PMID- 28399124 OWN - NLM STAT- MEDLINE DCOM- 20170905 LR - 20181113 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 12 IP - 4 DP - 2017 TI - Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures. PG - e0174989 LID - 10.1371/journal.pone.0174989 [doi] LID - e0174989 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. FAU - Shah, Jyotsna AU - Shah J AUID- ORCID: 0000-0001-9890-366X AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. FAU - Weltman, Helena AU - Weltman H AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. FAU - Narciso, Patricia AU - Narciso P AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. FAU - Murphy, Christina AU - Murphy C AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. FAU - Poruri, Akhila AU - Poruri A AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. FAU - Baliga, Shrikala AU - Baliga S AD - Kasturba Medical College, Mangalore, Manipal University, India. FAU - Sharon, Leesha AU - Sharon L AD - Kasturba Medical College, Mangalore, Manipal University, India. FAU - York, Mary AU - York M AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. FAU - Cunningham, Gail AU - Cunningham G AD - University of California, San Francisco, California, United States of America. FAU - Miller, Steve AU - Miller S AD - University of California, San Francisco, California, United States of America. FAU - Caviedes, Luz AU - Caviedes L AD - Labotorio de Investigacion en Enfermedades Infecciosas of the Universidad Peruana Cayetano Heredia, Iquitos, Peru. FAU - Gilman, Robert AU - Gilman R AD - Labotorio de Investigacion en Enfermedades Infecciosas of the Universidad Peruana Cayetano Heredia, Iquitos, Peru. AD - John Hopkins University, Baltimore, Maryland, United States of America. FAU - Desmond, Edward AU - Desmond E AD - Microbial Disease Laboratory, California Department of Public Health, Richmond, California, United States of America. FAU - Ramasamy, Ranjan AU - Ramasamy R AD - ID-FISH Technology Inc., Palo Alto, California, United States of America. LA - eng PT - Journal Article DEP - 20170411 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Fluorescent Dyes) RN - 0 (RNA, Ribosomal, 16S) RN - 0 (RNA, Ribosomal, 23S) SB - IM MH - Bacillus subtilis/cytology/genetics/isolation & purification/metabolism MH - Corynebacterium/cytology/genetics/isolation & purification/metabolism MH - Fluorescent Dyes MH - Genes, rRNA MH - *In Situ Hybridization, Fluorescence MH - Microscopy, Fluorescence MH - Mycobacterium avium Complex/classification/cytology/genetics/*isolation & purification MH - Mycobacterium tuberculosis/classification/cytology/genetics/*isolation & purification MH - Nocardia/cytology/genetics/isolation & purification/metabolism MH - RNA, Ribosomal, 16S/genetics MH - RNA, Ribosomal, 23S/genetics MH - Sensitivity and Specificity PMC - PMC5388335 COIS- Competing Interests: JS, HW are authors on patents listed below. JS has stocks in ID-FISH and IGeneX. PN, CM were employees of ID-FISH; RR is employee of ID-FISH; and AK is an employee of IGeneX and ID-FISH. They do not have any stock in these companies. This study was supported by NIH SBIR Phase I and Phase II Grants # A1056785. Following patents have been issued to ID-FISH. (1) METHOD FOR IMPROVING CELL PERMEABILITY TO FOREIGN PARTICLES. Inventors: Jyotsna S. Shah, Santa Clara, California (United States); Helena Weltman, Los Altos, California (United States); Patent Number: US 11/494,430. Issued Jan 21, 2014. (2) METHODS FOR DETECTING AND DIFFERENTIATING MYCOBACTERIUM GENUS AND MYCOBACTERIUM AVIUM COMPLEX IN A SAMPLE OR CULTURE, Inventors: Jyotsna S. Shah, Santa Clara, California (United States); Helena Weltman, Los Altos, California (United States); Patent Number US 11/975,306, Issued: June 24, 2014. The authors confirm that this does not alter their adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors. EDAT- 2017/04/12 06:00 MHDA- 2017/09/07 06:00 PMCR- 2017/04/11 CRDT- 2017/04/12 06:00 PHST- 2016/10/24 00:00 [received] PHST- 2017/03/17 00:00 [accepted] PHST- 2017/04/12 06:00 [entrez] PHST- 2017/04/12 06:00 [pubmed] PHST- 2017/09/07 06:00 [medline] PHST- 2017/04/11 00:00 [pmc-release] AID - PONE-D-16-42387 [pii] AID - 10.1371/journal.pone.0174989 [doi] PST - epublish SO - PLoS One. 2017 Apr 11;12(4):e0174989. doi: 10.1371/journal.pone.0174989. eCollection 2017.