ABSTRACT
The aim of this study was to determine whether Chlamydia trachomatis could be detected in saliva and if infection is specific to an anatomical site in the oropharynx. Men who have sex with men (MSM) who were diagnosed with oropharyngeal chlamydia at the Melbourne Sexual Health Centre in 2017-2018 were invited to participate upon returning for treatment. Swabs at the tonsillar fossae and posterior oropharynx and a saliva sample were collected. Throat samples were tested for C. trachomatis by the Aptima Combo 2 assay. The bacterial loads of C. trachomatis in all samples were assessed by quantitative PCR (qPCR) detecting the ompA gene. We calculated the positivity and bacterial load of C. trachomatis for all samples. Forty-two MSM were included. The median age was 28 years (interquartile range [IQR], 24 to 33 years). Thirty-two participants (76.2%; 95% confidence interval [CI], 60.5% to 87.9%) had C. trachomatis detected by qPCR at both the tonsillar fossae and the posterior oropharynx, followed by 9.5% (n = 4; 95% CI, 2.7% to 22.6%) positive at the posterior oropharynx only and 4.8% (n = 2; 95% CI, 0.58% to 16.2%) positive at the tonsillar fossae only. Twenty-nine MSM had C. trachomatis detected in saliva (69.0%; 95% CI, 52.9% to 82.3%). The median C. trachomatis load in saliva was 446 copies/ml (IQR, 204 to 1,390 copies/ml), that in the tonsillar fossae was 893 copies/swab (IQR, 390 to 13,224 copies/ml), and that in the posterior oropharynx was 1,204 copies/swab (IQR, 330 to 16,211). There was no significant difference in C. trachomatis load between the tonsillar fossae and the posterior oropharynx (P = 0.119). Among MSM with oropharyngeal chlamydia, nearly three-quarters had chlamydia DNA detected in saliva, although the viability and implications for transmission are unknown.
INTRODUCTION
Chlamydia trachomatis infection is a major health burden, with an estimated 131 million cases reported in 2012 worldwide (1). The majority of cases of oropharyngeal chlamydia are asymptomatic (2), with an estimated infection duration of 667 days (3). A study in Melbourne, Australia, reported an 1.5% prevalence of oropharyngeal chlamydia among gay, bisexual, and other men who have sex with men (MSM) attending a sexual health center between 2015 and 2016 (4) and data have shown there has been an increase in oropharyngeal chlamydia prevalence among MSM attending an sexual health clinic in Sydney, Australia (from 0.7% in 2011 to 1.6% in 2014) (5).
There has been evidence to suggest that saliva has played an important role in gonorrhea transmission, but the role of saliva in chlamydia transmission is not fully understood (6). Past studies have shown that Neisseria gonorrhoeae can be isolated from the tonsillar fossae, the posterior oropharynx, and the saliva (7, 8), suggesting that saliva exchange in sexual activities may be associated with gonorrhea acquisition. Epidemiological studies have found that using saliva as a lubricant for anal sex and rimming are strong risk factors for anorectal gonorrhea but not for anorectal chlamydia (9–11). There has been increased evidence showing that tongue kissing may be a risk factor for oropharyngeal gonorrhea (12); however, the evidence of the risk factors for oropharyngeal chlamydia is limited (11). The benefits of screening men who have sex with men (MSM) for oropharyngeal C. trachomatis are unclear, and in countries that do screen MSM for oropharyngeal C. trachomatis, such as Australia and the United Kingdom, the guidelines for what constitutes exposure to C. trachomatis are unclear (13, 14). It is important to understand whether saliva can act as a medium for chlamydia transmission, as it could impact screening recommendations for MSM. The first step in understanding saliva’s role in C. trachomatis transmission is to determine if C. trachomatis can be detected in saliva.
The aim of this study was to determine if C. trachomatis could be detected in saliva among untreated MSM who were diagnosed with oropharyngeal chlamydia. The secondary aim was to quantify and compare the bacterial loads of C. trachomatis at the tonsillar fossae and the posterior oropharynx and in saliva.
MATERIALS AND METHODS
Study population and setting.Participants were recruited at the Melbourne Sexual Health Centre (MSHC), Australia, between 1 August 2017 and 29 August 2018. MSM who tested positive for oropharyngeal chlamydia by nucleic acid amplification test using the Aptima Combo 2 (AC2) assay (Hologic, San Diego, CA, USA) as part of routine sexually transmitted infection (STI) screening at MSHC, who had no antibiotics in the past 4 weeks, and who returned for treatment within 14 days were invited to participate in the Chlamydia and Saliva (CAS) study. There were 76 MSM who were referred to the study.
Sexual behavior data collection.On the day of enrollment, participants were asked to complete a short questionnaire about sexual activity with any male partners in the days leading up to the initial screening of C. trachomatis infection. The questionnaire asked if participants had tongue kissed, performed oral sex on a partner with or without the partner ejaculating, or rimmed a partner’s anus and how many days before the screening they had done each activity. Analysis was restricted to activities performed in the 30 days prior to screening.
Sample collection.Eligible men were identified upon returning to clinic for treatment of oropharyngeal chlamydia.
On the day of treatment, two throat swabs were collected from the participants: the first one from the tonsillar fossae (swabbing first the left tonsil and then the right tonsil) and the second one from the posterior oropharynx. Tonsillar fossa and posterior oropharynx swabs were collected in the same order by four trained research nurses. Swabs were immediately placed into 2.9 ml of Aptima specimen transport medium (Hologic, San Diego, CA, USA). After AC2 testing, the remaining solution was aliquoted into a 2-ml sterile screw-cap microtube (Interpath Services Pty Ltd., Heidelberg West, Victoria, Australia) and stored at –80°C until further testing by quantitative PCR (qPCR). Participants were then asked to accumulate saliva for 30 s by rubbing their cheeks on their teeth and swirling their tongue around their mouth to generate the saliva and then expectorate into a 50-ml specimen jar (Sarstedt Australia Pty Ltd., Mawson Lakes, South Australia, Australia). The research nurse immediately collected the saliva with a UriSwab (Copan Innovations, Brescia, Italy) by swirling a sponge in the specimen jar. The UriSwabs were centrifuged at 1,200 rpm for 60 s. Up to 2 ml of saliva was then aliquoted into a 2-ml sterile screw-cap microtube (Interpath Services Pty Ltd., Heidelberg West, Victoria, Australia) and stored at −80◦C until further testing by qPCR.
Aptima C. trachomatis detection.Tonsillar fossa and posterior oropharynx swabs were tested for C. trachomatis using transcription-mediated amplification (TMA) of AC2, which replicates a specific region of the 23S rRNA from C. trachomatis (Gen-Probe Panther system; Hologic, San Diego, CA, USA). Samples that tested positive for chlamydia were confirmed with the Aptima CT assay (Gen-Probe PANTHER system; Hologic, San Diego, CA, USA), which replicates a specific region of the 16S rRNA from C. trachomatis. If either the tonsillar fossa swab or the posterior oropharynx swab tested positive for chlamydia, the samples from that participant (including the saliva sample) were assessed by qPCR for sample adequacy and bacterial load of C. trachomatis.
Chlamydia quantification.Nucleic acid extraction and chlamydia quantitation were performed on all stored samples at the end of the study. The MagNA Pure 96 system (Roche Molecular Diagnostics, Mannheim, Germany) was used to extract nucleic acid. For saliva samples, 200 μl of primary sample was extracted using the DNA and viral nucleic acid small-volume kit, per the manufacturer’s Pathogen Universal 200 3.1 protocol, and eluted in 50 μl. Aptima specimen transport medium (500 μl) was extracted with the DNA and viral nucleic acid large-volume kit, per the Pathogen Universal 500 3.1 protocol, and eluted in 50 μl. All extracted DNA was assessed for adequacy with qPCR amplification of a 260-bp product of the human beta globin gene (15).
Detection of C. trachomatis was performed using a qPCR assay targeting the ompA gene (primary CT PCR primers and HEX-labeled probe) (16) on the LightCycler 480 II real-time platform (Roche Molecular Diagnostics, Mannheim, Germany) (assay limit of detection, 10 to 40 genomes/reaction, depending on the genovar). Each 20-μl qPCR mixture consisted of a final concentration of 0.5 μM forward and reverse primer, 0.25 μM probe and 1× SensiFast Probe No-ROX kit (Bioline), and 5 μl of sample DNA, using the PCR cycling conditions outlined previously (16). The C. trachomatis load was determined using a standard curve generated with a gBlock of the full ompA gene (approximate genome location, bases 778879 to 780060 from reference strain D/UW-3/CX; GenBank accession no. AE001273.1), which underwent a 10-fold serial dilution to extinction prior to analysis in triplicate on the LightCycler 480. The calculated number of copies per microliter for each of the dilutions was confirmed using digital droplet PCR (Bio-Rad, Hercules, CA, USA). C. trachomatis load results were calculated to be reported in copies per milliliter for saliva or copies per swab for tonsillar fossa and posterior oropharyngeal swabs.
Statistical analysis.We calculated the proportion of men who tested C. trachomatis positive by (i) the AC2 assay and (ii) qPCR. The 95% confidence intervals (CI) of the proportions were also calculated using exact binomial methods.
Descriptive analyses were conducted to calculate the median numbers of chlamydia and beta globin load copies per ml for saliva and for each swab from tonsillar fossa and posterior oropharynx sites and the median number of days between diagnosis and enrollment. C. trachomatis and human beta globin load were log10 transformed for analysis. A two-sample t test was used to compare log-transformed human beta globin loads between samples with no C. trachomatis detected by qPCR and those that were C. trachomatis positive by qPCR for each site (saliva, tonsillar fossae, and posterior oropharynx). A paired sample t test was used to compare log-transformed C. trachomatis and beta globin loads between the tonsillar fossae and the posterior oropharynx. Participants who had engaged in sexual activity in the 30 days prior to screening were compared with those who had not. A two-sample t test was used to compare the log10 C. trachomatis bacterial loads between the two groups. Research nurses were divided between the one nurse who recruited most of the participants (n = 21) and the others who collectively recruited 21 participants, and a two-sample t test was used to compare log10 C. trachomatis bacterial loads between the two groups. Pearson’s correlation was conducted to examine the relationship between median C. trachomatis load in saliva and the median load in the oropharynx (i.e., posterior oropharynx and tonsillar fossae, separately). All statistical analyses were performed using Stata (version 14; Stata Corporation, College Station, TX, USA).
Ethical approval was obtained from the Alfred Hospital Ethics Committee, Melbourne, Australia (number 340/17).
RESULTS
Of the 76 men who were referred to the study team, 19 declined to participate (six declining due to time constraints, and the remaining declining with no reason given), one was ineligible because he returned to the clinic for treatment more than 14 days after being diagnosed, and one was ineligible because he had had antibiotics in the previous 2 weeks before being diagnosed. Of the 55 eligible men who were enrolled in the study, four (7.3%) were excluded because they tested negative for C. trachomatis from both oropharyngeal swabs by AC2 on the day of treatment. An additional nine were excluded due to failure to detect human beta globin, indicating sample inadequacy. There were 42 participants included in the final analysis, each with three samples (i.e., 126 samples).
The median age of the 42 participants was 28 (interquartile range [IQR], 24 to 33) years (see Table S1 in the supplemental material). The median time between the date of screening and the date of returning to MSHC for treatment was 5 days (IQR, 4 to 6 days; maximum, 12 days) (Table S1). On the day of treatment, 12 (28.6%; 95% CI, 15.7% to 44.6%) participants self-reported having a sore throat in the past 7 days (Table S1). No oral signs or lesions were observed in any participant upon examination.
Of the 42 participants, two (4.8%; 95% CI, 0.58% to 16.2%) tested positive by AC2 at tonsillar fossae only, one (2.4%; 95% CI, 0.06% to 12.6%) tested positive at the posterior oropharynx only, and 39 (92.9%; 95% CI, 80.5% to 98.5%) tested positive by AC2 at both the posterior oropharynx and the tonsillar fossae. C. trachomatis was detected by qPCR in both the tonsillar fossae and posterior oropharynx in the majority of participants (n = 32; 76.2%; 95% CI, 60.5% to 87.9%). Two participants (4.8%; 95% CI, 0.58% to 16.2%) had C. trachomatis detected by qPCR in the tonsillar fossae but not at the posterior oropharynx, and four participants (9.5%; 95% CI, 2.7% to 22.6%) had C. trachomatis detected in the posterior oropharynx but not the tonsillar fossae. C. trachomatis was detected by qPCR of saliva in 29 participants (69.0%; 95% CI, 52.9% to 82.3%) (Table 1). Of the 13 participants with no C. trachomatis detected in saliva, two (15%; 95% CI, 1.9% to 45.4%) had greater than median loads of C. trachomatis in the tonsillar fossae and two (15%; 95% CI, 1.9% to 45.4%) had greater than median loads of C. trachomatis in the posterior oropharynx.
Total median and log10 median loads of C. trachomatis and human beta globin detected by qPCR for each sitea
The median bacterial load of C. trachomatis in the tonsillar fossae was 893 copies/swab (IQR, 390 to 13,224) (Table 1), and that in the posterior oropharynx was 1,204 copies/swab (IQR, 330 to 16,211). There was no significant difference in median bacterial load of C. trachomatis between the tonsillar fossae and the posterior oropharynx (P = 0.119) (Fig. 1). The median bacterial load of C. trachomatis in saliva was 446 copies/ml (IQR, 204 to 1,390 copies/ml). The median load of human beta globin detected in the tonsillar fossa samples was 1.95× 105 copies/swab, that in the posterior oropharynx was 1.26 × 105 copies/swab, and that in the saliva samples was 5.33 × 104 copies/ml (Table 1). The mean human beta globin load detected in the posterior oropharynx was significantly lower than the beta globin load in the tonsillar fossae (P = 0.002). The mean human beta globin load in saliva was significantly lower than the mean beta globin load in the tonsillar fossa (P < 0.001) and posterior oropharynx (P < 0.001) swabs. There was no significant difference between the mean human beta globin levels detected in the saliva samples where C. trachomatis was detected by qPCR and those where C. trachomatis was not detected by qPCR (P = 0.292), nor was there a difference in the qPCR-positive and -negative posterior oropharynx (P = 0.390) or tonsillar fossa (P = 0.397) swabs.
Log-transformed median copies of C. trachomatis (CT) for each sample type. Note that the copies of C. trachomatis for the posterior oropharynx and the tonsillar fossae were measured per swab and those of the saliva samples were measured per milliliter.
There was no difference in C. trachomatis load in the posterior oropharynx (P = 0.593) or the tonsillar fossae (P = 0.692) between the research nurses who collected the swabs.
There was a positive correlation between C. trachomatis load in saliva and C. trachomatis load in the tonsillar fossae (r = 0.566; P < 0.001) and posterior oropharynx (r = 0.544; P < 0.001) (Fig. 2).
Correlation of log10 median saliva C. trachomatis (CT) load with log10 C. trachomatis load at the tonsillar fossae (A) and posterior oropharynx (B).
For the 30 days prior to the day of screening, 41 out of 42 participants provided sexual behavior information. Most participants (90.2%; 95% CI, 76.9% to 97.3%; n = 37) had tongue kissed, and the median number of days since last kissing was 5 (IQR, 4 to 10 days). A majority of participants (87.8%; 95% CI, 73.8% to 95.9%; n = 36) had receptive penile-oral sex (partner’s penis in participant’s mouth) in the 30 days prior to screening, and 36.6% (95% CI, 22.1% to 53.1%; n = 15) had receptive penile-oral sex with ejaculation. The median days since receptive penile-oral sex was 5 (IQR, 4 to 14 days), and the median days since receptive penile-oral sex with ejaculation was 7 (IQR, 6 to 14). About one-third (31.7%; 95% CI:,18.1% to 48.1%; n = 13) of the participants had rimmed (oroanal contact) their partner’s anus in the 30 days prior to screening, and the median number of days since rimming was 5 (IQR, 4 to 10 days). There were no significant differences in the median bacterial loads of C. trachomatis between those who had engaged in sexual activities in the 30 days prior to screening and those who had not (Table S2).
DISCUSSION
This study has shown that C. trachomatis DNA can be detected in saliva in most cases of oropharyngeal chlamydia, suggesting that saliva exchange has the potential to play a role in chlamydia transmission among MSM. Higher values of C. trachomatis load in saliva were associated with higher C. trachomatis loads at the tonsillar fossae and posterior oropharynx. There were no significant differences in the C. trachomatis loads between the tonsillar fossae and the posterior oropharynx. While more than 90% of MSM diagnosed with oropharyngeal chlamydia had C. trachomatis detected at both the tonsillar fossae and the posterior oropharynx, the fact that three cases (7%) had only one site with C. trachomatis detected by AC2 suggests that sampling both the tonsillar fossae and the posterior oropharynx is necessary for accurate diagnosis of oropharyngeal C. trachomatis. There is no standard sampling methodology currently in use for detecting oropharyngeal chlamydia. This study suggests that the use of one swab to sample both the tonsillar fossae and the posterior oropharynx should be adopted by clinicians as standard practice.
To our knowledge, this is the first study quantifying C. trachomatis load in saliva. It is expected that some C. trachomatis DNA would be found in saliva due to shedding of the bacteria from infected epithelial cells of the posterior oropharynx and tonsillar fossae (17); however, it is unclear if C. trachomatis detected in this study is viable. Recent evidence has suggested that Neisseria gonorrhoeae can be transmitted via saliva. Epidemiological studies have identified that saliva used as a lubricant for anal sex is a risk factor for anorectal gonorrhea (10) and that kissing is a risk factor for oropharyngeal gonorrhea (18). However, the role of saliva in chlamydia transmission is less clear (19, 20). Unlike with gonorrhea, saliva as a lubricant for anal sex is not associated with anorectal chlamydia after adjusting for condom use and other activities such as rimming (9). Our study showed that C. trachomatis can be detected in saliva and supports the possibility of C. trachomatis transmission via saliva; however, more research into the load of viable and therefore potentially infectious C. trachomatis is needed in order to better understand the implication of the load of DNA detected in saliva reported here.
There were several limitations to this study. First, we cannot determine whether the detected C. trachomatis in our samples is viable or not, as the samples were not assessed by culture. Future studies should determine if C. trachomatis can be cultured from saliva, though culture is less sensitive than AC2.
Second, due to the intracellular nature of C. trachomatis, load could be influenced by the sampling methodology; i.e., swabbing for a longer time may result in collection of more bacteria and swabs could be expected to yield higher loads than saliva alone. The load of human beta globin in the saliva was lower in our samples than the loads at the posterior oropharynx and tonsillar fossae, further supporting this theory. This study utilized uniform sampling methodology with respect to order and number of swabs per participant but did not set time limits for collection at each site. One research nurse collected the swabs for half of participants (n = 21), and the other half of participants (n = 21) had swabs collected by three different research nurses. There was no significant difference in C. trachomatis load from the swabs collected between these two groups.
Human beta globin load was higher in swabs taken from the tonsillar fossae than from swabs taken from the posterior oropharynx, which indicates that more cells were collected from the tonsillar fossa swabs than from the posterior oropharynx swabs. However, there was no significant difference in C. trachomatis load between the tonsillar fossae and the posterior oropharynx. A possible explanation for the lower load of beta globin in the posterior oropharynx samples is due to the difficulty in collecting swabs from this site, as well as this being a single site as opposed to the two-site tonsillar fossae.
Four participants who tested negative at both sites by AC2 when returning for treatment potentially had spontaneous clearance of oropharyngeal chlamydia. A study in the Netherlands showed that the spontaneous clearance of oropharyngeal chlamydia was high in MSM (80%), with a majority returning for treatment within 14 days; however, this was with a small sample size (n = 5) (21). It is also possible that differing sampling methods contributed to these four participants’ retesting negative, although this would be unexpected given that the research nurses collecting the samples for retesting were following the procedure described previously to ensure collection from both tonsillar fossae and posterior oropharynx. Additionally, nine participant samples of oropharyngeal swabs that tested positive by AC2 were not detected by qPCR, and human beta globin was not detected in these samples. The most likely reason for this discrepancy is sample inadequacy, as the AC2 assay has greater sensitivity.
This is the first study to examine C. trachomatis loads in both the posterior oropharynx and tonsillar fossae as well as in saliva among MSM. Our results indicate that there is no significant difference in C. trachomatis load between the posterior oropharynx and the tonsillar fossae. We found that the median loads of C. trachomatis in the posterior oropharynx and tonsillar fossae (1,204 and 893 copies per swab, respectively) are lower than reported loads at the anorectum (median, 14,800 copies per swab) and in the first-void urine (median, 3,720 copies/ml) in MSM (22). This finding is consistent with N. gonorrhoeae, which has been found to have significantly higher relative loads in the anorectum than in the posterior oropharynx (23). However, the median loads of C. trachomatis in the oropharynx and saliva reported here are lower than the median loads of N. gonorrhoeae reported in the pharynx (3.2 × 105 copies/swab) and saliva (1.1 × 105 copies/ml) among MSM returning within 7 days (24).
Our study showed no significant difference in C. trachomatis load at any oropharyngeal site between those who had specific sex practices involving the oropharynx and those who did not in the 30 days before screening; however, there were very few men in our sample who had not kissed (n = 4) or had receptive oral sex (n = 5). Our results indicate that receptive oral sex with ejaculation and insertive rimming may not contribute to a higher C. trachomatis load in the oropharynx; however, the broader implication of this finding is unclear, given the gaps in knowledge around the load of infectious chlamydia. Previous studies have shown that genital C. trachomatis load decreases with repeat infections in women, suggesting that partial immunity may develop (25); however, a retrospective study at MSHC showed that the median C. trachomatis load from anorectal samples in MSM was higher for those with repeat infections than for index cases (26). No similar study has been done, to our knowledge, to examine oropharyngeal C. trachomatis load in repeat infections. Further studies should determine the infectious load of C. trachomatis, as this will have implications for routes of oropharyngeal C. trachomatis transmission. As has been mentioned previously, partner studies would be particularly useful to determine the impact of load on transmission (27).
This study contributes to the growing knowledge of oropharyngeal C. trachomatis. Clarifying if it is possible for oropharyngeal C. trachomatis to be transmitted to other sites may have an impact on screening and treatment guidelines.
ACKNOWLEDGMENTS
T.R.P. conducted the data analysis and wrote the first draft of the manuscript. E.P.F.C. and C.K.F. conceived and designed the study and aided in data interpretation. E.P.F.C. oversaw the study and provided statistical advice. T.R.P., C.K.F., K.M., and E.P.F.C. designed the questionnaire. D.A.W., V.D.P., and B.P.H. oversaw laboratory testing using the Aptima Combo 2 assay and were involved in laboratory data interpretation. J.D. performed the qPCR testing. J.D. and G.M. contributed to the interpretation for laboratory data on qPCR bacterial load analyses. K.M., R.W., and D.L. were involved in study recruitment and sample collection. K.M. was involved in data entry and management and specimen processing, transportation, and storage. All authors were involved in data interpretation and revised the manuscript for intellectual content.
We acknowledge Mark Chung for designing the graphics for the study materials, Tiffany Rose for aiding in patient recruitment, Sabrina Trumpour for assisting with database management, and the laboratory staff of the microbiological diagnostic unit on site at MSHC for aiding in specimen testing.
This work was funded by a National Health and Medical Research Council (NHMRC) program grant (GNT568971). E.P.F.C. was supported by an NHMRC Early Career Fellowship (GNT1091226).
We declare no competing interests.
FOOTNOTES
- Received 21 August 2019.
- Returned for modification 11 September 2019.
- Accepted 31 October 2019.
- Accepted manuscript posted online 6 November 2019.
Supplemental material is available online only.
- Copyright © 2019 American Society for Microbiology.
