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Evaluation of multiplex real-time PCR for detection of Haemophilus ducreyi, Treponema pallidum, herpes simplex virus type 1 and 2 in the diagnosis of genital ulcer disease in the Rakai District, Uganda
  1. T R Suntoke1,
  2. A Hardick2,
  3. A A R Tobian2,
  4. B Mpoza4,
  5. O Laeyendecker1,2,
  6. D Serwadda3,
  7. P Opendi4,
  8. C A Gaydos2,
  9. R H Gray5,
  10. M J Wawer5,
  11. T C Quinn1,2,
  12. S J Reynolds1,2
  1. 1
    National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
  2. 2
    Division of Infectious Diseases, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA
  3. 3
    Institute of Public Health, Makerere University, Kampala, Uganda
  4. 4
    Rakai Health Sciences Program, Rakai, Uganda
  5. 5
    Department of Population and Family Health Sciences, Bloomberg School of Public Health, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA
  1. Dr Thomas C Quinn, Division of Infectious Diseases, Johns Hopkins Medical Institutions, 855 N. Wolfe Street, Room 531, Baltimore, Maryland 21205, USA; tquinn{at}


Objective: To develop a real-time PCR assay that reliably and accurately detects the predominant sexually transmitted aetiological agents of genital ulcer disease (GUD) (Haemophilus ducreyi, Treponema pallidum and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)) and to assess the use of real-time PCR diagnostic testing in a rural African field site.

Methods: Two multiplex real-time PCR reactions were used to detect H ducreyi/and HSV-1/HSV-2 in ulcer swabs from 100 people with symptomatic genital ulcers in rural Rakai, Uganda. Results were compared with syphilis, HSV-1 and HSV-2 serology.

Results: Of 100 GUD samples analysed from 43 HIV positive and 57 HIV negative individuals, 71% were positive for one or more sexually transmitted infection (STI) pathogens by real-time PCR (61% for HSV-2, 5% for T pallidum, 3% for HSV-1, 1% for H ducreyi and 1% for dual H ducreyi/HSV-2). The frequency of HSV in genital ulcers was 56% (32/57) in HIV negative individuals and 77% (33/43) in HIV positive individuals (p = 0.037). Assay reproducibility was evaluated by repeat PCR testing in the USA with 96% agreement (κ = 0.85).

Conclusions: STI pathogens were detected in the majority of GUD swab samples from symptomatic patients in Rakai, Uganda, by real-time PCR. HSV-2 was the predominant cause of genital ulcers. Real-time PCR technology can provide sensitive, rapid and reproducible evaluation of GUD aetiology in a resource-limited setting.

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  • Funding: This research was supported by The Division of Intramural Research, The National Institute of Allergy and Infectious Diseases, National Institutes of Health.

  • Competing interests: None.

  • Ethics approval: The study was approved by the Science and Ethics Committee of the Uganda Virus Research Institute and the Western Institutional Review Board for Johns Hopkins University.

  • Contributors: TRS, AH, AAT, BM, OL, CAG and SJR performed real-time PCR and serological testing and provided technical assistance on assay development. BM, PO and DS provided support for sample data and collection in Rakai, Uganda. TRS, AAT, OL, CAG, RHG, MJW, TCQ and SJR contributed to manuscript preparation and provided overall direction and support for the project.