Detection and identification of azithromycin resistance mutations on Treponema pallidum 23S rRNA gene by nested multiplex polymerase chain reaction
Background: Azithromycin-resistant strains of Treponema pallidum is associated with the mutation of 23S rRNA gene of T. pallidum. Although these strains are now prevalent in many countries, there is no laboratory test kit to detect and identify these mutations. Thus, in this study we developed a nested multiplex polymerase chain reaction (PCR) to detect and identify A2058G and A2059G mutations in 23S rRNA gene.
Methods: Three primer sets were designed for nested PCR reactions. To obtain maximum PCR reaction, all parameters were optimized. The specificity of the primer sets was evaluated towards some microorganisms. A sensitivity test was conducted to get detection limit of deoxyribonucleic acid (DNA). Forty-five whole blood specimens were tested by PCR, and positive results were confirmed by the DNA sequencing.
Results: The assay could detect at least 4,400 DNA copy number and showed no cross reaction with other microorganisms used in the specificity test. A total 13 of 45 whole blood specimens were PCR positive for T. pallidum, and no single mutations (either A2058G or A2059G) were detected. Two positive specimens were confirmed by the DNA sequencing and showed no mutation.
Conclusion: Nested multiplex PCR developed in this study showed a specific and sensitive test for the detection and identification of A2058G and/or A2059G mutations of 23S rRNA T. pallidum gene.
Stamm LV. Global challenge of antibiotic-resistant Treponema pallidum. Antimicrob Agents Chemother. 2010;54(2):583–9. https://doi.org/10.1128/AAC.01095-09
Stamm LV. Syphilis: antibiotic treatment and resistance. Epidemiol In fect. 2015; 143(8):1567–74. https://doi.org/10.1017/S0950268814002830
Katz KA, Klausner JD. Azithromycin resistance in Treponema pallidum. Curr Opin Infect Dis. 2008(1);21:83–91. https://doi.org/10.1097/QCO.0b013e3282f44772
Mitchell SJ, Engelman J, Kent CK, Lukehart SA, Godorne C, Klausner JD. Azithromycin-resistant syphilis infection: San Francisco, California, 2000–2004. Clin Infect Dis. 2006;42(3):337–45. https://doi.org/10.1086/498899
Marra CM, Colina AP, Godornes C, Tantalo LC, Puray M, Centurion-Lara A, et al. Antibiotic selection may contribute to increases in macrolide-resistant Treponema pallidum. J Infect Dis. 2006;194(2):1771–3. https://doi.org/10.1086/509512
Matejkova P, Flasarova M, Zakoucka H, Borek M, Kremenova S, Arenberger P, et al. Macrolide treatment failure in a case of secondary syphilis: a novel A2059G mutation in the 23S rRNA gene of Treponema pallidum subsp. Pallidum. J Med Microbiol. 2009; 58(Pt 6):832–6. https://doi.org/10.1099/jmm.0.007542-0
Lukehart SA, Godornes C, Molini BJ, Sonnett P, Hopkins S, Mulcahy F, et al. Macrolide resistance in Treponema pallidum in the United States and Ireland. N Engl J Med. 2004;351:154–8. https://doi.org/10.1056/NEJMoa040216
Vester B, Douthwaite S. Macrolide resistance conferred by base substitution in 23S rRNA. Antimicrob Agents Chemother. 2001;45:1–12. https://doi.org/10.1128/AAC.45.1.1-12.2001
Tipple C, Taylor GP. Syphilis testing, typing, and treatment follow-up: a new era for an old disease. Curr Opin Infect Dis. 2015;28(1):53–60. https://doi.org/10.1097/QCO.0000000000000124
Tipple C. Molecular studies of Treponema pallidum [thesis]. Imperial College London; 2013.
LaFond RE, Lukehart SA. Biological basis for syphilis. Clin Microbiol Rev. 2006; 19(1):29–49. https://doi.org/10.1128/CMR.19.1.29-49.2006
Grimes M, Sahi SK, Godornes BC, Tantalo LC, Roberts N, Bostick D, et al. Two Mutations associated with Macrolide Resistance in Treponema pallidum: Increasing Prevalence and Correlation with Molecular Strain Type in Seattle, Washington. Sex Transm Dis. 2012;39(12):954–8. https://doi.org/10.1097/OLQ.0b013e31826ae7a8
Zhu B, Bu J, Li W, Zhang J, Huang G, Cao J, et al. High resistance to azithromycin in clinical samples from patients with sexually transmitted diseases in Guangxi Zhuang Autonomous Region, China. PloS ONE. 2016;11(7):e0159787. https://doi.org/10.1371/journal.pone.0159787
Molini B, Tantalo LC, Sahi SK, Rodriguez V, Brandt SL, Fernandez MC. et al. Macrolide resistance in Treponema pallidum correlates with 23S rDNA mutations in recently isolated clinical strains. Sex Transm Dis. 2016;43(9):579–83. https://doi.org/10.1097/OLQ.0000000000000486
Harishankar A, Chandy M, Bhattacharya S. How to develop an in-house real-time quantitative cytomegalovirus polymerase chain reaction: Insights from a cancer centre in Eastern India. Indian J Med Microbiol. 2015;33(4):482–90. https://doi.org/10.4103/0255-0857.167351
Sboner A, Mu XJ, Greenbaum D, Auerbach RK, Gerstein MB. The real cost of sequencing: higher than you think! Genome Biol. 2011;12:125. https://doi.org/10.1186/gb-2011-12-8-125
Oh BH, Song YC, Lee YW, Choe YB, Ahn KJ. Comparison of nested PCR and RFLP for identification and classification of malassezia yeasts from healthy human skin. Ann Dermatol. 2009;21(4):352–7. https://doi.org/10.5021/ad.2009.21.4.352
Burstain JM, Grimprel E, Lukehart SA, Norgard MV, Radolf JD. Sensitive detection of Treponema pallidum by using the polymerase chain reaction. J Clin Microbiol. 1991;29(1):62–9.
Chen CY, Chi KH, Pillay A, Nachamkin E, Su JR, Ballard RC. Detection of the A2058G and A2059G 23S rRNA gene point mutations associated with azithromycin resistance in Treponema pallidum by use of a TaqMan real-time multiplex PCR assay. J. Clin. Microbiol. 2013;51(3):908–13. https://doi.org/10.1128/JCM.02770-12
Grunenwald H. Optimization of polymerase chain reaction. Dalam: Bartlett JMS, Stirling D, editor. Methods in molecular biology. Vol. 226: PCR protocols. 2nd Ed. Totowa, NJ: Humana Press Inc.; 2003. p. 89–100.
Innis MA, Gelfand DH. Optimization of PCRs. Dalam: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editor. PCR protocols: a guide to methods and applications. San Diego, CA: Academic Press, Inc.; 1990. p. 3–12. https://doi.org/10.1016/B978-0-12-372180-8.50005-6
Rylichlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 1990;18(21):6409–12. https://doi.org/10.1093/nar/18.21.6409
Liu H, Rodes B, Chien CY, Steiner B. New test for syphilis: rational design of a PCR method for detection of Treponema pallidum in clinical specimens using uniqe regions of the DNA plimerase I gene. J. Clin. Microbiol. 2001;39(5):1941–6. https://doi.org/10.1128/JCM.39.5.1941-1946.2001
Orle, K. A., C. A. Gates, D. H. Martin, B. A. Body, and J. B. Weiss. 1996. Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers. J. Clin. Microbiol. 1996;34(1):49–54.
Martin IE, Tsang RSW, Sutherland K, Tilley P, Read R, Anderson B, et al. Molecular characterization of syphilis in patients in Canada: azithromycin resistance and detection of Treponema pallidum DNA in whole-blood samples versus ulcerative swab. J. Clin. Microbiol. 2009;47(6):1668–73. https://doi.org/10.1128/JCM.02392-08
Grange PA, Gressier L, Dion PL, Farhl D, Benhaddou N, Gerhardt P, et al. Evaluation of a PCR test for detection of Treponema pallidum in swab and blood. J. Clin. Microbiol. 2011;50(3):546–52. https://doi.org/10.1128/JCM.00702-11
Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clin. Microbiol. Rev. 2000;13(4):559–70. https://doi.org/10.1128/CMR.13.4.559-570.2000
Copyright (c) 2017 Desy A. Gultom, Yeva Rosana, Ida Efendi, Wresti Indriatmi, Andi Yasmon
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Authors who publish with Medical Journal of Indonesia agree to the following terms:
- Authors retain copyright and grant Medical Journal of Indonesia right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial License that allows others to remix, adapt, build upon the work non-commercially with an acknowledgment of the work’s authorship and initial publication in Medical Journal of Indonesia.
- Authors are permitted to copy and redistribute the journal's published version of the work non-commercially (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Medical Journal of Indonesia.