Efficient Differentiation ofMycobacterium aviumComplex Species and Subspecies by Use of Five-Target Multiplex PCR

Abstract

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6,14,16,24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27 Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans.

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

“…If suspicious colonies were seen on HEY or if the MGIT 960 instrument was signal-positive, a 5-target multiplex PCR was performed to establish if MAP was present (Shin et al, 2010). The nature of the CMR material inoculated onto solid media tended to resemble bacterial growth, causing many cultures to be unnecessarily tested.…”

Infecção sistêmica por Mycobacterium avium em cão: relato de caso

“…Vet. Zootec., v.65, n.4, p.1111-1115, 2013 micobactérias saprófitas e a doença disseminada, que pode envolver múltiplos órgãos e constitui a manifestação mais comum de infecções humanas e em animais por M. avium (Thorel et al, 2001).…”

“…A infecção por M. avium é descrita em muitos mamíferos, incluindo cães (Shackelford e Reed, 1989;Miller et al, 1995;Thorel et al, 2001;Naughton et al, 2005;Glanemann et al, 2008) , gatos (Jordan e Cohn, 1993), primatas, suínos, bovinos, ovinos e equinos (Thorel et al, 2001). Essa infecção é considerada rara em cães e gatos devido às suas resistências natas a esse microrganismo (Shackelford e Reed, 1989); em contrapartida, vem aumentando o número de casos relatados, especialmente em felinos, nos últimos anos (Lappin, 2005).…”

“…A avaliação citológica ou a histopatológica dos tecidos afetados podem demonstrar o bacilo álcool-ácido resistente (coloração de Ziehl-Neelsen), entretanto não diferenciam as espécies de Mycobacterium. Neste caso, para a definição do diagnóstico, o indicado é a realização da cultura para Mycobacterium com diferenciação das espécies (Lappin, 2005) e/ou técnicas moleculares disponíveis para as principais micobactérias (Miller et al, 1995;Glanemann et al, 2008;Shin et al, 2010).…”

Persistence of Mycobacterium avium subspecies paratuberculosis in Endangered Florida Key Deer and Key Deer Habitat

“…Inoculated tubes were incubated in a BACTEC MGIT 960 instrument and monitored hourly for microbial growth over 49 days. A fivetarget multiplex PCR was used to test DNA extracted from culture-positive samples (Shin et al 2010). PCR methods were used to amplify each of five possible mycobacterial targets: a 16S rRNA target common to all mycobacteria, a chromosomal 1 DNA target (DT1) found in two serotypes of M. avium subsp.…”