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Abstract Background Determination of the prevalence and drug susceptibility of the M. tuberculosis strains is important in tuberculosis control. We determined the genetic diversity and susceptibility profiles of mycobacteria isolated from tuberculosis patients in Mbarara, South Western Uganda. Methods We enrolled, consecutively; all newly diagnosed and previously treated smear-positive TB patients aged ≥ 18 years. The isolates were characterized using regions of difference (RD) analysis and spoligotyping. Drug resistance against rifampicin and isoniazid were tested using the Genotype® MDRTBplus assay and the indirect proportion method on Lowenstein-Jensen media. HIV-1 testing was performed using two rapid HIV tests. Results A total of 125 isolates from 167 TB suspects (60% males) with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analyzed. Majority (92.8%) were new cases while only 7.2% were retreatment cases. All the 125 isolates were identified as M. tuberculosis strict sense with the majority (92.8%) of the isolates being modern strains while seven (7.2%) isolates were ancestral strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty two (49.6%) of the isolates formed 16 clusters consisting of 2-15 isolates each. A majority (59.2%) of the isolates belong to the Uganda genotype group of strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 15 isolates and SIT 128 (T2) with 3 isolates. Sixty nine (87%) of the 79 patterns had not yet been defined in the SpolDB4.0.database. Resistance mutations to either RIF or INH were detected in 6.4% of the isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpoβ gene mutations seen in the sample were D516V, S531L, H526Y H526D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations only while one strain showed the inhA promoter gene mutation. Conclusion The present study shows that the TB epidemic in Mbarara is caused by modern M. tuberculosis strains mainly belonging to the Uganda genotype and anti-TB drug resistance rate in the region is low.

Abstract 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.

Abstract Background Drug resistant tuberculosis (TB) is a growing concern worldwide. Rapid detection of resistance expedites appropriate intervention to control the disease. Several technologies have recently been reported to detect rifampicin resistant Mycobacterium tuberculosis directly in sputum samples. These include phenotypic culture based methods, tests for gene mutations and tests based on bacteriophage replication. The aim of the present study was to assess the feasibility of implementing technology for rapid detection of rifampicin resistance in a high disease burden setting in Africa. Methods Sputum specimens from re-treatment TB patients presenting to the Mulago Hospital National TB Treatment Centre in Kampala, Uganda, were examined by conventional methods and simultaneously used in one of the four direct susceptibility tests, namely direct BACTEC 460, Etest, "in-house" phage test, and INNO- Rif.TB. The reference method was the BACTEC 460 indirect culture drug susceptibility testing. Test performance, cost and turn around times were assessed. Results In comparison with indirect BACTEC 460, the respective sensitivities and specificities for detecting rifampicin resistance were 100% and 100% for direct BACTEC and the Etest, 94% and 95% for the phage test, and 87% and 87% for the Inno-LiPA assay. Turn around times ranged from an average of 3 days for the INNO-LiPA and phage tests, 8 days for the direct BACTEC 460 and 20 days for the Etest. All methods were faster than the indirect BACTEC 460 which had a mean turn around time of 24 days. The cost per test, including labour ranged from $18.60 to $41.92 (USD). Conclusion All four rapid technologies were shown capable of detecting rifampicin resistance directly from sputum. The LiPA proved rapid, but was the most expensive. It was noted, however, that the LiPA test allows sterilization of samples prior to testing thereby reducing the risk of accidental laboratory transmission. In contrast the Etest was low cost, but slow and would be of limited assistance when treating patients. The phage test was the least reproducible test studied with failure rate of 27%. The test preferred by the laboratory personnel, direct BACTEC 460, requires further study to determine its accuracy in real-time treatment decisions in Uganda.

Abstract Background One of the challenges facing the tuberculosis (TB) control programmes in resource-limited settings is lack of rapid techniques for detection of drug resistant TB, particularly multi drug resistant tuberculosis (MDR TB). Results obtained with the conventional indirect susceptibility testing methods come too late to influence a timely decision on patient management. More rapid tests directly applied on sputum samples are needed. This study compared the sensitivity, specificity and time to results of four direct drug susceptibility testing tests with the conventional indirect testing for detection of resistance to rifampicin and isoniazid in M. tuberculosis. The four direct tests included two in-house phenotypic assays – Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS), and two commercially available tests – Genotype® MTBDR and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany). Methods A literature review and meta-analysis of study reports was performed. The Meta-Disc software was used to analyse the reports and tests for sensitivity, specificity, and area under the summary receiver operating characteristic (sROC) curves. Heterogeneity in accuracy estimates was tested with the Spearman correlation coefficient and Chi-square. Results Eighteen direct DST reports were analysed: NRA – 4, MODS- 6, Genotype MTBDR® – 3 and Genotype® MTBDRplus – 5. The pooled sensitivity and specificity for detection of resistance to rifampicin were 99% and 100% with NRA, 96% and 96% with MODS, 99% and 98% with Genotype® MTBDR, and 99% and 99% with the new Genotype® MTBDRplus, respectively. For isoniazid it was 94% and 100% for NRA, 92% and 96% for MODS, 71% and 100% for Genotype® MTBDR, and 96% and 100% with the Genotype® MTBDRplus, respectively. The area under the summary receiver operating characteristic (sROC) curves was in ranges of 0.98 to 1.00 for all the four tests. Molecular tests were completed in 1 – 2 days and also the phenotypic assays were much more rapid than conventional testing. Conclusion Direct testing of rifampicin and isoniazid resistance in M. tuberculosis was found to be highly sensitive and specific, and allows prompt detection of MDR TB.

Abstract 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 (≥ 5 copies) of the IS6110 element. When strains with P = 0.615), patients aged 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.

Abstract Background The poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated in the increase of tuberculosis (TB). Presence of different lineages of Mycobacterium tuberculosis has been described in different parts of the world. This study determined the predominant strain lineages that cause TB in Rubaga division, Kampala, Uganda, and the prevalence of resistance to key anti-tuberculosis drugs in this community. Methods This was a cross-sectional study of newly diagnosed sputum smear-positive patients aged ≥ 18 years. A total of 344 isolates were genotyped by standard spoligotyping and the strains were compared with those in the international spoligotype database (SpolDB4). HIV testing and anti-tuberculosis drug susceptibility assays for isoniazid and rifampicin were performed and association with the most predominant spoligotypes determined. Results A total of 33 clusters were obtained from 57 spoligotype patterns. According to the SpolDB4 database, 241 (70%) of the isolates were of the T2 family, while CAS1-Kili (3.5%), LAM9 (2.6%), CAS1-Delhi (2.6%) were the other significant spoligotypes. Furthermore, a major spoligotype pattern of 17 (4.5%) strains characterized by lack of spacers 15–17 and 19–43 was not identified in SpolDB4. A total of 92 (26.7%) of the patients were HIV sero-positive, 176 (51.2%) sero-negative, while 76 (22.1%) of the patients did not consent to HIV testing. Resistance to isoniazid was found in 8.1% of strains, while all 15 (4.4%) strains resistant to rifampicin were multi-drug resistant. Additionally, there was no association between any strain types in the sample with either drug resistance or HIV sero-status of the patients. Conclusion The TB epidemic in Kampala is localized, mainly caused by the T2 family of strains. Strain types were neither associated with drug resistance nor HIV sero-status.

Background: Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients.Method: Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing.Results: The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage.Conclusion: The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients. [ABSTRACT FROM AUTHOR]

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

Background Despite sustained exposure to a person with pulmonary tuberculosis (TB), some M. tuberculosis (Mtb) exposed individuals maintain a negative tuberculin skin test (TST). Our objective was to characterize these persistently negative TST (PTST-) individuals and compare them to TST converters (TSTC) and individuals who are TST positive at study enrollment. Methods During a TB household contact study in Kampala, Uganda, PTST-, TSTC, and TST + individuals were identified. PTST- individuals maintained a negative TST over a 2 year observation period despite prolonged exposure to an infectious tuberculosis (TB) case. Epidemiological and clinical characteristics were compared, a risk score developed by another group to capture risk for Mtb infection was computed, and an ordinal regression was performed. Results When analyzed independently, epidemiological risk factors increased in prevalence from PTST- to TSTC to TST+. An ordinal regression model suggested age (p < 0.01), number of windows (p < 0.01) and people (p = 0.07) in the home, and sleeping in the same room (p < 0.01) were associated with PTST- and TSTC. As these factors do not exist in isolation, we examined a risk score, which reflects an accumulation of risk factors. This compound exposure score did not differ significantly between PTST-, TSTC, and TST+, except for the 5-15 age group (p = 0.009). Conclusions Though many individual factors differed across all three groups, an exposure risk score reflecting a collection of risk factors did not differ for PTST-, TSTC and TST + young children and adults. This is the first study to rigorously characterize the epidemiologic risk profile of individuals with persistently negative TSTs despite close exposure to a person with TB. Additional studies are needed to characterize possible epidemiologic and host factors associated with this phenotype. [ABSTRACT FROM AUTHOR]

Background Previous studies have shown that Mycobacterium tuberculosis (MTB) Uganda family, a sublineage of the MTB Lineage 4, is the main cause of tuberculosis (TB) in Uganda. Using a well characterized patient population, this study sought to determine whether there are clinical and patient characteristics associated with the success of the MTB Uganda family in Kampala. Methods A total of 1,746 MTB clinical isolates collected from1992-2009 in a household contact study were genotyped. Genotyping was performed using Single Nucleotide Polymorphic (SNP) markers specific for the MTB Uganda family, other Lineage 4 strains, and Lineage 3, respectively. Out of 1,746 isolates, 1,213 were from patients with detailed clinical data. These data were used to seek associations between MTB lineage/sub-lineage and patient phenotypes. Results Three MTB lineages were found to dominate the MTB population in Kampala during the last two decades. Overall, MTB Uganda accounted for 63% (1,092/1,746) of all cases, followed by other Lineage 4 strains accounting for 22% (394/1,746), and Lineage 3 for 11% (187/1,746) of cases, respectively. Seventy-three (4 %) strains remained unclassified. Our longitudinal data showed that MTB Uganda family occurred at the highest frequency during the whole study period, followed by other Lineage 4 strains and Lineage 3. To explore whether the long-term success of MTB Uganda family was due to increased virulence, we used cavitary disease as a proxy, as this form of TB is the most transmissible. Multivariate analysis revealed that even though cavitary disease was associated with known risk factors such as smoking (adjusted odds ratio (aOR) 4.8, 95% confidence interval (CI) 3.33-6.84) and low income (aOR 2.1, 95% CI 1.47-3.01), no association was found between MTB lineage and cavitary TB. Conclusion The MTB Uganda family has been dominating in Kampala for the last 18 years, but this longterm success is not due to increased virulence as defined by cavitary disease. [ABSTRACT FROM AUTHOR]

Background: The poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated in the increase of tuberculosis (TB). Presence of different lineages of Mycobacterium tuberculosis has been described in different parts of the world. This study determined the predominant strain lineages that cause TB in Rubaga division, Kampala, Uganda, and the prevalence of resistance to key anti-tuberculosis drugs in this community. Methods: This was a cross-sectional study of newly diagnosed sputum smear-positive patients aged ≥ 18 years. A total of 344 isolates were genotyped by standard spoligotyping and the strains were compared with those in the international spoligotype database (SpolDB4). HIV testing and anti-tuberculosis drug susceptibility assays for isoniazid and rifampicin were performed and association with the most predominant spoligotypes determined. Results: A total of 33 clusters were obtained from 57 spoligotype patterns. According to the SpolDB4 database, 241 (70%) of the isolates were of the T2 family, while CAS1-Kili (3.5%), LAM9 (2.6%), CAS1-Delhi (2.6%) were the other significant spoligotypes. Furthermore, a major spoligotype pattern of 17 (4.5%) strains characterized by lack of spacers 15-17 and 19-43 was not identified in SpolDB4. A total of 92 (26.7%) of the patients were HIV sero-positive, 176 (51.2%) sero-negative, while 76 (22.1%) of the patients did not consent to HIV testing. Resistance to isoniazid was found in 8.1% of strains, while all 15 (4.4%) strains resistant to rifampicin were multidrug resistant. Additionally, there was no association between any strain types in the sample with either drug resistance or HIV sero-status of the patients. Conclusion: The TB epidemic in Kampala is localized, mainly caused by the T2 family of strains. Strain types were neither associated with drug resistance nor HIV sero-status. [ABSTRACT FROM AUTHOR]

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

Background: Despite sustained exposure to a person with pulmonary tuberculosis (TB), some M. tuberculosis (Mtb) exposed individuals maintain a negative tuberculin skin test (TST). Our objective was to characterize these persistently negative TST (PTST-) individuals and compare them to TST converters (TSTC) and individuals who are TST positive at study enrollment.Methods: During a TB household contact study in Kampala, Uganda, PTST-, TSTC, and TST + individuals were identified. PTST- individuals maintained a negative TST over a 2 year observation period despite prolonged exposure to an infectious tuberculosis (TB) case. Epidemiological and clinical characteristics were compared, a risk score developed by another group to capture risk for Mtb infection was computed, and an ordinal regression was performed.Results: When analyzed independently, epidemiological risk factors increased in prevalence from PTST- to TSTC to TST+. An ordinal regression model suggested age (p < 0.01), number of windows (p < 0.01) and people (p = 0.07) in the home, and sleeping in the same room (p < 0.01) were associated with PTST- and TSTC. As these factors do not exist in isolation, we examined a risk score, which reflects an accumulation of risk factors. This compound exposure score did not differ significantly between PTST-, TSTC, and TST+, except for the 5-15 age group (p = 0.009).Conclusions: Though many individual factors differed across all three groups, an exposure risk score reflecting a collection of risk factors did not differ for PTST-, TSTC and TST + young children and adults. This is the first study to rigorously characterize the epidemiologic risk profile of individuals with persistently negative TSTs despite close exposure to a person with TB. Additional studies are needed to characterize possible epidemiologic and host factors associated with this phenotype. [ABSTRACT FROM AUTHOR]