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Chung and Lee: Comparative Evaluation of Multiplex Real-Time PCR Assays for Six Pathogens of Sexually Transmitted Infections
Original Article

Annals of Clinical Microbiology 2017; 20(1): 1-6.

Published online: 21 March 2017

DOI: https://doi.org/10.5145/ACM.2017.20.1.1

Comparative Evaluation of Multiplex Real-Time PCR Assays for Six Pathogens of Sexually Transmitted Infections

Hae-Sun Chung, Miae Lee

Department of Laboratory Medicine, Ewha Womans University College of Medicine, Seoul, Korea.

Correspondence: Miae Lee, Department of Laboratory Medicine, Ewha Womans University College of Medicine, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 07985, Korea. (Tel) 82-2-2650-5222, (Fax) 82-2-2650-5091, miae@ewha.ac.kr

Received 16 November 2016       Revised 27 February 2017       Accepted 28 February 2017

© 2017 The Korean Society of Clinical Microbiology

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The multiplex real-time PCR assay is a sensitive test for simultaneous detection of various pathogens of sexually transmitted infections (STIs). We evaluated the performance of two multiplex real-time PCR assays for six STI pathogens.

Methods

DNA samples after being used to conduct PCR for STI pathogens were stored below −70℃. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by multiplex real-time PCR with GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR Kits (Infopia, Korea; GeneFinder assay) and Real-Q CT&NG/MH&TV/MG&UU Kits (BioSewoom, Korea; Real-Q assay). Discrepant results were resolved by another multiplex real-time assay, Anyplex II STI-7 Detection (Seegene, Korea). Any two positive results for the assays were considered true positive.

Results

Among 81 samples, the GeneFinder assay detected 63 pathogens from 45 cases (16 CT, 2 NG, 6 MG, 20 MH, 18 UU, and 1 TV) and Real-Q assay detected 66 pathogens from 47 cases (16 CT, 2 NG, 8 MG, 20 MH, 19 UU, and 1 TV). For the results of positive cases and negative cases, the overall concordance rate between the two multiplex real-time assays was 93.8% (Kappa=0.87). For each pathogen, the agreement rates of the two assays ranged from 97.5 to 100% (Kappa>0.8).

Conclusion

There was no significant difference between the results of GeneFinder assay and Real-Q assay. Both multiplex real-time PCR assays can be useful methods for the detection of STI pathogens in clinical laboratories.

Keywords: Multiplex real-time polymerase chain reaction, Sexually transmitted infection

Figures and Tables

Table 1

Results of detection of single and multiple pathogens by GeneFinder and Real-Q assays

acm-20-1-i001

Abbreviations: GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/MG&UU.

Table 2

Overall agreement between GeneFinder and Real-Q assays

acm-20-1-i002

*Proportion of positive agreement (Ppos), calculated as twice the number of agreed positive cases/(total number of cases+number of agreed positive cases−number of agreed negative cases); and proportion of negative agreement (Pneg), calculated as twice the number of agreed negative cases/(total number of cases+number of agreed positive cases−number of agreed negative cases). †by McNemar test. ‡Number of cases of GeneFinder negative and Real-Q positive included one mixed infection case; one of three pathogens was missed.

Abbreviations: GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/MG&UU.

Table 3

Comparison of the results of GeneFinder and Real-Q assays according to the pathogens

acm-20-1-i003

*Proportion of positive agreement (Ppos), calculated as twice the number of agreed positives/(total number of specimens+number of agreed positives−number of agreed negatives); and proportion of negative agreement (Pneg), calculated as twice the number of agreed negatives/(total number of specimens+number of agreed positives−number of agreed negatives). †P values were calculated by McNemar test for pathogens with discrepant results.

Abbreviations: GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/MG&UU.

Table 4

Interpretation of discrepant results

acm-20-1-i004

*Ct ranges for positive specimens of M. genitalium, M. hominis, and U. urealyticumwere 23.09-35.99 (median 29.23), 19.70-36.43 (median 26.67), and 23.82-36.94 (median 32.49) by Real-Q assay. †Ct range for positive specimens of M. hominis was 20.64-37.50 (median 27.35) by GeneFinder assay.

Abbreviations: Anyplex, Anyplex II STI-7 Detection; GeneFinder, GeneFinder STD I (CT/NG/UU)/II (MG/MH/TV) Multiplex Real-time PCR; Real-Q, Real-Q CT&NG/MH&TV/MG&UU.

References

1. WHO. Guidelines for the management of sexually transmitted infections. Geneva: World Health Organization;2003. p. 1–5.
2. Korea Centers for Disease Control and Prevention and Korean Association of Urogenital Tract Infection and Inflammation. Sexually Transmitted Infections Korean Guidelines. Cheongju and Seoul: Korea Centers for Disease Control and Prevention and Korean Association of Urogenital Tract Infection and Inflammation;2011. p. 3–41.
3. Workowski KA, Bolan GA. Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2015. MMWR Recomm Rep. 2015; 64:1–137.
4. Hong SG. Genital infections. Korean Society of Infectoius Diseases. Infectious Diseases. 2nd ed. Seoul: KoonJa Publishing Inc.;2014. p. 239–253.
5. Lee SJ, Lee HJ, et al. Sexually transmitted infections. Korean Association of Urogenital Tract Infection and Inflammation. Urogenital Tract Infection and Inflammation. Seoul: Ilchokak Publishing Co., Ltd.;2013. p. 254–358.
6. Trembizki E, Costa AM, Tabrizi SN, Whiley DM, Twin J. Opportunities and pitfalls of molecular testing for detecting sexually transmitted pathogens. Pathology. 2015; 47:219–226.
7. Koo SH, Park KU, et al. Microbiological test methods. Korean Society for Laboratory Medicine. Laboratory Medicine. 5th ed. Seoul: Panmun Education;2014. p. 517–536.
8. Choe HS, Lee DS, Lee SJ, Hong SH, Park DC, Lee MK, et al. Performance of Anyplex™ II multiplex real-time PCR for the diagnosis of seven sexually transmitted infections: comparison with currently available methods. Int J Infect Dis. 2013; 17:e1134–e1140.
9. Kim Y, Kim J, Lee KA. Analytical performance of multiplex real-time PCR for six sexually transmitted pathogens. Clin Lab. 2015; 61:1749–1754.
10. LeFevre ML. U.S. Preventive Services Task Force. Screening for Chlamydia and gonorrhea: U.S. Preventive Services Task Force recommendation statement. Ann Intern Med. 2014; 161:902–910.
11. Lim DH, Lee SJ, Shim BS, Kim CS, Kim ME, Cho YH. The new Korean guideline for sexually transmitted infections. Korean J Urogenit Tract Infect Inflamm. 2011; 6:96–113.
12. Wei HB, Zou SX, Yang XL, Yang DQ, Chen XD. Development of multiplex real-time quantitative PCR for simultaneous detection of Chlamydia trachomatis and Ureaplasma parvum. Clin Biochem. 2012; 45:663–667.
13. Kim Y, Kim J, Lee KA. Prevalence of sexually transmitted infections among healthy Korean women: implications of multiplex PCR pathogen detection on antibiotic therapy. J Infect Chemother. 2014; 20:74–76.
14. Markoulatos P, Siafakas N, Moncany M. Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal. 2002; 16:47–51.
15. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev. 2000; 13:559–570.
16. Gunson RN, Bennett S, Maclean A, Carman WF. Using multiplex real time PCR in order to streamline a routine diagnostic service. J Clin Virol. 2008; 43:372–375.
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