Detection of Chlamydia trachomatis and herpes simplex virus type 1 or 2 in cervical samples in human papilloma virus (HPV)-positive and HPV-negative women


Corresponding author and reprint requests: W. Y. Almawi, Department of Medical Biochemistry, Arabian Gulf University, PO Box 22979, Manama, Bahrain


This study investigated whether the prevalence of human papilloma virus (HPV) in association with Chlamydia trachomatis, herpes simplex virus (HSV)-1 and/or HSV-2 was greater in high-grade than in low-grade or control cervical biopsy specimens. HPV-positive (n = 86) and HPV-negative (n = 213) women were screened for HPV, HSV and C. trachomatis by PCR. The most common HPV genotypes were HPV-16, HPV-6 and HPV-33; mixed HPV infection (n = 12) was also seen. A higher prevalence of C. trachomatis, HSV-1 and HSV-2 was found in HPV-positive samples. High-risk HPV genotypes and combined HPV + C. trachomatis or HPV + HSV-1, but not HSV-2, infections were associated with a greater risk of developing cervical carcinoma.

Human papilloma virus (HPV) is the main aetiological factor in cervical neoplasia [1]. However, the high incidence and long latency of HPV suggest that additional factors are involved in inducing cervical intra-epithelial neoplasia (CIN) [1,2], including herpes simplex virus (HSV) [3] and Chlamydia trachomatis[4,5]. These factors act by increasing the HPV-associated CIN risk through several mechanisms, including modulation of host immunity. Previous infection has been reported to be the most significant factor for HPV persistence [5], and a relationship between HPV and other sexually transmitted infections has been suggested. The presence of HSV-2 in HPV-positive cases suggests that it could initiate mutations and carcinogenesis [6,7], and may act with HPV in increasing the risk of CIN [7,8], although contradictory results have also been obtained [9]. C. trachomatis has also been associated with an increased risk of cervical neoplasia in HPV-positive patients [8], with the risk imparted by C. trachomatis being proportional to its titre, which is elevated in older women [4]. The present study investigated the association between HPV and HSV-1, HSV-2 and/or C. trachomatis, and correlated the results with other CIN risk-factors.

In total, 299 women (86 HPV-positive and 213 HPV-negative, based on Pap smear tests and confirmed by PCR [10]) attending obstetrics and gynaecology outpatient clinics were recruited to the study (Table 1), and were asked to complete and sign a standard questionnaire/consent form after the purpose of the study was explained and after all institutional ethical requirements were met. Endocervical scrapings were collected using a speculum-assisted Ayre's spatula. Where endocervical sampling could not be performed, vaginal specimens (n = 14) were collected using a sterile Ayre's spatula after separation of the labia minora and intra-vaginal 180° rotation of the spatula.

Table 1.  Characteristics of study participants
VariableHPV-positiveHPV-negativepOR95% CI
  • CIN, cervical intra-epithelial neoplasia; HPV, human papilloma virus.

  • a

    Based on 71 HPV-positive and 171 HPV-negative women for whom reliable information was collected.

Age (years)
 Mean ± SD33.6 ± 7.133.6 ± 7.40.961  
Smokers36 (41.9%)54 (25.4%)0.0022.36 1.38–4.03
Pregnancies65 (75.6%)151 (70.9%)0.4771.27 0.72–2.26
No. partnersa
 135 (49.3%)137 (80.1%)   
 2–529 (40.8%)17 (9.9%)0.0312.30 1.13–4.64
 > 57 (9.9%)17 (9.9%)0.4241.61 0.62–4.19
Pap smear
 Negative22 (25.6%)192 (90.1%)< 0.00126.6013.73–51.54
 Positive64 (74.4%)21 (9.9%)   
CIN grade
 022 (25.6%)192 (90.1%)   
 142 (48.8%)21 (9.9%)< 0.00117.46 8.80–34.62
 26 (7.0%)0< 0.0010.103 0.07–0.15
 316 (18.6%)0< 0.0010.103 0.07–0.15

HPV was amplified with the L1 consensus primers MY09 and MY11 [10]. HPV genotypes (HPV-6/11/16/18/33) were determined by hybridisation with genotype-specific biotinylated DNA probes, followed by visualisation with a DNA enzyme immunoassay (DiaSorin, Salluggia, Italy). C. trachomatis DNA was detected using the Amplicor STD Amplification and CT Detection systems (Roche Diagnostics, Mannheim, Germany), with detection by hybridisation with C. trachomatis-specific biotinylated probes. HSV-1 and HSV-2 were detected by nested PCR [11]. Statistical analysis was performed using SPSS v.13 software (SPSS Inc., Chicago, IL, USA), with Pearson's chi-square test used to assess intergroup significance, and Student's t-test used to determine differences in means.

While HPV-positive women were comparable to HPV-negative women in terms of age and previous pregnancies, a higher prevalence of smokers, women with multiple male sexual partners and women with positive Pap smears was seen among HPV-positive women (Table 1). Of the 64 HPV-positive women with abnormal cytology, 42 were graded CIN I, six were graded CIN II, and 16 were graded CIN III; all abnormal cytology results in HPV-negative women were graded CIN I (Table 1). The most prevalent genotypes were HPV-16 (n = 48), HPV-6 (n = 10) and HPV-33 (n = 6). Mixed HPV infections (n = 12) included eight patients with high-risk/high-risk (HR/HR) genotypes and four with high-risk/low-risk (HR/LR) genotypes. The mean age of C. trachomatis-positive (n = 29), HSV-1-positive (n = 40) and HSV-2-positive (n = 33) women was comparable to that of HPV-positive cases. Abnormal cytology was significantly greater in women with HPV-16 and mixed HR/HR infections, as well as in those with HSV-1 and C. trachomatis infections.

HSV-1, HSV-2 and C. trachomatis were more prevalent among HPV-positive women (Table 2). Combined HSV-1/HSV-2 and HSV-1/C. trachomatis, but not HSV-2/C. trachomatis infection, was significantly more prevalent among HPV-positive cases (Table 2). Predictors of abnormal cytology were determined by logistic regression analysis, with the dependent variable being a Pap smear and the independent variables being age, number of sexual partners, abortions, smoking, other infections and HPV, C. trachomatis, HSV-1 and HSV-2 infections. The only variables that were selected were HPV (p < 0.001; OR 22.340) and C. trachomatis (p 0.006; OR 5.910); HSV-1 or HSV-2 were not associated significantly with abnormal cytology.

Table 2.  Frequency of isolation of herpes simplex virus (HSV)-1, HSV-2 and Chlamydia trachomatis from women positive or negative for human papilloma virus (HPV)
 HPV-negativeHPV- positivepOR95% CI
HSV-1115.62943.3< 0.00112.845.9–27.9
HSV-1 + HSV-210.51011.6< 0.00127.903.5–221.6
C. trachomatis136.71625.8< 0.0014.842.2–10.8
C. trachomatis + HSV-210.522.30.2005.050.5–56.4
C. trachomatis + HSV-110.578.10.00118.792.3–155.1

It has been suggested by some studies [3,5,6], but not by others [9], that C. trachomatis and/or HSV are predisposing factors for HPV infection. The present study demonstrated an increased prevalence of C. trachomatis and HSV-1 among HPV-positive patients, as was expected because of the similarity in their mode of transmission [5,12]. HPV infection was more common among women with multiple sexual partners, and those who were smokers or had abnormal cytology [1]. Abnormal cytology was associated with a lower risk for CIN in HPV-negative women, as all the abnormal cytology seen in HPV-negative women was graded CIN 1, compared with 65.6% of HPV-positive women who were graded CIN 1 and 34.3% who were graded CIN 2/3. As demonstrated previously [13], including in Lebanon [14], HPV-16 was the most prevalent genotype among HPV-positive women with abnormal cytology.

The association of HSV-2 with abnormal cytology was modest compared with that of HPV [15]. While HSV-2 may cooperate with HPV, its effects appear to be indirect, indicating that HSV-2 is not a significant risk-factor for cervical carcinoma [6,9], in contrast with the association between HSV-1 and CIN in HPV-positive women [16]. Abnormal cytology was more prominent in women with HPV-16 and mixed HR/HR HPV, C. trachomatis and HSV-1 infection. While HSV-1 and HSV-2 are associated commonly with labial and genital lesions [17], concomitant HSV-1 infection may exert a protective role in HSV-2-infected individuals [18]. In contrast, it has also been suggested that HSV-2 induces CIN, which becomes more pronounced with HPV infection [16].

HPV-positive women harbouring HSV-1, or both HSV-1 and HSV-2, were more likely to have a positive Pap smear than HSV-2-positive patients, suggesting that HSV-2 acts by cross-immunising against HSV-1 [19], and/or by inhibiting HPV expression. Abnormal cytology was also significantly higher in patients infected with HR/HR than in those infected with HR/LR genotypes, suggesting that the timing of these infections was crucial in precipitating abnormal cytology, and that infection with an LR genotype may facilitate development of cell-mediated immunity. C. trachomatis was a risk-factor for HPV persistence [5], and may act as a co-factor with HPV in precipitating abnormal cytology by inducing expression of pro-inflammatory mediators [20], altering cell-to-cell adhesion, and affecting cellular differentiation [20]. The results of the present study are in agreement with studies that linked C. trachomatis with cervical neoplasia after controlling for HPV, but contradict others that found no association between C. trachomatis and low-grade CIN in HPV-adjusted analyses. Accurate determination of HPV and other co-infections in relation to cervical cancer is of relevance in assessing the role of these sexually transmitted infections in cervical diseases.