Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria.

Letícia Muraro Wildner,Paulo Cp Ferreira,Christiane Lourenço Nogueira,Erna G Kroon,Gabriela Segat,Maria Luiza Bazzo,Darcita B Rovaris,Simone Gonçalves Senna,Aline Daiane Schlindwein,Jaquelline Germano De Oliveira,Susie Coutinho Liedke,Claudio A Bonjardim

doi:10.1590/0074-0276130458

Abstract

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Highlights

2004, Duarte et al 2009)
The infections caused by nontuberculous mycobacteria (NTM) are clinically indistinguishable from those caused by M. tuberculosis, the therapeutic regimen differs between the Mycobacterium tuberculosis complex (MTC) and NTM, even among different species of NTM
We have previously described a polymerase chain reaction (PCR)-based method called a mycobacteria mobility shift assay (MMSA) that was designed for MTC and NTM species identification (Waléria-Aleixo et al 2000)

Summary

Introduction

2004, Duarte et al 2009). The infections caused by NTM are clinically indistinguishable from those caused by M. tuberculosis, the therapeutic regimen differs between the MTC and NTM, even among different species of NTM. The traditional identification of mycobacterial species is based on an analysis of phenotypic and biochemical characteristics using methods that are labour intensive, time consuming and poorly discriminative. These methods may take up 60 days and may result in ambiguous or erroneous identification. Molecular tests have been described as a rapid, highly discriminative and easy-to-perform alternative for mycobacterial identification. Mycobacterial species identification by the MMSA is based on micro-heterogeneities in nucleotide sequences of the 16S rRNA gene These micro-heterogeneities can be evaluated based on the mobility of heteroduplexes formed between 1,027 bp DNA fragments of the 16S rRNA mycobacterial gene from a test sample and from reference mycobacteria.

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Conclusion