|Year : 2017 | Volume
| Issue : 1 | Page : 74-79
Human papillomavirus and Chlamydia trachomatis infection in gyneco-obstetric outpatients from a mexican hospital
Laura Conde-Ferráez1, Jorge Ramiro Carrillo Martíez2, Guadalupe Ayora-Talavera1, María del Refugio González Losa1
1 Centro de Investigaciones Regionales, Universidad Autónoma de Yucatán, Mérida, Mexico
2 Centro Médico Nacional Lic. Ignacio García Téllez, Instituto Mexicano del Seguro Social Mérida, Mérida, Mexico
|Date of Web Publication||16-Mar-2017|
Centro de Investigaciones Regionales, Universidad Autónoma de Yucatán, Calle 96 s/n X Ave Jacinto Canek and Calle 47, Paseo de Las Fuentes, Merida, Yucatan, CP 97225
Source of Support: None, Conflict of Interest: None
Context: Human papillomavirus (HPV) and Chlamydia trachomatis are the most frequent sexually transmitted infections, usually asymptomatic. Persistent infection with high-risk HPV types and other cofactors such as the concomitant infection with C. trachomatis can represent a higher risk to develop cervical lesions; therefore, screening with sensitive methods could aid to identify women at risk. Aims: The aim is to determine the prevalence and concurrence of both infections, detected with in-house molecular methods, and to identify the risk factors associated to the infections in Mexican women. Subjects and Methods: This was a cross-sectional study including gynaecological-obstetrical medical outpatients from a Social Security Hospital in Southeast Mexico. After informed consent, cervicovaginal samples were collected and tested for HPV and C. trachomatis using polymerase chain reaction (PCR). HPV positives were further tested for high-risk HPV16, 18, 58 and low-risk 11 using real-time PCR. All methods employed were in-house. Data analyses included odds ratios (OR), Chi-square and linear regressions. Results: Women included were 233, aging 15–49 (mean 30 years), 52.8% were pregnant. For HPV and C. trachomatis testing, 230 samples were adequate, resulting in 48 (20.9%) and 15 (6.5%) positives, respectively; 4 (1.7%) were positive to both. The most frequent genotype identified was HPV58 (25% of typified samples). C. trachomatis positives were 73% asymptomatic, none had pelvic inflammatory disease or infertility histories. The only variable associated to HPV infection was the history of previous sexually transmitted disease (OR = 3.69,P= 0.0019). Conclusions: More than 25% of the population was infected with either agent. We successfully used in-house molecular methodologies for diagnosis and typing, showing HPV and C. trachomatis prevalence consistent to previous reports. Concomitant infections were found, HPV high-risk types were involved in half of these cases, representing a higher risk.
Keywords: Chlamydia, human papillomavirus, sexually transmitted infection, polymerase chain reaction
|How to cite this article:|
Conde-Ferráez L, Martíez JR, Ayora-Talavera G, Losa Md. Human papillomavirus and Chlamydia trachomatis infection in gyneco-obstetric outpatients from a mexican hospital. Indian J Med Microbiol 2017;35:74-9
|How to cite this URL:|
Conde-Ferráez L, Martíez JR, Ayora-Talavera G, Losa Md. Human papillomavirus and Chlamydia trachomatis infection in gyneco-obstetric outpatients from a mexican hospital. Indian J Med Microbiol [serial online] 2017 [cited 2020 Jul 13];35:74-9. Available from: http://www.ijmm.org/text.asp?2017/35/1/74/202324
| ~ Introduction|| |
Human papillomavirus (HPV) and Chlamydia trachomatis are the most frequent sexually transmitted infections in the world. Annually, there are 101 million of new cases of C. trachomatis infection in adults; and in the case of HPV, it is estimated that 209 million women are infected worldwide.
Both pathogens are the cause of important health problems. Untreated C. trachomatis infection can cause lymphogranuloma venereum or lead to complications including, pelvic inflammatory disease, ectopic pregnancy and infertility; whereas HPV high-risk genotypes can cause cervical cancer (CC) one of the most important cancers in women, it is estimated that 87% of CC related deaths occur in the less developed countries.
The true burden of C. trachomatis infection is unknown, mainly because of the high percentage of asymptomatic cases (up to 70%–80% in women) or symptoms being unspecific or improperly recognised, for many women remain untreated. In some settings, routine screening has been implemented for young women and high-risk populations, but the costs are high and most laboratory methods are technically demanding.
In the case of HPV, the molecular detection of high-risk genotypes can be used in conjunction to the cytological screening as the part of public CC early detection programs. Nevertheless, the infection with high-risk genotypes is the necessary cause of CC but it is not sufficient  and most of the HPV infections are spontaneously cleared without developing cervical lesions. Many cofactors have been suggested for the development of CC and precursor lesions, one of them is C. trachomatis infection, presumably as a consequence of the chronic inflammation of the endocervix, in conjunction to HPV infection. Mechanisms of carcinogenesis that might be enhanced by C. trachomatis are not fully understood (latest findings are reviewed in Silva et al., 2014).
Therefore, it is important to early identify the infection with these agents, using sensitive detection methods that can be scalable to high throughput with relatively low costs, such as polymerase chain reaction (PCR). In this work, we aimed to screen a group of Mexican women attending to a Social Security Hospital for gynaecological and obstetrical medical evaluation, to describe the prevalence and concurrence of both infections, and to identify the risk factors associated.
| ~ Subjects and Methods|| |
The studied population included outpatients of the gynaecological and obstetric service in a Regional Social Security Hospital in Mérida, Yucatán, during August 2010–January 2011. All participants signed an informed consent. The project was approved by the corresponding scientific and bioethical committee from the hospital (register 059-P/2009).
Sampling was performed by a gynaecology/obstetrics medical resident. Cervicovaginal samples were collected by a with a cytobrush and deposited in 5 ml of phosphate-buffered saline with penicillin 500 U/ml, streptomycin 500 µg/ml and gentamicin 4 mg/ml. Samples were kept at 4°C during transportation to the laboratory in the university for further analyses.
Cervicovaginal samples were processed for manual DNA extraction using a modified protocol. Briefly, 500 µl of transport media containing samples were centrifuged full speed (14,000 rpm) in a table top centrifuge and the cell pellets were resuspended in 200 µl of the lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM EDTA, 250 µg/ml Proteinase K), and incubated at 56°C for 2 h with agitation. The cell debris was further pelleted down and the nucleic acids were precipitated with two times total volume of ice cold absolute ethanol with 0.3M sodium acetate pH 5.2, followed by twice washing with 70% ethanol. After air drying, the pellets were finally diluted in nuclease-free water.
Human papillomavirus testing
Extracted DNAs were tested for HPV using My09-My11 universal primers to amplify a 450 bp region from the L1 gene. As internal control, a 268 bp region from the human b-globin gene was amplified using GH20 and PCO4 primers. PCRs were performed in a volume of 50 µl, with 200 µM dNTP's, 3 mM MgCl2, 50 pmol each oligonucleotide, 1 U GoTaq Polymerase (Promega, Madison, USA). Thermal cycler conditions as follows: 95°C, 9 min; 38 cycles of 95°C 1 min, 55°C 1 min, 72°C 1 min; final elongation 72°C, 5 min. The products of PCR were visualised in silver stained acrylamide gels (8%).
HPV genotypes were identified in HPV positive samples using real-time PCR in the equipment StepOne Real-Time PCR System (Applied Biosystems, Foster City, USA). Reaction mixes were performed with Platinum Sybr Green qPCR SuperMix-UDG, 50 nM de Rox Reference Dye 2 µL DNA, in a final volume of 25 µL and the corresponding specific primers for each genotype as follows. HPV16 and 18 were detected using 8 pmol of previously described primers  HPV-16Fw (5'-TTTGTTACTGTTGTTGATACTAC-3'); HPV-16Rev (5'-GAAAAATAAACT GTAAATCATATTC-3'); HPV-18Fw (5'-TTTGTTACTGTGGTAGATACC-3'), HPV-18Rev (5'-GAAAAATAAACTGCAAATCATATTC-3'), with the following conditions: 50°C 2 min, 94°C 10 min, 40 cycles of 95°C 20 s and 55°C 1 min. The Tm of the expected amplicons was 73.9°C for HPV16 and 76.4°C for HPV18.
HPV58 was detected using with 10 pmol of primers described by Chan et al., 2007 HPV-58E7Fw (5'-ATGTGACAGCTCAGACGAGG-3') and HPV-58E7Rev (5'-CAGCTGCTGTAGGGTTCGT-3') with the following conditions: 50°C 2 min, 95°C 3 min, 40 cycles of 95°C 15 s and annealing at 60°C 1 min. The Tm of the expected amplicons was 82.4°C.
HPV11 was detected with 10 pmol previously described primers  11S (5'-GGAATACATGCGCCATGTGG-3') and 11A (5'-CGAGCAGACGTCCGTCCTCG-3') with the following conditions: 50°C 2 min, 95°C 10 min, 40 cycles of 95°C 30 s and 55°C 30 min. The Tm of the expected amplicons was 79.5°C.
A melting curve was performed by default after competition of all real-time PCR runs, according to the equipment manufacturer. To perform a qualitative analysis of the PCR products obtained, the melt analysis allows to measure the Tm of the expected products and identify any possible non-specific amplification by comparison to the controls.
As positive controls, plasmids containing the complete genomes of each genotype were included; these were kindly donated by de Villiers (HPV16, 18 and 11) and Matsukura (HPV58). Other genotypes were not tested for.
Chlamydia trachomatis testing
C. trachomatis detection was performed using an in-house end-point PCR method developed in New Delhi, India, which amplify a 368 pb from the phospholipase D endonuclease superfamily. This method was chosen because it was reported to have a higher sensitivity than other assays targeting other genomic regions. Reactions were performed using 1 U GoTaq polymerase (Promega), 25 pmol each primer, 200 µM dNTPs, 1.5 mM MgCl2 and 2 µl template DNA in a final volume of 50 µl. DNA from a reference strain (UW-3/CX serovar D) was kindly donated by Dr. M.G. Aguilera Arreola, and used to amplify the fragment that was cloned in a TOPO-TA vector (Invitrogen, Thermo Fisher Scientific, Carlsbad, USA) following the manufacturer's protocol, and used subsequently as positive control of amplification. The products of PCR were visualised in silver stained acrylamide gels (8%).
An instrument was applied to collect data concerning sociodemographic data, clinical, sexual and reproductive histories in order to identify risk factors. The following variables were analysed: age, marital status, pregnancy, symptomatic vaginal discharge, gestations, abortions, sexual onset, number of total sexual partners, history of sexually transmitted diseases (STDs), condom use for family planning method, and socioeconomic level.
All information and laboratory results were included in a database using SPSS software (v. 19 IBM Company, USA). Frequency of variables, Chi-square and crude odds ratios (ORs) were calculated using Epi Info 2000 SPSS (v. 19, IBM Company, USA). Linear regression analyses were performed to all variables that resulted significant in the bivariate tests.
| ~ Results|| |
Analysis of the population
From the 235 women included in this study, two were eliminated because of insufficient sample or data, and therefore, 233 were analysed. Age ranges were 15–49 (mean 30 years), 123 were pregnant (52.8%). Most were asymptomatic to STDs (159 women, 68.2%). Symptomatic patients (31.8%) presented pain (17 women 7.3%), bleeding (19 women, 8.2%) and vaginal discharge (38 women, 16.3%).
About marital status, 186 were married (79.8%), 24 in cohabitation with partner (10.3%), 16 single (6.9%), seven divorced or separated (3%).
In respect to reproductive history, number of gestations ranged from 0 to 8 (mean 2.28 gestations); 40 had history of abortion (17.2%, three missing data). Sterility was referred by three women (1.3%). History of pelvic inflammatory disease in six women (2.6%).
Regarding sexual history, sexual onset ranged from 12 to 32 years old (mean 18.76 years, one missing); 153 had only one sexual partner (65.7%), 58 two sexual partners (24.5%) and 20 three or more sexual partners (8%) two were missing data. Referred family planning methods, included condom use in 47 (20.2%), sterilisation of woman or partner in 34 (14.6%), none in 96 (41.2%), hormonal contraceptives in 31 (13.3%), intrauterine device in 14 (6%), and other methods in 11 (4.7%).
History of STDs was reported by 27 women (11.6%, 46 missing data), being HPV the most frequent (24/27). Information about HIV serostatus was not available.
Socioeconomic level according to the European Society for Opinion and Marketing Research (ESOMAR) classification was defined as follows: 47 low (20.2%); 129 medium-low (55.4%), 37 medium (15.9%), 17 medium-high (7.7%), one high (0.4%), and one missing datum.
Molecular detection of human papillomavirus and Chlamydia trachomatis
The DNAs were obtained from the 233 included samples, three samples were considered inadequate because of not amplifying the internal control β-globin, and therefore, they were not tested for HPV or C. trachomatis.
Infections were found in 27.4% (63/230) of the tested women, as follows: 20.9% were HPV positive (48/230), and 6.5% were C. trachomatis positive (15/230). Both agents were found simultaneously in 1.7% (4/230), representing 6.3% of all positives were concomitant infections (4/63). Taking in to account HPV positives, 8.3% were positive to C. trachomatis (4/48), while in HPV negatives 6% were C. trachomatis positive (11/182).
Bivariate analysis showed that C. trachomatis infection was not a significant risk factor for HPV infection (OR = 1.24, confidence interval: 0.36–5.13, Fisher's P = 0.3838).
The age distribution of the infected women is presented in [Table 1]. As seen, the positivity to C. trachomatis tend to decrease with age while the age distribution of HPV increases in the middle ages ranges and decreases in the older range. Nevertheless, logistic regression analysis showed non-significant results.
|Table 1: Distribution of human papillomavirus and Chlamydia trachomatis infection by age ranges|
Click here to view
About HPV positives, 77% were low to medium-low socioeconomic level, 43.7% were pregnant, and 83.3% were married.
Regarding C. trachomatis positive women, 93% of them were low to medium-low socioeconomic level, 73% were asymptomatic, 66% were pregnant and 60% were married. None of the C. trachomatis positive women had pelvic inflammatory disease or infertility histories.
The analysis of variables associated to the infections is shown in [Table 2].
|Table 2: Analysis of variables associated to Chlamydia trachomatis and human papillomavirus infection|
Click here to view
As seen, the only significant result is the history of previous STD is associated to HPV positivity, being 3.69 times more frequently found in patients with previous STD diagnosis. The other analysed variables were non-significant.
From the 48 HPV positives, 32 (66.66%) were available for genotyping HPV high-risk types 16, 18, 58 and low-risk type 11, the rest had not sufficient sample. The results were as follows: HPV16 9.4%; (3/32); HPV18 3.2% (1/31 one was not determined); HPV58 25% (8/32). None resulted positive to HPV11.
| ~ Discussion|| |
More than a quarter of the population studied was infected (27.4%). In the case of HPV, the prevalence of HPV in women from Mexico vary. A meta-analysis identified a prevalence of 15% in women with normal cytology. In a recent study was reported a general prevalence of HPV of 19.8% in obstetric patients from the same hospital. This can be comparable to present results, considering that in this study included women were at reproductive age, and HPV prevalence in pregnant women was 17.35%. Interestingly, HPV prevalence in non-pregnant was 24.7%.
About C. trachomatis, the prevalence found in our study was relatively low (6.5%) in comparison with reports from other Latin American countries, such as 20.7% in sexually active women from Brazil  and 21% asymptomatic pregnant women in Trinidad and Tobago.
An important study of both infections reported a prevalence of 13.8% HPV and 9.7% C. trachomatis. This is probably the largest study (2436 women) detecting infection of both HPV and C. trachomatis by molecular methods, that included women living in the Mexican-United States border. C. trachomatis prevalence was 16% in HPV-positive women from this last population; similarly to what has been found in Brazil (13% prevalence in HPV positives) and in India (14% prevalence in HPV positives). In comparison, in our study we found 8.3% prevalence of C. trachomatis in HPV positives, and 6% in HPV negatives.
The prevalence of concomitant infection in the studied population was 1.7%, (4/230). C. trachomatis infection has been considered synergistic to HPV infection, contributing to an increased risk of developing cervical lesions throughout various proposed mechanisms., Concomitant infections have been reported in adolescents from the United States as frequent as 25%, and importantly C. trachomatis infection was associated to persistence of high-risk HPV infection.
In this work, women with HPV high-risk types HPV16 and HPV58 were found in half of the tested chlamydia positives and may represent a higher risk for the women as cofactor for high-risk HPV induced disease, and therefore, these concomitant infections, although infrequent, should not be disregarded.
At present, the syndromic management of STDs avoids the identification of asymptomatic cases such as observed in this study, because only 26% of the C. trachomatis positives were symptomatic, and only one woman out of the four identified having HPV and C. trachomatis concomitant infections was symptomatic. These women must be considered in a higher risk and should be subject to follow-up.
The only associated variable to HPV infection was a previous history of STDs. The most frequently reported STD was indeed HPV. Other referred infections were Trichomonas and Candida.
All methods performed were in-house, no commercial kits were employed as to reduce the costs for the feasibility of this project. The setup of in-house methodologies is important for future applications in other epidemiological research projects in our laboratory, because it is part of a public University with usually very limited budget. For this study, we employed a previously very well established MY-PCR  which is widely used internationally, and incorporated a method for C. trachomatis recently developed in India with promising results. In addition, we incorporated the use of real-time PCR for genotyping the HPV positives, to distinguish the women at the highest risk. In this respect, HPV58 was frequently found (25% of typified samples). This is in agreement with previous reports from Mexico, especially in our region,,, and show the importance of HPV58 which is also a frequent genotype in Asia  but is usually not contemplated in studies from other regions, and is not covered by the licensed vaccines Gardasil ® (Merck, Merck and Co. Whitehouse Station, USA) and Cervarix ® (GlaxoSmithKline, GlaxoSmithKline, Philadelphia, USA).
| ~ Conclusions|| |
More than a quarter of the population studied was infected with either agent. We successfully used affordable in-house molecular methodologies for these STI diagnoses and typing, showing prevalences of HPV and C. trachomatis consistent to previous reports. Concomitant infections were low in our population, and the risk factors were not significant; however, it is worth mentioning that women with HPV high-risk types were found in the tested C. trachomatis positives and therefore may represent a higher risk for the women. Therefore, these concomitant infections although infrequent, should not be disregarded. A limitation of our study is that we were not able to document the follow-up of the patients. Longitudinal studies addressing C. trachomatis as a cofactor for HPV induced lesions are needed.
We would like to thank our students and trainees: AA Araujo Vargas, G Vega Cepeda and EG Petul López for their support in sample collection and processing. We thank Dr. MG Aguilera Arreola (Escuela Nacional de Ciencias Biológicas-IPN) for donating control DNA from C. trachomatis; EM de Villiers (German Cancer Research Center-DKFZ) for HPV16, 18 and 11 plasmids controls, and T Matsukura (National Institute of Health, Tokyo) for HPV58 plasmid control.
Financial support and sponsorship
Universidad Autónoma de Yucatán.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]