Identification of Mycobacterium tuberculosis clinical isolates in Bangladesh by a species distinguishable multiplex PCR

<p>Abstract</p> <p>Background</p> <p>Species identification of isolates belonging to the <it>Mycobacterium tuberculosis </it>complex (MTC) seems to be important for the appropriate treatment of patients, since <it>M. bovis </it>is naturally resistant to a first line anti-tuberculosis (TB) drug, pyrazinamide, while most of the other MTC members are susceptible to this antimicrobial agent. A simple and low-cost differentiation method was needed in higher TB burden countries, such as Bangladesh, where the prevalence of <it>M. bovis </it>among people or cattle has not been investigated.</p> <p>Methods</p> <p>Genetic regions <it>cfp32</it>, RD9 and RD12 were chosen as targets for a species distinguishable multiplex PCR and the system was evaluated with twenty reference strains of mycobacterial species including non-tubercular mycobacteria (NTM). A total of 350 clinical MTC isolates obtained in Bangladesh were then analyzed with this multiplex PCR.</p> <p>Results</p> <p>All of the MTC reference strains gave expected banding patterns and no non-specific amplifications were observed in the NTM strains. Out of 350 clinical isolates examined by this method, 347 (99.1%) were positive for all of the <it>cfp32</it>, RD9 and RD12 and determined as <it>M. tuberculosis</it>. Two isolates lacked <it>cfp32 </it>PCR product and one lacked RD12, however, those three samples were further examined and identified as <it>M. tuberculosis </it>by the sequence analyses of <it>hsp65 </it>and <it>gyrB</it>.</p> <p>Conclusions</p> <p>The MTC-discrimination multiplex PCR (MTCD-MPCR) developed in this study showed high specificity and was thought to be very useful as a routine test because of its simplicity. In the current survey, all the 350 MTC isolates obtained from Bangladesh TB patients were determined as <it>M. tuberculosis </it>and no other MTC were detected. This result suggested the general TB treatment regimen including pyrazinamide to be the first choice in Bangladesh.</p

Validation of the ALS Assay in Adult Patients with Culture Confirmed Pulmonary Tuberculosis

BACKGROUND: We have earlier shown that Bacille Calmette-Guérin (BCG) vaccine-specific IgG Antibodies in Lymphocyte Supernatant (ALS) can be used for diagnosis of active tuberculosis (TB) in adults and children. METHODOLOGY/PRINCIPAL FINDINGS: The ALS method was validated in a larger cohort (n = 212) of patients with suspicion of pulmonary TB using multiple antigens (BCG, LAM, TB15.3, TB51A, CFP10-ESAT6-A, CFP, CW) from Mycobacterium tuberculosis. The sensitivity and specificity of the ALS assay was calculated using non-TB patients as controls. The sensitivity and the specificity were highest with BCG vaccine (90% and 88% respectively) followed by LAM (89% and 87% respectively). Simultaneous assessment of multiple antigen-specific antibodies increased sensitivity (91%) and specificity (88%). Using higher lymphocyte count in smaller volume of culture media increased detection and reduced the assay duration to ∼30 hrs. Twenty one patients with clinical findings strongly suggestive of TB finally diagnosed as non-TB patients were positive by the ALS assay, of which 9 (43%) were positive for 7 antigens and 19 (90%) for at least 3 antigens. CONCLUSIONS/SIGNIFICANCE: Our findings show that simultaneous detection of antigens improves the diagnostic potential of the ALS assay; the modified method increases sensitivity and can provide results in <48 hours, and enable detection of some cases of pulmonary TB that are not detectable by standard methods

Characterization of Mycobacterium orygis as M. tuberculosis Complex Subspecies

The oryx bacilli are Mycobacterium tuberculosis complex organisms for which phylogenetic position and host range are unsettled. We characterized 22 isolates by molecular methods and propose elevation to subspecies status as M. orygis. M. orygis is a causative agent of tuberculosis in animals and humans from Africa and South Asia

Operational Feasibility of Using Loop-Mediated Isothermal Amplification for Diagnosis of Pulmonary Tuberculosis in Microscopy Centers of Developing Countries▿

The characteristics of loop-mediated isothermal amplification (LAMP) make it a promising platform for the molecular detection of tuberculosis (TB) in developing countries. Here, we report on the first clinical evaluation of LAMP for the detection of pulmonary TB in microscopy centers in Peru, Bangladesh, and Tanzania to determine its operational applicability in such settings. A prototype LAMP assay with simplified manual DNA extraction was evaluated for accuracy and ease of use. The sensitivity of LAMP in smear- and culture-positive sputum specimens was 97.7% (173/177 specimens; 95% confidence interval [CI], 95.5 to 99.9%), and the sensitivity in smear-negative, culture-positive specimens was 48.8% (21/43 specimens; CI, 33.9 to 63.7%). The specificity in culture-negative samples was 99% (500/505 specimens; CI, 98.1 to 99.9%). The average hands-on time for testing six samples and two controls was 54 min, similar to that of sputum smear microscopy. The optimal amplification time was 40 min. No indeterminate results were reported, and the interreader variability was 0.4%. Despite the use of a single room without biosafety cabinets for all procedures, no DNA contamination was observed. The assay was robust, with high end-point stability and low rates of test failure. Technicians with no prior molecular experience easily performed the assay after 1 week of training, and opportunities for further simplification of the assay were identified

Microevolution of Mycobacterium tuberculosis in a Tuberculosis Patient▿

Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplified from one isolate) but were differentiated by small variations in IS6110 fingerprints, spoligotypes, 6 hypervariable MIRU-VNTR loci, and/or DiversiLab profiles, revealing patterns of microevolution in a clonal infection

Occurrence of tuberculosis among people exposed to cattle in Bangladesh

BackgroundTuberculosis (TB) has been an important public health concern in Bangladesh. The most common cause of human TB is Mycobacterium tuberculosis, while bovine TB is caused by Mycobacterium bovis.ObjectiveThe objective of this study was to determine the frequency of TB in individuals with occupational exposure to cattle and to detect Mycobacterium bovis among cattle in slaughterhouses in Bangladesh.MethodsBetween August 2014 and September 2015, an observational study was conducted in two government chest disease hospitals, one cattle market, and two slaughterhouses. [Correction added on 27 June 2023, after first online publication: In the preceding sentence, the year “2014” has been added after the word “August”.] Sputum samples were collected from individuals who met the criteria for suspected TB and had been exposed to cattle. Tissue samples were collected from cattle that had low body condition score(s). Both humans and cattle samples were screened for acid-fast bacilli (AFB) by Ziehl–Neelsen (Z-N) staining and cultured for Mycobacterium tuberculosis complex (MTC). Region of difference (RD) 9-based polymerase chain reaction (PCR) was also performed to identify Mycobacterium spp. We also conducted Spoligotyping to identify the specific strain of Mycobacterium spp.ResultsSputum was collected from a total of 412 humans. The median age of human participants was 35 (IQR: 25–50) years. Twenty-five (6%) human sputum specimens were positive for AFB, and 44 (11%) were positive for MTC by subsequent culture. All (N = 44) culture-positive isolates were confirmed as Mycobacterium tuberculosis by RD9 PCR. Besides, 10% of cattle workers were infected with Mycobacterium tuberculosis in the cattle market. Of all TB (caused by Mycobacterium tuberculosis) infected individuals, 6.8% of individuals were resistant to one or two anti-TB drugs. The majority of the sampled cattle (67%) were indigenous breeds. No Mycobacterium bovis was detected in cattle.ConclusionsWe did not detect any TB cases caused by Mycobacterium bovis in humans during the study. However, we detected TB cases caused by Mycobacterium tuberculosis in all humans, including cattle market workers

Zoonotic tuberculosis - the changing landscape

Despite slow reductions in the annual burden of active human tuberculosis (TB) cases, zoonotic TB (zTB) remains a poorly monitored and an important unaddressed global problem. There is a higher incidence in some regions and countries, especially where close association exists between growing numbers of cattle (the major source of Mycobacterium bovis ) and people, many suffering from poverty, and where dairy products are consumed unpasteurised. More attention needs to be focused on possible increased zTB incidence resulting from growth in dairy production globally and increased demand in low income countries in particular. Evidence of new zoonotic mycobacterial strains in South Asia and Africa (e.g. M. orygis), warrants urgent assessment of prevalence, potential drivers and risk in order to develop appropriate interventions. Control of M. bovis infection in cattle through detect and cull policies remain the mainstay of reducing zTB risk, whilst in certain circumstances animal vaccination is proving beneficial. New point of care diagnostics will help to detect animal infections and human cases. Given the high burden of human tuberculosis (caused by M. tuberculosis) in endemic areas, animals are affected by reverse zoonosis, including multi-drug resistant strains. This, may create drug resistant reservoirs of infection in animals. Like COVID-19, zTB is evolving in an ever-changing global landscape.the European and Developing Countries Clinical Trials Partnership (EDCTP2) pro- gramme which is supported under Horizon 2020, the European Union's Framework Programme for Research and Innovation. icddr, b is grateful to the Governments of Bangladesh, Canada, Sweden and the UK for providing core/unrestricted support. AZ is in receipt of a National Institutes of Health Research (NIHR) senior investigator award and the PANDORA-ID-NET.http://www.elsevier.com/locate/ijidhj2022Veterinary Tropical Disease