Study population
This was a prospective cohort study including all consecutive pregnant
women with laboratory confirmed SARS-CoV-2 infection by deep throat
saliva (DTS) or nasopharyngeal swab (NPS) real-time
reverse-transcriptase-polymerase-chain-reaction (rRT-PCR) test or by
rapid antigen-detection tests (Panbio™ Covid-19 Ag Rapid Test Device
(Abbott Realtime SARS CoV-2 Amplification reagent kit. Abbott molecular
Inc. Des Plaines, IL USA)17 , during pregnancy or at
the time of delivery, who consented to participate in the study. In
probable cases of COVID-19 where rRT-PCR was negative, if the symptoms
had started within seven days of testing, the rRT-PCR was repeated 24
hours after the first test, and if the symptoms had started beyond seven
days of testing, a serology test (ELISA) was
performed18.
Breast milk samples were collected from six recruiting units, Hospital
Universitario de Torrejón (Madrid, Spain), Hospital Universitario Vall
d´Hebrón (Barcelona, Spain), Hospital Universitario Clínico San Cecilio
(Granada, Spain), the Chinese University of Hong Kong COVID-19
collaborative network ( Hong Kong SAR China), Hospital
Clínico Universitario Virgen de la Arrixaca (Murcia, Spain) and Hospital
Universitario Príncipe de Asturias (Madrid, Spain) from March 2020 to
March 2021. The study was approved by each one of the Local Research
Ethics Committees at the participant centers. All women gave written
informed consent.
Each participant had one sample of colostrum (between the day of
delivery until day 4 postpartum), and one sample of mature milk (from
day 7 postpartum until 6 weeks postpartum) collected and stored at
-80ºC. Maternal blood for serological analysis was also collected at the
same time, serum was separated and stored at -80ºC.
Clinical data, including maternal age, body mass index (BMI) at the
beginning of pregnancy, date of last menstrual period (LMP) and COVID-19
severity, was recorded for every participant, pseudo-anonymized and
entered into a common secured database. The COVID-19 severity was
classified as asymptomatic, mild (when no hospitalization was required)
and pneumonia (when the diagnosis of pneumonia was established and
needing hospitalization)19. The time interval in days
between maternal diagnosis of COVID-19 and delivery was calculated.
Gestational age was estimated by first trimester sonographic assessment
of fetal crown-rump length20 or confirmed by LMP.
Virological sample
collection and analysis
Breast milk (from 0·1 to 1·0 mL) was collected by manual expression with
strict contact precautions to avoid contamination (facial mask and hand
cleaning). Blood samples were collected in serum sep clot activator 8 mL
tubes, which were then centrifuged for five minutes at 3500g and then
serum was collected. Both serum and breast milk samples were divided
into 0·5 mL aliquots (when possible) in separate Eppendorf tubes, which
were labelled with a unique patient identifier and stored at -80ºC until
subsequent analysis. Stored samples from Barcelona were analyzed locally
at the end of the recruitment period. Samples from all other sites were
sent without any further processing overnight on dry ice to Synlab
Diagnósticos Globales Laboratory (Alcobendas, Madrid, Spain) on monthly
basis from Spanish sites and in a single batch at the end of the
recruitment period from Hong Kong.
At the laboratory, breast milk samples were thawed, centrifuged at 800g
for 15 minutes, fat was removed, and supernatant transferred to a new
tube. Centrifugation was repeated twice to ensure removal of all cells
and fat12. Skimmed acellular milk was then tested
against SARS-CoV-2 specific immunoglobulin M (IgM), immunoglobulin A
(IgA) and immunoglobulin G (IgG) reactive to the receptor binding domain
(RBD) of the SARS-CoV-2 spike protein 1 (protS1)12,15.
Serum samples were thawed and tested against SARS-CoV-2 specific
antibodies. Determination of IgA and IgG antibodies were performed by
ELISA method (Enzyme-Linked Immunosorbent Assay) providing
semi-quantitative serology results against the S1 domain of the spike
protein of SARS-CoV-2 in serum samples (Anti-SARS-CoV-2 ELISA (IgG) and
Anti-SARS-CoV-2 ELISA (IgA), Euroimmunn Medizinische Labordiagnostika
AG, Lubeck, Alemania)21. IgM determination was
performed by chemiluminescencent microparticle immunoassays, using spike
protein specific (Abbott test, SARS-CoV-2 IgM (Abbott), Abbott Ireland
Diagnostics Division Finisklin, Ireland)22. Both
techniques used CE marked and validated kits.
IgA and IgG were considered positive, indeterminate and negative when
results were >1·1, 0·8-1·1 and <0·8,
respectively; IgM was considered positive, indeterminate and negative
when results were >1·1, 0·9-1·1 and <0·9,
respectively. IgM could not be analyzed in all the samples send to the
laboratory due to insufficient sample (82 colostrum samples and 12
mature milk samples).
Colostrum samples from mothers with active illness at the time of
collection were tested for SARS-CoV-2 by rRT-PCR. The Spanish samples,
viral RNA was extracted with Chemagic Viral DNA/RNA Kit using the
Chemagic 360 with integrated dispense, that includes lyophilized Poly(A)
RNA, lyophilized Proteinase K and a lysis/binding buffer, and were
analyzed with Euroinmune Kit (ORF1ab an N targets) and
TaqManTM 2019-nCov Assay kitv2 Thermofisher (s,ORF1ab
and N targets). For Hong Kong samples, viral RNA was extracted using
RNeasy® Mini Kit (QIAGEN) and the detection of SARS-CoV-2 RNA was
performed with the FDA-authorized CDC 2019-Novel Coronavirus (2019 nCoV)
Real-Time RT-PCR Diagnostic Panel (EUA 200001). The N gene (both N1 and
N2) was assayed, with the human RNase P (RP) as an endogenous reference
control. Samples that contained organic or inorganic contaminants that
interfered in the amplification of the PCR were considered inhibited.