Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

Abstract

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culturebased diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera. Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

“…Real-time PCR detected M. chimaera DNA up to a concentration of 10 pg/μL; this result is comparable with the sensitivity (two genomic copies) reported by Zozaya-Valdés et al [ 29 ]. A DNA search using PCR will detect dead as well as live bacteria, however.…”

“…DNA extraction was performed by heat treatment. The real-time PCR protocol was adapted from Zozaya-Valdés et al [ 29 ] to detect a 79 bp fragment of the SR1 region, identified as highly specific for M. chimaera . The SR1 region target for the TaqMan assay was selected by aligning 96 mycobacterial genome sequences, including 63 M. chimaera sequences from different countries, and it was 100% conserved among all M. chimaera templates.…”

Comparison of methods available for identification of Mycobacterium chimaera

“…Some isolates will remain unclassifiable except using whole-genome sequencing patterns, which need to be determined. A new assay based on a simplex PCR has been recently described and could also be used in such a situation to rapidly identify M. chimaera [29]. The strengths of our study were to provide clinical validation of commercially available tools for M. chimaera identification and differentiation from M. intracellulare.…”

Straight from the Horse’s “Mouth”: Genomic Epidemiology of an Icelandic Equine Epidemic

“…Bacterial factors carried by mobile genetic elements may drive a common fitness advantage across different bacterial genetic backbones, irrespective of sequence type, as has been reported in recent Streptococcus pyogenes outbreaks ( 15 , 19 ), and as such, comparative analyses may yield important insight into intersequence-type bacterial outbreaks. Such targeted analyses can be applied to advance diagnostics and surveillance of key clones, as has been applied to Mycobacterium chimaera ( 20 ). The authors of this research speculate that the infected equine population lacked protective immunity to the outbreak strain, and this remains an interesting hypothesis.…”