A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala

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. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1121-7) contains supplementary material, which is available to authorized users

Diagnostic accuracy of the Cepheid 3-gene host response fingerstick blood test in a prospective, multi-site study: interim results.

BACKGROUND: The development of a fast and accurate, non-sputum-based point-of-care triage test for tuberculosis (TB) would have a major impact on combating the TB burden worldwide. A new fingerstick blood test has been developed by Cepheid (the Xpert-MTB-Host Response (HR)-Prototype), which generates a 'TB score' based on mRNA expression of 3 genes. Here we describe the first prospective findings of the MTB-HR prototype. METHODS: Fingerstick blood from adults presenting with symptoms compatible with TB in South Africa, The Gambia, Uganda and Vietnam was analysed using the Cepheid GeneXpert MTB-HR prototype. Accuracy of the Xpert MTB-HR cartridge was determined in relation to GeneXpert Ultra results and a composite microbiological score (GeneXpert Ultra and liquid culture) with patients classified as having TB or other respiratory diseases (ORD). RESULTS: When data from all sites (n=75 TB, 120 ORD) were analysed, the TB score discriminated between TB and ORD with an AUC of 0·94 (CI, 0·91-0·97), sensitivity of 87% (CI, 77-93%) and specificity of 94% (88-97%). When sensitivity was set at 90% for a triage test, specificity was 86% (CI, 75-97%). These results were not influenced by HIV status or geographical location. When evaluated against a composite microbiological score (n=80 TB, 111 ORD), the TB score was able to discriminate between TB and ORD with an AUC of 0·88 (CI, 0·83-0·94), 80% sensitivity (CI, 76-85%) and 94% specificity (CI, 91-96%). CONCLUSIONS: Our interim data indicate the Cepheid MTB-HR cartridge reaches the minimal target product profile for a point of care triage test for TB using fingerstick blood, regardless of geographic area or HIV infection status

Distribution and transmission of mycobacterium tuberculosis complex lineages among children in peri-urban Kampala, Uganda

To gain insight into the transmission of tuberculosis (TB) in peri-urban Kampala-Uganda, we performed a household contact study using children as a surrogate for recent transmission of Mycobacterium tuberculosis (MTB). Using this approach, we sought to understand M. tuberculosis complex (MTBC) lineage diversity, distribution and how these relate to TB transmission to exposed children.; MTBC isolates from children aged ≤ 15 years, collected from 2002 to 2010 in a household-contact study, were analyzed using a LightCycler RT-PCR SNP genotyping assay (LRPS). The resultant genotypic data was used to determine associations between MTBC lineage and the children's clinical and epidemiological characteristics.; Of the 761 children surveyed, 9 % (69/761) had culture-positive TB an estimate in the range of global childhood TB; of these 71 % (49/69) were infected with an MTBC strain of the "Uganda family", 17 % (12/69) infected with MTBC lineage 4 strains other than MTBC Uganda family and 12 % (8/69) infected with MTBC lineage 3, thereby disproportionately causing TB in the study area. Overall the data showed no correlation between the MTBC lineages studied and transmission (OR = 0.304; P-value = 0.251; CI: 95 %; 0.039-2.326) using children a proxy for TB transmission.; Our findings indicate that MTBC Uganda family strains are the main cause of TB in children in peri-urban Kampala. Furthermore, MTBC lineages did not differ in their transmissibility to children

Perceptions about and reasons for participation in research bronchoscopy in Uganda: A qualitative analysis.

This study sought to assess perceptions towards and reasons for participation in research bronchoscopy studies in a high TB burden urban setting. Additionally, the study aimed to identify areas of pre- and post-procedural concern among healthy adults approached to participate in research bronchoscopy. A cross sectional qualitative study was undertaken at the Uganda-Case Western Reserve University Collaboration Tuberculosis Research Project Clinic at Mulago National Referral Hospital in Kampala, Uganda. In-depth interviews were conducted with participants at their pre-bronchoscopy visit (n = 17) and after they had undergone bronchoscopy (n = 23) to examine their perceptions and experiences with the procedure. Following consent, all interviews were audio recorded and later transcribed and typed in MS WORD. Local language interviews were translated into English by the social science interviewers. Qualitative analysis was performed manually following an inductive and emergent approach typical in thematic analysis. This study was approved by the Makerere University School of Social Sciences Research Ethics Committee (MAKSS REC 09.18.220) and registered with the Uganda National Council for Science and Technology (UNCST SS4785). Overall willingness to participate in bronchoscopy was high as many participants viewed the study as primarily a means of getting free health checks and determining their health status. Notably, despite extensive face to face counseling for this study coupled with the fact that our participants had been involved in prior research at the site, therapeutic misconception still played a pivotal role in willingness to participate in research bronchoscopy. Therapeutic misconception has important ethical and research implications in clinical research, which requires strategies to tackle it, even among a pool of potential participants who are knowledgeable about a disease or clinical care procedures. Continuous awareness and knowledge building about the difference between being a trial participant and therapeutic misconception must become a mainstay in trials to improve the process of informed consent for future research bronchoscopy studies

Impact of geographic distance on appraisal delay for active TB treatment seeking in Uganda: a network analysis of the Kawempe Community Health Cohort Study

Abstract Background Appraisal delay is the time a patient takes to consider a symptom as not only noticeable, but a sign of illness. The study’s objective was to determine the association between appraisal delay in seeking tuberculosis (TB) treatment and geographic distance measured by network travel (driving and pedestrian) time (in minutes) and distance (Euclidean and self-reported) (in kilometers) and to identify other risk factors from selected covariates and how they modify the core association between delay and distance. Methods This was part of a longitudinal cohort study known as the Kawempe Community Health Study based in Kampala, Uganda. The study enrolled households from April 2002 to July 2012. Multivariable interval regression with multiplicative heteroscedasticity was used to assess the impact of time and distance on delay. The delay interval outcome was defined using a comprehensive set of 28 possible self-reported symptoms. The main independent variables were network travel time (in minutes) and Euclidean distance (in kilometers). Other covariates were organized according to the Andersen utilization conceptual framework. Results A total of 838 patients with both distance and delay data were included in the network analysis. Bivariate analyses did not reveal a significant association of any distance metric with the delay outcome. However, adjusting for patient characteristics and cavitary disease status, the multivariable model indicated that each minute of driving time to the clinic significantly (p = 0.02) and positively predicted 0.25 days’ delay. At the median distance value of 47 min, this represented an additional delay of about 12 (95% CI: [3, 21]) days to the mean of 40 days (95% CI: [25, 56]). Increasing Euclidean distance significantly predicted (p = 0.02) reduced variance in the delay outcome, thereby increasing precision of the mean delay estimate. At the median Euclidean distance of 2.8 km, the variance in the delay was reduced by more than 25%. Conclusion Of the four geographic distance measures, network travel driving time was a better and more robust predictor of mean delay in this setting. Including network travel driving time with other risk factors may be important in identifying populations especially vulnerable to delay

Impact of geographic distance on appraisal delay for active TB treatment seeking in Uganda: a network analysis of the Kawempe Community Health Cohort Study

The study’s objective was to determine the association between appraisal delay in seeking tuberculosis (TB) treatment and geographic distance measured by network travel (driving and pedestrian) time (in minutes) and distance (Euclidean and self-reported) (in kilometers) and to identify other risk factors from selected covariates and how they modify the core association between delay and distance.Background: Appraisal delay is the time a patient takes to consider a symptom as not only noticeable, but a sign of illness. The study’s objective was to determine the association between appraisal delay in seeking tuberculosis (TB) treatment and geographic distance measured by network travel (driving and pedestrian) time (in minutes) and distance (Euclidean and self-reported) (in kilometers) and to identify other risk factors from selected covariates and how they modify the core association between delay and distance. Methods: This was part of a longitudinal cohort study known as the Kawempe Community Health Study based in Kampala, Uganda. The study enrolled households from April 2002 to July 2012. Multivariable interval regression with multiplicative heteroscedasticity was used to assess the impact of time and distance on delay. The delay interval outcome was defined using a comprehensive set of 28 possible self-reported symptoms. The main independent variables were network travel time (in minutes) and Euclidean distance (in kilometers). Other covariates were organized according to the Andersen utilization conceptual framework. Results: A total of 838 patients with both distance and delay data were included in the network analysis. Bivariate analyses did not reveal a significant association of any distance metric with the delay outcome. However, adjusting for patient characteristics and cavitary disease status, the multivariable model indicated that each minute of driving time to the clinic significantly (p = 0.02) and positively predicted 0.25 days’ delay. At the median distance value of 47 min, this represented an additional delay of about 12 (95% CI: [3, 21]) days to the mean of 40 days (95% CI: [25, 56]). Increasing Euclidean distance significantly predicted (p = 0.02) reduced variance in the delay outcome, thereby increasing precision of the mean delay estimate. At the median Euclidean distance of 2.8 km, the variance in the delay was reduced by more than 25%. Conclusion: Of the four geographic distance measures, network travel driving time was a better and more robust predictor of mean delay in this setting. Including network travel driving time with other risk factors may be important in identifying populations especially vulnerable to delay

Tuberculosis case finding in first-degree relative contacts not living with index tuberculosis cases in Kampala, Uganda

To assess the prevalence of pulmonary tuberculosis among first-degree relative (FDR) contacts not living with tuberculosis (TB) casesPurpose: To assess the prevalence of pulmonary tuberculosis among first-degree relative (FDR) contacts not living with tuberculosis (TB) cases. Methods: A cross-sectional analysis of household contacts living with an index TB case and FDR contacts living outside of households in Kampala, Uganda, is presented. Results: A total of 177 contacts (52 FDRs and 125 index household contacts) of 31 TB cases were examined. Compared with index household contacts, FDR contacts were older, more likely to be TB symptomatic (50% vs 33%), had a higher percentage of abnormal chest X-rays (19% vs 11%), sputum smear positive (15% vs 5%), and many similar epidemiologic risk factors, including HIV infection (13% vs 10%). Contact groups had similar pulmonary tuberculosis prevalence: 9.6% in FDR vs 10.4% in index household contacts and similar Mycobacterium tuberculosis infection: 62% in FDR vs 61% in index households. Conclusion: TB is common among FDR contacts. High TB prevalence justifies targeting FDRs during household contact investigations. Combining TB active-case finding among FDR contacts with household contact investigation in low-income setting is feasible. This should be part of national TB control program strategies for increasing TB case-detection rates and reducing community TB transmission and death

Wasting among Uganda men with pulmonary tuberculosis is associated with linear regain in lean tissue mass during and after treatment in contrast to women with wasting who regain fat tissue mass: prospective cohort study

Nutritional changes during and after tuberculosis treatment have not been well described. We therefore determined the effect of wasting on rate of mean change in lean tissue and fat mass as measured by bioelectrical impedance analysis (BIA), and mean change in body mass index (BMI) during and after tuberculosis treatment.Background: Nutritional changes during and after tuberculosis treatment have not been well described. We therefore determined the effect of wasting on rate of mean change in lean tissue and fat mass as measured by bioelectrical impedance analysis (BIA), and mean change in body mass index (BMI) during and after tuberculosis treatment. Methods: In a prospective cohort study of 717 adult patients, BMI and height-normalized indices of lean tissue (LMI) and fat mass (FMI) as measured by BIA were assessed at baseline, 3, 12, and 24 months. Results: Men with wasting at baseline regained LMI at a greater rate than FMI (4.55 kg/m2 (95% confidence interval (CI): 1.26, 7.83 versus 3.16 (95% CI: 0.80, 5.52)) per month, respectively during initial tuberculosis therapy. In contrast, women with wasting regained FMI at greater rate than LMI (3.55 kg/m2 (95% CI: 0.40, 6.70) versus 2.07 (95% CI: -0.74,4.88)), respectively. Men with wasting regained BMI at a rate of 6.45 kg/m2 (95% CI: 3.02, 9.87) in the first three months whereas women, had a rate of 3.30 kg/m2 (95% CI: -0.11, 6.72). There were minimal changes in body composition after month 3 and during months 12 to 24. Conclusion: Wasted tuberculosis patients regain weight with treatment but the type of gain differs by gender and patients may remain underweight after the initial phase of treatment

Long-term dominance of Mycobacterium tuberculosis Uganda family in peri-urban Kampala-Uganda is not associated with cavitary disease

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