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Journal of Clinical Microbiology, January 2009, p. 282-283, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.00703-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Impact of a Chemistry-Based DNA Extraction Method on Performance of a Commercial Amplification Assay for Detection of Mycobacterium tuberculosis Complex

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The worldwide spread of tuberculosis (TB) has created an urgent mandate for the rapid diagnosis and effective treatment of patients with active disease. A decline in expertise among clinicians and the low sensitivity of sputum smears have been claimed as being responsible for the delayed detection of a substantial number of TB cases in developed countries (1, 2). The ability of commercially available direct amplification assays to detect the Mycobacterium tuberculosis complex (MTB) in clinical samples is largely dependent upon the efficiency of the nucleic acid extraction, which presently relies upon procedures such as heat, mechanical lysis, and sonication, which are less effective than chemistry-based methods. The objective of this study was to compare the guanidinium thiocyanate-based DNA purification protocols developed by Boom et al. (NucliSens; bioMérieux BV, Boxtel, The Netherlands) (3) with the lysis extraction protocol provided by a commercial assay, the strand displacement amplification BDProbeTec ET Mycobacterium tuberculosis complex direct detection assay (DTB) (Becton Dickinson Biosciences, Sparks, MD). DTB is a fully automated walk-away system running an internal amplification control (IAC) with each sample, which couples strand displacement amplification to a fluorescent energy transfer detection system (7).

Eighty-three selected clinical specimens, collected from 70 patients, were retrospectively assayed by DTB after they underwent two different extraction procedures. Specimens, almost entirely collected from inpatients screened for TB, belonged to the following categories: smear positive, yielding MTB (n = 20); smear negative, yielding MTB (n = 21); smear positive, yielding nontuberculous mycobacteria (NTM) (n = 11); smear and culture negative, collected from patients whose clinical history provided enough evidence of active disease to undergo antituberculous chemotherapy (n = 3); and smear and culture negative, collected from non-TB patients showing clinical and X-ray pictures resembling active TB (n = 28). Investigated specimens included 24 sputum, 13 bronchoalveolar lavage, 23 bronchial washing, two gastric aspirate, and four urine samples, six normally sterile body fluid samples (pleural, pericardial, synovial, and cerebrospinal fluids and ascites), and 11 miscellaneous samples, such as pus and biopsy specimens. All the specimens underwent standard bacteriological procedures (5). Decontamination was performed by the N-acetyl-L-cysteine-sodium hydroxide method. A 0.5-ml portion of the processed sediment was cultivated by a combination of the Bactec MGIT 960 system (Becton Dickinson Diagnostic Instrument Systems) (9) and Löwenstein-Jensen solid medium. To detect acid-fast bacilli (AFB), smears from clinical samples as well as those from positive culture media were stained by the Ziehl-Neelsen stain. Mycobacterial isolates were identified by specific DNA probe assays, by standard biochemical tests, and by the high-performance liquid chromatography method.

DNA was prepared using NucliSens magnetic extraction reagents (NucliSens; bioMérieux BV, Boxtel, The Netherlands) (6). These reagents were applied to 500-µl sample portions lysed using a 5-mol/liter guanidinium thiocyanate lysis buffer (NucliSens lysis buffer) and a magnetic extraction instrument (NucliSens miniMag). The above-described procedure was performed according to the instructions supplied by the manufacturer. Briefly, under high-salt conditions, DNA binds to magnetic silica particles. These silica particles act as a solid phase, and nonnucleic acid components are removed by several washing steps performed in the miniMag instrument. DNA is then eluted from the solid phase into 50 µl of elution buffer. Finally, after addition of sample neutralization buffer, DNA samples were ready to be assayed.

On the other hand, 500-µl sample portions were added to a sample wash buffer to remove possible inhibitors and were centrifuged and heated at 105°C for 30 min (BDProbetc ET oven). Then the pellet was resuspended in a sample lysis buffer and sonicated for 45 min at 65°C in a water sonic bath (Branson Ultrasonic Corp., Dambury, CT). After the addition of a sample neutralization buffer, samples were ready to be assayed. DTB amplification and detection were performed according to the instructions provided by the manufacturer (8). Samples showing MTB MOTA (metric other than acceleration) values greater than 3,400 were considered positive for MTB, regardless of the IAC values. If the MTB MOTA value was less than 3,400 and the corresponding IAC MOTA value was greater than 5,000, the specimen was considered negative for MTB. Finally, if the MTB MOTA value was less than 3,400 and the corresponding IAC MOTA value was less than 5,000, the result was regarded as indeterminate. Patients' clinical records were carefully reviewed, aiming to set up the combination of culture and clinical diagnosis as the "gold standard." Two categories of samples were considered to be true positives—samples that were culture positive for MTB, and samples that were culture negative for MTB but shared one or both of the following conditions: (i) the sample originated from a patient with other culture-positive samples, and (ii) the patient's clinical history provided enough evidence of TB to initiate antituberculous chemotherapy. After this analysis, amplification results were reclassified, as appropriate. Statistical comparisons were calculated by using the chi-square test; a P value of <0.05 was considered significant. Sensitivity, specificity, and predictive values were determined accordingly.

From an analytical point of view, differences among cutoff values, positive and negative controls, and samples were broad enough to allow easy discrimination by both assays. Altogether, 41 specimens yielded cultures positive for MTB. A comparison of the amplification results, with smear, culture, and clinical data, is summarized in Table 1. Of the 20 samples which were smear and culture positive, all were positive by both assays. Sensitivity and specificity were excellent, and no inhibition has occurred so far. Moreover, the magnitude of the mean MOTA value was slightly higher for those samples undergoing NucliSens extraction (me-DTB; 70,218 MOTA ranging from 45,292 to 126,847) than for those processed by heat and sonication (st-DTB; 66,925 MOTA ranging from 26,603 to 97,411). Twenty-one samples were smear negative but culture positive for AFB; 18 were positive by both assays. However, two additional samples tested by me-DTB exhibited MTB MOTA values slightly below the cutoff value (MTB MOTA values of 3,247 and 3,311), and one more sample was flagged as indeterminate. We also observed that in this category, the magnitude of the mean MOTA value was lower for those undergoing me-DTB assay (MOTA value of 24,852, ranging from 60,875 to 4,267) than for those processed by st-DTB assay (MOTA value of 39,466, ranging from 76,685 to 3,564). A consequent adjustment of the cutoff value (from an MTB MOTA value of 3,400 to 3,000) increased sensitivity (two more positive samples) without reducing specificity (4). There were three smear- and culture-negative samples collected from patients for whom the diagnosis of TB was set on the basis of clinical, X-ray, and/or histological findings. All of these were st-DTB positive, but only two were me-DTB positive. From 11 smear-positive specimens, NTM were grown. All of these specimens were negative by both amplification assays. Of the 28 samples from patients with nontuberculous pulmonary disease that were smear and culture negative for AFB, 5 were st-DTB positive, while none were me-DTB positive. These samples, after resolution of discrepant results, were considered to be false positives. Although recent data seem to confirm our finding (11), we did not expect that a more efficient extraction would have improved both sensitivity and specificity. In our study, the overall inhibition rate was 2.4%. It occurred in two respiratory samples (one of them was smear negative and MTB yielding) undergoing me-DTB assay. Previous reports indicating that inhibition occurs more frequently after silica-guanidinium thiocyanate-based extraction (10, 11) are in agreement with our data. Sensitivity, specificity, and positive and negative predictive values of st-DTB assay were 93.2, 87.2, 89.1, and 91.9%, respectively, and those of me-DTB assay were 90.9, 100, 100, and 90.7%, respectively. After adopting the newly proposed cutoff value, the aforementioned me-DTB sensitivity, specificity, and positive and negative predictive values became 95.5, 100, 100, and 95.1%, respectively.

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TABLE 1. Comparison of st-DTB and me-DTB amplification assays with smear/culture results and clinical data

We conclude that in our study, the NucliSens guanidinium thiocyanate-based DNA extraction protocol has increased DTB sensitivity in the category of smear-negative MTB-yielding specimens. Moreover, this extraction method has significantly reduced the number of false-positive results. NucliSens extraction could be recommended for smear-negative samples collected from patients with high clinical suspicion of active TB and for sample retesting when a positive result does not match up to clinical expectations. Further research is needed to confirm our data.

 
Published ahead of print on 19 November 2008.

Top LETTER REFERENCES

 

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2. Asch, S., B. Leake, R. Anderson, and L. Gelberg. 1998. Why do symptomatic patients delay obtaining care for tuberculosis? Am. J. Respir. Crit. Care Med. 157:1244-1248.[Abstract/Free Full Text]
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3. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Nordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503.[Abstract/Free Full Text]
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4. Cloud, J. L., C. Shutt, W. Aldous, and G. Woods. 2004. Evaluation of a modified Gen-Probe amplified direct test for detection of Mycobacterium tuberculosis complex organisms in cerebrospinal fluid. J. Clin. Microbiol. 42:5341-5344.[Abstract/Free Full Text]
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5. CLSI. 2007. Laboratory detection and identification of mycobacteria; proposed guideline. M48-P. Clinical and Laboratory Standards Institute, Wayne, PA.
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6. Espy, M. J., J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter, J. D. C. Yao, N. L. Wengenack, et al. 2006. Real-time PCR in clinical laboratory: applications for routine laboratory testing. Clin. Microbiol. Rev. 19:165-256.[Abstract/Free Full Text]
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7. Little, M. C., J. Andrews, R. Moore, S. Bustos, L. Jones, C. Embres, G. Durmovicz, J. Harris, et al. 1999. Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET. Clin. Chem. 45:777-784.[Abstract/Free Full Text]
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8. Piersimoni, C., C. Scarparo, P. Piccoli, A. Rigon, G. Ruggiero, D. Nista, and S. Bornigia. 2002. Performance assessment of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex from respiratory and extrapulmonary specimens. J. Clin. Microbiol. 40:4138-4142.[Abstract/Free Full Text]
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9. Roberts, G. D., L. Hall, and D. M. Wolk. 2002. Mycobacteria, p. 256-273. In A. L. Truant (ed.), Manual of commercial methods in clinical microbiology. American Society for Microbiology, Washington, DC.
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10. Suffys, P., P. R. Vanderborght, P. Barros dos Santos, L. A. Pinto Correa, Y. Bravin, and A. L. Kritski. 2001. Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidinium thiocyanate DNA isolation procedure. Mem. Ist. Oswaldo Cruz 96:1137-1139.
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11. van Doorn, H. R., E. S. Bruijnesteijn van Coppenraet, B. Duim, C. M. J. E. Vandenbroucke-Grauls, J. F. Weel, J. Dankert, E. J. Kuijper, and M. D. de Jong. 2006. Silica-guanidinium thiocyanate-based nucleic acid isolation protocol does not improve sensitivity of two commercial tests for detection of Mycobacterium tuberculosis in respiratory samples. Eur. J. Clin. Microbiol. Infect. Dis. 25:673-675.[CrossRef][Medline]

						Claudio Piersimoni* Giancarlo Gherardi Domenico Nista Stefano Bornigia Department of Clinical Microbiology United Hospitals Via Conca 71 Ancona, Italy
						* Phone: 39-71-596.3049 Fax: 39-71-596.4184 E-mail: piersim{at}tin.it

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Journal of Clinical Microbiology, January 2009, p. 282-283, Vol. 47, No. 1
0095-1137/09/$08.00+0     doi:10.1128/JCM.00703-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Piersimoni, C. Articles by Bornigia, S. Search for Related Content PubMed PubMed Citation Articles by Piersimoni, C. Articles by Bornigia, S.