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

Jyotsna Shah, Helena Weltman, Patricia Narciso, Christina Murphy, Akhila Poruri, Shrikala Baliga, Leesha Sharon, Mary York, Gail Cunningham, Steve Miller, Luz Caviedes, Robert Gilman, Edward Desmond, Ranjan Ramasamy

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)e0174989
JournalPLoS One
Volume12
Issue number4
DOIs
Publication statusPublished - 2017

Fingerprint

Mycobacterium avium complex
Mycobacterium tuberculosis complex
Mycobacterium avium Complex
Mycobacterium tuberculosis
Pathogens
fluorescence in situ hybridization
Fluorescence In Situ Hybridization
Assays
Color
Fluorescence
color
pathogens
assays
probes (equipment)
Fluorescent Dyes
Mycobacterium
Nocardia
DNA sequences
23S Ribosomal RNA
Liquids

Cite this

Shah, Jyotsna ; Weltman, Helena ; Narciso, Patricia ; Murphy, Christina ; Poruri, Akhila ; Baliga, Shrikala ; Sharon, Leesha ; York, Mary ; Cunningham, Gail ; Miller, Steve ; Caviedes, Luz ; Gilman, Robert ; Desmond, Edward ; Ramasamy, Ranjan. / Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures. In: PLoS One. 2017 ; Vol. 12, No. 4. pp. e0174989.
@article{edcf72e3aaaa468ba4f4eb6259c3c897,
title = "Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures",
abstract = "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.",
author = "Jyotsna Shah and Helena Weltman and Patricia Narciso and Christina Murphy and Akhila Poruri and Shrikala Baliga and Leesha Sharon and Mary York and Gail Cunningham and Steve Miller and Luz Caviedes and Robert Gilman and Edward Desmond and Ranjan Ramasamy",
year = "2017",
doi = "10.1371/journal.pone.0174989",
language = "English",
volume = "12",
pages = "e0174989",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "4",

}

Shah, J, Weltman, H, Narciso, P, Murphy, C, Poruri, A, Baliga, S, Sharon, L, York, M, Cunningham, G, Miller, S, Caviedes, L, Gilman, R, Desmond, E & Ramasamy, R 2017, 'Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures', PLoS One, vol. 12, no. 4, pp. e0174989. https://doi.org/10.1371/journal.pone.0174989

Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures. / Shah, Jyotsna; Weltman, Helena; Narciso, Patricia; Murphy, Christina; Poruri, Akhila; Baliga, Shrikala; Sharon, Leesha; York, Mary; Cunningham, Gail; Miller, Steve; Caviedes, Luz; Gilman, Robert; Desmond, Edward; Ramasamy, Ranjan.

In: PLoS One, Vol. 12, No. 4, 2017, p. e0174989.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures

AU - Shah, Jyotsna

AU - Weltman, Helena

AU - Narciso, Patricia

AU - Murphy, Christina

AU - Poruri, Akhila

AU - Baliga, Shrikala

AU - Sharon, Leesha

AU - York, Mary

AU - Cunningham, Gail

AU - Miller, Steve

AU - Caviedes, Luz

AU - Gilman, Robert

AU - Desmond, Edward

AU - Ramasamy, Ranjan

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pone.0174989

DO - 10.1371/journal.pone.0174989

M3 - Article

C2 - 28399124

VL - 12

SP - e0174989

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 4

ER -