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Introduction of Haemophilus ducreyi

Presumptive Isolation of Haemophilus ducreyi using X, V, and XV disks on Muller-Hinton agar as shown above picture. The picture is suggestive for this organism because of growth around X and XV disks but not in the V disk. It is the causative agent of the sexually transmitted infection known as chancroid. Chancroid is thought to be the most common cause of genital ulcer disease (GUD).  Other causes of GUD include Treponema pallidum, Chlamydia trachomatis serovars L1, L2, and L3, Calymmatobacterium granulomatis, and herpes simplex virus. Although chancroid occurs commonly in parts of Africa, Asia, and Latin America of total 20% to 60% of GUD infections. This organism is fastidious, Gram-negative coccobacilli dies rapidly outside the human host, making diagnostic testing using culture methods difficult. Chancroid produces genital ulcerative lesions (soft ulcers that are painful) and may lead to bubo formation (swelling of inguinal lymph nodes) but there is no evidence of spreading systemically. Whereas it has also been found to cause chronic skin ulceration away from the genitalia, infect children and adults, and behave in a manner that seems yaws (Yaws is a contagious non-venereal endemic treponemal infection caused by Treponema pallidum subspecies pertenue. It affects mostly children in rural communities in developing countries and infection occurs after direct person-to-person contact).

Scientific classification

• Kingdom: Bacteria
• Phylum: Proteobacteria
• Class: Gamma Proteobacteria
• Order: Pasteurellales
• Family: Pasteurellaceae
• Genus: Haemophilus
• Species: H. ducreyi

Morphology 

Gram-negative coccobacilli that produce characteristic tan-yellow colonies that are highly self-adherent and can be ‘nudged’ intact over the surface of the agar.

Pathogenesis

Haemophilus ducreyi is an opportunistic pathogen that infects its host by way of breaks in the skin or epidermis. Inflammation then takes place as the area of infection is inundated with lymphocytes, macrophages, and granulocytes. This pyogenic inflammation causes regional lymphadenitis in the sexually transmitted disease (STD) chancroid.

Laboratory Diagnosis of Haemophilus ducreyi

Specimen Collection

Ulcer swab: Cleanse ulcer, swab base of the lesion, take four swab samples. Dacron, cotton, or calcium alginate swabs are acceptable for specimen collection.

Swab 1 is for culture by bedside inoculation on chocolate agar or gonococcal agar with added fetal calf serum (GC-HgS agar) or Mueller-Hinton agar supplemented with 5% chocolate horse blood (MH-HB agar), whereas in case of delay place swab in Amies transport media and transport to the laboratory.

Swab 2 is for  PCR.  Place dry swab in a sterile tube, keep at 4°C during transit. If the delay is long, freeze at –70°C until ready to send to a reference laboratory.

Swab 3 is for viruses and thus place in viral transport media for herpes culture.

Swab 4 is for syphilis and gram stain and so make two slides; one for Gram stain, one for DFA for syphilis.

Bubo aspirate: Aspirate above bubo through intact skin. Note: If bubo is draining, use a swab to collect material and process as per ulcer swabs.

Serum: Collect 5 mL blood in a red stopper tube.

Microscopy: Presence of Gram-negative coccobacilli but it is of limited value because of low sensitivity (5% to 63%) and specificity (51% to 99%).

Antigen detection: Hansen et al developed an antigen detection assay to detect Haemophilus ducreyi lipooligosaccharide (LOS) using a LOS-specific monoclonal antibody and an adaptation of the Limulus amoebocyte assay. Direct immunofluorescent testing of ulcer material using an H. ducreyi-specific monoclonal antibody appears to be useful.

Culture: Culture is considered the ‘gold standard. It can not grow on an ordinary medium like nutrient agar because of being fastidious. Chocolate agar is the common non-selective medium and other media are  Gonococcal agar supplemented with 2% bovine hemoglobin and 5% fetal calf serum, 1% IsovitaleX supplement, 3 µ/mL of vancomycin (gonococcal agar supplemented with 2% bovine hemoglobin and 5% fetal calf serum), and Mueller-Hinton agar (MHA) supplemented with 5% chocolatized horse blood, 1% Isovitalex supplement and 3 µ/mL of vancomycin (MHA supplemented with 5% chocolatized horse blood) have been shown to be an optimal combination. Modification of this technique by the substitution of 0.2% activated charcoal for fetal calf serum has proven to be equally effective, and cheaper. All inoculated media should be incubated in 5% CO2 at 33°C to 35°C (it is critical that the temperature does not exceed 35°C) in a humid environment.

Biochemical test: Catalase test negative whereas oxidase test positive

Serology: Detection of antibody production as serological assays but low sensitivity and specificity rate i.e.  55% to 100% and 23% to 96%, respectively.

Molecular Test: genetic amplification methods are sometimes used to diagnose infections with H. ducreyi and the genetic tests have greater sensitivity

Treatment

Initial stages of the lesion, cleaning with a soapy solution is recommended and a sitz bath may be beneficial. Fluctuant nodules may require aspiration. Antibiotics, macrolides are often used to treat chancroid. The CDC recommendation is either a single oral dose (1 gram) of azithromycin, a single IM dose (250 mg) of ceftriaxone, oral (500 mg) of erythromycin three times a day for seven days, or oral (500 mg) of ciprofloxacin twice a day for three days. Aminoglycosides such as gentamicin, streptomycin, and kanamycin have been used to successfully treat chancroid; however, there have been reports of ciprofloxacin and erythromycin and aminoglycoside resistance. Although susceptibility testing of Haemophilus ducreyi has been performed using agar dilution or E-test methods. However, given the growing resistance of H. ducreyi to antibiotics as evidenced by therapy failures having clinical isolates for antibiotic therapy evaluation is important from an epidemiological perspective.

Keynotes for Haemophilus ducreyi

1. In suspected cases of chancroid, testing for herpes simplex virus and syphilis should also be performed.
2. Cultures are currently considered the “gold standard” test for H. ducreyi.
3. Some strains of H. ducreyi have been reported to be sensitive to vancomycin and therefore would not grow on selective media containing this antibiotic.
4. Presumptive identification tests to consider include oxidase (positive for H. ducreyi), catalase (negative for H. ducreyi), and X factor nutritional requirement (H. ducreyi requires X factor for growth and this can most easily be evaluated using the porphyrin test). For confirmation of species identification, the isolate can be sent to a reference laboratory for H. ducreyi-specific molecular tests.
5. The swab in transport medium (e.g. Amies or Amies with charcoal) needs to reach the laboratory within 4 hours because H. ducreyi will not survive well beyond this time frame.
6. Culture from intact buboes is often negative; ulcer swabs are better for culture.
7. H. ducreyi is distinct from other Haemophilus and Aggregatibacter species by 16S rRNA and housekeeping gene sequences.

References

1. https://en.wikipedia.org/wiki/Chancroid
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095004/
3. https://www.sciencedirect.com/topics/medicine-and-dentistry/haemophilus-ducreyi
4. https://journals.lww.com/coinfectiousdiseases/Abstract/2016/02000/Haemophilus_ducreyi__from_sexually_transmitted.9.aspx
5. https://www.osmosis.org/learn/Haemophilus_ducreyi_(Chancroid)
6. https://www.thelancet.com/journals/langlo/article/PIIS2214-109X(14)70197-4/fulltext