Vertical Transmission of Chlamydia trachomatis in Chongqing China
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- Yu, J., Wu, S., Li, F. et al. Curr Microbiol (2009) 58: 315. doi:10.1007/s00284-008-9331-5
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This is the first study to investigate vertical transmission of Chlamydia trachomatis in Chongqing China. For this study, 300 cervical swab samples from pregnant women and 305 nasopharygeal swab samples from their babies (605 specimens) were collected for nest polymerase chain reaction (nPCR) of the ompl gene, which encodes the major outer membrane protein (MOMP) and typed C. trachomatis using Cleavase fragment-length polymorphism (CFLP) labeled with digoxin. From these samples, 11% (33/300) of pregnant women samples were successfully amplified. The vertical transmission rate of C. trachomatis from mother to baby was 24% (8/33). The vertical transmission rates were 66.7% (6/9) for mothers with vaginal delivery and 8.3% (2/24) for those with cesarean section. The incidence of premature membrane rupture among C. trachomatis-positive pregnant women was 30.3% (10/33), which was greater than among those who were C. trachomatis-negative (13.5%, 36/267; χ2 = 4.2; p < 0.05). Four genotypes including type E (3 pairs), type F (2 pairs), type H (2 pairs), and type D (1 pair) were observed by CFLP assay labeled with digoxin and confirmed by DNA sequencing in the 16 C. trachomatis-positive samples from eight pregnant women and their eight infants. Each pair of matched maternal–infantile samples showed identical CFLP. This study showed the incidence of C. trachomatis infection in pregnant women, the vertical transmission rate for C. trachomatis, and the genotypes of C. trachomatis in Chongqing, China. The CFLP assay labeled at the 5′ end of the forward primer with digoxin was first used successfully to genotype of C. trachomatis. As a promising method for C. trachomatis genotyping, CFLP had good sensitivity, reproducibility, and simplicity and no radioactive contamination.
Chlamydia trachomatis infection is the most prevalent sexually transmitted disease. It is estimated that 89 million cases occur annually worldwide . The incidence of genitourinary tract C. trachomatis infection among pregnant women was 3.62% to 46.2% during the recent 5 years in China, and the incidence of cervical C. trachomatis infection among parturient pregnant women was 11.3% to 14.0% in Chongqing . No subjective symptoms are manifested by 85% to 90% of infected pregnant women. If investigation and prevention are not initiated promptly, C. trachomatis infection will cause infertility, abortion, eccyesis, and etc., it may even result in fetal infection by vertical transmission, which could be very dangerous.
The prevalence of C. trachomatis infection vertical transmission was 34.8% to 55.0% in China during the recent 5 years. Because 50% of infected men and 80% of infected women are asymptomatic, the number of reported cases represents only a fraction of the infected population .
Currently, 19 human serovars and related variants have been identified by using polyclonal and monoclonal antibodies against the major outer-membrane protein (MOMP), which is encoded by the ompl gene . Among the serovars, A, B, Ba, and C are commonly associated with trachoma, serovars D to K are commonly associated with urogenital infections, and serovars L1 to L3 are commonly associated with lymphogranuloma venereum . Moreover, serovars G, I, and D are associated with cervical squamous cell carcinoma, and serotype K is associated with infertility [5, 6].
Recently, many techniques have used to genotype C. trachomatis such as DNA sequencing and restriction fragment-length polymorphism (RFLP) . Cleavase fragment-length polymorphism (CFLP) analysis was demonstrated to be effective, reproducible, rapid, and discriminatory for Neisseria meningitidis epidemiology . Although many studies have reported the distribution and genotyping of C. trachomatis, few have focused on the characteristics of C. trachomatis vertical transmission. In this study, we first demonstrated the characteristics of C. trachomatis vertical transmission in Chongqing China. More importantly, we evaluated CFLP as a genotyping method for C. trachomatis, which proved to be a promising means that might be used for DNA sequence-based subtyping.
Materials and Methods
A total of 605 specimens including 300 endocervical swab specimens from pregnant women and 305 nasopharygeal swabs of their neonates (5 pairs of twins) were collected from April 2003 to February 2004 in Chongqing Women and Children’s Health Care Institute. For collection of the vaginal swabs, a sterile swab was inserted 1 to 2 cm into the endocervical canal, then rotated for 2 cycles and withdrawn, avoiding contact with vaginal surfaces. The swab was temporarily stored in saline solution. Similarly, nasopharygeal swabs were collected using a cotton pledget covered by a sterile plastic tube after they were inserted into the nasopharynx and rotated for 2 cycles, withdrawn, and stored in saline solution.
Ompl Gene DNA Extraction and Nest Polymerase Chain Reaction
The ompl gene DNA extraction was performed according to the C. trachomatis DNA extraction kit. Samples were collected and centrifuged at 12,000 rpm for 10 min. The supernatant was removed, and 50 μl of Tris-ethylenediaminetetraacetic acid (EDTA) was added to the pellet and boiled for 10 min after mixing. The samples were centrifuged at 10,000 rpm for 5 min and stored at −20°C for ompl polymerase chain reaction (PCR) as a template.
The ompl gene nest PCR (nPCR) comprised two steps of gene amplification, conducted with outer primers C-1 (5′-ACTTGGTGTGACGCTATCAGC-3′) and C-2 (5′-GTTCCTACTGCAATACCGCAAGA-3), the stable domains outside the variable domains (DV) 1 to 4 including the variable domains 1 to 4 and the inner primers C-3 (5′- GGCAAGCAAGTTTAGCTCTCTCT-3′) and C-4 (5′- GTTCCTACTGCAATACCGCAAGA-3′), which were the s domains outside DV4. The reaction system included 2.5 μl 10 × PCR buffer, 2 mmol/l magnesium chloride, 200 μmol/l dNTPs, 0.5 μmol/l sense primer, 0.5 μmol/l antisense primer, 1 U Taq DNA polymerase, 5 μl template, and 13.8 μl diethylpyrocarbonate (DEPC) solution.
Primary and nested omp1 PCRs were performed using the PTC200 thermocycler (MJ Research, Waltham, MA, USA) under the following conditions: 6 min of denaturation at 94°C, followed by 35 cycles of amplification at 93°C for 45 s, 58°C for 45 s, and 72°C for 1 min. As a positive control, DNA extracted from reference serovar L2 strain culture was used, whereas double-distilled water was used as a negative control in each omp1 amplification. The omp1 nPCR products were visualized after electrophoresis in 2% agarose gels by ethidium bromide straining.
The opml gene DNA from eight pairs of the C. trachomatis-positive matched maternal–infantile sample was extracted and amplified using the primers with the same sequence under the same condition described earlier. However, the forward primers were labeled at the 5′-end with digoxigenin. The PCR products were purified with a QIAquick PCR purification kit (Qiagen, Venlo, The Netherlands, Germany) for CFLP. The Cleavase 1 reaction was performed according to the CFLP kit manufacturer’s instructions (Third-Wave Technology, Madison, WI, USA) with optimization. Briefly, 4 μl of purified DNA was reconstituted in 9 μl of DNA buffer heated to 95°C for 15 s. Next, the temperature was dropped to 35°C, at which temperature 9 μl of a master mixture containing 2 mmol/l of MnCl2 (2 μl), 2 μl of 10 × CFLP buffer (10 mmol/l morpholinepropanesulfonic acid [MOPS], pH 7.5), and 25 U of Cleavase I (1 μl) was added to the DNA solution. The solution was heated to 85°C at 0.1°C /s, and the reaction was terminated with a 16-μl stop solution. All the samples in this study were analyzed at this incubation temperature and time frame.
Cleaved products were electrophoresed on 8% denaturing acrylamide gel (10 × 15 × 0.06 cm) at 600 V and 40 to 50 mA, then transferred onto a nylon membrane with a pore size of 0.2 μm (Schleicher & Schuell, Keene, NH, Germany) by dry blotting overnight. The membranes were agitated for 15 min and blocked in 1% blocking agent (Boehringer Mannheim, Ingelheim am Rhein, Germany) for 15 min twice. After the blocking agent was removed, antidigoxigenin antibody labeled with alkaline phosphatase (Roche Co., Palo Alto, CA, USA) was added at a 1:5,000 dilution in antibody buffer (0.1 mol/l tris-HCl and 0.1 mol/l NaCl, pH 7.5) and shaken for 60 min. The membranes were washed three times (5 min each) with 0.1% sodium dodecyl sulfate in Genius Buffer I (Marshal Osaka, Japan) (0.15 mol/l NaCl and 0.1 mol/l Tris-HCl, pH 7.5) followed by three washes (5 min each) with Genius buffer (0.1% sodium dodecyl sulfate [SDS], 0.15 mol/l NaCl, and 0.1 mol/l tris-HCl, pH 7.5). To each membrane, nitroblue tetrazolium (NBT)/BCIP diluted with buffer (0.1 mol/l tris-HCl and 0.1 mol/l NaCl, pH 9.5) at a dilution of 1:200 was applied as a chemiluminiscent substrate in the detection system. The membranes were carefully wrapped in clean Saran wrap without drying, exposed to a radiographic film, and developed in an automatic film developer.
The opml gene DNA from eight pairs of C. trachomatis-positive matched maternal–infantile sample were DNA sequenced using the Sanger method on an automated 377A sequencing instrument (Applied Biosystems Inc., Foster City, CA, USA). Sequences from both strands were assembled with the DNAassit software (Hugh Patterton, Version 1.02). The data generated were compared with the CFLP fingerprints of each isolate. The DNA sequence data were compared with the sequence published by Yuan et al. .
Data were analyzed using SPSS11.5 (SPSS, Inc., Chicago, IL, USA). Statistical analysis of the data was performed using the chi-square or Fisher’s exact test. A p value less than 0.05 was considered statistically significant.
Vertical Transmission of C. trachomatis in Chongqing, China
Genotyping by CFLP Analysis and DNA Sequencing
Chlamydia trachomatis currently is the most common organism causing sexually transmitted disease worldwide. In pregnant women, C. trachomatis infection is unique in that it can cause infection in the fetus and infant through vertical transmission, resulting in neonatal conjunctivitis and pneumonia. In this study, we found that the prevalence of vertical transmission with cesarean section was lower than with vaginal delivery. The prevalence of vertical transmission in this study (24.2%) was lower than that previously reported, which may be explained by the high rate of cesarean section (among the 33 pregnant women whose cervical scrapes tested positive for C. trachomatis, 24 underwent cesarean section).
Findings show that PROM may occur due to ascending infection of C. trachomatis in the cervix . In this study, we found that the incidence of PROM among the C. trachomatis-positive pregnant women was greater than among the C. trachomatis-negative pregnant women. Organism colonizing of the cervix is known to be connected with PROM, including group B streptococcus, diploccoccus of Neisser, bacillusoid, C. trachomatis, and mycoplasma urealytium. Proteinase secreted by bacterium hydrolyzes extracellular material of the fetal membrane, which reduces the tensile strength of tissues. The bacterium enhances its proteinase activity by activating peroxidase of leucocyte, which weakens the resistance force of the fetal membrane, with the result that the number of collagen fibers decreases and the friability of the membrane increases.
The production of prostaglandin can be increased by the increased phosphatidase produced by bacteria and the bacterial endotoxin. The increased prostaglandin results in uterine contraction, an increase in intrauterine pressure, and PROM.
Although the McCoy cell culture is considered to be the gold standard for detecting C. trachomatis, it has limited application in clinical practice because the sensitivity can be affected by the extraction, transportation, preservation, and culture of specimens. It also has limitations for general laboratories because cell culture needs not only a long time but also rigorous procedures. The molecular biologic technique is simple, rapid, accurate, and practical, thus showing promise for use in C. trachomatis detection.
Many studies and literature reviews have suggested that nucleic acid amplification tests are better than other diagnostic methods for detecting C. trachomatis. Polymerase chain reaction, the most widely used method, currently can amplify three types of target DNA fragment including the commom plasmid of C. trachomatis, 16SrRNA of C. trachomatis , and the ompl gene . One strain of C. trachomatis harbors only 1 matched copy of the ompl gene but 7 to 10 copies of the common plasmid gene. The more copies the DNA template has, the easier it is to amplify and thus the higher sensitivity of the plasmid amplification. Although 16SrRNA has a great copy number, it is complicated to prepare the RNA template, and thus the sensitivity of amplification is 10 times lower than that of plasmid .
The sensitivity and specificity of PCR can be improved with nPCR, which first increases the quantum of the initial template by amplifying the external nest primer of nPCR and then multiplies the target gene to a visible level by amplifying the internal nest primer. The nPCR needs two different pairs of primer and has a secondary amplification function. Therefore, the sensitivity and specificity of nPCR is higher than that of the traditional PCR. Because ompl-nPCR has high sensitivity and specificity and needs no other special instruments, it is a good technique for detecting C. trachomatis.
Many methods for C. trachomatis genotyping are reported such as PCR-based RFLP and sequencing of the amplified omp1 gene, which encodes the MOMP [14, 15]. The CFLP procedure is another rapid, simple genotyping technique besides single-strand conformation polymorphism (SSCP) and RFLP.
To our knowledge, our study is the first to genotype C. trachomatis using CFLP labeled with digoxin. The CFLP analysis is a DNA-based subtyping method with the capacity for direct assignment of alleles based on the nucleotide sequences of genes. Because of its sensitivity in detecting nucleotide changes, CFLP can be applied to the rapid screening of many strains during investigations of outbreaks or to surveillance systems .
The discrimination power of CFLP is dependent on the DNA sequence of the locus examined such as gene mutation, gene insert, deletion mutation, and so on. Furthermore, CFLP can relatively position the mutated gene according to the mark. The CFLP technique has been applied to detect gene mutation such as human gene p53, which suppresses the growth of tumor ; CYp21, whose mutation can cause congenital adrenal cortical hyperplasia ; BRCA, one of the hereditary breast cancer susceptibility genes ; and the like. It is reported that no less than 70 types of DNA fragments of human bacterium and virus were detected for gene mutation using CFLP with a sensitivity of 96.5% to 98% and a specificity of 100% .
The CFLP technique has been applied to the genotyping of hepatitis C virus (HCV) and trichina worm. Sreevatsan et al.  examined the distribution for 85% of the HCV genotype in America using CFLP and DNA sequencing. Sreevatsan et al.  investigated the feasibility and validity of using CFLP for their large-scale screening study on the genotyping of patients with C hepatitis who are resistant to α-interferon (α-IFN). Cross matching of data generated in two different laboratories for 42 samples also produced identical CFLP fingerprints for each isolate. In addition, analysis of the same amplicon cleaved under different conditions (45°C or 55°C for 4 min) yielded identical patterns, thus confirming that CFLP is reproducible.
Compared with SSCP, CFLP can be used to analyze DNA fragments larger than 2 kbp . The sensitivity of SSCP decreases significantly with DNA fragments larger than 250 to 300 bp. As for RFLP, only the genomic DNA fragments that hybridize to the probes are visible with RFLP analysis, thereby limiting the ability of the technique to distinguish between closely related strains such as the genotypes of C. trachomatis. Furthermore, not only is it unable to reflect the antigenic variation caused by the alteration of amino acids produced by nucleotide substitution, but it also cannot distinguish types Da, Ia, and L2a of C. trachomatis. Therefore, it is not precise or sufficiently sensitive .
We previously analyzed the genotype of C. trachomatis from five neonatal nasopharyngeal swabs testing positive for C. trachomatis with traditional CFLP whose ompl VD 4 of C. trachomatis was labeled with [α-35S]dATP. This was foundational in establishing a new C. trachomatis genotying system.
On the basis of the study, we labeled the 5′ termini of primer with digoxin without the drawback of a short half-life, radioactive contamination, or difficulty with manipulation of the isotope. Furthermore, we also applied temperature-switched CFLP and optimized the reaction conditions of the technique for detecting the fragments of ompl VD 4 of C. trachomatis .We have detected four genotypes (E, F, H, and D) of C. trachomatis in the clinical specimens successfully using CFLP, with confirmation by DNA sequencing, which reflects the genotype of C. trachomatis in Chongqing China to some degree. In the study, we analyzed CFLP fingerprints of the same specimens in the different batches and different specimens with the same genotype, showing that CFLP has good reproducibility .
This study had several limitations. First, the clinic sample size of the study was small. Second, the standard strain of C. trachomatis was unavailable to us, so the genotype of C. trachomatis by CFLP had to be determined by DNA sequencing, which we also found effective. Third, the standard CFLP map of C. trachomatis had not been established, and more clinical samples are needed.
Despite these limitations, CFLP labeled with digoxin proved to be a promising method for C. trachomatis genotyping, with good sensitivity, reproducibility, no radioactive contamination, and simplicity.
We thank Bing Deng and Xiaoping Zhang for their technical assistance. In addition, we are grateful to Xiaoyun Zhong for helpful discussions. This work was supported by the National Natural Science Foundation of China (30170991).