Comparison of GC-Lect and modified Thayer-Martin media for isolation of Neisseria gonorrhoeae.

A new selective medium, GC-Lect, was compared with modified Thayer-Martin medium (MTM) for isolation of Neisseria gonorrhoeae. Cultures from 620 sexually transmitted disease clinic patients were directly inoculated onto both media, placed in candle extinction jars, and incubated. N. gonorrhoeae was isolated from 175 (29%) of 607 genital cultures, 3 (3%) of 88 pharyngeal cultures, and 6 (29%) of 21 rectal cultures. Ten cultures were positive only on GC-Lect, and 3 were positive only on MTM. In 3 of the 10 cultures positive only on GC-Lect, overgrowth of a Capnocytophaga sp. may have obscured growth on MTM. In this study, vancomycin-susceptible (MIC, less than 4.0 micrograms/ml) N. gonorrhoeae was isolated on both media, and none of the isolates missed by either medium were susceptible to vancomycin. No differences were noted between the two media in time required for isolation of N. gonorrhoeae. While isolation rates of N. gonorrhoeae were similar, suppression of nongonococcal bacterial species by GC-Lect was superior to that by MTM. GC-Lect is equal to MTM for detection of N. gonorrhoeae and is superior for suppression of normal flora.     

Effect of holding temperature on isolation of Neisseria gonorrhoeae.

The effect of holding temperature on the recovery of Neisseria gonorrhoeae was studied. From 300 specimens tested, Thayer-Martin medium plates inoculated and incubated in the presence of CO2 at 35, 22, and 4 degrees C for 24 h before incubation at 35 degrees C yielded 100, 96, and 95% of all isolates ultimately recovered from 82 positive specimens. Although there was a decrease in the quantity of organisms recovered, initial incubation of specimens under refrigeration or at room temperature yielded greater than or equal to 95% of the positive specimens.     

Comparison of hemoglobin-free culture media and thayer-martin medium for the primary isolation of Neisseria gonorrhoeae.

Translucent culture media are essential for the determination of gonococcal colony types. In this study two hemoglobin-free, translucent culture media were compared with Thayer-Martin (TM) medium for the primary isolation of Neisseria gonorrhoeae from male urethral specimens. The antibiotic-free translucent medium (GCB) and the translucent medium containing vancomycin, colistin, and nystatin (VCN) were as efficient as TM medium in detecting positive specimens. In fact, VCN medium gave positive results significantly more often than TM medium(P less than 0.05). The amount of growth on positive plates was comparable for all three media. Contaminants were noted most often on GCB medium (P less than 0.0001), and the frequency of contaminants with TM plates exceeded that of VCN plates (P less than 0.0001). The high isolation rate of N. gonorrhoeae with VCN hemoglobin-free culture medium and its lower cost and greater ease of preparation make it a suitable alternative to TM medium for use with male urethral specimens.     

Comparison of ligase chain reaction and culture for detection of Neisseria gonorrhoeae in genital and extragenital specimens.

In addition to the urogenital tract, Neisseria gonorrhoeae infects extragenital sites such as the pharynx and anorectal canal. Culture and a ligase chain reaction (LCR)-based assay were compared for their performance for the diagnosis of N. gonorrhoeae infection with specimens from various urogenital and extragenital sites of 200 men and 125 women. The sensitivity and specificity of the LCR assay with male urethral swabs were both 100%, compared to values of 95.9 and 100%, respectively, for culture of urethral swabs or 98.0 and 100%, respectively, for LCR with first-void urine (FVU). For women, LCR with FVU showed the highest sensitivity (94.7%), and culture of urethral samples showed the lowest sensitivity (63.2%) (P < 0.05). In a selected subgroup of 47 men and 22 women at increased risk, the rates of pharyngeal infection were 15 and 18%, respectively, and those of anorectal infection were 13 and 45%, respectively. The sensitivity of LCR was greater than that of culture for both pharyngeal and anorectal specimens. Thus, the overall performance of LCR testing with swabs or FVU was better than that of culture for the diagnosis of genital or extragenital gonorrhea.     

Evaluation of the GO Slide (Roche) growth transport system for isolation of Neisseria gonorrhoeae from clinical specimens.

A new growth transport system for the isolation of Neisseria gonorrhoeae from clinical specimens was evaluated. The system, GO Slide (Roche) of F. Hoffmann-La Roche & Co., Basel, Switzerland, showed 88% sensitivity for male urethral specimens and 59% sensitivity for endocervical specimens compared with transport in the Amies transport medium combined with culturing on modified Thayer-Martin medium. The media used appear not to support growth of certain strains of N. gonorrhoeae. recovery and growth-supporting capabilities of this system need to be improved before the system can be used routinely.     

New selective medium for the isolation of Neisseria gonorrhoeae.

GC-Lect, a new selective medium for the isolation of Neisseria gonorrhoeae which contains five antimicrobial agents, was evaluated with stock cultures and with 500 clinical specimens. With stock cultures, vancomycin-resistant Staphylococcus epidermidis that grew on modified Thayer-Martin medium (MTM) was inhibited on the new medium. Also, vancomycin-susceptible strains of N. gonorrhoeae were much less inhibited on the new medium than on Martin-Lewis agar or MTM. With oropharyngeal cultures of healthy volunteers, Capnocytophaga species were frequently isolated on MTM from two of three manufacturers but were completely inhibited on GC-Lect. In the clinical study, visible growth of N. gonorrhoeae occurred within 24 h in 72% of the positive cultures on GC-Lect, compared with only 52% on the reference medium. A total of 50 positive cultures were obtained with GC-Lect, compared with 49 obtained with MTM. The selectivity of GC-Lect was superior, with only 19 cultures producing growth of normal flora, compared with 78 cultures on MTM after 24 h of incubation. The selectivity was especially improved on GC-Lect with regard to yeasts (2 versus 30 cultures) and gram-positive cocci (5 versus 31 cultures).     

Detection of Neisseria gonorrhoeae from air-dried genital samples by single-tube nested PCR.

A single-tube nested PCR method was developed for the detection of Neisseria gonorrhoeae. The optimized assay had a detection limit of less than 0.3 cell. Five different storage conditions for gonococcal specimens were compared with respect to the PCR detection of bacteria. For air-dried gonococcal slides containing three bacteria, DNA was detected after 8 weeks at ambient temperature, and for slides containing 300 bacteria, DNA could be detected after 24 weeks at ambient temperature. Air-dried storage combined with analysis by the single-tube nested PCR and a commercially available PCR (Amplicor) was used to test 350 cervical specimens from women in the West African island nation of Cape Verde. The in-house PCR detected 17 cases of N. gonorrhoeae infection, while the Amplicor system detected 14 cases of N. gonorrhoeae infection. No specimen was negative by the in-house PCR assay and positive by the Amplicor PCR. This sensitive nested PCR assay, combined with air-dried storage, allows for the detection of gonococci when specimen storage and transport times are extended and freezing conditions are not available.     

Comparison of Transgrow and Gonozyme for the detection of Neisseria gonorrhoeae in mailed specimens

The Transgrow culture system and Gonozyme (Abbott Laboratories, North Chicago, Ill.), an enzyme-linked immunosorbent assay procedure, were compared by examining 510 patients (320 females, 190 males) from whom duplicate genital swabs were obtained for the diagnosis of Neisseria gonorrhoeae infection. Both Transgrow and the Gonozyme swabs were mailed to the laboratory. Clinical, epidemiological, and laboratory data for the 30 specimens for which there were discrepancies were evaluated to determine the probability of gonorrhea. At the same time, Gonozyme was compared to on-site Thayer-Martin cultures from 258 of the 510 patients, with a 93% agreement. When sensitivity and specificity were calculated on the basis of clinical, epidemiological, and on-site laboratory data, Gonozyme had a sensitivity of 95% and a specificity of 99%. Transgrow culture was considered to have a 100% specificity and a sensitivity of 69%. Gonozyme appeared to be a superior method for the diagnosis of gonorrhea by means of mailed specimens.     

Comparative evaluation of New York City and modified Thayer-Martin media for isolation of Neisseria gonorrhoeae.

Commercially manufactured New York City (NYC) medium and modified Thayer-Martin (MTM) medium were compared for their ability to isolate Neisseria gonorrhoeae from clinical specimens. Twenty-seven public health laboratories throughout California evaluated 4,802 specimens collected from patients attending either sexually transmitted disease or family planning clinics. Total of 726 and 737 N. gonorrhoeae isolates were recovered from NYC and MTM medium, respectively. Although less contamination was noted on NYC medium, MTM medium was equivalent to commercially prepared NYC medium for the isolation of N. gonorrhoeae from clinical specimens.     

Comparison of modified New York City medium with Martin-Lewis Medium for recovery of Neisseria gonorrhoeae from clinical specimens.

A modified formulation of New York City medium was comparatively evaluated with Martin-Lewis medium for the recovery of Neisseria gonorrhoeae from clinical specimens. A total of 240 strains of gonococci were recovered from 1,250 specimens collected from walk-in patients attending a sexually transmitted disease clinic. N. gonorrhoeae was cultivated on both of these media from 182 clinical specimens with an additional 58 gonococcal strains isolated on either of the media. Of these discrepant gonococcal isolates, 27 strains were recovered on only modified New York City medium, whereas the remaining 31 strains were recovered on only Martin-Lewis agar. The differences in these isolation rates were not statistically significant. The overall results showed that modified New York City and Martin-Lewis media were comparable in their ability to grow gonococci from clinical material. Since modified New York City medium is capable of supporting the growth of N. gonorrhoeae, Mycoplasma pneumoniae, and urogenital mycoplasmas and inhibiting the growth of commensal microorganisms, it is possible that it may have considerable application as a multifunctional plating medium within the clinical laboratory.     

Primary isolation of Neisseria gonorrhoeae on hemoglobin-free New York City medium.

New York City medium and New York City medium without hemoglobin were comparatively evaluated for their ability to support the growth of Neisseria gonorrhoeae isolated from 1,010 clinical specimens. Although hemoglobin in the form of lysed horse erythrocytes stimulated gonococcal growth, the absence of this component from New York City medium did not have a detrimental effect on the recovery of gonococci isolated from clinical specimens. Both media were comparable in their ability to cultivate gonococci from clinical material, with a total of 187 gonococcal isolates being recovered on each of the media. The results of this study showed that the preparation of New York City medium can be facilitated and that its cost can perhaps be reduced by the elimination of the hemoglobin component from the formulation without adverse effect on the recovery of N. gonorrhoeae isolated from clinical specimens.     

Comparison of Gen-Probe DNA probe test and culture for the detection of Neisseria gonorrhoeae in endocervical specimens.

A 2-h nonisotopic DNA probe assay for the direct detection of Neisseria gonorrhoeae in urogenital specimens has recently been modified (PACE 2; Gen-Probe, San Diego, Calif.). The new assay format was developed to increase the sensitivity of the assay and simplify procedural steps. In this study, the new DNA probe test was compared with a culture reference method for the detection of N. gonorrhoeae in endocervical specimens. The results of the DNA probe test were expressed as a ratio of relative light units (RLU) of the specimen/RLU of the cutoff recommended by the manufacturer. All patient samples with sample RLU/cutoff RLU ratios less than 0.7 were interpreted as negative, and ratios greater than 2.0 were interpreted as positive for gonorrhea. Samples with sample RLU/cutoff RLU ratios between 0.7 and 2.0 were repeated until two or more consistent negative or positive ratios were obtained. A total of 469 specimens were tested with an overall disease prevalence of 6.1%. Of the 469 patients tested, 5 specimens (1.0%) fell in this borderline region and were retested. If the manufacturer's recommended cutoff value had been used, the original DNA probe results would have resulted in two false-positives. Our data were analyzed for both symptomatic (prevalence, 11.7%) and asymptomatic (prevalence, 2%) women. The study indicated that with our modification of the manufacturer's endpoint interpretation, the DNA probe test was essentially equivalent to the culture method in terms of sensitivity, specificity, and positive and negative predictive values in both symptomatic and asymptomatic patient populations. The new DNA probe test can serve as a suitable screening and diagnostic test for the diagnosis of gonorrheal genital infections in women. Additionally, it offers the advantages of rapid turnaround time and ease of use and allows simultaneous testing for Chlamydia trachomatis on the same specimen.     

Survey of the use of selective culture for Neisseria gonorrhoeae in specimens from the female genital tract sent by general practitioners to a microbiology laboratory.

A retrospective survey of the number of cultures found to be positive for Neisseria gonorrhoeae in genital specimens from female patients sent by general practitioners (GPs) over a three-year period was carried out. The organism could be detected in only four specimens out of over 28,000 specimens sent. Specifically, additional selective culture for N gonorrhoeae had been carried out in 8529 of these specimens. An estimate of the cost savings achievable if this laboratory was no longer to culture routinely for N gonorrhoeae was made. GPs should be aware of their local laboratory's normal practice when processing such specimen and should request specific culture if appropriate. The low number of specimens from which N gonorrhoeae could be cultured might suggest that GPs are referring 'at-risk' patients to genitourinary medicine clinics already.     

Neisseria gonorrhoeae colonises the genital tract of oestradiol-treated germ-free female mice

Germ-free BALB/c mice treated with oestradiol and inoculated intravaginally with a serum-resistant strain or a freshly isolated, piliated strain of Neisseria gonorrhoeae were colonised vaginally. The organisms were recovered intermittently for a month or longer and there was evidence that they could reach the upper genital tract. Mice given progesterone and those not treated with either hormone did not become colonised. This is the first evidence of sustained mucosal colonisation in animals other than chimpanzees.     

Toxic effect of calcium alginate swabs on Neisseria gonorrhoeae.

Calcium alginate (CA)-tipped swabs have been reported to interfere with the recovery of herpes simplex virus, Chlamydia trachomatis, and Ureaplasma urealyticum and may cause cytotoxicity in cell culture. To determine whether CA swabs also inhibit the growth of Neisseria gonorrhoeae, we carried out a series of experiments using either CA swabs that were toxic or nontoxic in a cell culture cytotoxicity assay or nontoxic rayon or cotton swabs. Leaving a toxic CA swab in 3 ml of Mueller-Hinton broth inoculated with 10(4) CFU/ml caused rapid killing within 6 h at 37 degrees C; colony counts of five strains were less than 1% of those of Mueller-Hinton broth controls. When the tips of toxic CA swabs were inoculated directly and kept at 37 degrees C without holding medium, the swabs were sterile at 6 h. If the same swabs were placed in Amies medium with charcoal, organisms could still be recovered at 6 h. Toxicity was less at room temperature than at 37 degrees C. Inhibition of growth of N. gonorrhoeae was not seen with rayon or cotton swabs. The toxic component was neither the CA fiber nor the aluminum wire but probably the glue used to attach the fibers. We concluded that some lots of CA swabs kill N. gonorrhoeae in vitro. Survival of N. gonorrhoeae is improved with nontoxic swabs, particularly cotton swabs, and Amies medium with charcoal regardless of swab type.     

Use of New York City medium for improved recovery of Neisseria gonorrhoeae from clinical specimens.

New York City (NYC) and Martin-Lewis (ML) media were evaluated comparatively for their ability to support the growth of Neisseria gonorrhoeae from clinical specimens. A total of 1,010 urethral, cervical, pharyngeal, and rectal specimens were collected from walk-in patients attending a clinic for sexually transmitted diseases. A total of 187 and 165 isolates of gonococci were cultivated on NYC and ML media, respectively, with 161 of these isolates being recovered on both media. Overall, the use of NYC medium resulted in a 13.3% increased recovery rate of gonococci. When gonococci were recovered on both media from primary isolation, the NYC medium supported a more luxuriant growth and a greater number of colonies, which usually resulted in the detection of positive cultures 1 day sooner than on ML medium. Both media were comparable in their ability to suppress the growth of saprophytic microorganisms. The results of this study demonstrated that the use of NYC medium markedly enhanced the recovery of N. gonorrhoeae from clinical specimens as compared to ML medium.     

Multiple selective media for the isolation of anaerobic bacteria from clinical specimens.

Using fresh clinical material, a comparison of a number of anaerobic selective media was made. For Gram-negative anaerobes nalidixic acid tween agar (NAT), neomycin agar (NA), and neomycin-vancomycin agar (NVA) all performed equally well. Kanamycin-containing media were more inhibitory to all Gram-negative anaerobes other than Bacteroides fragilis and B. melaninogenicus. When the recovery of Gram-positive anaerobes was examined NAT performed better than any of the other selective media used. No single selective medium could recover all anaerobes. Better isolation was achieved using a combination of two selective media (the best combinations being NAT and NVA or NAT and NA). Only a combination of three selective media gave the maximum recovery of anaerobes in this study (NAT, NVA, and NA or KA).     

Effect of off-site transportation on detection of Neisseria gonorrhoeae in endocervical specimens

OBJECTIVES: To evaluate both the effect of off-site transportation on detection of Neisseria gonorrhoeae in cultured endocervical specimens and the impact of transportation on viability of N. gonorrhoeae by comparison of culture with a nucleic acid probe assay. DESIGN: Three endocervical swabs were randomly collected; one was tested on-site using a nucleic acid-based assay (PACE 2NG System, Gen-Probe, Inc, San Diego, Calif), one was tested off-site following inoculation to modified Thayer-Martin agar (Remel, Lenexa, Kan), and a third swab was tested on-site by culture isolation. A nucleic acid amplification assay of the original swab for PACE 2NG testing was used to resolve discrepancies. SETTING: The emergency department of a university medical center. PATIENTS: Four hundred two patients were evaluated. The test population consisted of both asymptomatic and symptomatic patients. MAIN OUTCOME MEASURE: Positivity for N. gonorrhoeae by one or more of the test procedures, with discrepancy analysis when warranted. RESULTS: Of 402 specimens evaluated, the sensitivities for on-site and off-site testing using culture isolation for N. gonorrhoeae were 88.9% and 77.8%, respectively, in a population prevalence of 6.7%. However, the sensitivity for on-site PACE 2NG testing for N. gonorrhoeae was 96.3%. CONCLUSIONS: A decrease in sensitivity between on-site and off-site culture was found, which suggested transportation may have an adverse effect on the detection of N gonorrhoeae. However, with the limited population and prevalence, the difference was not found to be statistically significant. Further studies indicated that the nucleic acid probe assay was significantly more sensitive (P = .05) when compared with off-site testing using a culture isolation method, demonstrating that viability is an important consideration. These results suggested that a molecular probe assay should be considered in testing specimens for N. gonorrhoeae, especially when the specimen is to be transported off-site.     

Comparison of commercially available New York City medium and Martin-Lewis medium for recovery of Neisseria gonorrhoeae from clinical specimens

In 100 clinical specimens containing Neisseria gonorrhoeae, commercially prepared New York City medium in JEMBEC plates was superior to Martin-Lewis medium for recovery of gonococci in 49 specimens, equal to Martin-Lewis medium in 43 specimens, and less effective in 8 specimens.     

Immunogenicity of ribosomal preparations from Neisseria gonorrhoeae.

Protection against gonococcal infection was obtained by immunization with ribosomal preparations from Neisseria gonorrhoeae. Ribosomes were isolated from disrupted cells by differential ultracentrifugation and treatment of the microsomal fraction with 0.25% sodium dodecyl sulfate. The isolated ribosomal preparations contained 55% ribonucleic acid, 39% protein, and 0.35% carbohydrate. The ribosomal preparations contained small amounts of endotoxin as determined by thiobarbituric acid- and lead acetate-sensitized mice assays. Guinea pigs immunized subcutaneously with ribosomal preparations were challenged intrachamberially with 10(7) colony-forming units of N. gonorrhoeae, and protection was assessed by clearance of the organism from subcutaneous chambers. The ribosomal preparations elicited significant protection, which was enhanced by incoporation of the immunogen into adjuvant. This protection was comparable to that obtained with whole cells. Treatment with proteolytic enzymes destroyed the protective effect of the ribosomal preparations, but ribonuclease had no measurable effect. Passive hemagglutination and immunodiffusion tests with sera from immunized animals demonstrated the presence of antibody to the ribosomal antigens. Results of adsorption of antiribosomal sera with enzyme-treated ribosomal preparations also indicated the protein nature of the immunogen. These results indicate that protein associated with the gonococcal ribosomal preparation is the major protective immunogen. The role of endotoxin contamination in the immunogenicity of gonococcal ribosomal preparations warrants further investigation.