In vitro tests are increasingly applied in chemical hazard assessment. Basic culture conditions may affect the outcome of in vitro tests and should be optimised to reduce false predictions. The neural embryonic stem cell test (ESTn) can predict early neurodevelopmental effects of chemicals, as it mimics the differentiation of stem cells towards the neuroectodermal lineage. Normal early neural differentiation depends crucially on folic acid (FA) and methionine (MET), both elements of the one-carbon (1C) cycle. The aim of this study was to assess the concentration-dependent influence of FA and MET on neural differentiation in the ESTn, and its consequences for assay sensitivity to methotrexate (MTX), a compound that interferes with the 1C cycle. Neural differentiation was inhibited below 0.007 mM and above 0.22 mM FA, while both stem cell viability (< 0.097 mM, > 1.52 mM) and neural differentiation (< 0.181 mM, > 1.35 mM) were affected when changing MET concentrations. A 10-day exposure to 13 nM MTX inhibited neural differentiation, especially in FA- and MET-deficient conditions. However, a 24-hour exposure to 39 nM MTX decreased neural cell and neural crest cell differentiation markers only when the concentration of FA in the medium was three times the standard concentration, which was expected to have a protective effect against MTX. These results show the importance of nutrient concentrations, exposure scenarios and timing of read-outs for cell differentiation and compound sensitivity in the ESTn. Caution should be taken when interpreting results from a single in vitro test, especially when extrapolating to effects on complex morphogenetic processes, like neural tube development.

We observed an increase in notifications of puerperal group A Streptococcus (GAS) infections in July and August 2018 throughout the Netherlands without evidence for common sources. General practitioners reported a simultaneous increase in impetigo. We hypothesised that the outbreak of puerperal GAS infections resulted from increased exposure via impetigo in the community.We conducted a case-control study to assess peripartum exposure to possible, non-invasive GAS infections using an online questionnaire. Confirmed cases were recruited through public health services while probable cases and controls were recruited through social media. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) with logistic regression analysis.We enrolled 22 confirmed and 23 probable cases, and 2,400 controls. Contact with persons with impetigo were reported by 8% of cases and 2% of controls (OR: 3.26, 95% CI: 0.98-10.88) and contact with possible GAS infections (impetigo, pharyngitis or scarlet fever) by 28% and 9%, respectively (OR: 4.12, 95% CI: 1.95-8.68). In multivariable analysis, contact with possible GAS infections remained an independent risk factor (aOR: 4.28, 95% CI: 2.02-9.09).We found an increased risk of puerperal fever after community contact with possible non-invasive GAS infections. Further study of this association is warranted.