Abstract
Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35°C (63.27 to 65.42°C); M. kansasii, 59.20°C (58.07 to 60.33°C); M. avium, 57.82°C (57.05 to 58.60°C); M. intracellulare, 54.46°C (53.69 to 55.23°C); M. marinum, 58.91°C (58.28 to 59.55°C); rapidly growing mycobacteria, 53.09°C (50.97 to 55.20°C) or 43.19°C (42.19 to 44.49°C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.
Mycobacteria cause a variety of illnesses, including tuberculosis, which has profound individual and public health implications (28). The identification of the mycobacteria responsible for disease has important ramifications for infection control and the selection of antimicrobial therapy. Identification, however, is hampered by the slow growth of most mycobacteria, which may take as long as 2 months, even if the organism is present in pure culture (11).
Several rapid diagnostic modalities for the identification of mycobacteria have been developed. The first major advance in the rapid identification of mycobacteria was the commercial availability of genetic probes (Accuprobe; Gen-Probe Incorporated, San Diego, Calif.) for four commonly encountered and/or clinically significant mycobacteria: Mycobacterium tuberculosis complex, M. avium/M. intracellulare complex, M. kansasii, and M. gordonae. Although these assays significantly diminish the time to identification, the recommended use of this product requires growth of an organism in culture. In addition, the use of more than one probe is often necessary, if the mycobacterium is recovered from broth culture and colony morphology information is not available to aid in probe selection.
The next major advance was the detection of M. tuberculosis by nucleic acid amplification tests (NAT). These technologies are attractive because of the possibility of directly detecting M. tuberculosis in clinical respiratory specimens. PCR-based assays for the detection of M. tuberculosis approach the sensitivity and specificity of conventional culture but have the added advantage of being rapid (5). Unfortunately, because of the possibility of false-negative reactions among other reasons, the use of NAT for M. tuberculosis has come under scrutiny and their usefulness has been questioned (4). In addition, the Centers for Disease Control and Prevention (CDC) have suggested an algorithm for the use of these tests that includes repeat testing, which is costly and unattractive to the laboratory (4).
More recently, the INNO-LiPA line probe assay (Innogenetics N.V., Ghent, Belgium) for detection of mycobacteria and their identification to the species level has been developed. This assay utilizes reverse hybridization of the PCR product onto a paper strip that contains probes for a variety of mycobacteria. Although this test is a technically excellent method of detecting and differentiating the majority of clinically important mycobacteria, it is costly and is not readily available in North America (18, 27; M. J. Tuohy, M. Sholtis, G. W. Procop, and G. S. Hall, presented at the 102nd General Meeting of the American Society for Microbiology, 2002).
Recent innovations in real-time PCR have simplified and expedited PCR considerably. The LightCycler system (Roche Diagnostics, Indianapolis, Ind.) combines real-time PCR with product detection using fluorogenic hybridization probes that employ the principle of fluorescence resonance energy transfer to achieve rapid PCR results (30). This system has been used successfully for the rapid detection or identification of a variety of microorganisms (8-10, 15, 16, 19-21, 23). Real-time PCR with fluorogenic probes has also been shown to be useful for the detection of mycobacteria, including M. tuberculosis (12, 13, 17). An added feature of the LightCycler real-time PCR system is the postamplification melting point analysis. The ability of the LightCycler system to perform melting curve analyses also allows for differentiation of closely related organisms (24, 29; L. Doyle, C. Starkey, B. Yen-Lieberman, and G. W. Procop, presented at the 102nd General Meeting of the American Society for Microbiology, 2002). This feature has been used to distinguish between closely related organisms, such as the polyomaviruses JC virus and BK virus, Bartonella species, and Bordetella pertussis and Bordetella parapertussis (24, 29; Doyle et al., presented at 102nd Gen. Meet. Am. Soc. Microbiol.). Therefore, we designed and tested a LightCycler real-time PCR assay that was expected to detect all mycobacteria and also to distinguish M. tuberculosis from nontuberculous mycobacteria (NTM) by melting curve analysis.
MATERIALS AND METHODS
Characterization of mycobacterial isolates.
Approval for the study was obtained from the Cleveland Clinic Foundation Institutional Review Board. The mycobacterial isolates tested were those that had been obtained from clinical specimens at the Cleveland Clinic Foundation over the preceding 5 years, as well as type strains. Eighteen reference strains of mycobacteria and 168 clinical isolates were tested (Table 1). The isolates were identified by their growth characteristics and biochemical reactions and by the Accuprobe assays (for species for which probes were available) according to routine laboratory protocol. The identity of the M. kansasii isolates was also confirmed by the INNO-LiPA MYCOBACTERIA line probe assay (Innogenetics N.V.). Additionally, all isolates were also tested by pyrosequencing of a portion of hypervariable region A of the mycobacterial 16S rRNA gene and comparison of the sequences obtained with those in the GenBank database (National Center for Biotechnology Information, National Institutes of Health) (22, 26). This procedure served as an independent check on the identity of the mycobacteria in addition to routine laboratory testing and was used to resolve discrepancies. Lysates of cultured isolates were obtained as follows: by using a 10-μl disposable plastic loop, approximately 1 loopful of bacteria growing in Lowenstein-Jensen or 7H11 medium was suspended in 200 μl of a lysis buffer (21) containing 1% Triton X-100, 0.5% Tween 20, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA and was incubated in a heating block at 100°C for 30 min. The resulting lysate was diluted 10,000 times in the same buffer and used for PCR.
TABLE 1.
Mycobacterial strains tested
| Species (n) | Strain(s)a | Characterization method(s)b |
|---|---|---|
| Reference strains | ||
| M. tuberculosis (8) | ATCC 25177, ATCC 25618, ATCC 27294, ATCC 35822, ATCC 35823, ATCC 35828, ATCC 35837, ATCC 35838 | Reference strains |
| M. bovis (1) | ATCC 35734 | Reference strain |
| M. avium-intracellulare (2) | ATCC 25291, ATCC 700898 | Reference strains |
| M. scrofulaceum (1) | ATCC 19981 | Reference strain |
| M. kansasii (2) | ATCC 12478, TMC 1201 | Reference strains |
| M. marinum (1) | TMC 927 | Reference strain |
| M. gordonae (1) | ATCC 14470 | Reference strain |
| M. fortuitum (1) | TMC 6841 | Reference strain |
| M. chelonae (1) | TMC 1542 | Reference strain |
| Clinical isolates | ||
| M. tuberculosis (28) | Clinical isolates | GC, BR, AP, PS |
| M. kansasii (24) | Clinical isolates | GC, BR, AP, LiPA, PS |
| M. avium (17) | Clinical isolates | GC, BR, AP, PS |
| M. intracellulare (8) | Clinical isolates | GC, BR, AP, PS |
| M. marinum (22) | Clinical isolates | GC, BR, PS |
| M. gordonae (17) | Clinical isolates | GC, BR, AP |
| M. fortuitum (19) | Clinical isolates | GC, BR, PS |
| M. chelonae/abscessusc (23) | Clinical isolates | GC, BR, PS |
| Rapid growers NFId (10) | Clinical isolates | GC, BR, PS |
TMC, Trudeau Mycobacterial Cultures.
GC, growth characteristics; BR, biochemical reactions; AP, Accuprobe; LiPA, line probe assay; PS, pyrosequencing.
Categorized here as identified by the laboratory, whose results did not always agree with pyrosequencing results.
NFI, not further identified.
Real-time PCR.
The oligonucleotide primers and hybridization probes for PCR targeted for amplification a portion of the mycobacterial 16S rRNA gene (GenBank accession no. X52917) that included hypervariable region A (26). One author (G.W.P.) had previous experience using this region for the sequence-based identification of mycobacteria. The primers and probes were obtained from BioChem (Salt Lake City, Utah). The sequence of the forward primer (positions 29 to 45) was 5′-ACGGAAAGGTCTCTTCG-3′, whereas the sequence of the reverse primer (positions 242 to 227) was 5′-GTCGTCGCCTTGGTAG-3′. The fluorogenic hybridization probes were chosen to specifically hybridize with hypervariable region A. The probes used had 100% sequence homology with M. tuberculosis complex but had ≥1-bp mismatches with the other clinically relevant mycobacteria. The first hybridization probe (positions 151 to 171) was 5′-CATGTCTTGTGGTGGAAAGCG-fluorescein isothiocyanate-3′, whereas the second hybridization probe (positions 174 to 194) was 5′-LC Red 640-TTAGCGGTGTGGGATGAGCCC-phosphate-3′. PCR was performed using the LightCycler system (30). The reaction volume of 20 μl was a mixture of 5 μl of extracted target DNA and 15 μl of hybridization probe master mix, with the primers, probes, and magnesium chloride present in concentrations of 0.25 μM, 0.4 μM, and 3.0 mM, respectively. A solution of Tris-EDTA was used as the negative control. An extract of M. tuberculosis ATCC 27294 (American Type Culture Collection, Manassas, Va.) was used as the positive control.
The reaction protocol was as follows: an initial FastStart Taq DNA polymerase activation phase at 95°C for 10 min; a 45-cycle amplification phase consisting of a 95°C denaturation segment for 10 s, a 50°C annealing segment for 10 s, and a 72°C extension segment for 20 s; a melting phase from 45 to 75°C with a temperature transition rate of 0.1°C/s; and a rapid cooling phase. The quantity of amplified product was monitored in the F2 mode by detection of energy emitted at 640 nm.
Melting curve analysis.
The temperature at which the hybridization probes dissociated from their target sites was determined by melting curve analysis. This allowed for differentiation between species based on differences in the avidity of the hybridization probes for the complementary sequences in the amplified DNA. The melting curve for each specimen was analyzed manually to determine the melting temperature (Tm). Tm is the peak of the curve of the derivative of fluorescence with respect to temperature (−dF/dT). By using the manual Tm function of the LightCycler software, Tm was defined as that temperature at which the cursor just covered the highest point on the melting curve (the −dF/dT curve). If the highest point was a plateau, the midpoint of the plateau was taken to be the Tm. After the Tm for each isolate was determined in this manner, the mean, standard deviation (SD), and confidence intervals for the Tm of each species of mycobacterium were calculated.
Clinical specimens.
Nucleic acid extracts from 50 clinical specimens that had been proven by culture to contain M. tuberculosis were collected and stored frozen. The sites and acid-fast bacillus (AFB) smear results of the clinical specimens are given in Table 3. Extracts from the clinical specimens were made by using the COBAS Amplicor M. tuberculosis assay (Roche Diagnostics) lysis protocol according to the manufacturer's instructions. The extracts were tested using the LightCycler assay and the COBAS Amplicor M. tuberculosis PCR assay (Roche Diagnostics) by one investigator (U.R.). The assay protocol used differed slightly from that described above and employed an asymmetric PCR. This modification used 0.25 μM forward primer, 0.50 μM reverse primer, 0.2 μM each hybridization probe, and 4 mM MgCl2. In addition, three clinical specimens that were AFB smear positive but proven by culture to contain NTM were tested by the LightCycler assay at the Cleveland Clinic Foundation, using the conditions described above for the testing of isolates.
TABLE 3.
Results of AFB smears, LightCycler assay, and COBAS Amplicor assay for 50 clinical specimens proven by culture to contain M. tuberculosis
| Specimen type (no. tested) | LightCycler assay result | COBAS Amplicor M. tuberculosis PCR result |
|---|---|---|
| Smear-positive specimens (45) | ||
| Sputum (33), respiratory secretion (4), bronchoalveolar lavage (3) | All positive | All positive |
| Urine (3) | 2 positive, 1 negativea | 2 positive, 1 negativea |
| Gastric aspirate (1) | Positive | Positive |
| Biopsy (1) | Negativea | Negativea |
| Smear-negative specimens: sputum (2), urine (1), gastric aspirate (1), stool (1) | All positive | All positive |
The initial (i.e., fresh; never frozen) extracts from both the urine specimen and the biopsy specimen that were PCR negative in this parallel series were positive by the COBAS Amplicor M. tuberculosis assay. In this study, however, the extracts from all the specimens were thawed and retested by both the COBAS Amplicor M. tuberculosis and LightCycler assays. One urine specimen and one biopsy specimen were PCR negative by both methods. Inhibition was not detected by the COBAS internal control; therefore, we suspect that degradation may have been responsible for these two false-negative results.
RESULTS
In testing of cultured isolates, the PCR assay as described above was 100% sensitive for the detection of all mycobacterial species tested except M. gordonae, for which it detected 10 of 18 isolates (sensitivity, 55.56%). When detected, however, all the M. gordonae isolates had the same unique melting temperature. PCR of these isolates using SYBR Green I dye instead of the hybridization probes demonstrated amplification of the nucleic acid from these organisms in every instance, indicating that the nucleic acids were not detected by the hybridization probes. In the quantitation analysis, the mycobacteria were consistently detected between amplification cycles 25 and 30.
The Tms for isolates of any particular species or complex remained within a narrow range. Characteristic melting curves for M. tuberculosis, M. kansasii, and M. intracellulare are shown in Fig. 1. The Tm of M. tuberculosis was 64.35 ± 0.83°C (mean ± 2 SDs), which was unequivocally higher than that of any other mycobacterial species. The most commonly encountered mycobacterial species tested all had separate and distinct Tms, except for M. kansasii and M. marinum, which had overlapping Tms (Table 2). The assay differentiated M. avium from M. intracellulare. The one isolate of M. bovis tested had the same Tm as M. tuberculosis, and the one isolate of M. scrofulaceum tested had the same Tm as M. avium.
FIG. 1.
Representative graph showing the differential melting curves for M. tuberculosis (TB), M. kansasii (MK), and M. intracellulare (MI).
TABLE 2.
Tms for different mycobacteria
| Organism (no. of isolates) | No. of isolates yielding a melting curve | Mean Tm ± 2 SDs (°C) | 99.9% CIa |
|---|---|---|---|
| M. tuberculosisb (37) | 37 | 64.35 ± 0.83 | 63.27-65.42 |
| M. kansasii (26) | 26 | 59.20 ± 0.87 | 58.07-60.33 |
| M. aviumc (20) | 20 | 57.82 ± 0.60 | 57.05-58.60 |
| M. intracellulare (8) | 8 | 54.46 ± 0.60 | 53.69-55.23 |
| M. gordonae (18) | 10 | 49.52 ± 0.50 | 48.87-50.17 |
| M. marinum (23) | 23 | 58.91 ± 0.49 | 58.28-59.55 |
| Rapid growersd (54) | 54 | ||
| Group I (36) | 36 | 53.09 ± 1.64 | 50.97-55.20 |
| Group II (19) | 19 | 43.19 ± 0.78 | 42.19-44.19 |
CI, confidence interval.
Includes one M. bovis isolate.
Includes one M. scrofulaceum isolate.
Includes 20 M. fortuitum isolates, 24 M. chelonae/abscessus isolates, and 10 isolates not further identified.
The reference strain and all 20 isolates identified in the laboratory as M. fortuitum melted at a Tm of 53.09 ± 1.64°C (mean ± 2 SDs) (here designated group I). Seventeen of the 24 isolates identified as M. abscessus, M. chelonae, or M. chelonae/abscessus complex, as well as the reference strain of M. chelonae, melted at a Tm of 43.19 ± 0.78°C (mean ± 2 SDs) (here designated group II). The remaining seven isolates characterized in the laboratory as belonging to one of these species melted at the Tm characteristic of the group I rapid growers. Pyrosequencing analysis of four of these isolates disclosed the sequences of M. sphagni, and the remaining three had sequences of M. fortuitum. Nine of the 10 rapid growers that had not been further identified in the laboratory had a Tm characteristic of group I, while the remaining one had a Tm characteristic of group II.
When the individual data points for the Tms were examined, there was no overlap among the different species of mycobacteria that commonly cause systemic illness. Similarly, there was no Tm overlap among the different species of mycobacteria that commonly cause cutaneous illness. No species in either group had a Tm that overlapped that of M. gordonae (Fig. 2).
FIG. 2.
Individual data points for the Tms for common mycobacteria that cause systemic illness (top row) or cutaneous illness (bottom row) and for a common contaminant (middle row). TB, M. tuberculosis; MK, M. kansasii; MA, M. avium; MI, M. intracellulare; MG, M. gordonae; MM, M. marinum; MF, M. fortuitum; Mabs/chel, M. abscessus/chelonae.
There was 100% correlation in the parallel study between the COBAS Amplicor M. tuberculosis assay (Roche Diagnostics) and the LightCycler assay. Both these assays detected 48 of the 50 specimens that contained M. tuberculosis (Table 3), yielding a sensitivity of 96%. The melting points for isolates identified by the LightCycler assay as M. tuberculosis were all within the range given for M. tuberculosis in Table 2. Similarly, LightCycler PCR products from the three clinical specimens shown by culture to contain NTM all had melting points indicative of non-M. tuberculosis mycobacteria. Culture with Accuprobe (Gen-Probe) confirmation demonstrated that two of the specimens contained a member of the M. avium/M. intracellulare complex, whereas the third contained M. kansasii. Melting curve analysis of the LightCycler PCR results for these specimens demonstrated melting points that were within the defined ranges (Table 2) of M. avium, M. intracellulare, and M. kansasii.
DISCUSSION
M. tuberculosis infects one-third of the world's population (6), and is the leading cause of death due to any infectious agent worldwide (31). Over the past few decades, the incidence of infections caused by NTM has also increased, and in the United States, isolates of NTM are now more common than those of M. tuberculosis (1). The CDC guidelines call for strict isolation of patients suspected of having tuberculosis in order to prevent spread to health care workers and other patients (3). Isolation precautions are not deemed necessary for NTM infections. Effective adherence to these guidelines requires a microbiologic diagnosis of the species of mycobacterium causing the clinical illness in a given patient. Furthermore, treatment of tuberculosis is different from that of NTM infections (1, 25). Early identification of the species of mycobacterium causing illness in a patient would have significant clinical impact.
Real-time PCR has been used to differentiate members of the M. tuberculosis complex from other mycobacteria (12, 13, 17). These assays, however, either detect only M. tuberculosis or require additional hybridization probe sets to detect other mycobacteria (12, 13, 17). Taking advantage of differential melting characteristics measured with a strategically designed probe set, our assay identified M. tuberculosis and distinguished it from NTM with a single probe set. This represents an advance in our ability to rapidly identify M. tuberculosis isolates and differentiate these from NTM, without the added costs of additional hybridization probes. In addition, the presence of a reaction indicative of an NTM concurrent with a negative reaction for M. tuberculosis helps assure the user that the negative result for M. tuberculosis is a true negative, rather than a false negative, which has been one of the drawbacks of many of the nucleic acid amplification methods for M. tuberculosis developed to date. In this validation study, this assay accurately detected all clinically significant organisms and consistently distinguished M. tuberculosis from NTM. It had a lower sensitivity for the detection of M. gordonae, but this lack of sensitivity is of limited clinical consequence, since this organism is usually a laboratory contaminant (2, 7, 14). The only discordant results for organisms initially characterized by growth characteristics and biochemical reactions were seen with the M. abscessus/M. chelonae complex. The concordance of the real-time PCR results with those of sequencing of these isolates, however, confirmed that the initial identification of these isolates by the laboratory by traditional methods was erroneous.
Given the limited range of temperatures for which hybridization probe disassociation or melting may occur and the wide variety of NTM, overlapping of the melting curves among the NTM was expected. This assay was designed to answer the most important clinical question: is the mycobacterium present M. tuberculosis or not? This objective was achieved. Although the melting curves in the region where the NTM occur may overlap for some of these organisms, they do not for others. Therefore, information suggestive of the type of NTM may be gleaned from the melting curve analysis, particularly if information regarding the clinical presentation of disease is known. For example, although the hybridization probes give essentially the same Tm for M. kansasii and M. marinum, these organisms cause very different types of diseases; clinical information would help in the differentiation of these organisms. In addition, although the Tms of the common rapid growers M. fortuitum and M. chelonae/M. abscessus complex are distinct from those of the systemic pathogens, as well as from one another, M. chelonae cannot be distinguished from M. abscessus by this assay.
The role of this test in the clinical microbiology laboratory may be to identify M. tuberculosis and distinguish it from NTM as soon as the culture demonstrates growth of an AFB. Rapid and accurate identification of M. tuberculosis, regardless of the method, facilitates decisions about isolation and the institution or cessation of therapy. This test would also give information suggestive of the type of NTM present, which conceivably could aid in subsequent test selection. Such information would hopefully decrease diagnostic delays by reducing unnecessary testing. If an isolate with a Tm corresponding to that of M. gordonae is detected, one could conclude that the organism is likely a contaminant. Finally, the possibility of sequencing the amplicon to further characterize the NTM remains an option and has been done (data not shown), as the amplified region has been shown to be useful for sequence-based identification of mycobacteria (26).
The wait for culture positivity is often the most time-consuming portion of the culture and identification of mycobacteria. Although molecular methods for the identification of cultured isolates are useful, the optimal use of these technologies would be direct identification of mycobacteria from clinical specimens. The Amplified Mycobacterium tuberculosis Direct Test (MTD) (Gen-Probe) and the COBAS Amplicor M. tuberculosis assay (Roche Diagnostics) are two NAT that have been approved by the U.S. Food and Drug Administration for the direct detection of M. tuberculosis in respiratory specimens (4). A real-time PCR using the LightCycler system has been shown to be comparable to the COBAS Amplicor M. tuberculosis assay by using clinical specimens (17). The LightCycler real-time PCR assay described here was also shown to be comparable to the COBAS Amplicor M. tuberculosis assay in a parallel study. In addition, our LightCycler assay also appropriately characterized three AFB-positive clinical specimens that contained M. avium, M. intracellulare, or M. kansasii as containing NTM rather than M. tuberculosis.
These data suggest that the LightCycler assay described here would be useful for the rapid differentiation of M. tuberculosis from NTM by using cultured isolates and that it may be useful for the direct detection and differentiation of M. tuberculosis and NTM in clinical specimens.
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