JOURNAL OF CLINICAL MICROBIOLOGY, July 1998, p. 19641968 0095-1137/98/$04.00 0 Copyright 1998, American Society for Microbiology.

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Vol. 36, No. 7

Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantication of IS6110 DNA in Sputum during Treatment of Tuberculosis
L. E. DESJARDIN,1 Y. CHEN,1 M. D. PERKINS,2 L. TEIXEIRA,2 M. D. CAVE,1,3 AND K. D. EISENACH1,3* University of Arkansas for Medical Sciences1 and J. L. McClellan Memorial Veterans Affairs Hospital,3 Little Rock, Arkansas, and Duke University Medical Center/Universidade Federal do Esp `rito Santo, Vitoria, Brazil2
Received 21 January 1998/Returned for modication 9 March 1998/Accepted 28 March 1998

Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantication were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantication with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a uorogenic probe in which the intensity of uorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantied in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantication of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efcacy. The emergence of drug-resistant strains of Mycobacterium tuberculosis has led to the need to rapidly measure treatment efcacy. Drug susceptibility proles can take several weeks. Additionally, in vitro drug susceptibility test results do not reect in vivo drug absorption difculties that can also lead to the development of drug resistance and the spread of disease. Currently, sputum smear and culture are used to monitor the response to therapy for patients with pulmonary tuberculosis. Conversion of positive results to negative results after 1 to 2 months of therapy is indicative of effective treatment (7, 30). The objective of this work was to determine whether quantitative changes in the amount of mycobacterial DNA present in sputum reect bactericidal drug activity. Competitive PCR is a standard method for the quantication of DNA and has been used successfully in a number of studies (6, 9, 21, 25, 29, 33). However, this technique is labor intensive and requires results from multiple reactions to be analyzed for each sample. Recently, the ABI Prism 7700 Sequence Detection System (TaqMan) has been shown to be a rapid and sensitive method for quantication of PCR and reverse transcription-PCR products (8, 10). The system uses a uorogenic probe, and the amount of uorescence detected is proportional to the amount of accumulated PCR product. The amount of target DNA in a sample is interpolated from a standard curve run simultaneously with the unknown samples. Quantication of PCR products occurs in real time during the amplication process, with no postamplication handling being necessary. This eliminates potential sources of carryover contamination and reduces the handling time. In this report,
* Corresponding author. Mailing address: Medical Research Service, Slot LR-151, J. L. McClellan Memorial VA Hospital, 4300 W. 7th St., Little Rock, AR 72205. Phone: (501) 660-2062. Fax: (501) 6646748. E-mail: eisenachkathleend@exchange.uams.edu. 1964

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we compare two methods for the quantication of mycobacterial DNA in patient samples. Sputum samples were collected before and during the course of chemotherapy to evaluate whether the measure of M. tuberculosis DNA can be used as an indicator of treatment efcacy.
MATERIALS AND METHODS Patients and specimens. Sputa were collected from two patients with newly diagnosed, smear-positive pulmonary tuberculosis at the Universidade Federal do Esp `rito Santo Hospital in Vitoria, Brazil. Subjects presented with cavitary disease and were human immunodeciency virus negative. Patients harbored drug-sensitive M. tuberculosis strains and were treated with a standard four-drug regimen of isoniazid, rifampin, ethambutol, and pyrazinamide for 2 months, followed by isoniazid and rifampin treatment for an additional 4 months. The patients responded to therapy and showed no clinical signs of relapse for up to 8 months following the cessation of treatment. Noninduced spot sputum samples were collected in triplicate before the start of chemotherapy (day 0) and in duplicate on days 2, 4, 7, 14, 30, 60, and 120. Sputa were stored at 4C until homogenization with 2.5% N-acetyl-L-cysteine and 4-mm glass beads as described previously (2). The specimens were divided into aliquots of 0.5 ml of homogenate and were frozen at 70C until nucleic acid extraction. Sputum was further processed by 2% NaOH-sodium citrate decontamination and centrifuge sedimentation for quantitative microscopy and culture (16). Quantitative AFB microscopy and culture. For quantitative AFB microscopy, 20 l of homogenized and decontaminated sputum was xed onto a 78-mm2 area on a slide and subjected to auramine O staining, and the number of AFB per eld was counted microscopically for an average of 50 elds per sample. AFB enumeration was shown to be reproducible by repeated counting on separate days. Quantitative culture was performed by plating serial dilutions of homogenized-decontaminated sputum on 7H10/7H10S agar (S 200 U of polymyxin B per ml, 10 g of amphotericin B per ml, 50 g of carbenicillin per ml, and 20 g of trimethoprim per ml) (19). A series of 10-fold dilutions was made from 0.5 ml of decontaminated specimen with a 0.85% NaCl0.25% Tween 80 solution. A total of 30 l of each dilution was plated in duplicate onto separate quadrants of the biplate. Colonies were counted after incubation for 3 to 4 weeks at 37C in 5% CO2, with the ideal dilution considered to be between 10 and 50 colonies per quadrant. DNA isolation. DNA was isolated from 0.5 ml of homogenized sputum by a combination of organic and mechanical lysis with glass beads. The entire 0.5-ml aliquot of sputum was mixed with 1 ml of TriZOL-LS (Gibco BRL Life Tech-

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FIG. 1. Sequence of the region of M. tuberculosis IS6110 amplied in both competitive and TaqMan PCRs. The primers for competitive PCR are labeled T4 and T5. The primers used in the ABI 7700 system are labeled IS6 and IS7. The BglI site is the site of insertion of the 80-bp M. avium fragment to make the internal control plasmid pQIS6110.

nologies, Grand Island, N.Y.), transferred to a 2-ml matrix tube (Bio 101, Inc., La Jolla, Calif.), and processed twice for 45 s each time in a FastPrep 120 processor (Bio 101, Inc.). The aqueous phase was removed and DNA was extracted from the interface and organic fractions. A total of 400 l of back extraction solution (100 mM Tris [pH 8], 1 mM EDTA [pH 8], 50 mM NaCl, 1% sodium dodecyl sulfate) was added to the organic phase-interface fractions and was mixed by vortexing for 2 min. The samples were incubated at 65C for 15 min, mixed for 2 min by vortexing, and centrifuged for 15 min at 18,000 g. The newly formed, DNA-containing aqueous phase was transferred to a 1.5-ml microcentrifuge tube. Extraction of the organic phase-interface fractions was repeated as described above to remove any residual DNA from the organic fraction. Aqueous phases from both the rst and second extractions were combined and 2 l of linear acrylamide (Ambion, Austin, Tex.), a 1/10 volume of 3 M sodium acetate, and an equal volume of isopropanol were added, followed by precipitation of the samples overnight at 20C. The tubes were centrifuged at 18,000 g for 30 min, and the pellets were washed twice with 75% ethanol before drying and resuspension in 100 l of distilled H2O. Mycobacterial genomic DNA was isolated from cultured Mycobacterium bovis 410, which contains a single copy of the IS6110 insertion element, by the method of Beggs et al. (1). The concentration and purity of the DNA were determined spectrophotometrically, and the DNA had an A260/A280 of 1.9. Recombinant plasmids pIS6110 and pQIS6110 were isolated from DH5 cells (GibcoBRL Life Technologies) by the PERFECTprep DNA isolation kit (5 Prime f 3 Prime, Inc., Boulder, Colo.). Competitive PCR. (i) Internal control. The entire sequence of IS6110 from a clone kindly provided by Ndingsa Fomukong was subcloned into pBluescript (Stratagene, La Jolla, Calif.) and was named pIS6110. An internal control plasmid, named pQIS6110, was constructed by ligating an 80-bp Mycobacterium avium DNA fragment with the same G C content as IS6110 into a BglI site (see Fig. 1). (ii) PCR. The appropriate dilution of test DNA that would fall within the range of dilutions of DNA for the internal controls was initially determined empirically by PCR analysis of serial dilutions without the pQIS6110 competitor. Dilutions of the internal control pQIS6110, from 10 to 10,000 molecules/ l, were made in a 10-ng/ l yeast RNA solution (Ambion) and were coamplied with the appropriate dilution of test DNA. PCR was performed with oligonucleotide primers T4 and T5, which amplify a 123-bp region of the IS6110 sequence (5). PCR primers were radiolabeled with 32 P, and a total of 5 105 cpm of labeled primers, in addition to 0.4 M unlabeled oligonucleotide primers, was added to each reaction mixture. The PCR conditions were as described previously (2). Amplication was performed with a GeneAmp PE9600 thermal cycler (Applied BioSystems/Perkin-Elmer [ABI/PE], Foster City, Calif.) with the prole of 1 cycle of 50C for 5 min (for uracil DNA glycosylase decontamination); 1 cycle of 94C for 5 min for denaturation; 32 cycles of 94C for 30 s, 68C for 1.5 min, and 72C for 1 min; and 1 cycle of 72C for 20 min (nal extension). Target DNA concentrations were chosen so that the DNA was still in exponential amplication after 32 cycles of PCR (data not shown). (iii) Quantication of products. PCR products were electrophoresed on 8% nondenaturing polyacrylamide gels, dried, and exposed for 4 h to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, Calif.). The intensities of the internal control bands and of authentic products were quantied with the PhosphorImager by using ImageQuant, version 3.1, software (Molecular Dynamics). ABI 7700 quantication. (i) Oligonucleotide primers and probes. The primers for M. tuberculosis DNA quantication for use with the ABI Prism 7700 Sequence Detection System (ABI/PE) were in the same region of the IS6110 sequence as the primers used for competitive PCR, but different sequences were selected for the oligonucleotide primers in order to optimize the PCR for a two-step prole, which is preferred by TaqMan chemistry. Selection of new primers also allowed adjustment of the melting temperatures to those optimal for the ABI 7700 system (PCR primers and uorogenic probe) (see Fig. 1). The upstream PCR primer (IS6) corresponds to the region from base 807 to base 824 (sequence, 5 -GGCTGTGGGTAGCAGACC-3 [bases are numbered as for the sequence with GenBank accession no. X52471]). The reverse primer (IS7) corresponds to the region of IS6110 from base 952 to base 969 (sequence, 5 -CG GGTCCAGATGGCTTGC-3 ). The internal oligonucleotide probe was labeled with the uorescent dyes 5-carboxyuoroscein (FAM) on the 5 end and N,N,N ,N -tetramethyl-6-car-

boxyrhodamine (TAMRA) on the 3 end. The internal probe hybridizes within the 163-bp region amplied by the PCR primers and has the sequence 5-(FAM)TGTCGACCTGGGCAGGGTTCG-(TAMRA)-3 . When the two dyes are in close proximity, as with an intact oligonucleotide probe, TAMRA acts as a quencher for FAM by absorbing at the FAM emission spectra. The 5 exonuclease activity of Taq polymerase will degrade an internally hybridizing probe during the course of PCR (13). Degradation of the probe leads to separation of these two dyes in solution, with a subsequent increase in the level of uorescence in the reaction mixture. The amount of uorescence measured in a sample is proportional to the amount of specic PCR product generated (10). (ii) PCR conditions. The PCR mixture (50 l total volume) consisted of primers IS6 and IS7 (0.8 M each), 100 nM IS6110 TaqMan probe, dATP, dCTP, and dGTP (Pharmacia, Alameda, Calif.) each at a concentration of 200 nM, 400 nM dUTP (Pharmacia), 5 mM MgCl2, 100 ng of bovine serum albumin per l (Pharmacia), 10 ng of yeast RNA (Ambion) per l, 1 U of uracil DNA glycosylase (New England Biolabs, Beverly, Mass.), 1 U of Taq polymerase (Fisherbrand, Plano, Tex.), and 1 PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl). Amplication and detection were performed with the ABI 7700 system with the following prole: 1 cycle of 50C for 2 min, 1 cycle of 95C for 5 min, and 45 cycles of 94C for 30 s and 68C for 1 min. The TAMRA signal was used to standardize the reaction (10). (iii) Standard curve. The threshold is set at 10 times the standard deviation of the mean baseline emission calculated for PCR cycles 3 to 10. The fractional cycle number reecting a positive PCR result is called the cycle threshold (Ct). The Ct values for standards and samples were usually in the range of between 18 and 32 cycles of amplication. The amount of product in a particular reaction mixture is measured by interpolation from a standard curve of Ct values generated from known starting concentrations of DNA.

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RESULTS Competitive PCR quantication. IS6110 is a multicopy insertion element found in M. tuberculosis complex organisms and has been the target of numerous diagnostic assays. The region targeted for amplication in these studies has been shown to be specic for the M. tuberculosis complex (11, 27) and is diagrammed in Fig. 1. Puried DNA from M. bovis 410 and the plasmid pIS6110, each containing a single copy of IS6110, was quantied. An example of a competitive PCR gel is shown in Fig. 2A. The radioactivity in individual bands was measured with the PhosphorImager, and the values were plotted (Fig. 2B). The line has a slope of approximately 1, which indicates equivalent amplication efciencies of test and competitor DNAs. It has been reported that a slope of 1 is required for the accurate and reproducible quantication of unknown samples by competitive PCR (24). However, other factors must be involved since we and others have observed accurate and reproducible quantication with slopes of less than 1 (23). Quantitative competitive PCR analyses were performed in triplicate with an estimated 500 molecules of the target DNA described above. The calculated values for M. bovis and pIS6110 were 338 33 molecules (mean standard deviation) and 289 40, respectively. These replicate values demonstrate that the assay is reproducible, with coefcients of variation of 10% for M. bovis and 14% for pIS6110. The number of molecules measured ( 300) is lower than the expected 500 molecules of input template, perhaps due to the use of 10-fold dilutions of competitor. The use of large dilutions of

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FIG. 2. Competitive PCRs for M. bovis genomic DNA and pIS6110 plasmid DNA. (A) Autoradiograph of two competitive PCRs. PCR amplication of the internal control plasmid, pQIS6110, results in a 203-bp product. Amplication of test DNAs from both M. bovis and pIS6110 results in a 123-bp product. The number of pQIS6110 molecules added to each PCR mixture is listed under the lane. (B) Relative amounts of sample and internal control DNA for the data in the M. bovis gel in panel A. The equation and t of the line are shown.

FIG. 3. Standard curves generated by analysis of known amounts of template DNA with the ABI 7700 system. Puried DNA from M. bovis 410, plasmid pIS6110, and internal control plasmid pQIS6110, each containing one copy of the IS6110 insertion element, were assayed in parallel. The regression lines calculated for the datum points are shown. The R2 values for each of the lines was 0.99.

competitor allows for a greater dynamic range of the assay, although some accuracy may be sacriced (6, 33). While it is possible that an unequal amplication of the internal control versus the authentic IS6110 target occurred, further experiments suggest that this is not likely (see below). Importantly, the assay is reproducible and IS6110 molecules are quantied similarly whether they are in a genomic or a plasmid background. This suggests that in this case, the DNA structure surrounding the target does not appear to affect PCR amplication efciency. Automated DNA quantication. A quantitative assay was also developed for IS6110 using the ABI Prism 7700 Sequence Detection System (TaqMan). Known amounts of M. bovis 410 genomic DNA, from 50 to 500,000 molecules per reaction mixture, resulted in Ct values ranging from 18 to 31 cycles, with coefcients of variation of less than 1% between replicate standards (Table 1). Standard curves generated with DNA from M. bovis 410, plasmid pIS6110, and the internal control pQIS6110 plasmid were assayed in parallel. All standard curves were linear over 6 orders of magnitude, with the R2 values of the lines being greater than 0.99 in each case (Fig. 3). The slopes of the curves were similar for all three sources of control DNA, with no appreciable differences between genomic and plasmid DNA. These data suggest that amplication efciencies were similar for mycobacterial genomic DNA, plasmid DNA, and the engineered plasmid DNA used as an internal control in the competitive assay. Measurements of M. tuberculosis DNA in sputum. The number of IS6110 molecules present in DNA isolated from sputum
TABLE 1. Measurement of replicate standard curves ranging from 50 to 500,000 molecules of M. bovis 410 genomic DNA per reaction mixture
Quantity (no. of molecules) Ct for the following reaction: 1 2 3 4 Mean SD % CVa

specimens was quantied by both the competitive and the ABI 7700 PCR systems. The results for multiple samples collected from two patients during the rst 2 to 3 months of antituberculosis therapy are presented in Fig. 4. Both DNA quantication assays resulted in the measurement of similar levels of IS6110 DNA. Duplicate quantications of IS6110 DNA with the ABI 7700 system showed a coefcient of variation of less than 10% for all

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50 500 5,000 50,000 500,000

31.3 27.3 24.6 21.6 18.2

31 27.7 24.8 21.4 18.3

31 27.7 24.5 21.5 18.2

31.5 27.77 24.56 21.46 18.24

31.19 27.63 24.62 21.48 18.22

0.27 0.22 0.16 0.08 0.06

0.87 0.78 0.64 0.37 0.31

CV, coefcient of variation.

FIG. 4. Comparison of TaqMan and competitive PCR assays for two patients receiving tuberculosis chemotherapy. IS6110 DNA levels were determined with both assay systems for DNA isolated from sputum collected during the course of chemotherapy. The same DNA sample was tested in both systems. Specimens were collected in triplicate on day 0, before the onset of antimicrobial chemotherapy, and duplicate samples were collected thereafter. The competitive assay was performed once per sample. Error bars show the standard deviations for duplicate TaqMan measurements.

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DISCUSSION The most common methods used to monitor response to therapy for patients with pulmonary tuberculosis are conversion of positive results of smears for AFB or culture of sputum specimens to negative results. Culture can take up to 4 weeks for a result, and AFB staining lacks sensitivity and specicity. Although some reports indicate that PCR of M. tuberculosis DNA in sputum is an effective means of measuring treatment efcacy (15, 18, 31, 32), our work showed that a qualitative assessment of M. tuberculosis DNA in sputum does not correspond to the stage or severity of disease in patients receiving standard chemotherapy (12). It has been suggested that quantitative levels of DNA might reect bacterial load and thus indicate the efcacy of chemotherapy (12, 14, 22). To test this idea, we used a new PCR system, the ABI 7700 system (TaqMan), to determine DNA levels in a real-time format. This system measures the amount of target DNA produced during each cycle of an amplication reaction; thus, all samples are quantied during the exponential phase of amplication, the phase considered to produce the most accurate results. The system also allows for a large dynamic range, and we have achieved linearity over 6 logs of input DNA. There are also advantages of speed and ease of use with this system. In our laboratory, it would require a person a minimum of 1 week to analyze a set of serially collected samples from a single patient by the standard competitive method, whereas less than a day is required with the automated system. When either the competitive PCR or the automated assay is performed with puried control DNA, the coefcients of variation are typically 10% or less. Quantication of DNA isolated from sputa showed a similar reproducibility with the ABI 7700 system, although by the competitive PCR sample DNA tended to have a larger variation than control DNA. This has been noted in the past for competitive PCR (6, 26). A possible reason for the larger sample variation obtained by competitive PCR is the increased handling necessary to assay multiple dilutions of samples. It is also possible that when dealing with relatively impure DNA, such as that isolated from sputum, the presence of contaminants in these preparations may interfere with uniform and consistent dilution and amplication of target DNA. This is supported by the uniformity of replicate measurements with puried DNA compared to that with sample DNA (Table 1). The competitive assay is also more labor intensive than the assay with the ABI 7700 system, so replicate measurements of a single sample are not often performed, potentially leading to errors in quantication. The data show that measurement of IS6110 DNA before and during the course of chemotherapy corresponds to the number of bacilli enumerated by staining for AFB. For one patient, the numbers of AFB and the amount of IS6110 DNA were nearly the same. For the other patient, the amount of IS6110 DNA was consistently 10-fold higher than the number of AFB, perhaps because the insertion element was present in multiple copies in the chromosome of this strain of M. tuberculosis. Importantly, neither the AFB count nor the IS6110 DNA level corresponded to the number of cultivable bacilli (number of CFU) after the initiation of chemotherapy, and thus, these measures do not reect the bactericidal activities of chemotherapeutic agents. These data conict with observations by others who have shown a strong correlation between AFB and culture positivity, in which the AFB result becomes negative at the same time as or before the sputum becomes culture negative (20, 32). However, patients who have advanced cavitary disease and who are treated with a rifampincontaining regimen often become culture negative but remain

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FIG. 5. Amounts of AFB, numbers of CFU, and IS6110 DNA levels in sputum collected during the rst 2 to 3 months of chemotherapy. Data for two patients are shown. Values represent the highest measurement for replicate specimens collected on a given day.

samples, demonstrating the reproducibility of this assay. Due to the laborious nature of the competitive assay, only two patient samples were tested in duplicate by this technique, resulting in higher coefcients of variation (98 and 74%, respectively) than those observed with the ABI 7700 system. The variation between replicate spot sputum specimens collected sequentially before and after the initiation of chemotherapy can also be seen in Fig. 4. In most cases, replicate spot samples resulted in similar numbers of IS6110 molecules; however, differences of up to 10-fold were observed. Comparison of M. tuberculosis DNA to quantitative microscopy and culture. The levels of M. tuberculosis DNA, the numbers of AFB found on microscopy, and the numbers of CFU were measured in sputum collected at each time point, and relative levels were compared during the course of chemotherapy for two patients. For the rst patient, 108 IS6110 DNA molecules per sample were found on day 0 and more than 106 IS6110 DNA molecules per sample were measured after 120 days of treatment (Fig. 5A). Quantitative microscopy for AFB revealed a similar trend, but with values about 10-fold lower than those for IS6110 at each time point. The higher levels of IS6110 DNA may be due to the fact that IS6110 is a multicopy element, with up to 25 copies per M. tuberculosis genome (28). At pretreatment (day 0), the numbers of CFU were similar to the total numbers of AFB present and within 60 days declined to below the limit of detection ( 102 CFU), a decrease of at least 106-fold. In contrast, AFB and IS6110 DNA levels decreased by less than 100-fold during the same time period. A similar analysis for the second patient was performed with samples collected over a course of 60 days from the initiation of chemotherapy (Fig. 5B). Bacterial load estimates for day 0 were similar by AFB microscopy, quantitative culture, and IS6110 PCR, the numbers being approximately 106 per sample. AFB and IS6110 DNA levels decreased by approximately 10to 100-fold over the rst 30 days of treatment, while the numbers of CFU fell to undetectable levels at this same point in treatment, a decrease of at least 105-fold.

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smear positive (17), as was the case in this investigation. Although only two patients were initially examined, we are testing an additional 18 patients and thus far have observed similar results (3). The result that neither AFB counts nor M. tuberculosis DNA levels correlated with the numbers of viable organisms suggests that neither is a good indicator of bactericidal effect. Similar data have been observed in mice, in which quantitative M. tuberculosis DNA levels did not correspond to the numbers of bacilli cultured from either the spleens or the lungs of animals treated with antituberculosis drugs (4). We are currently testing other microbial markers which might more appropriately reect the bactericidal effect of antituberculosis treatment and nd an excellent correlation between cell viability and M. tuberculosis mRNA levels (reference 3 and unpublished data). Levels of specic mRNA targets can be readily measured by reverse transcription-PCR, with quantication performed with the ABI 7700 system. We have observed a precipitous reduction in specic M. tuberculosis mRNA levels within as few as 2 days after the start of antituberculosis agents. Furthermore, ratios of M. tuberculosis DNA levels, as measured here, to specic mRNA levels may provide the most meaningful assessment of the effects of antituberculosis agents. Although the quantities of M. tuberculosis DNA in sputum may not be an appropriate measure of bactericidal effect, the excellent correlation between the numbers of CFU and DNA levels in patients prior to treatment indicates that quantitative PCR could be used to estimate the initial bacterial load. Since the test for IS6110 DNA is M. tuberculosis complex specic, it would also remove the ambiguities of a positive AFB smear. The speed and sensitivity of this technique would support its use for routine tuberculosis diagnostics, in particular, when the numbers of bacilli are limiting.
ACKNOWLEDGMENTS We thank Maria Winters, Shirley Haun, Barbie Demchuck, and Robert Pruss for expert technical assistance; Ndingsa Fomukong for the IS6110-containing plasmid; and Joseph Bates, Tobin Hellyer, Jerrold Ellner, Shirley Haun, and Gery Hehman for critical reading of the manuscript and helpful discussions. This work was funded by the Tuberculosis Research Unit (NIH contract no. NO-AI-45244).
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