Investigation of SARS-CoV-2 in vaginal secretions of women with coronavirus disease 2019

Objectives: The present study investigates the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the vaginal swabs of female patients diagnosed with coronavirus disease 2019 (COVID-19) based on a positive real-time reverse transcription polymerase chain reaction (RT-PCR) test on a combined throat and nasopharyngeal swab. Methods: This study included 48 female patients hospitalized in two tertiary hospitals diagnosed with COVID-19 based on a positive RT-PCR test of the combined throat and nasopharyngeal swab samples, along with clinical and radiological ﬁ ndings. The IBM SPSS software package was used for the statistical analysis of the study data. Results: SARS-CoV-2 positivity was detected in only one patient (2.08 %) in the present study from RT-PCR tests of vaginal swab samples. This patient was a 64-year-old, post-menopausal woman who tested positive for SARS-CoV-2 in a RT-PCR test of a vaginal swab sample six days after having tested positive in an RT-PCR test of a combined throat and nasopharyngeal swab. The patient ’ s partner also tested positive for SARS-CoV-2 in an RT-PCR of a combined throat and nasopharyngeal swab. Conclusions: The present study is the ﬁ rst to report the presence of SARS-CoV-2 in vaginal secretions in Türkiye. The authors believe there is a need for studies investigating the presence of SARS-CoV-2 in the semen samples of the male partners of female patients to establish whether the presence of SARS-CoV-2 in vaginal secretions can play a role in the transmission of the virus.


Introduction
Coronavirus disease 2019 (COVID- 19) is caused by the alpha variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is responsible for the current global pandemic.
According to the World Health Organization (WHO), there have been 581,686,197 confirmed cases of COVID-19, and 6,410,961 reported deaths worldwide as of August 8, 2022.Furthermore, as of August 1, 2022, a total of 12,308,330,588 doses of vaccine have been administered against COVID-19 around the world [1].
The most common symptoms of COVID-19 are fever, cough, and dyspnea; the most commonly reported comorbidities are hypertension, diabetes mellitus, and cardiovascular system and respiratory disorders [2][3][4].
The definitive diagnosis of COVID-19 is based on the detection of SARS-CoV-2 in a real-time reverse transcription polymerase chain reaction (RT-PCR) test of respiratory nasopharyngeal, oropharyngeal or combined throat and nasopharyngeal swab samples, or on bronchoalveolar lavage fluid and deep tracheal aspirate along with clinical and radiological findings suggestive of the disease [3,5,6].
Aside from the upper and lower respiratory tracts, the virus has also been isolated from anal swabs, urine, and tears [7].There have also been studies investigating the presence of the virus in the vaginal secretions of women [8][9][10][11][12][13][14][15][16], and this finding can be considered worthy of further investigation to elucidate the potential risk of contraction through sexual intercourse and the transmission of the virus from mother to fetus [7].There have also been studies reporting the presence of SARS-CoV-2 in the semen and testes of males infected by the virus in both the acute and recovery phases of the infection [16,17], and this finding may support the notion that SARS-CoV-2 could be contracted from the male partner [16,18,19].
The dominant transmission route of SARS-CoV-2 is exposure to respiratory droplets.However, the virus can also be transmitted through contact with a surface or object contaminated by the virus and then touching the mouth, nose, or eyes.Recent studies have also identified the fecaloral route as another transmission mode [20,21].Aside from the main routes of transmission, the identification of other transmission routes is crucial for the prevention of the spread of the disease and for controlling the spread of infection.
The present study reports on investigating SARS-CoV-2 in the vaginal swabs of female patients diagnosed with COVID-19 after having tested positive in a RT-PCR test of upper respiratory tract samples.

Materials and methods
In this descriptive prospective study, the diagnosis of COVID-19 was based on the guidelines of the Ministry of Health.Among the cases matching the definition of a possible case, those who tested positive for SARS-CoV-2 based on molecular methods (RT-PCR) using combined throat and nasopharyngeal swab samples were classified as definitive cases [22].
Included in the study were 48 patients who were treated as in-patients with a diagnosis of COVID-19 in Ankara Training and Research Hospital and Ankara City Hospital.The demographic data of the patients, as well as comorbid conditions, imaging findings and the time to the RT-PCR testing of the virus from vaginal swab samples, were recorded on a data collection form prepared on an MS Excel sheet.
The vaginal swab samples were collected from the posterior fornix after the insertion of a speculum.The swab was rotated for 5 s and immediately placed into a transport medium after removal from the vagina.
The presence of SARS-CoV-2 in vaginal fluid samples was investigated using the real-time RT-PCR method (Bioeksen, Türkiye), targeting the RNA-dependent RNA polymerase (RdRp) gene.The RT-PCR test kit used in the present study was suitable for identifying SARS-CoV-2 in vaginal fluid samples.
The study was approved by a tertiary Ankara City Hospital, with Ethics Committee approval number E1/1187/2020, dated December 14, 2020.

Sample collection
Cervical samples were collected using flexible, plastic Dacron swabs.After the sample collection, the swabs were plunged into a 2 mL Viral Transport Medium (VTM) and dispatched to the Molecular Biology Laboratory for testing.

Real-time reverse transcriptase PCR
The detection of SARS-CoV2 in vaginal fluid samples was investigated with the Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) method targeting the RdRp (RNA-dependent RNA polymerase) gene.
The RNA extraction from the vaginal fluid samples was performed using a Biospeedy Viral Nucleic Acid Isolation Kit (Bioeksen, Istanbul, Turkey), according to the manufacturer's instructions; The swab samples in the VTM were vortexed for 15 s, and 100 μL samples were transferred to 1.5 mL microcentrifuge tubes containing 100 μL of a viral nucleic acid extraction buffer supplied by the manufacturer.After vortexing again, the tube was ready for the PCR reaction test.
The real-time reverse-transcription PCR was performed with a Bio-Speedy COVID-19 RT-qPCR Detection Kit (Bioeksen, Istanbul, Turkey).A 20 μL reaction volume contained 5 μL RNA, 5 μL Oligo Mix (RdRp gene for SARS-CoV-2 detection, Rnase P gene for internal control), 10 μL 2 × Primer Script Mix containing Taq Polymerase, each deoxyribo triphosphates (dNTP), reverse transcriptase and ribonuclease inhibitor.Thermal cycling was performed at 45 °C for 10 min for the reverse transcription, followed by 95 °C for 3 min and then 45 cycles of 95 °C for 5 s and 55 °C for 35 s in a Rotor-Gene Q device (Qiagen, Hilden, Germany).Cycle threshold (Ct) values less than 40 were defined as positive.

Statistical analysis
IBM SPSS Statistics (Version 21.0.Armonk, NY: IBM Corp.) was used for all statistical analyses.A Shapiro-Wilk test and histograms were used to test whether the variables were normally distributed.Continuous variables without a normal distribution were expressed as medians (minimum-maximum), while variables with a normal distribution were expressed as mean ± standard deviation.Categorical variables were expressed as numbers and percentages (n, %).
Laboratory tests revealed lymphopenia in 29 %, leukocytosis in 6.25 %, and elevated C-reactive protein (CRP) levels in 79 % of the patients; and 71 % of the patients were identified with lung involvement either on chest X-ray or computed tomography of the thorax.In 34 patients (71 %), the typical appearance of pneumonia associated with COVID-19 was observed on computed tomography scans of the thorax.No patient required invasive mechanical ventilation, three patients were discharged from intensive care, and one was under follow-up in the intensive care unit (ICU).The laboratory and imaging findings and clinical outcomes of the patients are presented in Table 2.
Of the total, 11 patients (23 %) had a history of sexual intercourse within one month before diagnosis, and 37 patients (77 %) had no history of sexual intercourse.The partners of eight out of the 11 (73 %) patients with a history of sexual intercourse also tested positive for the virus by RT-PCR.The median time from a positive RT-PCR test on nasopharyngeal and/or throat swabs until the vaginal swab sampling was 5 days (1-22 days).The patient's age and menopausal status and the results of the RT-PCR testing of combined throat/nasopharyngeal swabs and vaginal fluid samples are presented in Table 3. SARS-CoV-2 was identified from an RT-PCR test of vaginal swab samples in one of the 48 patients (2.08 %), a 64-year-old patient with hypertension.Laboratory tests of this patient revealed leukocytosis (white blood cell count: 11,080/mm 3 ), while a chest X-ray revealed lung involvement.
In a prospective, observational study, the ribonucleic acid (RNA) associated with SARS-CoV-2 was not identified in 102 peritoneal fluid samples and 98 vaginal swab samples of 113 patients who were to undergo abdominal or lower genital tract surgery [8].
In a Turkish study, Aslan et al. [10] reported negative RT-PCR test results from vaginal fluid samples of 12 pregnant women with a mean age of 32 ± 7.9 years with a confirmed diagnosis of COVID-19 based on RT-PCR.
In a study conducted in India, Khoiwal et al. [11] investigated the presence of SARS-CoV-2 using RT-PCR testing on the vaginal and cervical smear samples of 61 women (28 of reproductive age and 33 postmenopausal) with Rubin et al. [9] identified the presence of SARS-CoV-2 in a vulvar lesion in a 26-year-old female patient based on the RT-PCR method.In the present study, the RT-PCR testing of

The day of RT-PCR testing after diagnosis
In throat/nasopharyngeal swab SARS-CoV- RT-PCR result

SARS-CoV- in vaginal fluid
The results of SARS-CoV- RT-PCR on combined throat and nasopharyngeal swab of sexual partner In a meta-analysis of studies examining the vertical transmission of the virus from the mother to the fetus, Yang et al. [12] reviewed 83 neonates born to mothers with COVID-19 in a total of 22 studies.They reported positive RT-PCR test results from nasopharyngeal swabs in three neonates 16, 36, and 72 h after birth.However, the RT-PCR testing of amniotic fluid, placenta, breast milk and cord blood revealed negative results, suggesting the nonoccurrence of the intrauterine vertical transmission of SARS-CoV-2.
In a study of 10 postmenopausal women with severe COVID-19 pneumonia based on clinical and radiological findings confirmed by RT-PCR testing, Qiu et al. [13] reported negative RT-PCR tests for SARS-CoV-2 17 days after the onset of the disease and onwards.
In the present study, the median time from a positive RT-PCR test based on the combined throat and nasopharyngeal swabs of 48 female patients until vaginal swab sampling was 5 days (1-22 days).
In a Chinese study of 35 women with a median age of 61.5 years, Cui et al. investigated the presence of SARS-CoV-2 using RT-PCR on rectal and vaginal swab samples.They reported negative RPT-PCR test results for SARS-CoV-2 from the vaginal swab samples, while one patient tested positive for SARS-CoV-2 on a rectal swab sample [14,15].The rate of SARS-CoV-2 positivity was 43 % in the sexual partners of the patients.In the two patients who reported sexual intercourse with their partners during the possible incubation period, one partner tested positive, while the other tested negative for SARS-CoV-2.It was suggested that this finding might be attributable to the scarce expression, or perhaps the lack of the angiotensin-converting enzyme-2 (ACE-2) expression, in the vagina and cervix, where the virus binds to the host cell through its spike protein [14].
In contrast to their findings, there are also studies reporting an abundant expression of ACE-2 in the ovary, uterus, vagina, and placenta., which suggests the possibility of viral transmission from the mother to the fetus and through sexual intercourse, aside from transmission through droplets and contact [23].
No studies conducted in Türkiye have reported SARS-CoV-2 positivity using RT-PCR on vaginal swab samples [10].
The present study can be considered worth publication as the first study in Türkiye to report SARS-CoV-2 positivity in a vaginal swab sample using RT-PCR.
The limitations of the present study include the small number of patients who underwent RT-PCR testing for SARS-CoV-2 on vaginal swab samples, the nonhomogeneous age distribution of the study patients, and the lack of testing of genital samples of the male partners for SARS-CoV-2 to support the suggested transmission through sexual intercourse.

Conclusions
In conclusion, the authors consider further clinical studies involving a larger number of patients and investigating the presence of SARS-CoV-2 in the semen samples of male partners using RT-PCR are required to elucidate whether the presence of SARS-CoV-2 in vaginal secretions has a role in virus transmission through sexual intercourse.
Research funding: None declared.Author contributions: All authors have accepted respon sibility for the entire content of this manuscript and approved its submission.Competing interests: Authors state no conflict of interest.Informed consent: Informed consent was obtained from all individuals included in this study.Ethical approval: The study was approved by a tertiary Ankara City Hospital, with Ethics Committee approval number E1/1187/2020, dated December 14, 2020.

Table  :
Patient history of contact with COVID--positive family member prior to diagnosis, comorbidities and symptoms related to COVID-.

Table  :
Laboratory and imaging findings and clinical outcomes of the patients.

Table  :
Patients' age and menopausal status, rRT-PCR test results, and rRT-PCR test results of their partners.