Mutations seen in nucleotides 521, 716, and 1063 of genotype K are similar to those observed in sequences already deposited in GenBank with accession numbers AY937267, AY937268, and AY937269. Mutation at nucleotide 521 was also observed in a sequence with accession number AY535168.2 Genotype F showed the highest level of conservation, with only three out of the 26 mutations and one of them silent (Table 2). Interestingly, mutations observed in genotypes D, E, and F did not match any other already deposited in the database.
To confirm the grouping of our sequences with the sequences of each serotype already deposited in GeneBank, we constructed a phylogenetic tree using the nucleotide sequence of the omp1 gene described here (Fig. 2). Bootstrap values are shown at the nodes. Values lower than 50 are considered insignificant and are not shown. The analysis produced trees that segregated the identified Brazilian genotypes of C. trachomatis into three clades, consistent with serocomplexes. The three main clusters consisted of B-complex (genotype D and E), C-complex (genotype K), and an intermediate complex (genotype F).
This study involved women attending a public service for treatment and prevention of sexually transmitted diseases in one of the major cities in Brazil, having an estimated population of around 2.4 million people. The age of the women enrolled in this study varied between 15 and 54 years old and, as expected, the higher number of positive results for C. trachomatis infection was found in young women. Our study confirmed that younger age is an important risk factor for chlamydial infection, because 68% of the infected women were between 15 and 25 years old. It is clear that this is the age group of higher sexual promiscuity and, consequently, is at a higher risk for STD. The association of C. trachomatis infection with young age may be a matter of sexual behavior but, as pointed out by Norman, studies must be done to investigate how individual factors, such as immune immatureness, can contribute to the initial establishment of C. trachomatis infection and its progress.24
PCR using clinical specimens is tricky and can be unsuccessful because of inhibitors sometimes found in significant amounts in samples such as endocervical fluids, urine, and blood. Futhermore, contamination can also reduce reproducibility of results.31–34 To reduce inhibitory factors and improve PCR sensitivity, purified DNA was used as the template, instead of the crude lysate suggested by others.23 Purified DNA was initially tested for β-globin normalizing gene amplification and only those positive were screened for C. trachomatis cryptic plasmid, improving the detection.
A phylogenetic analysis of the 12 sequences identified in this study was done using C. psittaci (AY762613) as an outgroup strain. Three main clusters were identified, as described by others4,47,50: one cluster of B complex, one of F-G, and other of C complex.
An important result was the detection of two genotypes (D and E) in one woman, characterizing a mixed infection. This same association was the most common mixed infection found by Molano et al. in a study also evaluating women attending a STD Clinic.9 It has to be kept in mind that although, today, DNA sequencing is the way to detect a mixed infection, it is rarely done, because of the necessity of cloning amplicons followed by sequencing of multiple clones. For this reason, we can presume that mixed infections could be much more common than reports show, because sequencing of multiple clones from positive samples is not always done. This can be an important point to consider when evaluating treatment effectiveness and reinfection as well as drug resistance.
1. Yuan Y, Zhang YX, Watkins NG, et al. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis
serovars. Infect Immun 1989; 57:1040–1049.
2. Millman K, Black CM, Johnson RE, et al. Population-based genetic and evolutionary analysis of Chlamydia trachomatis
urogenital strain variation in the United States. J Bacteriol 2004; 186:2457–2465.
3. Lofy KH, Hofmann J, Mosure DJ, et al. Chlamydial infections among female adolescents screened in juvenile detention centers in Washington State, 1998–2002. Sex Transm Dis 2006; 33:63–67.
4. Lysen M, Osterlund A, Rubin CJ, et al. Characterization of ompA genotypes by sequence analysis of DNA from all detected cases of Chlamydia trachomatis
infections during 1 year of contact tracing in a Swedish County. J Clin Microbiol 2004; 42:1641–1647.
5. Jurstrand M, Falk L, Fredlund H, et al. Characterization of Chlamydia trachomatis
omp1 genotypes among sexually transmitted disease patients in Sweden. J Clin Microbiol 2001; 39:3915–3919.
6. Morre SA, Rozendaal L, van Valkengoed IG, et al. Urogenital Chlamydia trachomatis
serovars in men and women with a symptomatic or asymptomatic infection: An association with clinical manifestations? J Clin Microbiol 2000; 38:2292–2296.
7. Batteiger BE, Lin PM, Jones RB, et al. Species-, serogroup-, and serovar-specific epitopes are juxtaposed in variable sequence region 4 of the major outer membrane proteins of some Chlamydia trachomatis
serovars. Infect Immun 1996; 64:2839–2841.
8. Brunham RC, Kimani J, Bwayo J, et al. The epidemiology of Chlamydia trachomatis
within a sexually transmitted disease core group. J Infect Dis 1996; 173:950–956.
9. Molano M, Meijer CJ, Morre SA, et al. Combination of PCR targeting the VD2 of omp1 and reverse line blot analysis for typing of urogenital Chlamydia trachomatis
serovars in cervical scrape specimens. J Clin Microbiol 2004; 42:2935–2939.
10. Dean D, Oudens E, Bolan G, et al. Major outer membrane protein variants of Chlamydia trachomatis
are associated with severe upper genital tract infections and histopathology in San Francisco. J Infect Dis 1995; 172:1013–1022.
11. Nelson HD, Helfand M. Screening for chlamydial infection. Am J Prev Med 2001; 20(3 Suppl):95–107.
12. Awwad ZM, Al-Amarat AA, Shehabi AA. Prevalence of genital chlamydial infection in symptomatic and asymptomatic Jordanian patients. Int J Infect Dis 2003; 7:206–209.
13. Hsu MC, Tsai PY, Chen KT, et al. Genotyping of Chlamydia trachomatis
from clinical specimens in Taiwan. J Med Microbiol 2006; 55 (Part 3):301–308.
14. Gaydos CA, Theodore M, Dalesio N, et al. Comparison of three nucleic acid amplification tests for detection of Chlamydia trachomatis
in urine specimens. J Clin Microbiol 2004; 42:3041–3045.
15. Cheng H, Macaluso M, Vermund SH, et al. Relative accuracy of nucleic acid amplification tests and culture in detecting chlamydia in asymptomatic men. J Clin Microbiol 2001; 39:3927–3937.
16. Pinto VM, Tancredi MV, Tancredi Neto A, et al. Sexually transmitted disease/HIV risk behaviour among women who have sex with women. AIDS 2005; 19(Suppl 4):S64–S69.
17. de Lima Soares V, de Mesquita AM, Cavalcante FG, et al. Sexually transmitted infections in a female population in rural north-east Brazil: Prevalence, morbidity and risk factors. Trop Med Int Health 2003; 8:595–603.
18. Amaral MG, Kulay L Jr, Granato C, et al. [Chlamydia trachomatis
infection and risk factors in pregnant women.] Rev Assoc Med Bras 1995; 41:193–196.
19. de Codes JS, Cohen DA, de Melo NA, et al. [Screening of sexually transmitted diseases in clinical and non-clinical settings in Salvador, Bahia, Brazil]. Cad Saude Publica 2006; 22:325–334.
20. Miranda AE, Szwarcwald CL, Peres RL, et al. Prevalence and risk behaviors for chlamydial infection in a population-based study of female adolescents in Brazil. Sex Transm Dis 2004; 31:542–546.
21. Santos C, Teixeira F, Vicente A, et al. Detection of Chlamydia trachomatis
in endocervical smears of sexually active women in Manaus-AM, Brazil, by PCR. Braz J Infect Dis 2003; 7:91–95.
22. Fioravante FC, Costa Alves Mde F, Guimaraes EM, et al. Prevalence of Chlamydia trachomatis
in asymptomatic Brazilian military conscripts. Sex Transm Dis 2005; 32:165–169.
23. Lan J, Walboomers JM, Roosendaal R, et al. Direct detection and genotyping of Chlamydia trachomatis
in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis. J Clin Microbiol 1993; 31:1060–1065.
24. Norman J. Epidemiology of female genital Chlamydia trachomatis
infections. Best Pract Res Clin Obstet Gynaecol 2002; 16:775–787.
25. Mahony JB, Luinstra KE, Sellors JW, et al. Comparison of plasmid- and chromosome-based polymerase chain reaction assays for detecting Chlamydia trachomatis
nucleic acids. J Clin Microbiol 1993; 31:1753–1758.
26. Everett KD, Andersen AA. Identification of nine species of the Chlamydiaceae using PCR-RFLP. Int J Syst Bacteriol 1999; 49(Part 2):803–813.
27. Everett KD, Hornung LJ, Andersen AA. Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: Three PCR tests. J Clin Microbiol 1999; 37:575–580.
28. Roosendaal R, Walboomers JM, Veltman OR, et al. Comparison of different primer sets for detection of Chlamydia trachomatis
by the polymerase chain reaction. J Med Microbiol 1993; 38:426–433.
29. Millman KL, Tavare S, Dean D. Recombination in the ompA
gene but not the omcB
gene of Chlamydia contributes to serovar-specific differences in tissue tropism, immune surveillance, and persistence of the organism. J Bacteriol 2001; 183:5997–6008.
30. Hsieh YH, Bobo LD, Quinn TC, et al. Determinants of trachoma endemicity using Chlamydia trachomatis
ompA DNA sequencing. Microbes Infect 2001; 3:447–458.
31. Wilcox MH, Reynolds MT, Hoy CM, et al. Combined cervical swab and urine specimens for PCR diagnosis of genital Chlamydia trachomatis
infection. Sex Transm Infect 2000; 76:177–178.
32. Toye B, Woods W, Bobrowska M, et al. Inhibition of PCR in genital and urine specimens submitted for Chlamydia trachomatis
testing. J Clin Microbiol 1998; 36:2356–2358.
33. Coutlee F, de Ladurantaye M, Tremblay C, et al. An important proportion of genital samples submitted for Chlamydia trachomatis
detection by PCR contain small amounts of cellular DNA as measured by β-globin gene amplification. J Clin Microbiol 2000; 38:2512–2515.
34. Hamdad-Daoudi F, Petit J, Eb F. Assessment of Chlamydia trachomatis
infection in asymptomatic male partners of infertile couples. J Med Microbiol 2004; 53(Part 10):985–990.
35. Araujo RS, Guimaraes EM, Alves MF, et al. Prevalence and risk factors for Chlamydia trachomatis
infection in adolescent females and young women in central Brazil. Eur J Clin Microbiol Infect Dis 2006; 25:397–400.
36. Kohl KS, Sternberg MR, Markowitz LE, et al. Screening of males for Chlamydia trachomatis
and Neisseria gonorrhoeae
infections at STD clinics in three US cities—Indianapolis, New Orleans, Seattle. Int J STD AIDS 2004; 15:822–828.
37. Falk L, Lindberg M, Jurstrand M, et al. Genotyping of Chlamydia trachomatis
would improve contact tracing. Sex Transm Dis 2003; 30:205–210.
38. Vall-Mayans M, Villa M, Saravanya M, et al. Sexually transmitted Chlamydia trachomatis
, Neisseria gonorrhoeae
, and HIV-1 infections in two at-risk populations in Barcelona: Female street prostitutes and STI clinic attendees. Int J Infect Dis, in press.
39. George JA, Panchatcharam TS, Paramasivam R, et al. 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 2003; 56:88–92.
40. Pajaro MC, Barberis IL, Godino S, et al. Epidemiology of sexually transmitted diseases in Rio Cuarto, Argentina. Rev Latinoam Microbiol 2001; 43:157–160.
41. Esquivel CA, Briones Ezcarzaga ML, Castruita Limones DE, et al. Prevalence of Chlamydia trachomatis
infection in registered female sex workers in northern Mexico. Sex Transm Dis 2003; 30:195–198.
42. Pedersen LN, Kjaer HO, Moller JK, et al. High-resolution genotyping of Chlamydia trachomatis
from recurrent urogenital infections. J Clin Microbiol 2000; 38:3068–3071.
43. van de Laar MJ, van Duynhoven YT, Fennema JS, et al. Differences in clinical manifestations of genital chlamydial infections related to serovars. Genitourin Med 1996; 72:261–265.
44. Geisler WM, Suchland RJ, Whittington WL, et al. The relationship of serovar to clinical manifestations of urogenital Chlamydia trachomatis
infection. Sex Transm Dis 2003; 30:160–165.
45. Ngandjio A, Clerc M, Fonkoua MC, et al. Screening of volunteer students in Yaounde (Cameroon, Central Africa) for Chlamydia trachomatis
infection and genotyping of isolated C. trachomatis
strains. J Clin Microbiol 2003; 41:4404–4407.
46. Brunham R, Yang C, Maclean I, et al. Chlamydia trachomatis
from individuals in a sexually transmitted disease core group exhibit frequent sequence variation in the major outer membrane protein (omp1) gene J Clin Invest 1994; 94:458–463.
47. Sturm-Ramirez K, Brumblay H, Diop K, et al. Molecular epidemiology of genital Chlamydia trachomatis
infection in high-risk women in Senegal, West Africa. J Clin Microbiol 2000; 38:138–145.
48. Frost EH, Deslandes S, Gendron D, et al. Variation outside variable segments of the major outer membrane protein distinguishes trachoma from urogenital isolates of the same serovar of Chlamydia trachomatis
. Genitourin Med 1995; 71:18–23.
49. Sylvan SP, Von Krogh G, Tiveljung A, et al. Screening and genotyping of genital Chlamydia trachomatis
in urine specimens from male and female clients of youth-health centers in Stockholm County. Sex Transm Dis 2002; 29:379–386.
50. Stothard DR, Boguslawski G, Jones RB. Phylogenetic analysis of the Chlamydia trachomatis
major outer membrane protein and examination of potential pathogenic determinants. Infect Immun 1998; 66:3618–3625.
51. Rodriguez P, de Barbeyrac B, Persson K, et al. Evaluation of molecular typing for epidemiological study of Chlamydia trachomatis
genital infections. J Clin Microbiol 1993; 31:2238–2240.
52. Dean D, Millman K. Molecular and mutation trends analyses of omp1 alleles for serovar E of Chlamydia trachomatis.
Implications for the immunopathogenesis of disease. J Clin Invest 1997; 99:475–483.