Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe.

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.

Genetic polymorphism of toll-like receptors in HIV-I infected patients with and without tuberculosis co-infection.

Background: Toll-like receptors (TLRs) are identified as one of the key components of innate immune system due to their ability to sense conserved molecular motifs associated with several pathogens. It has been implicated from several evidence that mutations in genes encoding TLRs are associated with increased or decreased susceptibility to various infectious diseases. Methods: The study was prospective, cross-sectional, as well as longitudinal in nature, which includes 223 HIV-positive patients, 150 HIV-positive patients with latent tuberculosis (TB) infection, 150 HIV-positive patients with active TB, 200 HIV-negative newly diagnosed sputum smear positive pulmonary TB patients, and 205 healthy subjects. Results: A statistically significant difference was observed in allelic frequencies of TLR4 between healthy subjects and HIV + TB patients (P < 0.001), healthy subjects, and pulmonary TB (PTB) Category-I patients (P < 0.01) and between healthy subjects and HIV + TB patients (P < 0.001). TLR4 genotype frequencies were also significantly different between healthy subjects and PTB Cat I patients (P < 0.001) and HIV + and HIV + TB patients (P < 0.01). A statistically significant difference was also observed between HIV + and PTB Cat I patients (P = 0.04), HIV + LTBI and HIV + TB patients (P = 0.01), and between HIV + TB and PTB Cat I patients (P < 0.01). Conclusion: This study implicates that Asp299Gly polymorphism in TLR4 gene is associated with increased susceptibility to active TB in HIV-seropositive patients. Increased frequency of 'A' allele in TLR9 gene was also discovered at the time of active TB development in ART naïve HIV + patients, who developed active TB on follow-up.

Laboratory Cost Analysis of Conventional and Newer Molecular Tests for Diagnosis of Presumptive Multidrug-Resistant Tuberculosis Patients.

Introduction: Tuberculosis (TB) remains a deadliest infectious disease. Lack of rapid test with low cost is one of the important challenges to eradicate the TB. The objective of the study was to analyze the laboratory costs of conventional and newer molecular tests, for diagnosis of presumptive multidrug-resistant TB (MDR-TB) patients. Methods: A detailed laboratory cost of various conventional tests (Ziehl - Neelsen [ZN] microscopy, light-emitting diode-fluorescent microscopy [LED-FM], culture and drug susceptibility testing [DST] using solid Lowenstein-Jensen media and liquid media [BACTEC MGIT 960]) was compared with rapid methods (GenoType MTBDRplus line probe assay [LPA] and GeneXpert MTB/RIF assay). Laboratory cost was also calculated in terms of cost per TB and MDR-TB case detected by using different diagnostic scenarios. Results: Cost per test for ZN microscopy, LED-FM, LPA, GeneXpert MTB/RIF assay, solid culture plus DST, liquid culture plus DST was found as $2.5 (INR 156.8), $2.0 (INR128.9), $18.6 (INR1210), $13.8 (INR 895.2), $21.5 (INR 1396.6), and $29.1 (INR 1888.2), respectively. The laboratory cost for detecting TB and MDR-TB by diagnostic scenarios involving molecular DST was found to be less as compared to involving only conventional liquid culture-based test. Conclusions: The implementation of rapid molecular tests with selective use of liquid culture-based DST may be less in cost as compared to the use of culture-based DST alone, at high burden reference TB laboratory.

Multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains in geriatrics: An analysis and its implications in tuberculosis control.

Objective: This study aimed to analyze the trends of tuberculosis (TB) disease, drugs susceptibility patterns in geriatric TB over a period of three years (from 2010 to 2012). Materials & methods: In this study, laboratory data on diagnosis of geriatric tuberculosis suspected patients (age ≥60 years) was analyzed retrospectively at National Reference Laboratory (NRL). Results: Among 12,140 geriatric TB suspects, 1621 (13%) were acid-fast bacillus (AFB) smear-positive and 10,519 (87%) were smear-negative. Analysis of 915 culture results showed 470 (51%) as positive for Mycobacterium tuberculosis complex (MTBC), 63 (7%) contaminated and 36 (4%) identified as mycobacteria other than tuberculosis (MOTT). A total 210/470 (45%) were multidrug-resistant TB (MDR-TB) strains. Among the mono-resistant strains, isoniazid mono-resistant was found more frequently (134/470, 28%) whereas, it was least among rifampicin mono-resistant 5/470 (1%). The second-line drug susceptibility testing (DST) results showed 7% (17/240) extensively drug-resistant TB (XDR-TB) strains. Most common second line mono-resistant strain was observed with ofloxacin, 16% (38/240). Conclusion: This study shows high number of MDR/XDR geriatric TB patients at tertiary care TB hospital. The study highlighted the need of separate line of early identification, diagnosis and treatment of geriatric TB patients. However, further study with improved sample size may needed to confirm the findings.

Diagnostic utility of GenoType MTBDRsl assay for the detection of moxifloxacin-resistant mycobacterium tuberculosis, as compared to phenotypic method and whole-genome sequencing.

Background: Recently, moxifloxacin (MFX)-resistant results of Mycobacterium tuberculosis (Mtb) obtained by GenoType MTBDRsl (second-line line probe assay [SL-LPA]) have been stratified to determine their resistance level; however, its accuracy has not been well studied. Therefore, the study aimed to evaluate the diagnostic accuracy of SL-LPA, with phenotypic drug susceptibility testing (pDST) and whole-genome sequencing (WGS) for the detection of MFX-resistant Mtb and their resistance level. Methods: A total of 111 sputum samples were subjected to SL-LPA according to the diagnostic algorithm of the National Tuberculosis Elimination Program. Results were compared with pDST of MFX (at critical concentration [CC, 0.25 µg/ml] and clinical breakpoint [CB, 1.0 µg/ml] using BACTEC mycobacterial growth indicator tube-960), and WGS. Results: At CC, SL-LPA and pDST yielded concordant results of MFX for 104 of 111 (94%). However, at CB, 23 of 30 (77%) isolates carrying gyrA mutation known to confer low-level resistance to MFX were scored as susceptible by pDST. Among 46 Mtb isolates carrying gyrA mutations known to confer high-level resistance to MFX, 36 (78%) isolates yielded concordant results, while 10 (22%) isolates were scored as susceptible at CB by pDST. WGS identified gyrA mutations in all isolates suggested by SL-LPA. Conclusion: It is concluded that the stratification of MFX-resistant results by SL-LPA/genotypic method is not very well correlated with pDST (at CB), and hence, pDST may not be completely replaced by SL-LPA. gyrA D94G and gyrAA90V are the most prevalent mutations in MFX-resistant Mtb.

Comparative Performance of Line Probe Assay (Version 2) and Xpert MTB/RIF Assay for Early Diagnosis of Rifampicin-Resistant Pulmonary Tuberculosis.

BACKGROUND: The emergence of drug-resistant tuberculosis (TB), is a major menace to cast off TB worldwide. Line probe assay (LPA; GenoType MTBDRplus ver. 2) and Xpert MTB/RIF assays are two rapid molecular TB detection/diagnostic tests. To compare the performance of LPA and Xpert MTB/RIF assay for early diagnosis of rifampicin-resistant (RR) TB in acid-fast bacillus (AFB) smear-positive and negative sputum samples. METHODS: A total 576 presumptive AFB patients were selected and subjected to AFB microscopy, Xpert MTB/RIF assay and recent version of LPA (GenoType MTBDRplus assay version 2) tests directly on sputum samples. Results were compared with phenotypic culture and drug susceptibility testing (DST). DNA sequencing was performed with rpoB gene for samples with discordant rifampicin susceptibility results. RESULTS: Among culture-positive samples, Xpert MTB/RIF assay detected Mycobacterium tuberculosis (Mtb) in 97.3% (364/374) of AFB smear-positive samples and 76.5% (13/17) among smear-negative samples, and the corresponding values for LPA test (valid results with Mtb control band) were 97.9% (366/374) and 58.8% (10/17), respectively. For detection of RR among Mtb positive molecular results, the sensitivity of Xpert MTB/RIF assay and LPA (after resolving discordant phenotypic DST results with DNA sequencing) were found to be 96% and 99%, respectively. Whereas, specificity of both test for detecting RR were found to be 99%. CONCLUSION: We conclude that although Xpert MTB/RIF assay is comparatively superior to LPA in detecting Mtb among AFB smear-negative pulmonary TB. However, both tests are equally efficient in early diagnosis of AFB smear-positive presumptive RR-TB patients.

Alpinia nigra fruits mediated synthesis of silver nanoparticles and their antimicrobial and photocatalytic activities.

In the present systematic study, silver nanoparticles have been synthesized using the fruits of Alpinia nigra. Apart from the presence of saponins, glycosides, alkaloids, steroids, the extract of A. nigra fruits are rich in polyphenols. The Total Flavonoid and Phenol Content of A. nigra fruits extract is 718 mgRE/g extract and 74.9 mgGAE/g extract respectively. The formation of the nanoparticles was validated through characterization techniques like UV-Vis spectroscopy, X- ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Energy dispersive X-ray spectroscopy (EDX). The spherical shape of silver nanoparticles is observed in Transmission Electron Microscopy (TEM) images. The average particle size of the silver nanoparticles is 6 nm. The biomolecules of the fruit extract played the dual role of reducing and capping agents which is evident from Fourier Transform Infrared (FTIR) spectrometer and Scanning Electron Microscopy (SEM) image analysis. The A. nigra capped silver nanoparticles exhibited promising antimicrobial activity against gram negative bacteria Klebsiella pneumoniae, gram positive bacteria Staphylococcus aureus and the pathogenic fungus, Candida albicans. Amongst the three pathogens, Klebsiella pneumoniae is the most susceptible to silver nanoparticles. Furthermore, the nanoparticles efficiently catalysed the degradation of the anthropogenic dyes Methyl orange, Rhodamine B and Orange G in the presence of sunlight. The photocatalytic degradation process follows the pseudo-first order kinetics. These results confirm that the silver nanoparticles can be efficiently synthesized via a green route using A. nigra fruits with applications as antimicrobial and catalytic agents.

Biogenic synthesis of gold nanoparticles and their application in photocatalytic degradation of toxic dyes.

Plants and their extracts play an important role in the green synthesis of nanoparticles mainly because of their environmental benignity. Based on plant extracts number of metal nanoparticles have been synthesized. In our study, we report a green technique for the synthesis of gold nanoparticles using the aqueous extracts of Alpinia nigra leaves and their photocatalytic activities. The antioxidant, antibacterial and antifungal potential of the synthesized nanoparticles were also evaluated. The aqueous extract of the plant is rich in flavonoids with Total Flavonoid Content of 491mgRE/g extract. The presence of flavonoids was further confirmed through analytical High Performance Liquid Chromatography (HPLC) analysis. The A. nigra mediated syntheses of gold nanoparticles (ANL-AuNPs) were characterized by UV-Vis spectrophotometer, Fourier Transform Infrared (FTIR) Spectroscopy, X-ray Diffraction (XRD) and Transmission Electron Microscopy (TEM). The crystalline nature of the ANL-AuNPs was confirmed by the powder XRD analysis. The TEM micrographs showed that the ANL-AuNPs was predominantly spherical in shape and the average particle size was 21.52 nm. The polyphenolics and other functional groups present in the aqueous extract that acted as reducing and capping agent in the synthesis of the Au-NPs were identified via FTIR spectral analysis. These green synthesized nanoparticles exhibited antioxidant activity with IC50 value of 52.16 µg/ml and showed inhibition in the growth of both gram-positive and gram-negative bacteria. The pathogenic fungus, Candida albicans was also susceptible to these nanoparticles. The ANL-AuNPs in the presence of sunlight catalyzed the degradation of the anthropogenic pollutant dyes, Methyl Orange and Rhodamine B with percent degradation of 83.25% and 87.64% respectively. The photodegradation process followed pseudo first order kinetic model. These results confirm that Alpinia nigra is a potential bioresource for the synthesis of Au-NPs with versatile applications.

Evaluating the Diagnostic Accuracy of Xpert MTB/RIF Assay in Pulmonary Tuberculosis.

Pulmonary tuberculosis still remains a major communicable disease worldwide. In 2013, 9 million people developed TB and 1.5 million people died from the disease. India constitutes 24% of the total TB burden. Early detection of TB cases is the key to successful treatment and reduction of disease transmission. Xpert MTB/RIF, an automated cartridge-based molecular technique detects Mycobacterium tuberculosis and rifampicin resistance within two hours has been endorsed by WHO for rapid diagnosis of TB. Our study is the first study from India with a large sample size to evaluate the performance of Xpert MTB/RIF assay in PTB samples. The test showed an overall sensitivity and specificity of 95.7% (430/449) and 99.3% (984/990) respectively. In smear negative-culture positive cases, the test had a sensitivity of 77.7%. The sensitivity and specificity for detecting rifampicin resistance was 94.5% and 97.7% respectively with respect to culture as reference standard. However, after resolving the discrepant samples with gene sequencing, the sensitivity and specificity rose to 99.0% and 99.3% respectively. Hence, while solid culture still forms the foundation of TB diagnosis, Xpert MTB/RIF proposes to be a strong first line diagnostic tool for pulmonary TB cases.