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.
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Jyotsna
authors.identifier: http://orcid.org/0000-0001-9890-366X
authors.initials: J
authors.lastname: Shah
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Helena
authors.initials: H
authors.lastname: Weltman
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Patricia
authors.initials: P
authors.lastname: Narciso
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Christina
authors.initials: C
authors.lastname: Murphy
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Akhila
authors.initials: A
authors.lastname: Poruri
authors.affiliation: Kasturba Medical College, Mangalore, Manipal University, India.
authors.forename: Shrikala
authors.initials: S
authors.lastname: Baliga
authors.affiliation: Kasturba Medical College, Mangalore, Manipal University, India.
authors.forename: Leesha
authors.initials: L
authors.lastname: Sharon
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: York
authors.affiliation: University of California, San Francisco, California, United States of America.
authors.forename: Gail
authors.initials: G
authors.lastname: Cunningham
authors.affiliation: University of California, San Francisco, California, United States of America.
authors.forename: Steve
authors.initials: S
authors.lastname: Miller
authors.affiliation: Labotorio de Investigación en Enfermedades Infecciosas of the Universidad Peruana Cayetano Heredia, Iquitos, Peru.
authors.forename: Luz
authors.initials: L
authors.lastname: Caviedes
authors.affiliation: Labotorio de Investigación en Enfermedades Infecciosas of the Universidad Peruana Cayetano Heredia, Iquitos, Peru.|John Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Gilman
authors.affiliation: Microbial Disease Laboratory, California Department of Public Health, Richmond, California, United States of America.
authors.forename: Edward
authors.initials: E
authors.lastname: Desmond
authors.affiliation: ID-FISH Technology Inc., Palo Alto, California, United States of America.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Ramasamy
mesh_terms: D001412:Bacillus subtilis / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003352:Corynebacterium / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005456:Fluorescent Dyes; D020459:Genes, rRNA; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D015269:Mycobacterium avium Complex / Q000145:classification / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification*; D009615:Nocardia / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S