Urethritis in men is clinically characterized by a urethral discharge and/or dysuria, but asymptomatic infection with Neisseria gonorrhoeae or Chlamydia trachomatis occurs frequently. If untreated, gonococcal and chlamydial urethritis may progress to epididymitis. Rectal and oropharyngeal infection with N. gonorrhoeae and C.
trachomatis may occur in homosexual men.
For the purpose of patient management, urethritis should be divided into gonococcal urethritis and nongonococcal urethritis (NGU). Approximately half the cases of NGU are caused by C. trachomatis, but the etiology of the majority of the remaining cases has not been fully elucidated. According to some studies, Ureaplasma urealyticum may be a cause of urethritis, and Trichomonas vaginalis can be found in 1 - 3% of cases of NGU. Intra-urethral infection with human herpesvirus may yield a urethral discharge. Bacterial agents such as staphylococci, various Enterobacteriaceae, Acinetobacter spp, and Pseudomonas spp can be isolated from the urethra of healthy men, and have not been shown to cause urethritis.
The examination of specimens for C. trachomatis is complicated and will not be discussed in this section. Besides isolation in cell culture systems, non-culture methods for the detection of chlamydial antigens by enzyme immunoassay and immunofluorescence assay have recently become available. These methods, while promising, remain prohibitively expensive.
Collection and transport of specimens
For the collection of urethral specimens, a swab with a narrow diameter or a sterile bacteriological loop should be inserted 3-4 cm into the urethra and gently rotated before withdrawal. Purulent discharge can be collected directly on a swab or on the inoculating loop. The composition of both the tip and the shaft of the swab is important. For the culture of N.
gonorrhoeae, charcoal-treated cotton tips or calcium alginate or Dacron tips are preferred. If these special, commercially prepared, sampling swabs are not available and regular cotton swabs are used, the specimen should be inoculated immediately. A prostatic massage does not increase the rate of isolation of gonococci or chlamydiae in cases of urethritis.
Anorectal specimens are obtained by inserting a swab 4 - 5 cm into the anal canal. For oropharyngeal specimens, the posterior pharynx and the tonsillar crypts should be swabbed and plated immediately.
Ideally the inoculation of specimens for the isolation of N. gonorrhoeae should be made directly on to the culture medium in the clinic. Inoculated plates should be placed in a candle jar or into an atmosphere containing 5 - 10% carbon dioxide, with high humidity. If immediate plating and incubation are not possible, a transport medium such as Amies or Stuart transport medium should be used. The transport time should be as short as possible, and must be less than 12 hours in ambient temperatures up to 30 °C. Refrigeration is to be avoided.
Direct examination and interpretation
Most studies have shown that the presence of four or more polymorphonuclear (PMN) leukocytes per oil-immersion field is strongly indicative of urethritis in men. This criterion is particularly useful to the clinician who has to decide whether to treat patients with vague urethral complaints.
In most cases of gonorrhoea in the male, the discharge is purulent, and numerous polymorphonuclear leukocytes (> 10 per oil-immersion field) can be seen in the urethral smear. However, this is not always the case in NGU, which yields a less severe inflammatory reaction. Smears with more than 4 PMN leukocytes per oil-immersion field, and without intracellular Gram-negative diplococci, are highly suggestive of NGU.
A thinly spread smear, prepared by rolling a swab over a slide, should be heat-fixed and Gram-stained. This method minimizes distortion of polymorphonuclear leukocytes. The presence of intracellular Gram-negative diplococci in PMN leukocytes in a urethral smear is strongly suggestive of gonorrhoea. Both the sensitivity and specificity of a Gram-stained smear are greater than 98% for the diagnosis of gonorrhoea in the male.
Gram-stained smears of intra-urethral specimens from asymptomatic males, from blind rectal swabs, or from oropharyngeal samples are not recommended. However, microscopic examination of purulent material obtained under anoscopy has a fairly high diagnostic value.
Culture of Neisseria gonorrhoeae
Inoculated plates with modified Thayer-Martin (MTM)1 agar (or New York City medium (NYC)2) should be incubated at 35 °C in a humid atmosphere enriched with carbon dioxide (candle jar), and should be observed daily for two days. Laboratories processing a large number of specimens for N. gonorrhoeae often prefer to use a nonselective chocolate agar enriched with IsoVitaleX, or an equivalent supplement, in addition to the selective MTM, because as many as 3 - 10% of gonococcal strains in a given area may be susceptible to the concentration of vancomycin used in selective media.
1 Modified Thayer-Martin agar is prepared by adding at 50 °C an antibiotic mixture and Iso VitaleX, or an equivalent supplement, to chocolate agar prepared from GC agar or Columbia agar as basal medium. Antibiotic mixtures containing 3 or 4 antibiotics are commercially available from several sources; VCN mixture contains vancomycin, colistin, and nystatin; VCNT mixture also contains trimethoprim.
The final concentrations of the antibiotics in the prepared medium are:
2 Modified New York City medium is prepared by adding to 500 ml of sterile GC agar base cooled to 50 °C, the following supplements:
- 50 ml of horse blood lysed by adding 5 ml/litre saponin,
- sterile yeast autolysate,
- an antibiotic mixture containing vancomycin, colistin, amphotericin, and trimethoprim.
The ingredients are commercially available from Oxoid Ltd, Wade Rd, Basingstoke, England.
Gonococcal colonies are 0.5 - 1 mm in diameter after 24 hours, and appear grey to white, opaque, raised, and glistening. Particularly after further incubation, colonies of different sizes and morphology can be observed, representing the five different colony types of N.
Identification of Neisseria gonorrhoeae
A presumptive identification of N. gonorrhoeae isolated from urogenital specimens is based on a positive oxidase reaction and a Gram-stained smear showing Gram-negative diplococci. Confirmation of the identification can be obtained by carbohydrate degradation assays or other tests, using methods and media discussed extensively 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.
Antimicrobial susceptibility testing
There is considerable geographical variation in the susceptibility of N. gonorrhoeae strains to penicillin. In some areas, such as sub-Saharan Africa or south-east Asia, most gonococcal strains are now β-lactamase-producing. Chromosomally mediated resistance to penicillin not based on β-lactamase production is also becoming more common in many countries. However, the disc diffusion test is not reliable in detecting such strains.
In areas where penicillin, ampicillin, or amoxicillin is still used for the treatment of gonococcal infections, N. gonorrhoeae isolates (particularly from cases of treatment failure) should be routinely screened for (β-lactamase production by one of the recommended tests, such as the Nitrocefin test.2 For the Nitrocefin test, a dense suspension from several colonies is prepared in a small tube with 0.2 ml saline; 0.025 ml of Nitrocefin is then added to the suspension and mixed for one minute. A rapid change in the colour, from yellow to pink or red, indicates that the strain produces β-lactamase.
2 The Nitrocefin reagent is obtainable from Oxoid (Basingstoke, England), and consists of 1 mg of Nitrocefin (SR112) and 1 vial of rehydration fluid (SR112A). The tube test can be replaced by a disc test, using Nitrocefin-impregnated paper discs (Cefinase discs, available from BBL Microbiology Systems Division, Becton & Dickinson & Co., PO Box 243, Cockeysville, MD, USA).
Antimicrobial susceptibility testing of N. gonorrhoeae by the disc diffusion assay is not recommended.