1887

Journal of Medical Microbiology

Volume 58, Issue 7

Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR kit for detection of Free

Abstract

To improve the control of infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by . The method should facilitate early detection leading to better prevention and treatment of genital infection in India.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): DFA, direct fluorescence assay , EBs, elementary bodies , FISH, fluorescence in situ hybridization , NAAT, nucleic acid amplification test , NPV, negative predictive value , PPV, positive predictive value and STD, sexually transmitted disease
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2009-07-01
2019-11-21
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References

  1. An, Q., Radcliffe, G., Vassallo, R., Buxton, D., O'Brien, W. J., Pelletier, D. A., Weisburg, W. G., Klinger, J. D. & Olive, D. M. ( 1992; ). Infection with a plasmid-free variant chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection. J Clin Microbiol 30, 2814–2821.
     [Google Scholar]
  2. Bass, C. A., Jungkind, D. L., Silverman, N. S. & Boodi, J. M. ( 1993; ). Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens. J Clin Microbiol 31, 2648–2653.
     [Google Scholar]
  3. Black, C. M. ( 1997; ). Current methods of laboratory diagnosis of Chlamydia trachomatis infection. Clin Microbiol Rev 10, 160–184.
     [Google Scholar]
  4. Black, C. M. ( 1998; ). “But Doctor, I'm Celibate!” The potential for, sources and implications of false-positive (and negative) results of tests for Chlamydia trachomatis. Clin Microbiol Newsl 20, 120–124.[CrossRef]
    [Google Scholar]
  5. Catry, M. A., Borrego, M. J., Cardoso, J., Azevedo, J. & Santo, I. ( 1995; ). Comparison of the Amplicor Chlamydia trachomatis test and cell culture for the detection of urogenital chlamydial infections. Genitourin Med 71, 247–250.
     [Google Scholar]
  6. CDC ( 2002; ). Sexually transmitted diseases treatment guidelines 2002. Centers for Disease Control and Prevention. MMWR Recomm Rep 51, (RR-6). 1–78.
     [Google Scholar]
  7. Chaudhry, U. & Saluja, D. ( 2002; ). Detection of Neisseria gonorrhoeae by PCR using orf1 gene as target. Sex Transm Infect 78, 72–78.[CrossRef]
    [Google Scholar]
  8. Chout, R., Vaton, S., Quist, D., Bucher, R., Leguyader-Desprees, P. & Sayada, C. ( 1995; ). Improvement of cervical Chlamydia detection in asymptomatic family planning attendees by using Amplicor Chlamydia trachomatis assay. Cell Mol Biol 41, 731–736.
     [Google Scholar]
  9. Crotchfelt, K. A., Pare, B., Gaydos, C. & Quinn, T. C. ( 1998; ). Detection of Chlamydia trachomatis by the Gen-Probe AMPLIFIED Chlamydia trachomatis assay (AMP CT) in urine specimens from men and women and endocervical specimens from women. J Clin Microbiol 36, 391–394.
     [Google Scholar]
  10. Divekar, A. A., Gogate, A. S., Shivkar, L. K., Gogate, S. & Badhwar, V. R. ( 2000; ). Disease prevalence in women attending the STD clinic in Mumbai (formerly Bombay), India. Int J STD AIDS 11, 45–48.[CrossRef]
    [Google Scholar]
  11. Farencena, A., Comanducci, M., Donati, M., Ratti, G. & Cevenini, R. ( 1997; ). Characterization of a new isolate of Chlamydia trachomatis which lacks the common plasmid and has properties of biovar trachoma. Infect Immun 65, 2965–2969.
     [Google Scholar]
  12. Gaydos, C. A., Theodore, M., Dalesio, N., Wood, B. J. & Quinn, T. C. ( 2004; ). Comparison of three nucleic acid amplification tests for detection of Chlamydia trachomatis in urine specimens. J Clin Microbiol 42, 3041–3045.[CrossRef]
    [Google Scholar]
  13. George, J. A., Panchatcharam, T. S., Paramasivam, R., Balasubramanian, S., Chakrapani, V. & Murugan, G. ( 2003; ). Evaluation of diagnostic efficacy of PCR methods for Chlamydia trachomatis infection in genital and urine specimens of symptomatic men and women in India. Jpn J Infect Dis 56, 88–92.
     [Google Scholar]
  14. Jaschek, G., Gaydos, C. A., Welsh, L. E. & Quinn, T. C. ( 1993; ). Direct detection of Chlamydia trachomatis in urine specimens from symptomatic and asymptomatic men by using a rapid polymerase chain reaction assay. J Clin Microbiol 31, 1209–1212.
     [Google Scholar]
  15. Joshi, J. V., Playekar, S., Hazari, K. T., Shah, R. S. & Chitlange, S. M. ( 1994; ). The prevalence of Chlamydia trachomatis in young women. Natl Med J India 7, 57–59.
     [Google Scholar]
  16. Kapur, S., Ahmed, M., Singh, V., Guido, K., Mittal, A. & Salhan, S. ( 2006; ). Fluorescent in situ hybridization can reliably detect rRNA sequences of Chlamydia trachomatis without amplification. Acta Cytol 50, 277–283.[CrossRef]
    [Google Scholar]
  17. Kessler, H. H., Pierer, K., Stuenzner, D., Aner-Grumbach, P., Haller, E. M. & Marth, E. ( 1994; ). Rapid detection of Chlamydia trachomatis in conjunctival, pharyngeal, and urethral specimens with a new polymerase chain reaction assay. Sex Transm Dis 21, 191–195.[CrossRef]
    [Google Scholar]
  18. Loeffelholz, M. J., Lewinski, C. A., Silver, S. R., Purohit, A. P., Herman, S. A., Buonagurio, D. A. & Dragon, E. A. ( 1992; ). Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbiol 30, 2847–2851.
     [Google Scholar]
  19. Mahony, J. B. & Chernesky, M. A. ( 1985; ). Effect of swab type and storage temperature on the isolation of Chlamydia trachomatis from clinical specimens. J Clin Microbiol 22, 865–867.
     [Google Scholar]
  20. Mahony, J. B., Luinstra, K. E., Sellors, J. W. & Chernesky, M. A. ( 1993; ). Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis nucleic acids. J Clin Microbiol 31, 1753–1758.
     [Google Scholar]
  21. Mahony, J. B., Luinstra, K. E., Sellors, J. W., Pickard, L., Chong, S., Jang, D. & Chernesky, M. A. ( 1994; ). Role of confirmatory PCRs in determining performance of Chlamydia Amplicor PCR with endocervical specimens from women with a low prevalence of infection. J Clin Microbiol 32, 2490–2493.
     [Google Scholar]
  22. Mårdh, P. A., Weström, L., Colleen, S. & Wølner-Hanssen, P. ( 1981; ). Sampling, specimen handling, and isolation techniques in the diagnosis of chlamydial and other genital infections. Sex Transm Dis 8, 280–285.[CrossRef]
    [Google Scholar]
  23. Niederhauser, C. & Kaempf, L. ( 2003; ). Improved sensitivity of the Chlamydia trachomatis Cobas Amplicor assay using an optimized procedure for preparation of specimens. Eur J Clin Microbiol Infect Dis 22, 118–121.
     [Google Scholar]
  24. Ostergaard, L. ( 2002; ). Microbiological aspects of the diagnosis of Chlamydia trachomatis. Best Pract Res Clin Obstet Gynaecol 16, 789–799.[CrossRef]
    [Google Scholar]
  25. Palmer, L. & Falkow, S. A. ( 1986; ). Common plasmid of Chlamydia trachomatis. Plasmid 16, 52–62.[CrossRef]
    [Google Scholar]
  26. Schachter, J., Stamm, W. E. & Quinn, T. C. ( 1996; ). Discrepant analysis and screening for Chlamydia trachomatis. Lancet 348, 1308–1309.[CrossRef]
    [Google Scholar]
  27. Semeniuk, H., Zentner, A., Read, R. & Church, D. ( 2002; ). Evaluation of sequential testing strategies using non-amplified and amplified methods for detection of Chlamydia trachomatis in endocervical and urine specimens from women. Diagn Microbiol Infect Dis 42, 43–51.[CrossRef]
    [Google Scholar]
  28. Shattock, R. M., Patrizio, C., Simmonds, P. & Sutherland, S. ( 1998; ). Detection of Chlamydia trachomatis in genital swabs: comparison of commercial and in house amplification methods with culture. Sex Transm Infect 74, 289–293.[CrossRef]
    [Google Scholar]
  29. Singh, V., Rastogi, S., Garg, S., Kapur, S., Kumar, A., Salhan, S. & Mittal, A. ( 2002; ). Polymerase chain reaction for detection of endocervical Chlamydia trachomatis infection in Indian women attending a gynecology outpatient department. Acta Cytol 46, 540–544.[CrossRef]
    [Google Scholar]
  30. Singh, V., Salhan, S., Das, B. C. & Mittal, A. ( 2003; ). Predominance of Chlamydia trachomatis serovars associated with urogenital infections in females in New Delhi, India. J Clin Microbiol 41, 2700–2702.[CrossRef]
    [Google Scholar]
  31. Smith, I. W., Morrison, C. L., Patrizio, C. & McMillan, A. ( 1993; ). Use of a commercial PCR kit for detecting Chlamydia trachomatis. J Clin Pathol 46, 822–825.[CrossRef]
    [Google Scholar]
  32. Sriprakash, K. S. & Macavoy, E. S. ( 1987; ). Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis. Plasmid 18, 205–214.[CrossRef]
    [Google Scholar]
  33. Thejls, H., Gnarpe, J., Gnarpe, H., Larsson, G., Platz-Christensen, J., Ostergaard, L. & Victor, A. ( 1994; ). Expanded gold standard in the diagnosis of Chlamydia trachomatis in a low prevalence population: diagnostic efficacy of tissue culture, direct immunofluorescence, enzyme immunoassay, PCR and serology. Genitourin Med 70, 300–303.
     [Google Scholar]
  34. Thomas, B. J., MacLeod, E. J. & Taylor-Robinson, D. ( 1993; ). Evaluation of sensitivity of 10 diagnostic assays for Chlamydia trachomatis by use of a simple laboratory procedure. J Clin Pathol 46, 912–914.[CrossRef]
    [Google Scholar]
  35. Wu, C. H., Lee, M. F., Yin, S. C., Yang, D. M. & Cheng, S. F. ( 1992; ). Comparison of polymerase chain reaction, monoclonal antibody based enzyme immunoassay, and cell culture for detection of Chlamydia trachomatis in genital specimens. Sex Transm Dis 19, 193–197.[CrossRef]
    [Google Scholar]
  36. Wylie, J. L., Moses, S., Babcock, R., Jolly, A., Giercke, S. & Hammond, G. ( 1998; ). Comparative evaluation of Chlamydiazyme, PACE 2, and AMP-CT assays for detection of Chlamydia trachomatis in endocervical specimens. J Clin Microbiol 36, 3488–3491.
     [Google Scholar]
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Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis
J. Med. Microbiol. 58, 867 (2009); https://doi.org/10.1099/jmm.0.008698-0
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vol. , part 7, pp. 867-873

PCR amplification with P1/P2 primers using purified genomic DNA of serovars A, L2 and D; and CLUSTAL W showing the homology between amplified sequences of plde in various serovars of and clinical samples. [PDF](42 KB)

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