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.