Dataset Information

Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae.

ABSTRACT: Multidrug-resistant gonorrhea has become an urgent issue for global public health. As the causative agent of gonorrhea, Neisseria gonorrhoeae, has been progressively developing resistance to nearly all prescribed antimicrobial drugs, monitoring its antimicrobial resistance on a broader scale has become a crucial agenda for effective antibiotic stewardship. Unfortunately, gold standard antimicrobial susceptibility testing (AST) relies on time and labor-intensive phenotypic assays, which lag behind the current diagnostic workflow for N. gonorrhoeae identification based on nucleic acid amplification tests (NAAT). Newer assay technologies based on NAAT can rapidly identify N. gonorrhoeae from clinical specimen but fundamentally lack the capacity to provide phenotypic AST information. Herein, we propose a direct-quantitative PCR (direct-qPCR) assay that enables pathogen-specific identification and phenotypic AST via quantitative measurement of N. gonorrhoeae growth directly from a liquid medium without any sample preprocessing. The assay has an analytical sensitivity of 102 CFU/mL and is highly specific to N. gonorrhoeae in the presence of urogenital flora and clinical swab eluent. We tested seven N. gonorrhoeae strains against three antibiotic agents, penicillin, tetracycline, and ciprofloxacin, and achieved 95.2% category agreement and 85.7% essential agreement with the FDA-approved E-test. The assay presented in this work has the unique ability to identify N. gonorrhoeae and provide phenotypic AST directly from the liquid medium with cell densities as low as 102 CFU/mL, demonstrating an accelerated, sensitive, and scalable workflow for performing both identification and AST of N. gonorrhoeae.

SUBMITTER: Chen L

PROVIDER: S-EPMC6138521 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae.

Chen Liben L Shin Dong Jin DJ Zheng Shuyu S Melendez Johan H JH Gaydos Charlotte A CA Wang Tza-Huei TH

ACS infectious diseases 20180730 9

Multidrug-resistant gonorrhea has become an urgent issue for global public health. As the causative agent of gonorrhea, Neisseria gonorrhoeae, has been progressively developing resistance to nearly all prescribed antimicrobial drugs, monitoring its antimicrobial resistance on a broader scale has become a crucial agenda for effective antibiotic stewardship. Unfortunately, gold standard antimicrobial susceptibility testing (AST) relies on time and labor-intensive phenotypic assays, which lag behin ...[more]

PMID: 29999304

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Project description:OBJECTIVE:To investigate the in vitro antimicrobial susceptibility and resistant trends of Neisseria gonorrhoeae strains isolated in Guangzhou, from 1996 to 2001. METHODS:The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) to four antimicrobials, penicillin G, ciprofloxacin, ceftriaxone, and spectinomycin. The resistance of all strains to four antibiotics was interpreted according to criteria used in the project of surveillance of gonococcal antibiotic susceptibility in the WHO Western Pacific Region. Penicillinase producing N gonorrhoeae (PPNG) was analysed by the paper acidometric method. RESULTS:793 consecutive N gonorrhoeae isolates collected in Guangzhou were studied from 1996 to 2001. A total of 55 strains of PPNG were identified and the prevalence rapidly spread from 2% to 21.8%. Of the four antibiotics examined, ceftriaxone and spectinomycin appeared to be the most effective agents although two spectinomycin resistant strains were isolated in 1996. Their MIC(50), MIC(90), and geometric mean MIC (MICmean) were all between the sensitive ranges of the interpretative criteria and remained stable over the years. However, resistance increased continuously to penicillin G and dramatically to ciprofloxacin. In 1996-2001, MIC(50), MIC(90), and MICmean of penicillin G increased from 1 micro g/ml to 2 micro g/ml, 4 micro g/ml to 32 micro g/ml, and 0.68 micro g/ml to 2.35 micro g/ml, respectively; those of ciprofloxacin steeply increased from 0.12 micro g/ml to 4 micro g/ml, 2 micro g/ml to 32 micro g/ml, and 0.14 micro g/ml to 2.62 micro g/ml in 1996-9, respectively, and then declined slightly in 2000-1. The prevalence of resistant isolates spread from 57.2% to 81.8% for penicillin G and from 17.6% to 72.7% for ciprofloxacin over the 6 years. CONCLUSION:Resistance to penicillin and ciprofloxacin increased greatly during 1996-2001. Ceftriaxone and spectinomycin should be used as the first line agents in treating gonorrhoea. It is of great importance to continuously survey the susceptibilities of N gonorrhoeae to antibiotics in controlling the spread of gonococcal infections.

Project description:Antimicrobial resistance (AMR) and genetic determinants of resistance of N. gonorrhoeae isolates from Hefei, China, were characterized adding a breadth of information to the molecular epidemiology of gonococcal resistance in China.126 N. gonorrhoeae isolates from a hospital clinic in Hefei, were collected between January, 2014, and November, 2015. The minimum inhibitory concentration (MIC) of N. gonorrhoeae isolates for seven antimicrobials were determined by the agar dilution method. Isolates were tested for mutations in penA and mtrR genes and 23S rRNA, and also genotyped using N. gonorrhoeae multi-antigen sequence typing (NG-MAST).All N. gonorrhoeae isolates were resistant to ciprofloxacin; 81.7% (103/126) to tetracycline and 73.8% (93/126) to penicillin. 39.7% (50/126) of isolates were penicillinase producing N. gonorrhoeae (PPNG), 31.7% (40/126) were tetracycline resistant N. gonorrhoeae (TRNG) and 28.6% (36/126) were resistant to azithromycin. While not fully resistant to extended spectrum cephalosporins (ESCs), a total of 14 isolates (11.1%) displayed decreased susceptibility to ceftriaxone (MIC ? 0.125 mg/L, n = 10), cefixime (MIC ? 0. 25 mg/L, n = 1) or to both ESCs (n = 3). penA mosaic alleles XXXV were found in all isolates that harbored decreased susceptibility to cefixime, except for one. Four mutations were found in mtrR genes and mutations A2143G and C2599T were identified in 23S rRNA. No isolates were resistant to spectinomycin. Gonococcal isolates were distributed into diverse NG-MAST sequence types (STs); 86 separate STs were identified.N. gonorrhoeae isolates from Hefei during 2014-2015, displayed high levels of resistance to antimicrobials that had been recommended previously for treatment of gonorrhea, e.g., penicillin, tetracycline and ciprofloxacin. The prevalence of resistance to azithromycin was also high (28.6%). No isolates were found to be fully resistant to spectinomycin, ceftriaxone or cefixime; however, 11.1% isolates, overall, had decreased susceptibility to ESCs.

Project description:BackgroundA variety of techniques are available for antimicrobial susceptibility testing of Neisseria gonorrhoeae.ObjectiveThe aim of this study was to find a cost-effective, reliable and easily applicable microbiological method to detect antimicrobial susceptibilities of N. gonorrhoeae in resource-poor countries.DesignProspective study.SettingMale and female STD clinic of Regional STD Teaching, Training and Research Centre, New Delhi, India.ParticipantsN. gonorrhoeae isolates from all male and female patients presenting with acute gonococcal urethritis and cervical discharge.Material and methodsA total of 295 consecutive N. gonorrhoeae isolates during 2005-2010 was used to compare the Clinical and Laboratory Standards Institute (CLSI) and CDS disc diffusion technique with Etest by performing antimicrobial susceptibility testing in parallel for penicillin, tetracycline, ceftriaxone, ciprofloxacin and spectinomycin. WHO reference strains were used as controls.ResultsCDS disc diffusion zones of inhibition showed that complete percentage agreement for penicillin, ciprofloxacin and tetracycline was high with their analogous Etest minimal inhibitory concentrations in comparison to CLSI disc diffusion technique, that is, 91.5%, 92.9% and 99.3% versus 87.5%, 88.5% and 74.9%, respectively. CDS results had less number of major and minor category discrepancies in comparison to CLSI and CDS method showed excellent correlation coefficient (r=1) with Etest for all five antimicrobial agents tested in comparison to CLSI (r=0.92). It was very poor (r=0.61) by CLSI method for tetracycline. The correlation coefficients between the two methods and the Etest were identical if tetracycline was removed from the CLSI analysis.ConclusionsThe CDS technique is an attractive alternative for N. gonorrhoeae susceptibility testing and is recommended for monitoring the antimicrobial susceptibility in less developed and resource-poor settings to facilitate enhanced antimicrobial resistance surveillance when the WHO Gonococcal Antimicrobial Surveillance Programme is undergoing expansion to meet the ongoing challenges of surveillance and control of gonococcal antimicrobial resistance.

Dataset Information

Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae.

Publications

Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae.

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