Analytical performance of multiplex real-time PCR for six sexually transmitted pathogens

Yoonjung Kim, Juwon Kim, Kyung A. Lee

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: Most organisms that cause sexual transmitted diseases (STDs) are fastidious pathogens that are difficult to detect with conventional microbiological methods and the proportions of multiple infections were noted up to 39.3% among the STI-positive subjects. However, only a few multiplex PCR and multiplex real-time PCR tests that can screen more than six microorganisms that cause STDs have been assessed. Methods: A total of 114 endocervical swabs (Thin Prep® PAPTEST™ PreservCyt® Solution®, Hologic Inc., Marlborough, MA, USA) were collected from healthy Korean women. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by uniplex PCR with Seeplex® kits and by multiplex real-time PCR with Real-Q Kits (Biosewoom Inc., Seoul, Korea). To evaluate analytical sensitivity, plasmids containing target genes from CT, NG, MG, MH, UU, and TV were serially diluted five times with saline buffer and replicated eight times per dilution. Results: Real-Q STIs Kit assays showed 100% sensitivity for detecting MH, MG, CT, TV, NG and 94.1% sensitivity for detecting UU. In addition, it showed 100% specificity for UU, MH, MG, CT, TV, and NG. The analytic sensitivity of UU (95% probit = 17.3 copy/μL, 95% CI = 11.6 to 138.6) and MH (95% probit = 30.9 copy/μL, 95% CI = 20.6 to 169.9) was relatively lower than for others pathogens. Thus, the cutoff Ct value of < 45 for UU and MH and a cutoff Ct value of < 38 for CT, MH, NG, TV could minimize differences in detection limit among the six STIs (95% probit values = 5.3 to 14.6) and to optimize overall diagnostic performance. Conclusions: For medical applications of a multiplex real-time PCR assay, one kind of cutoff value, which is according to manufacturer's instructions, was generally used without the consideration of lowest actual detectable concentration of each target substance. However, analytical performance at the low concentration limit often defines the ability of the test to diagnose disease and determine treatment endpoints. Therefore, suitable cutoffs for negative or positive screens by multiplex real-time PCR should be evaluated for accurate diagnosis.

Original languageEnglish
Pages (from-to)1749-1754
Number of pages6
JournalClinical Laboratory
Volume61
Issue number11
DOIs
Publication statusPublished - 2015 Jan 1

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Mycoplasma hominis
Ureaplasma urealyticum
Multiplex Polymerase Chain Reaction
Pathogens
Trichomonas vaginalis
Mycoplasma genitalium
Real-Time Polymerase Chain Reaction
Neisseria gonorrhoeae
Chlamydia trachomatis
Assays
Sexually Transmitted Diseases
Medical applications
Microorganisms
Dilution
Buffers
Plasmids
Genes
Korea
Limit of Detection
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

@article{dc343f7cd2da4d2dacbf1069052fe881,
title = "Analytical performance of multiplex real-time PCR for six sexually transmitted pathogens",
abstract = "Background: Most organisms that cause sexual transmitted diseases (STDs) are fastidious pathogens that are difficult to detect with conventional microbiological methods and the proportions of multiple infections were noted up to 39.3{\%} among the STI-positive subjects. However, only a few multiplex PCR and multiplex real-time PCR tests that can screen more than six microorganisms that cause STDs have been assessed. Methods: A total of 114 endocervical swabs (Thin Prep{\circledR} PAPTEST™ PreservCyt{\circledR} Solution{\circledR}, Hologic Inc., Marlborough, MA, USA) were collected from healthy Korean women. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by uniplex PCR with Seeplex{\circledR} kits and by multiplex real-time PCR with Real-Q Kits (Biosewoom Inc., Seoul, Korea). To evaluate analytical sensitivity, plasmids containing target genes from CT, NG, MG, MH, UU, and TV were serially diluted five times with saline buffer and replicated eight times per dilution. Results: Real-Q STIs Kit assays showed 100{\%} sensitivity for detecting MH, MG, CT, TV, NG and 94.1{\%} sensitivity for detecting UU. In addition, it showed 100{\%} specificity for UU, MH, MG, CT, TV, and NG. The analytic sensitivity of UU (95{\%} probit = 17.3 copy/μL, 95{\%} CI = 11.6 to 138.6) and MH (95{\%} probit = 30.9 copy/μL, 95{\%} CI = 20.6 to 169.9) was relatively lower than for others pathogens. Thus, the cutoff Ct value of < 45 for UU and MH and a cutoff Ct value of < 38 for CT, MH, NG, TV could minimize differences in detection limit among the six STIs (95{\%} probit values = 5.3 to 14.6) and to optimize overall diagnostic performance. Conclusions: For medical applications of a multiplex real-time PCR assay, one kind of cutoff value, which is according to manufacturer's instructions, was generally used without the consideration of lowest actual detectable concentration of each target substance. However, analytical performance at the low concentration limit often defines the ability of the test to diagnose disease and determine treatment endpoints. Therefore, suitable cutoffs for negative or positive screens by multiplex real-time PCR should be evaluated for accurate diagnosis.",
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Analytical performance of multiplex real-time PCR for six sexually transmitted pathogens. / Kim, Yoonjung; Kim, Juwon; Lee, Kyung A.

In: Clinical Laboratory, Vol. 61, No. 11, 01.01.2015, p. 1749-1754.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Analytical performance of multiplex real-time PCR for six sexually transmitted pathogens

AU - Kim, Yoonjung

AU - Kim, Juwon

AU - Lee, Kyung A.

PY - 2015/1/1

Y1 - 2015/1/1

N2 - Background: Most organisms that cause sexual transmitted diseases (STDs) are fastidious pathogens that are difficult to detect with conventional microbiological methods and the proportions of multiple infections were noted up to 39.3% among the STI-positive subjects. However, only a few multiplex PCR and multiplex real-time PCR tests that can screen more than six microorganisms that cause STDs have been assessed. Methods: A total of 114 endocervical swabs (Thin Prep® PAPTEST™ PreservCyt® Solution®, Hologic Inc., Marlborough, MA, USA) were collected from healthy Korean women. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by uniplex PCR with Seeplex® kits and by multiplex real-time PCR with Real-Q Kits (Biosewoom Inc., Seoul, Korea). To evaluate analytical sensitivity, plasmids containing target genes from CT, NG, MG, MH, UU, and TV were serially diluted five times with saline buffer and replicated eight times per dilution. Results: Real-Q STIs Kit assays showed 100% sensitivity for detecting MH, MG, CT, TV, NG and 94.1% sensitivity for detecting UU. In addition, it showed 100% specificity for UU, MH, MG, CT, TV, and NG. The analytic sensitivity of UU (95% probit = 17.3 copy/μL, 95% CI = 11.6 to 138.6) and MH (95% probit = 30.9 copy/μL, 95% CI = 20.6 to 169.9) was relatively lower than for others pathogens. Thus, the cutoff Ct value of < 45 for UU and MH and a cutoff Ct value of < 38 for CT, MH, NG, TV could minimize differences in detection limit among the six STIs (95% probit values = 5.3 to 14.6) and to optimize overall diagnostic performance. Conclusions: For medical applications of a multiplex real-time PCR assay, one kind of cutoff value, which is according to manufacturer's instructions, was generally used without the consideration of lowest actual detectable concentration of each target substance. However, analytical performance at the low concentration limit often defines the ability of the test to diagnose disease and determine treatment endpoints. Therefore, suitable cutoffs for negative or positive screens by multiplex real-time PCR should be evaluated for accurate diagnosis.

AB - Background: Most organisms that cause sexual transmitted diseases (STDs) are fastidious pathogens that are difficult to detect with conventional microbiological methods and the proportions of multiple infections were noted up to 39.3% among the STI-positive subjects. However, only a few multiplex PCR and multiplex real-time PCR tests that can screen more than six microorganisms that cause STDs have been assessed. Methods: A total of 114 endocervical swabs (Thin Prep® PAPTEST™ PreservCyt® Solution®, Hologic Inc., Marlborough, MA, USA) were collected from healthy Korean women. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by uniplex PCR with Seeplex® kits and by multiplex real-time PCR with Real-Q Kits (Biosewoom Inc., Seoul, Korea). To evaluate analytical sensitivity, plasmids containing target genes from CT, NG, MG, MH, UU, and TV were serially diluted five times with saline buffer and replicated eight times per dilution. Results: Real-Q STIs Kit assays showed 100% sensitivity for detecting MH, MG, CT, TV, NG and 94.1% sensitivity for detecting UU. In addition, it showed 100% specificity for UU, MH, MG, CT, TV, and NG. The analytic sensitivity of UU (95% probit = 17.3 copy/μL, 95% CI = 11.6 to 138.6) and MH (95% probit = 30.9 copy/μL, 95% CI = 20.6 to 169.9) was relatively lower than for others pathogens. Thus, the cutoff Ct value of < 45 for UU and MH and a cutoff Ct value of < 38 for CT, MH, NG, TV could minimize differences in detection limit among the six STIs (95% probit values = 5.3 to 14.6) and to optimize overall diagnostic performance. Conclusions: For medical applications of a multiplex real-time PCR assay, one kind of cutoff value, which is according to manufacturer's instructions, was generally used without the consideration of lowest actual detectable concentration of each target substance. However, analytical performance at the low concentration limit often defines the ability of the test to diagnose disease and determine treatment endpoints. Therefore, suitable cutoffs for negative or positive screens by multiplex real-time PCR should be evaluated for accurate diagnosis.

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