Generalist and specialist species differ in the breadth of their ecological niche. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis Lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that Lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that while the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of Lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Multidrug-resistant tuberculosis (MDR-TB), caused by drug resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. In this study, we examined a dataset of 5,310 M. tuberculosis whole genome sequences from five continents. Despite great diversity with respect to geographic point of isolation, genetic background and drug resistance, patterns of drug resistance emergence were conserved globally. We have identified harbinger mutations that often precede MDR. In particular, the katG S315T mutation, conferring resistance to isoniazid, overwhelmingly arose before rifampicin resistance across all lineages, geographic regions, and time periods. Molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of pre-MDR polymorphisms, particularly katG S315, into molecular diagnostics will enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

The human-and animal-adapted lineages of the Mycobacterium tuberculosis complex (MTBC) are thought to have expanded from a common progenitor in Africa. However, the molecular events that accompanied this emergence remain largely unknown. Here, we describe two MTBC strains isolated from patients with multidrug resistant tuberculosis, representing an as-yet-unknown lineage, named Lineage 8 (L8), seemingly restricted to the African Great Lakes region. Using genome-based phylogenetic reconstruction, we show that L8 is a sister clade to the known MTBC lineages. Comparison with other complete mycobacterial genomes indicate that the divergence of L8 preceded the loss of the cobF genome region-involved in the cobalamin/vitamin B12 synthesis-and gene interruptions in a subsequent common ancestor shared by all other known MTBC lineages. This discovery further supports an East African origin for the MTBC and provides additional molecular clues on the ancestral genome reduction associated with adaptation to a pathogenic lifestyle.

f For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first-and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was >92% for 7 of 12 drugs when a strict definition was used and >96% for 10 of 12 drugs when agreement was defined by allowing a ؎ one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was >95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first-and second-line drugs except ethambutol, and results were available sooner than those determined by APM.T he emergence and spread of drug-resistant strains of Mycobacterium tuberculosis comprise a serious threat to tuberculosis (TB) control (1, 2). Knowledge of M. tuberculosis drug susceptibility is important in optimizing individual patient management and TB control in populations. Genotypic methods have the potential for a very short time to results, but to date, the knowledge of the full spectrum of genetic loci and mutations associated with resistance to many antituberculosis drugs is incomplete (3,4,5,6,7). Phenotypic methods therefore remain important. The reference phenotypic method-the indirect agar proportion method (APM) using Middlebrook solid media-is qualitative and based on drug critical concentrations. Limitations of the APM and related methods include lack of standardization and in some cases the need for in-laboratory preparation of drug stocks and agar plates, which can be a source of variability over time and between laboratories. Critical concentrations are based on historical epidemiological data and for some drugs are not well-aligned with achievable drug serum concentrations or accurate in predicting clinical failure (8,9,10). Studies on molecular drug resistance mechanisms in M. tuberculosis have shown that, at least for some antibiotics, different mutations are associated with different MICs, furthe...

BackgroundSlow decline in the incidence of tuberculosis (TB) has been observed in most high TB burden countries. Knowledge of the prevalence of different TB risk factors can help expand TB control strategies. However with the exception of Human Immunodeficiency Virus (HIV) the prevalence of the other TB risk factors are poorly studied in Uganda. We aimed to determine the prevalence of different TB risk factors and TB disease presentation among TB patients in Kampala Uganda.MethodsWe assessed 365 adult TB patients and used descriptive statistics to summarize their socio-demographic, clinical, radiological, sputum mycobacteriology and TB risk factors (HIV, diabetes, TB contact, alcohol use, tobacco smoking, poverty and overcrowding) data.ResultsA total of 158 (43.3%) patients were male and the median age was 29 (IQR 28–30). Majority of the patients (89.2%) had pulmonary TB, 86.9% were new and 13.2% were retreatment. Wasting (i.e. body mass index of <18.5 kg/m2) was found in 38.5% of the patients and 63% presented with cough. Constitutional symptoms (fever, anorexia, night sweats and weight loss) were reported by 32.1%. Most patients (78.6%) presented with non-cavity lung parenchyma disease (infiltrates, nodules, masses) but 35.2% had cavity disease. Pleural disease was detected in 19.3% of patients. Positive smear microscopy and culture (irrespective of month of treatment) was found in 52.7% and 36.5% of patients respectively. Any drug resistance was detected in 21.1% of patients while multidrug resistance (MDR) TB defined as resistance to rifampicin and isoniazid was detected in 6.3% of patients. All MDR patients were new patients.The prevalence of TB risk factors were as follows: HIV 41.4%, diabetes 5.4%, close contact 11.5%, family history 17.5%, smoking 26.37%, poverty 39.5%, overcrowding 57.3% and alcohol use 50.7%. Overcrowding increased smear positive rate, prevalence ratio 1.22, p = 0.09 but all the other studied risk factors did not affect clinical, radiological and mycobacteriological study patient characteristics.ConclusionsAmong TB patients in Kampala, Uganda, there is high prevalence of the known TB risk factors. Targeting reducing their prevalence may lead to better TB control in the country. Tuberculosis, risk factors, Uganda.

BackgroundThe diagnosis of childhood tuberculosis remains a challenge worldwide. The Xpert MTB/RIF test, a rapid mycobacteria tuberculosis diagnostic tool, was recommended for use in children based on data from adult studies. We evaluated the performance of the Xpert MTB/RIF test for the diagnosis of childhood pulmonary tuberculosis using one induced sputum sample and described clinical characteristics associated with a positive Xpert MTB/RIF test. The sputum culture on both Lowenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) was the gold standard.MethodsWe consecutively enrolled 250 Ugandan children aged 2 months to 12 years with suspected pulmonary tuberculosis between January 2011 and January 2012 into a cross-sectional diagnostic study at a tertiary care facility in Uganda.ResultsWe excluded data from 15 children (13 contaminated culture and 2 indeterminate MTB/RIF test results) and analysed 235 records. The Xpert MTB/RIF test had a sensitivity of 79.4% (95% CI 63.2 - 89.7) and a specificity of 96.5% (95% CI 93 – 98.3). The Xpert MTB/RIF test identified 13 of the 14 (92.9%) smear positive-culture positive and 14 of the 20 (70%) smear negative -culture positive cases. The Xpert MTB/RIF identified twice as many cases as the smear microscopy (79.4% Vs 41.2%). Age > 5 years (OR 3.3, 95% CI 1.4 – 7.4, p value 0.005), a history of Tuberculosis (TB) contact (OR 2.4, 95% CI 1.1 – 5.2, p value 0.03), and a positive tuberculin skin test (OR 4.1, 95% CI 1.7 – 10, p value 0.02) was associated with a positive Xpert MTB/RIF test. The median time to TB detection was 49.5 days (IQR 38.4-61.2) for LJ, and 6 days (IQR 5 – 11.5) for MGIT culture and 2 hours for the Xpert MTB/RIF test.ConclusionThe Xpert MTB/RIF test on one sputum sample rapidly and correctly identified the majority of children with culture confirmed pulmonary tuberculosis with high specificity.

BackgroundIntroduction of Xpert® MTB/RIF assay has revolutionalised the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (rif), a surrogate marker for multi-drug resistant TB (MDR-TB) as well as one of the principal first-line anti-tuberculosis drugs. In general, rpoB mutations can be found in 96.1% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide and these mutations usually are located in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, which is referred to as Rifampicin-resistance-determining region (RRDR). In this study, we determined the frequency of MDR-TB in Kampala using Xpert® MTB/RIF in comparison with the agar proportion method using Middlebrook 7H11and further determined the frequency of probes for different rpoB gene mutations using Xpert® MTB/RIF assay in the 81 bp RRDR.MethodsA total of 1501 specimens received at Mycobacteriology laboratory, Makerere University for Xpert testing between May 2011 and May 2014 were analysed by Xpert® MTB/RIF assay. Specimens that were positive for both MTB and rifampicin resistance were further subjected to a complete first line anti-mycobacterial drug susceptibility testing using Middlebrook 7H11 agar proportion method (APM).ResultsXpert® MTB/RIF assay detected 313 MTB positive specimens and out of which 12 specimens had both MTB and rifampicin- resistance conferred by four different rpoB gene mutations in the 81 bp-RRDR of MTB, further one (1/12), specimen was found to be rifampicin mono-resistant on APM while the 11 were found to be MDR-TB. Probes associated with the observed rif- resistance were as follows: E (7/12), B (3/12), A (1/12), D (1/12) and no rif-resistance was associated with probe C. No specimen yielded rif-resistance associated with more than one probe failure (mutation combinations). Probe D was associated with rifampicin mono-resistant.ConclusionsMDR-TB was at 3.5% in the studied population. Mutations associated with Probe E (58%) also known as codons 531and 533 are the commonest rpoB gene mutation identified by Xpert® MTB/RIF assay in this setting and mutations identified by probe E of the assay, turned out to be MDR-TB strains by agar proportion method antimicrobial susceptibility testing. No mutation was detected in the codon 522.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2334-14-481) contains supplementary material, which is available to authorized users.

BackgroundA high prevalence of tuberculosis (TB) in children presenting with severe pneumonia has previously been reported in South Africa. However, little is known about TB among children with pneumonia in Uganda and other resource limited countries. Moreover, TB is associated with high morbidity and mortality among such children. We conducted this study to establish the burden of pulmonary TB in children admitted with severe pneumonia in our setting.MethodsA cross-sectional study was conducted at Mulago, a National Referral and teaching hospital in Uganda. Hospitalised children 2 months to 12 years of age with severe pneumonia based on WHO case definition were enrolledfrom February to June 2011. Children with a previous TB diagnosis or receiving anti-TB treatment were excluded. Each child was screened for TB using Tuberculin skin test, Chest X-ray, induced sputum samples and blood culture for mycobacterium. Sputum smears were examined using fluorescent microscopy, and cultured on both Lowenstein Jensen media (LJ) and Mycobacterial Growth Indicator Tubes (MGIT).ResultsOf the 270 children with severe pneumonia who were recruited over a 5-month period in 2011, the incidence ratio of pulmonary TB in children admitted with severe pneumonia was 18.9% (95% CI 14.6 – 23.9). The proportion of culture confirmed PTB was 6.3% (95% CI 3.8 – 9.7). Age group under 1 year and 1 to 5 years (OR 2.8 (95% CI 1.7 – 7.4) and OR 2.4 (95% CI 1.05 – 5.9) respectively) were more likely to be associated with pulmonary TB compared to those children over 5 years of age. A history of TB smear positive contact was associated with pulmonary TB (OR 3.0 (95% CI 1.3–6.5).ConclusionsWe found a high burden of pulmonary TB in children admitted with severe pneumonia. These data highlight the need for TB screening in children admitted with severe pneumonia so as to improve TB case finding and child survival.