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

Date Published: April 11, 2017

Publisher: Public Library of Science

Author(s): 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, Olivier Neyrolles.


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 5.1×104 cfu per ml and for M. avium 1.5×104 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.

Partial Text

The World Health Organization (WHO) estimates that one third of the human population is infected with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis [1]. A small proportion of those infected, estimated to be 10.4 million new cases in 2015, then develop active disease resulting in 1.4 million deaths in 2015 [1]. An additional 0.4 million deaths from tuberculosis among persons with HIV infection was also reported in 2015 [1]. While most human pulmonary mycobacterial infections are caused by MTB and closely related species such as Mycobacterium bovis that together comprise the Mycobacterium tuberculosis complex or MTBC, clinically relevant infections with non-tuberculous mycobacteria (NTM) also occur in the USA and elsewhere [2, 3]. The management and treatment of patients with tuberculosis varies from that of patients infected with NTM. Pulmonary disease, the most commonly reported localized manifestation for NTM infections, is most often associated with mycobacterial species related to Mycobacterium avium (the M. avium complex or MAC), but other Mycobacteria such as M. kansasii, M. fortuitum, M. xenopi, M. abscessus, and M. simiae can be responsible in some instances [4–8]. Although NTM-associated pulmonary disease has been described primarily among immunocompromised persons [2, 3], it is now recognized in some persons without predisposing conditions [5–8]. Norcardiosis caused by Norcadia species also needs to be differentiated from MTB in human lung disease [9]. Norcadia are Gram positive, partially acid fast-staining bacteria closely related to the genus Mycobacterium. They are found in soil and water and norcardiosis can manifest in pulmonary, cutaneous or disseminated forms [9]. An estimated 500–1000 new cases of nocardiosis are diagnosed each year in the USA, with approximately 60% of cases being associated with immune-compromise, e.g. due to HIV infection, cancer and diabetes [9].

The findings suggest that a combination of the MN Genus-MTBC and MTBC-MAC FISH assays can be effective diagnostic tools for detecting Mycobacteria from solid and liquid cultures and for their identification as MTBC, MAC or NTM other than MAC. The MN Genus-MTBC and MTBC-MAC FISH assays are best performed sequentially in that order on cultures so that non-MTBC mycobacteria identified in the MN Genus-MTBC assay can subsequently be classified as MAC or other NTM by the MTBC-MAC assay. The MTBC-MAC FISH assay described here is the first molecular test that can detect and differentiate the MTBC and MAC in a single smear. Our findings further suggest that the FISH assays are useful for simultaneously detecting mixed infections of MTBC and MAC. The two FISH assays have a LOD of at least 5.1×104 cfu of bacilli per ml which can help minimize delays in diagnosis by being applicable relatively early after initiation of cultures. The two assays are also rapid, with results becoming available in less than two hours. The likely universal applicability of the MN Genus-MTBC and MTBC-MAC FISH assays is demonstrated by the results with clinical samples from three geographically disperse countries viz. India, Peru and the USA.




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