Utility of a lateral flow assay for culture confirmation of Mycobacterium tuberculosis complex.

BACKGROUND: Therapy for the clinical management of patients with Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) is different. Prompt detection and discrimination is necessary for administration of suitable therapy. METHODS: The aim of the study was to evaluate the performance of a Immunochromatographic Test (ICT) in rapid differentiation of MTBC from NTM grown in Lowenstein-Jensen medium and MGIT broth in comparison to molecular methods. RESULTS: Of the 106 isolates in this study, 96 and 95 were identified as MTBC by 16S rRNA PCR and MPT64 respectively. The sensitivity and specificity of MPT64 was found to be 99% and 100% respectively. CONCLUSION: The Lateral Flow Assay Test is a useful and specific tool in rapid differentiation of M. tuberculosis complex from culture. Therefore proper identification avoids unnecessary ATT to patients infected with NTM.

Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection.

Molecular techniques have revolutionized tuberculosis (TB) diagnosis by offering a faster and more sensitive approach, detecting Mycobacterium tuberculosis (Mtb) DNA directly from samples. Single-tube nested PCR (STNPCR) combines two PCR reactions with separate oligonucleotide sets in a single tube. Moreover, colorimetric methods in PCR products have been studied for pathogen detection. Thus, this study aimed to establish a novel system based on colorimetric STNPCR for Mtb detection using microtiter plates with IS6110-amplified fragments. The results showed a general colorimetric STNPCR detection limit of 1 pg/µl. Its general sensitivity and specificity were 76.62 and 60.53%, respectively, with kappa index agreement of 0.166.

A total of 318 biological samples (urine, plasma, peripheral blood mononuclear cells, pleural fluid and sputum) from pulmonary/extrapulmonary TB and non-TB patients were used in this study. The colorimetric STNPCR assay using IS6110 as the target gene was developed and optimized for Mtb detection based on similar validated systems. Cut-off values based on receiver operator characteristic curve analysis were defined to determine the sensitivity and specificity for each sample type. The technique's performance was assessed according to kappa index calculations and interpretation.

Evaluating the Sensitivity of Different Molecular Techniques for Detecting Mycobacterium tuberculosis Complex in Patients with Pulmonary Infection.

This study aimed to evaluate the accuracy of detecting drug-resistant Mycobacterium tuberculosis complex (MTBC)-specific DNA in sputum specimens from 48 patients diagnosed with pulmonary tuberculosis. The presence of MTBC DNA in the specimens was validated using the GeneXpert MTB/RIF system and compared with a specific PCR assay targeting the IS6110 and the mtp40 gene sequence fragments. Additionally, the results obtained by multiplex PCR assays to detect the most frequently encountered rifampin, isoniazid, and ethambutol resistance-conferring mutations were matched with those obtained by GeneXpert and phenotypic culture-based drug susceptibility tests. Of the 48 sputum samples, 25 were positive for MTBC using the GeneXpert MTB/RIF test. Nevertheless, the IS6110 and mtp40 single-step PCR revealed the IS6110 in 27 of the 48 sputum samples, while the mtp40 gene fragment was found in only 17 of them. Furthermore, multiplex PCR assays detected drug-resistant conferring mutations in 21 (77.8%) of the 27 samples with confirmed MTBC DNA, 10 of which contained single drug-resistant conferring mutations towards ethambutol and two towards rifampin, and the remaining nine contained double-resistant mutations for ethambutol and rifampin. In contrast, only five sputum specimens (18.5%) contained drug-resistant MTBC isolates, and two contained mono-drug-resistant MTBC species toward ethambutol and rifampin, respectively, and the remaining three were designated as multi-drug resistant toward both drugs using GeneXpert and phenotypic culture-based drug susceptibility tests. Such discrepancies in the results emphasize the need to develop novel molecular tests that associate with phenotypic non-DNA-based assays to improve the detection of drug-resistant isolates in clinical specimens in future studies.

Performance of lipid fingerprint by routine matrix-assisted laser desorption/ionization time of flight for the diagnosis of Mycobacterium tuberculosis complex species.

OBJECTIVES: Rapid detection of bacterial pathogens at species and sub-species levels is crucial for appropriate treatment, infection control, and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial lipid-based approach to identify MTBC cultures and sub-species. METHODS: A blinded study was conducted using 90 mycobacterial clinical isolate strains comprising MTBC strains sub-cultured in Middlebrook 7H11 medium supplemented with 10% oleic-acid, dextrose, catalase growth supplement and incubated for up to 6 weeks at 37°C and using the following seven reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, and M. bovis BCG) grown under the same conditions, to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the matrix-assisted laser desorption/ionization target followed by the addition of the matrix. Acquisition of the data was performed using the positive ion mode. RESULTS: Based on the identification of clear and defined lipid signatures from the seven reference strains, the method that we developed was fast (<10 minutes) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the matrix-assisted laser desorption/ionization time of flight were 94.4 (95% CI, 86.4-98.5) and 94.4 (95% CI, 72.7-99.9), respectively. CONCLUSIONS: Mycobacterial lipid profiling provides a means of rapid, safe, and accurate discrimination of species within the MTBC.

Incubation time of Mycobacterium tuberculosis complex sputum cultures in BACTEC MGIT 960: 4weeks of negative culture is enough for physicians to consider alternative diagnoses.

We retrospectively analyzed time to detection of 3747 positive MGIT sputum cultures at a laboratory in a country with heavy burden of tuberculosis. Ninety-nine percent of diagnostic cultures turned positive within 28days, suggesting that physicians may consider alternative diagnoses if sputum cultures remain negative after 4weeks of incubation.

An Integrated Approach Using Liquid Culture System Can it Make an Impact for Clinical Diagnosis of Genitourinary Tuberculosis?

Tuberculosis (TB) is one of the major health problems in India. Genitourinary TB (GUTB) comprises 20% of all extrapulmonary TB, and is the most common extrapulmonary site to be affected by this disease. The spectrum of varied pathological changes occur in GUTB, hence a thorough knowledge is required to prevent complications related to GUTB. Diagnostic dilemma is a common problem faced as culture and polymerase chain reaction results vary in their sensitivity and specificity. A thorough knowledge of epidemiology, immunopathogenesis, and spectrum of the disease and importance of including liquid culture system for the diagnosis of this disease are presented in three cases.