, and Ultra and MGIT liquid culture were done on resuspended pellet from sputum 2. Results of MGIT and solid media cultures done on sputum 3 were considered the reference standard. The pre-defined non-inferiority margin was 5·0%. FINDINGS: Between Feb 18, 2016, and Dec 4, 2019, we enrolled 2906 participants. 2600 (89%) participants were analysed, including 639 (25%) of 2600 who were positive for tuberculosis by the reference standard. Of the 2357 included in the non-inferiority analysis, 877 (37%) were HIV-positive and 984 (42%) were female. Sensitivity of Ultra performed directly on sputum 1 was non-inferior to that of sputum 2 MGIT culture (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; 95% CI -2·8 to 1·1). Sensitivity of Ultra performed on sputum 2 pellet was also non-inferior to that of sputum 2 MGIT (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; -2·7 to 1·0). INTERPRETATION: For the detection of M tuberculosis in sputum from adults with respiratory symptoms, there was no difference in sensitivity of a single Ultra test to that of a single MGIT culture. Highly sensitive, rapid molecular approaches for M tuberculosis detection, combined with advances in genotypic methods for drug resistance detection, have potential to replace culture. FUNDING: US National Institute of Allergy and Infectious Diseases.

Use of in silico approaches, synthesis and profiling of Pan-filovirus GP-1,2 preprotein specific antibodies.

Intermolecular interactions of protein-protein complexes play a principal role in the process of discovering new substances used in the diagnosis and treatment of many diseases. Among such complexes of proteins, we have to mention antibodies; they interact with specific antigens of two genera of single-stranded RNA viruses belonging to the family Filoviridae-Ebolavirus and Marburgvirus; both cause rare but fatal viral hemorrhagic fever in Africa, with pandemic potential. In this research, we conduct studies aimed at the design and evaluation of antibodies targeting the filovirus glycoprotein precursor GP-1,2 to develop potential targets for the pan-filovirus easy-to-use rapid diagnostic tests. The in silico research using the available 3D structure of the natural antibody-antigen complex was carried out to determine the stability of individual protein segments in the process of its formation and maintenance. The computed free binding energy of the complex and its decomposition for all amino acids allowed us to define the residues that play an essential role in the structure and indicated the spots where potential antibodies can be improved. Following that, the study involved targeting six epitopes of the filovirus GP1,2 with two polyclonal antibodies (pABs) and 14 monoclonal antibodies (mABs). The evaluation conducted using Enzyme Immunoassays tested 62 different sandwich combinations of monoclonal antibodies (mAbs), identifying 10 combinations that successfully captured the recombinant GP1,2 (rGP). Among these combinations, the sandwich option (3G2G12* - (rGP) - 2D8F11) exhibited the highest propensity for capturing the rGP antigen.

Accuracy of the tuberculosis molecular bacterial load assay to diagnose and monitor response to anti-tuberculosis therapy: a longitudinal comparative study with standard-of-care smear microscopy, Xpert MTB/RIF Ultra, and culture in Uganda.

BACKGROUND: In 2018, the tuberculosis molecular bacterial load assay (TB-MBLA), a ribosomal RNA-based test, was acknowledged by WHO as a molecular assay that could replace smear microscopy and culture for monitoring tuberculosis treatment response. In this study, we evaluated the accuracy of TB-MBLA for diagnosis and monitoring of treatment response in comparison with standard-of-care tests. METHODS: For this longitudinal prospective study, patients aged 18 years or older with presumptive tuberculosis (coughing for at least 2 weeks, night sweats, and weight loss) were enrolled at China-Uganda Friendship Hospital Naguru (Kampala, Uganda). Participants were evaluated for tuberculosis by TB-MBLA in comparison with Xpert MTB/RIF Ultra (Xpert-Ultra) and smear microscopy, with Mycobacteria Growth Indicator Tube (MGIT) culture as a reference test. Participants who were positive on Xpert-Ultra were enrolled on a standard 6-month anti-tuberculosis regimen, and monitored for treatment response at weeks 2, 8, 17, and 26 after initiation of treatment and then 3 months after treatment. FINDINGS: Between Nov 15, 2019, and June 15, 2022, 210 participants (median age 35 years [IQR 27-44]) were enrolled. 135 (64%) participants were male and 72 (34%) were HIV positive. The pretreatment diagnostic sensitivities of TB-MBLA and Xpert-Ultra were similar (both 99% [95% CI 95-100]) but the specificity was higher for TB-MBLA (90% [83-96]) than for Xpert-Ultra (78% [68-86]). Ten participants were Xpert-Ultra trace positive, eight (80%) of whom were negative by TB-MBLA and MGIT culture. Smear microscopy had lower diagnostic sensitivity (75% [65-83]) but higher specificity (98% [93-100]) than TB-MBLA and Xpert-Ultra. Among participants who were smear microscopy negative, the sensitivity of TB-MBLA was 96% (95 CI 80-100) and was 100% (95% CI 86-100) in those who were HIV positive. 129 (61%) participants were identified as tuberculosis positive by Xpert-Ultra and these individuals were enrolled in the treatment group and monitored for treatment response. According to TB-MBLA, 19 of these patients cleared bacillary load to zero by week 2 of treatment and remained negative throughout the 6-month treatment follow-up. Positivity for tuberculosis decreased with treatment as measured by all tests, but the rate was slower with Xpert-Ultra. Consequently, 31 (33%) of 95 participants were still Xpert-Ultra positive at the end of treatment but were clinically well and negative on TB-MBLA and culture at 6 months of treatment. Two patients were still Xpert-Ultra positive with a further 3 months of post-treatment follow-up. The rate of conversion to negative of the DNA-based Xpert-Ultra was 3·3-times slower than that of the rRNA-based TB-MBLA. Consequently for the same patient, it would take 13 weeks and 52 weeks to reach complete tuberculosis negativity by TB-MBLA and Xpert-Ultra, respectively. Participants who were positive on smear microscopy at 8 weeks, who received an extra month of intensive treatment, had a similar TB-MBLA-measured bacillary load at 8 weeks to those who were smear microscopy negative. INTERPRETATION: TB-MBLA has a similar performance to Xpert-Ultra for pretreatment diagnosis of tuberculosis, but is more accurate at detecting and characterising the response to treatment than Xpert-Ultra and standard-of-care smear microscopy. FUNDING: European and Developing Countries Clinical Trials Partnership, Makerere University Research and Innovation Fund, US National Institutes of Health.

A stool based qPCR for the diagnosis of TB in children and people living with HIV in Uganda, Eswatini and Mozambique (Stool4TB): a protocol for a multicenter diagnostic evaluation.

BACKGROUND: Tuberculosis (TB) is a major cause of mortality worldwide. Children and people living with HIV (PLHIV) have an increased risk of mortality, particularly in the absence of rapid diagnosis. The main challenges of diagnosing TB in these populations are due to the unspecific and paucibacillary disease presentation and the difficulty of obtaining respiratory samples. Thus, novel diagnostic strategies, based on non-respiratory specimens could improve clinical decision making and TB outcomes in high burden TB settings. We propose a multi-country, prospective diagnostic evaluation study with a nested longitudinal cohort evaluation to assess the performance of a new stool-based qPCR, developed by researchers at Baylor College of Medicine (Houston, Texas, USA) for TB bacteriological confirmation with promising results in pilot studies. METHODS: The study will take place in high TB/HIV burden countries (Mozambique, Eswatini and Uganda) where we will enroll, over a period of 30 months, 650 PLHIV (> 15) and 1295 children under 8 years of age (irrespective of HIV status) presenting pressumptive TB. At baseline, all participants will provide clinical history, complete a physical assessment, and undergo thoracic chest X-ray imaging. To obtain bacteriological confirmation, participants will provide respiratory samples (1 for adults, 2 in children) and 1 stool sample for Xpert Ultra MTB/RIF (Cepheid, Sunnyvale, CA, USA). Mycobacterium tuberculosis (M.tb) liquid culture will only be performed in respiratory samples and lateral flow lipoarabinomannan (LF-LAM) in urine following WHO recommendations. Participants will complete 2 months follow-up if they are not diagnosed with TB, and 6 months if they are. For analytical purposes, the participants in the pediatric cohort will be classified into "confirmed tuberculosis", "unconfirmed tuberculosis" and "unlikely tuberculosis". Participants of the adult cohort will be classified as "bacteriologically confirmed TB", "clinically diagnosed TB" or "not TB". We will assess accuracy of the novel qPCR test compared to bacteriological confirmation and Tb diagnosis irrespective of laboratory results. Longitudinal qPCR results will be analyzed to assess its use as treatment response monitoring. DISCUSSION: The proposed stool-based qPCR is an innovation because both the strategy of using a non-sputum based sample and a technique specially designed to detect M.tb DNA in stool. PROTOCOL REGISTRATION DETAILS: ClinicalTrials.gov Identifier: NCT05047315.

Performance evaluation of Truenat MTB and Truenat MTB-RIF DX assays in comparison to gene XPERT MTB/RIF ultra for the diagnosis of pulmonary tuberculosis in Uganda.

BACKGROUND: The World Health Organization endorsed Truenat MTB rapid molecular assay in 2020 and recommended additional in-country evaluation studies before uptake. We evaluated the accuracy and operational feasibility of Truenat MTB assay (Truenat) in comparison with GeneXpert Ultra and culture. METHODS: In a cross-sectional study of 250 presumptive TB patients, participants were requested to provide a sputum sample on the day of their visit to the clinic. The sputum sample was homogenized and a portion was tested using GeneXpert Ultra as per the routine standard procedure and the other portion was tested using Truenat assay at the clinic laboratory. The second sample portion was processed for Concentrated Fluorescent smear Microscopy (CFM), LJ, and MGIT cultures. Truenat sensitivity and specificity were compared to GeneXpert Ultra and culture. Test performance characteristics and operational feasibility assessment data through interview of the study laboratory staff were also collected and summarized as proportions and percentages. RESULTS: Of the 250 participants recruited in the study, the sensitivity and specificity of Truenat was n/N (%, 95%CI); 66/82 (80.5, 70.2-88.4) and 156/159 (98.1, 94.5-99.6) when compared with Ultra, 50/64 (89.3, 66.0-87.4) and 166/180 (92.2, 87.2-95.6) when compared with LJ, 58/71 (81.7,70.7-89.8) and 131/138 (94.9, 89.8-97.9) when compared to MGIT culture and 59/73 (80.8, 69.9-89.1) and 159/169 (94.1,89.3-97.1) when compared to LJ and/or MGIT culture. The sensitivity of Truenat was lower, 14/23 (60.9, 40.6-82.8) among smear-negative compared to 45/50 (90.0, 78.1-96.6) among smear-positive participants but not different by HIV status. There were no special training needs especially among laboratory personnel with previous GeneXpert /molecular test experience, 19/242 (7.8%) error/invalid, and 12 (17,4%) uninterpretable/indeterminate results mainly for rifampicin resistance determination. However, there were 3 (3.5%) of GeneXpert Ultra indeterminate results. CONCLUSION: Among presumptive TB patients in Uganda, the Truenat assay has high sensitivity and specificity. The Truenat assay has acceptable operational feasibility attributes when compared with the GeneXpert Assay.

The burden of severe asthma in sub-Saharan Africa: Findings from the African Severe Asthma Project.

Background: Severe asthma is associated with high morbidity, mortality, and health care utilization, but its burden in Africa is unknown. Objective: We sought to determine the burden (prevalence, mortality, and activity and work impairment) of severe asthma in 3 countries in East Africa: Uganda, Kenya, and Ethiopia. Methods: Using the American Thoracic Society/European Respiratory Society case definition of severe asthma, we analyzed for the prevalence of severe asthma (requiring Global Initiative for Asthma [GINA] steps 4-5 asthma medications for the previous year to achieve control) and severe refractory asthma (remains uncontrolled despite treatment with GINA steps 4-5 asthma medications) in a cohort of 1086 asthma patients who had been in care for 12 months and had received all GINA-recommended medications. Asthma control was assessed by the asthma control questionnaire (ACQ). Results: Overall, the prevalence of severe asthma and severe refractory asthma was 25.6% (95% confidence interval [CI], 23.1-28.3) and 4.6% (95% CI, 3.5-6.0), respectively. Patients with severe asthma were (nonsevere vs severe vs severe refractory) older (39, 42, 45 years, P = .011), had high skin prick test reactivity (67.1%, 76.0%, 76.0%, P = .004), had lower forced expiratory volume in 1 second percentage (81%, 61%, 55.5%, P < .001), had lower quality of life score (129, 127 vs 121, P < .001), and had higher activity impairment (10%, 30%, 50%, P < .001). Factors independently associated with severe asthma were hypertension comorbidity; adjusted odds ratio 2.21 (1.10-4.47), P = .027, high bronchial hyperresponsiveness questionnaire score; adjusted odds ratio 2.16 (1.01-4.61), P = .047 and higher ACQ score at baseline 2.80 (1.55-5.08), P = .001. Conclusion: The prevalence of severe asthma in Africa is high and is associated with high morbidity and poor quality of life.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda.

BACKGROUND: Mixed M. tuberculosis (MTB) infection occurs when one is infected with more than one clonally distinct MTB strain. This form of infection can assist MTB strains to acquire additional mutations, facilitate the spread of drug-resistant strains, and boost the rate of treatment failure. Hence, the presence of mixed MTB infection could affect the performance of some rapid molecular diagnostic tests such as Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays. METHODS: This was a cross-sectional study that used sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed. RESULTS: A total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among male's 3/31 (9.7%) and among middle-aged adults, 4/30 (33.3%). Lineage 4 of MTB contributed 3/5 (60.0%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88-64.44) but not for LPA. Being HIV-positive (P = 0.04) independently predicted the presence of mixed MTB infection. CONCLUSIONS: The presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

Long-term non-progression and risk factors for disease progression among children living with HIV in Botswana and Uganda: A retrospective cohort study.

OBJECTIVES: We utilize a large retrospective study cohort derived from electronic medical records to estimate the prevalence of long-term non-progression (LTNP) and determine the factors associated with progression among children infected with HIV in Botswana and Uganda. METHODS: Electronic medical records from large tertiary HIV clinical centers in Botswana and Uganda were queried to identify LTNP children 0-18 years enrolled between June 2003 and May 2014 and extract demographic and nutritional parameters. Multivariate subdistribution hazard analyses were used to examine demographic factors and nutritional status in progression in the pre-antiretroviral therapy era. RESULTS: Between the two countries, 14,246 antiretroviral therapy-naïve children infected with HIV were enrolled into clinical care. The overall proportion of LTNP was 6.3% (9.5% in Botswana vs 5.9% in Uganda). The median progression-free survival for the cohort was 6.3 years, although this was lower in Botswana than in Uganda (6.6 vs 8.8 years; P <0.001). At baseline, the adjusted subdistribution hazard ratio (aHRsd) of progression was increased among underweight children (aHRsd 1.42; 95% confidence interval [CI]: 1.32-1.53), enrolled after 2010 (aHRsd 1.32; 95% CI 1.22-1.42), and those from Botswana (aHRsd 2; 95% CI 1.91-2.10). CONCLUSIONS: In our study, the prevalence of pediatric LTNP was lower than that observed among adult populations, but progression-free survival was higher than expected. Underweight, year of enrollment into care, and country of origin are independent predictors of progression among children.

Genomic characterization of SARS-CoV-2 from Uganda using MinION nanopore sequencing.

SARS-CoV-2 undergoes frequent mutations, affecting COVID-19 diagnostics, transmission and vaccine efficacy. Here, we describe the genetic diversity of 49 SARS-CoV-2 samples from Uganda, collected during the COVID-19 waves of 2020/2021. Overall, the samples were similar to previously reported SARS-CoV-2 from Uganda and the Democratic Republic of Congo (DRC). The main lineages were AY.46 and A.23, which are considered to be Delta SARS-CoV-2 variants. Further, a total of 268 unique single nucleotide variants and 1456 mutations were found, with more than seventy percent mutations in the ORF1ab and S genes. The most common mutations were 2042C>G (83.4%), 14143C>T (79.5%), 245T>C (65%), and 1129G>T (51%), which occurred in the S, ORF1ab, ORF7a and N genes, respectively. As well, 28 structural variants-21 insertions and 7 deletions, occurred in 16 samples. Our findings point to the possibility that most SARS-CoV-2 infections in Uganda at the time arose from local spread and were not newly imported. Moreover, the relatedness of variants from Uganda and the DRC reflects high human mobility and interaction between the two countries, which is peculiar to this region of the world.

Apolipoprotein-E4: risk of severe malaria and mortality and cognitive impairment in pediatric cerebral malaria.

BACKGROUND: The relationship of apolipoprotein-E4 (APOE4) to mortality and cognition after severe malaria in children is unknown. METHODS: APOE genotyping was performed in children with cerebral malaria (CM, n = 261), severe malarial anemia (SMA, n = 224) and community children (CC, n = 213). Cognition was assessed over 2-year follow-up. RESULTS: A greater proportion of children with CM or SMA than CC had APOE4 (n = 162, 31.0%; n = 142, 31.7%; n = 103, 24.2%, respectively, p = 0.02), but no difference was seen in APOE3 (n = 310, 59.4%; n = 267, 59.6%; n = 282, 66.2%, respectively, p = 0.06), or APOE2 (n = 50, 9.6%; n = 39, 8.7%; and n = 41, 9.6%, respectively, p = 0.87). APOE4 was associated with increased mortality in CM (odds ratio, 2.28; 95% CI, 1.01, 5.11). However, APOE4 was associated with better long-term cognition (ß, 0.55; 95% CI, 0.04, 1.07, p = 0.04) and attention (ß 0.78; 95% CI, 0.26, 1.30, p = 0.004) in children with CM < 5 years old, but worse attention (ß, -0.90; 95% CI, -1.69, -0.10, p = 0.03) in children with CM ≥ 5 years old. Among children with CM, risk of post-discharge malaria was increased with APOE4 and decreased with APOE3. CONCLUSIONS: APOE4 is associated with higher risk of CM or SMA and mortality in children with CM, but better long-term cognition in CM survivors <5 years of age.

Building blocks for better biorepositories in Africa.

BACKGROUND: Biorepositories archive and distribute well-characterized biospecimens for research to support the development of medical diagnostics and therapeutics. Knowledge of biobanking and associated practices is incomplete in low- and middle-income countries where disease burden is disproportionately high. In 2011, the African Society of Human Genetics (AfSHG), the National Institutes of Health (NIH), and the Wellcome Trust founded the Human Heredity and Health in Africa (H3Africa) consortium to promote genomic research in Africa and established a network of three biorepositories regionally located in East, West, and Southern Africa to support biomedical research. This manuscript describes the processes established by H3Africa biorepositories to prepare research sites to collect high-quality biospecimens for deposit at H3Africa biorepositories. METHODS: The biorepositories harmonized practices between the biorepositories and the research sites. The biorepositories developed guidelines to establish best practices and define biospecimen requirements; standard operating procedures (SOPs) for common processes such as biospecimen collection, processing, storage, transportation, and documentation as references; requirements for minimal associated datasets and formats; and a template material transfer agreements (MTA) to govern biospecimen exchange. The biorepositories also trained and mentored collection sites in relevant biobanking processes and procedures and verified biospecimen deposit processes. Throughout these procedures, the biorepositories followed ethical and legal requirements. RESULTS: The 20 research projects deposited 107,982 biospecimens (76% DNA, 81,067), in accordance with the ethical and legal requirements and established best practices. The biorepositories developed and customized resources and human capacity building to support the projects. [The biorepositories developed 34 guidelines, SOPs, and documents; trained 176 clinicians and scientists in over 30 topics; sensitized ethical bodies; established MTAs and reviewed consent forms for all projects; attained import permits; and evaluated pilot exercises and provided feedback. CONCLUSIONS: Biobanking in low- and middle-income countries by local skilled staff is critical to advance biobanking and genomic research and requires human capacity and resources for global partnerships. Biorepositories can help build human capacity and resources to support biobanking by partnering with researchers. Partnerships can be structured and customized to incorporate document development, ethics, training, mentorship, and pilots to prepare sites to collect, process, store, and transport biospecimens of high quality for future research.

Evaluating the effectiveness of Uganda's Supranational TB Reference Laboratory quality management system training program.

BACKGROUND: Achieving the targeted organizational goals through effective training can increase employee satisfaction. Since 2015, the Supranational Reference Laboratory Uganda (SRL Uganda) has trained National Tuberculosis Reference Laboratories (NTRLs) from 21 countries in a variety of areas that cover both technical and programmatic aspects pertinent to TB laboratories. The Laboratory Quality Management System (LQMS) under SRL coordinates actions intended to ensure sustained quality of the laboratory services offered by the National TB Reference Laboratories. In order for laboratory results to be helpful in a clinical or public health setting, they must be accurate, reliable, and timely. The LQMS course aims to provide learners with knowledge on how to attain and maintain this quality. Prior to this study, there was hardly any data available on the effectiveness of LQMS trainings provided by SRL Uganda; using Kirkpatrick model, which is popular among researchers for evaluating the efficacy of the training program, this paper seeks to establish the effectiveness of the LQMS training offered by the SRL Uganda. METHOD: We evaluated the effectiveness of LQMS training within the Uganda's SRL network for courses offered during the period 2017 and 2021 for participants from the Southern and East African sub-Saharan region. RESULTS: In 2017 and 2021, respectively, test results from 10/17 and 9/17 showed overall post-test scores above 80%. Of the 18 labs evaluated, 14 showed improvement; of these, 7 labs were from the Eastern region and the other 7 were from Southern Africa; one facility in this region also maintained its accreditation. In the post-evaluation assessment, attendees of the LQMS course gave feedback of strongly agree and agree variety. CONCLUSION: More SRL Uganda network laboratories in the regions achieved a 5-star SLIPTA level rating and among these, 5 NTRLs got ISO 15189:2012 accredited by the end of 2021, while one maintained its accreditation status. This proves that the Laboratory Quality Management System training program was an effective tool in improving the quality of laboratory services, work practices, and processes.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda.

Background: We evaluated the effect of mixed-MTB strain infection on the performance of Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays among patients initiating MDR-TB treatment in Uganda. Methods: This was a cross-sectional study using sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from the peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed. Results: A total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among males 3/33 (9.7%) and among middle-aged adults, 4/30 (13.3%). Lineage 4 of MTB contributed 3/33 (9.1%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88-64.44) but not for LPA. Being HIV-positive (P=0.04) independently predicted the presence of mixed MTB infection. Conclusions: The presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

Unraveling virulence determinants in extended-spectrum beta-lactamase-producing Escherichia coli from East Africa using whole-genome sequencing.

Escherichia coli significantly causes nosocomial infections and rampant spread of antimicrobial resistance (AMR). There is limited data on genomic characterization of extended-spectrum ß-lactamase (ESBL)-producing E. coli from African clinical settings. This hospital-based longitudinal study unraveled the genetic resistance elements in ESBL E. coli isolates from Uganda and Tanzania using whole-genome sequencing (WGS). A total of 142 ESBL multi-drug resistant E. coli bacterial isolates from both Tanzania and Uganda were sequenced and out of these, 36/57 (63.1%) and 67/85 (78.8%) originated from Uganda and Tanzania respectively. Mutations in RarD, yaaA and ybgl conferring resistances to chloramphenicol, peroxidase and quinolones were observed from Ugandan and Tanzanian isolates. We reported very high frequencies for blaCTX-M-15 with 11/18(61.1%), and blaCTX-M-27 with 12/23 (52.1%), blaTEM-1B with 13/23 (56.5%) of isolates originating from Uganda and Tanzania respectively all conferring resistance to Beta-lactam-penicillin inhibitors. We observed chloramphenicol resistance-conferring gene mdfA in 21/23 (91.3%) of Tanzanian isolates. Extraintestinal E. coli sequence type (ST) 131 accounted for 5/59 (8.4%) of Tanzanian isolates while enterotoxigenic E. coli ST656 was reported in 9/34 (26.4%) of Ugandan isolates. Virulence factors originating from Shigella dysenteriae Sd197 (gspC, gspD, gspE, gspF, gspG, gspF, gspH, gspI), Yersinia pestis CO92 (irp1, ybtU, ybtX, iucA), Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (csgF and csgG), and Pseudomonas aeruginosa PAO1 (flhA, fliG, fliM) were identified in these isolates. Overall, this study highlights a concerning prevalence and diversity of AMR-conferring elements shaping the genomic structure of multi-drug resistant E. coli in clinical settings in East Africa. It underscores the urgent need to strengthen infection-prevention controls and advocate for the routine use of WGS in national AMR surveillance and monitoring programs.Availability of WGS analysis pipeline: the rMAP source codes, installation, and implementation manual can free be accessed via https://github.com/GunzIvan28/rMAP .

An open-source digital contact tracing system tailored to haulage.

Digital contact tracing presents numerous advantages compared to manual contact tracing methods, especially in terms of enhanced speed and automation. Nevertheless, a lack of comprehensive evaluation regarding functionality, efficiency, benefits, and acceptance within communities remains. Here we primarily focus on the functionality of THEA-GS, an open-source digital contact tracing tool developed through consultation with stakeholders. Additionally, we provide insights from its implementation on a limited sample of haulage drivers in Uganda, serving as a representative case for a low- and middle-income country. THEA-GS comprises two primary components: (a) a smartphone application, and (b) a suite of server-programs responsible for data processing and analysis, including databases and a web-based interface featuring dashboards. In essence, the mobile application records the timestamped location of haulage drivers within the road network and identifies possible transmission hotspots by analyzing factors such as the duration of stops and the communities associated with them. The tool can be integrated with national infrastructure to compare drivers' diagnostic results and contact structure, thereby generating individual and community risk assessments relative to the road network. During the Omicron-variant wave of the COVID-19 pandemic, a total of 3,270 haulage drivers were enrolled between October 2021 and October 2022. Around 75% of these drivers utilized THEA-GS for approximately two months. Based on an analysis of 3,800 test results, which included 48 positive cases, 125 contacts, and 40 million time-stamped GPS points, THEA-GS shows a significant speed improvement, being approximately 90 times faster than MCT. For instance, the average time from sample collection to notifying a case and their contacts was approximately 70 and 80 min, respectively. The adoption of this tool encountered challenges, mainly due to drivers' awareness of its purpose and benefits for public health. THEA-GS is a place-based digital contact tracing tool specifically designed to assist National Public Health Institutions in managing infectious disease outbreaks involving the haulage industry as a high-risk group. While its utility, acceptance, and accuracy have not been fully evaluated, our preliminary tests conducted in Uganda indicate the tool's functionality is robust, but social acceptance and adoption are heavily reliant on establishing trust among users.

Evaluation of Xpert MTB/XDR test for susceptibility testing of Mycobacterium tuberculosis to first and second-line drugs in Uganda.

BACKGROUND: Drug-Resistant Tuberculosis (DR-TB) is one of the major challenges to TB control. DESIGN AND METHODS: This was a blinded, laboratory-based cross-sectional study using sputum samples or culture isolates. Samples were from patients with rifampicin-resistant-TB and/or with high risk for isoniazid (INH) resistance and/or 2nd line fluoroquinolones (FQ) and injectable agents (IAs). The diagnostic accuracy of the Xpert® MTB/XDR test was compared to MGIT960 and the Hain Genotype® MTBDRplus and MDRsl assays (LPA) as reference DST methods. Factors for laboratory uptake of the Xpert® MTB/XDR test were also evaluated. RESULTS: Of the 100 stored sputum samples included in this study, 65/99 (65.6%) were resistant to INH, 5/100 (5.0%) were resistant to FQ and none were resistant to IAs using MGIT960. The sensitivity and specificity, n (%; 95% Confidence Interval, CI) of Xpert® MTB/XDR test for; INH was 58 (89.2; 79.1-95.5) and 30 (88.2; 72.5-96.6) and for FQ; 4 (80.0; 28.3-99.4) and 95 (100; 96.2-100), respectively. Using LPA as a reference standard, a total of 52/98 (53.1%) were resistant to INH, 3/100 (3.0%) to FQ, and none to IA. The sensitivity and specificity, n (%; 95%CI) of Xpert® MTB/XDR test compared to LPA for; INH was 50 (96.1; 86.7-99.5) and 34 (74.0; 58.8-85.7) for FQ 3 (100; 29.2-100) and 96 (99.0; 94.3-99.9) respectively. The factors for laboratory uptake and roll-out of the Xpert® MTB/XDR test included: no training needed for technicians with, and one day for those without, previous Xpert-ultra experience, recording and reporting needs were not different from those of Xpert-ultra, the error rate was 4/100 (4%), one (1%) indeterminate rate and test turn-around-time were 1hr/45 minutes. CONCLUSION: There is high sensitivity and specificity of Xpert® MTB/XDR test for isoniazid and fluoroquinolones. There are acceptable Xpert® MTB/XDR test attributes for the test uptake and roll-out.

HIV specific Th1 responses are altered in Ugandans with HIV and Schistosoma mansoni coinfection.

BACKGROUND: Fishing communities surrounding Lake Victoria in Uganda have HIV prevalence of 28% and incidence rates of 5 per 100 person years. More than 50% of the local fishermen are infected with Schistosoma mansoni (S. mansoni). We investigated the role of S. mansoni coinfection as a possible modifier of immune responses against HIV. Using polychromatic flow cytometry and Gran-ToxiLux assays, HIV specific responses, T cell phenotypes, antibody-dependent cell-mediated cytotoxic (ADCC) potency and titres were compared between participants with HIV-S. mansoni coinfection and participants with HIV infection alone. RESULTS: S. mansoni coinfection was associated with a modified pattern of anti-HIV responses, including lower frequency of bifunctional (IFNγ + IL-2 - TNF-α+) CD4 T cells, higher overall CD4 T cell activation and lower HIV ADCC antibody titres, compared to participants with HIV alone. CONCLUSIONS: These results support the hypothesis that S. mansoni infection affects T cell and antibody responses to HIV in coinfected individuals.