Genitourin Med 1997;73:245-252 245 Microbiological diagnosis of gonorrhoea Review A E Jephcott Gonorrhoea is caused by Neisseria gonorrhoeae. laboratory facilities, and the technical exper- This is a delicate and fastidious organism tise available in these, can be very different which dies rapidly if exposed to desiccating or from those normally found in the UK, and the oxidising conditions and requires a moist car- prevalence of infection may well be far higher. bon dioxide enriched atmosphere and a nutri- All these variables will affect the choice of the ent medium if it is to be cultured successfully. optimal test to employ-as will financial con- For these reasons diagnosis by culture was siderations and the expectations of the popula- regarded as difficult and uncertain, but many tion involved. Problems of maintaining decades ago the problems were overcome, and viability of the organism until cultured are culture became the method of first choice for unlikely to present problems in the usual UK diagnosis, and remains the "gold standard" clinic situation, whereas in field exercises or in against which others are measured.' However, rural surveys elsewhere in the world these may culture is by no means the only method avail- be of cardinal importance. able for diagnosis of gonorrhoea, and others offer alternative advantages such as speed, robustness, or technical simplicity, and no sin- Diagnostic strategies gle method is appropriate in all situations. 1 Direct visualisation of N gonorrhoeae cells in In the typical UK microbiology laboratory biological samples. specimens to be examined for gonococci will (i) Methylene blue stained smear usually have been taken from patients in (ii) Gram stained smear whom there is a significant likelihood of infec- (iii) Fluorescent antibody stained smear tion, and, on the results obtained, therapy is (a) Polyclonal antibodies likely to be administered. Prevalences will (b) Monoclonal antibodies vary, but are likely to be highest in patients attending genitourinary medicine clinics and 2 Direct detection of N gonorrhoeae cell compo- somewhat lower in other patient groups. nents in biological samples. However, specimens may also be received as (i) Solid phase immunoassay part of continuous or intermittent monitoring (ii) Nucleic acid hybridisation of particular population groups such as ante- (a) Nucleic acid probing natal clinic patients. Here the aim, while (b) Nucleic acid amplification including the treatment of any infected patients identified, is primarily to establish 3 Cultural detection of N gonorrhoeae in biologi- background knowledge of the prevalence of cal samples. infection in that community. Tests employed (i) Direct inoculation of culture medium in these differing situations will need to meet (ii) Use of transport medium and delayed different performance criteria. inoculation of culture medium Where a clinician is seeking to identify (iii) Transport cum culture systems infection in an individual patient the sensitivity Identification of cultured gonococci. of the test (that is, the likelihood of a genuine (a) Presumptive gonococcus identifica- infection being detected by the test) is of para- tion mount importance, whereas the risk of (b) Carbohydrate utilisation encountering a false positive result-which (c) Fluorescent antibody staining relates to the specificity of the test, will be of (d) Co-agglutination lesser importance.2 However, in a situation (e) Lectin agglutination where infection rates are low the problems of (f) Chromogenic substrates of constitu- encountering false positive results will become tive enzymes more significant, so that the specificity of the (g) Genetic probing test system employed will take on increasing importance and some sensitivity may have to 4 Specimens and sites for examination be sacrificed. Moreover, in any situation the (A) Genital secretions likelihood of any test result, either positive or (i) Urethral swab negative, being accurate (the so called positive (ii) Cervical swab and negative predictive values) will depend on (iii) Rectal/anal swab the number of genuine cases present in the (B) Distant infection sites population tested, as well as on the sensitivity (i) Conjunctival swab and specificity of the test.2 Thus, when num- (ii) Pharyngeal swab Public Health bers are low specificity becomes increasingly (iii) Salpinges, peritoneal cavity, etc Laboratory, Bristol important, whereas in a population with a high (C) Metastatic infection sites BS2 8EL A E Jephcott prevalence of infection, optimal sensitivity (i) Skin Accepted for publication should be sought. (ii) Joint aspirate 9 May 1997 Elsewhere in the world the availability of (iii) Blood 246 6Jephcott (iv) Cerebrospinal fluid Moreover, sensitivity of the test for men (D) First voided urine dropped to 53.9% when asymptomatic sexually active men were examined, and also fell dra- matically when a less experienced operator read Direct methods the slides. These figures are similar to those of ADVANTAGES other reports.5 6 1 Rapid-diagnosis can be reached before patient leaves clinic DIRECT VISUALISATION OF GONOCOCCI IN 2 Often cheap CLINICAL MATERIALS BY FLUORESCENT 3 Independent of constraints necessary for ANTIBODY STAINED SMEARS maintaining viability of organisms These are considered to be more specific, using antibody to detect gonococci and a bright green DISADVANTAGES label bonded onto this to make them stand out 1 Technical skill usually required in in the visual field. The technique originally7 clinic/survey centre received favourable reports, but some found it 2 Varying levels of accuracy (specificity) or to be little better than a Gram stained smear of sensitivity and discussed theoretical disadvantages.8 9 3 Absence of antibiotic sensitivity informa- These include problems of non-specific absorp- tion tion of fluorescein, autofluorescence, and diffi- culty of large antibody molecules penetrating through the macromolecular gel of genital Cultural methods secretions dried onto the slide. It also requires ADVANTAGES more technical skill than is usually available in a 1 Greater sensitivity clinic situation, and availability of an expensive 2 Almost 100% specificity fluorescence microscope. 3 Antibiotic sensitivity information obtainable Older antisera were raised in rabbits and were so called "polyclonal". These had to be DISADVANTAGES made specific by absorption with other species 1 Delay inevitable of bacteria and with tissue powders. They have 2 Cost been superseded by mixtures of monoclonal 3 Laboratory facilities required antibodies. Using these last Ison et al0 found 4 Technical expertise required (in a population with a high prevalence of infec- 5 Viability of organisms must be maintained tion) a sensitivity of 84.4% for men with a until culture is made specificity of 100% (being some 10% less sensi- tive than the Gram stain). In women the sensi- tivity was 65% and specificity 98% for urethral Direct methods samples with values of 72% and 94% respec- DIRECT VISUALISATION OF GONOCOCCI IN tively for cervical samples. These were better CLINICAL MATERIALS BY MICROSCOPY OF than the 40% sensitivity but 100% specificity STAINED SMEARS achieved by examination of Gram stained films This is the traditional method. Gonococci by the same team, but fall far short of those appear as bean-shaped diplococci, located in obtainable by culture. pairs with their long axes parallel, characteristi- In view of the technical difficulties, costs, cally lying inside polymorphonuclear cells. and lack of significantly increased diagnostic They are Gram negative. Originally a methyl- gain over the Gram film, this rapid method is ene blue stain was considered to demonstrate not used widely. these characteristic intracellular diplococci very clearly.' The likelihood of intracellular bean- SOLID PHASE IMMUNOASSAY DETECTION OF N shaped diplococci being anything other than GONORRHOAE IN CLINICAL MATERIALS gonococci is low (staphylococci can occur intra- A solid phase enzyme linked immunoassay cellularly but do so with a low frequency and (ELISA) is commercially available and is are not the same shape); nevertheless the Gram sold as "Gonozyme" (Abbott Laboratories, stain has now generally replaced methylene Chicago, IL, USA). Its advantages are that it blue staining.' This can demonstrate the Gram gives a rapid result, is relatively technically negative character of visualised microbes, but a undemanding, and can be performed without lack of clarity and precise definition is the price expensive equipment. Moreover, it can be car- paid. However, a Gram stained smear exam- ried out on dead organisms. This can allow it to ined by an experienced worker will detect be employed after prolonged transportation of almost all gonococcal infections in symptomatic the specimen, but complicates matters if used men with a very low false positive rate. Thus as a test of cure. Danielsson et all" assessed it in Goodhart et a14 reported the probability of gon- a clinic population and found a sensitivity of orrhoea to be 94-8% in smear positive sympto- 87% for men and 91% for women (specificity matic men, with only a 7.4% likelihood of 94.3% and 100% respectively). Whereas the infection in smear negative men. In women cer- results in men were no better than could be vical smear examination is less reliable. Thus, obtained by Gram film examination, in women the same authors reported that whereas there they were almost as good as those obtained by was a 97.3% probability of infection in patients culture. They suggested that