Abstract
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.
TEXT
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-AGAAGGCCGTCGAGAAGGTCA-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.
FIG 1.
Primer and probe target sites. Primer binding sites for previously described NTM detection assay primers are also shown, as follows: #1, Telenti et al. (30); #2, Kim et al. (31).
In silico analysis indicated complete homology to the targeted hsp65 gene region for 77 Mycobacterium species. Fourteen species had ≤2 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 nonmycobacterial species had >3 nucleotide mismatches to the primer sequences, requiring an annealing temperature of <55.5°C (see Fig. S2 and Table S3 in the supplemental material).
The assay's performance was assessed using DNA extracts from 15 NTM strains and negative controls that included closely related nonmycobacterial species, common respiratory pathogens, nine M. tuberculosis strains, Mycobacterium bovis, Escherichia coli, and human DNA (Table 1). The assay's sensitivity was assessed using a dilution series of purified Mycobacterium abscessus DNA (selected based both on its clinical importance and its position within NTM phylogeny). The correlation between template concentration and cycle threshold (CT) values was linear between 3.34 × 103 and 2.65 × 108 CFU/ml equivalents (slope, −3.31; R2 = 0.99), with a reaction efficiency of 100%. Analysis using Mycobacterium intracellulare DNA, a species with a single-base primer mismatch, resulted in a linear range of 6.26 × 103 to 4.39 × 108 CFU/ml equivalents (slope, −3.403; R2 = 0.99), with a reaction efficiency of 97%.
TABLE 1.
Amplification data for reference and control strains
| Species (strain) | Sourcec | CT value | CFU/ml equivalent |
|---|---|---|---|
| Mycobacterial speciesa | |||
| M. abscessus | ATCC 19977 | 16.9 | 2.65 × 108 |
| M. avium | Clinical strain | 20.1 | 7.56 × 107 |
| M. chelonae | ATCC 35752 | 22.6 | 1.26 × 107 |
| M. flavescens | Collection strain | 28.3 | 2.42 × 105 |
| M. fortuitum | ATCC 9820 | 18.6 | 2.08 × 108 |
| M. goodii | Clinical strain | 21.3 | 3.21 × 107 |
| M. gordonae | Clinical strain | 23 | 9.93 × 106 |
| M. interjectum | Clinical strain | 21.2 | 3.34 × 107 |
| M. intracellulare | Clinical strain | 23 | 9.99 × 106 |
| M. kansasii | Clinical strain | 24.9 | 2.68 × 106 |
| M. lentiflavum | Clinical strain | 21 | 3.81 × 107 |
| M. marinum | Collection strain | 23.3 | 7.72 × 106 |
| M. simiae | Clinical strain | 19 | 1.62 × 108 |
| M. smegmatis | Clinical strain | 20.8 | 4.39 × 107 |
| M. bovis (BCG) | Collection strain | NDd | ND |
| M. tuberculosis (H37Rv) | Clinical strain | ND | ND |
| M. tuberculosis (Uganda 1) | Clinical strain | ND | ND |
| M. tuberculosis (Orygis) | Clinical strain | ND | ND |
| M. tuberculosis (MDR) | Clinical strain | ND | ND |
| M. tuberculosis (LAM/Uganda 1) | Clinical strain | ND | ND |
| M. tuberculosis (EAI) | Clinical strain | ND | ND |
| M. tuberculosis (EAI 1) | Clinical strain | ND | ND |
| M. tuberculosis (BJ Delhi CAS) | Clinical strain | ND | ND |
| M. tuberculosis (BJ Delhi CAS 1) | Clinical strain | ND | ND |
| Nonmycobacterial speciesb | |||
| Rhodococcus equi | Collection strain | ND | ND |
| Nocardia farcinica | Collection strain | ND | ND |
| Corynebacterium glucuronolyticum | Collection strain | ND | ND |
| Staphylococcus aureus | Clinical strain | ND | ND |
| Pseudomonas aeruginosa | Clinical strain | ND | ND |
| Haemophilus influenzae | Clinical strain | ND | ND |
| Escherichia coli | Clinical strain | ND | ND |
| Streptococcus pneumoniae | Clinical strain | ND | ND |
| Human | Placental DNA | ND | ND |
South Australian pathology collection.
Flinders Medical Centre pathology laboratory.
ATCC, American Type Culture Collection.
ND, not detected.
The potential for carryover of clinical sample components to influence assay performance was assessed in three ways. First, the amplification efficiency and dynamic range of M. abscessus DNA were determined following the addition of DNA extracts from culture- and qPCR-negative bronchoalveolar lavage (BAL) and sputum samples. Second, the assay's performance was assessed following the addition of purified human DNA at a concentration that substantially exceeded the levels in respiratory clinical samples. Third, the impact of the addition of horse blood prior to DNA extraction on M. abscessus DNA amplification efficiency was assessed, using a dilution series starting at 50% (vol/vol). In each case, no significant change in assay performance was observed (Mann Whitney test, P > 0.3) (see Fig. S3 to S5 in the supplemental material).
Assay validation was performed using 42 respiratory samples from patients suspected of respiratory NTM infection, including 30 BAL samples and 12 sputum samples (of which 8 were NTM positive according to standard diagnostic testing; see Table S4 in the supplemental material). Positive results from NTM culture were confirmed by qPCR, and species identity was confirmed by DNA sequencing. However, in three cases, samples were NTM culture negative but qPCR positive. Mycobacterium avium was detected in the BAL sample at a concentration of 8.7 × 104 CFU/ml equivalents, while Mycobacterium flavescens and M. avium were detected in the two sputum samples at 2.1 × 104 and 6.4 × 103 CFU/ml equivalents, respectively (Table 2; see also Table S5).
TABLE 2.
NTM-positive respiratory samples as determined by qPCR, and corresponding diagnostic culture results
| Sample type | CT value | CFU/ml equivalent | Identification obtained with: |
|
|---|---|---|---|---|
| qPCR/sequencing | Diagnostic microbiology | |||
| BAL fluid | 30.5 | 1.36 × 105 | M. avium | M. avium |
| BAL fluid | 30.9 | 1.02 × 105 | M. avium | M. avium |
| BAL fluid | 32.1 | 4.58 × 104 | M. intracellulare | M. intracellulare |
| BAL fluid | 35.4 | 1.13 × 104 | M. avium | M. avium |
| BAL fluid | 34 | 8.70 × 104 | M. avium | —a |
| BAL fluid | 32.7 | 3.02 × 104 | M. avium | M. avium |
| Sputum | 31.1 | 3.22 × 104 | M. massiliense | M. massiliense |
| Sputum | 31.7 | 2.08 × 104 | M. flavescens | —a,b |
| Sputum | 31.5 | 2.44 × 104 | M. abscessus | M. abscessus |
| Sputum | 33.4 | 6.43 × 103 | M. avium | —a |
| Sputum | 35.5 | 1.49 × 103 | M. avium | M. avium |
—, not detected.
Sample was recorded as “insufficient for adequate assessment” for standard diagnostic analysis.
Negative culture results in patients with suspected NTM infection are not uncommon, with NTM isolated from subsequent samples in some instances (26). A number of factors could contribute to discrepancies between culture-dependent and molecular analysis. For example, culture overgrowth by nonmycobacterial species can substantially reduce NTM detection, while sample decontamination techniques used to prevent this can reduce mycobacterial viability (12). In addition, NTM recovery can be reduced in patients receiving commonly used antibiotics, such as macrolides and quinolones (12).
In the case of the culture-negative, qPCR-positive BAL sample, high levels of Haemophilus influenzae growth were reported. While this species is fastidious, the finding suggests the potential presence of other nonmycobacterial species that might have contributed to the failure to isolate NTM through bacterial overgrowth. In the case of the culture-negative sputum sample in which M. flavescens was detected by PCR, M. abscessus had been isolated from this patient on a previous occasion (although definitive typing had not been performed), and the patient had received apparently successful eradication therapy. While the basis for discordance remains unclear, it is important to highlight that, as with all PCR-based assays, a positive result does not rely on the presence of viable bacterial cells (27, 28). DNA in nonviable bacteria or present in the extracellular environment (as might occur following successful antibiotic therapy) can also act as a PCR template (29), a factor that must be taken into account when interpreting disparities between culture and PCR-based results. In the case of the patient in whose sample M. avium was detected by qPCR alone, the corresponding sample was recorded as being “insufficient for adequate assessment” by culture (an outcome that is sometimes interpreted wrongly at the clinical level as a culture-negative result). However, sputum samples collected both prior to and following this sample were also found to be culture negative, suggesting a genuine discrepancy between culture and qPCR results.
Our study was unable to assess the assay's ability to detect a number of rare or recently described NTM species for which high-quality DNA sequence data are not yet available. It was further limited by a requirement for DNA sequencing to identify the source of positive PCR results, a technology that is not available in all laboratories. However, the single-reaction qPCR assay described offers substantial advantages over other molecular assays in terms of time and cost. Importantly, the assay does not amplify DNA from M. tuberculosis, a range of closely related nonmycobacterial species, or common airway bacteria and is unaffected by the presence of high concentrations of human DNA or blood derivatives. Our assay provides a specific, sensitive, and robust means to rapidly screen respiratory clinical samples for the presence of NTM and represents an important adjunct to conventional diagnostic approaches.
Supplementary Material
ACKNOWLEDGMENTS
We are grateful to David Gordon, Microbiology and Infectious Diseases, Flinders Medical Centre, who provided clinical isolates and bacterial type strains.
The authors declare no conflict of interest.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Funding Statement
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit-sectors.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.01410-16.
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