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Journal of Clinical Microbiology, August 2007, p. 2398-2403, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00292-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Test for Tuberculosis in Live European Badgers (Meles meles) Based on Measurement of Gamma Interferon mRNA by Real-Time PCR

J. Sawyer,¹ D. Mealing,¹ D. Dalley,² D. Davé,² S. Lesellier,² S. Palmer,² J. Bowen-Davies,^3, T. R. Crawshaw,⁴ and M. A. Chambers^2*

Technology Transfer Unit, Biotechnology Department,¹ TB Research Group, Department of Statutory and Exotic Bacteria, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom,² TBD Wildlife Unit, Aston Down, Stroud, Gloucestershire GL6 8GA, United Kingdom,³ Veterinary Laboratories Agency Starcross, Staplake Mount, Starcross, Exeter EX6 8PE, United Kingdom⁴

Received 6 February 2007/ Returned for modification 14 March 2007/ Accepted 21 May 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A real-time PCR assay for the measurement of gamma interferon (IFN-) mRNA in European badger (Meles meles) blood cultures was developed. The levels of IFN- mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens were compared with those in a nonstimulated control blood culture as the basis for determining the tuberculosis (TB) status of live badgers. The assay was validated by testing 247 animals for which there were matching data from postmortem examination and culture of tissues. Relative changes in the levels of IFN- mRNA in response to bovine tuberculin and specific antigens were found to be greater among badgers with tissues positive for TB on culture. The test was at its most accurate (87% of test results were correct) by using blood cultures containing bovine tuberculin as the antigen and when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response from the bovine tuberculin response. At a specificity of 90.7%, the test was 70.6% sensitive. At the same specificity, the current serological enzyme-linked immunosorbent assay for TB in badgers was only 53% sensitive. This work demonstrates that measurement of IFN- mRNA by real-time PCR is a valid method for the detection of TB in live badgers and may provide an alternative to the current serological methods of diagnosis, the Brock test. The testing procedure can be completed within 5 h of receipt of the blood culture samples. In addition, the use of a molecular biology-based test offers the potential to fully automate the testing procedure through the use of robotics.

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There has been a sharp rise in the incidence of bovine tuberculosis (TB) in cattle in the United Kingdom since the early 1990s (1). This adversely affects animal health and welfare and is a cause of considerable economic loss to farmers and the government. The European badger (Meles meles) has been implicated in the spread of disease by acting as a source of Mycobacterium bovis infection in cattle (9, 10). The accurate diagnosis of M. bovis infection in badgers can be achieved by bacteriological culture, but only for samples obtained postmortem. In vitro diagnostics that can be used to test live animals are required, for instance, to further develop vaccination strategies. Antibody-based assays have been developed (13, 14), but they lack sensitivity (3, 14), as they do for cattle (23). In comparison, assays based on measurement of cellular responses produce better results (6, 23). However, those developed to date for badgers are impractical for routine use (6). Tests analogous to that developed for the diagnosis of TB in cattle by the detection of gamma interferon (IFN-) in whole-blood cell cultures (21) have yet to be validated for badgers, although the badger IFN- gene has been cloned and a polyclonal antiserum to the cytokine has been produced (7). Having cloned the badger IFN- gene, we investigated the possibility of using IFN- mRNA levels as a measure of the cell-mediated response to mycobacterial antigens. A recent report on the use of a quantitative real-time PCR (qRT-PCR) method for the detection of IFN- in human TB patients demonstrated that the method gave good agreement with the results of the enzyme-linked immunosorbent assay (ELISA) and the enzyme-linked immunospot assay methods of detecting the cytokine (8). Our data demonstrate that a qRT-PCR approach can be used as the basis for a test for M. bovis infection in live badgers which produces better results than the current serological test.

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Badger samples. Blood samples were collected from badgers trapped as part of the Randomized Badger Culling Trial (RBCT) (10). Blood was collected as described on project and personal Home Office licenses from either the heart or the jugular vein in heparinized Vacutainer tubes (BD, Plymouth, United Kingdom) while the badger was under deep terminal anesthesia. Anesthesia was induced by the intramuscular injection of a mixture of ketamine (Vetalar V; Pharmacia Animal Health Ltd., United Kingdom) and medetomidine (Domitor; Pfizer Animal Health) at doses of 150 mg per badger (dose range, 10 to 44 mg/kg of body weight) and 1.5 mg per badger (dose range, 0.10 to 0.44 mg/kg), respectively. The time of blood collection was recorded on the tubes. The blood tubes were then transported to the laboratory at ambient temperature on the same day and were set up for culture within an average of 7 h from the time of collection (range, 3.75 to 10.83 h).

Badger necropsy. The badgers were submitted to nine of the Veterinary Laboratories Agency's (VLA's) regional laboratories for necropsy. All animals described in this report were subjected to a standardized detailed necropsy protocol. Badger carcasses were stored at 4°C, and in nearly all cases the badgers were examined within 3 days of submission. Following gross examination, the following tissues were examined visually and sectioned: a comprehensive range of lymph nodes, including those of the head, thoracic region, abdomen, and skeleton; the tonsils; the mandibular salivary glands; intestine; liver; kidney; and mammary gland and uterine horn (if available). Three pools of tissues that comprised most of the tissues described above were always taken and placed into 1% cetylpyridinium chloride (CPC) for separate culture, as follows. One comprised the bronchial, mediastinal, and retropharyngeal lymph nodes ("standard pool"). The second was additional tissues that were from the gastrointestinal tract (GIT pool), and the third was additional tissues that were not from the gastrointestinal tract ("non-GIT pool"). Any bite wounds were sampled and placed into a separate container of 1% CPC and cultured separately.

M. bovis culture. Tissues collected in 1% CPC were posted to the culture laboratory and in nearly all cases were processed within 72 h of the time that the tissues had been placed in CPC. At the culture laboratory the CPC was discarded and the tissues were rinsed in sterile 0.85% saline. Twenty milliliters of sterile 0.85% saline was added, and the tissues and saline were emulsified for about 1 min at high speed in a stomacher. Approximately 250 µl of the resulting suspension was inoculated onto slopes of modified Middlebrook 7H11 agar from two different batches. Suspensions from the standard pool of lymph nodes were cultured on 12 slopes, and the tissue suspensions from the GIT and the non-GIT pools were cultured on six slopes each. The slopes were flooded by tilting and were incubated at 37 ± 2°C. The remaining suspension was archived at –20°C. The slopes were examined weekly for 12 weeks, and if in the first 2 weeks there was overgrowth with contaminants, the archived suspension was thawed and an equal volume of 5% sulfuric acid was added. After 10 min it was centrifuged at 1,100 x g for not more than 10 min. The supernatant was discarded and a volume of sterile 0.85% saline equal to the volume of the original archived suspension was added. The pellet was disrupted by shaking, and the slopes were inoculated as described for the primary culture. Any growth of organisms characteristic of mycobacteria were identified by spoligotyping (11).

Badger blood culture. Whole heparinized blood was mixed in a 24-well culture plates in a 1:1 ratio with RPMI 1640 medium (Invitrogen) so that each culture (1.5-ml volume) contained 50 units/ml penicillin (Invitrogen), 50 µg/ml streptomycin (Invitrogen), 26 IU/ml heparin (Roche), and one or more of six antigens consisting of 30 µg/ml of purified protein derivative from M. bovis (PPD-B), 30 µg/ml of purified protein derivative from M. avium (PPD-A), 5 µg/ml of pokeweed mitogen (Sigma), 5 µg/ml of CFP-10, or 5 µg/ml each of ESAT-6 and CFP-10 or no antigen (control culture). The cultures were mixed by swirling the culture plates on a smooth flat surface, before incubation at 37°C in 5% CO₂ for 16 to 24 h. Following incubation, the plasma was removed and stored at –80°C. One milliliter of RLT lysis buffer (QIAGEN) was then added to the remaining sedimented cells. The samples were mixed by swirling and were frozen at –80°C in the culture plates.

RNA extraction. The lysed cells were defrosted at room temperature, and 200-µl aliquots were added to the wells of MagNA Pure LC sample cartridges. If necessary, the cartridges were sealed and refrozen at –80°C prior to RNA extraction. RNA was extracted from the 200-µl aliquots of cells by using the MagNA Pure instrument and the MagNA Pure LC RNA isolation kit—high performance (Roche Applied Science), according to the manufacturer's instructions. The elution volume was 100 µl. The extracted RNA was stored at –80°C.

Primers and probes. The ß-actin primers and probe sequences (Table 1) were based on canine sequences and were taken from a previously published study (12). The IFN- primers and probe (Table 1) were adapted from a previously published study (12) by substituting bases to match the published badger IFN- gene sequence (7).

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TABLE 1. Primers and probes used for amplification of badger IFN- and ß-actin mRNA

Real-time reverse transcriptase PCR. One-step real-time PCR was carried out by using the QuantiTect Probe RT-PCR kit (QIAGEN). ß-Actin reactions were carried out in a 25-µl volume containing 1x QuantiTect Probe RT-PCR master mix, 0.25 µl of QuantiTect reverse transcriptase mix, 0.4 µM primers Badger ß-actin (F) and Badger ß-actin (R), and 0.1 µM Badger ß-actin TaqMan. IFN- reactions were carried out as described for the ß-actin reactions; but the reaction mixtures contained 0.8 µM primers Badger IFN- (F) and Badger IFN- (R), 0.2 µM Badger IFN- TaqMan, and 2 mM MgCl₂ (in addition to the 4 mM present in the 1x QuantiTect master mix). Five microliters of template RNA was added to all reaction mixtures. All reactions were run in triplicate and were carried out in white 96-well PCR plates (ABgene) sealed with optical caps (ABgene) on an MX3000P instrument (Stratagene). The instrument was programmed to cycle at 50°C for 30 min; 95°C for 15 min; and then 50 cycles of 15 s at 95°C, 30 s at 55°C, and 30 s at 60°C.

PCR data generation. The average threshold cycle (C_T) values of triplicate reactions were determined with MX3000P software (version 3.0) by use of the amplification-based threshold determination, adaptive baseline analysis, and moving-average-algorithm enhancement options. The C_T data were exported to an Access database (Microsoft). The change in the level of expression of IFN- mRNA in the antigen-stimulated blood cultures relative to that in the control cultures was calculated by using the delta C_T method (17). In addition, the change in the level of expression following PPD-B stimulation divided by the change in the level of expression following PPD-A stimulation (pcr B/A) was calculated, as was the change in the level of expression following PPD-B stimulation minus the change in the level of expression following PPD-A (pcr B– A) stimulation. Sample processing and submission of C_T data were carried out by personnel who were blind to the TB status of the animal, with calculation of changes in the level of expression of IFN- mRNA being carried out by separate personnel.

Brock test. Badger serum samples (collected as described above) were subjected to the Brock test, the current indirect ELISA for the detection of antibodies to M. bovis, according to the published protocol (13) and by using a cutoff validated internally by VLA.

Data analysis. Selection of the best antigen for use in badger blood cultures was carried out by using receiver operating characteristic (ROC) analysis (15). The performance of the real-time reverse transcriptase PCR test was assessed by comparison with the results of the current "gold standard" (culture of postmortem tissue samples for M. bovis) and with those of the current immunological test for the detection of M. bovis in live badgers, the Brock test. Comparison of the tests was carried out by ROC analysis, calculation of standard test performance characteristics, and the one-tailed McNemar test for paired samples by using Analyze-it software (version 1.71; Analyze-it Software Ltd.). Tests of significance between means and proportions were calculated by using GraphPad InStat software (version 3.06) for Windows (GraphPad Software, San Diego, CA) and the tests referred to by name when they were used.

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The levels of IFN- mRNA in blood cultures stimulated with either bovine or avian tuberculin or specific mycobacterial antigens obtained by real-time reverse transcriptase PCR were compared with those in a nonstimulated control blood culture as the basis for the determination of the TB status of an individual badger. The assay was validated by testing 247 animals for which matching data on the results of culture of tissues for M. bovis were available (53 were culture positive). Brock test results were available for 244 of these 247 animals.

Selection of the best antigen(s) for use in badger blood cultures for IFN- PCR analysis. ROC analysis (Fig. 1) was used to compare the performance of the IFN- PCR test relative to that of culture when different antigens were used. Based on calculation of the area under the curve (AUC), a summary statistic of diagnostic accuracy (15), pcr B – A was marginally, although not significantly, better than pcr B/A or measurement of the change in the level of expression following PPD-B stimulation (pcr B) directly (Fig. 1A). A similar analysis (Fig. 1B) demonstrated that the use of a single specific mycobacterial antigen (CFP-10) appeared to produce better results than the use of a cocktail of CFP-10 and ESAT-6 (but the difference was not statistically significant), whereas the use of avian and bovine tuberculins was significantly better than the use of either mycobacterial antigen. For further comparison of the IFN- PCR test with the Brock test, the pcr B – A measure was used.

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FIG. 1. ROC curves comparing detection of TB in badgers based on measurement of the levels of IFN- mRNA in blood cultures stimulated with either bovine tuberculin (pcr B), bovine tuberculin when the response to avian tuberculin was taken into account by either subtracting (pcr B – A) or dividing by (pcr B/A) the avian tuberculin response, mycobacterial antigen CFP-10 (pcr cfp10), or a cocktail of CFP-10 and ESAT-6 (pcr cocktail). The AUC is shown, as are the numbers of badgers positive or negative by the culture method used in the analysis. A test of significance between curves is shown as the P value.

Performance of the IFN- PCR and Brock tests singly. The PCR test was at its most accurate (i.e., the largest proportion of test results agreed with those of culture) when a cutoff of pcr B – A of 14.04 was used. By use of this cutoff, 87.0% of the test results agreed with the culture results and the PCR test was 64.2% sensitive and 93.3% specific (Tables 2 and 3), with positive and negative predictive values of 72.3% and 90.5%, respectively.

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TABLE 2. Proportion of culture-positive and -negative badgers positive by the IFN- PCR test

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TABLE 3. Summary of performance of the IFN- PCR and Brock tests for diagnosis of M. bovis infection in badgers

By use of the cutoff for the Brock test internally validated by VLA, the sensitivity and specificity of the Brock test compared to the results of culture were 52.9% and 90.7%, respectively, giving an overall accuracy of 82.8% (i.e., 82.8% of the test results agreed with the culture results) (Tables 3 and 4). The positive and negative predictive values were 60.0% and 87.9%, respectively.

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TABLE 4. Proportion of culture-positive and -negative badgers positive by Brock test

Comparison of IFN- PCR test with Brock test. Comparison of the results of the Brock test and the IFN- PCR test to those of culture by ROC analysis (Fig. 2) for the 244 badgers for which the results of both tests were available showed that the AUC was significantly greater for the PCR test (difference = 0.089; P < 0.03) and demonstrates that the IFN- PCR results agree more closely with the culture data for the same animals than do the results of the Brock test. The AUC value of 0.906 is regarded as a highly accurate test result, according to suggested guidelines on the interpretation of AUC values (15), indicating that the IFN- PCR test is a useful test for the detection of TB in live badgers.

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FIG. 2. ROC curves comparing detection of TB in badgers based on the Brock test (BT) or measurement of the levels of IFN- mRNA in blood cultures stimulated with bovine tuberculin when the response to avian tuberculin was taken into account by subtracting the avian tuberculin response (pcr B – A). The AUC is shown, as are as the numbers of badgers positive or negative by the culture method used in the analysis.

To aid direct comparison with the Brock test, ROC analysis was used to select a different cutoff for the PCR test (pcr B – A value, 11.13) that gave the same specificity as the Brock test. By use of this cutoff, the IFN- PCR test has a sensitivity of 70.6% and an overall accuracy of 86.4% compared to the results of culture. Following from this, one-tailed McNemar's analysis demonstrated that the PCR test differed significantly (P = 0.046) from the Brock test in the proportion of samples found to be positive; i.e., the PCR test is more sensitive.

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We have shown that the measurement of IFN- mRNA by qRT-PCR in a cell-mediated assay can be used to determine the TB status of badgers in a way analogous to measurement of the IFN- protein in cattle (22).

Although we have developed antibodies for testing for IFN- protein in badgers (7) and are in the process of assessing a sandwich ELISA approach to the detection of badger IFN-, the ability to carry out testing without the need for the production of antibodies could be advantageous for testing in other species. This approach might allow the rapid development of IFN- reverse transcription-PCR assays for species for which few serological reagents are available (for example, some wildlife species) if some gene sequence information is known. In other cases the use of primer and probe sets from related species would allow testing to be carried out without sequence information; for example, the ß-actin primers and probes used in this study were originally designed for use in dogs. The use of an assay system based on DNA oligonucleotides, which can be ordered routinely and in bulk, would also avoid the need for the complex laboratory procedures associated with antibody production and would remove the batch consistency challenges that are associated with the use of polyclonal antibody-based assays in a routine testing situation. In addition, the method reported here used whole blood rather than isolated peripheral blood mononuclear cells. Although we made no direct comparison, the use of whole blood in the ELISA and the qRT-PCR methods for humans gave results comparable to those obtained with isolated peripheral blood mononuclear cells (8).

Although the PCR test is clearly an improvement over the current Brock test, the sensitivity of the test is still lower than that of an ideal screening test. At a specificity of 90.7%, the PCR test failed to detect 15 of 51 animals that were positive by culture (a 29% false-negative rate). There is some evidence from TB in other species (e.g., cattle) that cell-mediated immunity, typified by the IFN- response, is significantly reduced in animals with severe disease (19); and we have reported previously that seropositivity is associated with more advanced stages of disease in badgers (2). However, although 6 of the 15 badgers with false-negative results were positive by the Brock test, there were no obvious differences in the severity of the disease between the badgers with false-negative results by PCR and those that had true-positive results by PCR. In fact, almost half of the badgers in each category had no visible lesions at postmortem examination, even though they were found to have TB by culture (data not shown). Doherty et al. observed that, depending on the cytokine being studied, delays in the processing of blood can have a significant effect on the sensitivity of cytokine assays, including a reverse transcriptase PCR-based method (8). Examination of the 15 samples with false-negative results in this respect revealed that the blood had been processed after a mean of 396 min (approximately 6.5 h; 95% confidence interval [CI], 350 to 443 min), compared with an average of 424 min (approximately 7 h; 95% CI, 397 to 450 min) for the 36 samples with true-positive results. This difference was not significant (P = 0.2697, unpaired t test). Nonetheless, delays in the processing of the blood could have resulted in a reduced sensitivity for the PCR test more generally. In our study, the maximum delay was about 10 h, with an average delay of 7 h. In the study of Doherty et al. (8) at least, the IFN- reverse transcriptase PCR method was unaffected by a delay of up to 24 h between blood collection and use. This is encouraging, since rapid processing of blood will be a challenge in many field settings.

The 18 samples with false-positive test results (7% of the total) could be explained by the fact that the PCR test detected early cell-mediated events before any significant bacillary load or pathology had developed. In support of this, only one of these samples was also positive by the Brock test. This analysis also assumes that the current gold standard of culture of tissues is a definitive test. However, due to the nature of mycobacterial infections, a limited number of tissues may be infected and only a small number of organisms may be present in them, especially in the early stages of infection. Even by the use of the extensive range of tissues represented by the three culture pools, it is still possible that organisms were missed and that lesions in tissues affected with tuberculosis may not be visible grossly, as has been observed for both cattle (5) and possums (Trichosurus vulpecula) (16) at postmortem examination. Interestingly, in this case, 9 of the 18 badgers (50%) with false-positive results by PCR had visible lesions at postmortem examination in contrast to only 42 of the 175 badgers (24%) with true-negative results by PCR. The number of PCR-positive animals with visible lesions was disproportionately high (P = 0.0246, Fisher's exact test). This suggests either that culture failed to confirm the presence of TB in these animals with visible lesions animals (in which case the PCR test is more accurate than we have reported) or that the TB-like pathology was caused by some other infection that resulted in false positivity by the PCR test.

The improvement of the results of the IFN- PCR test relative to those of the Brock test was marginal statistically (P = 0.046) for this relatively limited sample. A larger sample for testing would increase the power of the analysis. However, the collection of large numbers of samples from badgers is logistically difficult, as is often the case for wildlife species. In this case we were able to obtain blood samples alongside the trapping that was carried out for RBCT. Even so, we were limited by the need to take blood from animals while they were under anesthesia. We had shown previously that blood taken immediately after death, even though it is easier to obtain during the RBCT, was unsuitable for the stimulation of IFN- (unpublished data). The same was demonstrated for cattle by others (18).

Experiences with cattle TB tests point to a situation in which different tests (for example, the tuberculin skin test and the IFN- test) produce different, if overlapping, results. In practice, the greatest sensitivity was obtained by the use of a combination of tests (4, 20, 22). Given this complicated background, set against different rates of disease progression and individual variation (all of which are poorly understood in badgers), it is probably unrealistic to expect one assay to provide an ideal test. A possible use for the IFN- PCR may be to use it alongside other tests to provide more confidence or to use it as a confirmatory test. For example, in this case, the use of a combination of the IFN- PCR and Brock test gave a 13.7% increase in sensitivity over that by use of the IFN- PCR alone (or a 31.4% increase over that by use of the Brock test alone) to 84.3% (95% CI, 71.4% to 93.0%). This gain in sensitivity was achieved with a relatively modest decrease in specificity of 8.8% to 81.9% (95% CI, 75.7% to 87.1%).

The design of the IFN- PCR test was driven by the need for a test system which could be realistically carried out in a routine laboratory environment. The automation of the RNA extraction procedure by the use of robotics and the use of a one-step reverse transcriptase PCR master mixture were essential in making this a potential method for routine testing for TB. Although the preparation of PCR master mixtures and the loading of samples were carried out by using manual and multichannel pipettes in this study, the further use of robotics could be envisaged to streamline and automate more of the testing process. The ability to freeze lysed cell samples and store them, prior to processing and analysis, was also an important factor in being able to batch samples and test them in an efficient manner. The testing procedure can be completed within 5 h of receipt of the antigen-stimulated blood culture samples, offering the prospect of rapid testing in comparison to the time of bacterial culture, which takes several weeks. The results of this study suggest that the test could be a useful additional tool for the diagnosis of TB in this species, although the need to stimulate blood in the laboratory means that other methods will need to be developed for use when greater speed and simplicity are the key requirements of the test (14).

 
This work was funded by the Department for Environment, Food and Rural Affairs, Great Britain.

We thank the staff of the Defra Wildlife Unit and VLA Starcross for assisting with sample collection, blood culture, and bacteriology; Joshua Mawdsley (CERA, VLA Weybridge) for the sample database; and the Independent Scientific Group for permission to use samples from RBCT. Anesthesia and sampling of the blood of the badgers alongside the RBCT were carried out under a license issued according to A(SP)A, following local ethical review. We thank Glyn Hewinson, VLA, for critical review of the manuscript.

 
* Corresponding author. Mailing address: TB Research Group, Department of Statutory and Exotic Bacteria, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: 44 (0) 1932 357494. Fax: 44 (0) 1932 357260. E-mail: m.a.chambers{at}vla.defra.gsi.gov.uk

Published ahead of print on 30 May 2007.

Present address: Home Office, P.O. Box 1138, Swindon SN1 2RZ, United Kingdom.

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Journal of Clinical Microbiology, August 2007, p. 2398-2403, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00292-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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