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Lee, Kim, and Lee: Performance Evaluation of Anyplex Plus MTB/NTM and MDR-TB Detection Kit for Detection of Mycobacteria and for Anti-Tuberculosis Drug Susceptibility Test
Original Article

Ann Clin Microbiol 2014;17(4):115-122.

Published online: 5 December 2014

DOI: https://doi.org/10.5145/ACM.2014.17.4.115

Performance Evaluation of Anyplex Plus MTB/NTM and MDR-TB Detection Kit for Detection of Mycobacteria and for Anti-Tuberculosis Drug Susceptibility Test

Jun Hyung Lee, Bo Hyun Kim, Mi-Kyung Lee

Department of Laboratory Medicine, Chung-Ang University College of Medicine, Seoul, Korea

Correspondence: Mi-Kyung Lee, Department of Laboratory Medicine, Chung-Ang University College of Medicine, Heukseok-ro 102, Dongjak-gu, Seoul 156-755, Korea. (Tel) 82-2-6299-2719, (Fax) 82-2-6298-8630, (E-mail) cpworld@cau.ac.kr

Received 15 October 201413 November 2014       Accepted 27 November 2014

Copyright © 2014 The Korean Society of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

초록

Background

The Anyplex plus MTB/NTM and MDR-TB Detection kits (Seegene, Korea) a real-time PCR assays for direct detection of Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) and for identification of rifampin (RIF) and isoniazid (INH) resistance of MTB in various specimens. We evaluated the diagnostic performance of the Anyplex plus MTB/NTM and MDR-TB Detection kit.

Methods

To determine the ability of the kit to detect MTB and NTM, 557 samples were tested. The diagnostic performance of the Anyplex plus MTB/NTM Detection kit was determined based on the results of culture, nucleic acid amplification tests (NAAT), radiologic analysis, and clinical features suggestive of mycobacterial infection. The performance of the kit was compared with those of other real-time PCR kits. For the drug susceptibility test (DST), 51 MTB isolates were tested. The diagnostic performance of the Anyplex plus MDR-TB Detection kit was determined based on the conventional DST and compared with other molecular DST kits.

Results

Sensitivity and specificity for MTB detection of the Anyplex plus MTB/NTM Detection kit were 82.9% (63/76) and 99.4% (478/481), respectively, while those for NTM detection were 76.5% (13/17) and 89.6% (484/540). Sensitivity and specificity for RIF resistance detection of the Anyplex plus MDR-TB Detection kit were 100% (3/3) and 97.9% (47/48), respectively, while those for INH resistance detection were 83.3% (5/6) and 100% (45/45).

Conclusion

The Anyplex plus MTB/NTM Detection kit showed good diagnostic performance for detection of MTB and NTM. Especially in MTB-positive cases, the Anyplex plus MDR-TB Detection kit provided rapid and reliable results of drug resistance to RIF and INH.

Keywords: Anyplex plus, Molecular diagnostic techni-ques, Multidrug-resistant tuberculosis, Mycobacterium tuberculosis, Nontuberculous Mycobacteria

REFERENCES

1.The Korean Society of Clinical Microbiology and Korea Centers for Disease Control and Prevention. Manual of Laboratory Tests for Tuberculosis (2013). http://www.cdc.go.kr/CDC/cms/cmsFile-Download.jsp?fid=51&cid=21373&fieldName=attach1&index=1. [Online] (last visited on 16 December 2014).
2.The Korean Society of Clinical Microbiology and Korea Centers for Disease Control and Prevention. Practical Guidelines for Laboratory Diagnosis of Tuberculosis (2011). https://kslm.org/board/lib/down.php?boardid=board_notice&no=722. [Online] (last visited on 16 December 2014).
3.Diagnostic Standards and Classification of Tuberculosis in Adults and Children. Am J Respir Crit Care Med. 2000. 161:1376–95.
4.Levidiotou S., Vrioni G., Galanakis E., Gesouli E., Pappa C., Stefanou D. Four-year experience of use of the Cobas Amplicor system for rapid detection of Mycobacterium tuberculosis complex in respiratory and nonrespiratory specimens in Greece. Eur J Clin Microbiol Infect Dis. 2003. 22:349–56.
5.Report of an expert consultation on the use of nucleic acid amplification tests for the diagnosis of tuberculosis: Centers for Disease Control and Prevention. http://www.cdc.gov/tb/publications/guidelines/am-plification_tests/default.htm. [Online] (last visited on 1 September 2012).
6.Lim J., Kim J., Kim JW., Ihm C., Sohn YH., Cho HJ, et al. Multicenter evaluation of Seegene Anyplex TB PCR for the detection of Mycobacterium tuberculosis in respiratory specimens. J Microbiol Biotechnol. 2014. 24:1004–7.
7.Steingart KR., Henry M., Ng V., Hopewell PC., Ramsay A., Cunningham J, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis. 2006. 6:570–81.
crossref
8.Hwang S., Oh KJ., Jang IH., Uh Y., Yoon KJ., Kim HY, et al. Evaluation of the diagnostic performance of the advansure TB/NTM real-time PCR kit for detection of mycobacteria. Korean J Clin Microbiol. 2011. 14:55–9.
crossref
9.Telenti A., Imboden P., Marchesi F., Lowrie D., Cole S., Colston MJ, et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993. 341:647–50.
10.Telenti A. Genetics and pulmonary medicine. 5. Genetics of drug resistant tuberculosis. Thorax. 1998. 53:793–7.
11.Heym B., Alzari PM., Honoré N., Cole ST. Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis. Mol Microbiol. 1995. 15:235–45.
12.Zhang Y., Heym B., Allen B., Young D., Cole S. The catalase-per-oxidase gene and isoniazid resistance of Mycobacterium tuberculosis Nature. 1992. 358:591–3.
13.Banerjee A., Dubnau E., Quemard A., Balasubramanian V., Um KS., Wilson T, et al. inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science. 1994. 263:227–30.
14.Rozwarski DA., Grant GA., Barton DH., Jacobs WR Jr., Sacchettini JC. Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis. Science. 1998. 279:98–102.
15.Ma X., Wang H., Deng Y., Liu Z., Xu Y., Pan X, et al. rpoB Gene mutations and molecular characterization of rifampin-resistant Mycobacterium tuberculosis isolates from Shandong Province, China. J Clin Microbiol. 2006. 44:3409–12.
16.Ohno H., Koga H., Kohno S., Tashiro T., Hara K. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob Agents Chemother. 1996. 40:1053–6.
17.Taniguchi H., Aramaki H., Nikaido Y., Mizuguchi Y., Nakamura M., Koga T, et al. Rifampicin resistance and mutation of the rpoB gene in Mycobacterium tuberculosis. FEMS Microbiol Lett. 1996. 144:103–8.
18.Yue J., Shi W., Xie J., Li Y., Zeng E., Wang H. Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China. J Clin Microbiol. 2003. 41:2209–12.

Table 1.
Mutation targets in Anyplex plus MDR-TB Detection kit
Drug-resistance Related gene Mutation targets
Rifampin-resistance rpoB L511P (CTG→ CCG) Q513L (CAA→ CTA) 3 a.a. deletion in 513-516 D516G (GAC→ GGC)
D516V (GAC→ GTC) D516Y (GAC→ TAC) S522L (TCG→ TTG) H526D (CAC→ GAC)
H526L (CAC→ CTC) H526N (CAC→ AAC) H526R (CAC→ CGC) H526Y (CAC→ TAC)
S531L (TCG→ TTG) S531W (TCG→ TGG) L533P (CTG→ CCG) 　
Isoniazid-resistance katG S315N (AGC→ AAC) S315T (AGC→ ACC) S315T (AGC→ ACA) 　
inhA promoter -8 (T→ A) -15 (C→ T) -17 (G→ T) 　

Abbreviation: a.a., amino acid.

Table 2.
Detection frequencies of Mycobacterium tuberculosis and nontuberculous mycobacteria
Decision* Organisms No. case (%)
Positive Mycobacterium tuberculosis 76(13.6)
Nontuberculous mycobacteria 17(3.1)
M. avium 6
M. intracellulare 5
M. abscessus 1
M. flavescens 1
M. massiliense 1
Not identified 3
Negative   464(83.3)
Total   557(100.0)

∗ True-positive specimens were defined as those that met the following criteria: (a) culture positive on 2% Ogawa medium and/or in the BACTEC MGIT 960 system; or (b) culture negative but positive in NAAT, provided one or more of the following additional criteria were met: (i) the specimen originated from a patient whose other specimens were culture positive;(ii) the specimen originated from a patient receiving TB treatment; (iii) the specimen originated from a patient whose history of TB included clinical symptoms, positive radiographic findings, positive Mantoux tuberculin skin test, positive histology, and improvement under treatment with anti-TB chemotherapy.

Table 3.
Performance of Anyplex plus MTB/NTM Detection and Advansure TB/NTM real-time PCR
Target AFB smear Assay Positive sample* Negative sample Sensitivity (95% CI)/ Specificity (95% CI) PPV (95% CI)/ NPV (95% CI)
PCR + PCR - PCR + PCR -
MTB All Anyplex plus 63 13 3 478 82.9 (72.5-90.6)/99.4 (98.2-99.9) 95.5 (87.3-99.1)/97.4 (95.5-98.6)
Advansure 68 8 1 480 89.5 (80.3-95.3)/99.8 (98.9-100.0) 98.6 (92.2-99.9)/98.4 (96.8-99.3)
Positive Anyplex plus 11 1 0 0 91.7 (61.5-99.8)/NA 1 100.0 (71.5-100.0)/0.0 (0.0-97.5)
Advansure 12 0 0 0 100.0 (73.5-100.0)/NA 100.0 (73.5-100.0)/NA
Negative Anyplex plus 52 12 3 478 81.3 (69.5-89.9)/99.4 (98.2-99.9) 94.6 (84.9-98.9)/97.6 (95.8-98.7)
Advansure 56 8 1 480 87.5 (76.9-94.5)/99.8 (98.9-100.0) 98.3 (90.6-100.0)/98.4 (96.8-99.3)
NTM All Anyplex plus 13 4 56 484 76.5 (50.1-93.2)/89.6 (86.7-92.1) 18.8 (10.4-30.1)/99.2 (97.9-99.8)
Advansure 7 10 6 534 41.2 (18.4-67.1)/98.9 (97.6-99.6) 53.9 (25.1-80.8)/98.2 (96.7-99.1)
Positive Anyplex plus 0 0 1 11 NA/91.7 (61.5-99.8) 0.0 (0.0-97.5)/100.0 (71.5-100.0)
Advansure 0 0 0 12 NA/100.0 (73.5-100.0) NA/100.0 (73.5-100.0)
Negative Anyplex plus 13 4 55 473 76.5 (50.1-93.2)/89.6 (86.7-92.1) 19.1 (10.6-30.5)/99.2 (97.8-99.8)
Advansure 7 10 6 522 41.2 (18.4-67.1)/98.9 (97.5-99.6) 53.9 (25.1-80.8)/98.1 (96.6-99.1)

∗ Criteria of positive sample is the same as Table 2.

Abbreviations: CI, confidence interval; PPV, positive-predictive value; NPV, negative-predictive value; Anyplex plus, Anyplex plus MTB/NTM Detection; Advansure, Advansure TB/NTM real-time PCR; MTB, M. tuberculosis; NTM, nontuberculous mycobacteria; NA, not applicable.

Table 4.
Culture based performance of Anyplex plus MTB/NTM Detection and Advansure TB/NTM real-time PCR
Target Assay Culture positive* Culture negative Sensitivity (95% CI)/ Specificity (95% CI) PPV (95% CI)/ NPV (95% CI)
PCR + PCR - PCR + PCR -
MTB Anyplex plus 46 6 20 485 88.5 (76.6-95.7)/96.0 (94.0-97.6) 69.7 (57.2-80.4)/98.8 (97.4-99.6)
Advansure 50 2 19 486 96.2 (86.8-99.5)/96.2 (94.2-97.7) 72.5 (60.4-82.5)/99.6 (98.5-100.0)
NTM Anyplex plus 9 7 60 481 56.3 (29.9-80.3)/88.9 (86.0-91.4) 13.0 (6.1-23.3)/98.6 (97.1-99.4)
Advansure 4 12 9 532 25.0 (7.3-52.4)/98.3 (96.9-99.2) 30.8 (9.1-61.4)/97.8 (96.2-98.9)

∗ Culture positive on 2% Ogawa medium and/or in the BACTEC MGIT 960 system.

Abbreviations: CI, confidence interval; PPV, positive-predictive value; NPV, negative-predictive value; Anyplex plus, Anyplex plus MTB/NTM Detection; Advansure, Advansure TB/NTM real-time PCR.

Table 5.
Performance of Anyplex plus MDR-TB Detection and Advansure MDR-TB GenoBlot Assay on detection of antituberculosis resistance
Drug Assay Resistant isolates∗ Susceptible isolates Sensitivity (95% CI)/ Specificity (95% CI) PPV (95% CI)/ NPV (95% CI)
R† S R† S
Rifampin Anyplex plus 3 0 1 47 100.0 (29.2-100.0)/97.9 (88.9-100.0) 75.0 (19.4-99.4)/100.0 (92.5-100.0)
Advansure 3 0 1 47 100.0 (29.2-100.0)/97.9 (88.9-100.0) 75.0 (19.4-99.4)/100.0 (92.5-100.0)
Isoniazid Anyplex plus 5 1 0 45 83.3 (35.9-99.6)/100.0 (92.1-100.0) 100.0 (47.8-100.0)/97.8 (88.5-99.9)
Advansure 5 1 0 45 83.3 (35.9-99.6)/100.0 (92.1-100.0) 100.0 (47.8-100.0)/97.8 (88.5-99.9)

∗ Resistant isolates were determined by conventional drug susceptibility test;

† Resistance was determined by each molecular based assay.

Abbreviations: R, resistant; S, susceptible; CI, confidence interval; PPV, positive-predictive value; NPV, negative-predictive value; Anyplex plus, Anyplex plus MTB/NTM Detection; Advansure, Advansure TB/NTM real-time PCR.

Table 6.
Discrepancies between molecular and conventional drug susceptibility test
Sample No. Drug Molecular Conventional DST
Anyplex plus MDR-TB Advensure MDR-TB Sequencing Absolute concentration method∗ Automated liquid culture†
7 Rifampin R R rpoB S531L R R
Isoniazid S S wild R R
47 Rifampin R R rpoB L533P S S
Isoniazid S S wild S S

∗ Absolute concentration method used L-J media;

† Automated liquid culture used BACTEC MGIT 960 system.

Abbreviations: Anyplex plus MDR-TB, Anyplex plus MDR-TB Detection; Advansure MDR-TB, Advansure MDR-TB GenoBlot Assay; DST, Drug Susceptibility Test; S, susceptible; R, Resistant.

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