VALIDATION THE OPTIMAL CONDITION AND EVALUATION EFFICIENCY OF IN-HOUSE REAL TIME PCR FOR NEISSERIA GONORRHOEAE DETECTION AT BANGRAK STIS CENTER
DOI:
https://doi.org/10.14456/taj.2022.7Keywords:
Neisseria gonorrhoeae, In-house real time PCR, CultureAbstract
Neisseria gonorrhoeae is one of the most common sexually transmitted pathogens. Fast and effective laboratory diagnostics are very important. The gold standard for N.gonorrhoeae diagnostic is culture method. Meanwhile nucleic acid amplification tests (NAATs) are critical new tools to diagnose N.gonorrhoeae infections. The aim of this study is to validate and assess the efficiency together with assessing the cost of the validated in-house real time PCR as compare to culture method.
The urethral, cervical, rectal and pharyngeal specimens from patients attended at Bangrak STIs Center, Thailand were used in this study. Sixty samples were used for validation experiment, while two hundred and forty samples were used in the comparison. All three hundred samples were tested by in-house real time PCR in which primers and probes were designed to specify at PorA pseudogene of N. gonorrhoeae and human-beta globin gene as an internal control. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were analyzed. AnyplexTM CT/NG Real-time Detection, commercial NAAT kit, was used to analyze the discrepancy result.
The suitable condition of PCR reaction for N. gonorrhoeae detection was at concentration 200 nM and also 200 nM for internal control. Validated duplex in-house real time PCR showed 100% correlation with culture method for genital specimens, while sensitivity, specificity, PPV and NPV were 100%, 86.67%, 88.24%, 100%, respectively for rectal swab and 100%, 96.67%, 96.77%, 100%, respectively for pharyngeal swab specimens. All five discrepancy results were negative by culture, but positive by in-house real time PCR and the commercial NAAT kit. The cost of the duplex in-house real time PCR was 165 Baht/test, while culture was 76 Baht/test. Although in-house real time PCR assay is more expensive than culture, but in term of diagnosis it is more cost-effective. The in-house real time PCR can detect N. gonorrhoeae
Based on our finding, in-house real time PCR is a rapid, sensitivity and specificity on various types of specimen with high accuracy similar to culture method. Our validated duplex in-house real-time PCR for N. gonorrhoeae detection can be used in routine service.
Downloads
References
HIV info hub. Division of AIDS and STIs [Internet]. [cited 2021 Oct 12]. Available from: https://hivhub.ddc.moph.go.th/epidemic.php (in Thai)
Wilawan Thipmontree, Chenchit Chayachinda, Rossaphorn Kittiyoawamarn. STI surveillance and prevention: gonorrhea. Siriraj Med Bull. 2020; 13(3): 196-204. (in Thai)
Stephanie N. Taylor, Oliver Liesenfeld, Rebecca A. Lillis, Barbara A. Body, Melinda Nye, James Williams, et al. Evaluation of the Roche cobas® CT/NG test for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in male urine. Sex Transm Dis. 2012; 39(7): 543-9. doi: 10.1097/OLQ.0b013e31824e26ff
Boyadzhyan B, Yashina T, Yatabe JH, Patnaik M, Hill CS. Comparison of the APTIMA CT and GC assays with the APTIMA combo 2 assay, the Abbott LCx assay, and direct fluorescentantibody and culture assays for detection of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol. 2004; 42(7): 3089-93. doi: 10.1128/JCM.42.7.3089-3093.2004
C. A. Gaydos, T. C. Quinn, D. Willis, A. Weissfeld, E. W. Hook, D. H. Martin, et al. Performance of the APTIMA combo 2 assay for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens. J Clin Microbiol. 2003; 41(1): 304-9. doi: 10.1128/JCM.41.1.304-309. 2003
Mark J Hopkins, Lynne J Ashton, Fath Alloba, Anura Alawattegama, Ian J Hart. Validation of a laboratory-developed real-time PCR protocol for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine. Sex Transm Infect. 2010; 86(3): 207-11. doi: 10.1136/sti.2009.040634
Johanna M E Venter, Precious M Mahlangu, Etienne E Müller, David A Lewis, Kevin Rebe, Helen Struthers, et al. Comparison of an in-house real-time duplex PCR assay with commercial HOLOGIC® APTIMA assays for the detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urine and extra-genital specimens. BMC Infect Dis. 2019, 19: 1-7. doi: 10.1186/s12879-018-3629-0
Arifin WN. Sample size calculator [Internet]. Wan Nor Arifin c2017-2021 [cited 2021 October 1]. Available from: https://wnarifin.github.io/ssc/sssnsp.html
National AIDS Management Center Bureau of AIDS, TB and STIs. 2017 Thailand Progress Report Prevention and Control of AIDS. Nonthaburi: National AIDS Management Center, Department of Disease Control, Ministry of Public Health. 2017. (in Thai)
Busara Bamrungsak, Siwimol Phoomniyom, Rungnapa Luengprasit. Development and validation of an in-house real-time PCR assay for the detection of Chlamydia trachomatis in sexually transmitted infections clinic. Thai AIDS Journal. 2020; 32(1): 28-41. (in Thai)
Siwimol Phoomniyom, Busara Bamrungsak, Kornsiri Boonprathueng, Pongsathorn Sangprasert, Rungnapa Luengprasit, Nattapon Ngarmjiratam, et al. Efficiency evaluation of an in-house real-time PCR assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens. Thai AIDS Journal. 2020; 33(1): 9-20. (in Thai)
Kehl SC, Georgakas K, Swain GR, Sedmak G, Gradus S, Singh A, et al. Evaluation of the Abbott LCx Assay for Detection of Neisseria gonorrhoeae in Endocervical Swab Specimens from Females. J. CLIN. MICROBIOL. 1998; 36(12): 3549-51.
Ewa Skulska, Beata Młynarczyk-Bonikowska, Szymon Walter de Walthoffen, Grażyna Młynarczyk, Magdalena Malejczyk, Sławomir Majewski. The Comparison of Real-Time PCR and bacterial culture in laboratory diagnostics of gonorrhoea in patients of Department of Dermatology and Venereology Medical University of Warsaw. MED. DOŚW. MIKROBIOL. 2015; 67(1): 29-38. (in Polish)
David M Whiley, John W Tapsall, Theo P Sloots. Nucleic Acid Amplification Testing for Neisseria gonorrhoeae an Ongoing Challenge. J Mol Diagn. 2006; 8(1): 3-15. doi: 10.2353/jmoldx.2006.050045
John R. Papp, Julius Schachter, Charlotte A. Gaydos, Barbara Van Der Pol. Recommendations for the Laboratory-Based Detection of Chlamydia trachomatis and Neisseria gonorrhoeae -2014. MMWR Recomm Rep. 2014; 63(2): 1-19.
