DNA probe confirmatory test for Neisseria gonorrhoeae.

A DNA probe test for the culture confirmation of Neisseria gonorrhoeae in clinical isolates was evaluated with 156 isolates of N. gonorrhoeae and 120 isolates of nongonococcal Neisseria species, organisms representative of other genera within the family Neisseriaceae, and organisms isolated on media selective for N. gonorrhoeae. The 30-min test used a chemiluminescent DNA probe that was homologous to rRNA sequences of N. gonorrhoeae. We report here a specificity and a sensitivity of 100% with the 276 clinical isolates tested, including 43 gonococcal strains that had been misidentified by other methods.     

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.     

Improved transport system for Neisseria gonorrhoeae in clinical specimens.

Protective transport media have to be used to preserve Neisseria gonorrhoeae in clinical specimens during their transit to the laboratory. In this study, a CO2- environment chamber, the Jembec chamber, was used for transport of clinical speciments requiring examination for gonococci. The survival of N. gonorrhoeae present in clinical speciments when placed in Amies charcoal transport medium was compared to their survival when inoculated into Jembec chambers containing either modified Thayer-Martin medium (MTM) or modified New York City transport medium (MNYC). For a period of up to 2 days in transit, the three systems were not significantly different. However, after 3 days in transit, MNYC/Jembec chambers preserved significantly more gonococci than Amies charcoal transport meduim (P less than 0.0001) or MTM/Jembec chambers (P=0.006). MNYC/Jembec chambers withstood 241 miles (386 km) of postal transit during winter months; 80% of the gonococci present in clinical specimens remained viable from 2 to 5 days under these conditions. The CO2 generated by the tablet in the Jembec chamber was suggicient to support the growth of N. gonorrhoeae if the chambers were incubated at 36 C immediately after inoculation. However, if delayed in transit, the chambers had to be incubated in 5 to 10% CO2 to promote the growth of N. gonorrhoeae. MNYC/Jembec chambers provide a selective environment that will protect and maintain the viability of N. gonorrhoeae for extended periods, allowing a reasonable time for postal transit of clinical specimens to the laboratory.     

Use of gen-probe probe competition assay as a supplement to probes for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urogenital specimens.

The potential for development of a cost-effective protocol for selective use of the Gen-Probe probe competition assay (PCA) in conjunction with PACE 2 for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urogenital specimens that would not compromise patient care was investigated. To accomplish this, PCA data from testing performed over 12 months were retrospectively reviewed. Of 237 samples that were presumptively positive for C. trachomatis by initial probe assay and could be tested by PCA, positive PCA results were obtained for 100, 79, and 59%, respectively, of specimens that gave a signal of more than 1,500, 1,000 to 1,500, and less than 1,000 relative light units (RLU). For the 141 specimens that were presumptively positive for N. gonorrhoeae and could be tested by PCA, positive PCA results were obtained for 99, 80, and 42%, respectively, of samples with a signal of more than 1,500, 1,000 to 1,500, and less than 1,000 RLU. These data indicate that PCA should be a routine supplement to Gen-Probe PACE 2 for specimens with an initial signal by probe assay of less than 1,500 RLU and may not be necessary for samples yielding a signal of more than 1,500 RLU.     

Evaluation of a non-radioactive DNA probe for confirmatory identification of Neisseria gonorrhoeae

A biotinylated DNA probe combined with a streptavidin-peroxidase complex for the identification of culture isolates of N. gonorrhoeae (Ortho diagnostic systems, Neckargemünd, FRG) was compared with the conventional carbohydrate utilisation test as reference. All 118 strains identified by the reference method as N. gonorrhoeae also gave positive reactions with the DNA hybridisation assay. However, with this test 2 of 23 non-gonococcal Neisseria or Branhamella species were identified as N. gonorrhoeae as well. The study shows that the DNA hybridisation technique can principally be used for the confirmatory identification of N. gonorrhoeae, but since specificity is particularly essential for confirmatory identification, the DNA hybridisation assay evaluated cannot be recommended for routine diagnosis.     

Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens.

We developed a multiplex PCR (M-PCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. M-PCR employed C. trachomatis-specific primers KL1-KL2 and N. gonorrhoeae-specific primers HO1-HO3 and produced products of 241 and 390 bp, respectively. PCR products were easily detected by agarose gel electrophoresis and confirmed by Southern hybridization using labelled oligonucleotide probes. M-PCR had a sensitivity of 10 fg of C. trachomatis and N. gonorrhoeae DNA (equivalent to 1 to 2 genome copies). M-PCR detected the presence of C. trachomatis and N. gonorrhoeae DNA in 15 male urethral and 12 female endocervical specimens, 3 of which were positive for C. trachomatis, 18 of which were positive for N. gonorrhoeae and 6 of which were positive for both organisms. M-PCR was evaluated further by testing 200 male first void urine (FVU) specimens, of which 18 were positive by C. trachomatis PCR and Chlamydiazyme and 4 were positive by C. trachomatis PCR but negative by Chlamydiazyme. All 22 FVU specimens were positive by a confirmatory PCR using a second plasmid target and were positive by M-PCR. Ten of 11 men with cultures that were positive for N. gonorrhoeae had FVU specimens that were positive by both N. gonorrhoeae PCR and M-PCR. Two other men with negative N. gonorrhoeae urethral cultures had FVU specimens that were positive by N. gonorrhoeae PCR, by two confirmatory N. gonorrhoeae PCR assays using 165 rRNA and cytosine methyltransferase primers, and by M-PCR. The sensitivity of M-PCR for detecting C. trachomatis was 100% (22 of 22 specimens), compared with 81.8% (18 of 22 specimens) for enzyme immunoassay. Sensitivity of M-PCR for N. gonorrhoeae was 92.3% (12 of 13 specimens) compared with 84.6% (11 of 13 specimens) for urethral culture. The specificity of M-PCR was 100% for both C. trachomatis (178 of 13 specimens) and N. gonorrhoeae (187 of 187 specimens). M-PCR testing of FVU specimens provided a sensitive and noninvasive method for detecting C. trachomatis and N. gonorrhoeae infection in men.     

Comparison of Gram stain with DNA probe for detection of Neisseria gonorrhoeae in urethras of symptomatic males.

The comparison of Gram-stained urethral smears with Gen-Probe for the detection of Neisseria Gonorrhoeae in the urethras of males with symptomatic urethritis revealed a 99.6% correlation between the two methods. A simple Gram stain would appear to be the method of choice for the detection of gonorrhea in symptomatic males, because it is much less expensive and much more rapid than the Gen-Probe method.     

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis in genitourinary specimens from men and women by a coamplification PCR assay.

A coamplification PCR test for the direct detection of Neisseria gonorrhoeae and Chlamydia trachomatis in urethral and endocervical swabs and urine samples from men and women was compared to standard culture techniques. Processed specimens were amplified in single reaction tubes containing primers for both organisms, and PCR products were detected by a colorimetric microwell plate hybridization assay specific for each pathogen. Of 344 specimens from men, 45 (13.1%) urine specimens were PCR positive for C. trachomatis, 51 (14.8%) urethral swab specimens were PCR positive, and 29 urethral swab specimens (8.4%) were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis were 96.2 and 99.3%, respectively, in urethral swab specimens, compared to 88.2 and 98.6% for urine specimens. Of the 192 specimens from women, 28 (14.6%) urine specimens were PCR positive for C. trachomatis, 32 (16.7%) endocervical specimens were PCR positive, and 19 (9.9%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for C. trachomatis for endocervical specimens were both 100% compared to 100 and 99.4%, respectively, for urine specimens from women. In men, 68 (19.8%) urine specimens were PCR positive for N. gonorrhoeae, 73 (21.2%) urethral swabs were PCR positive, and 59 (17.2%) urethral swabs were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 97.3 and 97.0%, respectively, for urethral specimens compared to 94.4 and 98.5% for urine specimens. In women, 18 (9.4%) urine specimens were PCR positive for N. gonorrhoeae, 23 (12.0%) were endocervical swab PCR positive, and 15 (7.8%) endocervical specimens were culture positive. After analysis of discrepancies, the resolved sensitivity and specificity of PCR for N. gonorrhoeae were 100 and 99.4%, respectively, for endocervical specimens compared to 90.0 and 95.9% for female urine specimens. These results indicate that a multiplex PCR is highly sensitive for detecting both C. trachomatis and N. gonorrhoeae from a single urine or genital swab, providing a more cost-effective way of screening multiple pathogens.     

Evaluation of a biotinylated probe for identification of Neisseria gonorrhoeae

We evaluated a DNA biotinylated probe for the rapid identification of Neisseria gonorrhoeae in culture (Ortho Diagnostics System, Raritan, NJ). Twenty-one strains of N. gonorrhoeae, including type strain, 57 strains of other Neisseria species, and 104 strains of other genera were studied. The probe was highly sensitive (100%) and specific (96%). All N. gonorrhoeae strains gave strong signals, and only two cross-reaction were observed with N. lactamica, which has a close genetic relationship to N. gonorrhoeae. Our results indicate that specific recombinant DNA probe should offer a reliable and rapid method for routine diagnosis of Neisseria gonorrhoeae.     

Evaluation of an rRNA-derived oligonucleotide probe for culture confirmation of Neisseria gonorrhoeae.

The reliability of an rRNA-derived oligonucleotide probe for Neisseria gonorrhoeae was tested with 187 N. gonorrhoeae isolates, 81 Neisseria meningitidis isolates, and several strains of other bacterial species. The probe proved to be 100% specific and 100% sensitive. N. gonorrhoeae cells could also be reliably identified in contaminated cultures with the oligonucleotide probe. The 2.6-megadalton cryptic plasmid used as a probe for N. gonorrhoeae was shown to be less sensitive, detecting 179 of 181 N. gonorrhoeae isolates.     

Validation of roche COBAS Amplicor assay for detection of Chlamydia trachomatis in rectal and pharyngeal specimens by an omp1 PCR assay

Screening guidelines for men who have sex with men (MSM) recommend testing of extragenital sites (pharyngeal and rectal) for gonorrhoea and chlamydia. Testing of specimens from these sites is not validated by most commercial nucleic amplification tests, such as the COBAS Amplicor assay. To investigate the utility of the COBAS Amplicor assay for detection of Chlamydia trachomatis in extragenital specimens, this study developed and evaluated confirmatory tests using the omp1 gene as an alternative target for amplification by PCR. Of anal and throat swabs collected from men in male-only saunas, 52 swabs that tested C. trachomatis positive by COBAS Amplicor and 30 swabs that tested as negative were included for confirmatory omp1 PCR testing. A total of 49 (94%) COBAS Amplicor-positive samples were confirmed by the omp1 PCR. A substantial proportion of specimens were confirmed by using a nested omp1 PCR (27%). Not confirmed by any omp1 PCR were three anal swabs (6%). It is most probable that these samples contained lower bacterial levels that were near or below the detection level of the omp1 PCR assays. The findings of this study support the confident reporting of C. trachomatis detected by COBAS Amplicor in extragenital specimens and support the utility of this assay as a screening test for MSM.     

Duplex PCR system for simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical specimens.

AIMS--To evaluate the use of a duplex polymerase chain reaction (PCR) assay for the simultaneous detection of Neisseria gonorrhoeae and Chlamydia trachomatis in clinical samples. METHODS--Genital swab specimens were obtained from both China (203 swabs) and Hong Kong (202 swabs). N gonorrhoeae and C trachomatis were detected in each specimen with a number of tests including enzyme immunoassays (IDEIA) and PCR assays using both single and double primer pairs. The primer pair for N gonorrhoeae was derived from the cppB gene on its cryptic plasmid and the PCR product was 390 base pairs long. For C trachomatis, the PCR product was 473 base pairs long, resulting from amplification of a sequence in the common 7.4 kilobase plasmid present in all serovars. For N gonorrhoeae, PCR results were also compared with those obtained by culture and Gram's smear of the discharges. RESULTS--For the 203 specimens collected in China, similar numbers of positive results (177) were obtained by both Gonozyme and duplex PCR for the detection of N gonorrhoeae. No discrepant results were found among the cultured specimens when Gonozyme and duplex PCR were compared. C trachomatis was detected in 47 specimens by duplex PCR, but was detected in only 28 by IDEIA. Of the 202 Hong Kong specimens, 46 were positive for N gonorrhoeae, detected by both Gonozyme and duplex PCR; 34 were positive for C trachomatis, 25 of which were detected by IDEIA and the remainder by duplex PCR. CONCLUSIONS--The duplex PCR assay is a satisfactory diagnostic tool for the simultaneous detection of N gonorrhoeae and C trachomatis in clinical swab samples. Further evaluation is suggested.     

Comparison of mismatch amplification mutation assay with DNA sequencing for characterization of fluoroquinolone resistance in Neisseria gonorrhoeae

A mismatch amplification mutation assay (MAMA) was developed for identification of point mutations in quinolone resistance-determining region (QRDR) of gyrA at codons 91 and 95. MAMA PCR was used to detect mutations at codons 91 and 95 of gyrA in 117 Neisseria gonorrhoeae isolates (with ciprofloxacin MICs of 0.004 to >32 microg/ml) from Bangladesh during 1997 to 2001. The QRDR regions of the gyrA genes from 31 randomly selected isolates were sequenced, and the results were compared with those of MAMA PCR. Using mismatch PCR, a mutation at Ser91 could be detected in all 27 (resistant and intermediate) isolates, and an Asp95-to-Gly95 mutation could be detected in all 15 isolates, as detected by sequencing. MAMA PCR offers a simple, inexpensive, rapid, and easier alternative for detection of point mutations in fluoroquinolone resistance in N. gonorrhoeae.     

Direct detection of Mycobacterium tuberculosis complex DNA and rifampin resistance in clinical specimens from tuberculosis patients by line probe assay

The INNO-LiPA.Rif TB test (LiPA) has only been applied to a limited number of clinical specimens. To assess the utility of this test for detecting Mycobacterium tuberculosis complex DNA and rifampin (RMP) resistance, 420 sputum samples comprising specimens from untreated (n=160) and previously treated (n=260) patients from 11 countries in Asia, Africa, Europe, and Latin America were tested. DNA was extracted from sputum samples by using a modification of the Boom's method, while the rpoB core region was amplified by nested PCR. The results were analyzed in conjunction with those obtained by Ziehl-Neelsen (ZN) microscopy and by culture on solid media. The LiPA test was positive for M. tuberculosis complex DNA in 389 (92.9%) specimens, including 92.0% (286 of 311) ZN-positive and 94.5% (103 of 109) ZN-negative specimens. Of these, 30.6% were RMP resistant. In contrast, 74.3% of the specimens were positive for M. tuberculosis by culture, and 30.8% of them were RMP resistant. LiPA detected M. tuberculosis complex DNA in 92.4% (110 of 119) of the culture-positive and 100.0% (41 of 41) of the culture-negative specimens from untreated patients. There was a 99.6% concordance between the RMP resistance as determined by culture and by the LiPA test. With an optimal DNA extraction method, LiPA allows rapid detection of M. tuberculosis complex DNA and RMP resistance directly from sputum specimens. LiPA can still provide useful information when culture fails for various reasons. The rapid availability of this information is necessary to adjust patient treatment and avoid the risk of amplification of drug resistance.     

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.     

Organism resembling Neisseria gonorrhoeae and Neisseria meningitidis.

A problem isolate resembling Neisseria gonorrhoeae and Neisseria meningitidis is reported. Growth and biochemical characteristics indicated the organism to be N. meningitidis, whereas serological characteristics indicated it to be N. gonorrhoeae. This vaginal isolate may be a genetically transformed gonococcus with the ability to utilize maltose. Conversely, it may be a meningococcus which has acquired antigenic determinants of N. gonorrhoeae.     

Rapid in situ generation of DNA restriction endonuclease patterns for Neisseria gonorrhoeae.

Restriction endonuclease (RE) digestion patterns of 26 isolates of Neisseria gonorrhoeae representing different serovars of serogroups WI, WII, and WIII were generated by agarose pellet entrapment and in situ digestion with HinfI and BglII. The method was fast, simple, and reproducible, and stable RE patterns were produced from subsequent in vitro passages. The cost of culture materials was reduced considerably, and no toxic or flammable solvents needed to be used. Excellent resolution of DNA fragments of higher molecular weights was obtained as a result of minimal mechanical shearing of the DNA. The REs HinfI and BglII were discriminative in the fragment length ranges of 2 to 6.5 and 2.5 to 21.5 kilobases, respectively. On the basis of densitometric scanning of electrophoretograms generated by HinfI digestion, the 26 isolates representing 12 serovars were divided into seven groups. BglII was found to be more discriminative; 15 RE patterns were established among the 26 isolates. Patterns generated by both REs showed that there was no correlation between a particular RE pattern and a serovar, since strains with identical RE patterns were from different serovars. With the exception of two strains (D3 and D14), which demonstrated positive correlation when both enzymes were used, all strains with identical serovar patterns had different RE patterns.     

Real-time PCR assay for detection of quinolone-resistant Neisseria gonorrhoeae in urine samples

A need exists for the development of applicable surveillance tools to detect fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) in urine samples. We describe here a real-time PCR assay for detecting mutations in the Ser91 codon of the gyrA gene of N. gonorrhoeae in urine specimens. We tested 96 urine samples collected along with Gonorrhea Isolate Surveillance Project (GISP) urethral swab samples and compared the results with matched MICs of ciprofloxacin, as reported by the regional GISP laboratory. We then tested 100 urine specimens, known to be gonorrhea positive by nucleic acid amplification testing, provided by females to challenge the real-time PCR assay with urine specimens containing potentially less target DNA content than specimens from symptomatic males. With an MIC threshold of 0.125 mug of ciprofloxacin/ml, our assay correctly identified resistance in 41 of 44 (93.2%; 95% confidence interval [CI] = 81.3 to 98.6%) corresponding resistant culture specimens and correctly identified 51 of 51 (100%; 95% CI = 93.0 to 100%) susceptible specimens. One specimen did not amplify. The assay successfully amplified the gyrA amplicon and determined a susceptibility genotype in 72 of 100 (72%) urine specimens collected from female patients. We developed an assay for detecting QRNG in urine specimens that correlated well with MIC results of cultured specimens and had moderate sensitivity with urine specimens. This methodology might fulfill the need for a QRNG detection system for urine specimens, a useful characteristic in the age of nucleic acid amplification testing for gonococcal infection.     

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.