title: A multiplex PCR assay for the simultaneous detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis.
abstract: BACKGROUND
  For developing countries, sexually transmitted infections (STIs) and their complications are ranked in the top 5 disease categories for which adults seek medical treatment. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) are the three most common STIs worldwide, with TV accounting for over half of the cases. In developing countries, traditional methods for diagnosing STIs are laborious, often not very sensitive, and have a long turnaround time with most recent commercially available diagnostic tests targeting one or, at most, two of these STIs at a time. Here, we describe the development of a highly sensitive, rapid and affordable sample-to-answer multiplex PCR-based assay for the simultaneous detection of Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis.
  
  
  METHODS
  We designed a multiplex PCR assay for the detection of 4 targets (CT, TV, NG, and process/PCR control) using melt curve analysis. To establish the limit of detection (LOD) for each pathogen, we used previously extracted and quantified TV, NG, and CT genomic DNA (Vircell, Spain). For each target, the LOD was determined by lowering its copy number while increasing the other two STI loads in a stepwise fashion. The process/PCR control remained constant in the optimized assay and was spiked into each sample before extraction. For a concordance study, we tested urine, vaginal and rectal swab specimens from 26 patients positive for one or more of the tested STIs. In addition, 56 liquid cytology specimens (Thinprep) were used to assess specificity.
  
  
  RESULTS
  This assay has a turnaround time of less than 2h and has a limit of detection as low as 7-31 copies for each STI in the presence of the other 2 targets. Our assay also demonstrated 100% concordance with 26 known clinical samples from urine, vaginal and rectal swab specimens. TV, NG, CT, and our process/PCR control were consistently identified at 78°C, 82.3°C, 85.7°C, and ~92°C, respectively. When applied to DNA extracted from residual Thinprep specimens, the assay was negative in 54/56 samples. Two samples were found to be co-infected with CT.
  
  
  CONCLUSIONS
  Our multiplex assay combines a rapid and cost-effective approach to molecular diagnostics with the versatility required for use within a variety of laboratory settings. These performance characteristics make this multiplex STI assay highly suitable for use in a clinical laboratory.
authors.affiliation: Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States; Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH, United States.
authors.forename: Ahmad N
authors.initials: AN
authors.lastname: Abou Tayoun
authors.affiliation: Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States; Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH, United States.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Burchard
authors.affiliation: Department of Medicine, Warren Alpert Medical School of Brown University, Providence, RI, United States.
authors.forename: Angela M
authors.initials: AM
authors.lastname: Caliendo
authors.affiliation: Department of Electrical Engineering, California Institute of Technology, Pasadena, CA, United States.
authors.forename: Axel
authors.initials: A
authors.lastname: Scherer
authors.affiliation: Department of Pathology, Geisel School of Medicine at Dartmouth, Hanover, NH, United States; Dartmouth Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH, United States. Electronic address: gregory.j.tsongalis@hitchcock.org.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Tsongalis
mesh_terms: D002690:Chlamydia Infections / Q000175:diagnosis*; D002692:Chlamydia trachomatis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D016054:DNA, Protozoan / Q000032:analysis; D005260:Female; D006069:Gonorrhea / Q000175:diagnosis*; D006801:Humans; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009344:Neisseria gonorrhoeae / Q000235:genetics; D014247:Trichomonas Vaginitis / Q000175:diagnosis*; D014246:Trichomonas vaginalis / Q000235:genetics; D016482:Urinalysis; D014626:Vaginal Smears
chemical_list: D004269:DNA, Bacterial; D016054:DNA, Protozoan
keywords: Chlamydia; Neisseria; PCR; Trichomonas