Multiplex real-time PCR assays for the prediction of cephalosporin, ciprofloxacin and azithromycin antimicrobial susceptibility of positive Neisseria gonorrhoeae nucleic acid amplification test samples.

BACKGROUND: The incidence of antimicrobial-resistant Neisseria gonorrhoeae (GC) is rising in Canada; however, antimicrobial resistance (AMR) surveillance data are unavailable for infections diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs), representing over 80% of diagnoses. We developed a set of 10 improved molecular assays for surveillance of GC-AMR and prediction of susceptibilities in NAAT specimens. METHODS: Multiplex real-time PCR (RT-PCR) assays were developed to detect SNPs associated with cephalosporin (ponA, porB, mtrR -35delA, penA A311V, penA A501, N513Y, G545S), ciprofloxacin (gyrA S91, parC D86/S87/S88) and azithromycin [23S (A2059G, C2611T), mtrR meningitidis-like promoter] resistance. The assays were validated on 127 gonococcal isolates, 51 non-gonococcal isolates and 50 NAATs with matched culture isolates. SNPs determined from the assay were compared with SNPs determined from in silico analysis of WGS data. MICs were determined for culture isolates using the agar dilution method. RESULTS: SNP analysis of the 50 NAAT specimens had 96% agreement with the matched culture RT-PCR analysis. When compared with MICs, presence of penA A311V or penA A501 and two or more other SNPs correlated with decreased susceptibility and presence of three or more other SNPs correlated with intermediate susceptibility to cephalosporins; presence of any associated SNP correlated with ciprofloxacin or azithromycin resistance. NAAT-AMR predictions correlated with matched-culture cephalosporin, ciprofloxacin and azithromycin MICs at 94%, 100% and 98%, respectively. CONCLUSIONS: We expanded molecular tests for N. gonorrhoeae AMR prediction by adding new loci and multiplexing reactions to improve surveillance where culture isolates are unavailable.

Estimated prevalence of Hepatitis C Virus infection in Canada, 2011.

BACKGROUND: Prevalence estimates contribute to our understanding of the magnitude of a particular health condition and in planning appropriate public health interventions. OBJECTIVE: To estimate the prevalence of chronic Hepatitis C virus (HCV) infection, anti-HCV-positive status (anti-HCV) and the proportion of undiagnosed HCV infections in Canada. METHODS: A combination of back-calculation and workbook methods was used. The back-calculation method estimated prevalent chronic HCV infection and the proportion undiagnosed using the Canadian Cancer Registry's data on hepatocellular carcinoma reported between 1992 and 2008 and the Canadian Notifiable Disease Surveillance System's data on Hepatitis C virus (HCV) cases reported between 1991 and 2009 in a Markov multi-state disease progression model with parameters adjusted to Canada. The workbook method divided the total population of Canada into population subsets and developed estimates of population size and anti-HCV prevalence for each. Sub-population size estimates were multiplied by anti-HCV prevalence measures to calculate the prevalence of anti-HCV by sub-population. A measure of spontaneous clearance was used to estimate the number of persons with chronic HCV from estimates of the number of anti-HCV-positive persons. RESULTS: The back-calculation method estimated the prevalence of chronic HCV infection at 0.64% and the proportion of undiagnosed chronic HCV infection at 44% in 2011. The workbook method estimated the anti-HCV prevalence at 0.96% (plausibility range: 0.61% to 1.34%) and chronic HCV infection at 0.71% (0.45 - 0.99%). INTERPRETATION: By combining mid-point estimates from both methods, it is estimated that between 0.64% to 0.71% of the overall Canadian population was living with chronic HCV infection in 2011 and 44% of these individuals were undiagnosed.