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close this bookBasic Laboratory Procedures in Clinical Bacteriology (WHO; 1991; 128 pages)
View the documentPreface
View the documentIntroduction
open this folder and view contentsQuality assurance in microbiology
close this folderPart I. Bacteriological investigations
open this folder and view contentsBlood
open this folder and view contentsCerebrospinal fluid
open this folder and view contentsUrine
open this folder and view contentsStool
open this folder and view contentsLower respiratory tract infections
open this folder and view contentsUpper respiratory tract infections
close this folderSexually transmitted diseases
View the documentIntroduction
View the documentUrethritis in men
View the documentGenital specimens from women
View the documentSpecimens from genital ulcers
open this folder and view contentsPurulent exudates, wounds, and abscesses
open this folder and view contentsAnaerobic bacteriology
open this folder and view contentsAntimicrobial susceptibility testing
open this folder and view contentsPart II. Essential media and reagents for isolation and identification of clinical pathogens
View the documentSelected further reading
View the documentSelected WHO publications of related interest
View the documentBack Cover
 

Specimens from genital ulcers

Genital ulcers are a very common problem in many developing countries. Their etiological diagnosis and management are a challenge to the clinician as well as to the laboratory. Mixed infections are common. Genital ulcerative lesions can be caused by a variety of sexually transmissible agents:

 

- human herpesvirus
- Treponema pallidum
- Haemophilus ducreyi
- Calymmatobacterium granulomatis, the agent of granuloma inguinale
- Chlamydia trachomatis serovars L1, L2, L3

Genital herpes is the most common cause of genital ulcer disease in most industrialized countries, and is a cause of life-threatening complications in immunodeficient patients and neonates born to women with the infection. Its laboratory diagnosis will not be discussed here.

Syphilis is still the most serious disease associated with genital lesions, since it can give rise to severe late sequelae and to congenital syphilis. While serological tests play an important role in the diagnosis of all stages of syphilis, only the dark-field examination will be discussed here. Techniques and interpretation of serological tests for syphilis have been extensively reviewed elsewhere.1

 

1Bench level laboratory manual for sexually transmitted diseases. Unpublished document WHO/VDT/89.443; available on request from Programme of Sexually Transmitted Diseases, World Health Organization, 1211 Geneva 27, Switzerland.

Chancroid is the major cause of genital ulceration in many developing areas. The clinical features include painful, purulent ulcer(s) accompanied by painful and occasionally suppurative inguinal buboes. Late sequelae are not known to occur. The clinical differentiation from other genital ulcer diseases is difficult.

Granuloma inguinale is characterized by extensive beefy, red, granulated genital ulcers. Bubo formation is rare.

Chlamydial lymphogranuloma is typically associated with inguinal and/or femoral lymphadenopathy, and, less frequently, with small ulcers which heal spontaneously. Its diagnosis is based on serological tests and isolation of C. trachomatis serovars L1, L2, L3.

Collection of specimens

Treponema pallidum: Protective surgical gloves should be worn. Squeeze the ulcer between two fingers and clean the surface of the lesion with saline, using gauze swabs. Crusts should be removed if present. After wiping away the first drops of blood (if present), collect a sample of serous exudate by touching a completely clean glass slide to the surface of the lesion. Immediately place a clean coverslip firmly on the drop of exudate. Alternatively, the specimen may be aspirated from the lesion, or from an enlarged lymph node, using a sterile needle and syringe. The preparation should be examined immediately by a microscopist experienced in dark-field microscopy.

Haemophilus ducreyi: Specimens should be obtained from the base of the ulcer with a swab and inoculated directly on to the isolation medium. Material may also be aspirated from inguinal buboes, but isolation of H. ducreyi is less successful than from genital lesions. Transport media for H. ducreyi have not been evaluated.

If granuloma inguinale is suspected, ideally a biopsy of subsurface tissue from an area of active granulation should be made. Fresh smears should be made from a crushed piece of biopsy material. Alternatively, one may make a smear by scraping off the surface of the lesion.

Direct examination

Demonstration of treponemes in lesion material is the method of choice for the diagnosis of primary syphilis. Although T. pallidum can be stained (for instance with silver nitrate), dark-field microscopy is recommended because it is more sensitive and specific. A microscope equipped with a good light source and a dark-field condenser must be available for dark-field examination. Dark-field condensers block out the direct light rays, allowing only the peripheral rays (deflected by objects such as treponemes) to pass through.

Place a few drops of immersion oil on the condenser of a dark-field microscope. Lower the condenser slightly so that the oil is below the level of the stage. Place the slide on the microscope and raise the condenser until there is good contact between the oil and the underside of the slide. Carefully avoid trapping air bubbles in the oil.

Using the low-power objective (x10) bring the specimen into focus. Centre the light in the field by adjusting the centring screws located on the condenser, and focus the condenser by raising or lowering it until the smallest diameter of light is obtained. Recentre the light if necessary. Then use the dry x 40 objective, bring the specimen into focus, and examine the slide carefully. The contrast will be better when the microscopy is done in the dark. Avoid bright daylight.

T. pallidum appears white, illuminated on a dark background (Fig. 5). It is identified by its typical morphology, size, and movement. It is a thin (0.25-0.3 μm) organism, 6 - 16 μm long, with 8 - 14 regular, tightly wound deep spirals. It exhibits quick and rather abrupt movements. It rotates relatively slowly about the longitudinal axis (like a corkscrew). This rotation is accompanied by bending (twisting) in the middle and is executed rather stiffly. Lengthening and shortening (like an elastic expander spiral) may be observed. Distortion may occur in tortuous convolutions. When the organism is attached to, or obstructed by, heavier objects, the resulting vigorous struggling distorts the coils. Other non-syphilis spirochaetes may be loosely coiled, thick, and coarse; the movements are different (not like a corkscrew), but take the form of a more writhing motion, with marked flexion and frequent relaxation of the coils.

The demonstration of treponemes with morphology and motility characteristic of T. pallidum constitutes a positive diagnosis for primary and secondary syphilis. Patients with a primary chancre, which is dark-field positive, may be serologically negative. They normally become serologically reactive within a few weeks.


Fig. 5. Appearance of T. pallidum under dark-field microscopy

Failure to find the organism does not exclude a diagnosis of syphilis. Negative results may mean that:

 

• An insufficient number of organisms was present (a single dark-field examination has a sensitivity of no more than 50%).

• The patient had already taken antibiotics.

• The lesion was approaching natural resolution.

• The lesion was not syphilitic.

Whatever the result of the dark-field examination, a blood sample should always be taken for serological tests.

In the diagnosis of granuloma inguinale, when acetone-fixed smears are stained with Giemsa, typical, intracellular, encapsulated bacilli can be seen within histiocytes. The diagnosis of this disease is described in full elsewhere.1

 

1Bench level laboratory manual for sexually transmitted diseases. Unpublished document WHO/VDT/89.443; available on request from Programme of Sexually Transmitted Diseases, World Health Organization, 1211 Geneva 27, Switzerland.

For the diagnosis of chancroid, a Gram-stained smear is not recommended, since the sensitivity and specificity are both less than 50%. For the diagnosis of chlamydial lymphogranuloma, Giemsa-stained smears are not recommended. However, material from the ulcer should also be examined by dark-field microscopy to search for T. pallidum.

Culture

Gonococci are occasionally isolated from genital ulcers, but their significance in such specimens is unclear. Apart from H. ducreyi, no other bacterial species - neither facultative aerobes nor obligate anaerobes - have been shown to cause genital ulcer disease.

Specimens to be examined for H. ducreyi should be inoculated directly on to a selective, enriched agar plate.1 The medium used should not be older than one week. The plates should be incubated at 33-35 °C in a candle jar with a moistened towel at the bottom. After 48-72 hours’ incubation, small, nonmucoid, yellow-grey, semiopaque or translucent colonies appear that can be pushed intact across the agar surface. The sensitivity of a single culture for the isolation of H. ducreyi is 70-80%.

 

1 Mueller-Hinton agar base, supplemented with 5% sterile horse blood heated to 75 °C, 1% IsoVitaleX, and 3 μg/ml vancomycin.

A presumptive diagnosis of H. ducreyi can be made on the basis of the characteristic morphology of colonies on selective medium, and the demonstration of small Gram-negative, pleomorphic coccobacilli, often in single chains (streptobacilli), parallel chains (“school of fish”), or clumps in the suspect colonies. Although H. ducreyi is haemin-dependent, most clinical isolates fail to grow on the media used for the determination of X and V factor requirement. Nearly all recent isolates from developing countries produce β-lactamase.

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