DIAGN MICROBIOLINFECTDIS 1992;15:657-662
657
MYCOBACTERIOLOGY
Evaluation of Syngene D N A - D N A Probe Assays for the Identification of the Mycobacterium tuberculosis Complex and the Mycobacterium avium Complex Charles L. Woodley, Margaret M. Floyd, and Vella A. Silcox
Two hundred mycobacterial cultures were used to evaluate two alkaline-phosphatase-labeled DNA probe (SNAP) kits developed by Syngene (San Diego, CA) for identification of Mycobacterium tuberculosis complex and M. avium complex. The M. tuberculosis complex SNAP probe, when compared with standard biochemical identification tests, gave results that were in agreement at 100% sensitivity and 98.7% specificity. Ninety-nine M. avium complex strains that were previously tested by the Gen-Probe M. avium complex probe assays and mycolic acid analysis were included to evaluate the M. avium
complex SNAP assay which contained three probes, A (avium), I (intracellulare), and X. Eight strains identified as members of the M. avium complex by biochemical tests did not react with the three SNAP probes. These strains were also negative by the Gen-Probe assays. However, 23 strains identified as M. avium complex by biochemical tests and mycolic acid analysis and negative with the Gen-Probe assays gave positive results with the X probe and negative results with the A and I probes of the SNAP assay.
INTRODUCTION
available that are both rapid a n d reliable for identification of the major species of Mycobacterium. These procedures include the use of monoclonal antibodies for detection of mycobacterial antigens (Mauch et al., 1988; Nishimori et al., 1987; SchOningh et al., 1990), radiometric m e t h o d s to detect radiolabeled metabolites for identification (Siddiqi et al., 1984), and i m m u n o a s s a y s for detection of specific e n z y m e s (Udou et al., 1987; W a y n e and Diaz, 1987). The detection of mycolic acids unique to Mycobacterium and some closely related genera have been u s e d for identification of species of this group. Detection of mycolic acids, first by thin-layer c h r o m a t o g r a p h y (Minnikin et al., 1984), then by gas-liquid chromatography (Lambert et al., 1986) a n d high-performance liquid chromatography (HPLC) (Butler a n d Kilburn, 1988), has been reported as highly accurate for identification of all Mycobacterium species. Identification by nucleic acid analysis was once t h o u g h t to be impractical because of the difficulty in
For m a n y decades the mycobacteriology laboratory was plagued by the slow bacterial growth rate that often translated into m o n t h s of waiting for specific identification of an acid-fast bacillus isolated from a diseased patient. In recent years, however, n e w research and commercial procedures have become From the MycobacteriologyLaboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Human Services, Atlanta, Georgia, USA, Address reprint requests to Dr. C.L. Woodley, Mycobacteriology Laboratory (F08), National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA. Received 11 September 1991; revised and accepted 7 April 1992. Published 1992 by Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$0.00
658
lysing these bacteria. However, Gen-Probe (San Diego, CA) produced DNA probes that hybridize with rRNA, which they were able to release from mycobacterial cells. These proved to be highly accurate, allowing identification of cultures of mycobacteria within hours (Drake et al., 1987; Gonzales and Hanna, 1987; Kiehn and Edwards, 1987; Musial et al., 1988; Sherman et al., 1989; Woodley et al., 1989). The present single-stranded DNA probes available are targeted for detection of rRNA from the M. tuberculosis complex, M. avium complex, and M. gordonae. The 12SI-labeledprobes (Gen-Probe) are being replaced by their nonradioactive ACCU-PROBESystem that uses chemiluminescent-labeled probes. A probe specific for M. tuberculosis complex (that is, M. tuberculosis, M. bovis, M. africanum, M. microti, and Calmette-Gu6rin bacillus [BCG] variants of M. bovis) is valuable for diagnosis since M. tuberculosis accounts for 65% of the pathogenic mycobacterial isolates in the United States and represents 50% of the acidfast bacilli isolated by state public health laboratories (Good and Snider, 1982). Members of the M. avium complex (that is, M. avium and M. intracellulare) represent the second most commonly isolated Mycobacterium species in the United States (Good and Snider, 1982). These species, principally M. avium, have been isolated from up to 53% of patients who have acquired immunodeficiency syndrome (AIDS) (Yakrus and Good, 1990). The two species can generally be separated by the time-consuming and not too readily available method of serotyping (McClatchy 1981; Good and Beam, 1984), but not by standard in vitro biochemical test methods (Kent and Kubica, 1985). An alkaline-phosphatase-labeled oligonucleotide DNA probe SNAP, for identification of mycobacteria was developed by Syngene (San Diego, CA) that targeted DNA from members of the M. tuberculosis complex and M. avium complex. We report here the results of our evaluation of the SNAP probe using mycobacterial strains identified in our laboratory by standard biochemical procedures and other accepted methods.
MATERIALS A N D METHODS Bacterial Strains Cultures used in this study were selected from clinical isolates identified by the Mycobacteriology Laboratory, Centers for Disease Control (Atlanta, GA). Control strains were obtained from either the Trudeau Mycobacterial Culture Collection (TMC) or the American Type Culture Collection (ATCC). A total of 200 cultures were studied: 175 clinical isolates and 25 ATCC or TMC strains. Many of the cultures submitted to our laboratory were previ-
C.L. Woodley et al.
TABLE 1 List of Species Used to Test the Mycobacterium SNAP Probes
Species a
Number of Strains Tested
Mycobacterium tuberculosis bovis boris BCG avium complex scrofulaceum asiaticum kansasii simiae gordonae xenopi terrae fortuitum complex szulgai marinum triviale smegmatis malmoense gastri Tsukamurella paurometabolum Nocardia asteroides Rhodococcus spp.
Unidentified mycobacteria
36 3
5 99 7 4 2 6 5 5 5
4 3 2 1 4 1 1 1 2
2 3
°Identifiedby standard biochemicalprocedures. ously identified by state laboratories using both Gen-Probe and biochemical methods. The number of strains of each species tested in the study is listed in Table 1.
Biochemical Identification Strains were identified by standard biochemical test procedures (Kent and Kubica, 1985).
Serology Some M. avium complex strains were selected for serotyping by the Serology Unit of the Mycobacteriology Laboratory. The agglutination method of Schaefer as modified by Good and Beam (1985) was used. Serovars 1-6 and 8-11 are currently recognized as M. avium, whereas serovars 7 and 12-25 are M. intracellulare (Baess, 1983; Yakrus and Good, 1990).
Mycolic Acid Analysis Identification of selected strains using mycolic acid analysis by HPLC was performed in the Mycobac-
659
Evaluation of Syngene D N A - D N A Probe Assays
teriology Laboratory as described by Butler and Kilburn (1988).
was interpreted as positive. Absence of color was interpreted as negative.
Gen-Probe Procedures The Gen-Probe rapid diagnostic kits for the M. tuberculosis complex and the M. avium complex were purchased from Gen-Probe. The procedures described in the package insert accompanying each kit were followed precisely. Cultures previously identified by the submitting laboratories as M. avium complex biochemically and positive or negative by Gen-Probe were reidentified using all tests.
SNAP Culture Identification Procedures The SNAP Culture Identification Test Kits were provided by Syngene. Two separate kits were provided, M. tuberculosis complex and M. avium complex. The M. avium complex kit contained three probes; m. avium, M. intracellulare, and a third probe identified by the company as X. These could be used separately or combined. One or two colonies from cultures on solid (Lowenstein-Jensen) medium were transferred to lysis tubes that were provided with SNAP kits. The lysis tube contained 1 ml of lysis buffer, 300 ~1 of chloroform, and glass beads. The caps were tightened, and the tubes were placed in a Minibead Beater (Biopec Products, Bartleville, OK) and shaken for 3 min to disrupt the mycobacterial cells. Lysis tubes were then centrifuged for 5 min at 3000 g. Of the top (aqueous) layer of the sample 300 ~1 was added to 300 ~1 of the denaturation reagent in the designated well of a Centri-Dot apparatus provided in the kit. The Centri-Dot apparatus, which can hold up to six samples, was centrifuged at 1000 g for 5 rain, and the Centri-Dot membrane was removed and rinsed in deionized water for 15 sec. The membrane was transferred to a reaction bag containing 1 ml of hybridization buffer and 30 p,1 of the desired probe (or 10 ~1 for the individual M. avium complex probes). The bag was sealed and incubated at 49°C in a water bath for 15 min. The membrane was removed from the bag, placed in a jar containing 200 ~1 of wash solution (WASH-I), and incubated for 15 rain in the 40°C water bath. The membrane was transferred to a second wash solution (WASH-2), shaken for 5 sec, then incubated for 15 rain at 49°C. The membrane was removed, blotted, and placed in a new reaction bag containing 1.0 ml alkaline phosphatase substrate buffer with 5 ~1 of nitro-blue tetrazolium chloride substrate and 5-bromo-4-chloro-3-indolyphosphate substrate. The bag was then sealed and incubated for 1 hr at 37°C. The membrane was removed, rinsed with deionized water, and examined while wet for a uniform blue or purple dot on the membrane which
RESULTS All 200 strains of mycobacteria were used to compare the M. tuberculosis complex SNAP probe with the standard biochemical identification tests. Overall agreement was 99% (Table 2). Two (TMC 803 and a clinical strain) of the four M. asiaticum strains tested gave weak positive reactions with the probe for M. tuberculosis. We failed to isolate M. tuberculosis from these cultures, and they were negative with the GenProbe for M. tuberculosis complex, as shown in Table 3. Both strains were identified as M. asiaticum by mycolic acid analysis, and there was no indication of a mixed culture. These same 200 strains were tested with the SNAP probe for the M. avium complex (containing the M. avium, M. intracellulare, and X probes mixed as one probe) (Table 4). Eight clinical strains identified biochemically as M. avium complex did not react with the probe. Two strains identified biochemically as M. scrofulaceum were positive when tested. These 10 strains were also tested with Gen-Probe M. avium complex probes and mycolic acid analysis, along with 140 other strains selected from the 200 strains (Table 5). The eight strains negative by the SNAP Probe were negative with the Gen-Probe, and mycolic acid analysis showed that only four of these strains were members of the M. avium complex. One of the two clinical strains identified as M. scrofulaceum by biochemical tests was positive by the SNAP and GenProbe M. avium complex probes and identified as the M. avium complex by mycolic acid analysis. The other strain of M. scrofulaceum was negative by GenProbe and identified as M. avium complex by mycolic analysis. When strains were positive with the combined probes for M. avium complex, we retested them with the individual probes (Table 6). The M. scrofulaceum strain positive with the SNAP and negative with the Gen-Probe reacted with the SNAP X probe. All strains identified as M. avium complex biochemically and positive for either the Gen-Probe M. avium probe or M. intracellulare probe were positive for the SNAP counterpart. Of the Gen-Probe-negative M. avium complex strains, twenty-two reacted with the SNAP X probe; all 22 strains were identified as members of the M. avium complex by mycolic acid analysis. Included in this group were three culture collection strains: ATCC 29555 (serotype 6); ATCC 35847 (serotype 7); and ATCC 35770 (serotype 18). Also included were three clinical strains that were serotyped 6, 9, and 16. Eight of the 22 strains did not react with any of the 28 antisera used for serotyping.
660
TABLE 2
C.L. Woodley et al.
Comparison of SNAP Mycobacterium tuberculosis Complex Culture Identification with Standard Biochemical Identification SNAP Identification as Mycobacterium tuberculosis Complex
Biochemical Identification as Mycobacterium tuberculosis Complex
Positive
Negative
44 2
0 154
Positive Negative Sensitivity = 100.0%; and specificity = 98.7%. TABLE 3
Comparison of SNAP Probe with Gen-Probe with Selected Strains Positive for Mycobacterium tuberculosis
Species ~
Number Tested
SNAP
Gen-Probe
Mycobacterium tuberculosis bovis bovis BCG asiaticumb Other species
36 3 5 4 42
36 3 5 2 0
36 3 5 0 0
qdentified by biochemicaltests. bldentified also by mycolicacid analysis. TABLE 4
Comparison of SNAP Mycobacterium avium Complex Culture Identification with Standard Biochemical Identification SNAP Identification as Mycobacterium avium Complex
Biochemical Identification as Mycobacterium avium Complexa Positive Negative
Positive
Negative
91 2b
8 99
~Sensitivity = 91.9%; and specificity = 98.0%. bIdentified as M. scrofulaceum.
Four of the strains could not be tested because of autoagglutination and the last four could not be confirmed.
DISCUSSION The Syngene SNAP assay for M. tuberculosis showed an accurate identification with sensitivity of 100% and specificity of 98.7% compared with standard biochemical identification. These numbers are similar to those reported by other investigators for the present commercial D N A - R N A probes (Kiehn and Edwards, 1987; Sherman et al., 1989). Two falsepositive results with M. asiaticum indicate there may be a degree of cross-reaction with this species. Lim
et al. (1991) recently reported two discrepant reactions while evaluating the SNAP assay for M. tuberculosis complex. Two strains identified as M. terrae complex by mycolic acid analysis were M. tuberculosis-probe positive. They noted no other discrepancies with the M. tuberculosis complex probe. The SNAP M. avium complex assay included a probe component (X) that detected strains identified by standard biochemical methods as M. avium complex, but negative by the Gen-Probe assay. We have collected a number of these strains since our initial studies with Gen-Probe (Woodley et al., 1989), and these are well represented in the present study creating a bias result in a direct comparison of probes from the two manufacturers (Woodley et al., 1989). Characterization of these strains in our laboratory using
Evaluation of Syngene DNA-DNA Probe Assays
TABLE 5
661
Comparison of SNAP Probe with Gen-Probe with Selected Strains a Positive
Species b
Number of Strains b
SNAP
Gen-Probe
99
91
69
7 4 1 6 5
2 0 0 0 0
1 0 0 0 0
28
0
0
Mycobacterium avium complex scrofulaceum xenopi malmoense simiae terrae
Other species
aApproximatelyequal number of M. avium, M. intracellulare, and Gen-Probe-negativeM. avium complex strains were selected. bldentifiedby biochemicaltests.
TABLE 6
Strains Positive to SNAP Assay Tested with the Individual Probes of Mycobacterium avium Complex Kit
Source
No.
Strains Collection Clinical
10 83 93
Total
"A. . . .
I. . . .
X"
4 29
3 30
3 20
33a
33b
23c
"Positivefor Gen-ProbeM. avium (includesATCC25291, 35767, 35768, and 35773). bpositive for Gen-ProbeM. intracellulare(includesATCC35769, 13950, and 35782). CNegativefor both Gen-Probes (includesATCC35847, 29555, and 35770).
mycolic acid analysis and biochemical identification has shown most to be typical of the M. avium complex. Because type strains of serotypes 6, 7, and 18 reacted with the SNAP X probe, both M. avium and M. intracellulare serotypes can be classified as X-positive strains by SNAP probes. Saito et al. (1990) found that some reference strains for serotypes 23, 24, and 28 were negative with both M. avium and M. intracellulare Gen-Probes. We also found strains (not included in the 200 strains) belonging to serotypes 23, 24, and 28 that were Genprobe negative for M. avium complex, but that reacted with the SNAP X probe. Two ATCC strains that reacted with the SNAP X probe, ATCC 35770 and ACTCC 35847, had previously been reported by Wayne and Diaz (1987) as the only strains that did not give a positive reaction among 11 M. intracellulare isolates tested with T-catalase dot-blot immunoas-
say. Tasaka et al. (1985 and 1986), using oLantigens as markers for serologic identification of the M. avium complex, reported the absence of the specific M. avium complex, e¢ antigen with the culture identified as ATCC 35847. In addition, Picken et al. (1988) showed that these two strains and ATCC 29555 have a unique restriction-fragment-length polymorphism pattern that differs significantly from the patterns of M. avium or M. intracellulare. Hawkins (1977) described a number of M. avium complex and related strains that exhibited a perfect fit on the basis of biochemical reaction for M. avium complex, though deeply pigmented. On the other hand, there are reports on "intermediate" strains that possess biochemical features of both M. avium complex and M. scrofulaceum (Murphy and Hawkins, 1975). These strains have high catalase activity with negative or occasional positive urease test result. Several of the strains that react with the SNAP X probe fit into the "intermediate" category between M. avium complex and M. scrofulaceum, and although the majority produce yellow pigment, only two have been identified biochemically as M. scrofulaceum. Presently, our studies indicate that strains positive with the Syngene SNAP X probe are members of M. avium complex by standard identification, serology, and mycolic acid identification with some strain variations. On the other hand, further studies, such as DNADNA homologies, may position these strains as either one or more new species. The SNAP DNA-DNA probe identification procedure was an effective method for identifying two important groups of mycobacteria. The procedure required no special equipment (except the minibead beater) and thus may prove especially suited for small laboratories.
662
C.L. W o o d l e y et al.
REFERENCES Baess I (1983) Deoxyribonucleic acid relationships between different serovars of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum. Acta Pathol Microbiol Scand [B] 91:201-203. Butler WR, Kilburn JO (1988) Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids. J Clin Microbiol 26:50-53. Drake TA, Hindler JA, Berlin OGW, Bruckner DA (1987) Rapid identification of Mycobacterium avium complex in culture using DNA probes. J Clin Microbiol 25:14421445. Gonzalez R, Hanna BA (1987) Evaluation of Gen-Probe DNA hybridization systems for the identification of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare. Diagn Microbiol Infect Dis 8:69-77. Good RC, Beam RE (1984) Seroagglutination. In The Mycobacteria: A Sourcebook. Eds, G Kubica and L Wayne. New York: Marcel Dekker, pp 105-122. Good RC, Snider DE (1982) Isolation of nontuberculosis mycobacteria in the United States. J Infect Dis 146:829833. Hawkins JE (1977) Scotochromogenic mycobacteria which appear intermediate between Mycobacterium avium-intracellulare and Mycobacterium scrofulaceum. Am Rev Respir Dis 116:963-964. Kent PT, Kubica GP (1985) Public Health Mycobacteriology: A Guide for the Level III Laborato~. Atlanta GA: Centers for Disease Control. Kiehn TE, Edwards FF (1987) Rapid identification using a specific DNA probe of Mycobacterium avium complex from patients with acquired immunodeficiency syndrome. ] Clin Microbiol 25:1551-1552. Lambert MA, Brehmer W, Sonneborn HH, Horn J, Wagner S (1986) Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography. J Clin Microbiol 23:733-736. Lira SD, Todd J, Lopez J, Ford E, Janda JM (1991) Genotypic identification of pathogenic Mycobacterium species by using a nonradioactive oligonucleotide probe. ] Clin MicrobioI 29:1276-1278. Mauch HW, Brehmer W, Sonneborn HH, Horn J, Wagner S (1988) Monoclonal antibodies selectively directed against the cell wall surface of Mycobacterium tuberculosis. J Clin Microbiol 26:1691-1694. McClatchy JK (1981) The seroagglutination test in the study of nontuberculous mycobacteria. Rev Infect Dis 3:867870. Minnikin DE, Minnikin SM, Parlett JM, Goodfellow M, Magnusson M (1984) Mycolic acid patterns of some species of Mycobacterium. Arch MicrobioI 139:225-231. Murphy DB, Hawkins JE (1975) Use of urease test disks in the identification of mycobacteria. J Clin Microbiol 1:465-468. Musial CE, Tice LS, Stockman LS, Roberts GD (1988) Identification of mycobacteria from cultures by using the
Gen-Probe rapid diagnostic system for Mycobacterium avium complex and the Mycobacterium tuberculosis complex. ] Clin Microbiol 26:2120-2123. Nishimori E, Yugi H, Naiki M, Sugimura T, Tanaka F, Nonomura I, Yokomizi Y, Kubo S (1987) Production and characteristics of serovar-specific monoclonal antibodies to serovars 4, 8, and 9 of Mycobacterium intracellulare. Infect Immun 55:711-715. Picken RW, Tsang AY, Yang HL (1988) Speciation of organisms within the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex based on restriction fragment length polymorphism. Mol Cell Probes 2:289-304. Saito H, Timioka H, Sato K, Tasaka H, Dawson DJ (1990) Identification of various serovar strains of Mycobacterium avium complex by using DNA probes specific for Mycobacterium avium and Mycobacterium intracellulare. J Clin Microbiol 28:1694-1697. Sch6ningh R, Verstijnen, CPH, Kuijper S, Kolk AHJ (1990) Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures. ] Clin Microbiol 28:708-711. Sherman I, Harrington N, Rothrock A, George H (1989) Use of a cutoff range in identifying mycobacteria by the Gen-Probe rapid diagnostic system. J Clin Microbiol 27:241-244. Siddiqi SH, Hwangbo CC, Silcox V, Good RC, Snider JR, DE, Middlebrook G (1984) Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species. Am Rev Respir Dis 130:634-640. Tasaka H, Nomura T, Matsuo Y (1985) Specificity and distribution of alpha antigens of Mycobacterium aviumintracellulare, Mycobacterium scrofulaceum, and related species of mycobacteria. Am Rev Respir Dis 132:173-174. Tasaka R (1986) Development of serological identification of slowly growing mycobacteria with alpha antigen as a marker. Kekkaku 61:663-669. Udou T, Mizuguhi Y, Wallace Jr RJ (1987) Patterns and distribution of aminoglycoside-acetylating enzymes in rapidly growing mycobacteria. Am Rev Respir Dis 136:338343. Wayne LC, Diaz CA (1987) Intrinsic catalase dot blot immunoassay for identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. J Clin Microbiol 25:1687-1690. Woodley CL, Silcox VA, Floyd MM, Kubica GP (1989) The use of DNA probes for rapidly identifying cultures of Mycobacterium. In Rapid Methods in Clinical Microbiology: Present Status and Future Trends (Advances in Experimental Medicine and Biology Series). Eds, B Kleger, D Jungkivid, E Hinks, and LA Miller. New York: Plenum, pp 51-56. Yakrus MA, Good RC (1990) Geographic distribution frequency, and specimen source of Mycobacterium avium complex serotype isolated from patients with acquired immunodeficiency syndrome. J Clin Microbiol 28:926929.
657
MYCOBACTERIOLOGY
Evaluation of Syngene D N A - D N A Probe Assays for the Identification of the Mycobacterium tuberculosis Complex and the Mycobacterium avium Complex Charles L. Woodley, Margaret M. Floyd, and Vella A. Silcox
Two hundred mycobacterial cultures were used to evaluate two alkaline-phosphatase-labeled DNA probe (SNAP) kits developed by Syngene (San Diego, CA) for identification of Mycobacterium tuberculosis complex and M. avium complex. The M. tuberculosis complex SNAP probe, when compared with standard biochemical identification tests, gave results that were in agreement at 100% sensitivity and 98.7% specificity. Ninety-nine M. avium complex strains that were previously tested by the Gen-Probe M. avium complex probe assays and mycolic acid analysis were included to evaluate the M. avium
complex SNAP assay which contained three probes, A (avium), I (intracellulare), and X. Eight strains identified as members of the M. avium complex by biochemical tests did not react with the three SNAP probes. These strains were also negative by the Gen-Probe assays. However, 23 strains identified as M. avium complex by biochemical tests and mycolic acid analysis and negative with the Gen-Probe assays gave positive results with the X probe and negative results with the A and I probes of the SNAP assay.
INTRODUCTION
available that are both rapid a n d reliable for identification of the major species of Mycobacterium. These procedures include the use of monoclonal antibodies for detection of mycobacterial antigens (Mauch et al., 1988; Nishimori et al., 1987; SchOningh et al., 1990), radiometric m e t h o d s to detect radiolabeled metabolites for identification (Siddiqi et al., 1984), and i m m u n o a s s a y s for detection of specific e n z y m e s (Udou et al., 1987; W a y n e and Diaz, 1987). The detection of mycolic acids unique to Mycobacterium and some closely related genera have been u s e d for identification of species of this group. Detection of mycolic acids, first by thin-layer c h r o m a t o g r a p h y (Minnikin et al., 1984), then by gas-liquid chromatography (Lambert et al., 1986) a n d high-performance liquid chromatography (HPLC) (Butler a n d Kilburn, 1988), has been reported as highly accurate for identification of all Mycobacterium species. Identification by nucleic acid analysis was once t h o u g h t to be impractical because of the difficulty in
For m a n y decades the mycobacteriology laboratory was plagued by the slow bacterial growth rate that often translated into m o n t h s of waiting for specific identification of an acid-fast bacillus isolated from a diseased patient. In recent years, however, n e w research and commercial procedures have become From the MycobacteriologyLaboratory, Respiratory Diseases Branch, Division of Bacterial Diseases, National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Human Services, Atlanta, Georgia, USA, Address reprint requests to Dr. C.L. Woodley, Mycobacteriology Laboratory (F08), National Center for Infectious Diseases, Centers for Disease Control, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA. Received 11 September 1991; revised and accepted 7 April 1992. Published 1992 by Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$0.00
658
lysing these bacteria. However, Gen-Probe (San Diego, CA) produced DNA probes that hybridize with rRNA, which they were able to release from mycobacterial cells. These proved to be highly accurate, allowing identification of cultures of mycobacteria within hours (Drake et al., 1987; Gonzales and Hanna, 1987; Kiehn and Edwards, 1987; Musial et al., 1988; Sherman et al., 1989; Woodley et al., 1989). The present single-stranded DNA probes available are targeted for detection of rRNA from the M. tuberculosis complex, M. avium complex, and M. gordonae. The 12SI-labeledprobes (Gen-Probe) are being replaced by their nonradioactive ACCU-PROBESystem that uses chemiluminescent-labeled probes. A probe specific for M. tuberculosis complex (that is, M. tuberculosis, M. bovis, M. africanum, M. microti, and Calmette-Gu6rin bacillus [BCG] variants of M. bovis) is valuable for diagnosis since M. tuberculosis accounts for 65% of the pathogenic mycobacterial isolates in the United States and represents 50% of the acidfast bacilli isolated by state public health laboratories (Good and Snider, 1982). Members of the M. avium complex (that is, M. avium and M. intracellulare) represent the second most commonly isolated Mycobacterium species in the United States (Good and Snider, 1982). These species, principally M. avium, have been isolated from up to 53% of patients who have acquired immunodeficiency syndrome (AIDS) (Yakrus and Good, 1990). The two species can generally be separated by the time-consuming and not too readily available method of serotyping (McClatchy 1981; Good and Beam, 1984), but not by standard in vitro biochemical test methods (Kent and Kubica, 1985). An alkaline-phosphatase-labeled oligonucleotide DNA probe SNAP, for identification of mycobacteria was developed by Syngene (San Diego, CA) that targeted DNA from members of the M. tuberculosis complex and M. avium complex. We report here the results of our evaluation of the SNAP probe using mycobacterial strains identified in our laboratory by standard biochemical procedures and other accepted methods.
MATERIALS A N D METHODS Bacterial Strains Cultures used in this study were selected from clinical isolates identified by the Mycobacteriology Laboratory, Centers for Disease Control (Atlanta, GA). Control strains were obtained from either the Trudeau Mycobacterial Culture Collection (TMC) or the American Type Culture Collection (ATCC). A total of 200 cultures were studied: 175 clinical isolates and 25 ATCC or TMC strains. Many of the cultures submitted to our laboratory were previ-
C.L. Woodley et al.
TABLE 1 List of Species Used to Test the Mycobacterium SNAP Probes
Species a
Number of Strains Tested
Mycobacterium tuberculosis bovis boris BCG avium complex scrofulaceum asiaticum kansasii simiae gordonae xenopi terrae fortuitum complex szulgai marinum triviale smegmatis malmoense gastri Tsukamurella paurometabolum Nocardia asteroides Rhodococcus spp.
Unidentified mycobacteria
36 3
5 99 7 4 2 6 5 5 5
4 3 2 1 4 1 1 1 2
2 3
°Identifiedby standard biochemicalprocedures. ously identified by state laboratories using both Gen-Probe and biochemical methods. The number of strains of each species tested in the study is listed in Table 1.
Biochemical Identification Strains were identified by standard biochemical test procedures (Kent and Kubica, 1985).
Serology Some M. avium complex strains were selected for serotyping by the Serology Unit of the Mycobacteriology Laboratory. The agglutination method of Schaefer as modified by Good and Beam (1985) was used. Serovars 1-6 and 8-11 are currently recognized as M. avium, whereas serovars 7 and 12-25 are M. intracellulare (Baess, 1983; Yakrus and Good, 1990).
Mycolic Acid Analysis Identification of selected strains using mycolic acid analysis by HPLC was performed in the Mycobac-
659
Evaluation of Syngene D N A - D N A Probe Assays
teriology Laboratory as described by Butler and Kilburn (1988).
was interpreted as positive. Absence of color was interpreted as negative.
Gen-Probe Procedures The Gen-Probe rapid diagnostic kits for the M. tuberculosis complex and the M. avium complex were purchased from Gen-Probe. The procedures described in the package insert accompanying each kit were followed precisely. Cultures previously identified by the submitting laboratories as M. avium complex biochemically and positive or negative by Gen-Probe were reidentified using all tests.
SNAP Culture Identification Procedures The SNAP Culture Identification Test Kits were provided by Syngene. Two separate kits were provided, M. tuberculosis complex and M. avium complex. The M. avium complex kit contained three probes; m. avium, M. intracellulare, and a third probe identified by the company as X. These could be used separately or combined. One or two colonies from cultures on solid (Lowenstein-Jensen) medium were transferred to lysis tubes that were provided with SNAP kits. The lysis tube contained 1 ml of lysis buffer, 300 ~1 of chloroform, and glass beads. The caps were tightened, and the tubes were placed in a Minibead Beater (Biopec Products, Bartleville, OK) and shaken for 3 min to disrupt the mycobacterial cells. Lysis tubes were then centrifuged for 5 min at 3000 g. Of the top (aqueous) layer of the sample 300 ~1 was added to 300 ~1 of the denaturation reagent in the designated well of a Centri-Dot apparatus provided in the kit. The Centri-Dot apparatus, which can hold up to six samples, was centrifuged at 1000 g for 5 rain, and the Centri-Dot membrane was removed and rinsed in deionized water for 15 sec. The membrane was transferred to a reaction bag containing 1 ml of hybridization buffer and 30 p,1 of the desired probe (or 10 ~1 for the individual M. avium complex probes). The bag was sealed and incubated at 49°C in a water bath for 15 min. The membrane was removed from the bag, placed in a jar containing 200 ~1 of wash solution (WASH-I), and incubated for 15 rain in the 40°C water bath. The membrane was transferred to a second wash solution (WASH-2), shaken for 5 sec, then incubated for 15 rain at 49°C. The membrane was removed, blotted, and placed in a new reaction bag containing 1.0 ml alkaline phosphatase substrate buffer with 5 ~1 of nitro-blue tetrazolium chloride substrate and 5-bromo-4-chloro-3-indolyphosphate substrate. The bag was then sealed and incubated for 1 hr at 37°C. The membrane was removed, rinsed with deionized water, and examined while wet for a uniform blue or purple dot on the membrane which
RESULTS All 200 strains of mycobacteria were used to compare the M. tuberculosis complex SNAP probe with the standard biochemical identification tests. Overall agreement was 99% (Table 2). Two (TMC 803 and a clinical strain) of the four M. asiaticum strains tested gave weak positive reactions with the probe for M. tuberculosis. We failed to isolate M. tuberculosis from these cultures, and they were negative with the GenProbe for M. tuberculosis complex, as shown in Table 3. Both strains were identified as M. asiaticum by mycolic acid analysis, and there was no indication of a mixed culture. These same 200 strains were tested with the SNAP probe for the M. avium complex (containing the M. avium, M. intracellulare, and X probes mixed as one probe) (Table 4). Eight clinical strains identified biochemically as M. avium complex did not react with the probe. Two strains identified biochemically as M. scrofulaceum were positive when tested. These 10 strains were also tested with Gen-Probe M. avium complex probes and mycolic acid analysis, along with 140 other strains selected from the 200 strains (Table 5). The eight strains negative by the SNAP Probe were negative with the Gen-Probe, and mycolic acid analysis showed that only four of these strains were members of the M. avium complex. One of the two clinical strains identified as M. scrofulaceum by biochemical tests was positive by the SNAP and GenProbe M. avium complex probes and identified as the M. avium complex by mycolic acid analysis. The other strain of M. scrofulaceum was negative by GenProbe and identified as M. avium complex by mycolic analysis. When strains were positive with the combined probes for M. avium complex, we retested them with the individual probes (Table 6). The M. scrofulaceum strain positive with the SNAP and negative with the Gen-Probe reacted with the SNAP X probe. All strains identified as M. avium complex biochemically and positive for either the Gen-Probe M. avium probe or M. intracellulare probe were positive for the SNAP counterpart. Of the Gen-Probe-negative M. avium complex strains, twenty-two reacted with the SNAP X probe; all 22 strains were identified as members of the M. avium complex by mycolic acid analysis. Included in this group were three culture collection strains: ATCC 29555 (serotype 6); ATCC 35847 (serotype 7); and ATCC 35770 (serotype 18). Also included were three clinical strains that were serotyped 6, 9, and 16. Eight of the 22 strains did not react with any of the 28 antisera used for serotyping.
660
TABLE 2
C.L. Woodley et al.
Comparison of SNAP Mycobacterium tuberculosis Complex Culture Identification with Standard Biochemical Identification SNAP Identification as Mycobacterium tuberculosis Complex
Biochemical Identification as Mycobacterium tuberculosis Complex
Positive
Negative
44 2
0 154
Positive Negative Sensitivity = 100.0%; and specificity = 98.7%. TABLE 3
Comparison of SNAP Probe with Gen-Probe with Selected Strains Positive for Mycobacterium tuberculosis
Species ~
Number Tested
SNAP
Gen-Probe
Mycobacterium tuberculosis bovis bovis BCG asiaticumb Other species
36 3 5 4 42
36 3 5 2 0
36 3 5 0 0
qdentified by biochemicaltests. bldentified also by mycolicacid analysis. TABLE 4
Comparison of SNAP Mycobacterium avium Complex Culture Identification with Standard Biochemical Identification SNAP Identification as Mycobacterium avium Complex
Biochemical Identification as Mycobacterium avium Complexa Positive Negative
Positive
Negative
91 2b
8 99
~Sensitivity = 91.9%; and specificity = 98.0%. bIdentified as M. scrofulaceum.
Four of the strains could not be tested because of autoagglutination and the last four could not be confirmed.
DISCUSSION The Syngene SNAP assay for M. tuberculosis showed an accurate identification with sensitivity of 100% and specificity of 98.7% compared with standard biochemical identification. These numbers are similar to those reported by other investigators for the present commercial D N A - R N A probes (Kiehn and Edwards, 1987; Sherman et al., 1989). Two falsepositive results with M. asiaticum indicate there may be a degree of cross-reaction with this species. Lim
et al. (1991) recently reported two discrepant reactions while evaluating the SNAP assay for M. tuberculosis complex. Two strains identified as M. terrae complex by mycolic acid analysis were M. tuberculosis-probe positive. They noted no other discrepancies with the M. tuberculosis complex probe. The SNAP M. avium complex assay included a probe component (X) that detected strains identified by standard biochemical methods as M. avium complex, but negative by the Gen-Probe assay. We have collected a number of these strains since our initial studies with Gen-Probe (Woodley et al., 1989), and these are well represented in the present study creating a bias result in a direct comparison of probes from the two manufacturers (Woodley et al., 1989). Characterization of these strains in our laboratory using
Evaluation of Syngene DNA-DNA Probe Assays
TABLE 5
661
Comparison of SNAP Probe with Gen-Probe with Selected Strains a Positive
Species b
Number of Strains b
SNAP
Gen-Probe
99
91
69
7 4 1 6 5
2 0 0 0 0
1 0 0 0 0
28
0
0
Mycobacterium avium complex scrofulaceum xenopi malmoense simiae terrae
Other species
aApproximatelyequal number of M. avium, M. intracellulare, and Gen-Probe-negativeM. avium complex strains were selected. bldentifiedby biochemicaltests.
TABLE 6
Strains Positive to SNAP Assay Tested with the Individual Probes of Mycobacterium avium Complex Kit
Source
No.
Strains Collection Clinical
10 83 93
Total
"A. . . .
I. . . .
X"
4 29
3 30
3 20
33a
33b
23c
"Positivefor Gen-ProbeM. avium (includesATCC25291, 35767, 35768, and 35773). bpositive for Gen-ProbeM. intracellulare(includesATCC35769, 13950, and 35782). CNegativefor both Gen-Probes (includesATCC35847, 29555, and 35770).
mycolic acid analysis and biochemical identification has shown most to be typical of the M. avium complex. Because type strains of serotypes 6, 7, and 18 reacted with the SNAP X probe, both M. avium and M. intracellulare serotypes can be classified as X-positive strains by SNAP probes. Saito et al. (1990) found that some reference strains for serotypes 23, 24, and 28 were negative with both M. avium and M. intracellulare Gen-Probes. We also found strains (not included in the 200 strains) belonging to serotypes 23, 24, and 28 that were Genprobe negative for M. avium complex, but that reacted with the SNAP X probe. Two ATCC strains that reacted with the SNAP X probe, ATCC 35770 and ACTCC 35847, had previously been reported by Wayne and Diaz (1987) as the only strains that did not give a positive reaction among 11 M. intracellulare isolates tested with T-catalase dot-blot immunoas-
say. Tasaka et al. (1985 and 1986), using oLantigens as markers for serologic identification of the M. avium complex, reported the absence of the specific M. avium complex, e¢ antigen with the culture identified as ATCC 35847. In addition, Picken et al. (1988) showed that these two strains and ATCC 29555 have a unique restriction-fragment-length polymorphism pattern that differs significantly from the patterns of M. avium or M. intracellulare. Hawkins (1977) described a number of M. avium complex and related strains that exhibited a perfect fit on the basis of biochemical reaction for M. avium complex, though deeply pigmented. On the other hand, there are reports on "intermediate" strains that possess biochemical features of both M. avium complex and M. scrofulaceum (Murphy and Hawkins, 1975). These strains have high catalase activity with negative or occasional positive urease test result. Several of the strains that react with the SNAP X probe fit into the "intermediate" category between M. avium complex and M. scrofulaceum, and although the majority produce yellow pigment, only two have been identified biochemically as M. scrofulaceum. Presently, our studies indicate that strains positive with the Syngene SNAP X probe are members of M. avium complex by standard identification, serology, and mycolic acid identification with some strain variations. On the other hand, further studies, such as DNADNA homologies, may position these strains as either one or more new species. The SNAP DNA-DNA probe identification procedure was an effective method for identifying two important groups of mycobacteria. The procedure required no special equipment (except the minibead beater) and thus may prove especially suited for small laboratories.
662
C.L. W o o d l e y et al.
REFERENCES Baess I (1983) Deoxyribonucleic acid relationships between different serovars of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum. Acta Pathol Microbiol Scand [B] 91:201-203. Butler WR, Kilburn JO (1988) Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids. J Clin Microbiol 26:50-53. Drake TA, Hindler JA, Berlin OGW, Bruckner DA (1987) Rapid identification of Mycobacterium avium complex in culture using DNA probes. J Clin Microbiol 25:14421445. Gonzalez R, Hanna BA (1987) Evaluation of Gen-Probe DNA hybridization systems for the identification of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare. Diagn Microbiol Infect Dis 8:69-77. Good RC, Beam RE (1984) Seroagglutination. In The Mycobacteria: A Sourcebook. Eds, G Kubica and L Wayne. New York: Marcel Dekker, pp 105-122. Good RC, Snider DE (1982) Isolation of nontuberculosis mycobacteria in the United States. J Infect Dis 146:829833. Hawkins JE (1977) Scotochromogenic mycobacteria which appear intermediate between Mycobacterium avium-intracellulare and Mycobacterium scrofulaceum. Am Rev Respir Dis 116:963-964. Kent PT, Kubica GP (1985) Public Health Mycobacteriology: A Guide for the Level III Laborato~. Atlanta GA: Centers for Disease Control. Kiehn TE, Edwards FF (1987) Rapid identification using a specific DNA probe of Mycobacterium avium complex from patients with acquired immunodeficiency syndrome. ] Clin Microbiol 25:1551-1552. Lambert MA, Brehmer W, Sonneborn HH, Horn J, Wagner S (1986) Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography. J Clin Microbiol 23:733-736. Lira SD, Todd J, Lopez J, Ford E, Janda JM (1991) Genotypic identification of pathogenic Mycobacterium species by using a nonradioactive oligonucleotide probe. ] Clin MicrobioI 29:1276-1278. Mauch HW, Brehmer W, Sonneborn HH, Horn J, Wagner S (1988) Monoclonal antibodies selectively directed against the cell wall surface of Mycobacterium tuberculosis. J Clin Microbiol 26:1691-1694. McClatchy JK (1981) The seroagglutination test in the study of nontuberculous mycobacteria. Rev Infect Dis 3:867870. Minnikin DE, Minnikin SM, Parlett JM, Goodfellow M, Magnusson M (1984) Mycolic acid patterns of some species of Mycobacterium. Arch MicrobioI 139:225-231. Murphy DB, Hawkins JE (1975) Use of urease test disks in the identification of mycobacteria. J Clin Microbiol 1:465-468. Musial CE, Tice LS, Stockman LS, Roberts GD (1988) Identification of mycobacteria from cultures by using the
Gen-Probe rapid diagnostic system for Mycobacterium avium complex and the Mycobacterium tuberculosis complex. ] Clin Microbiol 26:2120-2123. Nishimori E, Yugi H, Naiki M, Sugimura T, Tanaka F, Nonomura I, Yokomizi Y, Kubo S (1987) Production and characteristics of serovar-specific monoclonal antibodies to serovars 4, 8, and 9 of Mycobacterium intracellulare. Infect Immun 55:711-715. Picken RW, Tsang AY, Yang HL (1988) Speciation of organisms within the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex based on restriction fragment length polymorphism. Mol Cell Probes 2:289-304. Saito H, Timioka H, Sato K, Tasaka H, Dawson DJ (1990) Identification of various serovar strains of Mycobacterium avium complex by using DNA probes specific for Mycobacterium avium and Mycobacterium intracellulare. J Clin Microbiol 28:1694-1697. Sch6ningh R, Verstijnen, CPH, Kuijper S, Kolk AHJ (1990) Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the Mycobacterium tuberculosis and Mycobacterium avium complexes and derived from early cultures. ] Clin Microbiol 28:708-711. Sherman I, Harrington N, Rothrock A, George H (1989) Use of a cutoff range in identifying mycobacteria by the Gen-Probe rapid diagnostic system. J Clin Microbiol 27:241-244. Siddiqi SH, Hwangbo CC, Silcox V, Good RC, Snider JR, DE, Middlebrook G (1984) Rapid radiometric methods to detect and differentiate Mycobacterium tuberculosis/M. bovis from other mycobacterial species. Am Rev Respir Dis 130:634-640. Tasaka H, Nomura T, Matsuo Y (1985) Specificity and distribution of alpha antigens of Mycobacterium aviumintracellulare, Mycobacterium scrofulaceum, and related species of mycobacteria. Am Rev Respir Dis 132:173-174. Tasaka R (1986) Development of serological identification of slowly growing mycobacteria with alpha antigen as a marker. Kekkaku 61:663-669. Udou T, Mizuguhi Y, Wallace Jr RJ (1987) Patterns and distribution of aminoglycoside-acetylating enzymes in rapidly growing mycobacteria. Am Rev Respir Dis 136:338343. Wayne LC, Diaz CA (1987) Intrinsic catalase dot blot immunoassay for identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. J Clin Microbiol 25:1687-1690. Woodley CL, Silcox VA, Floyd MM, Kubica GP (1989) The use of DNA probes for rapidly identifying cultures of Mycobacterium. In Rapid Methods in Clinical Microbiology: Present Status and Future Trends (Advances in Experimental Medicine and Biology Series). Eds, B Kleger, D Jungkivid, E Hinks, and LA Miller. New York: Plenum, pp 51-56. Yakrus MA, Good RC (1990) Geographic distribution frequency, and specimen source of Mycobacterium avium complex serotype isolated from patients with acquired immunodeficiency syndrome. J Clin Microbiol 28:926929.
