Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

BACKGROUND: Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). METHODS: EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. RESULTS: We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. CONCLUSION: From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

EGFR mutations and ROS1 and ALK rearrangements in a large series of non-small cell lung cancer in South India.

BACKGROUND: Driver mutations are seen in 80% of lung adenocarcinomas, and they influence prognosis and choice of therapy. AIM: Aim of this study was to analyse the frequency of epidermal growth factor receptor (EGFR) mutations, ALK and ROS1 rearrangements and their association with age and gender in non-small cell lung cancer reported from a tertiary care center in South India. METHODS: Tumors from patients with non-small cell carcinoma of lung were evaluated for EGFR mutations, ALK and ROS1 rearrangements and their association with age and gender were studied. RESULTS: Two thirds of non-small cell carcinomas had driver mutations or rearrangements. EGFR mutation was common and seen in 34.1%, whereas ALK rearrangement was seen in 11.1% and ROS1 rearrangement in 2% patients. Among EGFR mutations, most common were Exon 19 deletion and L858R seen in 21.3% and 11% of patients, respectively. Adenocarcinoma was the histologic diagnosis in 81% to 85% of patients with exon 19 deletion and L858R mutation, respectively. EGFR mutation frequency in patients less than 36 years was 13.6%, whereas in older patients, it varied from 34% to 36%. Exon 19 deletion was seen in 29.8% females and 17.2% of males. CONCLUSION: EGFR mutations are more common than ALK and ROS1 rearrangements. They are more common in females. Patients less than 36 years have reduced frequency of EGFR mutations. Exon 19 deletion and L858R are most common and are more prevalent in lung adenocarcinomas. Rare EGFR mutations are seen in patients aged more than 50 years.

Culture and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Proven Mycobacterium Tuberculosis Endophthalmitis: A Case Series.

PURPOSE: To report early confirmation of Mycobacterium tuberculosis (MTB) endophthalmitis by detection of 85B mRNA in vitreous by a reverse transcriptase polymerase chain reaction (RT-PCR) technique. METHODS: Retrospective, interventional case series of 5 patients with MTB endogenous endophthalmitis. Vitreous aspirate was subjected to Ziehl-Neelsen (ZN) staining, BACTEC MicroMGIT culture, RT-PCR targeting the 85B gene, real-time PCR targeting the IS6110 region, and nested PCR targeting the MPB64 gene and IS6110 region. Correlation between detection of MTB RNA, culture positivity, and ZN staining was studied. RESULTS: Five patients with endophthalmitis with no history of tuberculosis revealed acid-fast bacilli on ZN staining of vitreous. RT-PCR detected 85B RNA within 24 h. Culture for MTB was positive in 3/5 patients after 1 month. None of the eyes recovered any useful vision. CONCLUSIONS: RT-PCR can detect viable MTB RNA and provide evidence of active infection much earlier than culture.

Nested reverse transcriptase-polymerase chain reactions targeting the messenger RNA of icl2, hspx, and rRNAP1 genes to detect viable Mycobacterium tuberculosis directly from clinical specimens.

There is an urgent need for a rapid and reliable test to detect actively multiplying Mycobacterium tuberculosis directly from clinical specimens for an early initiation of the appropriate antituberculous treatment. This study was aimed at the optimization and application of nested reverse transcriptase-PCR (nRT-PCR) targeting the messenger RNA of the icl2, hspx, and rRNAP1 genes directly from sputum specimens, and their evaluation against the culture by the BACTEC MicroMGIT mycobacterial culture system. 203 Sputum samples from clinically suspected tuberculosis patients and 30 control specimens (clinically proven viral or bacterial infections other than tuberculosis) were included in this study. The mycobacterial culture was performed by the BACTEC MicroMGIT system following the manufacturer's instructions. The primers for nRT-PCRs targeting icl2, hspx, and rRNAP1 genes were indigenously designed using the Primer-BLAST software, and optimized for sensitivity and specificity. The icl2, hspx, and rRNAP1 genes were able to pick up 63.9%, 67.2%, and 58.75%, respectively, of culture-negative sputum specimens collected from clinically suspected tuberculosis patients. However, three (1.4%) were negative for nRT-PCR, but M. tuberculosis culture positive. All the 30 controls were negative for culture by the BACTEC MicroMGIT method and all three nRT-PCR. The novel nRT-PCRs targeting icl2, hspx, and rRNAP1 genes developed in this study are rapid and reliable diagnostic tools to detect viable M. tuberculosis directly from sputum specimens. However, further study by including a larger number of sputum specimens needs to be carried out to ascertain the diagnostic utility of the novel nRT-PCRs optimized in the study.

Detection of novel and reported mutations in the rpoB, katG and inhA genes in multidrug-resistant tuberculosis isolates: A hospital-based study.

The objective of this study was to detect mutations associated with isoniazid (INH) and rifampicin (RIF) resistance in Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients using a PCR-based DNA sequencing technique. Phenotypic drug susceptibility testing was performed using a BACTEC™ MicroMGIT Culture System in 354 M. tuberculosis isolates. Among the 354 isolates, 18 were multidrug-resistant tuberculosis (MDR-TB). PCR-based DNA sequencing was performed targeting the rpoB gene for RIF and the whole of the katG gene and the promoter and coding region of the inhA gene for INH. Results were analysed using MultAlin analysis to identify the presence of polymorphisms or mutations by comparing with already available GenBank sequences. Only 37.5% of RIF-resistant isolates showed the presence of the most commonly reported mutation (Ser531Leu). The most commonly reported mutation (Ser531Leu) was detected in six MDR-TB isolates. The frequency of mutations associated with INH resistance was 31.5% (17/54) and 29.6% (16/54) for katG and inhA, respectively. Comparing the relative distribution of mutations in the two target loci revealed that 12 isolates (22.2%) had a mutation in both katG and inhA. Apart from previously reported mutations in the katG gene, there were three novel deletion and six novel substitution mutations. As reported in previous studies, Ser531Leu was the most common mutation detected in RIF-resistant isolates. The genetic mechanism of INH resistance in M. tuberculosis is highly complex involving several genes, and much remains to be explored to achieve a better understanding of this complex mechanism.

In silico analysis and prioritization of drug targets in Fusarium solani.

Mycotic keratitis has emerged as a major ophthalmic problem and a leading cause of blindness, since its recognition in 1879. Filamentous fungi are major causative of mycotic keratitis. In India, the main etiological organism responsible for mycotic keratitis is Aspergillus species followed by Fusarium species. In South India, Fusarium based keratitis scales up to 43%. Nearly one-third of mycotic keratitis treatment results in failure, as fungal infections are highly resistant to antibiotic therapies. Therefore, there is need to determine novel and specific targets to constrain Fusarium infections in human eye. In this study, we implemented subtractive proteomics coupled with in silico functional annotation to prioritize potential and specific drug targets which can be used to modulate the virulence of Fusarium solani subsp.pisi (Nectria haematococca MPVI). The results infer that Thiamine thiazole synthase (Thi4), an intracellular membrane bound protein as the potential target, which is a core protein in biological and metabolic process of this pathogen. Moreover, this protein occurs in the thiamine thiazole biosynthesis pathway which is unique to F.solani and devoid in human. Hence, we predicted a plausible structure for this protein and also performed ligand-binding cavity analysis which can be for a strong base for drug designing studies. This study will pave way in better understanding of potential drug targets in F.solani and also leading to therapeutic interventions of fungal keratitis.

Isolation of Mycobacterium massiliense from a corneal biopsy in India.

INTRODUCTION: Rapidly growing mycobacteria (RGM) are ubiquitous and are usually considered as saprophytes, and have been recovered from the environment, particularly in dust, watery soil and water distribution systems. However, Mycobacterium massiliense is a rare causative agent of ocular infection. CASE PRESENTATION: We report a case of M. massiliense in a 44-year-old female with signs and symptoms of a corneal ulcer. We carried out PCR-based DNA sequencing targeting the hsp 65 gene for the identification of M. massiliense. To confirm the identification, we also performed PCR-based RFLP targeting the hsp65 gene and PCR-based DNA sequencing targeting the internal transcribed spacer region, which showed 97 % nucleotide identity with M. massiliense. CONCLUSION: To the best of our knowledge, this is the first study in India to report the detection of M. massiliense from a corneal biopsy.

Whole-Genome Sequencing of Streptomycin-Resistant Mycobacterium tuberculosis Isolate VRFCWCF MRTB 180 Reveals Novel and Potential Mutations for Resistance.

We announce the draft genome sequence of a streptomycin monoresistant Mycobacterium tuberculosis strain (VRFCWCF MRTB 180) isolated from sputum of a clinically suspected tuberculosis patient.

Whole Genome Sequence of Polyresistant Mycobacterium tuberculosis CWCFVRF PRTB 19 Sputum Isolate from Chennai, India, Closely Clustering with East African Indian 5 Genogroup.

We announce the draft genome sequence of a polyresistant Mycobacterium tuberculosis strain (CWCFVRF PRTB 19) isolated from the sputum of a clinically suspected tuberculosis patient, and it closely clusters to the East African Indian 5 (EAI5) lineage.

Draft Genome Sequence of Multidrug-Resistant Mycobacterium tuberculosis Strain CWCFVRF MDRTB 670, Isolated from the Sputum of a Patient from Chennai, India, with Clinically Suspected Tuberculosis.

We announce the draft genome sequence of a multidrug-resistant Mycobacterium tuberculosis strain (CWCFVRF MDRTB 670) isolated from sputum from a patient with clinically suspected tuberculosis.

Whole-Genome Sequencing and Mutation Analysis of Two Extensively Drug-Resistant Sputum Isolates of Mycobacterium tuberculosis (VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028) from Chennai, India.

We announce the draft genome sequence of two extensively drug-resistant Mycobacterium tuberculosis strains, VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028, isolated from the sputum samples of a patient clinically suspected to have tuberculosis, and we also report novel mutations that confer drug resistance.

Detection of novel coupled mutations in the katG gene (His276Met, Gln295His and Ser315Thr) in a multidrug-resistant Mycobacterium tuberculosis strain from Chennai, India, and insight into the molecular mechanism of isoniazid resistance using structural bioinformatics approaches.

This study reports on the structural basis of drug resistance targeting the katG gene in a multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strain with two novel mutations (His276Met and Gln295His) in addition to the most commonly reported mutation (Ser315Thr). A structural bioinformatics approach was used to predict the structure of the mutant KatG enzyme (MT). Subsequent molecular dynamics and docking studies were performed to explain the mechanism of isoniazid (INH) resistance. The results show significant conformational changes in the structure of MT leading to a change in INH binding residues at the active site, with a significant increase in the inhibition constant (Ki) of 5.67 µm in the mutant KatG-isoniazid complex (MT-INH) compared with the wild-type KatG-isoniazid complex (WT-INH). In the case of molecular dynamics studies, root mean square deviation (RMSD) analysis of the protein backbone in simulated biological conditions revealed an unstable trajectory with higher deviations in MT throughout the simulation process (1 ns). Moreover, root mean square fluctuation (RMSF) analysis revealed an overall increase in residual fluctuations in MT compared with the wild-type KatG enzyme (WT), whilst the INH binding residues of MT showed a decreased fluctuation that can be observed as peak deviations. Hence, the present study suggests that His276Met, Gln295His and Ser315Thr mutations targeting the katG gene result in decreased stability and flexibility of the protein at INH binding residues leading to impaired enzyme function.