Corina Manolea 1,2,*, Andrei Capitanescu3, Roxana Bors, 4, Ioana Rugescu5, Melihan Bechir2,6, Claudia Mehedintu1,7, and Valentin Varlas1,4
1Department of Obstetrics and Gynecology, ‘Carol Davila’ University of Medicine and Pharmacy, Bucharest, Romania 2Department of Assisted Reproduction, Columna Medical Center, Bucharest, Romania 3Hemodialysis Unit, ‘Marie Curie’ Pediatric Clinical Emergency Hospital, Bucharest, Romania 4Department of Obstetrics and Gynaecology, Filantropia Clinical Hospital, Bucharest, Romania 5Department of Cells, National Transplant Agency, Bucharest, Romania 6Dept of Obstetrics and Gynecology, Infertility Center, Regina Maria Medical Network, Bucharest, Romania 7Department of Obstetrics and Gynecology, Nicolae Malaxa Clinical Hospital, Bucharest, Romania
*Correspondence address. Department of Obstetrics and Gynecology, ‘Carol Davila’ University of Medicine and Pharmacy, 37 Dionisie Lupu St, Bucharest 020021, Romania. E-mail: corina.manolea@drd.umfcd.ro https://orcid.org/0000-0003-1230-1073
Submitted on March 13, 2021; resubmitted on June 10, 2021; editorial decision on June 18, 2021
STUDY QUESTION: What is the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in triage- negative patients undergoing ART and fertility care providers after lockdown release and throughout 2020?
SUMMARY ANSWER: Out of the triage-negative patients whose blood samples were assessed for SARS-CoV-2 antibodies over 6 months, 5.2% yielded positive results with a significantly higher rate in health care workers (HCWs) and a significant month-by- month increase in those with evidence of antibodies.
WHAT IS KNOWN ALREADY: Patients of reproductive age are more prone to asymptomatic or minimal forms of coronavirus disease 2019 (COVID-19) as compared to older age groups, and the identification of those with active infection and those already exposed (and probably immunized) is important for safety and cost-effective use of testing resources in the fertility setting. Data on the prevalence of SARS-CoV-2 in ART patients are limited and encompass short time frames; current rates are unknown. There is also no consensus on the optimal way of screening triage-negative ART patients in moderate/high-risk areas.
STUDY DESIGN, SIZE, DURATION: A prospective longitudinal unicentric study on triage negative ART patients (n ¼ 516) and clinical staff (n ¼ 30) was carried out. We analyzed 705 serological tests for SARS-CoV-2 sampled between 17 May 2020 (the first working day af- ter lockdown release) up to 1 December 2020, to assess the positivity rates for SARS-CoV-2 antibodies.
PARTICIPANTS/MATERIALS, SETTING, METHODS: We collected data on the serological status for IgM and IgG antibodies against SARS-CoV-2 in 516 triage-negative men (n ¼ 123) and women (n ¼ 393) undergoing ART at a private fertility center and 30 HCWs that were at work during the study period. Antibodies were detected with a capture chemiluminescence assay (CLIA) targeting the highly Immunogenic S1 and S2 domains on the virus spike protein. We also analyzed the molecular test results of the cases exhibiting a positive serology.
MAIN RESULTS AND THE ROLE OF CHANCE: The data showed that 5.2% of the triage-negative ART patients had a positive serological result for SARS-CoV-2, with an overall conversion rate of 2.1% for IgG and 4.6% for IgM. There was no significant difference in seroprevalence between sexes. The small cohort (n ¼ 30) of HCWs had a markedly increased seroprevalence (12.9% for Ig M and 22.6% for IgG). The highest seropositivity in our cohort was recorded in November (16.2%). The IgM positivity rates revealed significant monthly increments, paralleling official prevalence rates based on nasopharyngeal swabs. No positive molecular tests were identified in cases exhibiting a solitary positive IgG result. We show that despite a 6-fold increase in the number of ART patients with a positive serology between May and December 2020, most of our patients remain unexposed to the virus. The study was undertaken in a high-risk area for COVID-19, with a 20-times increase in the active cases across the study period.
LIMITATIONS, REASONS FOR CAUTION: The geographical restriction, alongside the lack of running a second, differently-targeted immunoassay (orthogonal testing), could limit the generalizability and translation of our results to other fertility settings or other immunoassays.
WIDER IMPLICATIONS OF THE FINDINGS: The low positivity rates for IgG against the SARS-CoV-2 spike protein seen at the end of 2020 imply that most of the fertility patients are still at risk for SARS-CoV-2 infection. Until mass vaccination and other measures effec- tively diminish the pandemic, risk mitigation strategies must be maintained in the fertility units in the foreseeable future. Patients with a solitary IgGþ status are most likely ‘non-infectious’ and can elude further testing without giving up the strict use of universal protective measures. With increasing seroprevalences owing to infection or vaccination, and with the consecutive increase in test performance, it is possible that serological screening of ART patients might be more cost-effective than PCR testing, especially for the many patients with re- peat treatments/procedures in a time-frame of months.
STUDY FUNDING/COMPETING INTERESTS: This research received no external funding. All authors declare having no conflict of interest with regard to this trial.
Key words: severe acute respiratory syndrome coronavirus 2 / SARS-CoV-2 antibodies / assisted reproduction / ART / serological test- ing / IgG seroprevalence / SARS-CoV-2 prevalence trends / coronavirus disease 2019
WHAT DOES THIS MEAN FOR PATIENTS?
This study looks at how many of the individuals attending a fertility clinic during the second half of 2020 show evidence of recent or past infection with the virus causing coronavirus disease 2019 (COVID-19). Infection was evaluated in over 500 people undergoing assisted re- production, by a blood test measuring specific antibodies (proteins made by the body in response to foreign invaders such as viruses and bacteria). All of those tested reported no COVID-19 symptoms or known exposure around the time of their fertility treatment.
We found that roughly 5% of the women and men tested returned positive antibody readings, with no significant difference between the sexes; 2% presented with antibodies suggesting past infection while twice as many had antibodies suggesting recent or present infection. Among those tested at the end of the study, one out of six were positive. Close to 2% of all the patients were detected as having an ac- tive infection and had to postpone the treatment.
This means that although the number of those with infection and immunity is increasing, the majority of the infertility patients from a high-risk area for COVID-19 remain unexposed to the virus at the end of 2020. The use of protective measures and testing for COVID- 19 in the fertility clinics will most probably go on throughout all 2021.
Introduction
Introduction
Most of the year 2020 has been consumed by a viral pandemic that
seems to linger on (ECDC, 2020) and to reshape healthcare
worldwide.
Infertility is a pressing medical condition, time- and pandemicsensitive
(Alviggi et al., 2020), which is diagnosed in about one-fifth of
reproductive-aged couples, amounting to 186 million couples globally
(WHO, 2020); many of those affected will succeed in their dream of
parenthood with the use of ART.
One of the main concerns surrounding the generation of medically
assisted pregnancies in the middle of a pandemic environment was to
prevent infection in the fertility clinic and, consequently, to identify optimal
screening algorithms for the detection of asymptomatic or presymptomatic
cases of infection with severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) (ARCS and BFS, 2020; ASRM, 2020;
ESHRE, 2020; Romanian MoH, 2020).
In non-emergent non-coronavirus disease 2019 (COVID-19) care,
tele triage and testing are considered two indispensable steps for ensuring
safety for patients and staff (AMA, 2021). Testing for viral RNA
(through quantitative real-time RT-PCR) is widely accepted for confirming
active infection with SARS-CoV-2 in symptomatic individuals,
while serological testing is used to complement and remedy the nucleic
acid amplification tests (NAAT) detection, to establish the
timeline of the infection, to screen asymptomatic populations and to
quantify vaccine responses (Bo¨ger et al., 2020; La Marca et al., 2020;
Petherick, 2020; Vandenberg et al., 2020; Wang et al., 2020).
The rates and trends of SARS-CoV-2 prevalence in the fertility practice
are unknown. Existing recommendations for SARS-CoV-2 screening
in the fertility setting issued by the professional bodies insist on
triage and symptom-driven testing, and are not aligned (La Marca and
Nelson, 2020; Sparks and Kresowik, 2021), which is understandable
considering the lack of evidence on best practice (Papathanasiou,
2020). One piece of the information upon which cost-effective screening
protocols and preventative measures can be devised and optimized
is knowledge on SARS-CoV-2 prevalence trends in ART patients with
a negative triage questionnaire. This study aims to assess SARS-CoV-2
IgG and IgM seroprevalence in triage-negative people who are attending
and working in a fertility setting throughout the second half of
2020.
Materials and methods
Study design and population
We performed a prospective longitudinal unicentric study with a duration
of 6months, aiming to report the serological status for IgG and immunogenic sites of the spike protein are more sensitive than the
ones targeting other viral antigens (Prendecki et al., 2020). Given the
correlation with neutralizing activities, spike antibodies are also relevant
for functional immunity (Premkumar et al., 2020; Wajnberg et al.,
2020).
Briefly, magnetic beads are coated with both S1 and S2 antigens to
which specific antibodies attach (solid phase). Complexes of mouse
monoclonal antibodies to human IgG and isoluminol are created and
put in contact with the solid phase. The complexes bind to the SARSCoV-
2 antibodies present in the sample, producing a light signal that is
read by a photomultiplier.
Samples with signal levels above the manufacturers cutoff of 15
arbitrary units (AU)/ml were defined as positive, and samples below
12 AU/ml were defined as negative. Results between 12 and
15 AU/ml are reported as equivocal, and re-testing is advised
(www.diasorin.com). We excluded equivocal results from our
analysis.
According to the manufacturer, the S1/S2 IgG assay has a clinical
sensitivity of 90.7% for samples tested 5–15 days after infection and
97.9% for later than 15 days; the specificity is 98.6%.
IgM against the receptor binding domain of SARS-CoV-2 was evaluated
qualitatively by the same approach, with results interpreted as
positive or negative against an index of 1.1 described by the manufacturer.
The clinical sensitivity for the IgM assay used in this study is
91.5% for Days 8–14, and 94% for Days 15–30 post-infection; the
specificity is 99.3%. The combined assays offer a clinical sensitivity
that reaches 98.3% when testing is undertaken after Day 15
postinfection.
External validation studies of the assay used in this work provided
‘real-world’ analytical and clinical performances of the DiaSorin assay
that were close to the ones reported by the manufacturer, with the
time-dependent increase of accuracy inherent to the serological assays.
The populations used for external validation comprise adults over
18 years of age with a previous positive PCR test for SARS-CoV-2 and
different forms of infection ranging from asymptomatic to critical, with
no evidence of a difference in immunoassay sensitivity by infection severity
(Public Health England, 2020; The National SARS-CoV-2
Serology Assay Evaluation Group, 2020; Tre´-Hardy et al., 2020;
Turbett et al., 2020).
Ethical considerations
No personal data that could identify any person was included.
Approval from the Ethical Committee of Columna Medical Center
(reference: CMC-1330-15052020) was obtained before the initiation
of sampling and data collection.
Statistical analysis
We performed statistical analysis and graphs using Analyze IT 5.5
(Microsoft Office Excel Add-on, Leeds, UK). The data had a non-
Gaussian distribution and were presented as the median and the interval
between the quartiles. The differences in quantitative parameters
were tested using nonparametric tests. Qualitative data were compared
with the Chi-square test. We considered statistical significance
at a P-value lower than 0.05.
Results
A total of 713 blood tests for SARS-CoV-2 were analyzed and 98.8%
of them produced unequivocal results. The median age of the individuals
included in the study (516 triage-negative patients and 30 HCWs
was 35 years, interquartile range [32; 40]) . Three quarters of them
(77.1%) were women. After consenting, no patient opted out of serological
testing.
Out of the 705 blood samples that produced valid readings for
SARS-CoV-2 antibodies, 42 yielded positive results, giving a raw seroprevalence
of 6%. After adjusting for test performance, the seroprevalence
across the study period was 6.93%.
The overall seroconversion rate was 3% for IgG and 5% for IgM,
with high variability in the antibody titers and a non-significant difference
between sexes (P value: 0.13). Two percent of the samples were
reactive for both antibodies.
In the patient group, 2.1% (n¼21) had evidence of IgG antibodies
against SARS-CoV-2 and 4.6% (n¼35) had positive IgM results.
Regarding the HCW group, the prevalence of antibodies was 12.9%
for Ig M and 22.6% for IgG, markedly increased as compared to the
patient population (Table I).
The reader must be aware that more than half of the HCWs were
tested in the first 2months after recommencing clinical activities (when
national data on HCWs revealed a high prevalence rate); half of the
remaining were tested in November, during a pandemic peak
(Supplementary Table SI).
The first cases of patients and staff with positive IgG results were
seen in May, immediately after lockdown release (1% seroprevalence)
with significantly higher numbers thereafter (6% during autumn).
Monthly antibodies seroprevalences in the patient group rose gradually
after lockdown release: from 1.7% in the second half of May
(n¼58), to 5.5% in August (n¼220), to 10.9% in November (n¼64)
(Fig. 1).
When analyzing the reactivity rates for IgG and IgM recorded
monthly, the difference between the time intervals was found to be
significant (P<0.01) (Supplementary Table SI).
The number of individuals exhibiting a positive serological result for
one or both antibodies increased 6-fold during the study period.
We analyzed official data regarding national monthly SARS-CoV-2
general prevalence rates recorded between 25 February 2020, when
the first case of infection was reported, and 25 February 2021 (www.
worldometers.info, 2021). We performed ANOVA between the official
prevalence rates and our data and found a correlation of statistical
significance (P-value 0.0158) with an Odds Ratio of 0.512 (95% CI:
0.145–0.878) (Supplementary Table SII).
We extracted data and adjusted our cohort for a 3-month followup
until the date of submission, estimating a plateau followed by a decrease
in the ART population testing positive for antibodies by the
end of February 2021, alongside the decrease in cases officially
recorded in the national population during this period (Fig. 2).
When evaluating the trend in IgM positivity, we found the monthly
increase in IgM positivity rates to be significant and to parallel the increase
in national PCR-based prevalence rate (Fig. 3 and
Supplementary Table SIII).
Although outside the scope of this antibody prevalence study, we
analyzed the PCR results in all individuals that tested positive for either
IgM or IgG. Out of the 42 PCR tests carried out in patients with a positive IgM or/and IgG serology, 9 (21.4%) were positive. Only
34.61% of the patients with IgMþ results had a corresponding positive
PCR, while no positive molecular tests were seen among the patients
that were positive exclusively for IgG (Supplementary Table SIV).
Close to 2% of the individuals in our cohort had a positive result for
both serology and PCR.
Concerning the form of infection, and based on the recollection of
COVID-19 symptoms, 65% of those with positive serology were
asymptomatic, 25% reported minimal symptoms, and none required
hospital care.