A real-time PCR assay for direct characterization of the<i>Neisseria gonorrhoeae</i>GyrA 91 locus associated with ciprofloxacin susceptibility

Buckley, Cameron; Trembizki, Ella; Donovan, Basil; Chen, Marcus Y; Freeman, Kate; Guy, Rebecca; Kundu, Ratan; Lahra, Monica M; Regan, David G.; Smith, Helen; Whiley, David M.

doi:10.1093/jac/dkv366

Cited by 28 publications

(17 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, due to the increasing antimicrobial resistance in NG, a POCT that provides a diagnostic result simultaneously with antibiotic resistance information would be ideal. The feasibility and accuracy of NAATs to determine antimicrobial resistance has been reported recently 29. Such a test would provide LMICs with antimicrobial resistance data which had previously been limited and also enable individualised treatment, allowing recycling of antibiotics such as ciprofloxacin in areas where ceftriaxone treatment is recommended 30…”

Section: Discussionmentioning

confidence: 97%

Performance and operational characteristics of point-of-care tests for the diagnosis of urogenital gonococcal infections

Guy¹,

Causer

Klausner

et al. 2017

Sex Transm Infect

View full text Add to dashboard Cite

background In 2012, there was an estimated 78 million new cases of gonorrhoea globally. Untreated infection may lead to reproductive and neonatal morbidity and facilitate HIV transmission. Diagnosis and treatment are a priority for control and prevention, yet use of point-of-care tests (POCTs) for Neisseria gonorrhoeae (NG) is limited. Objectives To review the performance and operational characteristics of NG POCTs for diagnosis of urogenital gonorrhoea. Methods We compiled and synthesised findings from two separate systematic reviews which included evaluations published until August 2015. results Six tests were included: five were immunochromatographic tests (ICTs) or optical immunoassay (OIAs) based on antigen detection; with 5-7 steps and results in 25-40 min, and one (GeneXpert CT/NG) was a 'near-patient test' based on nucleic acid amplification technique (NAAT); with three steps, electricity required, and results in 90 min. When compared with laboratory-based NAATs as the reference tests, sensitivities of ICT and OIA-based POCTs ranged from 12.5% to 70% when cervical/vaginal swabs were tested. Specificities ranged from 89% to 99.8%. The near-patient NAAT had sensitivities of >95% and specificities of >99.8% consistently across all specimen types (urine, cervical and vaginal swabs). conclusions Based on a limited number of evaluations, antigen detection POCTs for NG lacked sufficient sensitivity to be used for screening. A near-patient NAAT has acceptable performance, only involved a few steps, but needs electricity, a temperature-controlled environment and has a 90 min run time. To achieve wider scale up of NG POCTs, we need strong evidence of cost-effectiveness, which should inform guidelines and ultimately increase test development, demand and reduce costs.

show abstract

Section: Discussionmentioning

confidence: 97%

Performance and operational characteristics of point-of-care tests for the diagnosis of urogenital gonococcal infections

Guy¹,

Causer

Klausner

et al. 2017

Sex Transm Infect

View full text Add to dashboard Cite

show abstract

“…The high PPV, specificity and sensitivity values are encouraging, however, and indicate that molecular AMR diagnosis may be useful in surveillance particularly in settings where diagnosis relies on nucleic amplification tests (NAATS) and cultures are not available (Table 4). 43 …”

Section: Discussionmentioning

confidence: 99%

Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance

Harrison

Clemence

Dillard

et al. 2016

Journal of Infection

View full text Add to dashboard Cite

SummaryObjectivesAntimicrobial resistance (AMR) threatens our ability to treat the sexually transmitted bacterial infection gonorrhoea. The increasing availability of whole genome sequence (WGS) data from Neisseria gonorrhoeae isolates, however, provides us with an opportunity in which WGS can be mined for AMR determinants.MethodsChromosomal and plasmid genes implicated in AMR were catalogued on the PubMLST Neisseria database (http://pubmlst.org/neisseria). AMR genotypes were identified in WGS from 289 gonococci for which MICs against several antimicrobial compounds had been determined. Whole genome comparisons were undertaken using whole genome MLST (wgMLST).ResultsClusters of isolates with distinct AMR genotypes were apparent following wgMLST analysis consistent with the occurrence of genome wide genetic variation. This included the presence of the gonococcal genetic island (GGI), a type 4 secretion system shown to increase recombination and for which possession was significantly associated with AMR to multiple antimicrobials.ConclusionsEvolution of the gonococcal genome occurs in response to antimicrobial selective pressure resulting in the formation of distinct N. gonorrhoeae populations evidenced by the wgMLST clusters seen here. Genomic islands offer selective advantages to host bacteria and possession of the GGI may, not only facilitate the spread of AMR in gonococcal populations, but may also confer fitness advantages.

show abstract

“…Moreover, strategic target design (i.e., distinct T m s of the amplicons) also allows multiplexing of more than one reaction per tube (20). However, only multiple-step (e.g., requirement for additional steps after nucleic acid amplification for readout) (21,22) or single-antibiotic (e.g., resistance to CIP only) NAAT-based methodologies to characterize AMR gonorrhea have been proposed in the past (23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

et al. 2016

View full text Add to dashboard Cite

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10 3 to 10 4 genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. G onorrhea is the second most common bacterial sexually transmitted infection worldwide, with an estimated 78 million new cases in 2012 (1). Moreover, Neisseria gonorrhoeae has developed resistance to most current and past treatment options. Antimicrobial-resistant (AMR) gonorrhea is a major public health concern about which the World Health Organization (WHO) emphasizes the importance of global surveillance to identify emerging resistance, monitor trends, and inform revisions of treatment guidelines (2, 3).At a molecular level, the mechanisms that confer resistance to the most common treatment options have been well characterized. For instance, the acquisition of mosaic penA alleles, with or without substitutions at amino acid position 501 of the encoded penicillin-binding protein 2 (PBP2), has been linked to decreased susceptibility or resistance to the extended-spectrum cephalosporins (ESCs) cefixime (CFX) and ceftriaxone (CRO) (4, 5). In particular, strains harboring a mosaic pattern XXXIV penA gene, including the internationally spreading N. gonorrhoeae multiantigen sequence typing (NG-MAST) genogroup 1407, have been responsible for ESC treatment failures in severa...

show abstract

A real-time PCR assay for direct characterization of theNeisseria gonorrhoeaeGyrA 91 locus associated with ciprofloxacin susceptibility

Abstract: The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.

Cited by 28 publications

References 8 publications

Performance and operational characteristics of point-of-care tests for the diagnosis of urogenital gonococcal infections

Performance and operational characteristics of point-of-care tests for the diagnosis of urogenital gonococcal infections

Genomic analyses of Neisseria gonorrhoeae reveal an association of the gonococcal genetic island with antimicrobial resistance

Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae

Contact Info

Product

Resources

About