SARS-CoV-2 RNA and antibody detection in breast milk from a prospective multicentre study in Spain

Abstract

To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies.

Design and patients

This is a prospective, multicentre longitudinal study (April–December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included.

Setting

Seven health centres from different provinces in Spain.

Main outcome measures

Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)—IgA, IgG and IgM—in breast milk samples from patients with COVID-19.

Results

All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%–87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation).

Conclusions

Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence.

Trial registration number NCT04768244 .

SciScore for 10.1101/2021.05.06.21256766: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	Consent: All participants received oral and written information about the study and written consent was obtained. IRB: All protocols performed in the study were in accordance with the ethical standards approved by the Ethical Committee of the Hospital Clínico Universitario of Valencia (Ref. 2020/133) and by the rest of the Ethical and Research’s Committees.
Sex as a biological variable	Participants were pregnant with intended breastfeeding and nursing women with positive PCR for SARS-CoV-2 in nasopharynge or presence of SARS-CoV-2 antibodies in serum determined in hospitals.
Randomization	Those women were randomly selected from the MAMI birth cohort in Spain [15] (ClinicalTrials.gov Identifier: NCT03552939).
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Authentication: Women were excluded when COVID-19 symptomatology required specific treatment and/or hospitalization in intensive care units.

Table 2: Resources

Antibodies
Sentences	Resources
Breast milk SARS-CoV-2-specific antibody detection: Levels of antibodies directed to structural proteins as RBD of the SARS-CoV-2 spike protein and to non-structural viral proteins as cysteine-like protease, also known as the main protease (Mpro) or 3CLpro, were analyzed.	SARS-CoV-2 spike protein suggested: None
For detection of the different antibody isotypes, anti-human IgA (α-chain-specific) HRP antibody (Thermo-Fisher Scientific; A18781; 1:6.000), anti-human IgM (μ-chain-specific) HRP antibody (Sigma-Aldrich; A0420; 1:4.000), and anti-human IgG (Fc specific) HRP antibody (Sigma-Aldrich; A0170; 1:4.000) were used and incubated for 1 h in 1 % (w/v) milk powder in PBS-T.	HRP suggested: (Sigma-Aldrich Cat# A0420, RRID:AB_257886)
Briefly, an anti-human IgA antibody pre-adsorbed to the plate allowed to capture the IgA, which was later detected by the addition of a biotinylated detection antibody and streptavidin-conjugated horseradish peroxidase that catalyzed the colorimetric reaction with the chromogenic substrate TMB.	anti-human IgA suggested: None
Experimental Models: Cell Lines
Sentences	Resources
RBD protein was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein RBD from SARS-CoV-2, Wuhan-Hu-1 with C-Terminal Histidine Tag, Recombinant from HEK293T Cells, NR-52946.	HEK293T suggested: None
Software and Algorithms
Sentences	Resources
For detection of the different antibody isotypes, anti-human IgA (α-chain-specific) HRP antibody (Thermo-Fisher Scientific; A18781; 1:6.000), anti-human IgM (μ-chain-specific) HRP antibody (Sigma-Aldrich; A0420; 1:4.000), and anti-human IgG (Fc specific) HRP antibody (Sigma-Aldrich; A0170; 1:4.000) were used and incubated for 1 h in 1 % (w/v) milk powder in PBS-T.	Thermo-Fisher Scientific suggested: None
For detection of MPro-reactive antibodies, a commercial ELISA Kit (ImmunoStep, Salamanca, Spain) was used.	ImmunoStep suggested: (IMMUNOSTEP, S.L., RRID:SCR_013411)
Endpoint titers were calculated from log-transformed titration curves using 4-parameter non-linear regression function in GraphPad Prism 8.0 and the positive cut-off values obtained from the prepandemic control group for each antigen and isotype.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Regarding the potential limitations, we did not collect skin swabs to control for the potential presence of SARS-CoV-2 in the skin [7,33]. However, all milk samples as well as the infants were negative for SARS-CoV-2 presence. Thus, although there are still limited data, it is accepted that breastfeeding does not represent a vehicle for vertical transmission of SARS-CoV-2 [9]. There are still many open questions: when are SARS-CoV-2 antibodies produced after maternal infection, when can they be detected in breast milk, and how long do they persist. To cover some of these questions, we aimed to determine the presence of antibodies in breast milk samples from COVID-19 women and to compare these with milk samples collected prior to the pandemic as reference controls. While different studies have reported presence of specific IgA antibodies against SARS-CoV-2 [7,12,13,36], limited information is available on IgG and IgM. Our results showed presence of anti-SARS-CoV-2 antibodies in milk, primarily IgA but also IgG and IgM targeting RBD. Furthermore, IgA- and IgG against non-structural MPro were analyzed for the first time in human milk samples. High intra- and inter-individual variability was observed in antibody presence and significant differences were observed for all three antibody classes when compared to prepandemic samples in agreement with previous data [26]. In our dataset we found that 82·9 % of the milk samples tested positive for the RBD antigen at least in one of the ...

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

Identifier	Status	Title
NCT04768244	Recruiting	Impact of Maternal COVID-19 Disease on Breast Milk and Infan…
NCT03552939	Completed	The Impact of Maternal Microbes on Infant Health Programming

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

Read the original source