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.

Antibiotic resistance profiles and population structure of disease-associated Staphylococcus aureus infecting patients in Fort Portal Regional Referral Hospital, Western Uganda.

Tackling antimicrobial resistance (AMR) is particularly challenging in low-resource settings such as Fort Portal Regional Referral Hospital (FPRRH) in Western Uganda. Specific knowledge of local AMR epidemiology is required to inform evidence-based improvement of antibiotic stewardship measures in the hospital. To address this, we combined existing antimicrobial susceptibility testing (AST) from FPRRH, with whole genome sequencing (WGS) of 41 Staphylococcus aureus isolates (2017-2019). AST revealed 73 % (30 of 41) of isolates were resistant to one or more antibiotics and 29 % (12 of 41) were multi-drug resistant (MDR). Resistance phenotypes were largely explained by the presence of antibiotic resistance genes in WGS data. Five isolates were methicillin-resistant S. aureus (MRSA) and MDR. Although all isolates were susceptible to clindamycin, a 24 % carriage of erm genes suggests potential for rapid development of resistance. We inferred a population structure for the S. aureus isolates by comparing their core genomes. Twenty isolates formed a tight cluster corresponding to multilocus sequence typing clonal complex (CC) 152, a CC found to be particularly prevalent in northern Africa. The frequency of genes associated with methicillin, chloramphenicol and ciprofloxacin resistance were significantly lower among CC152 strains than non-CC152 strains; thus, in keeping with previous work, we find that CC152 is almost exclusively methicillin-sensitive S. aureus (MSSA). Also, in agreement with other studies, we observed that the occurrence of Panton-Valentine leukocidin toxin-encoding genes was significantly higher among CC152 strains than non-CC152 strains. However, we also observed that the coagulase gene was over-represented in this CC, further defining the virulence strategy of this important pathogen. By generating detailed information about the epidemiology of circulating S. aureus and their antibiotic susceptibility, our study has provided, for the first time, data on which evidence-based infection and AMR interventions at FPRRH can be based.

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.

Frequency and patterns of second-line resistance conferring mutations among MDR-TB isolates resistant to a second-line drug from eSwatini, Somalia and Uganda (2014-2016).

BACKGROUND: Pulmonary tuberculosis is a leading cause of morbidity and mortality in developing countries. Drug resistance, a huge problem in this contagious disease, is driven by point mutations in the Mycobacterium tuberculosis genome however, their frequencies vary geographically and this affects applicability of molecular diagnostics for rapid detection of resistance. Here, we report the frequency and patterns of mutations associated with resistance to second-line anti-TB drugs in multidrug-resistant (MDR) M. tuberculosis isolates from eSwatini, Somalia and Uganda that were resistant to a second-line anti-TB drug. METHODS: The quinolone resistance determining region (QRDR) of gyrA/gyrB genes and the drug resistance associated fragment of rrs gene from 80 isolates were sequenced and investigated for presence of drug resistance mutations. Of the 80 isolates, 40 were MDR, of which 28 (70%) were resistant to a second-line anti-TB injectable drug, 18 (45%) were levofloxacin resistant while 12 (30%) were extensively drug resistant (XDR). The remaining 40 isolates were susceptible to anti-TB drugs. MIRU-VNTR analysis was performed for M/XDR isolates. RESULTS: We successfully sub-cultured 38 of the 40 M/XDR isolates. The gyrA resistance mutations (Gly88Ala/Cys/Ala, Ala90Val, Ser91Pro, Asp94Gly/Asn) and gyrB resistance mutations (Asp500His, Asn538Asp) were detected in 72.2% (13/18) and 22.2% (4/18) of the MDR and levofloxacin resistant isolates, respectively. Overall, drug resistance mutations in gyrA/gyrB QRDRs occurred in 77.8% (14/18) of the MDR and levofloxacin resistant isolates. Furthermore, drug resistance mutations a1401g and g1484 t in rrs occurred in 64.3% (18/28) of the MDR isolates resistant to a second-line anti-TB injectable drug. Drug resistance mutations were not detected in drug susceptible isolates. CONCLUSIONS: The frequency of resistance mutations to second-line anti-TB drugs in MDR-TB isolates resistant to second line anti-TB drugs from eSwatini, Somalia and Uganda is high, implying that rapid molecular tests are useful in detecting second-line anti-TB drug resistance in those countries. Relatedly, the frequency of fluoroquinolone resistance mutations in gyrB/QRDR is high relative to global estimates, and they occurred independently of gyrA/QRDR mutations implying that their absence in panels of molecular tests for detecting fluoroquinolone resistance may yield false negative results in our setting.

Prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites isolated from adult symptomatic patients in northern Uganda.

BACKGROUND: In the absence of an effective vaccine, malaria treatment and eradication is still a challenge in most endemic areas globally. This is especially the case with the current reported emergence of resistance to artemisinin agents in Southeast Asia. This study therefore explored the prevalence of K13-propeller gene polymorphisms among Plasmodium falciparum parasites in northern Uganda. METHODS: Adult patients (≥18 years) presenting to out-patients department of Lira and Gulu regional referral hospitals in northern Uganda were randomly recruited. Laboratory investigation for presence of plasmodium infection among patients was done using Plasmodium falciparum exclusive rapid diagnostic test, histidine rich protein-2 (HRP2) (Pf). Finger prick capillary blood from patients with a positive malaria test was spotted on a filter paper Whatman no. 903. The parasite DNA was extracted using chelex resin method and sequenced for mutations in K13-propeller gene using Sanger sequencing. PCR DNA sequence products were analyzed using in DNAsp 5.10.01software, data was further processed in Excel spreadsheet 2007. RESULTS: A total of 60 parasite DNA samples were sequenced. Polymorphisms in the K13-propeller gene were detected in four (4) of the 60 parasite DNA samples sequenced. A non-synonymous polymorphism at codon 533 previously detected in Cambodia was found in the parasite DNA samples analyzed. Polymorphisms at codon 522 (non-synonymous) and codon 509 (synonymous) were also found in the samples analyzed. The study found evidence of positive selection in the Plasmodium falciparum population in northern Uganda (Tajima's D = -1.83205; Fu and Li's D = -1.82458). CONCLUSIONS: Polymorphism in the K13-propeller gene previously reported in Cambodia has been found in the Ugandan Plasmodium falciparum parasites. There is need for continuous surveillance for artemisinin resistance gene markers in the country.

Clinico-pathological features of tuberculosis due to Mycobacterium tuberculosis Uganda genotype in patients with tuberculous lymphadenitis: a cross sectional study.

BACKGROUND: Tuberculous lymphadenitis is next to pulmonary tuberculosis as the most common cause of tuberculosis. Uganda genotype, one of the sub-lineages of Mycobacterium tuberculosis, is the most prevalent cause of pulmonary tuberculosis in Uganda. We here investigate the clinicopathological characteristics of patients with tuberculous lymphadenitis infected with M. tuberculosis Uganda genotype compared with those infected with M. tuberculosis non-Uganda genotype strains. METHODS: Between 2010 and 2012, we enrolled 121 patients (mean age 28.5 yrs, male 48%; female 52%) with tuberculous lymphadenitis, and categorized them by their M. tuberculosis genotypes. The clinical features and lymph node cytopathological parameters were compared between patients in the Uganda and non-Uganda categories using a crude and multivariable logistic regression model with adjustment for confounding factors. RESULTS: Of the 121participants, 56 (46%) were infected with strains of Uganda genotype. Patients infected with this genotype had significantly lower frequency of abdominal lymphadenopathy (odds ratio 0.4, p = 0.046) after adjusting for sex, age and HIV. Abdominal lymphadenopathy was also significantly associated with abnormal chest X-ray (p = 0.027). CONCLUSION: Tuberculous lymphadenitis patients infected with M. tuberculosis Uganda genotype were significantly less prone to have abdominal lymphadenopathy indicating potential reduced ability to disseminate and supporting the concept that differences in M. tuberculosis genotype may have clinical implications.

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.

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.

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.

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.

Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda.

INTRODUCTION: Escherichia coli, Klebsiella pneumoniae and Enterobacter (EKE) are the leading cause of mortality and morbidity in neonates in Africa. The management of EKE infections remains challenging given the global emergence of carbapenem resistance in Gram-negative bacteria. This study aimed to investigate the source of EKE organisms for neonates in the maternity environment of a national referral hospital in Uganda, by examining the phenotypic and molecular characteristics of isolates from mothers, neonates, and maternity ward. METHODS: From August 2015 to August 2016, we conducted a cross-sectional study of pregnant women admitted for elective surgical delivery at Mulago hospital in Kampala, Uganda; we sampled (nose, armpit, groin) 137 pregnant women and their newborns (n = 137), as well as health workers (n = 67) and inanimate objects (n = 70 -beds, ventilator tubes, sinks, toilets, door-handles) in the maternity ward. Samples (swabs) were cultured for growth of EKE bacteria and isolates phenotypically/molecularly investigated for antibiotic sensitivity, as well as ß-lactamase and carbapenemase activity. To infer relationships among the EKE isolates, spatial cluster analysis of phenotypic and genotypic susceptibility characteristics was done using the Ridom server. RESULTS: Gram-negative bacteria were isolated from 21 mothers (15%), 15 neonates (11%), 2 health workers (3%), and 13 inanimate objects (19%); a total of 131 Gram-negative isolates were identified of which 104 were EKE bacteria i.e., 23 (22%) E. coli, 50 (48%) K. pneumoniae, and 31 (30%) Enterobacter. Carbapenems were the most effective antibiotics as 89% (93/104) of the isolates were susceptible to meropenem; however, multidrug resistance was prevalent i.e., 61% (63/104). Furthermore, carbapenemase production and carbapenemase gene prevalence were low; 10% (10/104) and 6% (6/104), respectively. Extended spectrum ß-lactamase (ESBL) production occurred in 37 (36%) isolates though 61 (59%) carried ESBL-encoding genes, mainly blaCTX-M (93%, 57/61) implying that blaCTX-M is the ideal gene for tracking ESBL-mediated resistance at Mulago. Additionally, spatial cluster analysis revealed isolates from mothers, new-borns, health workers, and environment with similar phenotypic/genotypic characteristics, suggesting transmission of multidrug-resistant EKE to new-borns. CONCLUSION: Our study shows evidence of transmission of drug resistant EKE bacteria in the maternity ward of Mulago hospital, and the dynamics in the ward are more likely to be responsible for transmission but not individual mother characteristics. The high prevalence of drug resistance genes highlights the need for more effective infection prevention/control measures and antimicrobial stewardship programs to reduce spread of drug-resistant bacteria in the hospital, and improve patient outcomes.

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.

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.

Diarrhoeagenic Escherichia coli isolated from children with acute diarrhoea at Rakai hospital, Southern Uganda.

Background: Diarrhoeagenic Escherichia coli (DEC) is a leading cause of childhood diarrhoea. This study estimated the prevalence of DEC and DEC pathotypes among children with acute diarrhoea in Southern Uganda. Methods: A cross-sectional study was conducted on 267 children less than 5 years with acute diarrhoea, admitted to Rakai General Hospital in Southern Uganda. Faecal samples were collected from the children and processed for isolation of E. coli. The presence of DEC and the distribution of DEC pathotypes were determined by polymerase chain reaction. Results: A total of 102 (38.2%, 102/267) children had DEC of various pathotypes - enteroaggregative E. coli (EAEC) (14.2%); enteropathogenic E. coli (EPEC) (6.7%); enterotoxigenic E. coli (ETEC) (6%); enteroinvasive E. coli (EIEC) (7.5%); enterohemorrhagic E. coli (EHEC) (3%); and cell-detaching E. coli (CDEC) (0.75%). The difference in the overall prevalence of DEC was not significant regarding HIV but individually, EAEC and CDEC were associated with HIV-positive status while ETEC was associated with HIV-negative status. Conclusions: DEC is prevalent in children with acute diarrhoea in Southern Uganda and its identification in children should be considered among strategies for combatting childhood diarrhoea in Africa.

Unique circulating microRNA profiles in epidemic Kaposi's sarcoma.

Background: Human herpesvirus 8 (HHV-8) causes Kaposi's sarcoma (KS). Kaposi sarcoma in HIV/AIDS patients is referred to as epidemic KS and is the most common HIV-related malignancy worldwide. The lack of a diagnostic assay to detect latent and early-stage disease has increased disease morbidity and mortality. Serum miRNAs have previously been used as potential biomarkers of normal physiology and disease. In the current study, we profiled unique serum miRNAs in patients with epidemic KS to generate baseline data to aid in developing a miRNA-based noninvasive biomarker assay for epidemic KS. Methods: This was a comparative cross-sectional study involving 27 patients with epidemic KS and 27 HIV-positive adults with no prior diagnosis or clinical manifestation of KS. DNA and RNA were isolated from blood and serum collected from study participants. Nested PCR for circulating HHV-8 DNA was performed on the isolated DNA, whereas miRNA library preparation and sequencing for circulating miRNA were performed on the RNA samples. The miRge2 pipeline and EdgeR were used to analyse the sequencing data. Results: Fifteen out of the 27 epidemic KS-positive subjects (55.6%) tested positive for HHV-8 DNA, whereas only 3 (11.1%) out of the 27 HIV-positive, KS-negative subjects tested positive for HHV-8 DNA. Additionally, we found a unique miRNA expression signature in 49 circulating miRNAs in epidemic KS subjects compared to subjects with no epidemic KS, with 41 miRNAs upregulated and 8 miRNAs downregulated. Subjects with latent KS infection had a differential upregulation of circulating miR-193a compared to HIV-positive, KS-negative subjects for whom circulating HHV-8 DNA was not detected. Further analysis of serum from epidemic KS patients revealed a miRNA signature according to KS tumor status and time since first HIV diagnosis. Conclusions: This study reveals unique circulating miRNA profiles in the serum of patients with epidemic KS versus HIV-infected subjects with no KS, as well as in subjects with latent KS. Many of the dysregulated miRNAs in epidemic KS patients were previously reported to have crucial roles in KS infection and latency, highlighting their promising roles as potential biomarkers of latent or active KS infection.

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.

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.

Biobanking: Strengthening Uganda's Rapid Response to COVID-19 and Other Epidemics.

Introduction: SARS-CoV-2 is a fatal disease of global public health concern. Measures to reduce its spread critically depend on timely and accurate diagnosis of virus-infected individuals. Biobanks can have a pivotal role in elucidating disease etiology, translation, and advancing public health. In this article, we show how a biobank has been a critical resource in the rapid response to coronavirus disease of 2019 (COVID-19) in Uganda. Materials and Methods: The Integrated Biorepository of H3Africa Uganda established a COVID-19 biobank. Standard Operating Procedures for sample and data collection, sample processing, and storage were developed. An e-questionnaire data tool was used to collect sociodemographic factors. Samples were collected at 7-day intervals from patients, analyzed for key parameters, processed, annotated, characterized, and stored at appropriate temperatures. Results: Stored samples have been used in validation of 17 diagnostic kits, the Cepheid Xpert Xpress SARS-CoV-2 assay, as well as a sample pooling technique for mass screening and polymerase chain reaction assay validation. Kits that passed validation were deployed for mass screening boosting early detection, isolation, and treatment of COVID-19 cases. Also, 10 applications from researchers and biotech companies have been received and approved and 4 grants have been awarded Conclusion: The CoV-Bank has proven to be an invaluable resource in the fight against the COVID-19 pandemic in Uganda, as samples have been resources in the validation and development of COVID-19 diagnostic tools, which are important in tracing and isolation of infected cases to confront, delay, and stop the spread of the SARS-CoV-2 virus.

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.

Sputum microbiota profiles of treatment-naïve TB patients in Uganda before and during first-line therapy.

Information on microbiota dynamics in pulmonary tuberculosis (TB) in Africa is scarce. Here, we sequenced sputa from 120 treatment-naïve TB patients in Uganda, and investigated changes in microbiota of 30 patients with treatment-response follow-up samples. Overall, HIV-status and anti-TB treatment were associated with microbial structural and abundance changes. The predominant phyla were Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria and Actinobacteria, accounting for nearly 95% of the sputum microbiota composition; the predominant genera across time were Prevotella, Streptococcus, Veillonella, Haemophilus, Neisseria, Alloprevotella, Porphyromonas, Fusobacterium, Gemella, and Rothia. Treatment-response follow-up at month 2 was characterized by a reduction in abundance of Mycobacterium and Fretibacterium, and an increase in Ruminococcus and Peptococcus; month 5 was characterized by a reduction in Tannerella and Fusobacterium, and an increase in members of the family Neisseriaceae. The microbiota core comprised of 44 genera that were stable during treatment. Hierarchical clustering of this core's abundance distinctly separated baseline (month 0) samples from treatment follow-up samples (months 2/5). We also observed a reduction in microbial diversity with 9.1% (CI 6-14%) of the structural variation attributed to HIV-status and anti-TB treatment. Our findings show discernible microbiota signals associated with treatment with potential to inform anti-TB treatment response monitoring.