Chlamydia trachomatis and Neisseria gonorrhoeae PCR detection in women treated for ectopic pregnancy

Abstract

The aim of this study was to determine the presence of Chlamydia trachomatis (ChT) and Neisseria gonorrhoae (NG) in the genital tract of women with ectopic pregnancy and to compare the positive results with patients’ self-reported history of PID. Overall 40 women were eligible for the study. The samples for the ChT and NG Polymerase Chain-reaction (PCR) detection were obtained from the cervix, endometrium and fallopian tube. The results of testing for NG at all sites were negative as were the results from cervical testing for ChT. The prevalence of ChT in the upper genital tract was 12.5%. No statistically significant correlation was found between the positive cases and the previous signs of PID and laparoscopic findings. We found statistically significant relationship between signs of pelvic inflammation in a pacients’ history and histopathological findings of tubal inflammation. 12.5% prevalence of ChT confirms the ascending genital infection. There was no association between the positive PCR result and clinical history of pelvic inflammation.

IMPACT STATEMENT

• What is already known on this subject? Pelvic inflammatory disease, Chlamydia trachomatis and Neisseria gonorrhoae infections are the main risks for ectopic pregnancy. Clinical history of PID and perioperative adhesions may suggest prior upper genital infection.

• What do the results of this study add? Chlamydia trachomatis positive PCR result can be found in the upper genital tract without the positivity of cervical smear in women with ectopic pregnancy. Our study is unique in assessing the endometrial biopsy for the presence of Chlamydia trachomatis and Neisseria gonorrhoae.

• What are the implications of these findings for future clinical practice and/or future clinical research? There is no statistically significant association between positive PCR result and clinical history of PID or pelvic adhesions found during laparoscopy for tubal pregnancy. Therefore there is no need for the preventive antibiotic treatment in patients with these findings.

Keywords:

• Ectopic pregnancy
• pelvic inflammatory disease
• laparoscopy
• Chlamydia trachomatis
• Neisseria gonorrhoae
• polymerase chain reaction

Introduction

Chlamydia trachomatis genital infection is the most prevalent bacterial sexually transmitted infection worldwide (Chemaitelly et al. 2019). Polymerase chain reaction proved chlamydial nucleic acid in endocervical specimen of 10.1% of Slovak women (Mikulova et al. 2013). The persisting chronic chlamydial infection can be associated with long-term problems such as dyspareunia and signs of dysuria (Peitsidis et al. 2012). However it may be completely asymptomatic in both partners and can manifest later in life as subfertility, tubal occlusion or ectopic pregnancy (Rantsi et al. 2019). Neisseria gonorrheae infection is less common and can manifest symptoms like pelvic inflammatory disease. However, it can also be a risk factor for ectopic pregnancy (Stevens and Criss 2018). While both pathogens can cause a chronic and inapparent infection, we presume that ChT and/or NG nucleic acid can be detected in cervical swab, endometrium or fallopian tube at the time of ectopic nidation. The aim of our study was to verify this hypothesis, confirm anamnestic risk factors for ChT/NG positivity and state appropriate antibiotic therapy of PID in case of statistically significant results.

Materials and methods

All women with diagnosed ectopic pregnancy indicated for laparoscopic salpingectomy attending the University Hospital Bulovka, Prague in the period June 2013 to June 2016 were enrolled in the study. The study was approved by the Ethical Committee and follows the principles of the Declaration of Helsinky.

The exclusion criteria were:

Patient did not sign the informed consent

declined participation in the study

haemodynamically unstable

clinical history of PID treated by antibiotics

other than tubal ectopic pregnancy

prior techniques of the assisted reproduction

indication for the expectant or medical treatment

antibiotic treatment in the past 3 months

clinical history or perioperative finding of endometriosis

Patients who underwent any techniques of assisted reproduction were also excluded, as there is an increased the risk of ectopic pregnancy and they are usually tested for both pathogens routinely by cervical swab.

Detailed clinical history was recorded, with a specific emphasis on chronic mucopurulent discharge, chronic pelvic pain in the lower abdomen and dyspareunia. The endocervical swab and dilation and curretage endometrial sampling was performed for PCR. After laparoscopic salpingectomy, a fresh tissue specimen cut approximately 10 mm from the affected tube was sent for the PCR analysis. The whole abdominal cavity was examined, and signs of possible chronic PID were recorded, such as pelvic periadnexal or perihepatic adhesions. Tubal ectopic pregnancy was confirmed histologically in all participians. The senior histopoathologist evaluated the affected tube for signs of chronic inflammation.

Laboratory analysis

All three samples from each participant (cervical sample, endometrial biopsy, and the fallopian tube specimen) were analysed in our hospital laboratory using the real-time PCR. The ChT and NG detection is based on the amplification of both the cryptic plasmid multicopy sequence and the 16S ribosomal RNA gene-specific for each pathogen, and on measuring the amplification product concentration in the course of the real-time PCR process utilising a fluorescence-marked probe. Detection of a multicopy sequence of the cryptic plasmid enables very high sensitivity of Chlamydia detection (including the Swedish variant) and the chromosomal gene detection at the same time enables high specificity and makes detection of plasmid-less strains possible. ChT and NG DNA presence is indicated by fluorophore fluorescence growth. The detection kit takes advantage of the ‘hot start’ technology, minimising non-specific reactions and assuring maximum sensitivity. It contains uracil-DNA-glycosylase (UDG), eliminating possible contamination of the PCR reaction by amplification products. Diagnostic kit sensitivity runs in single ChT/NG genome copies per PCR reaction. This assures very high sensitivity of the laboratory detection in clinical material. The kit can sensitively detect even recently discovered porA mutants of NG that may go undetected by other kits. In situ polymerase chain reaction of the acquired material has high diagnostic accuracy and correlates with the histopathological exams (Schlott et al. 1998).

Statistical analysis

The results were recorded using Microsoft Excel, and they were statistically processed and evaluated by MedCalc statistic software. Continuous data were analysed using Student’s t-test and binary data with Fisher’s exact test. A p value of .05 or lower was considered statistically significant.

Results

In the reporting period, there were 128 women diagnosed with ectopic pregnancy at the department Gynaecology and Obstetrics, University Hospital Bulovka, 1st Medical Faculty, Prague.

Exlusion criteria fullfill 82 patients, 52 women were treated either medically or under the expectant regime, 15 women underwent assisted reproduction techniques, 3 women were not willing to sign the informed consent and 6 were haemodynamically unstable. 1 woman had caesarean scar pregnancy and 2 patients had a recent antibiotic treatment. After exclusion criteria, 46 patients were enrolled in the study. Six participants had to be further excluded due to positivity of the PCR inhibitors and thus an inconclusive result. 40 women were included into the statistical analysis.

After the thorough medical history collection and assessment, 9 women (22.5%) showed signs of possible chronic pelvic inflammation (four patients complained of chronic mucopurulent discharge, three patients had chronic pelvic pain in the lower abdomen, and two women suffered from dyspareuia. Fifteen (37.5%) participants had some previous abdominal surgery during their lifetime. Four patients (10%) had undergone a contralateral salpingectomy in the past. Laparoscopic findings resembling chronic PID, such as peri-adnexal, pelvic or perihepatic adhesions were present in twenty-three women (57.5%). All the three materials from each patient (the endocervical swab, endometrial biopsy, and specimen of the Fallopian tube) were analysed by the PCR method. There were no positive results of NG, none of the endocervical swabs were positive for ChT PCR; however, three endometrial biopsies were ChT PCR positive (7.5%) and three samples of the fallopian tubes were also PCR positive (7.5%). One patient had a positive result both in the endometrium and in the fallopian tube. In total, five women with tubal ectopic pregnancy were ChT PCR positive (12.5%) in their upper genital tract. None of those with a positive result had histopathologic signs of chronic inflammation in the tubes.

Table 1 shows patients who were divided into two groups- the women with a positive clinical history of pelvic inflammatory disease and women with negative clinical history. We found neither statistically significant correlation with perioperative adhesions during laparoscopy, nor with the positive PCR result of ChT/NG. We then compared those who tested negative for ChT with patients that tested positive for ChT as seen in Table 2. We did not find any significant difference in the prevlence of previous sapingectomy or clinical history of pelvic inflammatory disease or a presence of adhesions in the abdominal cavity during laparoscopy. Table 3 shows the characteristics of patients with ChT PCR positive result in the upper genital tract. The positive PCR result of ChT did not have any correlation with the histopathological finding of chronic inflammation.

Table 1. Group I represents patients with clinical sings of chronic pelvic inflammatory disease, Group II represents remaining number of asymptomatic patients.

	Group I (n = 9)	Group II (n = 31)	p-Value
LPSK adhesions	7 (77.8%)	16 (51.6%)	.2557
Chronic inflammation on histopathology	6 (66.7%)	6 (19.4%)	.012
ChT positive	2 (22.2%)	3 (9.7%)	.3114
NG positive	0	0

Table 2. Comparison of group of patients with positive and negative PCR results.

	Positive ChT PCR (n = 5)	Negative ChT PCR (n = 35)	p-Value
History of previous salpingectomy	1 (20.0%)	3 (8.6%)	.4271
Signs of chronic pelvic inflammatory disease	3 (60.0%)	7 (20.0%)	.0893
Pelvic adhesions identified during Laparoscopy	2 (40.0%)	21 (60.0%)	.6340

Table 3. Characteristics of patients with positive Cht result in the upper genital tract.

Case number	1	2	3	4	5
Age	27	28	33	36	44
Clinical history of PID	No	Yes	No	No	Yes
Previous salpingectomy	No	Yes	No	No	No
Perioperative adhesions	No	Yes	No	No	No
ChT positive in endometrium	Yes	No	No	Yes	Yes
ChT positive in Fallopian tube	No	Yes	Yes	No	Yes
Chronic inflammation -histopathology	No	No	No	No	No

Discussion

Chronic persistent infection or reinfection of ChT or NG has been considered a key factor in the process causing adhesions and damage of falopian tube. Retrospective cohort study on more than 800,000 women proved that the previous ChT positive cervical swab rises to 2.36 HR of subsequent PID in these women and 1.87 HR for ectopic pregnancy (Den Heijer et al. 2020). The true link between ChT and ectopic pregnancy is still not sufficiently elucidated; there is a paucity of solid evidence to explain the underlying aetiology. Determining the exact mechanism by which ChT infection leads to ectopic pregnancy is difficult (Reekie et al. 2019). Direct cellular destruction by the infection plays a minor role, nevertheless the crucial role is played by the autoimmune inflammation caused by macrophage activation and proinflammatory signalling leading to subtle intraluminar tubal adhesions (Harvie et al. 2019).

The link between ChT and ectopic pregnancy is based mainly on seroepidemiological case-control studies. Serological testing for ChT-specific antibodies has previously been problematic as available assays had poor sensitivity and specificity, and were often cross-reactive with other Chlamydiae species. Therefore, direct testing by nucleic acid amplification is widely recommended for its high sensitivity and specificity. The major advantage is the possibility of the direct ChT nucleic acid detection in the bioptic material (Bryan et al. 2019). Although majority of the case control studies defines risks of following ectopic pregnancy after cervical ChT infection (Hoenderboom et al. 2017), we were not able to find any positive ChT cervical swab. This can be explained by ChT spontaeous resolution by cervical immune system or by direct ChT infection of endometrium and fallopian tubes (Price et al. 2016).

Using PCR, Lan et al. performed a retrospective study on the archival specimens of the tubes of 37 women and preceding endometrial and cervical smears and endometrial biopsies for the presence of ChT. Only one tubal positive specimen was identified (2.7%). However, in six of the 37 women (16.2%), ChT DNA was detected in the genital specimens (cervix and/or endometrial) taken more than three years before salpingectomy. In concordance with our results, no ChT DNA was found in cervical specimens at the time of the ectopic pregnancy. On the other hand, he found no evidence of ChT in endometrial specimens taken at the same time as the ectopic pregnacy. In our study, there were three positive endometrial biopsies (7.5%) (Lan et al. 1995). Recent Australian study did not confirm any ChT infection of 17 fallopian tube samples in women with ectopic pregnancy. They did not perform any cervical swab or endometrial biopsy (Stamatopoulos et al. 2012). Unlike other similar studies, we aimed to analyse also the endometrial tissue in patients with ectopic pregnancy; three samples tested ChT PCR positive (7.5%). In a study that examined menstrual tissue samples in infertile women, there was 25% prevalence of ChT (Michou et al. 2014). The lower number of positive cases in our findings can be due to the fact that our cohort of women was not infertile. The link between endometrial biopsy positivity and ectopic pregnancy can be purely incidental or the infection of the upper genital tract has been resolved and only persisted as a chronic endometrial inflammation.

In our study we did not find any risk factors from the clinical history that would be an indication for the PCR ChT/NG testing in women with ectopic pregnancy, there is no need for a routine cervical PCR ChT/NG swab. Clinical history correlated only with histopathological findings of the chronic tubal inflammation (p = .012), however did not relate to ChT nucleic acid positivity. Preventive antibiotic therapy of women with positive clinical history of Chlamydia trachomatic infection or laparoscopic finding of adhesions related to inflammatory process is not indicated according to our results. To improve reproductive health in young people, the primary and secondary preventive care strategies for sexually transmitted diseases are indisputable. More studies to determine the etiologic factors of PID and EP are needed..

Disclosure statement

The authors declare no conflict of interest and no funding was received.