Identification of SARS-CoV-2 in the vaginal fluid and cervical exfoliated cells of women with active COVID-19 infection: A pilot study.

SARS‐CoV‐2 has been identified in nasopharyngeal secretions, feces, urine, semen, and tears. Foundational research about the presence of SARS‐CoV‐2 in the female genital tract may help to determine the risk of sexual transmission, as well as the risk of mother‐to‐child transmission during labor. The present study aimed to assess the presence of SARS‐CoV‐2 in vaginal fluid and cervical exfoliated cells of women with active COVID‐19 infection. This study is registered with the Clinical Trial Registry of India (CTRI/2020/09/027618). Ethical approval for this study was obtained from the local institutional ethics committee (AIIMS/IEC/20/575).

This prospective study which was conducted at a tertiary care center included 15 women with active COVID‐19 infection (both symptomatic and asymptomatic), diagnosed by reverse transcriptase‐polymerase chain reaction (RT‐PCR) from nasopharyngeal (NP) and oropharyngeal (OP) samples from November 25, 2020, to December 12, 2020. After obtaining informed written consent, vaginal (n = 15) and cervical (n = 12) swabs were collected. Three women with surgically absent cervices only had vaginal swabs collected. Samples for vaginal fluid and cervical exfoliated cells were obtained from the posterior fornix of the vagina and ectocervix using a speculum. Swabs were rotated for at least five seconds and extracted. All samples were kept in a viral transport medium (VTM), immediately transported to the microbiology laboratory, and treated as per the WHO standard protocol for laboratory diagnosis of SARS‐CoV‐2. Briefly, RNA extraction was performed from the first aliquot of VTM fluid in a magnetic bead‐based automated system (Kingfisher™ Flex System; Thermo Fisher Scientific, Waltham, MA, USA) followed by real‐time RT‐PCR using the Taqpath™ (Thermo Fisher Scientific, Waltham, MA, USA) COVID‐19 combo kit (for N, S, and ORF1b targets) in a QuantStudio™ 7 thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, a second sample aliquot was processed for transcription mediated amplification (TMA) in an FDA‐approved closed system called the Hologic Panther System (M/s Hologic Ltd, Manchester, UK). Post amplification target (ORF1ab) was detected by nucleic acid hybridization in terms of relative light units (RLU).

Table 1 presents the baseline clinical characteristics and laboratory results of the 15 participants. Mean age of participants was 51.26 years (range: 27–70 years). SARS‐CoV‐2 virus was not detected in vaginal and cervical samples by RT‐PCR test; however, it was identified in the vaginal fluid of three (20%) participants with the TMA Panther System. Out of these three participants, two were premenopausal and one was postmenopausal; one was symptomatic with COVID‐19‐related pneumonia (moderate), and the other two had asymptomatic COVID‐19 infection and were admitted for gynecological indications.

TABLE 1

Baseline clinical characteristics and laboratory results.

Case	Age	Menopausal status	Parity	Co‐morbidities	Presenting complaints	Diagnosis at admission	Severity	SARS‐COV−2 (OP&NP swabs) by RT‐PCR		Vaginal swab by RT‐PCR	Cervical swab by RT‐PCR	Vaginal swab by Panther System (with RLU value)	Cervical swab by Panther System
								Result	CT value (lowest)
1	27	Premenopausal	2	‐	Asymptomatic	Ectopic pregnancy	‐	Positive	23	Negative	Negative	Positive ‐ 651 (RLUX1000)	Negative
2	58	Postmenopausal	5	‐	Asymptomatic	Cervical carcinoma	‐	Positive	29	Negative	Negative	Negative	Negative
3	70	Postmenopausal	2	‐	Sore throat	URTI	Mild	Positive	24	Negative	Negative	Negative	Negative
4	42	Surgical menopause	2	‐	Fever and sore throat	URTI	Mild	Positive	30	Negative	Negative	Negative	Negative
5	70	Postmenopausal	3	‐	Fever, breathlessness, and sore throat	COVID−19‐related pneumonia	Moderate	Positive	21	Negative	Negative	Negative	Negative
6	65	Postmenopausal	3	‐	Asymptomatic	Ovarian carcinoma	‐	Positive	28	Negative	Negative	Negative	Negative
7	54	Surgical menopause	3	DM, HTN	Fever and breathlessness	COVID−19‐related pneumonia	Moderate	Positive	28	Negative	Negative	Negative	Negative
8	48	Premenopausal	3	TB, hyperthyroidism	Asymptomatic	Ovarian carcinoma	‐	Positive	30	Negative	Negative	Negative	Negative
9	54	Postmenopausal	4	DM	Fever and sore throat	COVID−19‐related pneumonia	Moderate	Positive	28	Negative	Negative	Negative	Negative
10	44	Surgical menopause	4	‐	Asymptomatic	Ovarian carcinoma	‐	Positive	18	Negative	Negative	Negative	Negative
11	45	Premenopausal	0	‐	Asymptomatic	Ovarian carcinoma	‐	Positive	27	Negative	Negative	Negative	Negative
12	61	Postmenopausal	5	‐	Asymptomatic	Ovarian carcinoma	‐	Positive	27	Negative	Negative	Positive ‐ 1021 (RLUX1000)	Negative
13	40	Premenopausal	3	DM	Fever and breathlessness	COVID−19‐related pneumonia	Moderate	Positive	23	Negative	Negative	Positive ‐ 662 (RLUX1000)	Negative
14	52	Postmenopausal	3	DM, HTN	Fever and breathlessness	COVID−19‐related pneumonia	Moderate	Positive	24	Negative	Negative	Negative	Negative
15	39	Premenopausal	2	‐	Asymptomatic	Large uterine fibroids	‐	Positive	17	Negative	Negative	Negative	Negative

Abbreviations: CT, cycle threshold; DM, diabetes mellitus; HTN, hypertension; NP, nasopharyngeal; OP, oropharyngeal; RLU, relative light unit; RT‐PCR, reverse transcriptase‐polymerase chain reaction; TB, tuberculosis; URTI, upper respiratory tract infection.

We could only find one study which reported detection of SARS‐CoV‐2 in vaginal swabs, while other case studies did not identify the presence of SARS‐CoV‐2 in vaginal fluid. , The detection rate in the present study was 20%, which might be explained by the higher analytical sensitivity and specificity of TMA‐based technology than RT‐PCR testing. ,

The strengths of the study were its prospective nature, inclusion of both reproductive age and postmenopausal women with mild to moderate COVID‐19 disease severity, and utilization of two different testing techniques (RT‐PCR & TMA). However, the small sample size and short study period are potential limitations. A larger cohort and longer duration of study would produce more robust results.

This pilot study demonstrated that a negative RT‐PCR test result does not rule out the possibility of a low level of SARS‐CoV‐2 in the female genital tract of women with confirmed COVID‐19 infection.

CONFLICTS OF INTEREST

The authors have no conflicts of interest.

AUTHOR CONTRIBUTIONS

KK and DK proposed the idea for the analyses. RK and DD collected the samples and relevant data. DK and RS performed the laboratory analysis. KK drafted the manuscript with direction from DK and JC. AB, PP and JC critically evaluated the manuscript. All authors approved the final version of the manuscript.