Laboratory Diagnosis of Chancroid

Chancroid is a rather contagious sexually-transmitted disease caused by a bacterial pathogen Haemophilus ducreyi. According to the Centers for Disease Control and Prevention (CDC), a probable diagnosis is established in cases of one or more painful genital ulcers, typical presentation with regional lymphadenopathy, no evidence of syphilis, and a negative test for herpes simplex virus.

A history of sexual intercourse followed by the formation of painful ulcers on genitalia, as well as the development of fluctuant, unilateral bubo (i.e. regional inflammation and enlargement of one or more lymph nodes) are the classic indicators that point towards the diagnosis of chancroid.

Clinical diagnosis and recovery of Haemophilus ducreyi in the laboratory were both used as “gold standards” for diagnosing this condition in the past; however, the advent of molecular methods has shown that the true prevalence of chancroid was inaccurate when just those two aforementioned approaches were used.

Classical Culture Techniques

Haemophilus ducreyi is a fastidious microorganism that necessitates nutrient rich and relatively expensive base to grow on, thus it is considered a difficult bacterium to culture in laboratory conditions from clinical specimens. Consequently, that resulted in the lack of appropriate prevalence estimates.

Although the material taken from the ulcer or bubo can be put on a slide and stained with Giemsa stain or methylene blue, features of this microorganism are cumbersome to identify with certainty. In other words, such direct examination is neither sensitive nor specific.

Most strains of Haemophilus ducreyi show optimal growth at 33 °C in a humid atmosphere supplemented with 5% carbon dioxide. Improved isolation rates are observed in microaerophilic conditions when culture plates are incubated in a closed anaerobic system without a catalyst, but with two carbon dioxide and hydrogen generating sachets.

As different strains exhibit various growth morphotypes, the combination of different culture media (at least two of them) should be used to achieve optimal recovery rates. This is further increased if transport time is minimized, and if specimens are kept at 4 °C at all times during transit.

Successful culture of Haemophilus ducreyi is pivotal for establishing a definite diagnosis of chancroid, although it cannot rule out other potential concomitant infections. In addition, the importance of culture is evident when antimicrobial susceptibility is warranted (e.g. in instances of therapeutic failure).

Molecular and Serological Methods

Nucleic acid amplification techniques (often abbreviated as NAATs) have proven reliable for identifying Haemophilus ducreyi in clinical samples. Specific growth requirements of individual strains do not influence the outcome of these molecular methods, which is why detection rates are continually higher when compared to classical culturing.

A plethora of different in-house polymerase chain reaction (PCR) methods have been described in medical literature, some offering the advantage of simultaneous testing for other important sexually-transmitted pathogens (most notably herpes simplex and Treponema pallidum). The latter is known as multiplex polymerase chain reaction (M-PCR), and involves multiple primer pairs that are added to the reaction mixture for simultaneous amplification of distinct DNA sequences.

Unlike highly sensitive and specific molecular methods, serological approaches for antibody detection are not helpful for identifying Haemophilus ducreyi and diagnosing acute chancroid. In experimental settings the disease may be identified with DNA blotting or immunofluorescent examination, but none of those techniques are used in routine laboratory practice.