Skip to main content
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Jan;34(1):49–54. doi: 10.1128/jcm.34.1.49-54.1996

Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.

K A Orle 1, C A Gates 1, D H Martin 1, B A Body 1, J B Weiss 1
PMCID: PMC228728  PMID: 8748271

Abstract

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

Full Text

The Full Text of this article is available as a PDF (197.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boulos R., Ruff A. J., Nahmias A., Holt E., Harrison L., Magder L., Wiktor S. Z., Quinn T. C., Margolis H., Halsey N. A. Herpes simplex virus type 2 infection, syphilis, and hepatitis B virus infection in Haitian women with human immunodeficiency virus type 1 and human T lymphotropic virus type I infections. The Johns Hopkins University (JHU)/Centre pour le Developpement et la Santé (CDS) HIV Study Group. J Infect Dis. 1992 Aug;166(2):418–420. doi: 10.1093/infdis/166.2.418. [DOI] [PubMed] [Google Scholar]
  2. Burstain J. M., Grimprel E., Lukehart S. A., Norgard M. V., Radolf J. D. Sensitive detection of Treponema pallidum by using the polymerase chain reaction. J Clin Microbiol. 1991 Jan;29(1):62–69. doi: 10.1128/jcm.29.1.62-69.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bzik D. J., Debroy C., Fox B. A., Pederson N. E., Person S. The nucleotide sequence of the gB glycoprotein gene of HSV-2 and comparison with the corresponding gene of HSV-1. Virology. 1986 Dec;155(2):322–333. doi: 10.1016/0042-6822(86)90196-0. [DOI] [PubMed] [Google Scholar]
  4. Chui L., Albritton W., Paster B., Maclean I., Marusyk R. Development of the polymerase chain reaction for diagnosis of chancroid. J Clin Microbiol. 1993 Mar;31(3):659–664. doi: 10.1128/jcm.31.3.659-664.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen B. A., Rowley A. H., Long C. M. Herpes simplex type 2 in a patient with Mollaret's meningitis: demonstration by polymerase chain reaction. Ann Neurol. 1994 Jan;35(1):112–116. doi: 10.1002/ana.410350118. [DOI] [PubMed] [Google Scholar]
  6. Cone R. W., Hobson A. C., Brown Z., Ashley R., Berry S., Winter C., Corey L. Frequent detection of genital herpes simplex virus DNA by polymerase chain reaction among pregnant women. JAMA. 1994 Sep 14;272(10):792–796. [PubMed] [Google Scholar]
  7. Cone R. W., Hobson A. C., Palmer J., Remington M., Corey L. Extended duration of herpes simplex virus DNA in genital lesions detected by the polymerase chain reaction. J Infect Dis. 1991 Oct;164(4):757–760. doi: 10.1093/infdis/164.4.757. [DOI] [PubMed] [Google Scholar]
  8. Dallas W. S., Ray P. H., Leong J., Benedict C. D., Stamm L. V., Bassford P. J., Jr Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli. Infect Immun. 1987 May;55(5):1106–1115. doi: 10.1128/iai.55.5.1106-1115.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dewhirst F. E., Paster B. J., Olsen I., Fraser G. J. Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences. J Bacteriol. 1992 Mar;174(6):2002–2013. doi: 10.1128/jb.174.6.2002-2013.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Greisen K., Loeffelholz M., Purohit A., Leong D. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol. 1994 Feb;32(2):335–351. doi: 10.1128/jcm.32.2.335-351.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grimprel E., Sanchez P. J., Wendel G. D., Burstain J. M., McCracken G. H., Jr, Radolf J. D., Norgard M. V. Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid. J Clin Microbiol. 1991 Aug;29(8):1711–1718. doi: 10.1128/jcm.29.8.1711-1718.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hardy D. A., Arvin A. M., Yasukawa L. L., Bronzan R. N., Lewinsohn D. M., Hensleigh P. A., Prober C. G. Use of polymerase chain reaction for successful identification of asymptomatic genital infection with herpes simplex virus in pregnant women at delivery. J Infect Dis. 1990 Nov;162(5):1031–1035. doi: 10.1093/infdis/162.5.1031. [DOI] [PubMed] [Google Scholar]
  13. Hay P. E., Clarke J. R., Strugnell R. A., Taylor-Robinson D., Goldmeier D. Use of the polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid. FEMS Microbiol Lett. 1990 Mar 15;56(3):233–238. doi: 10.1111/j.1574-6968.1990.tb13943.x. [DOI] [PubMed] [Google Scholar]
  14. Jessamine P. G., Plummer F. A., Ndinya Achola J. O., Wainberg M. A., Wamola I., D'Costa L. J., Cameron D. W., Simonsen J. N., Plourde P., Ronald A. R. Human immunodeficiency virus, genital ulcers and the male foreskin: synergism in HIV-1 transmission. Scand J Infect Dis Suppl. 1990;69:181–186. [PubMed] [Google Scholar]
  15. Jethwa H. S., Schmitz J. L., Dallabetta G., Behets F., Hoffman I., Hamilton H., Lule G., Cohen M., Folds J. D. Comparison of molecular and microscopic techniques for detection of Treponema pallidum in genital ulcers. J Clin Microbiol. 1995 Jan;33(1):180–183. doi: 10.1128/jcm.33.1.180-183.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson S. R., Martin D. H., Cammarata C., Morse S. A. Alterations in sample preparation increase sensitivity of PCR assay for diagnosis of chancroid. J Clin Microbiol. 1995 Apr;33(4):1036–1038. doi: 10.1128/jcm.33.4.1036-1038.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kimura H., Shibata M., Kuzushima K., Nishikawa K., Nishiyama Y., Morishima T. Detection and direct typing of herpes simplex virus by polymerase chain reaction. Med Microbiol Immunol. 1990;179(4):177–184. doi: 10.1007/BF00195248. [DOI] [PubMed] [Google Scholar]
  18. Koutsky L. A., Stevens C. E., Holmes K. K., Ashley R. L., Kiviat N. B., Critchlow C. W., Corey L. Underdiagnosis of genital herpes by current clinical and viral-isolation procedures. N Engl J Med. 1992 Jun 4;326(23):1533–1539. doi: 10.1056/NEJM199206043262305. [DOI] [PubMed] [Google Scholar]
  19. Kreiss J. K., Coombs R., Plummer F., Holmes K. K., Nikora B., Cameron W., Ngugi E., Ndinya Achola J. O., Corey L. Isolation of human immunodeficiency virus from genital ulcers in Nairobi prostitutes. J Infect Dis. 1989 Sep;160(3):380–384. doi: 10.1093/infdis/160.3.380. [DOI] [PubMed] [Google Scholar]
  20. Lakeman F. D., Whitley R. J. Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease. National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group. J Infect Dis. 1995 Apr;171(4):857–863. doi: 10.1093/infdis/171.4.857. [DOI] [PubMed] [Google Scholar]
  21. Larsen S. A., Steiner B. M., Rudolph A. H. Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev. 1995 Jan;8(1):1–21. doi: 10.1128/cmr.8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loeffelholz M. J., Lewinski C. A., Silver S. R., Purohit A. P., Herman S. A., Buonagurio D. A., Dragon E. A. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbiol. 1992 Nov;30(11):2847–2851. doi: 10.1128/jcm.30.11.2847-2851.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morse S. A. Chancroid and Haemophilus ducreyi. Clin Microbiol Rev. 1989 Apr;2(2):137–157. doi: 10.1128/cmr.2.2.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Noordhoek G. T., Wolters E. C., de Jonge M. E., van Embden J. D. Detection by polymerase chain reaction of Treponema pallidum DNA in cerebrospinal fluid from neurosyphilis patients before and after antibiotic treatment. J Clin Microbiol. 1991 Sep;29(9):1976–1984. doi: 10.1128/jcm.29.9.1976-1984.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Parsons L. M., Waring A. L., Shayegani M. Molecular analysis of the Haemophilus ducreyi groE heat shock operon. Infect Immun. 1992 Oct;60(10):4111–4118. doi: 10.1128/iai.60.10.4111-4118.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Romanowski B., Forsey E., Prasad E., Lukehart S., Tam M., Hook E. W., 3rd Detection of Treponema pallidum by a fluorescent monoclonal antibody test. Sex Transm Dis. 1987 Jul-Sep;14(3):156–159. doi: 10.1097/00007435-198707000-00007. [DOI] [PubMed] [Google Scholar]
  27. Schulte J. M., Martich F. A., Schmid G. P. Chancroid in the United States, 1981-1990: evidence for underreporting of cases. MMWR CDC Surveill Summ. 1992 May 29;41(3):57–61. [PubMed] [Google Scholar]
  28. Sturm A. W., Stolting G. J., Cormane R. H., Zanen H. C. Clinical and microbiological evaluation of 46 episodes of genital ulceration. Genitourin Med. 1987 Apr;63(2):98–101. doi: 10.1136/sti.63.2.98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Telzak E. E., Chiasson M. A., Bevier P. J., Stoneburner R. L., Castro K. G., Jaffe H. W. HIV-1 seroconversion in patients with and without genital ulcer disease. A prospective study. Ann Intern Med. 1993 Dec 15;119(12):1181–1186. doi: 10.7326/0003-4819-119-12-199312150-00005. [DOI] [PubMed] [Google Scholar]
  30. Tsurumi T., Maeno K., Nishiyama Y. Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 2 and comparison with the type 1 counterpart. Gene. 1987;52(2-3):129–137. doi: 10.1016/0378-1119(87)90039-4. [DOI] [PubMed] [Google Scholar]
  31. Tung C. H., Rudolph M. J., Stein S. Preparation of oligonucleotide-peptide conjugates. Bioconjug Chem. 1991 Nov-Dec;2(6):464–465. doi: 10.1021/bc00012a015. [DOI] [PubMed] [Google Scholar]
  32. Wasserheit J. N. Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other sexually transmitted diseases. Sex Transm Dis. 1992 Mar-Apr;19(2):61–77. [PubMed] [Google Scholar]
  33. Weigel L. M., Brandt M. E., Norgard M. V. Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum. Infect Immun. 1992 Apr;60(4):1568–1576. doi: 10.1128/iai.60.4.1568-1576.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wicher K., Noordhoek G. T., Abbruscato F., Wicher V. Detection of Treponema pallidum in early syphilis by DNA amplification. J Clin Microbiol. 1992 Feb;30(2):497–500. doi: 10.1128/jcm.30.2.497-500.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES