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

Shrikala Baliga, Christina Murphy, Leesha Sharon, Suchitra Shenoy, Dhanashree Biranthabail, Helena Weltman, Steve Miller, Ranjan Ramasamy, Jyotsna Shah

Research output: Contribution to journalArticle

2 Citations (Scopus)

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 × 104 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.

Original languageEnglish
Pages (from-to)1-7
Number of pages7
JournalInternational Journal of Infectious Diseases
Volume75
DOIs
Publication statusPublished - 01-10-2018

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DNA Probes
Mycobacterium
Sputum
Fluorescence In Situ Hybridization
Mycobacterium tuberculosis
Nocardia
Tuberculosis
Bacillus
Staining and Labeling
Bacteria
Acids
DNA Sequence Analysis
Limit of Detection
India
Fluorescence
Sensitivity and Specificity
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Microbiology (medical)
  • Infectious Diseases

Cite this

@article{119732e4d4b74d1a80a422d20396542d,
title = "Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes",
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 × 104 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.",
author = "Shrikala Baliga and Christina Murphy and Leesha Sharon and Suchitra Shenoy and Dhanashree Biranthabail and Helena Weltman and Steve Miller and Ranjan Ramasamy and Jyotsna Shah",
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Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes. / Baliga, Shrikala; Murphy, Christina; Sharon, Leesha; Shenoy, Suchitra; Biranthabail, Dhanashree; Weltman, Helena; Miller, Steve; Ramasamy, Ranjan; Shah, Jyotsna.

In: International Journal of Infectious Diseases, Vol. 75, 01.10.2018, p. 1-7.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Rapid method for detecting and differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in sputum by fluorescence in situ hybridization with DNA probes

AU - Baliga, Shrikala

AU - Murphy, Christina

AU - Sharon, Leesha

AU - Shenoy, Suchitra

AU - Biranthabail, Dhanashree

AU - Weltman, Helena

AU - Miller, Steve

AU - Ramasamy, Ranjan

AU - Shah, Jyotsna

PY - 2018/10/1

Y1 - 2018/10/1

N2 - 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 × 104 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.

AB - 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 × 104 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.

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