Non-radioactive DNA probe for the rapid identification of Mycobacterium avium complex from clinical specimens

Molecular and Cellular Probes (1991) 5, 27 7-280

Non-radioactive DNA probe for the rapid identification of

Mycobacterium avium complex from clinical specimens C. H . Huang and D . L. Jungkind Department of Pathology, Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA (Received 17 September 1990, Accepted 19 January 1991)

We evaluated an alkaline phosphatase labelled oligonucleotide probe (SNAP", Syngene Co ., Molecular Biosystems, Inc ., San Diego CA) for the direct culture identification of Mycobacterium avium complex (MAC) isolated from clinical specimens . Mycobacterial species identified by conventional biochemical methods were retested with this DNA probe using the Centri-Dot"' format . The probe accurately identified all 69 pigmented M. avium complex and 15 non-pigmented isolates of M . avium complex . There were no false-positives with 45 non-MAC mycobacteria isolates (10 species) and 16 non-mycobacteria organisms (10 species) . The sensitivity and specificity of the SNAPT' culture identification for M . avium complex were 100% . The alkaline phosphatase labelled DNA probe is stable for at least 9 months . The procedure can be completed within 2 h and is easily adapted in the clinical laboratory . For the strains encountered in our laboratory, we conclude that the SNAP' hybridization is a rapid, specific, and reliable method for culture identification of M . avium complex .

KEYWORDS: Mycobacterium avium complex, alkaline phosphatase labelled oligonucleotide probe

(SNAP"'), Probes A, I, and X .

INTRODUCTION poorly to traditional antituberculosis medications .', ',' Rapid identification of MAC is important for early appropriate therapy which consists of rifampin, clofazimine, ethambutol, ciprofloxacin or amikacin ." A radioactive DNA probe is commercially available for rapid culture identification of MAC . The disadvantages of radioactive probes are short half-life of the radioisotope, and potential radiation hazard . In this study, we evaluated an alkaline phosphatase labelled oligonucleotide probe for rapid culture identification of M. avium complex.

complex (MAC) is the most common isolate in patients with the acquired immunodeficiency syndrome (AIDS) in the United States ." Disseminated MAC infection is frequent in AIDS patients . In a recent study, 96% of disseminated non-tuberculous mycobacterial infections were due to M. avium complex .' MAC infection in non-AIDS patients has also steadily increased in recent years .' Previously, typical non-AIDS patients with pulmonary disease caused by M . avium complex were most likely to be elderly white males with progressive chronic lung disease .' The incidence of MAC infection in patients without predisposing conditions, such as chronic obstructive pulmonary disease, immunosuppressive therapy or a defect of cellular immunity other than AIDS, was also observed with increasing frequency. ',' Patients with MAC infection respond Mycobacterium avium

MATERIALS AND METHODS Organisms A total of 84 M . avium complex, and 45 non-MAC mycobacteria were originally recovered from clinical

Presented in part at the ASM annual meeting, May 13-17, 1990, Anaheim, CA .

0890-8508/91/040277 + 04 $03 .00/0

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specimens by the Thomas Jefferson University Hospital (TJUH) Mycobacteriology Laboratory . These isolates were isolated from different patients during the periods from January to December 1989 . Mycobacteria cultures were stored on Lowenstein Jensen (LJ) medium at room temperature for not more than 1 year . The non-mycobacteria species were fresh isolates from respiratory specimens . All species were identified by conventional biochemical methods . 10'11 Pigmented MAC is sometimes an environment isolate associated with the hot water supply at TJUH . Therefore, we routinely report the pigmentation of MAC to clinicians to aid their interpretation regarding clinical significance of the isolate . Pigment production was determined according to the conventional method .' °

DNA probe method TM We evaluated the SNAP (Syngene Inc ., San Diego ., CA 92121) culture identification diagnostic kit for accuracy of M . avium complex identification . The SNAPTM olignucleotide probe consists of three probes designated as A, I, and X . Probes A and I react specifically to M. avium and M . intracellular respectively . Probe X is an additional probe designed to detect any strains which are not detected by either probe A or I, but identified as MAC, such as M . avium strain (ATCC 29555) or M . intracellular (ATCC 35847 and 35770) (B . C . Lin, L . Kung and K . Kleinbenz, program abstr . 1990, ASM annual meeting Abstr No . U-12) . The assay procedure consisted of four stages : (1) lysis of bacteria ; (2) denaturation and fixation of nucleic acids to a Centri-Dot TM membrane ; (3) hybridi-

brane wash 2 for 15 min each at 50 ° C . The membrane was then blotted gently with a paper towel . The hybridized membrane was placed in a fresh reaction bag containing 1 . 0 ml alkaline phosphatase substrate buffer and 5 µI NBT substrate (nitro blue tetrazolium chloride in 70% v/v dimethylformamide in water) and 5 µl BCIP substrate (5-bromo-4-chloro-3-indolylphosphate in 70% v/v dimethylformamide) . The reaction bag was incubated in the dark at 37 ° C . A positive purple colour usually developed within 15 min .

RESULTS SNAPTM Centri-DotTM colour reaction Figure 1 shows the typical positive and negative reactions of this oligonucleotide probe . Mycobacterium avium (ATCC 25291) and M. tuberculosis (ATCC 25177) were used as a positive and negative control for each run . A positive purple colour usually developed within 15 min . The colour development would take longer if the cultures were too dry to scrape enough organisms . Comparison of SNAP' DNA probe for MAC identification with biochemical methods : The total of 69 pigmented MAC and 15 non-pigmented MAC identified by conventional biochemical methods were retested with SNAP` DNA probe. There were no false-negative results with these clinical isolates .

zation of an alkaline phosphatase labelled probe ; and (4) detection with enzyme substrates . The DNA probe procedure was performed according to the recommendation of the manufacturer . A loopful (1 gl inoculating loop) of culture from LJ medium was transferred to a lysis tube which contained 1 . 0 ml lysis buffer, 0. 3 ml chloroform and glass beads . The lysis tube was placed in a MiniBead Beater (Biospec Products, Bartiesville, OK) and agitated at high speed for 3 min. The supernatant was obtained after centrifuging the lysis tubes for 5 min at 2000 X g . We transferred 0 . 3 ml of supernatant to each well of the Centri-Dot' which contained 0 . 3 ml of denaturation reagent. The Centri-DotTM was centrifuged at 1000 x g for 5 min . The membrane was removed from the Centri-Dot and ringed in distilled water for 15 s . The membrane was then hybridized in a reaction . bag containing 1 . 0 ml hybridization buffer and 30µl of working MAC probe mix (probes A, I, and X) at 50° C . After 15 min, the membrane was washed with working HI TEMP membrane wash 1 and HI TEMP mem-

Identification of Mycobacterium avium complex using Centri-Dot"' format by SNAP' oligonucleotide probe. The positive reaction was identified by the purple colour . No colour developed for negative reaction . Wells 1, 3 and 5 were positive.

Fig. 1 .

Rapid identification of

M . avium .

279

Cross reactivity with non-MAC mycobacteria

MAC . There were no false-positives with 45 non-MAC

The SNAPT' DNA probe showed no false-positives on 45 non-MAC mycobacteria representing 10 species, including M . tuberculosis (n = 20), M . kansasii (9), M .

mycobacteria and 16 non-mycobacteria organisms . The overall sensitivity and specificity of the SNAP TM culture identification system were 100% . The SNAPTM DNA probe can be easily adapted to the clinical laboratory . With the availability of a

fortuitum (9), M . szulgai (2), M. flavescens (1), M . gordonae (1), M . terrae (1), M. marinum (1), M . xenopi

(1) and M . scrofulaceum (10) .

Cross-reactivities with non-mycobacteria There were no false-positive reactions with Norcardia sp . (n = 4), Streptomyces sp . (1), Staphylococcus aur-

centrifuge and water bath in most mycobacteriology laboratories, the only additional equipment needed is the MiniBead Beater. The lysate is stable for a week at 0 ° C or at - 80°C, so samples can be batched for DNA probe testing. The advantage of this alkaline phosphatase oligonucleotide probe is long shelf-life . The

eus (3), Neisseria meningitidis (2), Enterobacter cloacae (1), Pseudomonas aeruginosa (1), Haemophilus influenzae (1), Streptococcus pneumoniae (1), Streptococcus pyogenes (1), and Proteus mirabilis (1) .

probe is stable for at least 9 months . Additional work would be useful to study geographical and strain variation and to evaluate the effect of other variables such as media . We concluded that the SNAPTM non-radioactive probe with the Centri-Dot T' hybridization procedure was a

Stability of the lysate

rapid, specific, and reliable method for MAC culture identification using strains isolated on LJ medium from our patient population .

We stored aliquots of the supernatant of the bacterial lysate at 0 ° C and -80 ° C . The intensity of the final colour developed after 7 days storage, either at 0 ° C or -80° C showed no significant difference from the

ACKNOWLEDGEMENT

fresh lysate .

We thank Molecular Biosystems, Inc . for providing SNAP` culture identification diagnostic kits . Special thanks to Sam Morishima for general assistance with the study .

Stability of the DNA probe

REFERENCES

The alkaline phosphatase labelled DNA probe was very stable . In our hands, the kit 6 months after expiration date performed as well as the fresh dated

7.

kit .

DISCUSSION Mycobacterium avium

1 . Horsburgh, C . R . Jr . & Selik, R. M . (1989) . The epidemiology of disseminated nontuberculous mycobacterial infection in the acquired immunodeficiency syndrome (AIDS) . American Review of Respiratory Disease 139, 4-

complex is a frequent isolate

in non-AIDS and AIDS patients with mycobacteria infection .`, ` The majority of these isolates are resistant in vitro to most conventional antimycobacterial drugs . The rapid diagnosis of MAC and the use of multiple drug regimens not only appears to prolong survival but also improve quality of life ." Conventional biochemical identification requires at least 2 weeks after a pure culture is obtained . The total procedure of the SNAP` non-radioactive probe test takes as little as 2 h after cultivation . Hands-on time is only 45 min for six samples . In comparing the SNAPTM culture identification with conventional biochemical methods, there were no false-negatives in the total number of 84 MAC including 69 pigmented MAC and 15 non-pigmented

2 . MacDonell, K . B . & Glassroth, J . (1989) . Mycobacterium avium complex and other nontuberculous mycobacteria in patients with HIV infection . Seminars in Respiratory Infection 4, 123-32 . 3 . Saubolle, M . A. (1989). Nontuberculous mycobacteria as agents of human disease in the United States . Clinical Microbiology News 11, 113-7 . 4 . Iseman, M . D . (1989) . Mycobacterium avium complex and the normal host . New England Journal of Medicine

321, 896-7 . 5 . Wolinsky, E . (1979). Nontuberculous mycobacteria and associated disease. American Review of Respiratory Disease 119, 107-59. 6 . Prince, D . S ., Peterson, D . D ., Steiner, R . M . et al . (1989) . Infection with Mycobacterium avium complex in patients without predisposing conditions. New England Journal of Medicine 321, 863-8 . 7 . Hawkins, C. C ., Gold, J . W . M ., Whimbey, E . et al. (1986) .

complex infections in patients with the acquired immunodeficiency syndrome . Annal of Internal Medicine 105, 184-8. 8. Safrin, S . & Jacobson, M . A . (1989) . Mycobacterial infection in AIDS : The growing challenge . Journal of Respiratory Diseases 10, 30-41 . Mycobacterium avium



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9 . Centers for Disease Control . (1987) . Diagnosis and management of mycobacterial infection and disease in persons with human immunodeficiency virus infection . Annals of Internal Medicine 106, 254-6 . 10 . Kent, P . T . & Kubica, C . P. (1985) . Public health mycobacteriology, A Guide for the level III laboratory . Centers for Disease Control, Atlanta, GA . 11 . Koneman E . W., Allen, S . D . Dowell Jr ., V . R ., Janda, W . M ., Sommers, H . M . & Winn Jr., W . C . (1988) . Color

Atlas and Textbook of Diagnostic Microbiology 3rd edn, Philadelphia, PA : J . B . Lippincott Co. 12 . Young, L . S ., Inderlied, C. B ., Berlin, 0 . C . & Gottlieb, S . (1986). Mycobacterial infections in AIDS patients, with an emphasis on the Mycobacterium avium complex . Reviews of Infectious Diseases 8, 1024-33 . 13 . Young, L . S . (1988) . Mycobacterium avium complex infection . Journal of Infectious Diseases 157, 863-7 .