Culture-Independent Detection of Nontuberculous Mycobacteria in Clinical Respiratory Samples

Abstract

f Culture-based detection of nontuberculous Mycobacteria (NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust. While rates of infection caused by members of the Mycobacterium tuberculosis complex continue to fall in developed countries (1, 2), disease caused by nontuberculous mycobacteria (NTM) is an area of growing concern (3-6). Pulmonary infection represents more than 90% of NTM cases (7) and has been described in a range of clinical contexts (8-11). Appropriate management of suspected pulmonary NTM infection requires the timely detection and identification of the etiological agent. The current "gold standard" for detection of NTM in respiratory samples relies on protracted in vitro culture, potentially delaying targeted therapy. It also requires samples to undergo decontamination prior to culture to lower levels of commensal microbiota (12) and is associated with variable sensitivity (13). The ability to perform a rapid quantitative screen for the presence of any NTM species would provide an important early indication of mycobacterial involvement and would be informative in cases where samples are culture negative, despite clinical or radiological signs.To prevent false-positive results arising from the detection of closely related species (14, 15), existing molecular assays target narrow phylogenetic groups or specific pathogens (16-21), require prior mycobacterial isolation by culture (22-24), or are unable to provide accurate species-level NTM identification (25). We describe a TaqMan quantitative PCR (qPCR) assay, based on the single-copy hsp65 gene, for the direct detection of NTM species in respiratory clinical samples.The assay design was based on the full-length hsp65 gene sequences that are available for 116 of the 174 currently described NTM species, including all 56 NTM species reported in respiratory disease (see Fig. S1 in the supplemental material). The PCR primers (forward, HSP171 [5=-CGCCAAGGAGATCGAGCTGG-3=], and reverse, HSP563 [5=-GGACAAGGTCGGCAACGAGGG-3=]) generate a 348-bp hsp65 amplicon and are used in conjunction with a TaqMan probe (5=-FAM-AGAAGGCCGTCGAGAAG GTCA-BHQ-3= [FAM, 6-carboxyfluorescein; BHQ, black hole quencher]) at an annealing temperature of 60°C (Fig. 1). A detailed description of assay development and methods is provided in the supplemental material.In silico analysis indicated complete homology to the targeted hsp65 gene region for 77 Mycobacterium species. Fourteen species had Յ2 nucleotide mismatches within the primer binding region, with a corresponding reduction in annealing temperature of up to 4.5°C. However, in all such cases, the corresponding primer binding region showed 100% sequence homology (see Tables S1 and S2 in the supplemental material). Twenty-one mycobacterial species (including M. tuberculosis and Mycobacterium leprae) and 40 assessed non...

Non-tuberculous mycobacterial pulmonary disease

“…Similar molecular methods to those used in species identification may be applied to DNA extracted from respiratory samples to directly detect and identify NTM without the need for culture [128][129][130][131][132][133]. However, the reported sensitivity is low, detecting NTM in only 29-76% of culture positive samples [128][129][130]132], although better detection rates are reported in more heavily smear-positive samples.…”

Approach to the diagnosis and treatment of non-tuberculous mycobacterial disease

“…However, several species may not be differentiated as there is not enough genomic divergence to allow for accurate diagnosis beyond species complex level. Similar to MALDI-TOF, test characteristics depend on the library of available sequences [19] .…”

Incorporating direct molecular diagnostics in management algorithms for nontuberculous mycobacteria: Is it high time?

“…This would subject patients to unnecessary toxic treatment and may promote resistance. However, this limitation is mitigated by pairing the molecular diagnostic test, which has high specificity [18] , to a previous culture, and confirming concordance of the results. This strategy, when applied in conjunction with the appropriate clinical context, should limit over-treatment.…”

“…Sequencing these regions allows for an additional spectrum of less frequently isolated NTMs to be identified and not included in commercial kits. The benefit of using these genes for detecting NTMs over traditional diagnostic culture-based methods is highlighted in a study performed on respiratory specimens in which quantitative PCR of the hsp65 gene identified three NTMs (two M. avium and one M. flavescens ) reported as culture-negative [18] . A recent approach employed a combination of loop-mediated isothermal amplification and lateral flow biosensors, specifically targeting the rpoB gene of M. kansassi .…”