Self-collected penile-meatal swabs are suitable for the detection of STIs in Ugandan men with high rates of STI coinfections

Abstract

Background:

Sexually transmitted infections (STIs) are a global health challenge, with low- and middle-income countries (LMICs), where testing is not routinely performed, bearing a disproportionate burden. Self-collected penile-meatal swabs (SCPMS) are an alternative to urine for STI testing, but data from LMICs are limited.

Methods:

Between October 2019 and September 2020, recruited participants with urethral discharge syndrome at government health facilities in Kampala, Uganda provided urine and SCPMS. Samples were retrospectively analyzed for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), and Trichomonas vaginalis (TV) using Aptima® assays. Paired SCPMS and urine samples with discordant results were analyzed using real-time PCR assays.

Results:

Of the 250 participants, 66.8% (n=167), 22.8% (n=57), 12.8% (n=32), and 2.4% (n=6) tested positive for NG, CT, MG, and TV, respectively, on testing of their SCPMS and urine samples. The overall agreement between urine and SCPMS for all STIs using the Aptima assays was 98.7% (987/1000). After adjudication with PCR assays, the agreement between urine and SCPMS for NG, CT, MG, and TV was 100% (250/250), 97.6% (244/250), 99.6% (249/250), and 99.6% (249/250), respectively. There was no significant difference between SCPMS and urine (p-value=0.9996). For paired samples with one STI (n=164) or ≥ 2 STIs (n=39), concordance was 100% (656/656) and 97.2% (175/180), respectively. There was no association between number of STIs and agreement between sample types (p=0.8606).

Conclusions:

This study revealed a high level of agreement between SCPMS and urine for detection of STIs in symptomatic Ugandan men, even in the presence of multiple concurrent STIs. SCPMS may be a suitable alternative to urine, with ease of collection, transporting, and processing of samples.

Introduction

Curable sexually transmitted infections (STIs) pose a significant global health burden, with the World Health Organization (WHO) estimating that over 1 million STIs are acquired daily worldwide.¹ Low- and middle-income countries (LMICs) bear a disproportionate burden, with Africa being notably affected by a high prevalence of STIs.² In addition to the high prevalence of STIs, access to diagnostics and treatment is limited in LMICs. Delayed treatment of these infections could lead to a health and economic burden.³ While STIs caused by Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), and Trichomonas vaginalis (TV) are treatable, they can have a significant impact on reproductive health and increase the risk of HIV acquisition.¹.

Several diagnostic methods are employed for STIs. For men, the Centers for Disease Control and Prevention (CDC) recommends first catch urine for detection of STIs.⁴ However, collection of urine may require a bathroom, which may not be readily available in LMICs, and transport to a reference laboratory for testing can be cumbersome. Urethral swabs are an alternative to urine but require a clinician for collection, and collection is associated with discomfort and/or pain in men.⁵ Self-collected penile-meatal swabs (SCPMS) have been shown to be as accurate as urethral swabs for detection of STIs,⁶ may perform better than urine,⁷ and can be collected in a variety of venues.⁸ Self-collected swabs are easier to collect, process, and transport compared to urine samples.⁹ Furthermore, many of the innovative devices developed during the COVID-19 pandemic used self-collected swab specimens and may have platform potential as STI diagnostics. Although the SCPMS would have to be validated, devices that accept swab sample types could be leveraged to accelerate STI diagnostic devices.¹⁰ Although the performance of SCPMS for the detection of STIs has been previously evaluated,^{6, 7, 11–14} there are limited data on the performance of this collection modality in LMIC settings with high rates of STIs and multiple co-infections.

In a previous study, using SCPMS we described high rates of STIs, with gonorrhea exceeding 60%, among Ugandan men with urethral discharge syndrome (UDS).¹⁵ In this study, we assessed the performance of SCPMS for detecting NG, CT, Mycoplasma genitalium (MG), and TV in comparison to paired urine using samples from the previously described study.¹⁵ Additionally, we explored whether concurrent STIs influence the accuracy of SCPMS.

Material and Methods

Participants’ characteristics and recruitment.

The participants and samples used for this analysis have been described elsewhere.¹⁵ Briefly, between October 2019 and September 2020 (recruitment was halted between March and July 2020 due to the COVID-19 pandemic), men presenting with UDS at six Enhanced Gonococcal Antimicrobial Surveillance Programme (EGASP)-participating government health clinics in Kampala, Uganda were recruited. The study aimed to characterize the burden of curable STIs and HIV in Ugandan men with UDS.¹⁵ Prior to study entry, men were treated for UDS using syndromic case management per Ugandan government guidelines.¹⁵ As previously described, in this group of participants with a median (interquartile range (IQR)) age of 24 (22.0–32.0) years, behaviors associated with poor sexual health outcomes were common, including low condom use (<0.5% reported always condom use), participation in transactional sex (61.6%), and alcohol use before sex (56.7% of alcohol users).¹⁵

Sample collection:

Upon obtaining their consent, participants were asked to self-collect two penile-meatal swabs and provide urine; participants were asked to collect the SCPMS prior to collection of urine.¹⁵ Prior to sample collection, a research nurse described the procedure for self-collection of samples to participants using a pictorial illustration and a penis model. Following collection, all samples were transferred to the Infectious Diseases Institute (IDI) Translational Research Laboratory in Kampala, Uganda for processing and testing.

SCPMS and urine processing.

SCPMS were eluted in 1X phosphate-buffered saline, and frozen at −80°C as previously described.¹⁵ Urine samples were aliquoted in cryovials and frozen at −80°C. SCPMS eluates and urine samples were sent to the Johns Hopkins University School of Medicine for nucleic acid amplification tests (NAATs) analysis as described below.

Aptima® STI assays.

At Johns Hopkins, 200 μL of each swab eluate was individually transferred to the Aptima® Multitest Swab tube. Urine samples were transferred to the Aptima® Urine Specimen Collection Tube following manufacturer’s instructions. NAATs were performed on both the SCPMS and paired urine samples using the Aptima® CT/NG, TV, and MG assays (Hologic Inc., Marlborough, MA, USA) as previously described.⁶

In-house real-time PCR for adjudication of discordant STI results.

Paired SCPMS and urine samples that produced discordant results when analyzed with the Aptima® STI assays were also analyzed using real-time PCR assays. Additional testing with DNA-based real-time PCR assays was performed to determine if target DNA from the STI in question was present in paired SCPMS and urine samples with discordant results. For detection of MG, NG, and TV, previously described methods^16–18 were used with minor modifications as follows. The final reaction volume for MG, NG, and TV assays was increased to 50 μL and the reaction components were replaced with 25 μL of TaqMan™ Universal PCR Master Mix (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA) and 2 μL of 25 mM MgCl. Primer and probe concentrations were identical, however, for the TV reactions the probes listed in the reference¹⁸ were replaced with a single probe 5’−6FAM-ACA CCA ACA TAC GGC GATBHQ13’. Cycling conditions were modified as follows: initial denaturation at 95°C for 20 seconds, 40 cycles of denaturation at 95°C for 3 seconds, and annealing and extension at 60°C for 30 seconds. All assays were performed on the Applied Biosystems 7500 Fast Dx (Applied Biosystems, Foster City, CA, USA) instrument. Positive reactions were defined as reactions with PCR cycle threshold (Ct) values <40.

For CT detection, we performed an assay to detect the pmpH gene, which is a gene common to all CT serovars. The primer sequences were as follows: 139F 5’ACG ATT ACC GGA CAA AAC CA3’ and 542R 5’TTC CCG GCA TTA TTA TCG AA3’. PCR reactions consisted of 15 μL PowerUp SYBR Green Master Mix (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA), 2.5 μL of each primer (Integrated DNA Technologies, Coralville, Iowa, USA), and 10 μL of the template DNA (total 30 μL per reaction). Cycling conditions were as follows: Initial denaturation at 95°C for 20 seconds, cycle denaturation at 95°C for 3 seconds, cycle annealing and extension at 60°C for 60 seconds, repeat 40 cycles. Melt curve denaturation at 95°C for 15 seconds, annealing at 60°C for 60 seconds, second denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds. The presence of PCR product was confirmed by evidence of amplification and/or evidence of a first-derivative dissociation curve.

Statistical analysis.

Pearson’s Chi-squared and chi-squared tests for independence were performed using R version 4.3.1. Cohen’s kappa (κ) statistics for attribute agreement analysis were performed using GraphPad (https://www.graphpad.com/quickcalcs/kappa1.cfm).

Ethical approval.

In Uganda, the Joint Clinical Research Center (protocol reference number JC0919), and the Ugandan National Council for Science and Technology (study number HS455ES) approved the study. Study procedures were also approved by The Johns Hopkins Institutional Review Board (IRB00215298). No study procedures were conducted until written informed consent had been obtained from participants.

Results

All eligible participants (n=250) agreed to participate in the study and successfully collected multiple SCPMS and provided a urine sample. The demographics of participants and the STI results using SCPMS have been previously described.¹⁵ Briefly, the proportion of participants with SCPMS NAAT positive for NG, CT, MG, and TV was 66.4% (164/247), 21.7% (54/249), 12.4% (31/250), and 2.0% (5/250).¹⁵ Following the analysis of urine samples for this study, the proportion of participants with positive NAATs for NG, CT, MG, and TV increased to 66.8% (n=167), 22.8% (n=57), 12.8% (n=32), and 2.4% (n=6) when SCPMS and urine NAAT results were combined.

Table 1 describes the agreement between SCPMS and urine samples when using the Aptima® NAATs. The number of NAAT-positive participants by both SCPMS and urine for NG, CT, MG, and TV were 162, 51, 31, and 5, respectively. The overall agreement between NAAT results between the two sample types (SCPMS and urine) for NG, CT, MG, and TV was 98.0%, 97.6%, 99.6%, and 99.6%, respectively. Discordant results between the two sample types were more common (n=6) for CT. In total, 5.2% (n=13) of paired samples (SCPMS and urine from the same participant) exhibited discordance between SCPMS and urine NAAT results for specific STIs.

Table 1.

Agreement between SCPMS and urine using the Aptima® STI assays.

	SCPMS/Urine				Overall agreement
	+/+	+/−	−/+	−/−
Neisseria gonorrhoeae (NG)	162	2	3	83	98.0%
Chlamydia trachomatis (CT)	51	3	3	193	97.6%
Mycoplasma genitalium (MG)	31	0	1	218	99.6%
Trichomonas vaginalis (TV)	5	0	1	244	99.6%
Total	249	5	8	738	98.7%

SCPMS – self-collected penile-meatal swab.

Table 2 describes the agreement in STI results following additional testing of discordant SCPMS and urine paired samples with in-house DNA-based real-time PCR assays. The overall agreement between the two specimen types for RNA- and DNA-based detection of STIs was 99.2% (992/1000 tests). Notably, all discrepancies of Aptima® NAAT NG results (n=5; (2 SCPMS-positive/urine-negative and 3 SCPMS-negative/urine-positive)) between the two specimen types were successfully resolved following analysis with DNA-based tests. Of the six pairs with discordant Aptima® NAAT CT results, four pairs exhibited the same discordant results with the DNA PCR assay as with the RNA-based Aptima® assay. For the remaining two pairs of samples, discrepancies in the CT results could not be adjudicated because samples were unavailable for further testing. For paired samples with discordant MG (n=1) and TV (n=1) results, the DNA PCR results were the same as those obtained with the Aptima® STI assay. Importantly, the differences between the SCPMS and urine samples STI results were not significant (χ² =0.013931, df = 3, p-value=0.9996) with an almost perfect agreement overall (κ=0.979, 95% CI = 0.965 – 0.994) and within each test (NG: κ=1.0, CI = 1.000–1.000; CT: κ=0.929, CI = 0.873–0.985; MG: κ=0.982, CI = 0.946–1.000; TV: κ=0.907, CI = 0.726 – 1.000).

Table 2.

Agreement between SCPMS and urine following additional analyses using in-house real-time PCR assays.

	SCPMS/Urine				Overall agreement
	+/+	+/−	−/+	−/−
Neisseria gonorrhoeae (NG)	167	0	0	83	100.0%
Chlamydia trachomatis (CT)	51	2*	2*	193	97.6%
Mycoplasma genitalium (MG)	31	0	1	218	99.6%
Trichomonas vaginalis (TV)	5	0	1	244	99.6%
Total	254	2	3	738	99.2%

SCPMS – self-collected penile-meatal swab.

^*Two pairs of discordant samples could not be adjudicated due to availability of samples.

Concurrent urethral STIs were common in men with UDS. Of the 250 participants, 164 (65.6%) had a single STI, and 39 (15.6%) had two or more STIs. Notably, there was a high concordance of STI results between SCPMS and urine samples regardless of the number of STIs. Specifically, 100% of paired SCPMS and urine samples from the 164 participants with a single STI showed agreement for STI results; in the 39 participants with ≥ 2 STIs, the agreement between paired samples for STI results was 97.2% (175/180 tests). The chi-squared test for independence revealed no significant association between the number of STIs and agreement between SCPMS and urine (χ² = 0.0308, df = 1, p = 0.8606).

Discussion

In this study, we assessed the performance of SCPMS for detection of four curable STIs in Ugandan men with UDS. The rate of STIs in this group of symptomatic men was very high, especially for gonorrhea. SCPMS had comparable performance to urine, with a 99.2% agreement for detection of STIs, and the difference between STIs results obtained from SCPMS and urine was not statistically significant. Furthermore, SCPMS proved suitable for the detection of multiple co-current STIs.

Our findings support data from previous studies^{6, 7, 11–14} that SCPMS are a suitable specimen type for detecting STIs in men. SCPMS require swabbing over the urethral meatus, an often pain-free procedure, unlike urethral swabs which can cause discomfort and pain.⁵ Our study, however, is one of the first to highlight the potential of SCPMS as an additional specimen type for STI testing in a LMIC in men with high rates of STIs. Given that testing is not performed due to a lack of affordable diagnostic tests, syndromic management is used in Uganda for the treatment of UDS-associated STIs.¹⁹ As such, data on the prevalence and rates of STIs is limited in this setting. As self-collected swabs might be easier to collect, transport, and process than urine⁹, SCPMS could be used in resource-limited clinical settings for STI testing and in surveillance programs aimed at understanding the epidemiology of STIs in these settings. Additionally, SCPMS could be used for NG cultures, which following additional validation, could help expand availability of gonococcal antimicrobial resistance testing for both clinical and surveillance purposes.²⁰ allowing for the assessment of local antimicrobial susceptibility trends. Furthermore, studies in Kampala, and Rakai, Uganda found that self-collected genital samples were acceptable to both male and female participants, regardless of STI symptoms, highlighting the potential of this specimen type to expand STI diagnostic services.^{21, 22}

Although chlamydia and gonorrhea co-infections are common, most of the previous studies assessing the performance of SCPMS,^{6, 7, 11–14} have evaluated their performance in populations with a low prevalence of chlamydia and gonorrhea co-infections. In our study, 15.6% of men were positive for two or more STIs. The performance of SCPMS for detection of co-occurring STIs in our study was comparable to that of urine. This finding provides further support for SCPMS as an accurate specimen collection method for the diagnosis of multiple STIs.

In our study, 5.2% of paired SCPMS and urine samples had discordant Aptima NAAT results for specific STIs. We hypothesized that freezing the samples, instead of placing them directly in the Aptima® collection tube, and later thawing them could have contributed to the discordant results (i.e. RNA degradation). Additional testing of the discordant paired samples with in-house assays targeting DNA resolved 38.5% (n=5; all NG) of discordant results, suggesting that RNA degradation in these samples could have contributed to the discrepant results. Discrepancies between SCPMS and urine for CT, MG, and TV were not resolved following additional testing. Other factors that could have contributed to the discrepancies between specimen types, include improper collection of the SCPMS by participants, failure to collect first-catch urine specimens, or laboratory error during the specimen storage process.

Self-collection for STI testing has been shown to have numerous advantages in different scenarios. In the WHO Consolidated Guideline on Self-Care Interventions for Health: Sexual and Reproductive Health and Rights, the WHO highlights that self-collection provides benefits such as privacy, confidentiality, speed, convenience, and access (when affordable), enabling patients to actively participate in their health care.²³ A meta-analysis of 11 studies conducted in Australia, Denmark, and the USA found that programs offering self-collection of samples increased overall uptake of STI testing services compared with clinician-collection.²⁴ Another study reported that non-invasive, self-collected male specimens that are stable, and easy to transport and process, would enhance the success of male screening programs, particularly difficult-to-test populations.¹⁴ In scenarios where access to clinics is limited, such as LMIC settings where the health facilities are overcrowded with long waiting times, self-collected samples could provide a way of reducing pressure on the health system. For example, home self-testing for HIV is being rolled out in Uganda and has been shown to be acceptable in mothers and key populations.²⁵ The COPHAS study has shown that self-collection for STI testing is feasible and acceptable in community pharmacies, which are often the main provider of health services for many in Africa.²⁶ However, self-collection has certain limitations, especially regarding linkage to care as seen in the Ugandan HIV self-testing work.²⁵ Other concerns regarding self-collection of samples outside the clinical setting include, suboptimal return rates of samples for testing,^27–29 the cost of performing NAATs,²⁹ and patient access to appropriate care and treatment services after testing.²³

A limitation of this study is the lack of asymptomatic participants. Future research should include participants without symptomatic UDS to assess the sensitivity of SCPMS in individuals with a lower bacterial load. The lower bacterial load could affect the sensitivity of the tests during early infection, particularly as the samples are self-collected and, therefore, the degree of care and skill during collection can differ among participants. Detecting asymptomatic cases is of crucial importance as a high prevalence and incidence of asymptomatic STIs were identified among men and women in a variety of settings (China, India, Peru, Russia, and Zimbabwe).³⁰ Another limitation of this study is the low number of participants with two or more STIs. Assessing more participants with multiple STIs could provide further insights into the efficacy of SCPMS in detecting multiple concurrent STIs. For example, this would help determine the reliability of the SCPMS where different infections may influence the detectability of each specific pathogen. Our analysis used samples from a study aimed at characterizing the burden of curable STIs and HIV in Ugandan men with UDS and therefore was not powered to look at differences between SCPMS and urine for detection of STIs. However, given the high number of NG- and CT-positive samples and the high concordance rate between SCPMS and urine for detection of these STIs, SCPMS are likely a suitable collection modality for detection of gonorrhea and/or chlamydia. Lastly, additional testing using DNA-based assays was required to adjudicate discordant STI results between paired SCPMS and urine. Samples should be collected and processed following manufacturers’ instructions for NAATs, including placing the sample directly in the collection tube immediately following collection.

In agreement with other studies,^{6, 7, 11–14} the data presented in this study demonstrated that SCPMS are a suitable and acceptable sample type for the detection of CT, NG, MG, and TV among symptomatic men, including populations with prevalent co-infections. Our results highlight the utility of these swabs for accurately detecting bacterial STIs in LMIC settings such as Uganda. Implementing SCPMS samples may allow easier hub-and-spoke transport across diverse clinical and community settings and also allow the qualification of swab-based POC diagnostic assays that may struggle to incorporate urine as a sample type.¹⁰ Further research is warranted to assess the quality and implementation of self-collected samples in low-resource environments, aiming to optimize STI detection strategies and improve public health outcomes.

What is already known on this topic

Prior studies have demonstrated that self-collected penile-meatal swabs (SCPMS) are a suitable alternative to urine for detection of sexually transmitted infections (STIs) in men and are acceptable to men as this sample type is less invasive than urethral sampling. Despite robust evidence regarding the utility and benefits of SCPMS, there are limited data for SCPMS performance in low-resource settings in men with high STI and STI co-infection rates.

What this study adds

Our comparative study in symptomatic Ugandan men illustrates that SCPMS provided the same detection rates for curable STIs caused by Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, and Trichomonas vaginalis as urine (overall agreement 98.7%). Notably, the performance of SCPMS for detection of STIs was not affected by multiple co-infecting STIs. All eligible participants agreed to participate in the study and successfully collected multiple SCPMS suggestive of high acceptability; this underscores the potential of self-collection to increase STI testing in underserved men.

How this study might affect research, practice or policy

Our study demonstrates that SCPMS, which are easier to collect and transport than urine, could be implemented in surveillance programs in settings with limited clinical and laboratory infrastructures and then transported to reference labs. New point-of-care STI testing platforms using swab samples may also be validated for use in men. The high STI and HIV burden in Ugandan men underscores the need to better define the epidemiology of STIs in this population in order to develop and implement STI prevention interventions.