Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.

Preservation of sputum samples with cetylpyridinium chloride (CPC) for tuberculosis cultures and Xpert MTB/RIF in a low-income country.

BACKGROUND: Culture contamination with environmental bacteria is a major challenge in tuberculosis (TB) laboratories in hot and humid climate zones. We studied the effect of cetylpyridinium chloride (CPC) preservation on culture results and performance of Xpert MTB/RIF. METHODS: Consecutive sputum samples from microscopy smear-positive TB patients were collected. Two-hundred samples were equally split in two aliquots, one aliquot was treated with CPC and stored at ambient temperature for 7 days. The second aliquot was immediately processed. Samples were decontaminated for 20, 15 or 10 min, and subsequently cultured on Löwenstein-Jensen medium. Furthermore, 50 samples were stored for 7, 14 and 21 days, and 100 CPC-pretreated samples tested by Xpert MTB/RIF. RESULTS: CPC pretreated samples showed a higher culture yield compared to non-treated sputum samples across all decontamination times: 94% vs. 73% at 10 min (p = 0.01), 94% vs. 64% at 15 min (p = 0.004), and 90% vs. 52% at 20 min (p < 0.001). The quantitative culture grading was consistently higher in CPC treated compared to non-CPC treated samples. The proportion of contaminated cultures was lower in CPC pretreated samples across all decontamination times (range 2-6%) compared to non-CPC treated samples (15-16%). For storage times of CPC treated samples of 7, 14, and 21 days, 84, 86, and 84% of the respective cultures were positive. Of 91 CPC treated samples with a positive culture, 90 were also Xpert MTB/RIF positive. CONCLUSIONS: CPC increases culture yield, decreases the proportion of contamination, and does not alter the performance of Xpert MTB/RIF.

Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System.

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.

Tuberculous spondylitis diagnosed through Xpert MTB/RIF assay in urine: a case report.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) is associated with high rates of morbidity and mortality. Diagnosis of EPTB is challenging in resource-limited settings due to difficulties in obtaining samples, as well as the paucibacillarity of the specimens. Skeletal tuberculosis accounts for 10-35 % of EPTB cases, with vertebral osteomyelitis (Pott's disease) representing 50 % of the cases. We present two cases of suspected Pott's disease, diagnosed through GeneXpert MTB/RIF assay in urine at a rural Tanzanian hospital. CASE PRESENTATION: Case I A 49-year old male, HIV-1 positive, on co-formulated tenofovir disoproxil fumarate/lamivudine/efavirenz since 2009 and CD4 counts of 205 cells/µL (13 %). He presented with lower back pain and progressive lower limb weakness for two weeks prior to admission. The physical examination revealed bilateral flaccid paraplegia with reduced reflexes, but otherwise unremarkable findings. A lateral lumbar X-ray showed noticeable reduction of intervertebral space between L4 and L5, and a small calcification in the anterior longitudinal ligament between L4 and L5, being compatible with focal spondylosis deformans but inconclusive with regard to tuberculous spondylitis. An abdominal ultrasound showed normal kidneys, bladder and prostate gland. The urinalysis and complete blood counts (CBC) were normal. M. Tuberculosis was detected through GeneXpert MTB/RIF in centrifuged urine, with no resistance to rifampicin. Case II A 76-year old female, HIV-1 negative, presented with lower back pain and progressive weakness and numbness of the lower limbs for two months prior to admission. The physical examination revealed paraplegia, but otherwise unremarkable findings. The lumbosacral X-ray findings were compatible with spondylosis deformans of the lumbar spine and possible tuberculous spondylitis in L3-L4. The abdominal and renal ultrasound showed normal kidneys and bladder. The urinalysis and CBC were normal. M. Tuberculosis was detected through GeneXpert MTB/RIF in centrifuged urine, with no resistance to rifampicin. CONCLUSION: We report two cases of suspected tuberculous spondylitis diagnosed through Xpert MTB/RIF in urine samples from a rural Tanzanian hospital. Urine testing using Xpert MTB/RIF reflects disseminated disease and renal involvement, and may offer a feasible additional diagnostic approach for Pott's disease in rural Africa.

Evaluation of Xpert® MTB/RIF and Ustar EasyNAT™ TB IAD for diagnosis of tuberculous lymphadenitis of children in Tanzania: a prospective descriptive study.

BACKGROUND: Fine needle aspiration biopsy has become a standard approach for diagnosis of peripheral tuberculous lymphadenitis. The aim of this study was to compare the performance of Xpert MTB/RIF and Ustar EasyNAT TB IAD nucleic acid amplification assays, against acid-fast bacilli microscopy, cytology and mycobacterial culture for the diagnosis of TB lymphadenitis in children from a TB-endemic setting in Tanzania. METHODS: Children of 8 weeks to 16 years of age, suspected of having TB lymphadenitis, were recruited at a district hospital in Tanzania. Fine needle aspirates of lymph nodes were analysed using acid-fast bacilli microscopy, liquid TB culture, cytology, Xpert MTB/RIF and EasyNAT. Latent class analysis and comparison against a composite reference standard comprising "culture and/or cytology" was done, to assess the performance of Xpert MTB/RIF and EasyNAT for the diagnosis of TB lymphadenitis. RESULTS: Seventy-nine children were recruited; 4 were excluded from analysis. Against a composite reference standard of culture and/or cytology, Xpert MTB/RIF and EasyNAT had a sensitivity and specificity of 58 % and 93 %; and 19 % and 100 % respectively. Relative to latent class definitions, cytology had a sensitivity of 100 % and specificity of 94.7 %. CONCLUSIONS: Combining clinical assessment, cytology and Xpert MTB/RIF may allow for a rapid and accurate diagnosis of childhood TB lymphadenitis. Larger diagnostic evaluation studies are recommended to validate these findings and on Xpert MTB/RIF to assess its use as a solitary initial test for TB lymphadenitis in children.

Detection of Mycobacterium tuberculosis by EasyNAT diagnostic kit in sputum samples from Tanzania.

The EasyNAT assay was evaluated for the detection of tuberculosis in sputum smears from presumptive pulmonary tuberculosis (TB) patients in an African high-TB and high-HIV setting. The sensitivity of the EasyNAT assay was 66.7%, and the specificity and positive predictive value were 100% for the culture-positive patients. The sensitivity was only 10% in the smear-negative and culture-positive patients.

The relative transmission fitness of multidrug-resistant Mycobacterium tuberculosis in a drug resistance hotspot.

Multidrug-resistant tuberculosis (MDR-TB) is among the most frequent causes of death due to antimicrobial resistance. Although only 3% of global TB cases are MDR, geographical hotspots with up to 40% of MDR-TB have been observed in countries of the former Soviet Union. While the quality of TB control and patient-related factors are known contributors to such hotspots, the role of the pathogen remains unclear. Here we show that in the country of Georgia, a known hotspot of MDR-TB, MDR Mycobacterium tuberculosis strains of lineage 4 (L4) transmit less than their drug-susceptible counterparts, whereas most MDR strains of L2 suffer no such defect. Our findings further indicate that the high transmission fitness of these L2 strains results from epistatic interactions between the rifampicin resistance-conferring mutation RpoB S450L, compensatory mutations in the RNA polymerase, and other pre-existing genetic features of L2/Beijing clones that circulate in Georgia. We conclude that the transmission fitness of MDR M. tuberculosis strains is heterogeneous, but can be as high as drug-susceptible forms, and that such highly drug-resistant and transmissible strains contribute to the emergence and maintenance of hotspots of MDR-TB. As these strains successfully overcome the metabolic burden of drug resistance, and given the ongoing rollout of new treatment regimens against MDR-TB, proper surveillance should be implemented to prevent these strains from acquiring resistance to the additional drugs.

Developing customized stepwise MIRU-VNTR typing for tuberculosis surveillance in Georgia.

INTRODUCTION: Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) typing has been widely used for molecular epidemiological studies of tuberculosis (TB). However, genotyping tools for Mycobacterium tuberculosis (Mtb) may be limiting in some settings due to high cost and workload. In this study developed a customized stepwise MIRU-VNTR typing that prioritizes high discriminatory loci and validated this method using penitentiary system cohort in the country of Georgia. METHODS: We used a previously generated MIRU-VNTR dataset from recurrent TB cases (32 cases) in Georgia and a new dataset of TB cases from the penitentiary system (102 cases) recruited from 2014 to 2015. A Hunter-Gaston Discriminatory Index (HGDI) was calculated utilizing a 24 standard loci panel, to select high discriminatory power loci, subsequently defined as the customized Georgia-specific set of loci for initial typing. The remaining loci were scored and hierarchically grouped for second and third step typing of the cohort. We then compared the processing time and costs of the customized stepwise method to the standard 24-loci method. RESULTS: For the customized Georgia-specific set that was used for initial typing, 10 loci were selected with a minimum value of 0.32 to the highest HGDI score locus. Customized 10 loci (step 1) typing of 102 Mtb patient isolates revealed 35.7% clustered cases. This proportion was reduced to 19.5% after hierarchical application of 2nd and 3rd step typing with the corresponding groups of loci. Our customized stepwise MIRU-VNTR genotyping approach reduced the quantity of samples to be typed and therefore overall processing time and costs by 42.6% each. CONCLUSION: Our study shows that our customized stepwise MIRU-VNTR typing approach is a valid alternative of standard MIRI-VNTR typing panels for molecular epidemiological investigation in Georgia that saves time, workload and costs. Similar approaches could be developed for other settings.

Prisons as ecological drivers of fitness-compensated multidrug-resistant Mycobacterium tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) accounts for one third of the annual deaths due to antimicrobial resistance1. Drug resistance-conferring mutations frequently cause fitness costs in bacteria2-5. Experimental work indicates that these drug resistance-related fitness costs might be mitigated by compensatory mutations6-10. However, the clinical relevance of compensatory evolution remains poorly understood. Here we show that, in the country of Georgia, during a 6-year nationwide study, 63% of MDR-TB was due to patient-to-patient transmission. Compensatory mutations and patient incarceration were independently associated with transmission. Furthermore, compensatory mutations were overrepresented among isolates from incarcerated individuals that also frequently spilled over into the non-incarcerated population. As a result, up to 31% of MDR-TB in Georgia was directly or indirectly linked to prisons. We conclude that prisons fuel the epidemic of MDR-TB in Georgia by acting as ecological drivers of fitness-compensated strains with high transmission potential.

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.