JOURNAL OF CLINICAL MICROBIOLOGY, June 1984, p. 949-951 Vol. 19, No. 6 0095-1137/84/060949-03$02.00/0 Copyright C) 1984, American Society for Microbiology Cost-Effective Method of Triple-Site Culturing for Neisseria gonorrhoeae TONI GARTNER' AND LARRY D. EDWARDS2t* Clinic for Sexually Transmitted Diseases, Winnebago County Health Department, Rockford, Illinois 61108,1 and Division ofInfectious Diseases, Rockford School of Medicine, Rockford, Illinois 611072 Received 5 December 1983/Accepted 20 March 1984 The efficacy of the use of a single modified Thayer-Martin triplate versus that of three separate modified Thayer-Martin plates for the recovery of Neisseria gonorrhoeae from pharyngeal, anal, and genital sites were studied. A total of 98 males and 74 females who showed intracellular gram-negative diplococci on genital Gram stain were studied. Of 172 patients, 164 had gonorrhea at one or more sites. In the first group of 45 males and 21 females, a single swab from each site was used to inoculate randomly both a standard plate (100 mm in diameter) and one-third of a triplate. In the second group of 53 males and 53 females, two swabs were used for collection from each site. One of each pair of swabs was randomly inoculated onto a standard plate and a triplate. There was no significant difference between the results obtained by inoculation with single swabs and those obtained by inoculation with two swabs. There were no significant differences between the positivity rates obtained with the triplate and those obtained with three standard plates used at each site. Cost (39% that of three separate plates) and acceptance by clinic and laboratory personnel make the triplate method an economical, accurate, and effective triple-site screening system. There is a need to develop a simple and inexpensive, yet diplococci resembling N. gonorrhoeae on urethral or endo- efficient and effective, system for screening extragenital and cervical Gram stain. genital sites for Neisseria gonorrhoeae (5, 12, 16; T. Gartner All 172 participants were carefully questioned regarding and L. D. Edwards, Abstr. Ann. Meet. Am. Soc. Microbiol. their sexual preferences and practices by experienced, 1979, C(H)58, p. 356). In the past, time and expense have trained interviewers. been deterrents to the routine culturing of pharyngeal and All specimens were obtained by standard, recommended anal sites. Some investigators have stated that multiple-site techniques (3). In the initial group of 66 patients, a single culturing is not justifiable on the basis of cost-effectiveness calcium alginate swab was used for each site. Pharyngeal (1, 6, 15). specimens were obtained by rubbing the swab across the In recent years, there has been an increased awareness of surface of the posterior pharynx and tonsillar crypts. Anal extragenital gonorrheal infections. Several studies have re- specimens were obtained by inserting a swab approximately ported significant rates of pharyngeal and anal infections in 1 inch (2.54 cm) into the anal canal and rotating the swab for conjunction with genital gonorrhea or, less often, as the only several seconds to allow for the absorption of organisms infected site(s) (1, 2, 8, 13). Pharyngeal gonococcal infec- from the anal crypts. (When fecal material was present, tions are usually asymptomatic, however, so unless there is swabs were discarded and another specimen was taken.) In a history of oral-genital sex, cultures of the pharynx are females, the genital specimen for culture and Gram stain was seldom obtained (12, 14). obtained from the endocervical canal, and that in males was Because of the potential epidemiological and clinical sig- obtained from the anterior urethra. In the subsequent group nificance of extragenital gonorrhea, the need for more- of 106 patients, two swabs were used for each of the above effective treatment regimens, and the unreliability of symp- sites (endocervix, pharynx, and anus simultaneously, male toms and history as an indication for the culture of different urethra sequentially). sites, we decided to study an alternative, potentially cost- Full plates (100 by 15 mm) were kindly furnished by effective system for the routine culturing of genital and GIBCO Diagnostics, Madison, Wis., and triplates (100 by 15 pharyngeal sites. mm) containing the same manufacturing lot number of From 1 September 1978 through 2 October 1978, the first modified Thayer-Martin medium were supplied by Falcon group of 66 patients (45 males and 21 females) attending the Labware Division, Becton Dickinson & Co., Oxnard, Calif. Sexually Transmitted Diseases Clinic at the Winnebago All specimens were immediately inoculated onto modified County Health Department, Rockford, Ill., was studied. A Thayer-Martin medium containing vancomycin, colistin, second group of53 males and 53 females was studied from 12 nystatin, and trimethoprim (GIBCO) (10). Each patient May 1980 through 30 July 1980. These patients were admit- specimen (anal, pharyngeal, and urethral or endocervical) ted to the study if they had gram-negative intracellular was inoculated onto an entire modified Thayer-Martin plate (100 mm in diameter) and onto a corresponding one-third of a modified Thayer-Martin triplate (100 mm in diameter) in a * Corresponding author. "Z" pattern by rolling the swab across the agar surface. In t Present address: Department of Internal Medicine, Oral Roberts the initial group (single-specimen swab), a table of random University School of Medicine, P.O. Box 707070, Tulsa, OK 74170- numbers was used to determine whether the triplate was 7070. inoculated first or last for each specimen site. In the second 949 950 NOTES J. CLIN. MICROBIOL. group (two-specimen swabs), a separate swab was used for reagent costs per patient were reduced by two-thirds (one each agar surface and either the triplate or the full plate was triplate versus three full plates). inoculated first by the random choice of the examiner. After During the study period, three-site culturing by use of a inoculation, the culture plates were promptly placed in a triplate cost $0.43 per patient (plate plus medium), whereas 37°C incubator with 5 to 10% Co2, where they were held for three full plates cost $1.11 per patient. The decreased cost of up to 3 h, and then transported under controlled conditions reagents, increased costs of swabs, and personnel time were to the Division of Infectious Diseases Laboratory at the not included in this calculation. Rockford School of Medicine, Rockford, Ill. The triplate method was efficient and effective in increas- Immediately upon receipt in the laboratory, the inoculated ing the detection of N. gonorrhoeae and resulted in the agar surfaces were streaked for isolation with a wire loop potential for reduction in health care costs. It was as and placed in a 35°C incubator with 5 to 10% CO2. After accurate as the method of three full plates in detecting N. overnight incubation, plates were examined for typical oxi- gonorrhoeae. The extensive study of Judson and Werness dase-positive colonies resembling N. gonorrhoeae. Gram (5), in which females were screened at anal and cervical sites stains were made of any colonies meeting the above criteria. on a single agar plate, and our study of all patients with Confirmation of N. gonorrhoeae was made on isolates from positive genital Gram stains cultured at genital, anal, and all sites by production of acid from glucose but not lactose, pharyngeal sites on a single plate both support the value of maltose, or sucrose in cystine tryptic agar (BBL Micro- multiple-site culturing on a single plate (5). In our study, biology Systems, Cockeysville, Md.). All culture plates were detection is enhanced by the screening of all three sites. examined every 24 h for up to 72 h before being discarded. Others have found that N. gonorrhoeae can be inhibited Before being discarded, all plates were flooded with oxidase by other agents which may be present in secretions, i.e., reagent and searched for microcolonies (7). yeasts, other microorganisms and their metabolites, antibiot- After the conclusion of the study period, questionnaires ics, and antibodies (4, 9-11). Theoretically, these secretions, were distributed among the clinic examiners who had partici- if not spread adequately on the plate, may prevent N. pated in the study. Each person was asked to cite advantages gonorrhoeae from growing on artificial media. However, in and disadvantages of both types of culture systems, i.e., the actual practice, we found no significant difference between triplate and the three full plates, and indicate the overall the positivity rates per site on the smaller agar surface of the acceptibility of each system. triplate section and those on the larger surface of the full A total of 74 females and 98 males with positive genital plate. Gram stains yielded N. gonorrhoeae in 163 full plates and The purpose of our investigation was the evaluation of an 162 triplates (Table 1). There was no significant difference by alternative, cost-effective method for routine triple-site cul- sex, body site, or isolation rate between the results obtained turing for gonorrhea. Further clinical trials should be con- with the three full plates and those obtained with the triplate. ducted to confirm our findings. The triplate system offers a There was no correlation between a history of extragenital more cost-effective method (39% of the cost of three full sexual practice and culture positivity at extragenital sites, plates) of screening cultures for N. gonorrhoeae at three nor was there a significant difference in isolation rates sites. the use of one swab and the use of two swabs for between We sincerely appreciate the cooperation of Barbara Gleasman, inoculation. Director of the Division of Disease Control, and her staff at the Subjective evaluation of the triplate by clinic personnel Winnebago County Health Department. We thank GIBCO Diagnos- was positive.