Simple Method for Detection of Penicillinase-Producing Neisseria gonorrhoeae

A simple, reliable, and economical method for detection of penicillinase-producing Neisseria gonorrhoeae using a penicillin disk and a penicillin-sensitive organism is described.     

Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae

In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.     

Strain Typing and Antimicrobial Resistance of Fluoroquinolone-Resistant Neisseria gonorrhoeae Causing a California Infection Outbreak

Antimicrobial-resistant Neisseria gonorrhoeae is an emerging public health problem as a result of the alarming limitation in treatment options. We examined an outbreak in California of fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) by evaluation of a combination of routine isolates from the Gonococcal Isolate Surveillance Project and isolates collected by expanded surveillance performed between April 2000 and June 2002. QRNG isolates were characterized by two methods: (i) determination of a combination of antibiogram, auxotype, serovar, Lip type, and patterns of amino acid alteration in the quinolone resistance-determining region of GyrA and ParC (ASLGP) and (ii) pulsed-field gel electrophoresis (PFGE). Strain typing was used to describe the QRNG outbreak strains and the associated antimicrobial resistance profiles. Among 79 isolates that were completely characterized, we identified 20 different ASLGP strain types, and 2 of the types were considered to belong to outbreak strains that comprised 65% (51/79) of the isolates. By PFGE typing, there were 24 different strain types, and 4 of these were considered outbreak types and comprised 66% (52/79) of the isolates. The overall agreement between the typing methods in distinguishing outbreak strains and non-outbreak strains was 84% (66/79). The most common QRNG ASLGP strain type had chromosomally mediated resistance to penicillin and tetracycline and an azithromycin MIC of 0.5 μg/ml. The occurrence of an outbreak caused by QRNG strains that could fail to be eradicated by most antibiotic classes reinforces the serious problem with antimicrobial resistance in Neisseria gonorrhoeae that the public health system faces. Adherence to a regimen with the recommended antibiotics at the appropriate dose is critical, and monitoring for antimicrobial susceptibility needs to be actively maintained to adapt treatment guidelines appropriately.Neisseria gonorrhoeae antimicrobial susceptibility in the United States has been monitored by the Gonococcal Isolate Surveillance Project (GISP) at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, since 1986. The need for routine surveillance for antimicrobial susceptibility was established with the emergence of chromosomal resistance to penicillin and tetracycline in Neisseria gonorrhoeae in the United States in the 1970s and 1980s (32). With the development of plasmid-mediated resistance to penicillin and tetracycline, the treatment recommendations were changed to ceftriaxone, with cefixime and ciprofloxacin being oral alternatives (29). Fluoroquinolone-resistant Neisseria gonorrhoeae (QRNG) was first isolated in Hawaii in 1991, but it was not until after 1998 that the frequency of QRNG started to escalate (5). In California, the frequency of QRNG isolates increased from less than 1% in 2000 to 25.4% in 2005 (2). At the onset of the occurrence of QRNG in California, expanded surveillance suggested an outbreak among men who have sex with men (MSM) in Southern California (1). A transmission network was constructed from the epidemiological connections determined from the sexual partnerships and venues where sexual partners met and could be supported by strain typing of the isolates from these cases among MSM (18). In this paper, we report on the complete strain typing and examine the antimicrobial resistance profiles among the isolates identified in this surveillance. This should provide important information on the origins of evolving Neisseria gonorrhoeae antimicrobial resistance in the United States by characterizing the strains that became established on the continent.     

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.     

Molecular Assay for Detection of Genetic Markers Associated with Decreased Susceptibility to Cephalosporins in Neisseria gonorrhoeae

The incidence of antimicrobial-resistant Neisseria gonorrhoeae continues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeae strains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of ≥2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponA and mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.     

Detection of quinolone-resistant Neisseria gonorrhoeae.

The present National Committee for Clinical Laboratory Standards (NCCLS) guideline for testing Neisseria gonorrhoeae quinolone susceptibility defines only a susceptible category for ciprofloxacin, enoxacin, lomefloxacin, and ofloxacin, while susceptible, intermediate, and resistant categories are defined for fleroxacin. To further define the criteria for detection of quinolone resistance in gonococci, by standard disk diffusion and agar dilution methodologies recommended by the NCCLS, we tested 29 strains of quinolone-resistant N. gonorrhoeae (QRNG) recently isolated from ofloxacin-treated patients who were considered clinical failures. Regression analyses were performed on these results together with those of another 20 strains showing reduced susceptibility and 13 fully susceptible strains (ofloxacin MICs of < or = 0.25 microgram/ml). With 5-micrograms ofloxacin disks, resistance in 27 (93.1%) of the QRNG strains (MICs of > 1 microgram/ml) was detected by the criterion of a zone diameter of < 22 mm, while in the remaining 2 (6.9%), the disks failed to detect resistance. A cluster of 15 highly resistant strains showed ofloxacin MICs of > 4 micrograms/ml and zone diameters of < 13 mm. When tested with 5-micrograms ciprofloxacin disks, the corresponding values for resistance and high-level resistance of these QRNG strains were < 25 mm (MICs of > 0.5 micrograms/ml) and < 15 mm (MICs of > 2 micrograms /ml), respectively. Six strains for which ofloxacin MICs were > or = 8 micrograms/ml showed no zones at all with both 5-micrograms ofloxacin and 5-micrograms ciprofloxacin disks. These QRNG strains are now firmly established in the Southeast Asia region, and it is important for clinical laboratories to recognize these clinically resistant strains and to monitor their spread.     

Antimicrobial susceptibility profile of Neisseria gonorrhoeae at STI clinic

A total of 100 consecutive patients who attended a sexually transmitted infections clinic were studied. Thirteen had gonococcal urethritis, of which 10 showed growth of Neisseria gonorrhoeae on culture. All the isolates were tested for antimicrobial susceptibility by Australian Gonococcal Surveillance Programme (AGSP) method and beta lactamase production by chromogenic cephalosporin test. Four patients were co-infected with each of the following: HIV, HBV and Chlamydia trachomatis . Gonococcal urethritis (13%) was found more in male patients. Ten percent gonococcal isolates were penicillinase-producing N. gonorrhoeae , and another 10% were tetracycline-resistant N. gonorrhoeae .     

Evaluation of the Abbott LCx Assay for Detection of Neisseria gonorrhoeae in Endocervical Swab Specimens from Females

The Abbott LCx Neisseria gonorrhoeae assay (Abbott Laboratories, Abbott Park, Ill.) uses a ligase chain reaction (LCR) amplification in the LCx probe system for detection of a specific nucleotide sequence in the Opa-encoding gene of N. gonorrhoeae. We evaluated the LCx assay in a comparison with conventional culture employing modified Thayer-Martin media for the detection of N. gonorrhoeae from female endocervical specimens obtained from patients attending a sexually transmitted disease clinic. Discordantly LCR-positive and culture-negative specimens were further evaluated by testing with another LCR assay which used an N. gonorrhoeae-specific pilin probe. Specimens positive by both LCR assays were considered confirmed LCx-positive specimens. A specimen was considered to contain N. gonorrhoeae when it was either culture positive or culture negative and confirmed LCx positive. A total of 403 female endocervical specimens were evaluated. The prevalence of N. gonorrhoeae in this population was 8.7%. The sensitivity and specificity of the LCx assay were 94.3 and 99.4%, and those of culture were 77.1 and 100%, respectively. The Abbott LCx assay is a rapid, sensitive method for detection of N. gonorrhoeae in female endocervical specimens.     

Molecular Surveillance of Clinical Neisseria gonorrhoeae Isolates in Russia

The choice of adequate methods for epidemiological purposes remains a challenging problem in Neisseria gonorrhoeae molecular monitoring. In this study, the collection of geographically unrelated gonococci (n = 103) isolated in Russian clinics was comparably tested by (i) a traditional serotyping scheme, (ii) por typing, (iii) Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST), and (iv) multilocus sequence typing (MLST). It is shown that, according to sequencing data, a third of the strains carried new porB1 alleles, as well as tbpB ones, and more than half of the samples had new sequence types (STs) as determined by NG-MAST or MLST. The discriminatory power for each typing method was calculated by using the Hunter-Gaston discriminatory index, D. Commonly, modern nucleic acid-based typing methods (por typing, NG-MAST, and MLST) appeared to be more efficient than the classical serotyping scheme. While the traditional serotyping gave a D value of 0.82, the por typing, NG-MAST, and MLST approaches yielded D values of 0.97, 0.98, and 0.91, respectively. Each typing technique revealed the distribution of gonococci slightly correlated with their geographical sources. However, only the MLST method STs were highly associated with certain phenotypes. Although ST1594, ST1892, and ST6720 were typical for susceptible gonococci, ST1901 and ST6716 were undoubtedly associated with a multidrug-resistant phenotype. We conclude that every tested nucleic acid-based typing method is suitable for N. gonorrhoeae molecular surveillance. However, the MLST method seems to serve large-scale epidemiological purposes, whereas the NG-MAST and por typing approaches are more appropriate for the investigation of local outbreaks.The choice of an optimal epidemiological approach for Neisseria gonorrhoeae typing remains a problem for public health control strategies. The worldwide spread of drug-resistant strains requires typing methods to be introduced into a national surveillance programs that are already realized within the Australian Gonococcal Surveillance Programme (33). In Russia, strains that are resistant to penicillin, tetracycline, and fluoroquinolones are common, reaching up to 60% prevalence in some regions (14). As is generally known, most resistance mechanisms in N. gonorrhoeae are linked to mutations in genomic DNA, and a wide dissemination of such mutations has been demonstrated in subjects with gonorrhea in the Russian population (11). In fact, chromosomally mediated drug resistance expands clonally in a bacterial population. Thus, tracing the spread of gonorrhea by using strain differentiation methods is of great importance. In addition, population genetics are important in understanding the evolutionary history, epidemiology, and population dynamics of pathogens.Widespread adoption of the molecular diagnosis of gonorrhea could compromise traditional bacteriological cultivation in routine practice and highlights the need for the development of molecular tools for N. gonorrhoeae typing. Several promising approaches exist in this field. One of them is por typing (7, 8, 27), based on comparative analysis of porB gene nucleotide sequences. It possesses rather high discriminatory power (39) and presents data in a format comparable with serotyping data. It should be mentioned that PorB1 protein plays a key role in the adhesion of gonococci to epitheliocytes and reflects the pathogenicity of isolates. Moreover, in earlier studies the multidrug-resistant phenotype of N. gonorrhoeae was bound to certain serotypes determined in accordance with the antigenic properties of PorB1 proteins (7, 21, 29).Today, the N. gonorrhoeae multiantigen sequence typing (NG-MAST) (19) appears to be a leading method for N. gonorrhoeae typing. It is based on the analysis of internal fragments of two hypervariable genes, porB and tbpB, encoding superficial gonococcus antigens and therefore under positive selection (19). An online database (www.ng-mast.net) collects current information on the sequence types of gonococcus isolates in different regions of the world, and it can be an efficient instrument for objective estimation of the genetic variability of the microbial population and tracing the spread of infection (2). Moreover, an association of the NG-MAST sequence type with the antibiotic resistance profile has been demonstrated by some investigators (20, 26).In addition, NG-MAST, which is usually carried out on DNA extracted from a pure bacterial culture, can be performed directly from noncultured samples such as a piece of clothing (18) or clinical specimens (40). Although some researchers have reported a successful application of NG-MAST to urogenital specimens (urine specimens and swabs from the cervix, urethra, and vagina) and to rectal swabs, this typing method was found to be less suitable for throat swabs due to cross-reaction with commensal Neisseria species. It seems that direct typing schemes utilizing probe hybridization methods (16, 17) for a broad spectrum of clinical samples are more convenient than sequencing.The opposite approach—analysis of conservative, presumably selective neutral housekeeping genes—is taken by multilocus sequence typing (MLST) (1, 38). Possessing a sufficient number of allelic variants and characterized by the slow accumulation of mutations, these genes reflect the natural evolution of the microbial population and common trends in the spread of gonorrhea.Often, the effectiveness of N. gonorrhoeae surveillance depends on the methods used for species identification and the typing of clinical isolates. However, considering the variety of typing systems but the lack of world experience in their application to monitoring gonococci, it has not yet been determined whether there is a unique objective method for revealing the relationships between clinical isolates. The goal of the present study was to evaluate a number of approaches, including por typing, NG-MAST, MLST, and serotyping for typing geographically unrelated gonococcal isolates in Russia.(This study was partially presented during the 16th International Pathogenic Neisseria Conference in 2008 in the Netherlands.)     

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.     

Evaluation of a Strand Displacement Amplification Assay (BD ProbeTec-SDA) for Detection of Neisseria gonorrhoeae in Urine Specimens

The performance of a strand displacement amplification assay (the BDProbeTec-SDA assay) in detecting Neisseria gonorrhoeae in urine specimens was evaluated. When performed under stringent quality control conditions, the BDProbeTec-SDA assay is a sensitive, specific, and efficient method for the screening of large numbers of noninvasively obtained specimens. Because the predictive value of an assay is a function of the prevalence of the disease, culture confirmation is needed for samples with positive results from populations in which the prevalence of a disease is low or in situations in which false-positive results may have important medical, psychosocial, or medicolegal consequences.     

Hen Fluorescein-Labeled Gonococcal Lipopolysaccharide Antibody in the Delayed Fluorescent Antibody Technique for the Confirmation of Neisseria gonorrhoeae

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.     

Utility of Specimens Positive for Neisseria gonorrhoeae by the Aptima Combo 2 Assay for Assessment of Strain Diversity and Antibiotic Resistance

In our jurisdiction, the Aptima Combo 2 assay (Gen-Probe, Inc.) is used to detect Neisseria gonorrhoeae from specimens collected at clinics for sexually transmitted infections (STI) and from select community patients. In addition, swabs are also collected for N. gonorrhoeae culture, susceptibility testing, and sequence typing (ST). Since only a small proportion of samples from provincial cases undergo culture, the available trends in antimicrobial susceptibility and predominant strain types may not be representative of all N. gonorrhoeae infections. Due to the limitations facing the use of N. gonorrhoeae culture to understand these trends in the general community, we performed a molecular analysis for markers of cephalosporin resistance and ST determination by using nucleic acid extracts of specimens sent for Aptima testing. Thirty-four samples submitted for both Aptima testing and N. gonorrhoeae culture from the same anatomic location (within 24 h) were included in the study. Sequence type was determined based on the sequence of the por and tbpB genes, and amino acid changes in the PBP 2 protein, encoded by the penA gene, were considered representative for the assessment of antimicrobial susceptibility. Sequence identity of 100% was observed between the sequences obtained from Aptima-analyzed samples and culture samples. Sequencing results showed an association between decreased susceptibility to extended-spectrum cephalosporins (ESC^ds), tbp allele 110, ST 1407, and amino acid changes (G545S, I312M, and V316T) in the PBP 2 protein. Our data, generated based on a few representative genes, suggest that gonococcal samples positive by Aptima testing can be used to determine single nucleotide polymorphisms associated with ESC^ds and the sequence type based on molecular strain typing. Confirmation of these findings may obviate the need for gonorrhea culture in the future.     

Resistance of mice to genital infection with Neisseria gonorrhoeae

Five strains of mice (C3H, CBA, BALB/c, TO and ICR) were inoculated intra-vaginally with Neisseria gonorrhoeae in an attempt to produce an animal model of gonorrhoea. Of a total of 68 mice inoculated, only three (4.4%) were culture-positive after 3 days. Histological examination of both the genital mucosa of inoculated animals, and the mucosa of genital tract organ cultures inoculated in vitro failed to show any evidence of gonococcal adherence or colonisation. Mice of these strains, therefore, appear resistant to gonococcal infection of the genital tract.     

Antimicrobial susceptibility testing of Neisseria gonorrhoeae and implications for epidemiology and therapy.

Antimicrobial susceptibility testing (AST) of Neisseria gonorrhoeae has been under development since the early days of antimicrobial agents. However, it is rarely applied to clinical isolates today. The history of the various in vitro tests to determine the susceptibility of N. gonorrhoeae to antibiotics is rich with evidence that these results predict response to therapy for almost all agents tested. Further, AST is a useful and important aspect of strain characterization and disease epidemiology in conjunction with the more specific but laborious techniques of auxotyping, serotyping, and plasmid analysis. Current technology has overcome many of the objections to AST for N. gonorrhoeae with standardization of test media and the development of an accurate disk diffusion AST method that is suited to most clinical laboratories regardless of volume or level of technical expertise. Ironically, the very low level of resistance to the current primary treatment strategy in the United States, ceftriaxone or another potent cephalosporin, makes the use of AST somewhat superfluous.     

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR Using cppB Nested PCR and 16S rRNA PCR

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence (~9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.     

Evaluation of the New BD Max GC Real-Time PCR Assay,Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay,for Molecular Detection of Neisseria gonorrhoeae

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection of Neisseria gonorrhoeae.     

Pathogenesis and Immunology of Experimental Gonococcal Infection: Virulence of Colony Types of Neisseria gonorrhoeae for Chicken Embryos

The virulence of gonococcal strains and colony types was evaluated in embryonated hen eggs of various ages inoculated by different routes. Striking differences in virulence of colony types were revealed by intravenous inoculation of 11-day embryos. T1 and T2 colony types were found to have high virulence for embryos, whereas T3 and T4 colony types were relatively avirulent. These observations are in accord with previous studies in volunteers. The differences in virulence were not related to differences in toxicity of killed gonococci, sonic lysates, or to the susceptibility of gonococci to cidal effects of chicken embryo blood. Rather, they appeared to involve differences in clearance of gonococci from the blood stream and subsequent multiplication of the virulent colony types. Infection with virulent colony types appears to be primarily bacteremic in this model. Preliminary experiments indicated that chicken embryos may be protected against the lethal infection by prior treatment of the inoculum with normal and immune rabbit serum. This protective effect was not associated with bactericidal activity. The chicken embryo model is potentially useful as a means of investigating attributes of virulence of gonococci and factors in immunity against gonorrhea.