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

Abstract

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. N eisseria gonorrhoeae is a common sexually transmitted infection that affects mucosal surfaces and causes a spectrum of conditions, including urethritis, endocervicitis, pelvic inflammatory disease, and infertility (1). Over the years, gonococci have developed resistance to multiple classes of antibiotics, including penicillins, tetracyclines, macrolides, and quinolones (2). Of recent concern is a steady increase in the MICs to extended-spectrum cephalosporins, including cefixime and ceftriaxone, in several countries including Canada, with reported levels at 0.12 g/ml, one doubling dilution away from the Clinical and Laboratory Standards Institute (CLSI) limit of 0.25 g/ml for susceptibility (3-7; CLSI standard M100-S23, January 2013). According to a recent U.S. Centers for Disease Control and Prevention (CDC) update, untreatable gonorrhea could soon be a reality in the United States (http://www.cdc.gov/mmwr/preview /mmwrhtml/mm6131a3.htm). The agency noted that N. gonorrhoeae seems to be developing resistance to the oral antibiotic cefixime, and it no longer recommends cefixime at any dose as a firstline regimen for treatment of gonococcal infec...

Chlamydia trachomatisgenotypes in a cross-sectional study of urogenital samples from remote Northern and Central Australia

“…The storage of specimens in Aptima transport medium may have affected typeability. However, it has previously been shown that such storage does not preclude downstream genotyping, 31 and in our study, the lack of correlation between time of storage at 4°C and typeability suggests that the RNA was not progressively degraded.…”

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

“…The mechanisms causing resistance are complex and multifactorial (25)(26)(27). N. gonorrhoeae is highly recombinogenic and naturally competent, allowing for transformation of DNA from other commensal species (28). Target sequences may cross-react with other similar species, thus causing decreased assay specificities.…”

Role of Molecular Biology in Diagnosis and Characterization of Vulvo-Vaginitis in Clinical Practice

“…This may hamper the possibility to identify the ideal antibiotic therapy for a certain strain as well as limit the possibilities for antibiotic surveillance programs, for which reason N. gonorrhoeae cultures are still required. Progressively, however, the genes responsible for antibiotic resistance are being unraveled and hence, tests based on the detection of specific DNA or mRNA of antibiotic resistance determinants are under way to at least provide a prediction of the antibiotic resistance [89][90][91][92][93]. As always, other and unknown mechanisms causing antibiotic resistance can be at hand, and MB techniques will always leave some doubt on the completeness of the diagnosis of resistance.…”