Molecular Detection by PCR

Molecular detection of Haemophilus ducreyi (chancroid) by PCR.

• Chancroid

The specimen of choice for the diagnosis of chancroid is a swab that has been taken from the base of the genital ulcer. A bubo aspirate is also a suitable specimen.

Specimens may be subject to rejection if they are not the appropriate sample type, have insufficient volume, or are not accompanied by relevant patient information.

Use Dacron or cotton swabs to obtain specimens from ulcers. This is best collected by cleansing the area by flushing with sterile physiological saline, and then collecting material from the base of the ulcer using a Dacron or cotton swab. A bubo aspirate is obtained using needle and syringe to aspirate pustular material from the bubo through normal tissue.

Swabs can shipped dry OR in 1 ml Universal Transport Medium (Copan International). Bubo aspirates should be placed in a sterile tube and shipped frozen. Store swabs frozen until shipped for testing. Ship frozen on dry ice.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

H. ducreyi is the causative agent of chancroid, an STI characterized by necrotizing genital ulceration which may be associated with inguinal lymphadenitis (“bubo”) formation.  Chancroid is endemic in Southeast Asia and Africa but occurs infrequently in the developed world, usually in individuals who have travelled to these endemic areas or in localized urban outbreaks often involving the sex trade.  Immunocompromised individuals (e.g. HIV-positive) may be at higher risk for H. ducreyi infection.

Streptococcus and STI Unit requisition form, clearly specify organism testing request on the requisition form. Append all relevant clinical background information and testing performed.

Duplicate samples should not be submitted without prior consent as they may not be processed. Isolates collected from the same patient within 3 weeks of each other are considered duplicate.

DNA from all H. ducreyi samples received at the NML for PCR testing is initially extracted using a commercially-available kit.  Results are based upon H.ducreyi specific qPCR assays targeting the highly conserved 16S rRNA gene and the H.ducreyi specific “hemolysin” gene (hhdA). A sample is interpreted to be positive for H. ducreyi if either H. ducreyi-specific PCR is positive. Sample is also tested for RNase-P to ensure DNA extraction was successful.

A positive PCR result detecting the presence of a specific target sequence to a bacterial pathogen of interest indicates it's presence within the submitted specimen.

A negative PCR result indicates the absence of detectable bacterial DNA in the specimen, but does not rule-out infection as false-negative results may occur due to: inhibition of PCR reactions, sequence variability underlying the target primers and probes, or the presence of bacterial DNA in quantities less than the limit of detection of the respective assay.

As with any laboratory test, the results of the test should be interpreted with consideration of all of the laboratory and clinical findings available.

14 calendar days. Urgent specimens will be granted priority status, and completed as soon as possible.

1. Alfa, M. The laboratory diagnosis of Haemophilus ducreyi.  Can J Infect Dis Med Microbiol.2005;16:31–34.
2. Lewis DA. Chancroid: clinical manifestations, diagnosis, and management. Sex Transm Infect. 2003;79:68-71.
3. Orle KA, Gates CA, Martin DH, Body BA, and Weiss JB. Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers. J Clin Microbiol. 1996;34:49–54.
4. Chen C, Ballard RC. The Molecular Diagnosis of Sexually Transmitted Genital Ulcer Disease. (2012) Diagnosis of Sexually Transmitted Diseases: Methods and Protocols, Methods in Molecular Biology  903:103-112.
5. Glatz M, et al. A multicenter prospective trial to asses a new real-time polymerase chain reaction for detection of Treponema pallidum, herpes simplex-1/2 and Haemophilus ducreyi in genital, anal and oropharyngeal ulcers. Clinical Microbiology and Infection (2014): 20:O1020-O1027.

Pathogen Safety Data Sheets: Infectious Substances – Haemophilus ducreyi

Canadian Guidelines on Sexually Transmitted Infections