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.

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.

A 49-year-old male HIV positive patient on treatment failure presented with complaints of fever and dysphagia of three weeks duration and later on developed cervical lymphadenopathy along with severe vomiting and abdominal pain. Liver function tests were found to be worsening with severe drop in CD4 counts. An extensive workup for pyrexia was done. FNAC and biopsy of lymph node showed features suggestive of granulomatous lymphadenitis. CBNAAT of the lymph node aspirate was negative for MTB. Blood culture and lymph node cultures were negative for Mycobacterium Avium Complex (MAC). MAC was however, finally detected and reported positive on Fluorescence in Situ Hybridization (FISH) of the cervical lymph node aspirate. Prompt treatment for MAC was initiated with Ethambutol 800 mg OD and Azithromycin 500 mg OD following which fever spikes subsided and lymph node resolved. The Patient’s condition gradually improved and was discharged shortly with a good recovery on subsequent follow ups. Fever is one of the common symptoms in patients with MAC infection. Some other clinical manifestations include weight loss, hepatosplenomegaly and intra-abdominal lymphadenopathy. Diagnostic evaluation should be aggressive. As there is a high risk for MAC infection in advanced HIV cases with poor HAART compliance, FISH can be a valuable and effective diagnostic tool in early detection and treatment of MAC.