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.

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.

Pertussis prevalence and its determinants among children with persistent cough in urban Uganda.

BACKGROUND: We determined prevalence of pertussis infection and its associated host and environmental factors to generate information that would guide strategies for disease control. METHODS: In a cross-sectional study, 449 children aged 3 months to 12 years with persistent cough lasting ≥14 days were enrolled and evaluated for pertussis using DNA polymerase chain reaction (PCR) and ELISA serology tests. RESULTS: Pertussis prevalence was 67 (15% (95% Confidence Interval (CI): 12-18)) and 81 (20% (95% CI: 16-24)) by PCR and ELISA, respectively among 449 participating children. The prevalence was highest in children with >59 months of age despite high vaccination coverage of 94% in this age group. Study demographic and clinical characteristics were similar between pertussis and non-pertussis cases. Of the 449 children, 133 (30%) had a coughing household member and 316 (70%) did not. Among 133 children that had a coughing household member, sex of child, sharing bed with a coughing household member and having a coughing individual in the neighborhood were factors associated with pertussis. Children that had shared a bed with a coughing household individual had seven-fold likelihood of having pertussis compared to children that did not (odds ratio (OR) 7.16 (95% CI: 1.24-41.44)). Among the 316 children that did not have a coughing household member, age <23 months, having or contact with a coughing individual in neighborhood, a residence with one room, and having a caretaker with >40 years of age were the factors associated with pertussis. Age <23months was three times more likely to be associated with pertussis compared to age 24-59 months (OR 2.97 (95% CI: 1.07-8.28)). CONCLUSION: Findings suggest high prevalence of pertussis among children with persistent cough at a health facility and it was marked in children >59 months of age, suggesting the possibility of waning immunity. The factors associated with pertussis varied by presence or absence of a coughing household member.

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

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

Evaluation of in-house PCR for diagnosis of smear-negative pulmonary tuberculosis in Kampala, Uganda.

BACKGROUND: Nucleic acid amplification tests (NAATs) have offered hope for rapid diagnosis of tuberculosis (TB). However, their efficiency with smear-negative samples has not been widely studied in low income settings. Here, we evaluated in-house PCR assay for diagnosis of smear-negative TB using Lowenstein-Jensen (LJ) culture as the baseline test. Two hundred and five pulmonary TB (PTB) suspects with smear-negative sputum samples, admitted on a short stay emergency ward at Mulago Hospital in Kampala, Uganda, were enrolled. Two smear-negative sputum samples were obtained from each PTB suspect and processed simultaneously for identification of MTBC using in-house PCR and LJ culture. RESULTS: Seventy two PTB suspects (35%, 72/205) were LJ culture positive while 128 (62.4%, 128/205) were PCR-positive. The sensitivity and specificity of in-house PCR for diagnosis of smear-negative PTB were 75% (95% CI 62.6-85.0) and 35.9% (95% CI 27.2-45.3), respectively. The positive and negative predictive values were 39% (95% CI 30.4-48.2) and 72.4% (95% CI 59.1-83.3), respectively, while the positive and negative likelihood ratios were 1.17 (95% CI 0.96-1.42) and 0.70 (95% CI 0.43-1.14), respectively. One hundred and seventeen LJ culture-negative suspects (75 PCR-positive and 42 PCR-negative) were enrolled for follow-up at 2 months. Of the PCR-positive suspects, 45 (60%, 45/75) were still alive, of whom 29 (64.4%, 29/45) returned for the follow-up visit; 15 (20%, 15/75) suspects died while another 15 (20%, 15/75) were lost to follow-up. Of the 42 PCR-negative suspects, 22 (52.4%, 22/42) were still alive, of whom 16 (72.7%, 16/22) returned for follow-up; 11 (26.2%, 11/42) died while nine (21.4%, 9/42) were lost to follow-up. Overall, more PCR-positive suspects were diagnosed with PTB during follow-up visits but the difference was not statistically significant (27.6%, 8/29 vs. 25%, 4/16, p = 0.9239). Furthermore, mortality was higher for the PCR-negative suspects but the difference was also not statistically significant (26.2% vs. 20% p = 0.7094). CONCLUSION: In-house PCR correlates poorly with LJ culture for diagnosis of smear-negative PTB. Therefore, in-house PCR may not be adopted as an alternative to LJ culture.

High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago hospital in Kampala, Uganda.

BACKGROUND: There is limited data on Methicillin resistant Staphylococcus aureus (MRSA) in Uganda where, as in most low income countries, the routine use of chromogenic agar for MRSA detection is not affordable. We aimed to determine MRSA prevalence among patients, healthcare workers (HCW) and the environment in the burns units at Mulago hospital, and compare the performance of CHROMagar with oxacillin for detection of MRSA. RESULTS: One hundred samples (from 25 patients; 36 HCW; and 39 from the environment, one sample per person/item) were cultured for the isolation of Staphylococcus aureus. Forty one S. aureus isolates were recovered from 13 patients, 13 HCW and 15 from the environment, all of which were oxacillin resistant and mecA/femA/nuc-positive. MRSA prevalence was 46% (41/89) among patients, HCW and the environment, and 100% (41/41) among the isolates. For CHROMagar, MRSA prevalence was 29% (26/89) among patients, HCW and the environment, and 63% (26/41) among the isolates. There was high prevalence of multidrug resistant isolates, which concomitantly possessed virulence and antimicrobial resistance determinants, notably biofilms, hemolysins, toxin and ica genes. One isolate positive for all determinants possessed the bhp homologue which encodes the biofilm associated protein (BAP), a rare finding in human isolates. SCCmec type I was the most common at 54% prevalence (22/41), followed by SCCmec type V (15%, 6/41) and SCCmec type IV (7%, 3/41). SCCmec types II and III were not detected and 10 isolates (24%) were non-typeable. CONCLUSIONS: Hyper-virulent methicillin resistant Staphylococcus aureus is prevalent in the burns unit of Mulago hospital.

Determination of circulating Mycobacterium tuberculosis strains and transmission patterns among pulmonary TB patients in Kawempe municipality, Uganda, using MIRU-VNTR.

BACKGROUND: Mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) genotyping is a powerful tool for unraveling clonally complex Mycobacterium tuberculosis (MTB) strains and detection of transmission patterns. Using MIRU-VNTR, MTB genotypes and their transmission patterns among patients with new and active pulmonary tuberculosis (PTB) in Kawempe municipality in Kampala, Uganda was determined. RESULTS: MIRU-VNTR genotyping was performed by PCR-amplification of 15 MTB-MIRU loci from 113 cultured specimens from 113 PTB patients (one culture sample per patient). To determine lineages, the genotypes were entered into the MIRU-VNTRplus database [http://www.miru-vntrplus.org/] as numerical codes corresponding to the number of alleles at each locus. Ten different lineages were obtained: Uganda II (40% of specimens), Uganda I (14%), LAM (6%), Delhi/CAS (3%), Haarlem (3%), Beijing (3%), Cameroon (3%), EAI (2%), TUR (2%) and S (1%). Uganda I and Uganda II were the most predominant genotypes. Genotypes for 29 isolates (26%) did not match any strain in the database and were considered unique. There was high diversity of MIRU-VNTR genotypes, with a total of 94 distinct patterns. Thirty four isolates grouped into 15 distinct clusters each with two to four isolates. Eight households had similar MTB strains for both index and contact cases, indicating possible transmission. CONCLUSION: MIRU-VNTR genotyping revealed high MTB strain diversity with low clustering in Kawempe municipality. The technique has a high discriminatory power for genotyping MTB strains in Uganda.

Detection of multiple strains of Mycobacterium tuberculosis using MIRU-VNTR in patients with pulmonary tuberculosis in Kampala, Uganda.

BACKGROUND: Many studies using DNA fingerprinting to differentiate Mycobacterium tuberculosis (MTB) strains reveal single strains in cultures, suggesting that most disease is caused by infection with a single strain. However, recent studies using molecular epidemiological tools that amplify multiple targets have demonstrated simultaneous infection with multiple strains of MTB. We aimed to determine the prevalence of MTB multiple strain infections in Kampala, and the impact of these infections on clinical presentation of tuberculosis (TB) and response to treatment. METHODS: A total of 113 consecutive smear and culture positive patients who previously enrolled in a house-hold contact study were included in this study. To determine whether infection with multiple MTB strains has a clinical impact on the initial presentation of patients, retrospective patient data (baseline clinical, radiological and drug susceptibility profiles) was obtained. To determine presence of infections with multiple MTB strains, MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeats) -PCR was performed on genomic DNA extracted from MTB cultures of smear positive sputum samples at baseline, second and fifth months. RESULTS: Of 113 patients, eight (7.1%) had infection with multiple MTB strains, coupled with a high rate of HIV infection (37.5% versus 12.6%, p = 0.049). The remaining patients (105) were infected with single MTB strains. The proportions of patients with MTB smear positive cultures after two and five months of treatment were similar. There was no difference between the two groups for other variables. CONCLUSION: Infection with multiple MTB strains occurs among patients with first episode of pulmonary tuberculosis in Kampala, in a setting with high TB incidence. Infection with multiple MTB strains had little impact on the clinical course for individual patients. This is the first MIRU-VNTR-based study from in an East African country.

Rhomboid homologs in mycobacteria: insights from phylogeny and genomic analysis.

BACKGROUND: Rhomboids are ubiquitous proteins with diverse functions in all life kingdoms, and are emerging as important factors in the biology of some pathogenic apicomplexa and Providencia stuartii. Although prokaryotic genomes contain one rhomboid, actinobacteria can have two or more copies whose sequences have not been analyzed for the presence putative rhomboid catalytic signatures. We report detailed phylogenetic and genomic analyses devoted to prokaryotic rhomboids of an important genus, Mycobacterium. RESULTS: Many mycobacterial genomes contained two phylogenetically distinct active rhomboids orthologous to Rv0110 (rhomboid protease 1) and Rv1337 (rhomboid protease 2) of Mycobacterium tuberculosis H37Rv, which were acquired independently. There was a genome-wide conservation and organization of the orthologs of Rv1337 arranged in proximity with glutamate racemase (mur1), while the orthologs of Rv0110 appeared evolutionary unstable and were lost in Mycobacterium leprae and the Mycobacterium avium complex. The orthologs of Rv0110 clustered with eukaryotic rhomboids and contained eukaryotic motifs, suggesting a possible common lineage. A novel nonsense mutation at the Trp73 codon split the rhomboid of Mycobacterium avium subsp. Paratuberculosis into two hypothetical proteins (MAP2425c and MAP2426c) that are identical to MAV_1554 of Mycobacterium avium. Mycobacterial rhomboids contain putative rhomboid catalytic signatures, with the protease active site stabilized by Phenylalanine. The topology and transmembrane helices of the Rv0110 orthologs were similar to those of eukaryotic secretase rhomboids, while those of Rv1337 orthologs were unique. Transcription assays indicated that both mycobacterial rhomboids are possibly expressed. CONCLUSIONS: Mycobacterial rhomboids are active rhomboid proteases with different evolutionary history. The Rv0110 (rhomboid protease 1) orthologs represent prokaryotic rhomboids whose progenitor may be the ancestors of eukaryotic rhomboids. The Rv1337 (rhomboid protease 2) orthologs appear more stable and are conserved nearly in all mycobacteria, possibly alluding to their importance in mycobacteria. MAP2425c and MAP2426c provide the first evidence for a split homologous rhomboid, contrasting whole orthologs of genetically related species. Although valuable insights to the roles of rhomboids are provided, the data herein only lays a foundation for future investigations for the roles of rhomboids in mycobacteria.

Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test.

BACKGROUND: The ideal identification of Staphylococcus aureus clinical isolates requires a battery of tests and this is costly in resource limited settings. In many developing countries, the tube coagulase test is usually confirmatory for S. aureus and is routinely done using either human or sheep plasma. This study evaluated Mannitol salt agar and the deoxyribonuclease (DNase) test for improving the efficiency of the tube coagulase test in resource limited settings. The efficiency of human and sheep plasma with tube coagulase tests was also evaluated. METHODS: One hundred and eighty Gram positive, Catalase positive cocci occurring in pairs, short chains or clusters were subjected to growth on Mannitol salt agar, deoxyribonuclease and tube coagulase tests. Of these, isolates that were positive for at least two of the three tests (n = 60) were used to evaluate the performance of the tube coagulase test for identification of S. aureus, using PCR-amplification of the nuc gene as a gold standard. RESULTS: Human plasma was more sensitive than sheep plasma for the tube coagulase test (sensitivity of 91% vs. 81% respectively), but both plasmas had very low specificity (11% and 7% respectively). The sensitivity and specificity of the tube coagulase test (human plasma) was markedly improved when Mannitol salt agar and DNase were introduced as a tri-combination test for routine identification of Staphylococcus aureus (100% specificity and 75% sensitivity). The specificity and sensitivity of Mannitol salt agar/DNase/tube coagulase (sheep plasma) combination was 100% and 67%, respectively. CONCLUSION: The efficiency of the tube coagulase test can be markedly improved by sequel testing of the isolates with Mannitol salt agar, DNase and Tube coagulase. There is no single phenotypic test (including tube coagulase) that can guarantee reliable results in the identification of Staphylococcus aureus.

Comparison of transformation frequencies among selected Streptococcus pneumoniae serotypes.

Although there are over 90 serotypes of Streptococcus pneumoniae, antimicrobial resistance is predominantly found in a limited number of serotypes/serogroups, namely 6, 9, 14, 19 and 23. There is no compelling mechanism to account for this restriction. We aimed to determine whether serotypes commonly associated with drug resistance have higher transformation frequencies than those that are susceptible to antimicrobial agents. An in vitro investigation of the genetic transformation frequency of drug-resistant serotypes compared with that of susceptible serotypes under the influence of synthetic competence-stimulating peptides was performed. The transforming DNA was genomic DNA carrying a Tn916-like transposon containing the mefE gene that confers resistance to erythromycin. It was observed that serotypes 6, 9, 14, 19 and 23, which are highly associated with drug resistance, do not exhibit a higher degree of transformation efficiency than other serotypes. These findings suggest that the association of serotype with drug resistance is likely due to prolonged exposure to transforming DNA resulting from longer nasopharyngeal carriage and to a greater selective pressure from antimicrobials, particularly in children. This is the first study to compare the transformation frequencies of pneumococcal clinical isolates using genomic DNA that carries the composite Tn916-like element.

DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda.

BACKGROUND: The identification and differentiation of strains of Mycobacterium tuberculosis by DNA fingerprinting has provided a better understanding of the epidemiology and tracing the transmission of tuberculosis. We set out to determine if there was a relationship between the risk of belonging to a group of tuberculosis patients with identical mycobacterial DNA fingerprint patterns and the HIV sero-status of the individuals in a high TB incidence peri-urban setting of Kampala, Uganda. METHODS: One hundred eighty three isolates of Mycobacterium tuberculosis from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS6110-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern. RESULTS: One hundred and eighteen different fingerprint patterns were obtained from the 183 isolates. There were 34 clusters containing 54% (99/183) of the patients (average cluster size of 2.9), and a majority (96.2%) of the strains possessed a high copy number (> or = 5 copies) of the IS6110 element. When strains with <5 bands were excluded from the analysis, 50.3% (92/183) were clustered, and there was no difference in the level of diversity of DNA fingerprints observed in the two sero-groups (adjusted odds ratio [aOR] 0.85, 95%CI 0.46-1.56, P = 0.615), patients aged <40 years (aOR 0.53, 95%CI 0.25-1.12, P = 0.100), and sex (aOR 1.12, 95%CI 0.60-2.06, P = 0.715). CONCLUSION: The sample showed evidence of a high prevalence of recent transmission with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristics, including HIV status.