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

<div><p>Two rapid dual color fluorescence <i>in situ</i> hybridization (FISH) assays were evaluated for detecting <i>M</i>. <i>tuberculosis</i> and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the <i>Mycobacterium tuberculosis</i> complex (MTBC) and a green fluorescent probe specific for the <i>Mycobacterium</i> and <i>Nocardia</i> 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 <i>M</i>. <i>avium</i> 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 <i>Mycobacterium</i> 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 <i>Corynebacterium</i> 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 <i>Mycobacterium</i> 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 <i>Nocardia</i>, (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 <i>M</i>. <i>tuberculosis</i> was determined to be 5.1x10⁴ cfu per ml and for <i>M</i>. <i>avium</i> 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.</p>