Performance of Xpert MTB/RIF Assay in Diagnosis of Pleural Tuberculosis by Use of Pleural Fluid Samples.

Prospectively, 162 pleural fluid samples from patients with probable tuberculous pleural effusion were tested by the Xpert MTB/RIF assay and the Bactec MGIT-960 culture system. Of these, 43 (26.5%) were positive in the MGIT-960 culture, and 23 (14.2%), in the Xpert MTB/RIF assay. The sensitivity and specificity of the Xpert MTB/RIF compared with the MGIT-960 culture were 54.8% and 100%, respectively.

Comparison of Xpert MTB/RIF with line probe assay for detection of rifampin-monoresistant Mycobacterium tuberculosis.

The MTBDRplus line probe assay (LPA) and Xpert MTB/RIF have been endorsed by the World Health Organization for the rapid diagnosis of drug-resistant tuberculosis. However, there is no clarity regarding the superiority of one over the other. In a double-blinded prospective study, we evaluated the efficacy of the Xpert MTB/RIF on samples that were first tested by LPA under the revised national tuberculosis control program of India. A total of 405 sputum samples from suspected drug-resistant tuberculosis patients were included. Of these, 285 smear-positive samples were subjected to LPA. Seventy-two (25.8%) samples showed multidrug resistance, 62 (22.2%) showed rifampin monoresistance, 29 (10.3%) showed isoniazid monoresistance, and 116 (41.5%) were pan-susceptible. Six (2.1%) of the samples gave invalid results. Of the 62 rifampin-monoresistant samples by LPA, 38 (61.4%) showed rifampin resistance, while 21 (33.8%) were found susceptible to rifampin by Xpert MTB/RIF using cartridge version G4. Three (4.8%) samples gave an error. Of the 116 pan-susceptible samples, only 83 were available for Xpert MTB/RIF testing; 4 (5.1%) were rifampin resistant, 74 (94.8%) were susceptible, and 5 (6.0%) showed an error. The 25 discrepant samples were further subjected to MGIT960 drug susceptibility testing. The MGIT960 results showed 100% agreement with LPA results but only 64.4% agreement with Xpert MTB/RIF results. Sequencing analysis of discrepant samples showed 91.3% concordance with LPA but only 8.7% concordance with the Xpert MTB/RIF assay. These findings indicate that by using Xpert MTB/RIF testing we might be underestimating the burden of drug-resistant tuberculosis and indicate that country-specific probes need to be designed to increase the sensitivity of the Xpert MTB/RIF.

Snapshot of Mycobacterium tuberculosis Phylogenetics from an Indian State of Arunachal Pradesh Bordering China.

Uncontrolled transmission of Mycobacterium tuberculosis (M. tuberculosis, MTB) drug resistant strains is a challenge to control efforts of the global tuberculosis program. Due to increasing multi-drug resistant (MDR) cases in Arunachal Pradesh, a northeastern state of India, the tracking and tracing of these resistant MTB strains is crucial for infection control and spread of drug resistance. This study aims to correlate the phenotypic DST, genomic DST (gDST) and phylogenetic analysis of MDR-MTB strains in the region. Of the total 200 samples 22 (11%) patients suspected of MDR-TB and 160 (80%) previously treated MDR-TB cases, 125 (62.5%) were identified as MTB. MGIT-960 SIRE DST detected 71/125 (56.8%) isolates as MDR/RR-MTB of which 22 (30.9%) were detected resistant to second-line drugs. Whole-genome sequencing of 65 isolates and their gDST found Ser315Thr mutation in katG (35/45; 77.8%) and Ser531Leu mutation in rpoB (21/41; 51.2%) associated with drug resistance. SNP barcoding categorized the dataset with Lineage2 (41; 63.1%) being predominant followed by Lineage3 (10; 15.4%), Lineage1 (8; 12.3%) and Lineage4 (6; 9.2%) respectively. Phylogenetic assignment by cgMLST gave insights of two Beijing sub-lineages viz; 2.2.1 (SNP difference < 19) and 2.2.1.2 (SNP difference < 9) associated with recent ongoing transmission in Arunachal Pradesh. This study provides insights in identifying two virulent Beijing sub-lineages (sub-lineage 2.2.1 and 2.2.1.2) with ongoing transmission of TB drug resistance in Arunachal Pradesh.

Complete Genome Sequence of Mycobacterium orygis Strain 51145.

We report the complete 4,352,172-bp genome sequence of Mycobacterium orygis strain 51145 assembled into a single circular chromosome. Comparative genomic analyses with other lineages of the Mycobacterium tuberculosis complex can provide insights into the biology, evolution, and epidemiology of this important group of pathogenic mycobacteria.

Performance of Genotype MTBDRsl V2.0 over the Genotype MTBDRsl V1 for detection of second line drug resistance: An Indian perspective.

Genotype MTBDRsl Version 1 (V1.0) was recommended as an initial test for rapid detection of pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB). However, in recent years a number of novel mutations are identified that confer resistance. Thus, Genotype MTBDRsl V2.0 was endorsed by WHO. Though, Genotype MTBDRsl V2.0 has been rolled out in national TB programme in 2018, there is dearth of data from India on its performance for second line drug susceptibility testing (DST). For this, performance of new version was evaluated on 113 MDR-TB isolates. The results showed that 39 (34.5%) of these isolates were resistant to FQ and 7 (6.2%) were XDR by Genotype MTBDRsl V2.0. Amongst the FQ resistant isolates most prevalent mutation was ΔWT3-D94G (17; 38.6%) and N538D (12; 85.7%). Among the AG/CP and KAN resistant isolates most common mutation in the rrs region was ΔWT1-A1401G (5; 71.4%) and C-14T (2; 28.5%) in eis gene. Second line Bactec MGIT-960 detected 40 (35.4%) isolates as resistant to FQ and 6 (5.3%) as XDR isolates, whereas Genotype MTBDRsl V1.0 also detected 39 (34.5%) as resistant to FQ but missed 2 isolates in correctly identifying as XDR (5; 4.4%). Thus, concordance of second line Bactec MGIT-960 with Genotype MTBDRsl V2.0 was similar (100%) for FQ detection but it has improvised the diagnostic sensitivity for correctly identifying XDR isolates. Nevertheless, the cost of Genotype MTBDRsl V2.0 remains an issue for screening of second line drug (SLDs) resistance from countries with high burden of MDR-TB.

Mutations in SARS-CoV-2 Leading to Antigenic Variations in Spike Protein: A Challenge in Vaccine Development.

Objectives The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus has been unprecedentedly fast, spreading to more than 180 countries within 3 months with variable severity. One of the major reasons attributed to this variation is genetic mutation. Therefore, we aimed to predict the mutations in the spike protein (S) of the SARS-CoV-2 genomes available worldwide and analyze its impact on the antigenicity. Materials and Methods Several research groups have generated whole genome sequencing data which are available in the public repositories. A total of 1,604 spike proteins were extracted from 1,325 complete genome and 279 partial spike coding sequences of SARS-CoV-2 available in NCBI till May 1, 2020 and subjected to multiple sequence alignment to find the mutations corresponding to the reported single nucleotide polymorphisms (SNPs) in the genomic study. Further, the antigenicity of the predicted mutations inferred, and the epitopes were superimposed on the structure of the spike protein. Results The sequence analysis resulted in high SNPs frequency. The significant variations in the predicted epitopes showing high antigenicity were A348V, V367F and A419S in receptor binding domain (RBD). Other mutations observed within RBD exhibiting low antigenicity were T323I, A344S, R408I, G476S, V483A, H519Q, A520S, A522S and K529E. The RBD T323I, A344S, V367F, A419S, A522S and K529E are novel mutations reported first time in this study. Moreover, A930V and D936Y mutations were observed in the heptad repeat domain and one mutation D1168H was noted in heptad repeat domain 2. Conclusion S protein is the major target for vaccine development, but several mutations were predicted in the antigenic epitopes of S protein across all genomes available globally. The emergence of various mutations within a short period might result in the conformational changes of the protein structure, which suggests that developing a universal vaccine may be a challenging task.

Whole-Genome Sequencing of Two Extensively Drug-Resistant Mycobacterium tuberculosis Isolates from India.

The emergence of extensively drug-resistant tuberculosis (XDR-TB) presents a considerable challenge and a public health concern due to the high mortality rate of this disease. Whole-genome sequencing (WGS) of XDR-TB isolates is thus essential for understanding the mechanism of drug resistance. Here we report whole-genome sequences of two XDR-TB strains of two lineages from India.

Association of gyrA and rrs gene mutations detected by MTBDRsl V1 on Mycobacterium tuberculosis strains of diverse genetic background from India.

There is limited data on the use of Genotype MTBDRslVersion 1 (MTBDRsl V1) as an initial rapid screening test to rule out XDR-TB and most importantly its performance in various genotypes of Mycobacterium tuberculosis is scarcely studied. A total of 359 MDR-TB isolates were tested for gene mutations representing second line drug resistance, using the MTBDRsl_V.1 and the results were compared with phenotypic method (Bactec MGIT-960 system) for second-line drug (SLD) susceptibility testing. Genetic lineages of all these isolates were also determined using spoligotyping and SITVIT2 WEB database. The MTBDRsl V1 detected mutations in the gyrA, rrs, and emb genes in 108 (30%), 2 (0.5%) and 129 (35.9%) isolates, respectively. Remaining 120 (33.4%) had no second line drug (SLD) resistance. In 17 (4.7%) isolates mutations were detected in both gyrA and rrs genes. Its concordance with MGIT-960 culture drug susceptibility testing (DST) was 97% and 94.1%, 93.5%, 60.5% and 50% for the detection of XDR-TB, pre-XDR, Ethambutol, and Aminoglycosides/Cyclopeptides resistance. The Beijing lineage was predominant (46%) between both the pre-XDR/XDR-TB isolates. We conclude that MTBDRsl is useful for rapid detection of SLD resistance. Also in pre-XDR and XDR-TB isolates the frequency of relevant genetic mutations was significantly higher in the Beijing strains.

Diagnostic usefulness of Xpert MTB/RIF assay for detection of tuberculous meningitis using cerebrospinal fluid.

OBJECTIVE: Tuberculous meningitis (TBM) is the most severe form of extra-pulmonary tuberculosis (TB) due to association of diseases with high rates of mortality and morbidity. Diagnosis continues to be a clinical challenge as microbiological confirmation is rare and time consuming resulting in delayed treatment. Xpert MTB/RIF assay is a rapid and simple test, which has been endorsed by World Health Organization as an initial diagnostic test for the diagnosis of TBM. However, evidence still lacks for its performance on cerebrospinal fluid (CSF) for the diagnosis of TBM especially from India. METHODS: A total of 267 CSF samples from patients with high clinico-radiological suspicion of TBM were included in this study. Ziehl-Neelsen (ZN) staining, BACTEC Mycobacterial Growth Indicator Tube (MGIT-960) culture system, and Xpert MTB/RIF assay (using cartridge version G4) were tested on all samples. RESULTS: Of total 267 samples, all were negative for smear AFB and 52 (19.5%) were culture positive by MGIT-960 culture system. However, out of 52 (19.5%) cultures detected positive by MGIT-960, 5 (9.6%) were detected as resistant to rifampicin. Xpert MTB/RIF assay was positive in 38 (14.2%) samples and negative in 223 (83.5%) samples. Cartridge error was detected in 6 (2.2%) samples, which could not be repeated due to insufficient sample volume. The sensitivity and specificity of Xpert MTB/RIF assay in comparison to MGIT-960 was 55.1% (95%, CI: 40.2-69.3) and 94.8% (95%, CI: 90.9-97.4) respectively. Overall, Xpert MTB/RIF assay detected 38 (14.2%) as positive for MTB of which 4 (10.5%), 31 (81.6%) and 3 (7.9%) were found to be rifampicin resistant, sensitive and indeterminate respectively. CONCLUSION: Xpert MTB/RIF assay showed lower sensitivity as compared to MGIT 960 culture for the diagnosis of TBM from CSF samples.

Performance of Xpert MTB/RIF on Ascitic Fluid Samples for Detection of Abdominal Tuberculosis.

BACKGROUND: Diagnosis of abdominal tuberculosis (TB) from ascitic fluid samples using routinely available diagnostic methods is challenging due to its paucibacillary nature. Although performance of Xpert MTB/RIF assay has been evaluated extensively on pulmonary samples, its performance on extrapulmonary samples is still under evaluation. OBJECTIVES: The objective of this study was to find out the performance of Xpert MTB/RIF on ascitic fluid samples obtained from suspected cases of abdominal TB. Performance was compared with Mycobacterium growth indicator tube-960 (MGIT-960) culture and in-house multiplex polymerase chain reaction (PCR). The latter detects and differentiates Mycobacterium tuberculosis and nontuberculous mycobacteria simultaneously. MATERIALS AND METHODS: Sixty-seven patients suspected of probable/possible abdominal TB were included in this observational, prospective study. All samples were tested by Ziehl-Neelsen staining, MGIT-960 culture, in-house multiplex PCR, and Xpert MTB/RIF assay. RESULTS: All 67 samples were smear negative. Seventeen (25.4%) were MGIT-960 culture positive while 12 (17.9%) were detected positive by the Xpert MTB/RIF assay and 9 (13.4%) by in-house multiplex PCR. Sensitivity and specificity of the Xpert MTB/RIF assay compared with the MGIT-960 culture were 70.6% (95%, confidence interval [CI]: 44.1-89.7) and 100% (95%, CI: 92.8-100) and that of in-house multiplex PCR were 52.9% (95%, CI: 30.9-73.8) and 100% (95%, CI: 92.8-100), respectively. CONCLUSIONS: Diagnostic yield of Xpert MTB/RIF assay on ascitic fluid samples was lower than MGIT-960 culture. We thus emphasize on the need for urgent discovery of new biomarkers for paucibacillary TB.

Predominance of Beijing lineage among pre-extensively drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis: A tertiary care center experience.

OBJECTIVE/BACKGROUND: Increasing resistance to various first-line and second-line drugs has become a major concern in India. However, it is not known if some genotypes are more associated with second-line drug resistance. Thus, the main aim of this study was to find out the predominant genotype associated with second-line drug resistance. METHODS: During the study, a total of 234 multidrug resistant (MDR) strains of Mycobacterium tuberculosis, isolated between 2008 and 2015, were randomly selected and screened for pre-extensively drug-resistant (XDR) and XDR patterns using second-drug susceptibility testing with BACTEC MGIT 960. All the MDR isolates were tested against ofloxacin (2µg/mL), kanamycin (2.5µg/mL), amikacin (1µg/mL), and capreomycin (2.5µg/mL). Based on the resistance pattern pre-XDR was defined as M. tuberculosis isolates resistant to fluoroquinolone alone. The identified pre-XDR and XDR isolates were further characterized using spoligotyping. The spoligo patterns obtained were compared and analyzed using SITVIT_WEB Unweighted Pair Group Method with Arithmetic Mean, and Minimum Spanning Tree was derived using MIRU-VNTRplus. RESULTS: Among the 234 MDR strains of M. tuberculosis, 85 (36.3%) were detected as pre-XDRs and 15 (6.4%) as XDRs. All the pre-XDR strains were ofloxacin resistant, whereas among the XDR strains, 10 (66.6%) were resistant to ofloxacin, kanamycin, and capreomycin, four (26.6%) were resistant to ofloxacin, kanamycin, and amikacin, and one (6.6%) isolate was resistant to ofloxacin and kanamycin. Upon spoligotyping analysis, the Beijing lineage was found to be the single most dominant lineage among the pre-XDR strains (38.8%) followed by CAS (30.5%), X (7%), T (5.8%), Haarlem (3.5%), EAI (2.3%), and MANU (2.3%). Among the XDR isolates, seven (46.6%) belonged to Beijing, three (20%) belonged to CAS, and one (6.6%) to each of the EAI, T, URAL, and X lineages. Within the Beijing family, ST1 was the most common in both pre-XDR (94%) and XDR isolates. All the isolates belonged to the ST1 sublineage. CONCLUSION: The Beijing lineage was found to be the single most dominant genotype among the pre-XDR and XDR isolates.

Heat fixed but unstained slide smears are infectious to laboratory staff.

BACKGROUND AND AIM: In a clinical microbiology laboratory, heat fixed slide smears are commonly transported from one place to another for staining with different stains and also for onsite proficiency testing of laboratory technicians for accreditation of the laboratories. These smears are frequently handled without gloves by the staff in developing countries. Therefore, this study was conducted to check the survivability of tubercle bacilli on smears after physical and chemical treatments. METHODS: A total of 196 AFB positive smears were analyzed. Of these, 116 were stained with Ziehl Neelsen (ZN), 60 with cold Kinyoun and 10 were unstained but heat fixed and 10 were neither stained nor heat fixed. The last 20 smears served as controls. The ZN and Kinyoun stained smears were 0-1.5-year-old and stored at room temperature in slide boxes, while control smears were freshly prepared. All smears were prepared from sputum samples positive for acid fast bacilli. All four sets were subjected to slide culture to see if mycobacteria could survive and grow in any. For slide culture, a new and safe device was used, which is designed for three in one purpose: cell cultivation, direct observation of the growth under microscope and cell harvesting inside the closed tube. The slide smears were directly dipped into this tube that contained liquid culture medium. The tubes were incubated at 37 degreeC for four weeks. The growth, if any, was confirmed by MPT-64 rapid test and subculture on LJ slants. RESULTS: No growth was observed in ZN and Kinyoun stained slide smears. However, significant growth was observed in both control sets; the unstained non heat fixed as well as heat fixed slide smears. CONCLUSIONS: The results of our study indicate that tubercle bacilli remain viable even after heat fixation and carry risk of infection by contact However, stained smears are safe for handling and storage.