Research Article: Development of a loop-mediated isothermal amplification coupled lateral flow dipstick targeting erm(41) for detection of Mycobacterium abscessus and Mycobacterium massiliense

Date Published: January 23, 2019

Publisher: Springer Berlin Heidelberg

Author(s): Dongxin Liu, Wencong He, Mingxia Jiang, Bing Zhao, Xichao Ou, Chunfa Liu, Hui Xia, Yang Zhou, Shengfen Wang, Yuanyuan Song, Yang Zheng, Qian Chen, Jiale Fan, Guangxue He, Yanlin Zhao.

http://doi.org/10.1186/s13568-019-0734-4

Abstract

Mycobacterium abscessus (M. abscessus) and Mycobacterium massiliense (M. massiliense) are major pathogens that cause post-surgical wound infection and chronic pulmonary disease. Although they are closely related subspecies of M. abscessus complex, their infections are associated with different drug-resistance and cure rate. In the present study, a loop-mediated isothermal amplification (LAMP) coupled with lateral flow dipstick (LFD) method was developed to simultaneous detect M. abscessus and M. massiliense, via specific erm(41) gene. The amplification was carried out at 65 °C for only 60 min, and the results could be visualized on a lateral flow strip. Positive results only occurred in M. abscessus and M. massiliense, no cross-reaction with other mycobacterial species was observed. Therefore, the cost-effective MABC (M. abscessus complex)–LAMP–LFD method developed here was able to correct the diagnose of M. abscessus and M. massiliense infection in a short time. Thus, this method could be used to guide clinicians in treatment of M. abscessus group infections.

Partial Text

Rapidly growing mycobacteria (RGM) are ubiquitous environmental microorganisms (Brown-Elliott and Wallace 2002) and the prevalence of pulmonary infection due to RGM is increasing worldwide. Within the RGM, the Mycobacterium abscessus complex is a prominent cause of lung disease in patients with chronic pulmonary disease and cystic fibrosis (Chan et al. 2010; Zelazny et al. 2009). Based on divergence of rpoB sequences, M. abscessus complex is thought to be comprised of three subspecies—Mycobacterium abscessus, Mycobacterium massiliense and Mycobacterium bolletii (Adekambi et al. 2006; Howard 2013). Of the three, M. bolletii is rarely isolated, while M. massiliense and M. abscessus are major pathogens (Benwill and Wallace 2014; Brown-Elliott et al. 2015).

Mycobacterium abscessus and M. massiliense are two important pathogens in the M. abscessus complex that cause human infections. The discriminatory detection of these species are associated with different drug-resistance and cure rate (Lee et al. 2015; Zelazny et al. 2009). The conventional methods for species identification based on phenotypic features cannot accurately delineate these two species, due to the close relationship between M. abscessus and M. massiliense, which makes differentiation of M. abscessus and M. massiliense challenging in most clinical microbiology laboratories. Recently, targeted gene (hsp65, rpoB, ITS and secA1) sequencing combined with phylogenomic analysis, erm(41) PCR-based assays, or multi-locus sequence analysis targeting 8 housekeeping genes have all been proposed as methods to identify them accurately (Macheras et al. 2011; Sassi et al. 2013). However, those methods require bacterial culture, PCR amplification, and/or gene sequencing, all of which are relatively time-consuming, costly, and may not be feasible in all clinical microbiology laboratories. In the current study, we described a simple, robust, accurate, rapid and cost-effective LAMP-based method to detect and distinguish M. abscessus and M. massiliense from clinical specimens directly. This method has several advantages over previously mentioned ones in terms of low equipment requirement (merely a heat block or water bath) and visualized results on a lateral flow strip, which increase the feasibility in resource-limited settings.

 

Source:

http://doi.org/10.1186/s13568-019-0734-4

 

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