Validity of a CB-NAAT assay in diagnosing tuberculosis in comparison to culture: A study from an urban area of South India.

•CB-NAAT performance compared in 831 suspected pulmonary and extrapulmonary suspected cases.•The conventional stained smear and CB-NAAT results were compared to the MGIT culture.•Sensitivity and specificity of CB-NAAT was 84.43% and 94.93%.•The rapid results from CB-NAAT confirms its use in the tuberculosis diagnostic algorithm.•The benefits of disease diagnosis and prevention outweighs the price tag of the CB-NAAT tests.•This is more so for the resource poor countries where the burden of the disease is high.

Role of fluorescence in situ hybridization in detecting mycobacterium avium complex presenting as fever in treatment failure HIV.

A 49-year-old male HIV positive patient on treatment failure presented with complaints of fever and dysphagia of three weeks duration and later on developed cervical lymphadenopathy along with severe vomiting and abdominal pain. Liver function tests were found to be worsening with severe drop in CD4 counts. An extensive workup for pyrexia was done. FNAC and biopsy of lymph node showed features suggestive of granulomatous lymphadenitis. CBNAAT of the lymph node aspirate was negative for MTB. Blood culture and lymph node cultures were negative for Mycobacterium Avium Complex (MAC). MAC was however, finally detected and reported positive on Fluorescence in Situ Hybridization (FISH) of the cervical lymph node aspirate. Prompt treatment for MAC was initiated with Ethambutol 800 mg OD and Azithromycin 500 mg OD following which fever spikes subsided and lymph node resolved. The Patient's condition gradually improved and was discharged shortly with a good recovery on subsequent follow ups. Fever is one of the common symptoms in patients with MAC infection. Some other clinical manifestations include weight loss, hepatosplenomegaly and intra-abdominal lymphadenopathy. Diagnostic evaluation should be aggressive. As there is a high risk for MAC infection in advanced HIV cases with poor HAART compliance, FISH can be a valuable and effective diagnostic tool in early detection and treatment of MAC.

Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes.

OBJECTIVE: In resource-limited tuberculosis-endemic countries, Mycobacterium tuberculosis in sputum is mainly detected by acid-fast bacillus (AFB) staining and the identification of sputum-derived cultures. PCR techniques are practical only in well-resourced laboratories. This study investigated the application of a rapid, simple, and inexpensive fluorescence in situ hybridization (FISH) assay to identify and differentiate M. tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) in sputum. METHODS: The Mycobacterium/Nocardia Genus (MN Genus)-MTBC FISH assay performed in this study utilizes two different DNA probes labeled with different fluorescent molecules that hybridize respectively with 16S rRNA of the genus Mycobacterium and 23S rRNA of MTBC. The assay was tested on 202 patient sputum samples in Mangaluru, Karnataka State, India. Sputa were first liquefied and bacteria concentrated before performing the FISH assay and parallel culturing and AFB staining. The identities of cultured bacteria from DNA sequencing were compared with FISH assay findings from corresponding sputa. RESULTS: Of the 202 sputum samples tested, 67 reacted with both MN Genus-specific and MTBC-specific probes, none reacted only with the MTBC-specific probe, and 22 reacted only with the MN Genus-specific probe. The FISH assay yielded results in 2h and had a limit of detection of 2.2×104CFU/ml in sputum spiked with cultured M. tuberculosis. The diagnostic sensitivity, specificity, and positive and negative predictive values of the FISH assay for MTBC in patient sputa were 89.7%, 95.5%, 88.0%, and 92.6%, respectively. NTM were a significant cause of tuberculosis-like infections in Mangaluru. CONCLUSIONS: The MN Genus-MTBC dual probe fluorescence FISH assay previously applied to cultures can also be utilized in resource-limited tuberculosis-endemic countries for rapidly identifying and differentiating MTBC and NTM in sputum samples.

Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures.

Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.