Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance

Ella Trembizki, Cameron Buckley, Basil Donovan, Marcus Chen, Rebecca Guy, John Kaldor, Monica M. Lahra, David G. Regan, Helen Smith, James Ward, David M. Whiley

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

OBJECTIVES: Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.

METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306).

RESULTS: Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87%) of the N. gonorrhoeae NAAT-positive clinical specimens.

CONCLUSIONS: Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.

LanguageEnglish
Pages3244-3249
Number of pages6
JournalThe Journal of antimicrobial chemotherapy
Volume70
Issue number12
DOIs
Publication statusPublished - 1 Dec 2015

ASJC Scopus subject areas

  • Pharmacology
  • Microbiology (medical)
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Trembizki, Ella ; Buckley, Cameron ; Donovan, Basil ; Chen, Marcus ; Guy, Rebecca ; Kaldor, John ; Lahra, Monica M. ; Regan, David G. ; Smith, Helen ; Ward, James ; Whiley, David M. / Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. In: The Journal of antimicrobial chemotherapy. 2015 ; Vol. 70, No. 12. pp. 3244-3249.
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title = "Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance",
abstract = "OBJECTIVES: Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306).RESULTS: Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87{\%}) of the N. gonorrhoeae NAAT-positive clinical specimens.CONCLUSIONS: Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.",
author = "Ella Trembizki and Cameron Buckley and Basil Donovan and Marcus Chen and Rebecca Guy and John Kaldor and Lahra, {Monica M.} and Regan, {David G.} and Helen Smith and James Ward and Whiley, {David M.}",
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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', The Journal of antimicrobial chemotherapy, vol. 70, no. 12, pp. 3244-3249. https://doi.org/10.1093/jac/dkv274

Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. / Trembizki, Ella; Buckley, Cameron; Donovan, Basil; Chen, Marcus; Guy, Rebecca; Kaldor, John; Lahra, Monica M.; Regan, David G.; Smith, Helen; Ward, James; Whiley, David M.

In: The Journal of antimicrobial chemotherapy, Vol. 70, No. 12, 01.12.2015, p. 3244-3249.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance

AU - Trembizki, Ella

AU - Buckley, Cameron

AU - Donovan, Basil

AU - Chen, Marcus

AU - Guy, Rebecca

AU - Kaldor, John

AU - Lahra, Monica M.

AU - Regan, David G.

AU - Smith, Helen

AU - Ward, James

AU - Whiley, David M.

PY - 2015/12/1

Y1 - 2015/12/1

N2 - OBJECTIVES: Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306).RESULTS: Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87%) of the N. gonorrhoeae NAAT-positive clinical specimens.CONCLUSIONS: Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.

AB - OBJECTIVES: Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306).RESULTS: Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87%) of the N. gonorrhoeae NAAT-positive clinical specimens.CONCLUSIONS: Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.

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U2 - 10.1093/jac/dkv274

DO - 10.1093/jac/dkv274

M3 - Article

VL - 70

SP - 3244

EP - 3249

JO - Journal of Antimicrobial Chemotherapy

T2 - Journal of Antimicrobial Chemotherapy

JF - Journal of Antimicrobial Chemotherapy

SN - 0305-7453

IS - 12

ER -