Mismatch Amplification Mutation Assay-Based Real-Time PCR for Rapid Detection of Neisseria gonorrhoeae and Antimicrobial Resistance Determinants in Clinical Specimens

Valentina Donà, Joost H Smid, Sara Kasraian, Dianne Egli-Gany, Ferah Dost, Fatime Imeri, Magnus Unemo, Nicola Low, Andrea Endimiani, Valentina Donà, Joost H Smid, Sara Kasraian, Dianne Egli-Gany, Ferah Dost, Fatime Imeri, Magnus Unemo, Nicola Low, Andrea Endimiani

Abstract

Molecular methods are often used for Neisseria gonorrhoeae detection, but complete definition of antimicrobial resistance (AMR) patterns still requires phenotypic tests. We developed an assay that both identifies N. gonorrhoeae and detects AMR determinants in clinical specimens. We designed a mismatch amplification mutation assay (MAMA)-based SYBR green real-time PCR targeting one N. gonorrhoeae-specific region (opa); mosaic penA alleles (Asp345 deletion [Asp345del], Gly545Ser) associated with decreased susceptibility to cephalosporins; and alterations conferring resistance to ciprofloxacin (GyrA Ser91Phe), azithromycin (23S rRNA A2059G and C2611T), and spectinomycin (16S rRNA C1192T). We applied the real-time PCR to 489 clinical specimens, of which 94 had paired culture isolates, and evaluated its performance by comparison with the performance of commercial diagnostic molecular and phenotypic tests. Our assay exhibited a sensitivity/specificity of 93%/100%, 96%/85%, 90%/91%, 100%/100%, and 100%/90% for the detection of N. gonorrhoeae directly from urethral, rectal, pharyngeal, cervical, and vaginal samples, respectively. The MAMA strategy allowed the detection of AMR mutations by comparing cycle threshold values with the results of the reference opa reaction. The method accurately predicted the phenotype of resistance to four antibiotic classes, as determined by comparison with the MIC values obtained from 94 paired cultures (sensitivity/specificity for cephalosporins, azithromycin, ciprofloxacin, and spectinomycin resistance, 100%/95%, 100%/100%, 100%/100%, and not applicable [NA]/100%, respectively, in genital specimens and NA/72%, NA/98%, 100%/97%, and NA/96%, respectively, in extragenital specimens). False-positive results, particularly for the penA Asp345del reaction, were observed predominantly in pharyngeal specimens. Our real-time PCR assay is a promising rapid method to identify N. gonorrhoeae and predict AMR directly in genital specimens, but further optimization for extragenital specimens is needed.

Keywords: AMR; NAAT; Neisseria gonorrhoeae; antibiotic resistance; antimicrobial resistance; clinical methods; clinical samples; diagnostics; gonococcus; rapid tests; real-time PCR; sexually transmitted diseases.

Copyright © 2018 Donà et al.

References

    1. Newman L, Rowley J, Vander Hoorn S, Wijesooriya NS, Unemo M, Low N, Stevens G, Gottlieb S, Kiarie J, Temmerman M. 2015. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting. PLoS One 10:e0143304. doi:10.1371/journal.pone.0143304.
    1. Ison CA, Alexander S. 2011. Antimicrobial resistance in Neisseria gonorrhoeae in the UK: surveillance and management. Expert Rev Anti Infect Ther 9:867–876. doi:10.1586/eri.11.103.
    1. Unemo M, Nicholas RA. 2012. Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea. Future Microbiol 7:1401–1422. doi:10.2217/fmb.12.117.
    1. Wi T, Lahra MM, Ndowa F, Bala M, Dillon JR, Ramon-Pardo P, Eremin SR, Bolan G, Unemo M. 2017. Antimicrobial resistance in Neisseria gonorrhoeae: global surveillance and a call for international collaborative action. PLoS Med 14:e1002344. doi:10.1371/journal.pmed.1002344.
    1. Low N, Unemo M, Skov Jensen J, Breuer J, Stephenson JM. 2014. Molecular diagnostics for gonorrhoea: implications for antimicrobial resistance and the threat of untreatable gonorrhoea. PLoS Med 11:e1001598. doi:10.1371/journal.pmed.1001598.
    1. World Health Organization. 2012. Global action plan to control the spread and impact of antimicrobial resistance in Neisseria gonorrhoeae. World Health Organization, Geneva, Switzerland.
    1. Unemo M, Shafer WM. 2011. Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, and lessons learned for the future. Ann N Y Acad Sci 1230:E19–E28. doi:10.1111/j.1749-6632.2011.06215.x.
    1. Workowski KA, Berman SM, Douglas JM Jr. 2008. Emerging antimicrobial resistance in Neisseria gonorrhoeae: urgent need to strengthen prevention strategies. Ann Intern Med 148:606–613. doi:10.7326/0003-4819-148-8-200804150-00005.
    1. Pond MJ, Hall CL, Miari VF, Cole M, Laing KG, Jagatia H, Harding-Esch E, Monahan IM, Planche T, Hinds J, Ison CA, Chisholm S, Butcher PD, Sadiq ST. 2016. Accurate detection of Neisseria gonorrhoeae ciprofloxacin susceptibility directly from genital and extragenital clinical samples: towards genotype-guided antimicrobial therapy. J Antimicrob Chemother 71:897–902. doi:10.1093/jac/dkv432.
    1. Whiley DM, Trembizki E, Buckley C, Freeman K, Baird RW, Beaman M, Chen M, Donovan B, Kundu RL, Fairley CK, Guy R, Hogan T, Kaldor JM, Karimi M, Limnios A, Regan DG, Ryder N, Su JY, Ward J, Lahra MM. 2017. Molecular antimicrobial resistance surveillance for Neisseria gonorrhoeae, Northern Territory, Australia. Emerg Infect Dis 23:1478–1485. doi:10.3201/eid2309.170427.
    1. Unemo M, Golparian D, Nicholas R, Ohnishi M, Gallay A, Sednaoui P. 2012. High-level cefixime- and ceftriaxone-resistant Neisseria gonorrhoeae in France: novel penA mosaic allele in a successful international clone causes treatment failure. Antimicrob Agents Chemother 56:1273–1280. doi:10.1128/AAC.05760-11.
    1. van Dam AP, van Ogtrop ML, Golparian D, Mehrtens J, de Vries HJ, Unemo M. 2014. Verified clinical failure with cefotaxime 1g for treatment of gonorrhoea in the Netherlands: a case report. Sex Transm Infect 90:513–514. doi:10.1136/sextrans-2014-051552.
    1. Unemo M, Golparian D, Potocnik M, Jeverica S. 2012. Treatment failure of pharyngeal gonorrhoea with internationally recommended first-line ceftriaxone verified in Slovenia, September 2011. Euro Surveill 17(25):pii=20200 .
    1. Unemo M, Golparian D, Stary A, Eigentler A. 2011. First Neisseria gonorrhoeae strain with resistance to cefixime causing gonorrhoea treatment failure in Austria, 2011. Euro Surveill 16(43):pii=19998 .
    1. Unemo M, Shafer WM. 2014. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future. Clin Microbiol Rev 27:587–613. doi:10.1128/CMR.00010-14.
    1. Shultz TR, Tapsall JW, White PA. 2001. Correlation of in vitro susceptibilities to newer quinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC. Antimicrob Agents Chemother 45:734–738. doi:10.1128/AAC.45.3.734-738.2001.
    1. Ng LK, Martin I, Liu G, Bryden L. 2002. Mutation in 23S rRNA associated with macrolide resistance in Neisseria gonorrhoeae. Antimicrob Agents Chemother 46:3020–3025. doi:10.1128/AAC.46.9.3020-3025.2002.
    1. Chisholm SA, Dave J, Ison CA. 2010. High-level azithromycin resistance occurs in Neisseria gonorrhoeae as a result of a single point mutation in the 23S rRNA genes. Antimicrob Agents Chemother 54:3812–3816. doi:10.1128/AAC.00309-10.
    1. Galimand M, Gerbaud G, Courvalin P. 2000. Spectinomycin resistance in Neisseria spp. due to mutations in 16S rRNA. Antimicrob Agents Chemother 44:1365–1366. doi:10.1128/AAC.44.5.1365-1366.2000.
    1. Dona V, Low N, Golparian D, Unemo M. 2017. Recent advances in the development and use of molecular tests to predict antimicrobial resistance in Neisseria gonorrhoeae. Expert Rev Mol Diagn 17:845–859. doi:10.1080/14737159.2017.1360137.
    1. Dona V, Kasraian S, Lupo A, Guilarte YN, Hauser C, Furrer H, Unemo M, Low N, Endimiani A. 2016. Multiplex real-time PCR assay with high-resolution melting analysis for characterization of antimicrobial resistance in Neisseria gonorrhoeae. J Clin Microbiol 54:2074–2081. doi:10.1128/JCM.03354-15.
    1. Cha RS, Zarbl H, Keohavong P, Thilly WG. 1992. Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl 2:14–20. doi:10.1101/gr.2.1.14.
    1. Endimiani A, Guilarte YN, Tinguely R, Hirzberger L, Selvini S, Lupo A, Hauser C, Furrer H. 2014. Characterization of Neisseria gonorrhoeae isolates detected in Switzerland (1998–2012): emergence of multidrug-resistant clones less susceptible to cephalosporins. BMC Infect Dis 14:106. doi:10.1186/1471-2334-14-106.
    1. Unemo M, Golparian D, Sanchez-Buso L, Grad Y, Jacobsson S, Ohnishi M, Lahra MM, Limnios A, Sikora AE, Wi T, Harris SR. 2016. The novel 2016 WHO Neisseria gonorrhoeae reference strains for global quality assurance of laboratory investigations: phenotypic, genetic and reference genome characterization. J Antimicrob Chemother 71:3096–3108. doi:10.1093/jac/dkw288.
    1. Unemo M, Fasth O, Fredlund H, Limnios A, Tapsall J. 2009. Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes. J Antimicrob Chemother 63:1142–1151. doi:10.1093/jac/dkp098.
    1. Bewick V, Cheek L, Ball J. 2004. Statistics review 13: receiver operating characteristic curves. Crit Care 8:508–512. doi:10.1186/cc3000.
    1. Clopper C, Pearson E. 1934. The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26:404–413. doi:10.1093/biomet/26.4.404.
    1. The European Committee on Antimicrobial Susceptibility Testing. 2016. Breakpoint tables for interpretation of MICs and zone diameters, version 6.0, 2016. .
    1. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI document M100-S25. Clinical and Laboratory Standards Institute, Wayne, PA.
    1. Jacobsson S, Golparian D, Cole M, Spiteri G, Martin I, Bergheim T, Borrego MJ, Crowley B, Crucitti T, Van Dam AP, Hoffmann S, Jeverica S, Kohl P, Mlynarczyk-Bonikowska B, Pakarna G, Stary A, Stefanelli P, Pavlik P, Tzelepi E, Abad R, Harris SR, Unemo M. 2016. WGS analysis and molecular resistance mechanisms of azithromycin-resistant (MIC >2 mg/liter) Neisseria gonorrhoeae isolates in Europe from 2009 to 2014. J Antimicrob Chemother 71:3109–3116. doi:10.1093/jac/dkw279.
    1. Bharat A, Demczuk W, Martin I, Mulvey MR. 2015. Effect of variants of penicillin-binding protein 2 on cephalosporin and carbapenem susceptibilities in Neisseria gonorrhoeae. Antimicrob Agents Chemother 59:5003–5006. doi:10.1128/AAC.05143-14.
    1. Pandori M, Barry PM, Wu A, Ren A, Whittington WL, Liska S, Klausner JD. 2009. Mosaic penicillin-binding protein 2 in Neisseria gonorrhoeae isolates collected in 2008 in San Francisco, California. Antimicrob Agents Chemother 53:4032–4034. doi:10.1128/AAC.00406-09.
    1. Lahra MM, Ryder N, Whiley DM. 2014. A new multidrug-resistant strain of Neisseria gonorrhoeae in Australia. N Engl J Med 371:1850–1851. doi:10.1056/NEJMc1408109.
    1. Ohnishi M, Golparian D, Shimuta K, Saika T, Hoshina S, Iwasaku K, Nakayama S, Kitawaki J, Unemo M. 2011. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone. Antimicrob Agents Chemother 55:3538–3545. doi:10.1128/AAC.00325-11.
    1. Nakayama S, Shimuta K, Furubayashi K, Kawahata T, Unemo M, Ohnishi M. 2016. New ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain with a novel mosaic penA gene isolated in Japan. Antimicrob Agents Chemother 60:4339–4341. doi:10.1128/AAC.00504-16.
    1. Demczuk W, Sidhu S, Unemo M, Whiley DM, Allen VG, Dillon JR, Cole M, Seah C, Trembizki E, Trees DL, Kersh EN, Abrams AJ, de Vries HJC, van Dam AP, Medina I, Bharat A, Mulvey MR, Van Domselaar G, Martin I. 2017. Neisseria gonorrhoeae sequence typing for antimicrobial resistance, a novel antimicrobial resistance multilocus typing scheme for tracking global dissemination of N. gonorrhoeae strains. J Clin Microbiol 55:1454–1468. doi:10.1128/JCM.00100-17.
    1. Jeverica S, Golparian D, Maticic M, Potocnik M, Mlakar B, Unemo M. 2014. Phenotypic and molecular characterization of Neisseria gonorrhoeae isolates from Slovenia, 2006-12: rise and fall of the multidrug-resistant NG-MAST genogroup 1407 clone? J Antimicrob Chemother 69:1517–1525. doi:10.1093/jac/dku026.
    1. Unemo M, Golparian D, Skogen V, Olsen AO, Moi H, Syversen G, Hjelmevoll SO. 2013. Neisseria gonorrhoeae strain with high-level resistance to spectinomycin due to a novel resistance mechanism (mutated ribosomal protein S5) verified in Norway. Antimicrob Agents Chemother 57:1057–1061. doi:10.1128/AAC.01775-12.
    1. Trembizki E, Buckley C, Donovan B, Chen M, Guy R, Kaldor J, Lahra MM, Regan DG, Smith H, Ward J, Whiley DM. 2015. Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. J Antimicrob Chemother 70:3244–3249.
    1. Ameyama S, Onodera S, Takahata M, Minami S, Maki N, Endo K, Goto H, Suzuki H, Oishi Y. 2002. Mosaic-like structure of penicillin-binding protein 2 gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime. Antimicrob Agents Chemother 46:3744–3749. doi:10.1128/AAC.46.12.3744-3749.2002.
    1. Tanaka M, Nakayama H, Huruya K, Konomi I, Irie S, Kanayama A, Saika T, Kobayashi I. 2006. Analysis of mutations within multiple genes associated with resistance in a clinical isolate of Neisseria gonorrhoeae with reduced ceftriaxone susceptibility that shows a multidrug-resistant phenotype. Int J Antimicrob Agents 27:20–26. doi:10.1016/j.ijantimicag.2005.08.021.
    1. Allen VG, Mitterni L, Seah C, Rebbapragada A, Martin IE, Lee C, Siebert H, Towns L, Melano RG, Low DE. 2013. Neisseria gonorrhoeae treatment failure and susceptibility to cefixime in Toronto, Canada. JAMA 309:163–170. doi:10.1001/jama.2012.176575.

Source: PubMed

赞助者和合作者

医疗条件

药物干预

3
订阅