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Journal of Clinical Microbiology, October 2003, p. 4565-4572, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4565-4572.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays

Francesco Broccolo,1 Paolo Scarpellini,2 Giuseppe Locatelli,1,{dagger} Anna Zingale,2 Anna M. Brambilla,2 Paola Cichero,3 Leonardo A. Sechi,4 Adriano Lazzarin,2 Paolo Lusso,1 and Mauro S. Malnati1*

Unit of Human Virology,1 Division of Infectious Diseases,2 Department of Microbiology, San Raffaele Scientific Institute, Milan,3 Department of Biomedical Science, Division of Clinical and Experimental Microbiology, University of Sassari, Sassari, Italy4

Received 28 March 2003/ Returned for modification 8 June 2003/ Accepted 5 July 2003


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ABSTRACT
 
Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.


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INTRODUCTION
 
Tuberculosis (TB) remains one of the major public health problems worldwide (5, 25), particularly due to the appearance of drug-resistant Mycobacterium tuberculosis strains that render TB control programs more cumbersome (16, 24). Diagnostic tests devoted to the rapid, sensitive, and specific identification of the causative agent are key elements for successful health programs aimed at disease control. Moreover, the accurate determination of mycobacterial burden might be beneficial for fast assessment of patient response to standard therapy, especially in those patients suspected of harboring resistant M. tuberculosis strains (24). Traditional laboratory techniques (22, 34), such as direct microscopy observation and Mycobacterium culture on semisolid or liquid medium, are far from being sensitive and specific or adequate for a fast M. tuberculosis identification. Moreover, the harsh decontaminating procedures combined with the lack of homogeneity of the sputum and the tendency of Mycobacterium to clump render even quantitative culture systems unreliable.

Detection of M. tuberculosis-specific DNA sequences might represent a more sensitive and fast diagnostic target (9, 27, 29, 36); however, the successful use of DNA amplification techniques is strongly dependent on the choice of the target sequence (12, 28). Moreover, since respiratory tract specimens are naturally contaminated by many different species of commensal and pathogenic microorganisms, a high degree of specificity for M. tuberculosis recognition is mandatory.

PCR-based systems require, in addition, an efficient extraction and purification procedure for the DNA, which is further complicated by the physical peculiarity of the sputum and by the high lipid content of the mycobacterial cell wall. Thus, all the available techniques for mycobacterial DNA extraction require manipulation steps, which result in an unpredictable loss of starting material. At present, there are few methods available for real-time quantification of M. tuberculosis DNA (3, 7, 15, 17), but none allow for the control of both the efficiency of the extraction procedure and the presence of PCR inhibitors.

Here, we describe the development of two real-time calibrated PCR assays for the rapid, sensitive, and accurate determination of M. tuberculosis DNA burden directly from clinical samples. In our assays, the problems of DNA extraction efficiency and PCR inhibitors have been solved by using a synthetic DNA molecule, termed calibrator, specifically detected by an ad hoc-designed probe which does not cross-react with Mycobacterium sequences. The calibrator permits us to control each sample for the presence of PCR inhibitors, to determine a cutoff value of sensitivity for negative samples, and to normalize positive samples for the efficiency of DNA recovery (1). These assays, which amplify two distinct regions of the M. tuberculosis genome, one fragment of the IS6110 multicopy element (32) and one of the senX3-regX3 intergenic region (IR) (30), have been tested, alone or in combination, on 71 bacterial strains and on 121 clinical samples. The usefulness of these new assays has been established by comparison with routine microbiological techniques.


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MATERIALS AND METHODS
 
Bacterial strains and bacterial DNA extraction. Thirty-one clinical isolates of M. tuberculosis obtained from the San Raffaele Hospital repository were selected on the basis of their different contents of IS6110 copies (between 0 and 17 per isolate). Seven Mycobacterium bovis strains (two derived from bovine lymph nodes, one from pig lymph nodes, and four from human specimens), the M. bovis BCG Tice vaccine strain and 22 atypical mycobacterial strains (four isolates each of Mycobacterium gordonae and Mycobacterium avium; three isolates each of Mycobacterium intracellulare, Mycobacterium xenopi, and Mycobacterium chelonae; two isolates of Mycobacterium fortuitum; and one isolate each of Mycobacterium kansasii, Mycobacterium paratuberculosis, and Mycobacterium scrofulaceum) were also included. DNAs from eight bacterial strains (two isolates each of Escherichia coli and Streptococcus pneumoniae and one isolate each of Pseudomonas aeruginosa, Klebsiella pneumoniae, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, and Streptococcus pyogenes) and one fungus (Aspergillus fumigatus) were analyzed as control.

Bacterial strains were grown on agar plates, and DNA was extracted by using the QIAamp tissue kit (Qiagen, Inc., Chatsworth, Calif.).

Patients and sample preparation. The discharge diagnoses of all patients were reviewed at San Raffaele Hospital, where specimens were obtained. For those patients who had discordant results on the acid-fast bacillus (AFB) culture and PCR, the clinical record was carefully reviewed to determine whether the samples were taken while patients were receiving antituberculosis therapy and the clinical likelihood that they had an active mycobacterial infection. The diagnosis of TB was established according to the diagnostic standard of the American Thoracic Society (4). One hundred twenty-one samples, sputum, urine, cerebrospinal fluid, nasogastric lavage fluid, and bronchoalveolar lavage fluid, from 83 patients with (n = 20) or without (n = 63) a diagnosis of tuberculosis were analyzed. Eleven of the 20 TB patients and 55 of the 63 control patients were coinfected with human immunodeficiency virus.

The respiratory specimens (1 ml) were digested and decontaminated by using the N-acetyl-L-cysteine-NaOH procedure. The entire aliquot of specimen was homogenized with an equal volume of a 2.5% N-acetyl-L-cysteine solution in 68 mM phosphate buffer, pH 6.7, and centrifuged (400 x g, 15 m) to remove cellular debris. After neutralization and centrifugation at 3,000 x g for 20 min, the supernatant was discarded and the sediment was used for direct microscopy, culture, and DNA extraction. Extrapulmonary specimens from closed and normally sterile sites were not decontaminated but used directly after a single centrifugation or without any centrifugation if the amount of sample was small (0.2 ml). Fixed smears were stained with auramine fluorochrome stain. The AFB-positive slides were confirmed by Ziehl-Neelsen staining. The cultures were inoculated on a BACTEC system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) and observed for 8 weeks before they were discarded. Routine biochemical methods and Accuprobe culture confirmation kits (Gen-Probe, San Diego, Calif.) were used to identify the isolates.

Sputum samples were resuspended in 450 µl of lysis solution containing 100 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 0.5% (vol/vol) Tween 20, and 0.5% (vol/vol) Nonidet P-40. Samples were incubated at 95°C for 30 min, mixed for 2 min, and digested with 50 µl of proteinase K (20 µg/µl). After overnight incubation at 56°C, samples were heated at 95°C for 10 min to inactivate proteinase K. Phenol-chloroform extraction followed by high-salt isopropanol precipitation was performed as described previously (18), and purified material was resuspended in a final volume of 100 µl of AE buffer (5 mM Tris-HCl-0.5 mM EDTA). Synthetic calibrator DNA (104 copies of calibrator/sample) was added prior to DNA extraction to control the efficiency of each step of the analytical procedure. Ten microliters of the purified material was tested in each PCR in triplicate to measure both the calibrator and the M. tuberculosis copy number. Data were normalized based on the recovery rate of the calibrator DNA by using the following formula:

VT indicates the total volume of the extracted material, VR is the volume assayed in the PCR, GER indicates the number of M. tuberculosis genome equivalents measured in each reaction, VS is the volume of the analytical sample (expressed in milliliters), CI is the calibrator input, and CO is the calibrator output. On negative samples, the cutoff of sensitivity was calculated as follows:

MGED indicates the minimal number of genome equivalents detectable, VS indicates the volume of the biological sample (expressed in milliliters), CI is the calibrator input, and CO is the calibrator output.

To exclude sample cross-contamination, one negative control was included with every two samples, and all the different steps required for real-time PCR analysis were done in three separate dedicated rooms.

Primers and TaqMan probes. Primers TAQM3 (5'-AGGCGAACCCTGCCCAG-3') and TAQM4 (5'-GATCGCTGATCCGGCCA-3') amplify a 122-bp fragment of the IS6110 multicopy element (GenBank accession no. X52471). A probe of 30 bp (5'-TGTGGGTAGCAGACCTCACCTATGTGTCGA-3') which recognizes a region downstream of primer TAQM3 was synthesized (PE Biosystems, Warrington, United Kingdom) with the reporter dye VIC and the 6-carboxytetramethylrhodamine quencher dye covalently linked to the 5' and 3' ends of the oligonucleotide, respectively. The targeted amplicon was selected within a central region of the IS6110 multicopy element sequence which is conserved among Mycobacterium species. Primers TAQregT2 (5'-GTAGCGATGAGGAGGAGTGG-3') and TAQreg2L (5'-ACTCGGCGAGAGCTGCC-3') amplify a 146-bp fragment of the senX3-regX3 region of the M. tuberculosis genome (EMBL accession no. G2190478). A 22-bp oligonucleotide probe (5'-ACGAGGAGTCGCTGGCCGATCC-3') was synthesized (PE Biosystems) and conjugated with the reporter dye 6-carboxy-fluorescein and the 6-carboxytetramethylrhodamine quencher dye, which were covalently linked, respectively, to the 5' and 3' ends of the oligonucleotide. An extensive search of two databases (EMBL and GenBank) indicated that neither the primers nor the probes shared significant homology with other known nucleotide sequences.

Plasmid preparation. DNA was extracted from the M. tuberculosis strain C46 and PCR amplified by primers J and K derived from the IS6110 sequence (26). Five microliters of DNA (approximately 0.1 µg/µl) was amplified in a thermal cycler (Applied Biosystems, Foster City, Calif.) with a reaction mixture containing 100 pmol each of primers J and K, MgCl2 (1.5 mM), buffer (1x), 125 µM concentrations of each deoxynucleoside triphosphate, and 1 U of Taq Gold DNA polymerase. After 10 min of incubation at 95°C, 30 cycles of amplification with a temperature profile of 94, 58, and 72°C (30 s each) were performed. A final extension step of 10 min at 72°C was added. The senX3-regX3 IR 343-bp fragment was generated by conventional PCR with the oligonucleotides C5 (5'-GCGCGAGAGCCCGAACTGC-3') and regT3 (5'-AGGACGATGTCGGCGCCG-3'). The PCR mixture contained 100 mM deoxynucleosides, 150 pmol of primers, MgCl2 (1.5 mM), buffer (1x), and 1 U of Taq Gold DNA polymerase. After 10 min of incubation at 95°C, 30 cycles of amplification were performed with a thermal profile of 94, 65, and 72°C (30 s each). A final extension step of 10 min at 72°C was performed. PCR products were cloned into the pCRII plasmid by using the TOPO-TA cloning kit (Invitrogen Corp., San Diego, Calif.) according to the manufacturer's instructions. Plasmids pIS6110 and psenX3-regX3 IR were purified with the Qiagen plasmid maxi kit (Qiagen, Inc.), sequenced, and used to quantify the mycobacterial DNA load.

Real-time PCR conditions and RFLP analysis. Three independent real-time quantitative TaqMan PCR methods (IS6110, senX3-regX3 IR, and calibrator) were performed on each sample. All reactions were optimized to obtain the best amplification kinetics under the same cycling conditions (2 min at 50°C, 15 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min per cycle at 60°C) and composition of the reaction mixture. All reactions were performed in a final volume of 25 µl containing 100 mM (each) dATP, dCTP, and dGTP; 200 mM dUTP; 4 mM MgCl2; 1x TaqMan buffer A; 0.625 U of AmpliTaq Gold, 0.25 U of uracil-N-glycosylase; and 10 µl of DNA template. Target-specific primers and probes were used at the final concentrations of 300 and 200 nM, respectively. The principle of the real-time PCR has been described elsewhere (10). Each sample was tested in triplicate, and the mean value was reported. IS6110 restriction fragment length polymorphism (RFLP) analysis was performed as described previously (33).

Statistical analysis. Accuracy, defined as the level of approximation of a measured value to a reference value taken as a ‘gold standard,’ was estimated by computing the arithmetic differences between the number of DNA copies evaluated by real-time PCR and the theoretical number calculated according to UV spectroscopy. The significance of systematic biases was assessed by a paired-sample Student's t test and adjusted by covariance analysis, when needed. Repeatability (i.e., the variability of a method when repeated measures are taken on the same material in a single experiment) and reproducibility (i.e., the variability of a method when repeated measures are taken in different experiments) were estimated by computing the coefficient of variation (CV; the ratio between the standard deviation and the mean of repeated measurements). The difference between reference curves was assessed by covariance analysis. The two-tailed Student t test was used to evaluate the significance of differences in M. tuberculosis load among groups of samples. The comparison between noncalibrated and calibrated M. tuberculosis load was made by the two-tailed Student t test for paired samples.


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RESULTS
 
Design of real-time PCR systems. The aim of this study was to design a rapid, high-throughput quantitative PCR-based method suitable for measuring the M. tuberculosis load directly in clinical specimens. In this regard, two independent quantitative PCR methods were developed and optimized. The primers and probes were designed in two distinct regions of the M. tuberculosis genome. The first one is situated in the central region of the IS6110 multicopy insertion element (Fig. 1A) (3, 6, 11, 13, 33), which is found in almost all M. tuberculosis isolates as well as in other species of mycobacteria (8, 13). The second one belongs to the senX3-regX3 IR, which contains mycobacterial interspersed repetitive units (MIRUs), which so far have been described only for mycobacterial species belonging to the M. tuberculosis complex (19, 20, 30). To minimize amplification problems due to the variability in the number of 77-bp MIRUs present in different M. tuberculosis strains, the forward primer was designed in the 53-bp MIRU. This fragment is present only once within the senX3-regX3 region in all the M. tuberculosis strains tested (2, 19); it is located immediately before the beginning of the regX3 gene, where the probe and 3' primer were designed (Fig. 1B).



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  		FIG. 1. Schematic representation indicating spacing, positions, and orientations of primers and the probe of mycobacterial IS6110 and senX3-regX3 IR. (A) Schematic drawing of IS6110 showing locations of sequences amplified by published PCR protocols and the international RFLP probe. The bars above the drawing represent sequences determined previously by Kent et al. (13), Desjardin et al. (3), Eisenach et al. (6), Hellyer et al. (11), and van Embden et al. (33). (B) PCR amplification strategy to identify mycobacteria belonging to the M. tuberculosis complex strains. The TaqregT2 primer is specific for the 53-bp MIRU present in one copy of M. tuberculosis strains but absent in BCG.

Cross-reaction with the 77-bp MIRUs was avoided, and specific recognition of the 53-bp MIRU was achieved by placing the TAQRegT2 primer toward the end of the 53-bp MIRU, where a major gap of 24 nucleotides differentiates the two forms of MIRU.

Dynamic range and analytical sensitivity. To generate the reference curves for M. tuberculosis quantification, plasmids pIS6110 and psenX3-regX3 IR were quantified by UV spectroscopy. Then three distinct sets of 10-fold dilutions for each construct were prepared and amplified by PCR in the same run of the 7700 ABI Prism sequence detector system, as described previously (18). A wide dynamic range characterized both assays, discriminating between 100 and 106 M. tuberculosis genome equivalents/reaction. For all the systems generated, a strong linear relationship between the log of the starting copy number and the Ct values was obtained (r2 > 0.99) (Fig. 2). Moreover, the sensitivity and dynamic range of the two assays were not affected by the presence of concentrations up to 1 µg of human genomic DNA (data not shown).



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  		FIG. 2. Comparison between reference curves of the two real-time TaqMan PCR systems. The reference curves were obtained by plotting the Ct values (on the y axis) against the plasmid copy number (IS6110, open squares; senX3-regX3 IR, open triangles). In all of the resulting equations [IS6110, y = 38.85 to 3.422 log (x); senX3-regX3 IR, y = 39.05 to 3.398 log (x)], all of the slope coefficients and the intercept values were similar and no significant differences were found by covariance analysis.

Accuracy, repeatability, and reproducibility. Next, we measured the accuracy of both the IS6110 and senX3-regX3 TaqMan assays to quantify the M. tuberculosis DNA load. For DNA inputs above 104 copies/reaction, the error observed (10 experiments performed with serial dilutions of the reference plasmids pIS6110 and psenX3-regX3 IR tested in triplicate) was negligible (2 to 6%), whereas it was greater (5 to 16%) for inputs below 104 copies/reaction (Table 1). The repeatability and reproducibility of our TaqMan assays were assessed by calculating the CVs of the copy numbers determined experimentally (180 measurements for each reference DNA). Intraexperimental variability of both the IS6110 and senX3-regX3 assays was always below 20%, except for inputs of 10 genome equivalents/reaction (30 and 42%, respectively). Interexperimental variability was always below 40%, except for inputs of 10 genome equivalents/reaction (46 and 71%, respectively) (Table 1).


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  		TABLE 1. Accuracy, repeatability, and reproducibility of IS6110 and senX3-regX3 IR assays

Comparison of the IS6110 and senX3-regX3 TaqMan assays. The sensitivity and specificity of the two TaqMan assays were established with DNA obtained from 61 clinical isolates of various mycobacteria, belonging to the M. tuberculosis complex or to atypical species, and from 10 nonmycobacterial pathogens. All 39 Mycobacterium strains belonging to the M. tuberculosis complex, except one M. tuberculosis isolate and the BCG Tice strain, were detected by both TaqMan assays. As expected, M. tuberculosis isolate 13, which contains no IS6110 repeated elements, was detected only by the senX3-reg X3 system, whereas BCG Tice, which lacks the 53-bp MIRU, was detected only by the IS6110 TaqMan assay.

Of 22 atypical mycobacterial strains tested (belonging to 8 mycobacterium species), all but five (3 of 3 M. xenopi strains and 2 of 3 M. chelonae strains) were detected by the IS6110 TaqMan assay, whereas the senX3-regX3 system amplified only one strain belonging to the M. kansasii species (Table 2). Therefore, each assay showed a distinct pattern of Mycobacterium recognition. A broader spectrum of Mycobacterium species was characterized by the IS6110 system, whereas an almost exclusive recognition of M. tuberculosis complex strains was achieved by the senX3-regX3 assay. None of the other 11 microbial field isolates tested (10 bacterial strains belonging to 8 species and 1 fungus) was amplified by either real-time PCR assay (up to 1 µg of total microbial DNA tested for each PCR).


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  		TABLE 2. Distinct pattern of Mycobacterium recognition by TaqMan-based assays

To evaluate whether genome variability among M. tuberculosis strains affected the accuracy of the two TaqMan assays, purified DNA from 30 M. tuberculosis strains containing a variable number of IS6110 multicopy elements (from 1 to 17) was measured by both systems. Quantification of each strain was performed in triplicate, and the mean of the measured values was calculated (Table 3). As expected, except for the M. tuberculosis strain containing a single copy of the IS6110 element, the copy number measured by the IS6110 system was always higher than the copy number estimated by the senX3-regX3 assay (Table 3).


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  		TABLE 3. Quantification of IS6110 copy numbers by TaqMan assays and RFLP analysis

Since the M. tuberculosis genome contains one 53-bp MIRU of the senX3-regX3 region (2, 19), we calculated the ratio between the copy number measured by the IS6110 and senX3-regX3 assays to estimate the number of IS6110 repeat elements present in each strain. These values were then compared with those obtained by RFLP analysis (Table 3). In all instances, a good concordance between the two independent measurements was observed, indicating that both PCR assays correctly quantified the M. tuberculosis DNA load irrespective of the strain tested.

Clinical sensitivity and specificity. A total of 121 clinical samples obtained from 83 patients with (n = 20) or without (n = 63) a diagnosis of TB were tested by AFB microscopy, culture, and the rapid TaqMan-based assays. Overall, both real-time PCR assays showed a high degree of specificity and sensitivity, even with AFB-negative specimens (Table 4). Of the 54 samples obtained from patients with TB, AFB was positive in 33 (61%) cases, culture was positive in 39 (72%) cases, the senX3-regX3 IR TaqMan assay was positive in 51 (94%) cases, and the IS6110 TaqMan assay was positive in 53 (98%) cases. Forty-three of the 54 samples were collected under antitubercular therapy. Among these, microscopy and culture techniques detected M. tuberculosis in 23 of 43 (53%) and 28 of 43 (65%) samples, respectively, whereas the senX3-regX3 and IS6110 assays measured the M. tuberculosis DNA load in 40 of 43 (93%) and 42 of 43 (98%) samples, respectively. In both cases with discordant PCR assay results, the M. tuberculosis load measured by the IS6110 system was extremely low (13 and 25 genome equivalents/ml). Since the analytical sensitivity of both systems was identical, this discrepancy might be explained by the different M. tuberculosis genome content of amplifiable target sequences.


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  		TABLE 4. Comparison of microscopy, bacterial culture, and TaqMan assays on clinical specimens

Of the 66 specimens obtained from patients affected by other infectious diseases, AFB microscopy and the IS6110 TaqMan assay were positive on the same two samples (3%). In both cases, a Mycobacterium belonging to the M. avium complex was isolated (Table 4).

Comparison of M. tuberculosis bacillary and DNA load. Next, we matched the results of the AFB smear test and Mycobacterium culture with the level of M. tuberculosis DNA load measured with the senX3-regX3 assay (either corrected or uncorrected by calibration). Fifty-one samples were then divided into three groups: group 1 contained samples positive by AFB microscopy and culture (30 samples), group 2 contained samples positive by one of the two assays (12 samples), and group 3 contained specimens negative by both assays (9 samples).

The M. tuberculosis load differed significantly among all groups (group 1 versus group 2, P < 0.01; group 1 versus group 3, P < 0.0005; group 2 versus group 3, P < 0.05), demonstrating a good level of correlation between bacillar and DNA load. Interestingly, all samples with the lowest M. tuberculosis DNA load (groups 2 and 3) were obtained from patients successfully treated with antitubercular therapy. Moreover, in all groups, the M. tuberculosis load measured in the absence of calibration was significantly underestimated (Fig. 3) (P < 0.0005, Student t test for paired samples). Thus, the use of the calibrator allowed for an appreciable correction of M. tuberculosis DNA load, determining for each negative sample a specific cutoff value of M. tuberculosis DNA and excluding the presence of PCR inhibitors (data not shown).



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  		FIG. 3. Distribution of M. tuberculosis (MTB) DNA loads by senX3-regX3 IR TaqMan assay. PCR-positive samples were classified into three groups according to the results of microscopy and bacterial culture (group 1, diamonds; group 2, triangles; group 3, circles), and the M. tuberculosis load was reported before (filled symbols) and after (empty symbols) the correction due to calibration. Bars indicate the median DNA load copy numbers in each group. The median M. tuberculosis loads measured in the presence and absence of calibration were, respectively, 46,000 (range, 760 to 10,000,000) and 10,400 (range, 500 to 9,000,000) for samples in group 1, 2,750 (range, 600 to 430,000) and 822 (range, 160 to 129,000) for samples in group 2, and 454 (range, 220 to 4,150) and 184 (range, 10 to 1,530) for samples in group 3. Statistically significant differences have been reported only for calibrated values. +, positive; -, negative.


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DISCUSSION
 
The application of molecular techniques for the diagnosis and clinical monitoring of M. tuberculosis infection is gaining increasing attention. Numerous rapid diagnostic tests employing a number of different M. tuberculosis genomic targets, including the IS6110 insertion sequences, have been recently described (2, 6, 8, 9, 11, 12, 14, 19, 23, 27-29, 36). However, the mainstays of the laboratory of microbiology are still microscopy and culture on solid or liquid media (22, 34, 35). Indeed, the PCR systems developed thus far show good levels of sensitivity (90 to 100%) only on AFB smear-positive samples (2). Therefore, their use is restricted, when M. tuberculosis-specific primers are used, to the rapid identification of M. tuberculosis, allowing clinicians to make therapeutics choices only in the case of acute severe disease (i.e., meningitis).

The purpose of this study was to develop, by using a multiplex real-time PCR approach, a high-quality analytical system applicable directly to clinical specimens, allowing a rapid identification of the infectious agent as well as an assessment of the mycobacterial burden. The strategy of amplifying two distinct regions of the M. tuberculosis genome, namely the IS6110 multicopy insertion element and the senX3-regX3 IR, has been chosen to obtain a high degree of sensitivity and specificity. Indeed, the sole amplification of the IS6110 repeat element, one of the most-utilized PCR targets of the M. tuberculosis genome, does not allow a specific identification of M. tuberculosis (8, 13). Moreover, M. tuberculosis strains lacking the IS6110 element have been described previously (21). Our approach utilizes the amplification of a fragment of the IS6110 element, which is highly conserved among Mycobacterium species, to obtain the gender identification. Conversely, the amplification of a DNA fragment belonging to the senX3-regX3 IR, a recently described region specific for the M. tuberculosis complex (2, 19, 30), allows for a more precise identification of the Mycobacterium species involved.

This combined approach offers three major advantages, namely the following. (i) The assay is extremely fast (within a day), sensitive, and specific, as demonstrated on both AFB-positive and, more importantly, AFB-negative clinical specimens. (ii) It allows for the detection of a wide spectrum of Mycobacterium species, including M. tuberculosis strains that lack the IS6110 multicopy elements. (iii) The IS6110-to-senX3-regX3 copy number ratio permits the calculation of, without additional testing, the number of IS6110 repeat element copies present in the M. tuberculosis strain, which is useful genotypic information for the identification of epidemic outbreaks.

An important feature of this real-time PCR assay is that quantification of the M. tuberculosis load is obtained by measuring the copy number of the senX3-regX3 fragment, which has been designed to amplify a unique sequence in each M. tuberculosis genome. Thus, regardless of the M. tuberculosis strain tested, quantification of the DNA load can be directly related to the bacillar load. Indeed, the levels of M. tuberculosis DNA load in clinical specimens shows a good correlation with both AFB staining and M. tuberculosis isolation results. Moreover, the strong decrease in M. tuberculosis load measured in specimens belonging to patients under antitubercular treatment suggests that this test may be appropriate for monitoring treatment efficacy.

The addition of a synthetic DNA molecule termed the calibrator measured by a third real-time PCR assay completes the diagnostic setup, as it permits the monitoring of both the sample manipulation procedures and the presence of PCR artifacts due to the presence of PCR inhibitors. Indeed, the DNA extraction procedure can significantly affect the recovery rate of the nucleic acids, introducing a statistically significant bias of target underestimation (1, 31), which occurs irrespective of the DNA load in the starting material. Moreover, whereas the detection and amplification profile of the calibrator reveal the presence of PCR inhibitors, the measurement of the sample recovery rate establishes a precise cutoff of sensitivity for each negative sample.

In conclusion, our multiplex calibrated real-time PCR approach represents a fully controlled, fast, high-throughput diagnostic tool for the rapid identification of Mycobacterium infection directly in clinical specimens, which could be useful also for the clinical monitoring of antitubercular treatment.


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ACKNOWLEDGMENTS
 
We thank G. Finazzi (Zooprofilattico Institute, Brescia, Italy) for providing M. bovis strains and P. Biswas for critically reviewing the manuscript.

F.B. and P.S. contributed equally to this work.

This study was supported by grants from the III and IV Italian National AIDS Project, Ministry of Health, Rome, Italy.


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FOOTNOTES
 
* Corresponding author. Mailing address: Unit of Human Virology, Via Olgettina 58, DIBIT, San Raffaele Scientific Institute, 20132 Milan, Italy. Phone: 39-02-2643-4903. Fax: 39-02-2643-4905. E-mail: malnati.mauro{at}hsr.it. Back

{dagger} Present address: Pharmacia, Pharmacology Dept., Gene Expression Unit, 20014 Nerviano, Milan, Italy. Back


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Journal of Clinical Microbiology, October 2003, p. 4565-4572, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4565-4572.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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