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A real-time quadriplex PCR assay for the diagnosis of rectal lymphogranuloma venereum and non-lymphogranuloma venereum Chlamydia trachomatis infections
  1. C-Y Chen1,
  2. K H Chi1,
  3. S Alexander2,
  4. C A Ison2,
  5. R C Ballard1
  1. 1
    Division of STD Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
  2. 2
    Sexually Transmitted Bacteria Reference Laboratory, Health Protection Agency Centre for Infections, London, UK
  1. Dr C-Y Chen, Division of STD Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention, Mail Stop: G-39, 1600 Clifton Road, Atlanta, GA 30333, USA; cyc1{at}


Objectives: To develop and evaluate a real-time quadriplex PCR for the diagnosis of lymphogranuloma venereum (LGV) and non-LGV chlamydial infections using rectal swab specimens.

Methods: The design of the real-time quadriplex PCR assay incorporates an LGV-specific, a non-LGV-specific target sequence, a Chlamydia trachomatis plasmid target, and the human RNase P gene as an internal control. The performance of the quadriplex PCR was compared with a previously reported real-time duplex PCR assay on which LGV diagnosis was based on exclusion.

Results: Very good agreement (85 of 89 specimens, 95.5%) was found between the two multiplex PCR assays for the detection of C trachomatis DNA (kappa value 0.93, 95% CI 0.86 to 0.99). Both assays identified 34 LGV, 35 non-LGV C trachomatis and 16 negative specimens. Of two specimens that tested positive for non-LGV by the duplex PCR, one was found to be a mixed infection and the other was positive only for plasmid and RNase P targets by the quadriplex PCR. Two additional specimens that had equivocal results for non-LGV by the duplex PCR also tested positive only for plasmid target and human DNA by the quadriplex PCR. In addition, six specimens that tested negative by the duplex PCR assay were found to be invalid when using the quadriplex PCR.

Conclusions: A real-time quadriplex PCR assay has been developed that is capable of detecting LGV, non-LGV, or mixed infections simultaneously in rectal specimens. The assay also contains a supplemental amplification target for the confirmation of C trachomatis infection as well as a human DNA control for monitoring sample adequacy and PCR inhibition.

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  • Competing interests: None declared.

  • Contributors: C-YC co-designed the study, supervised its execution, analysed the data and led the writing of the article. KHC was responsible for optimising and performing all multiplex real-time PCR assays. SA performed various nucleic acid amplification testing and genotyping. She also participated in drafting and revision of the article. CAI contributed to study supervision and made comments on drafts. RCB conceived the idea, co-designed the study and contributed substantially to the analysis and writing, especially in commenting on all drafts of the article.

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