Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda

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

Knowledge and perceptions of brucellosis in the pastoral communities adjacent to Lake Mburo National Park, Uganda

BACKGROUND: Brucellosis is one of the most common zoonotic infections globally. Lack of knowledge about brucellosis may affect the health-seeking behavior of patients, thus leading to sustained transmission in these communities. Our study assessed knowledge and perceptions of brucellosis among pastoral communities adjacent to Lake Mburo National Park (LMNP), Kiruhura District, Uganda. METHODS: A community cross-sectional questionnaire survey involving 371 randomly selected household heads from three sub-counties neighboring LMNP were interviewed between June and August 2012. Data collected included communities’ knowledge on causes, symptoms, transmission, treatment, prevention and risk factors of brucellosis. Multivariable logistic regression analysis was performed to explore strength of association between overall knowledge of brucellosis and various individual factors using odds ratios and 95% confidence intervals. RESULTS: Only 70 (19%) knew the symptoms of brucellosis in animals, and three quarters (279, 75.5%) mentioned joint and muscle pain as a common symptom in humans. Almost all participants (370, 99.3%) had ever heard about brucellosis, majority (311, 84.7%) believed it affects all sexes and two thirds (67.7%) of the respondents believed close proximity to wildlife contributes to the presence of the disease. Almost all (352, 95.4%) knew that brucellosis in humans could be treatable using modern drugs. The main routes of infection in humans such as consumption of unpasteurized dairy products were known by 97% (360/371); eating of half-cooked meat by 91.4% and eating contaminated pasture in animals by 97.4%. There was moderate overall knowledge of brucellosis 197 (53.1%). Factors associated with higher overall knowledge were being agro-pastoralists (aOR: 2.08, CI: 1.17-3.71) compared to pure pastoralists while those who reported that the disease was a health problem (aOR: 0.18, CI: 0.06-0.56) compared to those who said it was not were less likely to be knowledgeable. CONCLUSIONS: There was moderate overall knowledge of human and animal brucellosis among the participants. Majority of the participants believed that close proximity to wildlife contributes to the presence of the disease in the area. There is a need for collaboration between the public health, veterinary and wildlife sectors to provide health education on brucellosis for better management of the disease in the communities

Determinants of patients' choice of provider in accessing brucellosis care among pastoral communities adjacent to Lake Mburo National Park in Kiruhura district, Uganda

Brucellosis is the commonest zoonotic infection worldwide causing human suffering and economic losses through affected livestock. Symptoms are similar to other febrile syndromes such as malaria and typhoid fever and it is often easily misdiagnosed, resulting in underreporting and misdirected treatments. The study was conducted in pastoralist rangelands. Females and households with six to ten members were more likely to seek health care at government facilities, while those with tertiary education were less likely, after the first provider. Improved geographical access needs to be matched with good quality basic services, adequate medicines, qualified health personnel and regular supportive supervision

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

<p>Abstract</p> <p>Background</p> <p>Mycobacterial interspersed repetitive units - variable number of tandem repeats (MIRU-VNTR) genotyping is a powerful tool for unraveling clonally complex <it>Mycobacterium tuberculosis </it>(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.</p> <p>Results</p> <p>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-VNTR<it>plus </it>database [<url>http://www.miru-vntrplus.org/</url>] 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.</p> <p>Conclusion</p> <p>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.</p

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

<p>Abstract</p> <p>Background</p> <p>The identification and differentiation of strains of <it>Mycobacterium tuberculosis </it>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.</p> <p>Methods</p> <p>One hundred eighty three isolates of <it>Mycobacterium tuberculosis </it>from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS<it>6110</it>-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern.</p> <p>Results</p> <p>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 IS<it>6110 </it>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, <it>P </it>= 0.615), patients aged <40 years (aOR 0.53, 95%CI 0.25–1.12, <it>P </it>= 0.100), and sex (aOR 1.12, 95%CI 0.60–2.06, <it>P </it>= 0.715).</p> <p>Conclusion</p> <p>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.</p

Comparison of rapid tests for detection of rifampicin-resistant Mycobacterium tuberculosis in Kampala, Uganda

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

Use of the GenoType® MTBDRplus assay to assess drug resistance of Mycobacterium tuberculosis isolates from patients in rural Uganda

<p>Abstract</p> <p>Background</p> <p>Drug resistance levels and patterns among <it>Mycobacterium tuberculosis </it>isolates from newly diagnosed and previously treated tuberculosis patients in Mbarara Uganda were investigated.</p> <p>Methods</p> <p>We enrolled, consecutively, all newly diagnosed and previously treated smear-positive TB patients aged ≥ 18 years. Isolates were tested for drug resistance against rifampicin (RIF) and isoniazid (INH) using the Genotype^®MDRTBplus assay and results were compared with those obtained by the indirect proportion method on Lowenstein-Jensen media. HIV testing was performed using two rapid HIV tests.</p> <p>Results</p> <p>A total of 125 isolates from 167 TB suspects with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analysed. A majority (92.8%) of the participants were newly presenting while only 7.2% were retreatment cases. Resistance mutations to either RIF or INH were detected in 6.4% of the total isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The <it>rpoβ </it>gene mutations seen in the sample were D516V, S531L, H526Y H526 D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with <it>katG </it>(codon 315) gene mutations while only one strain showed the <it>inhA </it>promoter region gene mutation.</p> <p>Conclusion</p> <p>The TB resistance rate in Mbarara is relatively low. The GenoType^®MTBDRplus assay can be used for rapid screening of MDR-TB in this setting.</p

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

<p>Abstract</p> <p>Background</p> <p>Many studies using DNA fingerprinting to differentiate <it>Mycobacterium tuberculosis </it>(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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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%, <it>p </it>= 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.</p> <p>Conclusion</p> <p>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.</p