Sexually Transmitted Diseases:
Genotyping of Chlamydia trachomatis From Endocervical Specimens in Brazil
Lima, Haleta E. MSc*; Oliveira, Milena B. Pharm*; Valente, Brenda G. MSc*; Afonso, Daniela A. F. PhD*; DaRocha, Wanderson D. PhD†; Souza, Maria Carmo M. do Pharm‡; Alvim, Túlio C. MD‡; Barbosa-Stancioli, Edel F. PhD*; Noronha, Fátima Soares Motta PhD*
From the *Departamento de Microbiologia, Universidade Federal de Minas Gerais; †Departamento de Ciências Biológicas, Ambientaise da Saúde, Centro Universitário Uni-BH; ‡Centro de Treinamento em DoenÇas Sexualmente Transmissíveis-Prefeitura de' Belo Horizonte, Belo Horizonte, Minas Gerais, Brasil
This work was supported by Fundação de Amparo àPesquisa de Minas Gerais (FAPEMIG and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). H.E. Lima and E.F. Barbosa-Stancioli received fellowships from CNPq. We are thankful to NAGE (Núcleo de Análise de Genoma e Expressão Gênica) for the sequencing facilities. This research was approved by the Ethics Research Committee of the Federal University of Minas Gerais.
Correspondence: Fátima Soares Motta Noronha, PhD, Departamento de Microbiologia, Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Belo Horizonte, MG 31270-901. E-mail: email@example.com.
Received for publication October 11, 2006, and accepted December 22, 2006.
Background: There is no data concerning genotyping of Chlamydia trachomatis from Brazilian samples.
Goal: To characterize the genotype of C. trachomatis detected in women assisted at a STD public clinic and establish the prevalence of this infection in that population.
Study Design: Endocervical samples of a group of 100 women were tested for chlamydial infection with PCR directed to C. trachomatis cryptic plasmid. Genotyping of positive samples were done after omp1 amplification and sequencing.
Results: The overall prevalence of C. trachomatis infection was 19%, with the highest prevalence in women between 15 and 25 years old (68.4%). Four genotypes were found associated with endocervical infections: D, E, F, and K. Sequence analysis revealed a coinfection of genotypes D and E in 1 woman.
Conclusions: To our knowledge this is the first study to characterize Brazilian C. trachomatis endocervical samples and Brazilian C. trachomatis genotype coinfection. Our results also emphasize the importance of routine diagnosis of C. trachomatis for the control of this STD.
CHLAMYDIA TRACHOMATIS IS THE LEADING bacterial cause of sexually transmitted diseases (STD) in industrialized countries, perhaps worldwide, and is responsible for a wide range of inflammation in the urogenital tracts of men and women. In developing countries, these bacteria are also the agent of trachoma that occurs when infection attacks the ocular mucosa.
The major outer membrane protein (MOMP), a key constituent of the cell wall of this obligate intracellular bacteria, is encoded by the omp1 gene and contains 4 variable domains (VDs) interspaced with five highly conserved regions.1 Three of these VDs are surface exposed and allow serovars classification. Currently, there are 19 serovars recognized for C. trachomatis, all of them with their genotypic profile well established.
Sexually transmitted genotypes of C. trachomatis D, E, F, G, H, I, J, and K, are a major cause of cervicitis and urethritis in many countries1,2 and L1, L2, L2a, and L3 are associated with the systemic infection lymphogranuloma venereum. Cervicitis and urethritis can become persistent and may initiate a pathogenic process leading to chronic disease, such as pelvic inflammatory disease, ectopic pregnancy, and others.3–7 Recent data show that although most of cervicitis and urethritis are associated with just 1 genotype, 2% to 15% of them are related to two or more genotypes.6,8–10
Chlamydial genital infections are present at a high prevalence rate in all currently studied countries, especially in the young population. Recent data also indicate that the number of asymptomatic infections are very high, being around 50% in the male population and reaching 80% for females.11–14 This large group of asymptomatically infected carriers is not only at risk of serious long-term sequelae but also to sustaining transmission within communities.15
In Brazil, screening for C. trachomatis is not routinely offered, and the infection is not of compulsory notification. However, isolated studies carried out in this country have shown infection rates ranging from 1.8% to 20.7% among women.16–22 This large range among reported rates reflects different methods used and populations evaluated. Nonetheless, these studies indicate the importance of this silent infection in our country.
Although many studies establish the prevalence of this infection in Brazil, there is no information concerning C. trachomatis serovars and genotypes. In this study, we describe the genotyping of C. trachomatis detected in women assisted at a sexually transmitted disease public clinic in Belo Horizonte, one of the major cities in Brazil. We also establish the prevalence of this infection in this particular population, searching at the same time for relationships between genotypes, clinical signs, and symptoms.
Materials and Methods
Endocervical samples were collected from 100 women attending a government center for treatment and control of sexually transmitted disease, referred to here as STD clinic, in the city of Belo Horizonte, Minas Gerais, Brazil. Clinical samples were collected with endocervical brushes during routine pelvic examination, subsequently placed into sterile collection tubes containing 2 mL of transport buffer (NaH2PO4 20 mM, Na2HPO4 20 mM, sucrose 20 mM, glutamin 5 mM, gentamycin 50 μg/mL, vancomycin 100 μg/mL, 25 μg/mL anfotericin B supplemented with 10% of inactivated fetal calf serum), and transported to the laboratory for further processing. Before analysis, samples were vortexed and centrifuged for 30 minutes at 16,000g. After supernatant elimination, the resulting pellet was dissolved in 200 μL of 10 mM Tris-HCl (pH 7.5) and stored at −20°C until use. For PCR, the freeze suspension was thawed and digested with proteinase K (0.5 mg/mL) for 1 hour at 56°C and then 10 minutes at 95°C,23 followed by alcoholic precipitation.
PCR of Endocervical Samples
Cryptic Plasmid Detection.
To test DNA sample quality, the β-globin gene was amplified and analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining (Table 1).
Detection of C. trachomatis was done by a PCR designed in our laboratory to amplify a sequence in the cryptic plasmid generating a fragment of about 512 bp. PCR was performed with 2.5 mM MgCl2, 200 μM dNTPs, 10 pmol of each primer (H1/H2), 1.5 U of Platinum Taq DNA polymerase (Invitrogen, CA) and 2 μL of sample in a 20 μL final volume. The reaction mixture was incubated for 5 minutes at 94°C, followed by 40 cycles of 1 minute at 94°C, 1 minute at 45°C, and 1 minute at 72°C, and a final elongation step of 7 minutes at 72°C. The entire amplified PCR product was analyzed by polyacrylamide gel electrophoresis. C. trachomatis serovar L2 DNA was used as a positive control.
Omp1 Amplification and Sequencing.
Samples shown to be positive for C. trachomatis by PCR detection of cryptic plasmid were selected for omp1 genotyping. The DNA from endocervical samples was used to amplify a fragment, approximately 1100 bp, of the omp1 gene by PCR (primer pairs showed in Table 1) consisting of 3 minutes at 94°C followed by 35 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C. PCR products were analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining. When necessary, samples were subjected to a Nested-PCR designed to generate a DNA fragment of approximately 1015 bp. Briefly, 1 μL of the first omp1 PCR product was added to a PCR mixture containing 10 pmol of each primer (JP1/JP2) (Table 1), 2.5 mM MgCl2, 200 μM (each) deoxynucleoside triphosphate, and 1.5 U Platinum Taq DNA polymerase (Invitrogen). Nested PCR consisted of 3 minutes at 94°C followed by 40 cycles of 1 minute at 94°C, 1 minute at 45°C, and 1 minute at 72°C. At the end, a temperature delay step of 7 minutes at 72°C allowed the elongation step. PCR products (20 μL) were analyzed by agarose gel electrophoresis. A fragment of approximately 1 kb from each sample, corresponding to omp1 gene DNA, was cloned into pCR4-TOPO vector (Topo TA Cloning Kit for sequencing and TOPO10 one shot cells; Invitrogen). After being tested by omp1 PCR, positive clones were selected for plasmidial DNA purification and sequencing. At least two clones from each sample were sequenced (ABI PRISM—Applied Biosystems INC.) using M13 universal primers. Omp1 nucleotide sequences obtained were analyzed using the BLASTN, BLASTN gapped, and BLASTX software packages available at the National Center for Biotechnology Information.
Sequences Alignment and Phylogenetic Tree Construction.
Phylogenetic tree analyses were used to demonstrate the evolutionary relationships between clinical isolates and reference strains of C. trachomatis obtained from GenBank. MOMP sequence, representing C. psittaci (GenBank AY762613), was used as an outgroup to root the tree. Nucleotide sequences of the omp1 gene determined in this study were aligned using the MEGA program (Version 3.1). The mean genetic distance within a genotype group was estimated as the average of all p distances between sequences of the same genotype for all possible sequence pairs obtained with MEGA (Version 3.1). Standard error was obtained using the bootstrap procedure with 1000 replicates, and 95% confidence intervals (CIs) were calculated in the usual fashion.
Nucleotide Sequence Accession Numbers.
The nucleotide sequence data reported in this paper appear in the NCBI nucleotide sequence databases with the accession numbers listed in Table 2.
A group of 100 women attending an STD clinic during a period of six months had endocervical samples collected to investigate C. trachomatis infection. The women ranged in age from 15 to 54 years old with a mean age of 27.9 years. All declared an active sexual life and most (67%) were between 15 and 29 years old.
Chlamydia trachomatis Prevalence
Endocervical specimens of all 100 women were tested for chlamydial infection using an in-house PCR designed to amplify a 512 bp fragment of C. trachomatis cryptic plasmid. Of the 100 samples analyzed, 19 (19%) were positive for C. trachomatis. A clear age dependency was observed, showing a high prevalence rate among younger women. The overall prevalence rates of C. trachomatis were 68.4% in group I (age 15–25 years old), 26.3% in group II (26–35 years old) and 5.3% in group III (age 36–55 years old).
Positive clinical samples for the cryptic plasmid PCR were subjected to a Nested-PCR for omp1 gene amplification and sequencing. Fifteen samples were positive for omp1 amplification and 9 of them were cloned and sequenced, generating 12 analyzed clones. Omp1 amplified fragment containing all four VDs allowed the identification of nucleotide sequences for genotype characterization (Table 2). Sequences analysis of the four variable regions of each sample resulted in the identification of four genotypes (genotype E—four sequences [33.3%]; genotype D—four sequences [33.3%]; genotype F—two sequences [16.7%], and genotype K—two sequences [16.7%]) by comparison with prototypes of each genotype deposited in GenBank (Accession numbers X62918, X52557, X52080, AF063204) (Fig. 1). Blast similarity data are shown in Table 2. Two sequences are identical to those found in GenBank and the other 10 showed 26 point mutations (Table 2). Twenty mutations (nucleotide changes) (77%) resulting in amino acid substitutions were observed in the analyzed sequences. Twenty-five (96.2%) were located in constant domains and only one substitution was located in VD intravenously, which was nonsilent.
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.
Analysis of omp1 DNA sequences from two clinical samples revealed more than one genotype, characterizing mixed infections. We found one individual with three genotypes representing two serotypes, D and E. Another clinical sample presented two genotypes: both variants of serotype K (Table 2).
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).
The prevalence of C. trachomatis has been identified in various areas of the world and studies done in different countries show that C. trachomatis is the main bacterial agent of sexually transmitted diseases. These studies also show that two factors are important to consider: the population analyzed and the sensitivity and specificity of the evaluation test.
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
Nowadays, PCR is considered to be the gold standard for detection of C. trachomatis infection because of its high sensitivity and specificity. Studies done by different groups have shown that a variety of regions of the bacterial genome can be used as a target for the detection of infection using this technique. Regions such as ribosomal RNA and omp1 gene can be used to identify the infection,25–28 but cryptic plasmid is considered to be the most appropriate for clinical tests since C. trachomatis has 7 to 10 copies per organism. On the other hand, the omp1 gene is suitable for genotyping C. trachomatis because it has conserved regions that are ideal for anchoring primers and variable domains that are appropriate for differentiating genotypes.1,5,27–30 Currently, omp1 gene sequencing enables the identification of all 19 serotypes of the bacteria. During this work, we developed an in-house PCR targeting the cryptic plasmid for detection of C. trachomatis in endocervical specimens and a Nested-PCR for omp1 amplification that enabled genotyping of positive samples.
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.
Of the 100 women analyzed, 19 (19%) were positive for C. trachomatis, coinciding with previous studies done under similar conditions in Brazil, using nucleotide amplification tests as well, that showed a prevalence range of 8.9% to 20.7%.20,21,35 Our results are also in agreement with an expressive number of studies done in a diversity of countries stating a wide range of prevalence rates, 4.3% to 30.8%36–39 for C. trachomatis infection. This includes studies done in other South American countries, such as Argentina, that found a prevalence of 11.5%,40 and Peru, with a prevalence rate of 22% among young people.41 The diversity of results reflects the variety of populations studied and the methods employed for infection detection.
Our Nested-PCR, designed to amplify a fragment of the omp1 gene, showed almost 80% sensitivity. A high sensitivity of PCR directed to plasmid over omp1 gene is expected given that this gene has only one copy per organism, whereas plasmid DNA is present in approximately 7 to 10 copies. Our results can be favorably compared with failure rates reported by others, which achieve 24%, although Jurstrand et al. reported a failure rate of only 1%.5 Discrepancies in the rates of omp1 detection observed in different studies are somehow expected since there is a diversity of primers, which are directed to different sites of the gene and are designed to amplify DNA fragments to varied extents. It is important to mention that PCR is also sensitive to specimen quality and to its maintenance during the interval between collection and analysis.
Despite the significant number of studies demonstrating a high prevalence of C. trachomatis infection in Brazil, until now there have been no studies describing the characterization of Brazilian serotypes and genotypes. This is important for epidemiologic studies and for studies aimed at identifying an association between genotype and the diverse clinical manifestations of this infection in Brazilian people, as well as the association with drug resistance.
A heterogeneous population of C. trachomatis prevailing in the female population evaluated was found with the identification of genotypes D, E, F, and K. To our knowledge, this is the first study to describe the genotypic diversity of C. trachomatis associated with genital infection in Brazilian women. C. trachomatis genotyping revealed that genotypes D and E were found most frequently, as observed in other studies on genotypic diversity of cervical samples done in other countries.2,5,42
An association between C. trachomatis genotype and clinical manifestations related to the infection was also investigated. Some authors10,43 postulate a connection between serovars and virulence, but others report a lack of relationship between C. trachomatis serovars and genital signs, symptoms, and disease.4,44,45 Of the 19 women found to be positive for C. trachomatis infection, 14 (73.7%) showed signs of cervical inflammation, such as an elevated number of polymorphonuclear neutrophils at Gram stain (more than 10 per microscopic field). Nevertheless, a correlation could not be established between the identified genotypes and clinical signs or symptoms of infection. It is important to mention, though, that five of the women who tested positive for C. trachomatis infection were asymptomatic and presented no sign of infection. No association with a specific genotype could be identified for these women.
A high number of nonsilent substitutions was observed in the omp1 gene, and this fact can suggest the occurrence of phenotypic selection or host selection and bacterial adaptation. There are studies reporting omp1 genetic variability in the range of 10% to 81%.10,13,46–48 We found 10 genetic variants out of a total of 12 omp1 gene sequences. Minor sequence variations in genotype F were found (only 3 out of 25), results that agree with two studies done in Sweden reporting genotype F with 2% to 4% divergence of reference sequences.5,49 Genotype E is reported to be the most conserved in different geographic regions.4,5,50,51 Interestingly, in the current study, genotype E showed the highest mutation rate, 1.08%, which, however, is still lower than that reported by Dean et al.,52 who found a mutation rate of 16% in genotype E when analyzing 67 clinical samples.
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.
Our study presents the characterization of C. trachomatis genotypes identified in a population of women in Brazil. It also confirms the high prevalence of C.trachomatis infection among younger women and reinforces the importance of implementing better diagnostic methods for the detection of this infection in the routine of public service STDs laboratories.
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