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1.  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 [ORadj], 5.0; 95% CI, 2.0 to 11.9; P < 0.001) and CAS (ORadj, 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 (ORadj, 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.
PMCID: PMC4068514  PMID: 24777100
2.  Determinants of Patients' Choice of Provider in Accessing Brucellosis Care among Pastoral Communities Adjacent to Lake Mburo National Park in Kiruhura District, Uganda 
PLoS ONE  2014;9(8):e105276.
Brucellosis is the commonest zoonotic infection worldwide with symptoms similar to other febrile syndromes such as malaria and typhoid fever. It is often easily misdiagnosed, resulting in underreporting and misdirected treatments. Understanding of the factors that influence brucellosis care seeking is essential in enhancing its effective management. Our study sought to determine the factors associated with choice of provider in accessing care for brucellosis among pastoral communities in Uganda.
This was a cross-sectional survey involving 245 randomly selected respondents previously diagnosed and treated for brucellosis, two months before the study. They were enrolled from three sub-counties neighboring Lake Mburo National Park between December 2012 to April 2013. Data on socio-demographics, availability, accessibility, affordability and acceptability of health services were collected. A multivariable logistic regression model was fitted to determine association between independent and outcome variables using odds ratios and 95% confidence intervals with p-value≤0.05 considered statistically significant.
Of the 245 respondents, 127(51.8%) sought health care at government facilities and the rest at private. Respondents who were less likely to choose a government facility were either single (OR: 0.50, CI: 0.26–0.97), had general weakness (OR: 0.09, CI: 0.01–0.72) or whom family took a decision (OR: 0.52, CI: 0.28–0.97). At multivariable analysis, choice of government facility was influenced by primary education (aOR: 0.46, CI: 0.22–0.97), having six to ten household members (aOR:3.71, CI:1.84–7.49), family advice (aOR:0.64, CI: 0.23–0.91), distance ≥10 kms (aOR:0.44, CI: 0.21–0.92), high costs at private clinics (aOR:0.01, CI:0.02–0.15) and no diagnosis at government facility (aOR:0.11, CI:0.01–0.97). Females were more likely to seek health care at government facilities, while those with tertiary education were less likely, after the first provider.
Females and households with six to ten members were more likely to choose government facilities. Government facilities need to be equipped to attract more patients.
PMCID: PMC4136815  PMID: 25133548
3.  Knowledge and perceptions of brucellosis in the pastoral communities adjacent to Lake Mburo National Park, Uganda 
BMC Public Health  2014;14:242.
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.
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.
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.
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.
PMCID: PMC3975325  PMID: 24612845
4.  Long-term dominance of Mycobacterium tuberculosis Uganda family in peri-urban Kampala-Uganda is not associated with cavitary disease 
BMC Infectious Diseases  2013;13:484.
Previous studies have shown that Mycobacterium tuberculosis (MTB) Uganda family, a sub-lineage 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.
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.
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.
The MTB Uganda family has been dominating in Kampala for the last 18 years, but this long-term success is not due to increased virulence as defined by cavitary disease.
PMCID: PMC3853102  PMID: 24134504
Mycobacterium tuberculosis complex; Lineage; Single nucleotide polymorphism; Mycobacteria; Strain family; Cavitation; Virulence; Epidemiology; Evolution
5.  Health service delay among pulmonary tuberculosis patients presenting to a National Referral Hospital, Kampala, Uganda: a cross sectional study 
Delay in the diagnosis of pulmonary tuberculosis (PTB) is common in many countries in Sub-Saharan Africa. Timely diagnosis of active tuberculosis is crucial in minimizing morbidity and mortality in the community as well as nosocomial transmission in health care facilities. This study aimed at determining factors associated with health service delay in the diagnosis and initiation of treatment among new PTB patients presenting to the National Referral Hospital-Mulago.
This was a cross-sectional study among eligible new PTB patients presenting at the National referral TB treatment center Mulago hospital, between March to May 2009. The patients were consecutively recruited and interviewed using a semi-structured questionnaire to assess socio- demographic and health service factors. Multivariate logistic regression using odds ratios and 95% confidence intervals was done.
Two hundred and sixty six newly diagnosed PTB patients were enrolled, of which 65.4% experienced health systems delay. The median health service delay was 9days (IQR=8-19). Factors associated with health service delay were: 1n-patient (OR= 4.68, 95% CI: 1.91-11.45), secondary as highest level of education attained (OR= 3.56, 95% CI: 1.18-10.74), primary as highest level of education attained (OR= 6.70, 95% CI: 2.13-21.02), presence of fever (OR= 3.28, 95% CI: 1.05-10.79), and patient delay at health facility (OR= 5.01, 95% CI: 1.33-18.9).
The study found a significant proportion of Health service delay among pulmonary tuberculosis patients presenting at the referral hospital. Being an in-patient and having fever as a symptom of tuberculosis needs further attention in order to have timely diagnosis. There is need for awareness on TB especially that most of the TB symptoms present like other febrile illnesses such as malaria and needs consideration when patients present to a health facility.
PMCID: PMC3810284  PMID: 24198882
Tuberculosis; health service delay; treatment delay; diagnostic delay; pulmonary tuberculosis
6.  Species and genotypic diversity of non-tuberculous mycobacteria isolated from children investigated for pulmonary tuberculosis in rural Uganda 
Smear microscopy, a mainstay of tuberculosis (TB) diagnosis in developing countries, cannot differentiate M. tuberculosis complex from NTM infection, while pulmonary TB shares clinical signs with NTM disease, causing clinical and diagnostic dilemmas. This study used molecular assays to identify species and assess genotypic diversity of non-tuberculous mycobacteria (NTM) isolates from children investigated for pulmonary tuberculosis at a demographic surveillance site in rural eastern Uganda.
Children were investigated for pulmonary tuberculosis as part of a TB vaccine surveillance program (2009–2011). Two cohorts of 2500 BCG vaccinated infants and 7000 adolescents (12–18 years) were recruited and followed up for one to two years to determine incidence of tuberculosis. Induced sputum and gastric aspirates were processed by the standard N-acetyl L-cysteine (NALC)-NaOH method. Sediments were cultured in the automated MGIT (Becton Dickson) liquid culture system and incubated at 37°C for at least six weeks. Capilia TB assay was used to classify mycobacteria into MTC and NTM. The GenoType CM/AS assays were performed to identify species while Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR genotyping was used to assess genetic diversity of the strains within each species.
Among 2859 infants and 2988 adolescents screened, the numbers of TB suspects were 710 and 1490 infants and adolescents respectively. The prevalence of NTM in infant suspects was 3.7% (26/710) (95% CI 2.5–5.2) while that in adolescent suspects was 4.6% (69/1490) (95% CI 3.6–5.8). On culture, 127 isolates were obtained, 103 of which were confirmed as mycobacteria comprising of 95 NTM and eight M. tuberculosis complex. The Genotype CM/AS assay identified 63 of the 95 NTM isolates while 32 remained un-identified. The identified NTM species were M. fortuitum (40 isolates, 63.5%), M. szulgai (9 isolates, 14.3%), M. gordonae (6 isolates, 9.5%), M. intracellulare (3 isolates, 4.7%), M. scrofulaceum (2 isolates, 3.2%), M. lentiflavum (2 isolates, 3.2%), and M. peregrinum (1 isolate, 1.6%). Genotyping did not reveal any clustering in M. intracellulare, M. gordonae and M. szulgai species. M. fortuitum, on the other hand, had two clusters, one with three isolates of M. fortuitum 1 and the other with two isolates of M. fortuitum 2 subspecies. The remaining 35 of the 40 isolates of M. fortuitum had unique fingerprint patterns.
M. fortuitum is the most common cause of infection by NTM among Infants and adolescents in rural Uganda. There is a varied number of species and genotypes, with minimal clustering within species, suggesting ubiquitous sources of infection to individuals in this community.
PMCID: PMC3599115  PMID: 23413873
7.  An Early Morning Sputum Sample Is Necessary for the Diagnosis of Pulmonary Tuberculosis, Even with More Sensitive Techniques: A Prospective Cohort Study among Adolescent TB-Suspects in Uganda 
The World Health Organization (WHO) recommends collection of two sputum samples for tuberculosis (TB) diagnosis, with at least one being an early morning (EM) using smear microscopy. It remains unclear whether this is necessary even when sputum culture is employed. Here, we determined the diagnostic yield from spot and the incremental yield from the EM sputum sample cultures among TB-suspected adolescents from rural Uganda. Sputum samples (both spot and early-morning) from 1862 adolescents were cultured by the Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT) methods. For spot samples, the diagnostic yields for TB were 19.0% and 57.1% with LJ and MGIT, respectively, whereas the incremental yields (not totals) of the early-morning sample were 9.5% and 42.9% (P < 0.001) with LJ and MGIT, respectively. Among TB-suspected adolescents in rural Uganda, the EM sputum culture has a high incremental diagnostic yield. Therefore, EM sputum in addition to spot sample culture is necessary for improved TB case detection.
PMCID: PMC3529437  PMID: 23304491
8.  A first insight into the genotypic diversity of Mycobacterium tuberculosis from Rwanda 
Mycobacterium tuberculosis complex (MTC) is the causative agent of tuberculosis (TB). Globally, increasing evidence shows that in M. tuberculosis, transmission varies from strain to strain and that different strains exhibit a range of geographical and host specificities, pathogenicity, and drug susceptibility. Therefore rapid and accurate differentiation of the members of MTC is critical in guiding treatment and public health decisions. We carried out a study at different health units and the National Reference Laboratory in Rwanda identify Mycobacterium tuberculosis complex species prevalent in TB patients in Rwanda. We further characterized the isolates using spoligotyping in order to gain an insight into the strain diversity of drug resistant and susceptible isolates of M. tuberculosis in this setting.
A total of 151 isolates from culture positive sputum samples were harvested, heat killed at 80°C for two hours, and then shipped to Makerere University College of Health Sciences, Uganda, for speciation and typing. Species identification was achieved by regions of difference (RD) analysis, while Spoligotyping was done to identify strain types.
Region of difference analysis identified all the 151 isolates as M. tuberculosis. Spoligotyping revealed predominance of the T2 family (58.3%, 88/151), with SIT 52 being the most prevalent strain (31.8%, 48/151). Among the 151 isolates, 64 (42.4%) were multidrug resistant (MDR) with 3 cases on mono-resistance. Of 94 retreatment cases, 48 (51.1%) were MDR and of 46 newly presenting cases 14 (30.4%) were MDR. There was a significant difference (p=0.01) in anti-TB drug resistance between new and retreatment cases in the sample. However, there was no significant relationship between HIV serostatus and the two major strain types SIT 52 (p =0.15and SIT 152 (p = 0.41).
Mycobacterium tuberculosis is the most prevalent species of Mycobacterium tuberculosis complex in Rwanda, and SIT 52 (T2) the predominant strain. There is significantly more MDR in the retreatment cases but no significant difference was observed by HIV status in relation to any spoligotypes.
PMCID: PMC3520741  PMID: 23131092
9.  Performance of Frontloading for Smear Microscopy in the Diagnosis of Pulmonary Tuberculosis: A Cross-Sectional Study at a Referral Hospital in Uganda 
PLoS ONE  2012;7(10):e48531.
To compare the performance of frontloading and the standard WHO method for diagnosis of pulmonary TB at Mulago Hospital in order to validate the technique in this setting.
This was a cross-sectional study in which 229 adult (≥18 years) TB suspects were consecutively enrolled. Suspects submitted three sputum samples as follows: at initial presentation, one hour after the first sample, and the next morning. The first and next morning samples formed the standard WHO method, while the first and the one hour later samples formed the frontloading method. Sample processing was by the standard N-acetyl L-cystein (NALC)-NaOH method, and fluorescent microscopy was done for both methods, while cultures of the first sample on Lowenstein-Jensen slants acted as a gold standard. The sensitivity, specificity and predictive values for the WHO standard and frontloading methods were compared.
The sensitivity of both the frontloading and standard schemes was 91.1% while their specificities were 86.2% and 91.7% respectively. There was excellent agreement between the diagnostic capacity of the two methods (kappa statistic = 0.87, P<0.0001). The positive predictive value for the frontloading scheme was 87.2% and that for the standard approach was 91.9%, while the negative predictive values were 90.4% and 90.9%, respectively. Among the HIV positive patients, frontloading identified 59/79 (74.7%) culture positive samples while the standard approach identified 55/79 (69.6%). In the HIV sero-negative category, on the other hand, front-loading identified 48/110 (43.6%) culture positive samples compared to 45/110 (40.9%) by the standard approach.
Frontloading based on smear examination of two same-day sputum samples has a similar performance to the current standard method and would not be associated with any significant missed diagnosis. It may therefore be advocated for use in our setting so as to reduce time to completion of diagnosis and patient loss to follow-up.
PMCID: PMC3483226  PMID: 23144768
10.  Evaluation of in-house PCR for diagnosis of smear-negative pulmonary tuberculosis in Kampala, Uganda 
BMC Research Notes  2012;5:487.
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.
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).
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.
PMCID: PMC3497582  PMID: 22947399
Pulmonary tuberculosis; Smear-negative TB; HIV-infected; HIV-TB co-infection; CD4 cell counts; Nucleic acid amplification tests; In-house PCR; Lowenstein-Jensen culture; Sensitivity; Specificity; Resource limited settings
11.  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.
PMCID: PMC2902549  PMID: 20472405
Streptococcus pneumoniae; Drug-resistant serotypes/serogroups; Transformation frequency; Tn916 transposon; mefE gene
12.  Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda 
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.
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.
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.
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.
PMCID: PMC3100262  PMID: 21453482
13.  Detection of multiple strains of Mycobacterium tuberculosis using MIRU-VNTR in patients with pulmonary tuberculosis in Kampala, Uganda 
BMC Infectious Diseases  2010;10:349.
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.
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.
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.
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.
PMCID: PMC3004912  PMID: 21143966
14.  Use of the GenoType® MTBDRplus assay to assess drug resistance of Mycobacterium tuberculosis isolates from patients in rural Uganda 
Drug resistance levels and patterns among Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients in Mbarara Uganda were investigated.
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.
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 rpoβ 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 katG (codon 315) gene mutations while only one strain showed the inhA promoter region gene mutation.
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.
PMCID: PMC2924299  PMID: 20691055
15.  Comparison of rapid tests for detection of rifampicin-resistant Mycobacterium tuberculosis in Kampala, Uganda 
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.
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.
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).
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.
PMCID: PMC2744678  PMID: 19709423
16.  DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda 
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.
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.
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 <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).
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.
PMCID: PMC2645406  PMID: 19196450
17.  Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in peri-urban Kampala, Uganda 
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.
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.
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.
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.
PMCID: PMC2519071  PMID: 18662405

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