Draft genome sequence of Mycolicibacterium conceptionense isolated from the sputum sample of a patient with pulmonary tuberculosis.

We report the draft genome of Mycolicibacterium conceptionense, a rapidly growing nontuberculous mycobacterium, isolated from the sputum sample of a patient undergoing treatment for multidrug-resistant tuberculosis in Delhi, India. The 6,366,717-bp genome contains 6,124 coding sequences, one 5S rRNA, three 16S rRNAs, six 23S rRNAs, and 49 tRNAs.

Design and Synthesis of Isatin-Tagged Isoniazid Conjugates with Cogent Antituberculosis and Radical Quenching Competence: In-vitro and In-silico Evaluations.

In pursuit of potential chemotherapeutic alternates to combat severe tuberculosis infections, novel heterocyclic templates derived from clinically approved anti-TB drug isoniazid and isatin have been synthesized that demonstrate potent inhibitory action against Mycobacterium tuberculosis, and compound 4i with nitrophenyl motif exhibited the highest anti-TB efficacy with a MIC value of 2.54 µM/ml. Notably, the same nitro analog 4i shows the best antioxidant efficacy among all the synthesized compounds with an IC50 value of 37.37 µg/ml, suggesting a synergistic influence of antioxidant proficiency on the anti-TB action. The titled compounds exhibit explicit binding affinity with the InhA receptor. The befitting biochemical reactivity and near-appropriate pharmacokinetic proficiency of the isoniazid conjugates is reflected in the density functional theory (DFT) studies and ADMET screening. The remarkable anti-TB action of the isoniazid cognates with marked radical quenching ability may serve as a base for developing multi-target medications to confront drug-resistant TB pathogens.

Synthesis of indole-functionalized isoniazid conjugates with potent antimycobacterial and antioxidant efficacy.

Aim: Developing potent medicinal alternates for tuberculosis (TB) is highly desirable due to the advent of drug-resistant lethal TB strains.Methods & results: Novel indole-isoniazid integrates have been synthesized with promising antimycobacterial action against the H37Rv strain, and the nitro analogs 4e and 4j show the highest efficacy with a minimum inhibitory concentration of 1.25 µg/ml. The molecular docking studies against InhA support the experimental findings. Indole conjugates display remarkable radical quenching efficiency, and compounds 4e and 4j demonstrate maximum IC50 values of 50.19 and 52.45 µg/ml, respectively. Pharmacokinetic analysis anticipated appreciable druggability for the title compounds.Conclusion: The notable bioaction of the indole-isoniazid templates projects them as potential lead in developing anti-TB medications with synergetic antioxidant action.

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C-terminal region of Rv1039c (PPE15) protein of Mycobacterium tuberculosis targets host mitochondria to induce macrophage apoptosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein.

Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.

Identification and In silico analysis of proline-glutamate/proline-proline-glutamate proteins of Mycobacterium tuberculosis complex: A comparison of computational web-based tools.

Background: Understanding the protein's subcellular localization and secretory nature can greatly improve the target identification for diagnostic assays and drug discovery, although their identification in laboratory experiments is a time-consuming and labor-intensive process. In order to identify proteins that could be targeted for therapeutic intervention or the development of diagnostic assays, we used a variety of computational tools to predict the subcellular localization or secretory nature of mycobacterial proline-glutamate/proline-proline-glutamate (PE/PPE) proteins. Methods: PSORTb version 3.0.3, TBpred, and Gpos-mPLoc analyses were performed on 30 selected PE/PPE protein sequences, while, SignalP 6.0, SignalP 5.0, Phobius, PSORTb version 3.0.3 and TBpred were used for signal sequence predictions. Results: Gpos-mPLoc and TBpred had the highest concordance for extracellular prediction, while PSORTb and TBpred had the highest concordance for prediction of membrane localization. The tools for predicting the secretory nature of proteins had little agreement. Conclusion: Multiple computational tools must be considered to provide an indication of the subcellular localization of PE/PPE proteins. Laboratory experiments should be used to confirm the findings of the tools.

Initial COVID-19 Severity and Long-COVID Manifestations: An Observational Analysis.

OBJECTIVE: New-onset or persistent symptoms beyond after 4 weeks from COVID-19 are termed "long-COVID." Whether the initial severity of COVID-19 has a bearing on the clinicoradiological manifestations of long COVID is an area of interest. MATERIAL AND METHODS: We did an observational analysis of the long-COVID patients after categorizing them based on their course of COVID-19 illness into mild, moderate, and severe groups. The clinical and radiological profile was compared across these groups. RESULTS: Out of 150 long-COVID patients recruited in the study, about 79% (118), 14% (22), and 7% (10) had a history of mild, moderate, and severe COVID-19, respectively. Fatigue (P = .001), breathlessness (P = .001), tachycardia (P = .002), tachypnea (P < .001), raised blood pressure (P < .001), crepitations (P = .04), hypoxia at rest (P < .001), significant desaturation in 6-minute walk test (P = .27), type 1 respiratory failure (P = .001), and type 2 respiratory failure (P = .001) were found to be significantly higher in the long-COVID patients with a history of severe COVID-19. These patients also had the highest prevalence of abnormal chest X-ray (60%) and honeycombing in computed tomography scan thorax (25%, P = .027). CONCLUSION: The course of long COVID bears a relationship with initial COVID-19 severity. Patients with severe COVID-19 are prone to develop more serious long-COVID manifestations.

Comparative Genetic Association Analysis of Human Genetic Susceptibility to Pulmonary and Lymph Node Tuberculosis.

BACKGROUND: Tuberculosis (TB) manifests itself primarily in the lungs as pulmonary disease (PTB) and sometimes disseminates to other organs to cause extra-pulmonary TB, such as lymph node TB (LNTB). This study aimed to investigate the role of host genetic polymorphism in immunity related genes to find a genetic basis for such differences. METHODS: Sixty-three, Single nucleotide polymorphisms (SNPs) in twenty-three, TB-immunity related genes including eleven innate immunity (SLCA11, VDR, TLR2, TLR4, TLR8, IRGM, P2RX7, LTA4H, SP110, DCSIGN and NOS2A) and twelve cytokine (TNFA, IFNG, IL2, Il12, IL18, IL1B, IL10, IL6, IL4, rs1794068, IL8 and TNFB) genes were investigated to find genetic associations in both PTB and LNTB as compared to healthy community controls. The serum cytokine levels were correlated for association with the genotypes. RESULTS: PTB and LNTB showed differential genetic associations. The genetic variants in the cytokine genes (IFNG, IL12, IL4, TNFB and IL1RA and TLR2, 4 associated with PTB susceptibility and cytokine levels but not LNTB (p < 0.05). Similarly, genetic variants in LTA4H, P2RX7, DCSIGN and SP110 showed susceptibility to LNTB and not PTB. Pathway analysis showed abundance of cytokine related variants for PTB and apoptosis related variants for LNTB. CONCLUSIONS: PTB and LNTB outcomes of TB infection have a genetic component and should be considered for any future functional studies or studies on susceptibility to pulmonary and extra-pulmonary TB.

Ultrasensitive Detection of Multidrug-Resistant Mycobacterium tuberculosis Using SuperSelective Primer-Based Real-Time PCR Assays.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1-1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.

Quantitative detection of mycobacterial mannophosphoinositides in tuberculosis patients by real-time immuno-PCR assay.

A real-time immuno-PCR assay was deliberated to detect mycobacterial mannophosphoinositides (PIMs). A dynamic range of PIMs (0.9 pg/mL-10 ng/mL) was detected in TB patients, wherein 88.2% and 81.1% sensitivities were obtained in pulmonary TB and extrapulmonary TB respectively, with 96-96.4% specificity. This assay may translate into a diagnostic kit.

Origin and Global Expansion of Mycobacterium tuberculosis Complex Lineage 3.

Mycobacterium tuberculosis complex (MTBC) Lineage 3 (L3) strains are abundant in world regions with the highest tuberculosis burden. To investigate the population structure and the global diversity of this major lineage, we analyzed a dataset comprising 2682 L3 strains from 38 countries over 5 continents, by employing 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping (MIRU-VNTR) and drug susceptibility testing. We further combined whole-genome sequencing (WGS) and phylogeographic analysis for 373 strains representing the global L3 genetic diversity. Ancestral state reconstruction confirmed that the origin of L3 strains is located in Southern Asia and further revealed multiple independent introduction events into North-East and East Africa. This study provides a systematic understanding of the global diversity of L3 strains and reports phylogenetic variations that could inform clinical trials which evaluate the effectivity of new drugs/regimens or vaccine candidates.

Evaluating the efficacy of stool sample on Xpert MTB/RIF Ultra and its comparison with other sample types by meta-analysis for TB diagnostics.

Precise and timely detection of tuberculosis (TB) is crucial to reduce transmission. This study aims to assess the accuracy of Xpert MTB/RIF Ultra on stool samples and systematically review the performance of Xpert MTB/RIF Ultra with different sample types by meta-analysis. Stool samples of smear-negative pulmonary TB (PTB), cervical lymph node TB, and abdominal TB patients were tested on the Xpert MTB/RIF Ultra system. Meta-analysis was performed on a set of 44 studies. Data were grouped by sample type, and the pooled sensitivity and specificity of Xpert MTB/RIF Ultra were calculated. The sensitivity of Xpert MTB/RIF Ultra with stool samples was 100% for smear-negative PTB, 27.27% for cervical lymph node TB, and 50% for abdominal TB patients, with 100% specificity for all included TB groups. The summary estimate for all PTB samples showed 84.2% sensitivity and 94.5% specificity, and EPTB samples showed 88.6% sensitivity and 96.4% specificity. Among all sample types included in our meta-analysis, urine showed the best performance for EPTB diagnosis. This pilot study supports the use of stool as an alternative non-invasive sample on Xpert MTB/RIF Ultra for rapid testing, suitable for both PTB and EPTB diagnosis.

Whole genome sequencing of isoniazid monoresistant clinical isolates of Mycobacterium tuberculosis reveals novel genetic polymorphisms.

In an attempt to uncover genotypic indicators for isoniazid (INH) resistance in M. tuberculosis, in addition to the canonical mutations in genes associated with INH resistance, including katG, inhA and fabG promoter; we analyzed, two INH monoresistant isolates, ASTS24/13 (INHR1) and SHR1/14 (INHR2). Targeted Sanger sequencing detected a canonical mutation at katG315 only in INHR2. Infection of THP-1 cells and exposure to antituberculosis drugs led to two-fold increase in the minimum inhibitory concentration of INH in INHR2. Whole genome sequences revealed that INHR1 and INHR2 belonged to Delhi Central Asian Strain and East African Indian lineages, respectively. The sequences were compared with INH susceptible isolates with the same lineage as the INH monoresistant strains. INHR1 had a novel unique mutation STOP420Trp in the efflux pump gene Rv0849, while INHR2 had a novel mutation Arg579Ser in efflux pump gene mmpL5. Comparison of lipid associated genes showed novel mutations in INHR1 in fadE16, fadD3 and fbpD; while INHR2 had mutations in fadE1, Rv0145, Rv1425, fadD9 and mmaA3. Both isolates also demonstrated novel mutations in cell wall associated genes. Our study highlights the importance of searching for alternate mechanisms of INH resistance that may contribute to the development of more comprehensive diagnostic tools.

Skin and soft-tissue infections due to rapidly growing mycobacteria: An overview.

Background: Rapidly growing mycobacteria (RGM) are increasingly being recognized as potential pathogens. RGM, particularly Mycobacterium abscessus, Mycobacterium fortuitum, and Mycobacterium chelonae, have been observed in both pulmonary and extrapulmonary infections including cutaneous, soft-tissue, and wound infections. However, there are limited reports of these potential pathogens from skin and soft-tissue infections. Moreover, the drug susceptibility profile of RGM is largely unknown in several regions of the world. Methods: We analyzed reports on RGM isolated from skin and soft-tissue infections globally for details of RGM species and drug susceptibility profile. We also analyzed the drug susceptibility profile of four RGM isolates, obtained from skin and soft-tissue infections in our laboratory, by broth microdilution method. Results: In the reports reviewed, the most common RGM isolated from skin and soft-tissue infections were M. abscessus (184/475, 38.7%), M. fortuitum (150/475, 31.5%), M. chelonae (72/475, 15%), and M. chelonae-M. abscessus complex (46/475, 9.6%). However, drug susceptibility was tested only in 26/39 (66.6%) reports. In our own laboratory, we obtained three isolates of M. abscessus and one isolate of M. fortuitum from one case of breast abscess and three cases of postsurgical wound infections. Maximum susceptibility of M. abscessus was observed to clarithromycin, amikacin, and linezolid. The M. fortuitum isolate was susceptible to clarithromycin, amikacin, clofazimine, and linezolid. Conclusion: Paucity of information available on RGM isolated from skin and soft-tissue infections highlights the need to be aware of the pathogenic potential and the drug susceptibility profile of these organisms.

Draft Genome Sequence of Mycobacterium simiae, a Potential Pathogen Isolated from the Normal Human Oral Cavity.

We report the draft genome sequence of Mycobacterium simiae, a slowly growing nontuberculous mycobacterium (NTM) isolated from a mouthwash sample of a healthy person. This genome of 6,603,693 bp exhibited a 66.13% GC content and 6,391 genes with 6,257 coding sequences, 3 rRNAs, and 78 tRNAs.

Correlation of drug resistance with single nucleotide variations through genome analysis and experimental validation in a multi-drug resistant clinical isolate of M. tuberculosis.

BACKGROUND: Genome sequencing and genetic polymorphism analysis of clinical isolates of M. tuberculosis is carried out to gain further insight into molecular pathogenesis and host-pathogen interaction. Therefore the functional evaluation of the effect of single nucleotide variation (SNV) is essential. At the same time, the identification of invariant sequences unique to M. tuberculosis contributes to infection detection by sensitive methods. In the present study, genome analysis is accompanied by evaluation of the functional implication of the SNVs in a MDR clinical isolate VPCI591. RESULT: By sequencing and comparative analysis of VPCI591 genome with 1553 global clinical isolates of M. tuberculosis (GMTV and tbVar databases), we identified 141 unique strain specific SNVs. A novel intergenic variation in VPCI591 in the putative promoter/regulatory region mapping between embC (Rv3793) and embA (Rv3794) genes was found to enhance the expression of embAB, which correlates with the high resistance of the VPCI591 to ethambutol. Similarly, the unique combination of three genic SNVs in RNA polymerase ß gene (rpoB) in VPCI591 was evaluated for its effect on rifampicin resistance through molecular docking analysis. The comparative genomics also showed that along with variations, there are genes that remain invariant. 173 such genes were identified in our analysis. CONCLUSION: The genetic variation in M. tuberculosis clinical isolate VPCI591 is found in almost all functional classes of genes. We have shown that SNV in rpoB gene mapping outside the drug binding site along with two SNVs in the binding site can contribute to quantitative change in MIC for rifampicin. Our results show the collective effect of SNVs on the structure of the protein, impacting the interaction between the target protein and the drug molecule in rpoB as an example. The study shows that intergenic variations bring about quantitative changes in transcription in embAB and in turn can lead to drug resistance.

An overview of pulmonary infections due to rapidly growing mycobacteria in South Asia and impressions from a subtropical region.

Background: Rapidly growing mycobacteria (RGM) comprise nearly half of the validated species of nontuberculous mycobacteria (NTM) and have been reported to have a higher incidence in Asia as compared to Europe and America. There is limited information on RGM infections from South Asia. Hence, the present study aimed to ascertain the incidence of pulmonary infections due to RGM in Delhi and to review the status of available information on the prevalence of RGM in South Asia, a region endemic for tuberculosis. Methods: We analyzed 933 mycobacterial isolates obtained from pulmonary samples in Delhi and performed species identification by polymerase chain reaction (PCR)-restriction analysis (restriction fragment length polymorphism) and line probe assay. Drug susceptibility testing (DST) was performed by broth microdilution method. We also reviewed reports available on pulmonary infections in South Asia, attributed to RGM. Results: Of the 933 mycobacterial isolates studied, NTM were identified in 152 (16.3%). Of these, 65/152 (42.8%) were RGM comprising Mycobacterium fortuitum (34/65; 52.3%), Mycobacterium abscessus (25/65; 38.5%), Mycobacterium chelonae (3/65; 4.61%), Mycobacterium mucogenicum (2/65; 3.1%), and Mycobacterium smegmatis (1/65; 1.5%). On applying the American Thoracic Society/Infectious Diseases Society of America guidelines, 11/25 (44%) M. abscessus, 3/3 (100%) M. chelonae, and both isolates of M. mucogenicum were found to be clinically relevant. DST revealed that maximum susceptibility of the RGM was seen to linezolid, clarithromycin, and amikacin. Conclusions: Of the RGM isolated in the present study, 16/65 (24.6%) were found to be clinically relevant. Hence, it is important to recognize these organisms as potential pathogens to identify patients with RGM disease to initiate appropriate therapy.

Detection of mycobacterial CFP-10 (Rv3874) protein in tuberculosis patients by gold nanoparticle-based real-time immuno-PCR.

Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5-5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5-93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.

The MPB64 immunochromatography assay: an analysis of doubtful results.

Culture remains the gold standard for tuberculosis (TB) diagnosis, and the mycobacteria growth indicator tube (MGIT), endorsed by the World Health Organization (WHO), is widely used. Further identification of a positive culture is done with the help of an immunochromatography assay, which often shows faint bands that are difficult to interpret. We analysed 125 BACTEC MGIT culture positive results, of which 11/16 (68.7%) of the doubtful assays, analysed by MGIT™ TBc Identification test (TBcId), were positive for Mycobacterium tuberculosis complex (MTBC), the remaining being non-tuberculous mycobacteria as determined by an in-house duplex polymerase chain reaction and line probe assay. Guidelines on faint or doubtful bands in immunochromatography assays are important so as not to overlook true-positive cases of TB.

Diagnostic performance of non-invasive, stool-based molecular assays in patients with paucibacillary tuberculosis.

Timely diagnosis of paucibacillary tuberculosis (TB) which includes smear-negative pulmonary TB (PTB) and extra-pulmonary TB (EPTB) remains a challenge. This study was performed to assess the diagnostic utility of stool as a specimen of choice for detection of mycobacterial DNA in paucibacillary TB patients in a TB-endemic setting. Stool samples were collected from 246 subjects including 129 TB patients (62 PTB and 67 EPTB) recruited at TB hospital in Delhi, India. Diagnostic efficacy of stool IS6110 PCR (n = 228) was measured, using microbiologically/clinically confirmed TB as the reference standard. The clinical sensitivity of stool PCR was 97.22% (95% confidence interval (CI), 85.47-99.93) for detection of Mycobacterium tuberculosis in stool samples of smear-positive PTB patients and 76.92% (CI, 56.35-91.03) in samples from smear-negative PTB patients. Overall sensitivity of PCR for EPTB was 68.66% (CI, 56.16-79.44), with the highest sensitivity for stool samples from patients with lymph node TB (73.5%), followed by abdominal TB (66.7%) and pleural effusion (56.3%). Stool PCR presented a specificity of 95.12%. The receiver operating characteristic curve also indicated the diagnostic utility of stool PCR in TB detection (AUC: 0.882). The performance characteristic of the molecular assay suggests that stool DNA testing has clinical value in detection of TB.