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
SGM
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.008698-0
2009-07-01
2021-03-07
Loading full text...

Full text loading...

/deliver/fulltext/jmm/58/7/867.html?itemId=/content/journal/jmm/10.1099/jmm.0.008698-0&mimeType=html&fmt=ahah

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 orf 1 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]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.008698-0
Loading
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
/content/journal/jmm/10.1099/jmm.0.008698-0
/content/journal/jmm/10.1099/jmm.0.008698-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error