Diagnostic Performance of Different Laboratory Methods for the Detection of Extrapulmonary Tuberculosis.

Accurate and appropriate extrapulmonary tuberculosis (EPTB) diagnosis remains challenging due to its paucibacillary nature, requirement of invasive collection procedures, and lack of sensitive tests. This study investigated the diagnostic performance of different methods for the diagnosis of EPTB. A total of 1340 EPTB specimens were collected from presumptive EPTB patients from four different hospitals between November 2015 and March 2017. The collected specimens were tested with AFB microscopy, culture, Xpert MTB/RIF assay (Xpert), and MTBDRplus assay. Among the 1340 EPTB specimens, 49 (3.66%), 141 (10.52%), 166 (12.39%), and 154 (11.49%) were positive in AFB microscopy, culture, Xpert MTB/RIF, and MTBDRplus assay, respectively. A total of 194 (14.9%) cases were found positive in at least one of these methods. Using culture as a reference standard, the sensitivity and specificity of AFB microscopy, Xpert MTB/RIF, and MTBDRplus assay were: 27.0%/99.1%, 83.7%/96.0%, and 79.4%/96.5%, respectively. Compared to the composite reference standard, the sensitivity of culture, AFB microscopy, Xpert MTB/RIF, and MTBDRplus assay was 72.7%, 25.3%, 85.6%, and 79.4%, respectively, with a specificity of 100% for all the methods. The Xpert MTB/RIF assay showed the highest sensitivity compared to other methods. Considering the short turnaround time and promising findings, Xpert MTB/RIF assay should be integrated into national TB guidelines as a routine diagnostic test.

Diagnostic Yield of Xpert MTB/RIF Assay Using Bronchoalveolar Lavage Fluid in Detecting Mycobacterium tuberculosis among the Sputum-Scarce Suspected Pulmonary TB Patients.

Tuberculosis (TB) remains one of the leading causes of death worldwide and is caused by the single infectious agent Mycobacterium tuberculosis (Mtb). Although sputum is the most common specimen for pulmonary TB detection, some other respiratory specimens, such as bronchoalveolar lavage (BAL) fluid, gastric lavage (GL), and induced sputum (IS), are also collected from patients who are unable to deliver sputum. In this study, we aimed to evaluate the diagnostic performances of different test methods for TB diagnosis using BAL fluid specimens from sputum-scarce pulmonary TB patients. In this current study, a total of 210 BAL fluid specimens were collected and subjected to culture on Lowenstein-Jensen (L-J) medium, using an N-acetyl-L-cysteine-Sodium Hydroxide decontamination and digestion method, Xpert MTB/RIF (Xpert, Cepheid, Sunnyvale, CA, USA) assay, and acid-fast bacilli (AFB) microscopy with a Ziehl-Neelsen staining method for the detection of pulmonary TB. The sensitivity and specificity of these methods were then analyzed against the composite reference standard (CRS). Additionally, the receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of these assays. Among the 210 specimens, 39 (18.6%), 27 (12.8%), and 12 (5.7%) were found positive with Xpert assay, culture, and AFB microscopy, respectively. Considering the CRS, 42 (20%) were positive as the final diagnosis. The Xpert assay had a significantly higher sensitivity (92.9%, 95% CI: 80.5-98.5) compared to culture (64.3%, 95% CI: 48.0-78.4) and AFB microscopy (28.6%, 95% CI: 15.7-44.6) against the CRS. Additionally, the area under the ROC curve (AUC) for the Xpert assay, culture, and AFB microscopy accounted for 0.964, 0.821, and 0.655, respectively, when using CRS as the reference. In conclusion, our study findings demonstrated that the Xpert assay conferred a considerable diagnostic potential compared to other conventional methods for the diagnosis of pulmonary TB from BAL fluid specimens.

In-Depth Immunophenotyping With Mass Cytometry During TB Treatment Reveals New T-Cell Subsets Associated With Culture Conversion.

Tuberculosis (TB) is a difficult-to-treat infection because of multidrug regimen requirements based on drug susceptibility profiles and treatment observance issues. TB cure is defined by mycobacterial sterilization, technically complex to systematically assess. We hypothesized that microbiological outcome was associated with stage-specific immune changes in peripheral whole blood during TB treatment. The T-cell phenotypes of treated TB patients were prospectively characterized in a blinded fashion using mass cytometry after Mycobacterium tuberculosis (Mtb) antigen stimulation with QuantiFERON-TB Gold Plus, and then correlated to sputum culture status. At two months of treatment, cytotoxic and terminally differentiated CD8+ T-cells were under-represented and naïve CD4+ T-cells were over-represented in positive- versus negative-sputum culture patients, regardless of Mtb drug susceptibility. At treatment completion, a T-cell immune shift towards differentiated subpopulations was associated with TB cure. Overall, we identified specific T-cell profiles associated with slow sputum converters, which brings new insights in TB prognostic biomarker research designed for clinical application.

Performance of WHO-Endorsed Rapid Tests for Detection of Susceptibility to First-Line Drugs in Patients with Pulmonary Tuberculosis in Bangladesh.

The fast and accurate detection of susceptibility in drugs is a major challenge for a successful tuberculosis (TB) control programme. This study evaluated the performance of WHO-endorsed rapid diagnostic tools, such as BACTEC MGIT 960 SIRE (MGIT SIRE), GenoType MTBDRplus (MTBDRplus) and Xpert MTB/RIF (Xpert), for detecting susceptibility to first-line anti-TB drugs among pulmonary TB patients in Bangladesh. A total of 825 sputum samples with results from drug susceptibility testing (DST) against first-line anti-TB drugs in the MGIT SIRE, MTBDRplus and Xpert assays were evaluated and compared with the gold standard proportion susceptibility method of the Lowenstein-Jensen (LJ) medium. The overall sensitivities of MGIT SIRE were 97.6%, 90.0%, 61.3% and 44.9%, while specificities were 89.9%, 94.5%, 91.3% and 92.2% for detection of susceptibility to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (EMB), respectively. For MTBDRplus, the sensitivities were 88.0% and 88.7%, and the specificities were 97.4% and 97.8% for the detection of susceptibility to INH and RIF, respectively. Xpert demonstrated a sensitivity and specificity of 94.8% and 99.5%, respectively, for the detection of RIF susceptibility. All tests performed significantly better in retreated TB patients compared with primary TB cases. For detection of RIF and INH susceptibility, all three assays showed almost perfect agreement with the LJ method, although MGIT SIRE exhibited low agreement for STR and EMB. Considering the high performance, shorter turnaround time and ease of use, molecular-based approaches Xpert and MTBDRplus can be widely implemented throughout the country for the rapid detection of drug-resistant TB.

Molecular Epidemiology and Genetic Diversity of Multidrug-Resistant Mycobacterium tuberculosis Isolates in Bangladesh.

Although the number of multidrug-resistant (MDR) tuberculosis (TB) cases is high overall, a major gap exists in our understanding of the molecular characteristics and transmission dynamics of the MDR Mycobacterium tuberculosis isolates circulating in Bangladesh. The present study aims to characterize the MDR-TB isolates of Bangladesh and to investigate the mode of transmission. A total of 544 MDR-TB isolates were obtained from a nationwide drug-resistant TB surveillance study conducted between October 2011 and March 2017 covering all geographic divisions of Bangladesh. The isolates were characterized using TbD1 deletion analysis, spoligotyping, and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Deletion analysis showed that 440 (80.9%) isolates were the modern type, while the remainder were the ancestral type. The largest circulating lineage was the Beijing type, comprising 208 isolates (38.2%), followed by T, EAI, and LAM with 93 (17.1%), 58 (10.7%), and 52 (9.5%) isolates, respectively. Combined MIRU-VNTR and spoligotyping analysis demonstrated that the majority of the clustered isolates were of the Beijing and T1 lineages. The overall rate of recent transmission was estimated at 33.8%. In conclusion, the MDR M. tuberculosis isolates circulating in Bangladesh are mostly of the modern virulent type. The Beijing and T lineages are the predominant types and most of the transmission of MDR-TB can be attributed to them. The findings also suggest that, along with the remarkable transmission, the emergence of MDR-TB in Bangladesh is largely due to acquired resistance. Rapid and accurate diagnosis and successful treatment will be crucial for controlling MDR-TB in Bangladesh. IMPORTANCE Multidrug-resistant TB is considered to be the major threat to tuberculosis control activities worldwide, including in Bangladesh. Despite the fact that the number of MDR-TB cases is high, a major gap exists in our understanding of the molecular epidemiology of the MDR-TB isolates in Bangladesh. In our study, we characterized and classified the MDR-TB isolates circulating in Bangladesh and investigated their mode of transmission. Our results demonstrated that the MDR M. tuberculosis isolates circulating in Bangladesh are mostly of the modern virulent type. The Beijing and T lineages are the predominant types and are implicated in the majority of MDR-TB transmission. Our findings reveal that, along with the remarkable transmission, the emergence of MDR-TB in Bangladesh is largely due to acquired resistance, which may be due to nonadherence to treatment or inadequate treatment of TB patients. Rapid diagnosis and adherence to an appropriate treatment regimen are therefore crucial to controlling MDR-TB in Bangladesh.

Xpert MTB/RIF Ultra assay for the detection of Mycobacterium tuberculosis in people with negative conventional Xpert MTB/RIF but chest imaging suggestive of tuberculosis in Dhaka, Bangladesh.

BACKGROUND: The World Health Organization is considering substituting Xpert MTB/RIF (Xpert) with Xpert MTB/RIF Ultra (Ultra) for tuberculosis (TB) diagnosis, but supportive evidence is scarce, particularly among people more likely (presumptive) to have paucibacillary pulmonary TB (PTB). METHODS: During January-July 2018, presumptive PTB patients visiting TB Screening and Treatment Centres of Dhaka for routine chest X-ray (CXR) and conventional Xpert were enrolled. Sputum specimens were additionally tested with microscopy, culture, and Ultra. Specimens with "Trace call" by Ultra (Ultra-trace) were retested. Yield and diagnostic accuracy using various approaches to Ultra-trace and concordance of Ultra with bacteriological-positive PTB were assessed. RESULTS: Altogether, 1,083 participants (104 'Xpert-positive'; 979 'Xpert-negative and CXR-suggestive') were enrolled. All Xpert-positives and 900 (92%) Xpert-negatives were concordant with Ultra, however, seventy-nine (8.1%) Xpert-negative specimens tested positive with Ultra; 37 (46.8%) were categorically positives, and 42 (53.2%) were Ultra-trace. Sixteen of the 42 were retested, of whom eight (50.1%) Ultra-trace turned categorically positive, leading to 45 (4.6%) additionally detected by Ultra. Ultra sensitivity and specificity were 93.9% and 94.6%, and it additionally detected 5.4% more TB patients with a concordance of 94.6% (kappa, □=0.78) compared to any bacteriologically positive specimen (microscopy, culture, or Xpert). CONCLUSION: Ultra exhibited improved detection and accuracy among Xpert-negatives in a cohort with a high likelihood of PTB.

Correlation of gyr Mutations with the Minimum Inhibitory Concentrations of Fluoroquinolones among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Bangladesh.

Fluoroquinolone (FQ) compounds-moxifloxacin (MOX), levofloxacin (LEV), and ofloxacin (OFL)-are used to treat multidrug-resistant tuberculosis (MDR-TB) globally. In this study, we investigated the correlation of gyr mutations among Mtb isolates with the MICs of MOX, LEV, and OFL in Bangladesh. A total of 50 MDR-TB isolates with gyr mutations, detected by the GenoType MTBDRsl assay, were subjected to drug susceptibility testing to determine the MICs of the FQs. Spoligotyping was performed to correlate the genetic diversity of the gyr mutant isolates with different MIC distributions. Among the 50 isolates, 44 (88%) had mutations in the gyrA gene, one (2%) had a mutation in the gyrB gene, and five (10%) isolates had unidentified mutations. The substitutions in the gyrA region were at A90V (n = 19, 38%), D94G (n = 16, 32%), D94A (n = 4, 8%), D94N/D94Y (n = 4, 8%), and S91P (n = 1, 2%), compared to the gyrB gene at N538D (n = 1.2%). D94G mutations showed the highest MICs for MOX, LEV, and OFL, ranging between 4.0 and 8.0 µg/mL, 4.0 and 16.0 µg/mL, and 16.0 and 32.0 µg/mL, respectively; while the most common substitution of A90V showed the lowest ranges of MICs (1.0-4.0 µg/mL, 2.0-8.0 µg/mL, and 4.0-32.0 µg/mL, respectively). Spoligotyping lineages demonstrated no significant differences regarding the prevalence of different gyr mutations. In conclusion, the substitutions of codon A90V and D94G in the gyr genes were mostly responsible for the FQs' resistance among Mtb isolates in Bangladesh. Low levels of resistance were associated with the substitutions of A90V, while the D94G substitutions were associated with a high level of resistance to all FQs.

Genetic diversity and characterization of M. tuberculosis isolates causing extrapulmonary tuberculosis in Bangladesh.

Tuberculosis (TB) remains one of the leading causes of death and Bangladesh ranks 7th among the highest TB burden countries. Though molecular epidemiological data for pulmonary TB (PTB) have previously been described in Bangladesh, data on the molecular characterization and clinical association with different lineages among extrapulmonary TB (EPTB) is lacking. The aim of the study was to investigate the molecular characterization and lineage distribution of M. tuberculosis isolates obtained from patients with EPTB in Bangladesh. Between November 2015 and March 2017, a total of 1,340 EPTB specimens including lymph node, pus, tissue, ascitic fluid, cerebrospinal fluid, pleural fluid, abscess wall, urine etc. were collected from four tertiary care hospitals in Dhaka city, Bangladesh. Among the specimens, 141 were found positive on solid culture. Molecular characterization of the 141 isolates was done by deletion analysis, spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTR) analysis. Among the 141 isolates, 80 (56.7%) were found as 'modern' and the remaining 61 (43.3%) were 'ancestral' type. Spoligotyping results revealed 91 distinct patterns of which 74 isolates were unique and the remaining 67 were divided into 17 distinct clusters. East African- Indian (EAI) lineage was the most predominant, comprising 26 (18.4%) isolates, followed by the Beijing lineage (14.2%). 15-loci MIRU-VNTR analysis revealed that 132 isolates (93.5%) had unique patterns, whereas only 9 (6.5%) isolates were grouped into 4 distinct clusters. In conclusion, the study findings provide a first insight into genetic diversity of EPTB isolates in Bangladesh. The present study demonstrated that 'modern' strains were more prevalent among the EPTB cases, while EAI lineages were predominantly circulating in this region.

Distribution and Frequency of rpoB Mutations Detected by Xpert MTB/RIF Assay Among Beijing and Non-Beijing Rifampicin Resistant Mycobacterium tuberculosis Isolates in Bangladesh.

BACKGROUND: Rifampicin resistance (RR) is a key indicator of multidrug-resistant tuberculosis (MDR-TB) and 95% of the RR is associated with the mutation in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene of Mycobacterium tuberculosis complex (MTBC). The Xpert MTB/RIF (Xpert) assay uses five overlapping molecular beacon probes (A-E) complementary to RRDR region that detect MTBC and mutations associated with RR. The objective of the study was to investigate the distribution and frequency of mutations detected by Xpert assay among Beijing and non-Beijing RR-TB isolates. METHODS: A total of 205 randomly selected RR-TB specimens detected by Xpert assay were included in this study. A portion of specimens was further subjected to culture, MTBDRplus test and the positive culture isolates were genotyped by spoligotyping. RESULTS: We found that the most frequent mutation occurred at probe E (S531L) binding region in both Beijing and non-Beijing isolates (61.9% and 66.9%, respectively). The Beijing family had higher mutation rates than non-Beijing (19.0% vs 12.4%) at probe B (D516V) while the non-Beijing family had higher mutations at probe D (H526D or H526Y) than the Beijing (13.2% vs 10.7%) family. Mutations at probes Aand C were less common in both Beijing and non-Beijing isolates. There was no significant difference (P=0.36) in the occurrence of mutations at different probes between Beijing and non-Beijing isolates. CONCLUSIONS: The study results revealed that the most frequent mutation occurs in the region of probe E and the least common mutations at probe A and C among both Beijing and non-Beijing RR-TB cases. This first insight into the probe mutation variation and frequencies among the RR-TB cases in Bangladesh forms the baseline information for further investigation.