zyxw zyxw zyxwvutsrqpo zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA TECHNICAL REPORT ~~~~ Direct detection of Mycobacterium tuberculosis in respiratory samples from patients in Scandinavia by polymerase chain reaction zyxwv Tone T 0 n j u m ' ~ Lisbeth ~, Klintx3, Tom Beyan*,/0rann Baann', Geir Furubeyl, Maria Cristea4, Bjmn Petrini' and Sven Ho#ne$ Institute of Microbiology, University of Oslo, Rikshospitalet (National Hospital), Oslo, Norway; 'Department of Microbiology, Ullevil University Hospital, Oslo, Norway; 3Swedish Institute for Infectious Disease Control, Stockholm, Sweden; 4Clinical Microbiology Laboratory, Karolinska Hospital, Stockholm, Sweden Objective: To investigate the use of DNA amplification by the polymerase chain reaction (PCR) for the detection of Mycobacteriurn tuberculosis directly in human respiratory specimens. zy Methods: The PCR assay employed was the Amplicor M. tuberculosis Test (Roche Diagnostics, Switzerland), which uses the 16s rDNA as the target template. Nine hundred and sixty samples from 741 patients in t w o clinical microbiology laboratories in Norway and Sweden were processed by routine culture analysis and PCR. Results: Of the 56 specimens containing cultivatable M. tuberculosis, 49 (87.5%) were detected by PCR. Among the 904 culture-negative specimens, 897 samples were negative also b y PCR and seven (0.8%) were positive by PCR. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of PCR were 91.7%, 99.6%, 94.2% and 99.4% for laboratory 1 and 80.0%, 98.7%. 76.2% and 99.0% for laboratory 2, respectively. For both laboratories combined the values were 87.5%. 99.2%. 87.5% and 99.2%. Conclusions: These results indicate that multiple (two or three) respiratory samples from each patient should be tested, to allow sufficient accuracy in detecting M. tuberculosis in the specimens. Still, the labor-intensive format of this test necessitates strong clinical indications and patient prioritization t o provide a service feasible within the current limits of routine laboratories. zyxwvut Key words: Tuberculosis, polymerase chain reaction (PCR), 16s rDNA, rapid diagnostics detection and a high recovery rate of niycobacterial cultures [2,3].Species-specific nucleic acid probes have significantly improved the opportunity for rapid confirmation of culture results for several mycobacterial species [4]. Still, days to weeks may be required for suficient growth for identification. The use of the polymerase chain reaction (PCR) for species identification of mycobacteria, particularly A4. tuberculosis, from early BACTEC cultures has been favorably explored [5,61. Several groups have previously validated PCR assays for the identification of M . tuberculosis directly in clinical specimens [7-111. Several nucleic acid targets have rendered sufficient sensitivity and representative species-specific differentiation, such as the 16s rKNA gene 112,131, IS elements (14--161, and the genes INTRODUCTION There is clearly a demand for more rapid and reliable laboratory methods for the diagnosis of Mycobacterium tuberculosis infections for public health and therapeutic reasons [l]. The introduction of the radiometric BACTEC system represents a major improvement in the cultivation of mycobacteria by providing rapid zyx *Corresponding author and reprint requests: Tone Tgnjum, Institute of Microbiology, University of Oslo, Rikshospitalet (National Hospital), Oslo, Norway Tel: +47 22 86 95 20 Fax: +47 22 74 15 96 e-rnail: tone.tonjurn@rh.uio.no Accepted 15 April 1996 127 128 zyxwvutsrqponml zyx C l i n i c a l M i c r o b i o l o g y a n d I n f e c t i o n , V o l u m e 2 N u m b e r 2, O c t o b e r 1 9 9 6 zyxwvuts encoding the 32-kDa and 65-kDa proteins [7,17]. Nucleic acid amplification techniques other than P C R , such as transcription-mediated amplification [ 3.4,18] and, more recently, strand displacement [19] and Qbeta-replicase probe amplification assays [20], are also being widely used. We evaluated the Amplicor P C R assay (R.oche, Switzerland) for detection of M . tuberculosis directly in respiratory specimens. The target D N A to be amplified was the mycobacterial 16s rDNA signature region [13,21]. The aim of our study was to establish whether this test was useful for direct detection of M . tuberculosis in respiratory samples in Scandinavian laboratories, one in Norway and one in Sweden. MATERIALS AND METHODS Material All routine clinical specimens submitted for cultivation of mycobacteria were processed by standard procedures. The specimens in laboratory I were decontaminated by the N-acetyl-L-cysteine/NaOH method [22]. Two volumes of NALC/NaOH solution (2% NaOH, 1.45% sodium-citrate, 0.5% N-acetyl-Lcysteine) were mixed well with the specimen and allowed to digest for 15 to 30 min at room temperature. Ten volumes of 10 mM phosphate buffer (pH 6.8) were added for dilution, before centrifugation at 3000,g for 15 min. In laboratory 2 the samples were decontaminated by the sodium lauryl sulfate method [23] and centrifuged at 35008 for 30min. Sediments were resuspended in 3 to 5 ml of phosphate-buffered saline. Smears were prepared, stained according to ZiehlNielsen or with auramine, and examined for acid-fast bacilli (AFB) by microscopy. Five hundred and eightytwo sputum samples and 378 bronchioalveolar lavage (BAL) samples from 741 patients were processed. Five hundred and forty-four samples were investigated by laboratory 1 and 416 by laboratory 2. Culture protocol BACTEC Middlebrook 12B vials (Becton Dickinson Diagnostic Instruments, Sparks, Md, USA) [2,3] were inoculated with 0.5 mL of each specimen. The 12B vials were monitored by using the BACTEC 460 radiometric reader (Becton Dickinson Diagnostic Instruments) on a regular basis for 6 weeks. Once a 12B vial attained a growth index (GI) of > = l o o , the presence of AFB was confirmed by Ziehl-Nilsen staining. Laboratory 2 inoculated each sample on Lowenstein-Jenssen (LJ) medium. The P C R assay was incorporated into the laboratory routine without any change in practices, and specimens were processed 5 to 6 daydweek. Culture identification Hybridization assays were performed directly on lysed AFB using commercially available nucleic acid probes for the M . tuberculosis complex and M . avium-intracellulare (MAC) (Accuprobe, Gen-Probe, San Diego, CA, USA) [4]. Mycobacterial species other than the M. tuberculosis complex and MAC were identified by conventional procedures. PCR analysis Sample preparation One hundred microliters of the decontaminated sputum or BAL was added to 500 pL of Tris-HC1 with 1% Triton X-100 and 0.05% sodium azide, mixed and centrifuged at 1 2 , 5 0 0 ~for 10 min. The supernate was carefully removed, and 100 pL of the lysis solution containing 1% Triton X-100, 0.4% sodium hydroxide and 0.05% sodium azide was added. The pellet was dissolved by vortex mixing and incubated at 60 "C for 45 min. After centrifugation, 100 pL Tris-HC1 with 0.05% sodium azide was added. One positive control containing M . tuberculosis D N A and three negative buffer controls were included in each experiment for reference purposes. zyxwv PCR amplification reaction Genus-specific primers KY18 and KY75 derived to correspond to a highly conserved region of the 16s r R N A gene (rDNA) of mycobacteria were used to amplify a 584-base-pair fragment (bases 15 to 598 of the M. tuberculosis 16s rDNA sequence, accession no. 52917 in Genbank). Fifty microliters of each sample were added to 50 pL of P C R reaction mixture (Amplicor, Roche, Basel, Switzerland) containing 10 m M Tris-HC1,50 mh4 KCl, 2 mM MgC12,200 pM concentrations of each deoxynucleotide triphosphate (dATP, dCTP, dGTP and dUTP), 0.001% (w/v) gelatin, uracil-N-glycosylase (UNG), biotinylated primers, and 0.5 U of Ampli-Taq polymerase (PerkinElmer Cetus, Nonvalk, CT, USA). Each sample was first heated a t 50 "C for 2 min, and then amplified in two cycles of 20 s at 98"C, 20 s at 62°C and 45 s at 72"C, and then in 35 cycles of 20 s at 94"C, 20 s at 62°C and 45 s at 72°C in a thermal cycler (Perkin Elmer Cetus TA9600). dUTP, instead of dTTF', was used as a substrate for U N G in order to prevent carryover of the amplified D N A [24]. Finally, samples were heated at 72 "C for 5 min until further processing to complete the initiated D N A polymerase activity and not allow UNG, which could have survived the extensive heating, to have an effect on dUTPcontaining PCR products. zyxwvutsrqponm zyxwvutsrqpo zyxwvutsrqpon zyxwvu T @ n j u r ne t a l : P C R d e t e c t i o n o f M. t u b e r c u l o s i s 129 zyxwvutsrq Hybridization reaction After the PCK amplification process, the amplified products were chemically denatured and added to a microwell plate containing a bound, M . tubevculosisspecific oligonucleotide probe, KY 172T3. This probe was selected from the hypervariable region of the 16s rRNA gene 13,211. The biotin-labeled P C R products were then hybridized to the probe and thus 'captured'. Detection reaction After washing to remove unbound material, an avidinhorseradish peroxidase (Av-HKP) conjugate was added to the plate. After washing to remove the unbound conjugate, the bound Av-HKP was reacted with peroxide (H202) and tetraniethylbenzidine (TMB) to form a color complex. The reaction was stopped by the addition of weak acid. The optical density a t 450 nni was measured in an automated microwell plate reader and the results were compared to the cut-off value of 0.350. A clinical specimen with an reading equal to or greater than 0.35 is positive, and a specimen with a reading less than 0.35 is negative for the presence of M . tubwrulosis DNA. Detection of M. tuberculosis by PCR The same 960 respiratory specimens were tested for presence of M. tubcrc~losisby thy nucleic acid aniplification method (PCR). In total, 49 of the 56 specimens which yielded M . tuberculosis by culture were positive for M . tubevculosis DNA by P C R (Table 1). PCK was positive for an additional seven specinienr from four patients, which were negative by culture. Based on the findings in Table 1, the overall sensitivity, specificity, and positive predictive and negative predictive values of this particular P C R test in comparison with culture were 87.5%1, 99.2'3'1, 87.5% and 99.294, respectively (Table 2). The values of the P C R test were 91.7%, 99.6%, 94.2% and 99.4% for laboratory 1 and 80.096, 98.7%, 76.2% and 99.0% for laboratory 2, respectively (Table 2). N o positive result for the M . tuberculosis complex was obtained by the P C K system for the specimens which were positive for atypical niycobacteria or other bacterial species grown in the BACTEC 12B or LJ media. Four patients whose sputum specimens were culture negative, but M. tuberculosis PCR-positive, had other PCR-positive samples, a past history of tuberculosis and/or clinical response to recent antituberculosis chemotherapy. zyxwvutsr zyxwvuts Chemical prevention of PCR product contamination The recommended procedures to prevent false-positive reactions as a result of target or amplified product DISCUSSION contamination were followed. Detection of mycobacteria by culture In total, 88 specimens were positive by culture (9.2%) (Table 1 ) . O f these, 56 (5.80/;,) were M . tuberculosis isolates from 33 patients. Moreover, 15 isolates of MAC, 12 M . malinoense, two M . clielouae, and one M. xcnopi were detected. Table 1 Comparison of results obtained by culture and I'CR in detection of M . ruhevcrrlosk in respiratory speciniens in Scandinavia. The nunibers for laboratory 1 and 2 are given on the upper line, and thc total numbcrs for both laboratorics are given in bold type Culture Mtbf I'CRf PClITotal 33+16 49 3h+4 7 56 Culture M t b Culture MOTT- Culture MtbCulture MOTT+ 2 " . i o+n 7 487 + 378 865 872 0 19+13 32 32 In comparison with culture, the sensitivity of PCK was 91.7% for laboratory 1 and 80.0% for laboratory 2, respectively (Tables 1 and 2). In comparison with previously published studies for direct detection of L\f. tuberculosis by P C R , other groups have found that the sensitivity of their P C R assays when cornpared with culture ranged from 82% to 94% [7,14-171. Seven samples in five patients were 2.I. tubevc-trloris culture negative and P C R positive and can as such be strictly regarded as false positive. But when other hctors are taken into account for evaluating the patient as ''if. tcrbcvculosis positive' by having other samples poyitive by culture and/or P C R or other factors indicating tuberculosis, most of this specificity problem is resolved. zyx Total 35+22 56 509+395 904 960 .'Other s.iinplcs froin these two patients wcrc M. tcrbercrtlosi~culture po"t1ve. "lieteyting by PCIX (AmpliCor) gave a positive result in one of thew samples; PCK confirmation testing by Roche in Hasel (blind teTtirig) gave poqitive results in the remaining two wiiples. Table 2 The sensitivity, specificit); and positive predictivc value and negative predictive value of the PCR tmt as compared to culture, based on the results in Tablc 1 Laboratory Lhoratory 1 Laboratory 2 Laboratories 1 and 2 Sensitivity (%) Specificity (9%) zyx zyx Positivc Negative prcdictivc predictivc value ('H,) v.iluc ('HI) 91.7 80.0 99.6 98.7 94.2 76.2 09 1 87.5 99.2 87.5 90.2 09.0 130 C l i n i c a l M i c r o b i o l o g y a n d I n f e c t i o n , V o l u m e 2 N u m b e r 2, O c t o b e r 1 9 9 6 Different decontamination procedures may account for the major part of the discrepant sensitivity in the two laboratories. Laboratory 1 used the NALC decontamination procedure, while laboratory 2 used the sodium dodecylsulfate (SDS) method. The NALC procedure is clearly the decontamination method recommended by the Amplicor manufacturing company and for P C R and other amplification techniques [14,15,18]. The higher sensitivity of P C R in comparison with culture for laboratory 1 could also be due to less optimized culture techniques than in laboratory 2, laboratory 1 using only BACTEC detection and not including solid media [3]. Possibly, recently documented batches of BACTEC vials with reduced performance in cultivating both M . tuberculosis and M O T T might be involved 1251. Other factors include the time elapsed between sampling and processing, sample handling, and the technical quality of the sample preparation, lysis and P C K set-up. The samples examined by P C R by laboratory 2 were transported after decontamination and sample preparation and frozen at -70 "C before P C R analysis. The sample lysis and pretreatment procedure with Triton X-100 and optimized buffering clearly facilitates direct detection of M. tuberculosis by P C R . The U N G enzyme inactivates up to lo9 copies of uracilcontaining M. tuberculosis amplified D N A [24]. This reduced the likelihood of false-positive results arising due to contamination with pre-existing P C R products. Furthermore, the inclusion of the hybridization event ensured that only M . tuberculosis-specific P C R products were detected, increasing the overall specificity of the test. These actions, in addition to careful laboratory precautions in sample processing and work habits, have now minimized the occurrence of false-positive PCR results. Factors lowering the sensitivity of P C R are interfering substances present in clinical specimens [7,11,15] and inadequate amounts of the microbial DNA to be detected. An uneven distribution of bacteria or DNA, even after lysis of the material, as may particularly apply to mucous material in sputum, may cause an arbitrary sampling effect. In our hands, the P C R assay worked just as well directly on respiratory specimens which were not subjected to decontamination (unpublished results). The elimination of factors inhibitory for P C R in clinical specimens remains a challenge in the use and acceptance of all amplification assays in the diagnostic setting. Certainly, the inclusion of a positive amplification control test is useful to assess the inhibiting factors which may be present in clinical material. Despite promising results of numerous published reports, the routine use of P C R to detect M. tuberculosis zyx directly in clinical specimens has been hampered for a variety of reasons, such as contamination, expense, and lack of sensivity and/or specificity [7,9-11,13,15,17]. In addition, the routine use of P C R in the clinical laboratory sets limitations because of the complex procedures required for amplification, such as cumbersome sample preparation and detection methods. Still, using P C R in the identification of M. tuberculosis directly in clinical samples offers unique improvements in this diagnostic field. This P C R assay offers a sensitive and specific test for M. tuberculosis performed within 5 to 6 h. More automation and lower assay expenses are required. For the future, this and other amplification techniques can facilitate the direct detection of the infecting agent and its antibiotic susceptibility pattern [26], as well as epidemiological mapping [27]. Potentially, all of these goals can be achieved in one single multiplex assay. zyxwvutsr zyx References 1. Bloom BR, Murray CJL. Tuberculoqis: commentary on a reemergent killer. Science 1992; 257: 1055-63. 2. Middlebrook C , Reggiardo Z , Tigerit WD. Automatable radiometric detection of growth of Mycobacterium tuberculosis in selective media. Am Rev Respir Dis 1977; 115: 1066-9. 3. Morgan MA, Horstmeier CD, DeYoung D R , Roberts GD. Comparison of a radiometric method (BACTEC) and conventional culture media for recovery of niycobacteria from smear-negative specimens. J Clin Microbiol 1983; 18: 384-8. 4. 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