JOURNAL OF CLINICAL MICROBIOLOGY, July 1995, p. 1856–1859 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 33, No. 7
Direct Detection of Mycobacterium tuberculosis Complex in Respiratory Specimens by Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and Roche Amplicor Mycobacterium Tuberculosis Test PAULI VUORINEN,* ARI MIETTINEN, RISTO VUENTO,
AND
¨ LLSTRO ¨M OLAVI HA
Department of Clinical Microbiology, Tampere University Hospital, FIN-33521, Tampere, Finland Received 6 January 1995/Returned for modification 27 February 1995/Accepted 17 April 1995
Two hundred and fifty-six sputum, bronchoalveolar lavage, and bronchial and tracheal aspirate specimens from 243 patients were tested for the presence of Mycobacterium tuberculosis complex by auramine fluorochrome staining, rRNA target amplification (Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test [AMTD]), and PCR (Roche Amplicor Mycobacterium Tuberculosis Test [Amplicor PCR]). The results were compared with those of conventional Lo ¨wenstein-Jensen tube culture and BACTEC radiometric liquid culture. A total of 26 specimens from 18 patients were culture positive for M. tuberculosis. In addition, seven specimens were positive by staining and by culture for other Mycobacterium species but negative by nucleic acid amplification methods and were not included in the comparison. When compared with that for culture, the sensitivities of the techniques were as follows: for staining, 80.8%; for Gen-Probe AMTD, 84.6%; and for Roche Amplicor PCR, 84.6%. The specificities were 99.1, 98.7, and 99.1%, respectively. After resolution of discrepant results by review of the patients’ clinical data, 29 specimens from 21 patients were considered positive, and the overall sensitivities, specificities, and positive and negative predictive values were 89.7, 100, 100, and 98.7% for culture; 75.9, 99.5, 95.7, and 96.9% for staining; 86.2, 100, 100, and 98.2% for Gen-Probe AMTD; and 82.8, 100, 100, and 97.9% for Roche Amplicor PCR, respectively. It is concluded that both nucleic acid amplification methods are rapid, sensitive, and specific methods for the detection of M. tuberculosis in respiratory specimens. amplify a specific DNA region of the 16S rRNA gene. The purpose of the present study was to test the new nucleic acid amplification methods for the direct detection of M. tuberculosis complex in respiratory specimens and to compare these methods with standard culture and staining techniques.
The laboratory diagnosis of Mycobacterium tuberculosis is currently based on acid-fast staining and culture of decontaminated samples on solid media or radiometric liquid culture (BACTEC technique). Staining has low sensitivity but is, nevertheless, considered a rapid and specific screening test of primary samples (10). Culture on solid media has acceptable sensitivity and specificity (10) but is a very slow method, requiring up to 6 weeks to detect positive specimens. The use of BACTEC culture has considerably quickened the diagnosis of M. tuberculosis, but even this technique requires an average of 13 days’ culture to detect positive specimens (2). New diagnostic methods employing gene technology based on amplification and detection of bacterial DNA or RNA are expected to improve the speed, sensitivity, and specificity of mycobacterial detection. Several papers have described the use of PCR and of amplification of a specific part of M. tuberculosis insertion sequence IS6110 in the diagnosis of M. tuberculosis infection (1, 8, 17). However, a recent attempt at quality control of homemade PCR applications resulted in an unexpectedly high variation in sensitivity, and the reliability of PCR for the detection M. tuberculosis was questioned (13). Therefore, the need for rapid, standardized, and reliable commercial detection systems for M. tuberculosis is obvious. Two newly developed direct detection kits using amplification of genetic material are already available. The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) is based on amplification of rRNA, whereas the Roche Amplicor Mycobacterium Tuberculosis Test (Amplicor PCR) employs PCR to
MATERIALS AND METHODS Clinical specimens. A total of 256 sputum, bronchoalveolar lavage, and bronchial and tracheal aspirate specimens from 243 patients were examined at the Department of Clinical Microbiology, Tampere University Hospital, Tampere, Finland. When a specimen arrived at the laboratory, a direct smear was first prepared. All specimens were screened by using auramine fluorescent stain, and positive results were confirmed with the Ziehl-Neelsen stain. Specimens for the BACTEC culture, Gen-Probe AMTD, and Roche Amplicor PCR methods were digested and decontaminated by the N-acetyl-L-cysteine-NaOH (NALC-NaOH) method (10). An equal volume of NALC-NaOH solution (2% NaOH, 1.45% sodium citrate, 0.5% NALC) was mixed with a specimen and incubated at room temperature for 20 min. Phosphate buffer (67 mM, pH 6.8) was added up to 50 ml, and the mixture was centrifuged (3,500 3 g) for 20 min. Excess fluid was poured off, and the sediment was resuspended in 2.0 ml of the phosphate buffer. Part of the sediment (0.5 ml) from each specimen was used to inoculate a BACTEC 12B bottle supplemented with PANTA PLUS (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.), and the rest of the sediment was divided into two parts which were stored at 2208C until the amplification techniques were performed. Part of the initial specimen was also treated by using the Zephiran-trisodium phosphate method for culture. The specimen was mixed with a 10-fold volume of a solution containing 6.5% trisodium phosphate and 8.5% benzalkonium chloride, mixed for 20 min at room temperature, allowed to stand for 20 min, and centrifuged (3,500 3 g) for 20 min. Excess fluid was poured off, and the sediment (0.1 ml) was used to inoculate two Lo ¨wenstein-Jensen tubes for conventional culture. Culture identification. Mycobacterial cultures were incubated at 378C for 6 weeks. Lo ¨wenstein-Jensen tubes were examined weekly for positive culture, and BACTEC bottles were read twice a week, using a BACTEC 460 apparatus (Becton Dickinson). A growth index of .100 was considered positive. Positive cultures from tubes and bottles were stained with Ziehl-Neelsen stain to confirm the presence of acid-fast bacilli which were further identified by RNA-DNA hybridization.
* Corresponding author. Mailing address: Department of Clinical Microbiology, Tampere University Hospital, Box 2000, FIN-33521, Tampere, Finland. Phone: 358-31-2475647. Fax: 358-31-2475260. Electronic mail address:
[email protected]. 1856
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TABLE 1. Initial comparison of culture results of 249 specimens with those obtained by staining, Gen-Probe AMTD assay, and Roche Amplicor PCR for detection of M. tuberculosis No. of culture results
Test and result
Positive
Sensitivity (%)
absorbances were measured with a spectrophotometer (Multiskan; Labsystems Ltd., Helsinki, Finland) at a wavelength of 450 nm. Specimens giving an absorbance value of .0.350 were considered positive. A positive sediment sample containing diluted M. tuberculosis control strain prepared as described above, as well as positive and negative amplification controls, was included in each run.
Specificity (%)
RESULTS
Negative
Staining Positive Negative
21 5
2 221
80.8
99.1
Gen-Probe AMTD Positive Negative
22 4
3 220
84.6
98.7
Roche Amplicor PCR Positive Negative
22 4
2 221
84.6
99.1
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DNA probe assay. Cultures positive for acid-fast bacilli were identified by using AccuProbe reagents (Gen-Probe, Inc., San Diego, Calif.) as described by the manufacturer and by Evans et al. (6). Briefly, a pellet resulting from the centrifugation of BACTEC broth or bacterial inoculum from a Lo ¨wenstein-Jensen tube was directly suspended in a tube containing specimen diluent. Cell suspensions were sonicated with an ELMA Transsonic Digital sonicator (ELMA, Singen/ Hohentwiel, Germany) for 15 min and heat activated (958C, 10 min). Suspensions were transferred to M. tuberculosis probe tubes and Mycobacterium avium complex probe tubes. The tubes were incubated at 608C for 15 min, the selection reagent was added, and the tubes were incubated at 608C for 10 min (5 min for the M. avium complex probe tubes). Hybridization results were read with a luminometer (Leader 50, Gen-Probe, Inc.), and specimens exhibiting .50,000 relative light units (RLU) were considered positive. Specimens yielding positive M. avium complex RLU were further identified by M. avium and Mycobacterium intracellulare probes. Gen-Probe AMTD. Gen-Probe RNA amplification was done as described by the manufacturer and by Jonas et al. and Miller et al. (9, 11). Briefly, an aliquot of sediment was added to a tube containing glass beads and sample buffer and sonicated for 15 min at room temperature with an ELMA sonicator. An aliquot of lysate was added to a tube containing amplification reagent and silicon oil. The tube was incubated at 958C for 15 min and cooled to 428C for 5 min. The amplification enzyme mixture was added, and the tube was incubated at 428C for 2 h. Termination reagent was added, and the incubation was continued for 10 min at 428C. A specific labelled hybridization probe was added, and the hybridization assay was carried out as described above for the DNA probe assay. The samples were read with a Leader 50 luminometer, and a cutoff value of 30,000 RLU was used for positive specimens. In each run, negative and positive sediment samples were included as controls. The positive sediment sample was prepared from a control strain of M. tuberculosis first suspended to correspond to McFarland standard 1 and then further diluted to 1/60,000, as recommended by the manufacturer. Amplicor PCR. The Roche Amplicor PCR (F. Hoffmann-La Roche Ltd., Basel, Switzerland) was done according to the manufacturer’s instructions. The sediment sample was mixed with wash solution and centrifuged (13,000 3 g) for 10 min. The supernatant was discharged, a lysis reagent was added, and the tubes were incubated t 608C for 45 min. After incubation, a neutralization reagent was added and an aliquot of the mixture was transferred to a PCR tube containing a master mix with all the components necessary for amplification. The PCR procedure was accomplished by using a Perkin-Elmer Thermocycler TC 9600 (Perkin-Elmer Cetus, Norwalk, Conn.). After the amplification, the amplified nucleotide sequences were detected by an enzyme immunoassay technique. The
Analytical performance of Gen-Probe AMTD assay and Roche Amplicor PCR. The performance and reproducibility of Gen-Probe AMTD were monitored by including the M. tuberculosis control strain and the buffer sediment sample as controls in each run. The signal for the negative control was 3,628 6 234 RLU (mean value 6 standard error; n 5 20), and for the positive control the signal was 2,554,728 6 111,030 RLU (mean value 6 standard error; n 5 20). For the Roche Amplicor PCR, the signals for the M. tuberculosis control strain and the negative and positive amplification controls were 2,598 6 0.203, 0.054 6 0.003, and 2.822 6 0.178 absorbance units (mean value 6 standard error; n 5 10), respectively. Clinical results. 256 clinical respiratory specimens (132 sputum, 17 bronchoalveolar lavage, and 107 bronchial and tracheal aspirate specimens) from 243 patients were included in this study. Altogether, 33 specimens yielded a culture positive for acid-fast bacilli. A specimen was considered culture positive for M. tuberculosis when it was positive for either the Lo ¨wenstein-Jensen tube culture or BACTEC 12B bottle, confirmed by staining, and identified as M. tuberculosis complex by AccuProbe RNA-DNA hybridization. Of the 33 staining- and culture-positive specimens, 7 were identified by AccuProbe as belonging in M. avium complex (four M. avium and three M. intracellulare strains). They tested negative by both nucleic acid amplification methods and were not included in the final comparison. In total, 26 specimens from 18 patients were culture positive for M. tuberculosis. The initial results are presented in Table 1. Of the 26 positive specimens, two were negative by staining but positive by AMTD or Amplicor PCR, and three were negative by staining and also negative by amplification methods. One culture-positive staining-negative specimen was AMTD positive but Amplicor PCR negative, and one staining-negative specimen was AMTD negative but Amplicor PCR positive. When compared with that for culture, the sensitivities were as follows: for staining, 80.8%; for Gen-Probe AMTD, 84.6%; and for Roche Amplicor PCR, 84.6%. The specificities were 99.1, 98.7, and 99.1%, respectively. Four culture-negative specimens were found to be positive by other methods, as summarized in Table 2. These specimens were obtained from four patients whose medical records were reviewed, and the final results are based on medical history, previous laboratory findings, and recent clinical decisions regarding treatment for tuberculosis. Two culture-negative specimens positive by both nucleic acid amplification methods were obtained from patients who had a history positive for tubercu-
TABLE 2. Analysis of the culture-negative, staining-, Gen-Probe AMTD assay-, and Roche Amplicor PCR-positive specimensa Patient no.
Specimen type
Staining result
Gen-Probe result (RLU)b
PCR result (AU)c
Clinical diagnosis of tuberculosis
M. tuberculosis previously isolated
Conclusion from discrepant results
1 2 3 4
Sputum Sputum Sputum Aspirate
1 2 2 1
1,980,435 80,311 1,857,759 3,804
2.943 0.072 0.739 0.039
1 1 1 2
1 1 1 2
1 1 1 2
1, positive result; 2, negative result. The cutoff value is 30,000. c AU, absorbance units. The cutoff value is 0.350. a b
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VUORINEN ET AL.
TABLE 3. Comparison of confirmed results by culture, staining, Gen-Probe AMTD assay, and Roche Amplicor PCR No. of confirmed results Test and result
Sensitivity (%)
Specificity (%)
Positive predictive value (%)
Negative predictive value (%)
Positive
Negative
Culture Positive Negative
26 3
0 220
89.7
Staining Positive Negative
22 7
1 219
75.9
Gen-Probe AMTD Positive Negative
25 4
0 220
86.2
100
100
98.2
Roche Amplicor PCR Positive Negative
24 5
0 220
82.8
100
100
97.8
losis and previous M. tuberculosis isolates and were subjected to treatment for tuberculosis on the basis of clinical decisions after sampling. One AMTD-positive but culture-, staining-, and PCR-negative specimen was obtained from a patient who had clinically diagnosed tuberculosis and previous positive isolates, and it was considered to be a true positive. One culturenegative, staining-positive specimen was obtained from a patient who had no indication of past or recent tuberculosis in the medical records, and it was considered to be a false positive. As a result of the discrepant analysis, three of four specimens were from patients with a positive history and a clinical diagnosis of tuberculosis, and they were considered to be true positives although they were not found to be positive by culture. After analysis of the discrepant results, the initial results were recalculated, and the final results are presented in Table 3. Thus, the sensitivities and specificities were as follows: for culture, 89.7 and 100%; for staining, 75.9 and 99.5%; for GenProbe AMTD, 86.2 and 100%; and for Roche Amplicor PCR, 82.8 and 100%, respectively. DISCUSSION The increase in the incidence of tuberculosis in Western countries has prompted the development of rapid direct detection methods for the laboratory diagnosis of M. tuberculosis. PCR employing DNA amplification of a specific part of M. tuberculosis insertion sequence IS6110 has been used in several studies, revealing a high degree of sensitivity (4, 7, 8, 12, 18). However, the reliability and reproducibility of homemade PCR applications were shown to be insufficient in a recent blinded study between laboratories, and the usefulness of PCR in the detection of M. tuberculosis was questioned (13). New rapid and standardized commercial detection systems include GenProbe AMTD and Roche Amplicor PCR. The rRNA amplification method by Gen-Probe has already been used with a high degree of sensitivity (2, 9, 11, 15). The aim of the present study was to compare Gen-Probe ATMD with a new PCR application by Roche for the detection of M. tuberculosis in respiratory specimens. The analytical performance of both nucleic acid amplification methods was monitored by including negative and positive sediment samples in each run. Both methods had a good reproducibility, and the signal differences between negative control, cutoff value, and positive control were broad enough to allow good resolution. Both reagent kits contain all specific
100
99.5
100
95.7
98.7
96.9
reagents needed for specimen lysis, amplification, and detection of amplified products. The methods were well suited for use in an experienced routine clinical microbiology laboratory, and there were no problems due to sample or amplicon contamination. Gen-Probe AMTD had an assay time of 5 h and proved slightly faster than Amplicor PCR, which had an assay time of 7 h. The sensitivity of conventional culture methods for respiratory specimens has been generally considered very good, and the BACTEC 460 radiometric system has reduced the time required for detection of M. tuberculosis. However, it has been suggested that the BACTEC system is unsatisfactory for specimens with negative smear results (14). Therefore, in the present study, the BACTEC system was used in conjunction with Lo ¨wenstein-Jensen tube culture. This combination is currently considered to be the ‘‘gold standard’’ procedure (14, 16). After discrepant analysis, the sensitivity of the combined culture procedure was 89.7%, and three culture-negative specimens from patients were considered to be false negatives on the basis of a review of the medical records. All three specimens were positive by AMTD, two were positive by Amplicor PCR, but only one specimen was positive by staining. Microscopic examination of acid-fast smears is a simple and inexpensive method. In the detection of M. tuberculosis, the specificity of positive staining is near 100% but the sensitivity has been considered limited (14). In this study, auramine fluorescent staining missed 5 culture-positive specimens of 26, and the discrepant analysis revealed a total of 7 false-negative and 1 false-positive specimens. Thus, staining reached a sensitivity of 75.9% and a specificity of 99.5%. The value of positive staining results from primary specimens is, however, limited, because of the increasing frequency of nontuberculous mycobacteria. The current material contained seven specimens which were culture positive for nontuberculous mycobacteria, all of which were positive by staining but negative by both nucleic acid amplification methods. The benefit of AMTD comes from detecting M. tuberculosis rRNA, present at about 2,000 copies per cell, thus offering a good theoretical sensitivity for specimens with a low target sequence number. In this study, the sensitivity and specificity of AMTD were 86.2 and 100%, respectively, which are in good accordance with recent studies revealing a sensitivity of 82 to 94% and a specificity of 97 to 99% (2, 9, 11, 15). AMTD detected three culture-negative specimens that were considered to be true positives on the basis of patient review. Of the
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three, one specimen was AMTD positive but negative by other methods. Three culture-positive specimens were negative by AMTD, RLU values below 10,000. The new PCR application by Roche employs genus-specific primers located in a highly conserved region of the 16S rRNA gene of Mycobacterium species and the amplification of a 584-bp sequence. The biotin-labelled amplicons are captured onto a microwell plate with a probe specific for M. tuberculosis complex, detecting a hypervariable region of the 16S rRNA gene. Captured amplicons are detected by a standard enzyme immunoassay technique. In the present study, Amplicor PCR reached a sensitivity of 82.8% and a specificity of 100% after discrepant analysis. Amplicor PCR missed two culture-positive specimens representing low absorbance values in the detection reaction but yielded positive results for two culture-negative specimens from patients with previous positive isolates and a clinical diagnosis of current tuberculosis. The negative results obtained by the nucleic acid amplification methods for culture-positive specimens may perhaps be explained by the presence of inhibitors of enzymatic amplification reactions, as earlier reported for different PCR applications (3, 5, 19). The unequal distribution of bacteria in specimens with a low target number is another possible explanation. The gold standard culture procedure failed to detect M. tuberculosis in three specimens from patients with overt tuberculosis, while AMTD and Amplicor PCR yielded positive results. Both nucleic acid amplification methods proved to be rapid, sensitive, and specific methods for the detection of M. tuberculosis in respiratory specimens. ACKNOWLEDGMENTS ¨ isti for excellent technical We thank Riitta Nerve and Terttu O assistance. This study was supported by the Tampere Tuberculosis Foundation and by the Medical Research Fund of Tampere University Hospital, Tampere, Finland. REFERENCES 1. Abe, C., K. Hirano, M. Wada, Y. Kazumi, M. Takahashi, Y. Fukasawa, T. Yoshimura, C. Miyagi, and S. Goto. 1993. Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test. J. Clin. Microbiol. 31: 3270–3274. 2. Abe, C., S. Hosojima, Y. Fukasawa, Y. Kazumi, M. Takahashi, K. Hirano, and T. Mori. 1992. Comparison of MB-Check, BACTEC, and egg-based media for recovery of mycobacteria. J. Clin. Microbiol. 30:878–881. 3. Bauwens, J. E., A. M. Clark, and W. E. Stamm. 1993. Diagnosis of Chlamydia trachomatis endocervical infections by a commercial polymerase chain reac-
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