Treponema pallidum PCR screening at mucosal sites of asymptomatic men who have sex with men taking HIV pre-exposure prophylaxis

ABSTRACT Early detection and treatment of syphilis will reduce the infectious period and transmission. We aimed to determine whether screening men who have sex with men (MSM) taking HIV pre-exposure prophylaxis (PrEP) for syphilis using Treponema pallidum polymerase chain reaction (PCR) could detect syphilis before the appearance of syphilis antibodies in serology. MSM attending 3-monthly PrEP clinic visits in Melbourne, Australia, were screened with a PCR assay targeting the polA gene of T. pallidum from an anal swab and an oral rinse between November 2019 and March 2020. Participants were serologically screened for syphilis using chemiluminescence immunoassay. A total of 309 asymptomatic participants provided an anal swab and oral rinse sample for T. pallidum PCR screening. Two syphilis cases (0.6%) were detected: one man had a positive serology only; another man had T. pallidum detected by PCR from an anal swab and a positive serology. PCR positivity was 0.3% (n = 1) for anal swabs and 0% (n = 0) for oral rinse. In this study, T. pallidum PCR screening at routine PrEP clinic visits did not identify additional cases of early syphilis over serological screening performed at these visits. IMPORTANCE With the ongoing syphilis epidemic in men who have sex with men (MSM), we investigated the role of using Treponema pallidum polymerase chain reaction (PCR) testing at the oral cavity and anus in MSM taking pre-exposure prophylaxis for the early detection of syphilis. We evaluated whether the PCR tests from these mucosal sites can detect syphilis infection early, before the development of syphilis antibodies in serology. Our study found two syphilis cases among 309 MSM, and only one syphilis case had a positive anal PCR swab, although serology was positive. We conclude that additional PCR testing is likely to be expensive and would not be cost effective for individuals who regularly screen for syphilis. However, future studies with a larger sample size are required.

G ay, bisexual, and other men who have sex with men (MSM) have a high prevalence of syphilis, with an estimated global prevalence of about 8% (1), higher than the prevalence in the general population.In Australia, MSM are disproportionately affected by syphilis (2,3).Longstanding public health interventions aimed at controlling syphilis, such as serological screening and contact tracing and treatment, have not curtailed growing syphilis epidemics among MSM internationally, warranting novel interventions (4)(5)(6)(7).
In many high-income countries, 3-monthly screening for sexually transmitted infections (STIs) has been recommended for MSM taking HIV pre-exposure prophylaxis (PrEP) (8).Despite an increase in coverage and frequency of serological screening in Australia, syphilis incidence remains high among MSM (9) with the highest rates occurring in MSM taking HIV PrEP (10).The incidence of syphilis among MSM taking HIV PrEP was reported to be between 8 and 13 per 100 person-years (i.e., incidence rate between 8 and 13 syphilis diagnoses in 100 person per year).(10)(11)(12).A previous study on MSM taking HIV PrEP in Melbourne showed that a significant proportion of primary and secondary syphilis was detected between the routine 3-monthly PrEP monitoring visits, suggesting that 3-monthly serological screening for syphilis alone is unlikely to be sufficient to reduce syphilis transmission in this high incidence population (13).Moreover, the use of serology for screening has the potential to miss a proportion of primary syphilis infections.In a study analyzing 814 primary syphilis cases, 5% (n = 38) of cases detected by Treponema pallidum polymerase chain reaction (PCR) had negative syphilis serology (14).The finding implies that T. pallidum PCR might be able to detect syphilis infection early before a positive syphilis serology.Early detection of syphilis infection is required to reduce the duration of infectious period and reproductive rate of syphilis.
Nucleic acid amplification tests (NAAT) such as PCR for T. pallidum are highly sensitive and specific for the diagnosis of primary syphilis (15)(16)(17)(18).To date, most studies have examined the use of T. pallidum PCR for diagnosing syphilis from suspected early lesions rather than for screening (17)(18)(19)(20)(21).In these studies, the detection of syphilis depends on the clinically evident syphilis lesions.However, primary syphilis lesions can occur in the oral cavity and anal canal where they might be hidden and not clinically evident (22).Furthermore, unrecognized oral and anal shedding with or without lesions in the oral cavity and anus have been shown to occur in early syphilis (23).
Studies have shown that T. pallidum can be detected by PCR from oral and anal samples of MSM with early syphilis with or without symptoms (23)(24)(25).Oral detection was most frequent in those with secondary syphilis (23), although syphilis detection by PCR occurred in various sample types in all early stages of syphilis in the absence of lesions (25).Based on these findings, we hypothesized that PCR screening from the oral cavity and anus might detect syphilis before the appearance of syphilis antibodies in serology in the absence of syphilis lesions.
In this study, we aimed to determine whether screening MSM taking HIV PrEP using T. pallidum PCR from oral and anal samples could detect syphilis before they were serologically positive.We chose MSM taking HIV PrEP as the study population due to the high incidence of syphilis in this group.

MATERIALS AND METHODS
This was a cross-sectional study of MSM attending the HIV PrEP clinic at the Mel bourne Sexual Health Centre, Australia, between November 2019 and March 2020.Men attending the clinic were reviewed every 3 months to screen for HIV and STIs and to obtain their PrEP medication.The clinic has an electronic medical record system that collects demographic and epidemiological data.In this study, we defined MSM as gay, bisexual, and other men who had sex with men in the last 12 months.We included transgender women taking PrEP who were having sex with men.
Individuals who attended to the HIV PrEP clinic for their 3-monthly review were asked to self-collect an anal swab and an oral rinse specimen for T. pallidum PCR testing.Self-collection was chosen as the preferred method, as the majority of clients attending the HIV PrEP clinic were regular attendees and were already familiar with self-collect ing swabs for chlamydia and gonorrhea.Moreover, past studies have demonstrated comparable sensitivity between self-collected and clinician-collected swabs (26,27).Eligible individuals were given a participant information sheet explaining the study, and verbal consent was obtained from the participants.The anal swab was collected using ESwab (Copan Diagnostics Inc., Murrieta, CA, USA).An oral rinse was collected by gargling 10 mL of sterile water for injection (Pfizer, Bentley, WA, Australia) and swilled for 15 s.

DNA extraction
Total DNA was extracted from fresh specimens on the Freedom EVO 100 (Tecan, Mannedorf, Switzerland) using the Quick DNA/RNA MagBead Extraction Kit (Zymo Research, Irvine, California, USA), as per the manufacturer's instructions.DNA extracts were stored at −20°C, and the original lesion and non-lesion study samples were stored at −70°C.

PCR testing
Specimens were tested using a TaqMan real-time PCR assay targeting the polA gene of T. pallidum, which was previously validated for clinical use with lesion samples (28).Testing was performed at the Victorian Infectious Diseases Reference Laboratory, Melbourne, Australia (28).Details of the T. pallidum PCR testing are described elsewhere (28).For a positive T. pallidum PCR sample, a cycle threshold (Ct) of 38 was determined as a cut-off value.If the Ct value was greater than or equal to 38, a repeat testing was performed, and the result was reported as positive if the serology was also positive.The T. pallidum PCR assay in our study has a sensitivity of 80% and specificity of 98% for detecting early syphilis infection from lesion samples (28).The limit of detection was determined to be one DNA copy per microliter for this PCR assay.The limit of detection was determined through a process of a serial dilution using a standard control, known as Amplirun Treponema Control (Vircell Microbiologists, Madrid, Spain).
Serology for syphilis was obtained at each 3-monthly clinic visit with testing using chemiluminescence immunoassay (CLIA) (Diasorin, Saluggia, Italy), with confirmation by T. pallidum particle agglutination assay (TPPA) (Fujirebio, Tokyo, Japan) and rapid plasma reagin (RPR) (Becton Dickinson, New Jersey, USA).Routine screening of chlamydia and gonorrhea with NAAT was performed using Aptima Combo 2 assay (Hologic Panther system; Hologic Inc., San Diego, CA, USA) on urine specimens, pharyngeal swabs, and anal swabs, if collected.HIV screening was performed using the DiaSorin Liaison XL Murex HIV Ab/Ag CLIA (fourth generation) assay.Separate anal swabs were obtained for T. pallidum PCR and chlamydia/gonorrhea NAAT.
A syphilis diagnosis was defined as testing positive by PCR or serology or both (29).The Australian Department of Health Public Health Laboratory Network defined an active syphilis infection as having reactive non-treponemal tests or demonstrated seroconver sion of treponemal test within 12 months of a negative test or detection of T. pallidum by NAAT (i.e., positive PCR) (29).For participants with a history of syphilis, an active infection was defined as having more than a fourfold rise in RPR titer and/or a positive T. pallidum PCR.In cases where only treponemal-specific serology was positive (i.e., positive CLIA and TPPA) without a reactive RPR, positive T. pallidum PCR, or any signs and symptoms of syphilis, the clinical team would confirm the participants' past infection with syphilis and treatment history with the participants in order to determine the presence of an active syphilis infection and to stage the infection correctly.
Categorical variables were reported as frequencies and percentages, and continuous variables as median and interquartile range (IQR).The number needed to screen to obtain a positive T. pallidum PCR test (i.e., indicating a new syphilis infection) was calculated by dividing the number of participants tested for syphilis by the number of participants tested positive for syphilis (30).This calculation was performed separately for anal swab and oral rinse samples.We also calculated the 95% confidence intervals (CIs) for syphilis diagnoses using binomial proportion confidence interval.We anticipated to recruit 1,200 participants over the 36-month period of evaluation.The study was stopped prematurely due to the COVID-19 pandemic in March 2020.During the analysis, we performed a chart review to confirm that the participants included in the study were unique patients.This step was taken to align with the cross-sectional design of the study.All analyses were performed using Stata (Version 16, Texas, USA).

RESULTS
Between November 2019 and March 2020, 309 individuals taking HIV PrEP attended the PrEP clinic for their regular 3-monthly PrEP review appointments and consented to provide both anal swab and oral rinse specimens for T. pallidum screening and were included in the analysis.
The median age was 34 years (IQR: 29-42), and the median number of male sexual partners in the last 3 months was 15 (IQR: 7-23).Most participants (92%, n = 285) had condomless anal sex in the last 3 months.Thirty-two percent (n = 92) had a past history of syphilis infection.Most participants (98%, n = 303) were taking daily PrEP, with 4% (n = 12) reported taking doxycycline as prophylaxis for STIs in the last 1 month.None presented with signs and symptoms of syphilis at presentation or as a sexual contact with an individual with syphilis infection.Table 1 shows the characteristics of the 309 participants.
The man with positive syphilis serology and anal T. pallidum PCR had 40 male sexual partners in the last 3 months and did not report symptoms or signs of syphilis, includ ing at the anus.His RPR titer was 1:8, which increased from 1:1 when the serology was performed 3 months earlier.Six months prior to the current consultation, he had received treatment for early latent syphilis infection with an RPR titer of 1:16 at that time.T. pallidum PCR from the anal swab had a Ct value of 37.He was also diagnosed with anal and pharyngeal gonorrhea.The other man with positive syphilis serology alone had nine male sexual partners in the last 3 months, did not have symptoms or signs of syphilis, and had no past history of syphilis.The RPR titer was 1:8 and T. pallidum PCR was negative from both the anal swab and the oral rinse.

Number needed to screen for syphilis detection using T. pallidum PCR
We estimated that the number needed to screen to detect a syphilis infection using T. pallidum PCR testing on an anal swab was 309.However, we were unable to calculate the number needed to screen for syphilis using an oral rinse, as we did not obtain any positive results from the oral rinse samples.

Estimated cost of using T. pallidum PCR for syphilis detection
The estimated cost of one T. pallidum PCR test for syphilis detection is AUD $35, excluding transport and processing costs (31).In our study, we utilized two T. pallidum PCR tests-anal swab and oral rinse-resulting in an additional cost of AUD $70 per person per visit, in addition to the syphilis serology test.The cost per additional syphilis case detected using additional T. pallidum PCR tests is estimated to be $21,600, calculated based on the need to test at least 309 individuals to detect a single case of syphilis, multiplied by the cost of $70 per person.

DISCUSSION
We conducted this study to examine the role of T. pallidum PCR screening of the oral cavity and anus among individuals at high risk of syphilis infection.We recruited 309 asymptomatic individuals taking HIV PrEP who had previously been reported to have a relatively high incidence of syphilis.Only one case of asymptomatic rectal T. pallidum was detected, and this case had a positive syphilis serology.No participant tested positive by PCR from the oral cavity.Our data suggest that using our method for T. pallidum PCR screening of the oral cavity and anus among MSM taking PrEP may not be cost effective.
Our data are consistent with a previous study showing that PCR testing is unlikely to identify a significant number of cases earlier than serology (32).
To our knowledge, our study is the second study to examine the role of T. pallidum PCR screening of asymptomatic MSM at the mucosal sites.A previous study conducted in the USA found that 0.4% (2/441) of MSM tested positive on anal swab by both transcrip tion-mediated amplification (TMA) testing and PCR, targeting the tpp47 gene before seroconversion of syphilis antibodies (32).In contrast, in our study, the individual with a positive anal PCR swab also had a concurrent positive syphilis serology.Furthermore, in the US study, one individual with a positive anal PCR in the absence of a positive serological test was a syphilis contact with a rectal ulcer, and therefore, the risk of syphilis infection would have been much higher.In our study, none of the syphilis cases was a syphilis contact.It is uncertain whether T. pallidum PCR screening of MSM who are syphilis contacts might detect syphilis by PCR before serology.
Whether or not the detection of T. pallidum by PCR from oral or anal sites precedes the appearance of syphilis antibodies will depend on several factors.This includes the sensitivity of the PCR and serological assays used in the detection of very early asymptomatic infection.The in-house real-time T. pallidum PCR assay in our study has a sensitivity of 80% and specificity of 98% for detecting early syphilis infection from lesion samples (28).The assay has not been validated for non-lesion samples, and therefore, the sensitivity might be lower than the lesion samples.Moreover, when comparing T. pallidum PCR to TMA, the sensitivity of T. pallidum PCR was slightly lower with PCR positive in eight of nine (89%) TMA-positive rectal specimens (32).It is possible that the number of syphilis cases detected by PCR was lower in our study because of the less sensitive T. pallidum molecular assay.Additionally, the two molecular assays target different regions of T. pallidum, with TMA targeting 23S rRNA and PCR in our study targeting polA gene of T. pallidum, respectively.Another factor to consider is the use of CLIA as a serological screening test, which has been shown to have excellent sensitivity and specificity as a confirmatory and a screening test for syphilis (33).In a comparative study where CLIA and enzyme immunoassay (EIA) were evaluated, using TPPA as a confirmatory test, CLIA demonstrated higher sensitivity than EIA (94% vs 83%) (34).However, it remains uncertain whether CLIA assays are more sensitive than other immunoassays.If CLIA assays were to exhibit earlier reactivity than other serological assays, this could potentially diminish any additional advantages from T. pallidum PCR screening.
It is unlikely that adding T. pallidum PCR as a screening test would be cost-effective.An Australian study estimated AUD $35 per T. pallidum PCR test, not including transport and processing costs (31).That means an estimate of an extra AUD $70 per person per visit with two T. pallidum PCR tests added to the existing syphilis serology tests, which cost around AUD $15 (35).Our data suggest that the cost per extra case detected would exceed $20,000, and given that these individuals have serology every 3 months, it means infections are missed for a relatively short period of time.This calculation is dependent on the incidence of syphilis and the proportion of early cases that are PCR positive and serology negative.With a recommendation of 3-monthly STI testing for men taking HIV-PrEP, the costs for syphilis screening would rise if T. pallidum PCR tests were integrated into routine STI screening.
There are several other limitations that should be noted.First, we only recruited 309 participants in this cross-sectional study with very few men who tested positive for syphilis.Our initial protocol was to recruit 1,200 men for over 36 months; however, the study was stopped after 5 months due to the COVID-19 pandemic and lockdown in Victoria, Australia.A small sample size could affect the accuracy of the results.Second, the study period was short, and this could underestimate the incidence of syphilis.Third, none of the participants presented as a syphilis contact, who are at higher risk for syphilis infection.Fourth, it is possible that some participants may have had syphilis diagnosed elsewhere, such as general practice, between the regular review appointments for HIV PrEP.Fifth, it is possible that not all clinic attendees participated to perform T. pallidum screening tests, which could have underestimated the number of syphilis infections.We did not have the data on those who declined the additional screening tests.Last, we included only MSM and transwomen taking PrEP, whose risk of syphilis infection and STI screening practice might differ from other MSM or transwomen who were not taking PrEP or not regularly screened.Moreover, the findings might not be generalizable to heterosexual men and women who might not have STI screening as frequently as MSM.The value of PCR screening in heterosexual populations, particularly in high-risk women, should be evaluated in light of re-emergence of congenital syphilis and an increase in syphilis cases in heterosexual men and women (3,36).
Because of the limitations of our study, larger studies of high-risk populations, not limited to MSM on HIV PrEP, using more sensitive T. pallidum assays for screening are warranted.These studies should be accompanied by cost-effectiveness analyses to determine if the cost of NAAT screening is offset by earlier diagnosis and reductions in syphilis transmission and sequelae.

TABLE 1
Demographics, sexual practices, and medical history of 309 participants taking HIV PrEP