Performance of GenoType MTBDRsl assay for detection of second-line drugs and ethambutol resistance directly from sputum specimens of MDR-TB patients in Bangladesh

Background Rapid and early detection of drug susceptibility among multidrug-resistant tuberculosis (MDR-TB) patients could guide the timely initiation of effective treatment and reduce transmission of drug-resistant TB. In the current study, we evaluated the diagnostic performance of GenoType MTBDRsl (MTBDRsl) ver1.0 assay for detection of resistance to ofloxacin (OFL), kanamycin (KAN) and ethambutol (EMB), and additionally the XDR-TB among MDR-TB patients in Bangladesh. Methods The MTBDRsl assay was performed directly on 218 smear-positive sputum specimens collected from MDR-TB patients and the results were compared with the phenotypic drug susceptibility testing (DST) performed on solid Lowenstein-Jensen (L-J) media. We also analyzed the mutation patterns of gyrA, rrs, and embB genes for detection of resistance to OFL, KAN and EMB, respectively. Results The sensitivity and specificity of the MTBDRsl compared to phenotypic L-J DST were 81.8% (95% CI, 69.1–90.9) and 98.8% (95% CI, 95.6–99.8), respectively for OFL (PPV: 95.7% & NPV: 94.1%); 65.1% (95% CI, 57.5–72.2) and 86.7% (95% CI, 73.2–94.9), respectively for EMB (PPV: 94.9% & NPV: 39.4%); and 100% for KAN. The diagnostic accuracy of KAN, OFL and EMB were 100, 94.5 and 69.6%, respectively. Moreover, the sensitivity, specificity and diagnostic accuracy of MtBDRsl for detection of XDR-TB was 100%. The most frequently observed mutations were at codon D94G (46.8%) of gyrA gene, A1401G (83.3%) of rrs gene, and M306V (41.5%) of the embB gene. Conclusion Considering the excellent performance in this study we suggest that MTBDRsl assay can be used as an initial rapid test for detection of KAN and OFL susceptibility, as well as XDR-TB directly from smear-positive sputum specimens of MDR-TB patients in Bangladesh.

Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates—Chennai, India, 2013–2016

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

Efficacy of GeneXpert in Diagnosing Smear Negative Pulmonary Tuberculosis and Comparison to Culture

Objectives: Aim of present study is to observe the efficiency of GeneXpert MTB/RIF Assay in comparison to MTB culture on Lowenstein Jensen media in diagnosis of smear negative pulmonary tuberculosis cases. Methods: This descriptive study was carried out in The University of Lahore in collaboration with King Edward Medical University/Mayo Hospital Lahore 11th September, 2020 to 10th April 2021. Smear negative for acid fast bacilli patients enrolled for anti TB treatment were the target population. After taking informed consent, patients were asked to submit first morning sputum sample for culture on Lowenstein Jensen Medium and GeneXpert. Results: A total of 345 smear negative TB patients were diagnosed clinically and/or on the basis of radiological findings with mean age of 38.28±17.93,consisting of 47.5% male and 52.5% females recruited in this study. History of TB contact was present among 41.4% patients whereas history of smoking and diabetes remained to be 27.2% and 17.4% respectively. Culture showed significantly higher rate (35.1%) (p-value <0.05) of detection of MTB as compared to GeneXpert (21.5%). A sensitivity of 56.01% was calculated for GeneXpert whereas for culture on LJ medium it was 60.63%. Conclusion: Sensitivity of GeneXpert MTB/RIF Assay is a bit low in diagnosing the SNPT patients as compared to the culture but still registers itself as a handsome tool in terms of promptness and definite detection of MTB complex. Further provision of rifampicin susceptibility is bonus in same time.

Prevalence and molecular identification of Mycobacteria isolated from animals slaughtered at Sokoto modern abattoir, Sokoto State, Nigeria

This study investigated the molecular epidemiology of Mycobacteria isolated from animals slaughtered at Sokoto modern abattoir. During meat inspection, 104 suspected tuberculosis lesions were sampled from a total of 102,681 animals slaughtered between November 2016 and January 2018. These samples were subjected to Ziehl Neelsen staining, followed by culture on Lowenstein-Jensen media. Subsequently, polymerase chain reaction (PCR) and sequencing of the 65KDa heat shock protein (hsp65) gene were performed to identify and phylogenetically characterize the cultured organisms. Because sequencing of the hsp65 gene was unable to distinguish between Mycobacterium bovis (M. bovis) and M. tuberculosis, PCR was performed to amplify a genomic region-specific to M. bovis in order to differentiate them from M. tuberculosis. Results showed that, 14 samples yielded growth after culture. Furthermore, hsp65 was detected in 9 out of the 14 isolates screened, 5 of the amplicons were successfully sequenced. Similarity search using NCBI BLAST tool showed the five sequences to share highest identities with Mycobacterium novocastrense (95.99%), M. canettii (94.54%), and M. tuberculosis/M. bovis (100%). Two out of the 5 isolates were confirmed to be M. bovis after PCR amplification using M. bovis specific primers. Phylogenetic tree further confirmed the identity of these isolates by placing them close to species of their kind. Further studies should be conducted to establish the transmission dynamics of the zoonotic Mycobacteria between animals and their owners, to facilitate control and eradication of tuberculosis.

Diagnostic Value of Bronchoalveolar Lavage Examination in Sputum Negative Patients for AFB in Suspected Pulmonary Tuberculosis

Background: Pulmonary tuberculosis (PTB) is one of the most common infections worldwide, more commonly among the developing countries like Bangladesh. So its early detection and prompt treatment was a challenge and the burden of diagnostic challenge was higher if the patients smear negative for Acid Fast Bacilli (AFB). Objective: Evaluate the diagnostic value of Bronchoalveolar lavage (BAL) for diagnosis of suspected Pulmonary Tuberculosis (PTB) whose sputum for AFB smear neagtive. Materials and Methods: A cross-sectional observational reserach was undertaken where 50 patients were included on the basis of specific inclusion and exclusion criteria. All patients who had negative smear for AFB but highly suspected for PTB underwent fibreoptic bronchoscopy to collect Bronchoalveolar lavage (BAL) fluid for diagnostic testing in the form of BAL for AFB and mycobacterial culture in Lowenstein Jensen medium. Results: The Male predominacy 29 (58%) was obserevd among the smear negative PTB patients. Clinically more than seventy percent (72%) presents with fever then cough with sputum and haemoptysis 62% and 32% respectively. Radiological cavitation 33 (66%) was the most common x-ray findings. After analysis of BAL for AFB about 31 (62%) patient found positive and on culture about mycobacterial growth found in 29 (58%) patients. Conclusion: Bronchoalveolar lavage had a superior diagnostic value in patients with smear negative suspected pulmonary tuberculosis. KYAMC Journal.2021;12(02): 84-87

Validez diagnóstica del GeneXpert para Mycobacterium tuberculosis y prueba de resistencia a rifampicina

Introducción. El diagnóstico temprano de tuberculosis permite el control de la enfermedad y su transmisibilidad. Objetivo. Describir la validez diagnóstica del GeneXpert MTB/RIF para Mycobacterium tuberculosis en muestra bronquial, utilizando como referencia el cultivo Löwenstein Jensen. Metodología. Estudio transversal analítico, mediante revisión de 942 registros de la Unidad de Broncoscopia durante el año 2014 al 2018, de las cuales 320 cumplieron criterios de inclusión. Estos datos fueron exportados a un formato compatible con Epi Info versión 7 y analizados con parámetros estadísticos de sensibilidad, especificidad, valor predictivo positivo, valor predictivo negativo, prueba de concordancia e índice Kappa a través de Epidat 4,2. Resultado. De los 320 pacientes sometidos a fibrobroncoscopía diagnóstica para tuberculosis con GeneXpert MTB/RIF, los resultados negativos fueron 79 % (252) y positivo 21 % (68); el 1 % mostró resistencia a rifampicina. Se reportó una fuerte concordancia de GeneXpert MTB/RIF con el cultivo bacilo ácido alcohol resistente, que se determinó con un índice de kappa de 0,88 +/- (0,81-0,94) IC 95 %, una sensibilidad del 98 %, especificidad del 96 %, valor predictivo positivo 83 % (IC 95 %), valor predictivo negativo 99,6 % (CI 95 %).Conclusión. La prueba GeneXpert MTB/RIF tiene una capacidad altamente sensible y específica para el diagnóstico de tuberculosis en muestras obtenidas por fibrobroncoscopía.

Characterization of Drug-Resistant Lipid-Dependent Differentially Detectable Mycobacterium tuberculosis

An estimated 15–20% of patients who are treated for pulmonary tuberculosis (TB) are culture-negative at the time of diagnosis. Recent work has focused on the existence of differentially detectable Mycobacterium tuberculosis (Mtb) bacilli that do not grow under routine solid culture conditions without the addition of supplementary stimuli. We identified a cohort of TB patients in Lima, Peru, in whom acid-fast bacilli could be detected by sputum smear microscopy, but from whom Mtb could not be grown in standard solid culture media. When we attempted to re-grow Mtb from the frozen sputum samples of these patients, we found that 10 out of 15 could be grown in a glycerol-poor/lipid-rich medium. These fell into the following two groups: a subset that could be regrown in glycerol after “lipid-resuscitation”, and a group that displayed a heritable glycerol-sensitive phenotype that were unable to grow in the presence of this carbon source. Notably, all of the glycerol-sensitive strains were found to be multidrug resistant. Although whole-genome sequencing of the lipid-resuscitated strains identified 20 unique mutations compared to closely related strains, no single genetic lesion could be associated with this phenotype. In summary, we found that lipid-based media effectively fostered the growth of Mtb from a series of sputum smear-positive samples that were not culturable in glycerol-based Lowenstein–Jensen or 7H9 media, which is consistent with Mtb’s known preference for non-glycolytic sources during infection. Analysis of the recovered strains demonstrated that both genetic and non-genetic mechanisms contribute to the observed differential capturability, and suggested that this phenotype may be associated with drug resistance.

STUDY OF RESISTANCE TO ANTITUBERCULOSIS DRUGS IN PATIENTS WHO ARE IN STATIONARY TREATMENT IN THE DEPARTMENT OF RESISTANT FORMS OF PULMONARY TUBERCULOSIS

A comparative study of resistance to anti-tuberculosis drugs in patients with newly diagnosed pulmonary tuberculosis and in patients with chronic forms of tuberculosis who were treated in an anti-tuberculosis hospital was carried out. We examined 142 patients treated in the department of resistant forms of tuberculosis. A complete bacteriological study of sputum for Mycobacterium tuberculosis was carried out with the determination of sensitivity to anti-tuberculosis drugs during the cultivation of microorganisms on a liquid nutrient medium in a system with automated registration of bacterial growth and on a dense nutrient medium of Lowenstein-Jensen using the method of absolute concentrations. Methods of PCR analysis were also used. According to the anamnesis, two groups of patients were formed: 82 patients with newly diagnosed tuberculosis and 60 patients with a chronic course of the disease or with a relapse of the disease. According to the age structure, patients under the age of 50 prevailed in both groups. At the same time, in the first group there was a significant number of patients aged 18-29 years - 20.7%. In the second group, the number of patients aged 18-29 years was significantly less - 5%. Despite the fact that all patients were assigned to the category of multidrug-resistant pathogen and were treated in the unit of resistant forms of tuberculosis, it was important to assess the level of Mycobacterium tuberculosis resistance separately for patients in each of the selected groups. The largest differences between groups were found in the assessment of second-line drug resistance. Patients of the second group showed high resistance to fluoroquinolones - 30% and amikacin - 41.6%. In patients with newly diagnosed pulmonary tuberculosis (first group), resistance to fluoroquinolones was 9.7%, and to amikacin - 17.1%. Capreomycin was the most effective in both groups. The resistance of the causative agent of tuberculosis in patients of the first group was 4.9%, in the second - 11.6%.

Occurrence and risk factors of nontuberculous mycobacteria in tuberculosis-suspected patients in the north of Iran

Background and Objectives: Some Nontuberculous Mycobacteria (NTM) can occasionally infect the human population and cause infections having symptoms similar to tuberculosis (TB). This study tried to provide updated data about the frequency and diversity of NTM species. Materials and Methods: Suspicious samples of Mycobacterium tuberculosis (MTB) with both positive results in Ziehl-Neelsen (ZN) staining and Löwenstein-Jensen medium culturing were evaluated during January 2016 and December 2018 in Gorgan, Iran. After determination of MTB isolates by the growth rate, pigmentation status, the niacin test, and the insertion sequence 6110 (IS6110) PCR assay, other unknown isolates (presumably NTM) were detected by the 16S rDNA sequencing method and drawing the phylogenetic tree. Based on the patients’ demographic information, their risk factors were also assessed. Results: Among 226 culture-positive samples, obtained from 2994 individuals with suspected symptoms of TB, the analyses found 12 (5.3%) NTM and three Mycobacterium caprae isolates. Mycobacterium simiae (6/12) was the most prevalent NTM species. The average nucleotide similarity value was 98.2% ± 3.7. In comparison to patients with MTB (211 confirmed cases), other mycobacterium infections were more common in patients over 65 years old (Odd ratio (95% convenience interval): 2.96 (0.69 - 12.59), P = 0.14). Conclusion: Although the NTM species has a small portion in TB suspected patients, their prevalence has increased, mainly in elderly patients. Moreover, M. simiae was the most prevalent NTM species in our region. Therefore, identification of common species in each region is recommended and clinicians should pay more attention to them in each region.