Chlamydia Trachomatis Infection in High-Risk Human Papillomavirus Based on Cervical Cytology Specimen

Objective: High-risk human papillomavirus (HR HPV) was associated with the development of cervical cancer. Asymptomatic Chlamydia trachomatis (C. trachomatis) infection is the most common bacterial, sexually-transmitted infection. This study aimed to investigate the association of C. trachomatis in positive HR HPV and the cytological results from liquid-based cytology (LBC). Methods: 150 residual LBC specimens were collected; all of which had undergone cytology and HPV testing by Cobas. The samples were established as C. trachomatis using real-time PCR (RT-PCR) with Cryptic F/Cryptic R primers. Results: Of 150 positive HPV findings, the most common (72.7%, 109/150) were the 12 other HR HPVs (viz., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). The cervical cytology of those positive HR HPVs were mostly negative (70.0%, 105/150). The C. trachomatis infections in positive HR HPV were 16% (24/150) HPV. The analysis of the abnormal cytology revealed that 41.6% had C. trachomatis co-infection (C. trachomatis and HPV infection) viz., LSIL (20.8%), HSIL (12.5%), and ASC-US (8.3%). A comparison with positive HPV without C. trachomatis co-infection revealed that the highest prevalence was for LSIL, ASC-US, and HSIL (11.1%, 10.3%, and 6.4%, respectively). There was no difference between the abnormalities and negative cervical cytology with negative and positive C. trachomatis co-infection in HR HPV positive (p = 0.174). Conclusion: C. trachomatis infection was not significantly associated HR-HPV and abnormal cytology. This study confirms the increasing rate of C. trachomatis infection in asymptomatic women so routine screening for these infections has been suggested to (a) prevent complications such as the chronic pelvic pain associated with prolong infection and (b) reduce sexual transmission of the infection.


Introduction
. C. trachomatis can moreover establish asymptomatic, persistent infections by several mechanisms, including antibiotic resistance, immune evasion, and apoptosis (Chumduri et al., 2013). In addition, C. trachomatis is like other intracellular pathogens that can cause substantial changes in gene expression and protein production in the host at the transcriptional, translational, and post-translational levels. Whether the interaction between HPV and C. trachomatis infection will result in development of cervical cancer remains unclear (Elwell et al., 2016). The current study aimed to investigate the prevalence of C. trachomatis in persons positive and at high-risk of HPV (HR HPV) infection based on liquid-based cytology.

Specimen collection and preparation
The study protocol was reviewed and approved by the Ethics Committee of Khon Kaen University, Thailand (HE 591453). We collected 150 residual, routine, positive HPV DNA specimens based on liquid-based cytology (LBC) in ThinPrep ® between November, 2016 and November, 2018 using the Cobas ® 4800 system. The data comprised the individual results for HPV 16 and 18 and the pooled results for the 12 other HR HPV genotypes (viz., 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) processed at the Cytology Unit, Department of Pathology, Faculty of Medicine, Khon Kaen University. The cytological results were categorized following the 2014 Bethesda System as (a) negative for an intraepithelial lesion or malignant (NILM), (b) atypical squamous cells of undetermined significance (ASCUS), (c) low-grade squamous intraepithelial lesion (LSIL), (d) atypical squamous cells that cannot be ruled out as high-grade squamous intraepithelial lesion (ASC-H), (e) high-grade squamous intraepithelial lesion (HSIL), or (f) squamous cell carcinoma (SCC) (Nayar and Wilbur, 2015).

DNA extraction
The scraped cervical cells were washed twice with phosphate buffer saline (PBS). The cells were lysed in 200 µl of lysis buffer (10 mM Tris HCl, 0.1 mM EDTA pH 7.5, 1% SDS and 0.5 M NaCl), to which 15 µl of proteinase K was added. This was then mixed by vortex until clear then incubated at 60°C for 30 min. The protein was precipitated by addition of protein precipitation buffer (5 M potassium acetate, 11.5 ml of glacial acetic acid and 28.5 ml of distill water, pH 5.5): the mixture was centrifuged at 13,500 rpm for 5 min at 4°C. The DNA was precipitated with an equal volume of isopropanol and collected by centrifugation at 13,500 rpm for 5 min at 25°C and washed with 70% ethanol. The DNA pellet was air-dried for 15-30 min then re-suspended in 40 µl of distilled water and stored at -20°C until used.

C. trachomatis DNA detection
The quality of extracted DNA was checked by amplification of a housekeeping gene (Glyceraldehyde 3-phosphate dehydrogenase; GAPDH). The extracted DNA was determined using GAPDH gene detection. C. trachomatis DNA was detected using SYBR real-time PCR (RT PCR) with Cryptic F/Cryptic R primers. The primers were applied for a Chlamydia cryptic plasmid investigation, which amplifies the 71 bp DNA fragment between the open reading frame 1 and 2 (Jaton et al., 2006). The real-time master mix PCR for the cryptic plasmid region (amplifying ABI 7500 fast, Applied Biosystems) contained 5.0 µl of SYBR (SsoAdvancedTM Universal SYBR®Green Supermix), 0.3 µl of each primer, 2.4 µl of distilled water, and 2 µl of DNA sample. Amplification was performed by a pre-warming step at 50°C for 2 min, followed by denaturing at 95°C for 10 sec, 45 cycles at 95°C for 15 sec, and 60°C for 1 min.

Statistical analysis
The chi-square test was used to analyze the correlation between HPV and C. trachomatis DNA detection. A p-value < 0.05 was considered significant. All statistical tests were performed using STATA version 10.

Discussion
Sexually-transmitted viral infections of HPV in the genital tract are common. HPV infection may have transient symptoms, and spontaneous clearance occurs in 80% of cases as the virus is flushed by the host immune system without any cell changes. In approximately 20% of women, HPV infection may be persistent and later evolve into cervical cancer in up to 10% of cases (Abreu et al., 2012;Wohlmeister et al., 2016). Actually, the most important viral factor is the type of HPV. HR HPV genotypes may infect the epithelium persistently, inducing progression and contributing to carcinogenesis. Studies have shown that the most common HR HPV detected in carcinoma cases are HPV 16, 18, 31, 33, 39, and 45 (Muñoz 2000;Kraus et al., 2006;Parkin et al., 2008;Wohlmeister et al., 2016a). Kaliff et al., ( 2018) carried out a study and found that HPV 16 was the most prevalent genotype, followed by HPV 18: the two are the main genotypes detected in squamous cell carcinoma (Oliveira-Silva et al., 2011;Wohlmeister et al., 2016). The 12 other high-risk HPV genotypes (viz.,HPV 31,33,35,39,45,51,52,56,58,59,66,and 68) were highly prevalent in the present study; more in fact than HPV 16 or HPV 18.
The prevalence of C. trachomatis among asymptomatic women positive for HR HPV in the current study was 16%. The prevalence of genital chlamydia infection in the current study was higher than in previous studies. Thongkrajai et al., (1999) reported a respective prevalence of C. trachomatis using the ELISA method of 6.8%, 5.2%, and 6.7% in rural northeast Thai women attending antenatal, postpartum, and family planning clinics. The prevalence of C. trachomatis was investigated using multiplex PCR among students from the northern region of Thailand: the prevalence was between 3.2% to 7.5% (Whitehead et al., 2008). In Hanoi, Vietnam, the prevalence by ELISA technique of lower genital tract C. trachomatis infection was 3.8% in asymptomatic women attending maternal and child health and family planning clinics (Anh et al., 2003). By comparison, the prevalence of C. trachomatis infection in the United States was 1.7% (Torrone et al., 2014). In Iran, nested polymerase chain reaction (PCR) used to test the residual fluids of Pap smears: 12.5% were positive for C. trachomatis infection (Javanmard et al., 2018). Differences in study populations and methodologies used for C. trachomatis detection yield a wide variation in prevalence rates. The prevalence also varies by age and race/ethnicity. Common co-infections are often asymptomatic, so healthcare providers should routinely screen sexually active women and provide prompt treatment for infected persons (Torrone et al., 2014). Safaeian et al., (2010) C. trachomatis infection affects HPV persistence and progression to cervical pre-malignancy by a causal disruption of the epithelial tissue. De Paula et al., (2007) reported that although a significant association was found for HPV infection and the precursor lesions of cervical cancer, it was not possible to establish a significant association between these lesions and C. trachomatis co-infection. In the current study, no significance was found between the abnormalities and negative cervical cytology with C. trachomatis in HR HPV positive co-infection (p = 0.174). The highest OR presented in HSIL (2.62 [0.4-12.8]) followed by the LSIL and ASCUS groups (2.32 [0.6-8.2] and 1.00 [0.1-5.2]) ( Table 3). The small number of cases of abnormal cytology in HR HPV make it difficult to confirm whether there are significant differences among the different types of C. trachomatis infections.
In conclusion, there were no significantly associated risk factors between C. trachomatis infection and HR-HPV infection. The abnormalities of cervical premalignancy are thus not likely associated with C. trachomatis in HR HPV co-infection. Results from the current study confirmed the observation of increasing C. trachomatis infection among asymptomatic women. Routine screening and appropriate treatment for these infections should be performed to prevent complications such as (a) chronic pelvic pain in women, (b) prolonged C. trachomatis infection, and (c) reduced spreading to sexual partners. OR