Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes. (October 2018)

• Record Type: Journal Article
• Authors: Baliga, Shrikala; Murphy, Christina; Sharon, Leesha; Shenoy, Suchitra; Biranthabail, Dhanashree; Weltman, Helena; Miller, Steve; Ramasamy, Ranjan; Shah, Jyotsna
• Is Part Of: International journal of infectious diseases. Volume 75(2018)
• Page Start: 1
• Page End: 7
• Publication Date: 2018-10
• Subjects: Fluorescence in situ hybridization -- LED fluorescence microscopy -- Mycobacterium tuberculosis -- Non-tuberculous mycobacteria -- Tuberculosis diagnosis -- Sputum assay; Communicable diseases -- Periodicals; Communicable Diseases -- Periodicals; Communicable diseases; Periodicals; Electronic journals; 616.9
• DOI: 10.1016/j.ijid.2018.07.011
• Languages: English
• ISSNs: 1201-9712

Abstract

Highlights: Rapid and inexpensive identification and differentiation of Mycobacterium tuberculosis from non-tuberculous mycobacteria in sputum is needed. The genus and species-specific dual color FISH assay meets this need. The assay requires only a light microscope with a LED/filter unit. This assay is simple and suitable for resource-limited laboratories. Abstract: 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 2 h and had a limit of detection of 2.2 × 10 4 CFU/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.