Polymerase chain reaction to detect Mycobacterium tuberculosis in a clinical microbiology laboratory

Journal of Microbiological Methods, 16 (I 992) 139-147 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167 - 7012/92/$05.00

139

MIMET 00521

Polymerase chain reaction to detect Mycobacterium tuberculosis in a clinical microbiology laboratory E. Veringa a, B. van Harsselaar ~ a n d P. H e r m a n s b aDepartment of Clinical Microbiology, University Hospital Leiden, Leiden, Netherlands and bNationai Institute of Public Health and Environmental Protection, Bilthoven, Netherlands (Received 3 December 1991; revision received 13 April 1992; accepted 15 April 1992)

Summary Direct detection of DNA from Mycobacterium tuberculosis in clinical specimens was performed by in vitro amplification of DNA using the polymerase chain reaction (PCR), followed by specific detection of the amplified product using DNA hybridization with a non-radioactive detection system. Primers based on a recently described insertion element IS986 were used to detect M. tuberculosis. The results were compared with results from traditional culture techniques. A total number of 167 sl~'imens were examined. PCR and hybridization results were positive in 54 of 67 culture positive specimens. The number of PCR positive specimens was increased to 63 by concentrating the samples by ethanol p ~ p i t a t i o n prior to amplification. This treatment, however, decreased specificity. The negative PCR reaction in the remaining 4 culture positive samples could be explained by the presence of inhibitors. Of the 40 culture negative samples 5 were PCR positive, one of which was false positive. PCR and hybridization generated results significantly more rapidly than traditional culture techniques, but the former assay also appeared to be more expensive.

Key words: Polymerase chain reaction; Tuberculosis; Clinical specimen

Introduction Current methods for isolation and identification of Mycobacterium species rely primarily on culture and biochemical procedures [1]. These methods require considerable time due to the extremely slow growth of most myeobacteria. Microscopic examination of smears of specimens (stained with auramine or according to ZiehlNeelsen) is the most rapid method to detect the bacilli. Unfortunately, this method is neither sensitive nor specific and confirmation by time-consuming culture techniques is still required. Therefore, more rapid, specific and sensitive methods to detect and identify mycobacteria are highly desirable. Such techniques have recently become Correspondence to." E. Veringa, Department of Infectious Diseases and Immunology, SSDZ, Reinier de Graafweg 7, 2625 AD Delft, Netherlands.

140 available through nucleic acid probe technology. DNA probes have been used to identify mycobacteria in culture [2,3]. The sensitivity of DNA probe assays can be increased significantly b)' in vitro amplification of mycobacterial DNA prior to hybridization, for example by means of the polymerase chain reaction (PCR) [4-7]. PCR has been used successfully to identify Mycobacterium tuberculosis from cultures [8-10] as well as to rapidly detect Mycobacterium tuberculosis directly in clinical specimens [10-16]. Recently, Hermans and coworkers have described an insertion element (IS986) specific for Mycobacterium tuberculosis complex strains [17]. The aim of the present study was to extend the work by Hermans and associates by performing PCR, based on IS986, on clinical samples. Also the economic aspects of the assays will be discussed. Materials and Methods

Specimens Clinical specimens collected at nine different hospitals were submitted to two large clinical microbiology laboratories. The majority of specimens consisted of sputa from patients with pulmonary problems. However, other specimens were also included in the study (Table 1). Smears were prepared from all specimens, except urine, and stained with auramine and/or according to Ziehl-Neelsen (ZN). Mycobacteria were cultured on L6wenstein-Jensen (L-J) agar for all specimens. Prior to culture, specimens from non-sterile sites were decontaminated to prevent growth of microorganTABLE ! Specificity of the assay Organism

Number of strains tested

PCR +

M. M. M. M. M. M. M. M. M. M.

3 3 3 1 I 1 I i 2 2

3 3 0 0 0 0 0 0 0 0

3 1 1 1 1 1 1

0 0 0 0 0 0 0 0

tubercu!osis a boris (BCG) a:,ium kansasii fortuitum gordonae scrophulaceum terrae chelonei xenopi

P. eeruginosa E. coli S. aureus K. pneumoniae S. pneumoniae H. influenzae M. catarrhalis

Normal oral flora b

~Including strain H37Rv. bStreptococcus viridans strains, diphtheroids and apathogenic Neisseria species.

14I isms other than mycobacteria. Decontamination procedures were laboratory depen-: dent. In one laboratory, specimens were treated with 1 N NaOH and neutralized with 1 N H2SO4, followed by centrifugation after which the pellets were resuspended in physiologic salt solution (PSS). In the other laboratory, specimens were decontaminated using commercially available Zephyrol (Hospidex by, Nieuwkoop, Netherlands). Specimens from normally sterile sites such as blood, spinal fluid, etc., were cultured on sheep blood agar also. These specimens were decontaminated only when growth of (contaminating) microorganisms was observed on blood agar. Part of each specimen was used for culture, the remaining material was stored at - 2 0 ° C for PCR and hybridization studies. Prior to PCR and hybridization, the specimens were thawed, diluted 2- or 5-fold (depending on the decontamination procedure used) with sterile distilled water and heated for 5 min at 100°C to lyse the cells. After mixing and centrifugation (5 min at 16 000 rcf), the supernatants were used for PCR. On negative specimens the sensitivity of the assay was increased by concentration of DNA by ethanol precipitation, using glycogen as carrier, prior to amplification. A mixture of 100 ~1 of supernatant, 3 volumes of 95% ethanol, one-tenth volume of 3 M NaAc (pH 5.4) and 2 ltl of glycogen solution was placed on ice for 30 min. After centrifugation for 20 min at 16 000 rcf, the pellet was washed with 70% ethanol, dried and resuspended in 30 #1 sterile distilled water. To a duplicate sample of each of the, initially negative, specimens, D N A from Mycobacterium tuberculosis was added (spiked) to check for the presence of inhibitors of the PCR.

Bacteria Mycobacterium species were grown on L-J medium. Other bacteria were grown on blood agar plates. Colonies were suspended in sterile distilled water and heated for 5 min at 100°C. After vigorous mixing and centrifugation, the supernatants were used in the amplification reaction in appropriate dilutions.

PCR (1) Oligonucleotides.

The synthetic oligonucleotide primers INS1 (5'C G T G A G G G C A T C G A G G T G G C - Y ) and INS2 ( 5 ' - G C G T A G G C G T C G G TGACAAA-3') were used as primers in the amplification reaction [17]. In the presence of target DNA, amplification resulted in a 245 bp D N A fragment. As an IS-DNA probe for hybridization of amplified DNA, we used the oligonucleotide INS4 with the sequence 5'-TGGGTAGCAGACCTCACCTA-3', corresponding to an internal sequence of the 245 bp fragment. INS4 was labeled with digoxigenin-1 ldUTP using the D N A tailing kit marketed by Boehringer Mannheim B.V. (Almere, Netherlands). The method involved the tailing of 200 ng of oligonucleotide in a 20/~1 reaction (1 #1 (200 ng) INS4, 4 pl tailing buffer, 6 #1 CoCL2, 2/11 digoxigenin-lldUTP, 2 ~1 dATP (1:50 in 10 mM Tris-HC1, pH 7.5), 1 #1 TdT and 4 #1 sterile distilled water). The reaction mixture was incubated for 5 min at 37°C. The tailed oligonucleotide was purified from the labeling reaction by ethanol precipitation using one-tenth volume of 3 M NaAe (pH 5.4), 1 #1 of glycogen solution and 3 volumes of 95% ethanol. The mixture was then placed on ice for 5 min and centrifuged for 15 min at 16000 rcf. After removal of the supernatant, the pellet was

142 washed briefly in 95% ethanol, dried and resuspended in 200 /~1 sterile distilled water. (2) Reaction mixture. Amplification mixtures were prepared each day and consisted of the following ingredients: 0.5 U of Taq polymerase (Perkin Elmer Nederland B.V., Gouda, Netherlands), 150 ng of each primer, 3 mM MgCI2, 50 mM NaCI, 10 mM Tris, 2 mg/ml bovine serum albumin and 0.2 mM of each dNTP in sterile distilled water (~na! concentrations). The mixture was divided in 45 /zl aliquots and covered with sterile mineral oil. 5 /~1 of specimen suspension was pipetted through the mineral oil into the mixture. When spiking duplicate samples, 5 #! of DNA from M. tuberculosis was added to 40 #! of amplification mixture and 5/11 of specimen suspension. The amplification reaction was performed using an automated thermal cycler (DNA Thermal Cycler, Perkin Elmer). The samples were denatured for 3 min at 94°C, followed by 32 amplification cycli. The cycle consisted of denaturation for 30 s at 94°C, annealing of primers for 45 s at 60°C, and primer extension for 2 rain at 72°C. Prior to hybridization experiments, electrophoresis was carried out on 10/zl of each amplified sample in 2% agarose gels containing 500/~g. 1- t ethydium bromide (final concentration). Photographs of agarose gels were taken on a 302 nm ultraviolet transilluminator. (3) Controls. In each experiment the following controls were included: one negative control (sterile distilled water) for every specimen and two positive controls (DNA from Mycobacterium tuberculosis).

Hybridization of amplified material PCR products (40/zl) were denatured by heating for 10 min at 100°C in 200/zl EDTA/NaOH buffer (final concentrations 10 mM and 0.4 M, respectively). The samples were cooled rapidly and loaded into wells of a dot-spot apparatus (Bio-Rad Laboratories B.V., Veenendaal, Netherlands) fitted with a Hybond N ÷ membrane (Amersham Nederland B.V., Houten, Netherlands) previously wetted in sterile distilled water. After transferring the DNA to the membrane, the membrane was wetted in 0.4 M NaOH and subsequently washed twice in 2 x SSC and dried. Hybridization was usually carried out shortly afterwards (same day). If hybridization was not performed on the same day, the membranes were heated for 30 min at 80°C and stored at room temperature until use. Prehybridization of the membranes was performed in a sealed plastic bag in final concentrations of 0.6 M NaCI, 0.2 M Tris-HCl (pH 7.5), 0.1% SDS, 1 mM EDTA, 0.25% blocking agent (Boehringer Mannheim), and 300 mg. !--1 herring sperm DNA. After incubation for 30 rain at 40°C, 200 ng of digoxigenin-11-dUTP labeled probe, INS4, was denatured by heating for 5 min at 100°C and added to 10 or 20 ml (depending on the size of the membrane) prehybridization fluid. Hybridization was allowed to occur by incubation for 2 h in a 40°C shaking water bath. Washing procedures included: 10 min incubation at room temperature in 0.3 M NaCI, 0.1 M Tris, 0.5% SDS, 10 min incubation at 40°C in 0.1 M NaCI, 0.1 M Tris, 0.5% SDS, and 5 min incubation (twice) at room temperature in 0.3 M NaC1, 0.1 M Tris. Labeled hybridized probe was detected using the Nucleic Acid Detection kit marketed by Boehringer Mannheim. Detection of digoxigenin-11-dUTP labeled probe, after hybridization to target nucleic acid, is based on enzyme-linked immunoassay using

143 an antibody conjugate (anti-digoxigenin alkaline phosphatase conjugate). A ~ubsequent enzyme-catalyzed color reaction with 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) and nitroblue tetrazolium salt (NBT) produces an inso!ubte blue precipitate, which visualizes hybrid molecules. Detection of labeled, hybridized probe was carried out under continuous gentle shaking at room temperature. The membranes were washed briefly (1 min) in buffer A (100 mM Tris, 150 mM NaCI, pH 7.5). The membranes were subsequently incubated in 1% (w/v) blocking reagent in buffer A to prevent background color development. After the blocking step, the membranes were washed again briefly ;,, buffer t, Membranes were ~,,b~au~ntlv incubated for 30 min in freshly prepared diluted antibody conjugate solution (150 mU conjugate per ml buffer A). Unbound antibody conjugate was removed by washing the membranes for 3 x l0 rain in buffer A. Membranes were equilibrated for 2 min with buffer B (100 mM Trh-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5). Membranes were then incubated with approximately l0 or 20 ml (depending on the size of the membrane) freshly prepared color substrate solution (45 #1 NBT solution, 35/A Xphosphate solution and l0 ml of buffer B) sealed in a plastic bag in the dark at room temperature, without shaking. When the desired spots were detected (usually after 218 h) the reaction was stopped by washing the membranes for 5 min in l0 x TE buffer (100 mM Tris-HC1, l0 mM EDTA, pH 8.0). When desired, the results were documented by photography of the membranes dried at room temperature. Membranes were stored in 10 x TE buffer, sealed in plastic bags, in the dark. Results

To ensure that the primers used were specific the PCR was attempted in a number of Mycobacterium species and other bacteria. As shown in Table 1 the reaction was specific for M. tuberculosis and M. bovis, belonging to M. tuberculosis complex. A total of 107 clinical specimens were tested (Table 2). From 66 specimens Mycobacterium tuberculosis was cultured, one specimen grew Mycobacterium bovis TABLE2 Specimens tested Specimen

Number of samples

Sputum Broncheal lavage Broncheal brush Pleural aspirate Stomach aspirate Lymph node Cerebrospinal fluid Joint aspirate Abscess/pus Urine Ascites

59 18 2 3 i 6 1 3 7 7 1

Total

107

144 TABLE 3 Comparison of culture and PCR to detect mycobacteria in sputum

PCR + PCR --

Culture +

Culture -

54 (63)~ 13 ( 4 ) a

5 (10) a 35 (30) a

67

40

~After concentration by ethanol precipitation.

BCG. 54 of these specimens were PCR positive. Of the 40 culture negative specimens five proved to be PCR positive (Table 3). One of these specimens was from a patient of whom material taken 1 day later was positive. Two samples were taken after treatment and were Ziehl-Neelsen positive, indicating the presence of killed bacteria, which still yield positive PCR. One culture negative and PCR positive sample was from a patient in whom the clinical diagnosis tuberculosis was made and who responded to therapy. In only one sample the PCR positivity could not be explained; this can be considered a false positive PCR result. To increase the sensitivity, samples in which the PCR was negative were concentrated by ethanol precipitation and retested with PCR. By concentration another nine culture positive samples became PCR positive (Table 3). In three of the four culture positive materials which were negative after concentration also, the negative PCR result could be explained by the presence of inhibitors, because added mycobacterial DNA also could not be amplified. Unfortunately, the increased sensitivity after ethanol precipitation also resulted in an increase in the number of false positive results and a loss of specificity (Table 4). Table 5 shows a comparison of the costs of reagents and the time requited to r". . . . . . . . . . . . , . , ~ o ~ . a ~ a t a U l l n ~ p t u n ~ m t u r e o r r ~ K p l u s nyonmzation. I n e price of equipment, such as thermal cycler and pipettes, is not included. Excluded also are the finances that may be needed to achieve the required laboratory infraqructure for performing PCR. r't~r~r,trr-,a

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Discussion The aim of the present study was to evaluate the PCR assay to detect Mycobacterium tuberculosis in clinical specimens. PCR has been reported by others to detect mycobacteria in clinical sample3 [1 !-14]. However, these methods included extensive TABLE 4 Sensitivity and specificity of the assay

Sensitivity Specificity

Direct PCR

PCR after concentration

80.6% 87.5%

94.0% 75%

145 TABLE 5 Economics compared for 10 specimensa ZN+ Reagents ($) Labor (h) Final result after

culture

7.25 10 4-8 weeks

PCR + hybridization 71.00 16 2-3 days

~One positive, nine negative specimens; including i0 negative controls and two positive controls.

DNA extraction and purification procedures as well as the use of radioactively labeled probes. We used a relatively simple method which did not include hazardous materials such as phenol, chloroform or radioisotopes. We applied primers from the IS986 insertion sequences which we have shown to be exclusively present in M. tuberculosis complex [17, present paper]. PCR performed directly on the specimens had a sensitivity of 80.6% when compared with the culture methods. The specificity of the reaction was 87.5%. Only one sample which was culture negative showed a false positive result in the PCR. The sensitivity could be enhanced by concentrating the samples by ethanol precipitation prior to amplification, but this decreased the specificity. Recently Sritharan and Barker [18] showed that heating with TE-Triton is the best method to prepare samples for the PCR reaction. Also, this treatment reduced the presence of factors which inhibited the PCR in the majority of PCR negative, culture positive specimens. Treatment with guanidine thiocyanate has also been reported to reduce the presence of inhibitors [19]. Positive results could be read at the end of the second day. The final readings were performed in the early morning of the third day which is significantly more rapid than traditional detection and identification of mycobacteria which usually requires 4-8 weeks. Although PCR plus hybridization generated results much more rapidly than culture techniques, the former method appeared to be almost twice as labor intensive. Another aim of the present study was to determine whether PCR and hybridization could be justified to be brought on-line in our clinical laboratory. The costs of the assay depend on the number of specimens tested. It is equally expensive to generate results from a negative specimen as from a positive specimen when using PCR plus hybridization. "i'hid is not true for culture techniques be,zat,se significantly more reagents arc used on positive specimens than on negative specimens. In the present study, we ran one negative control for every specimen, which, naturally, increased the price of the assay. A similar problem occurs with running the specimen controls (i.e., duplicate samples to which DNA from Mycobacterium tuberculosis was added) in all negative specimens. Such controls ate necessary to discriminate between negative results due to the absence of mycobacterial DNA and those due to the presence of inhibitors of the reaction. This problem may be solved by introducing a second set of primers, based on highly conserved parts of 16S rRNA sequences. This duplicate primer set added to fne same specimen could serve as specimen control. The advantage of using primers based on IS986 for detection of M. tuberculosis complex, rather than other sequences described to identify M. tuberculosis [8,9,11-

146

16], is that most of the latter sequences appeared to be highly conserved or not well characterized resulting in aspecific reaction patterns. The D N A sequence of IS986, however, was found to be highly specific for M. tuberculosis complex. The disadvantage of using detection of IS986 in screening procedures in search of mycobacteria is that mycobacteria not belonging to M. tuberculosis complex will be missed. Detection of mycobacteria other than M. tuberculosis complex such as M. avium complex is becoming increasingly important in AIDS-related cases. Therefore, the authors feel that genus specific primers (e.g., against 16S rRNA) would be advantageous in screening for mycobacterial infections whereas species specific primers (e.g., against IS986) would be of use in more specific species or complex identification. Although at present direct detection of M. tuberculosis by PCR presents certain problems that still need to be solved, we feel that the PCR technology will be extremely useful for clinical microbiology laboratories in the future, especially for direct detection and identification of microorganisms that grow slowly or not at all on artificial media. Acknowledgements We thank Dr. B.P. Overbeek, St. Antonius Hospital Nieuwegein, for providing us with clinical samples. We are also grateful to Dr. C. Verstijnen, Royal Institute of Tropical Hygiene, Amsterdam, and Dr. M.M.M. Salimans, University Hospital Leiden for excellent advice during the study. We are much indebted to Dr. H. Schellekens for his critically reading the manuscript. References i Tsukamura, M. (1981) A review of the'methods of identification and differentiation of mycobacteria. Rev. Infect. Dis. 3, 841-861. ....................... yu, M.M. and Kubica, G.P. (1990) The use of DNA probes for rapidly identifying cultures of Mycobacteriurn. Rapid Methods Clin. Microbiol. 26:;, 51-56. 3 Peterson, E.M., Lu, R., Floyd, C., Nakasone, A., Friedly, G. and de la Maza, L.M. (1989) Direct identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare from amplified primary cultures in BACTEC media using DNA probes. J. Clin. Microbiol. 27, 15431547. 4 Peter, J.B. (1991) The polymerase chain reaction: amplifying our options. Rev. Infect. Dis. 13, 166-171. 5 Mullis, K., Faloona, F., Scharf, S., S~iki, R., Horn, G. and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51,263--273. 6 Erlich, H.A., Gelfand, D.H. and Saiki, R.K. (1988) Specific DNA amplification. Nature 331,461-462. 7 Tenover, F.C. (1988) Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbiol. Rev. 1, 82-101. 8 B~ddinghaus, B., Rogall' T., Flohr,. T., Blrcker, H. and Bfttger, E.C. (1990) Detection and identification of mycobacteria by amplification of rRNA. J. Cli~. Microbiol. 28, i 751-1759. 9 Eisenach, K.D., Cave, M.D., Bates, J.H. and Crawford, J.T. (1990) Polymerase chain reaction amplification of a repetitive DNA sequence specific for Mycobacterium tuberculosis. J. Infect. Dis. 161, 977-981. 10 Thierry, D., Brisson-No~:l, A., ~'" . " " , mcent-Levy-Frebault, V., Nguyen, S., Guesdon, J.-L. and Gicquel, B. (1990) Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its

147 application in diagnosis. J. Clin. Microbiol. 28, 2668-2673. 11 Hermans, P.W.M., Schuitema, A.R.J., Van Soolingen, D., Verstynen, C.P.H.J., Bik, E.M., Thole, J.E.R., Kolk, A.H.J. and Van Embden, J.D.A. (1990) Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction. J. Clin. Microbiol. 28, 1204-1213. 12 Brisson-No61, A., Lecossier, D., Nassif, X., Gicquel, B., L~vy-Fr6bault, V. and Hance, A.J. (1989) Rapid diagnosis of tuberculosis by amplification of mycobacterial DNA in clinical samples. Lancet ii, 1069-1071. 13 De Wit, D., Steyn, L., Shoemaker, S. and Sogin, M. (1990) Direct det~tion of M).cobacteriura tuberculosis in clinical specimens by DNA amplification. J. Clin. Microbiol. 28, 2437-2441. 14 Shankar, P., Manjunath, N., Mohan, K.K., Prasad, K., Behari, M. and Shriniwas, A.G.K. (i991) Rapid diagnosis of tuberculous meningitis by polymerase chain reaction. Lancet 337, 5-7. 15 Hance, A.J., Grandchamp, B., L6vy-Fr6bault, V., Lecossier, D., Rauzier, J., Bocart, D. and Gicquel, B. (1989) Detection and identification of mycobacteria by amplification of mycobaeterial DNA. Mol. Microbiol. 3, 843-849. 16 Pao, C.C., Yen, T.S.B., You, J.-B., Maa, J.-S., Fiss, E.H. and Chang, C.-H. (1990) Detection and identification of Mycobacterium tuberculosis by DNA amplification. J. Clin. Microbiol. 28, 1877-1880. 17 Hermans, P.W.M., Van Soolingen, D., Dale, J.W., Schuitema, A.R.J., McAdam, R.A., Catty, D. and Van Embden, J.D.A. (1990) Insertion element IS986 from Mycobacterium tuberculosis: a useful tool for diagnosis and epidemiology of tuberculosis. J. Clin. Microbiol. 28, 2051-2058. 18 Sritharan, V. and Barker, R.H. (1991) A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. Mol. Cell. Probes 5, 385-395. 19 Brisson-Noel, A., Aznar, C., Chureau, C., Nguyen, S., Pierre, C., Bartoli, M., Bonete, R., Pialoux, G., Cicquel, B., and Garrigue, G. (1991) Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation. Lancet 388, 364-366.