Fecal iron quantification in a randomized controlled trial of Lactiplantibacillus plantarum ATCC 202195 in newborns in Dhaka, Bangladesh

• Fecal iron concentrations can be measured in infant samples with acceptable accuracy and precision using atomic absorption spectrometry (AAS). • Fecal iron quantification using AAS is resource-intensive and does not replace conventional biomarkers of iron status or stable-isotope methods to measure iron absorption. • In a demonstration of the fecal iron AAS assay in a randomized placebo-controlled trial, Lactiplantibacillus plantarum ATCC202195 with or without fructooligosaccharide administered to newborns in Dhaka, Bangladesh was not found to significantly affect fecal iron concentrations at 2 weeks of age or serum ferritin concentrations at 2 months of age. Probiotics may enhance host iron bioavailability, offering a strategy to address iron deficiency. Fecal iron may be a useful non-invasive biomarker of such effects in infants. To examine the use of fecal iron quantification in a randomized placebo-controlled trial (RCT) of neonatal administration of Lactiplantibacillus plantarum ATCC 202195 (LP202195), with or without fructooligosaccharide (FOS), in Dhaka, Bangladesh. Fecal iron quantification using atomic absorption spectrometry (AAS) was optimized using standards and reference materials, and pilot-tested using pooled stool aliquots (n=32) from an observational cohort of young infants in Bangladesh (aged 0-64 days). The optimized AAS assay was then applied to individual stool samples collected at 14 days of age (n=307) in a RCT in which newborns aged 0-4 days were randomly allocated to one of five groups: placebo, 1- or 7-day regimens of LP202195, with or without FOS. Serum ferritin was measured at 2 months postnatal age (n=251). Effects of the 1- and 7-day LP202195 regimens were estimated using linear regression and expressed as mean percent differences relative to placebo, with 95% confidence intervals (95%CI). The optimized AAS fecal iron assay had acceptable accuracy (91-99%), precision (within- and between-run coefficients of variation <10%), and recovery (93-112%), with a reportable range of 0.2 to 80 mg Fe per 100 g dry stool. In pooled samples from the observational cohort, fecal iron varied with age and feeding status. In the RCT, fecal iron concentrations did not significantly differ following1-day (% difference=9.8%, 95%CI: -19%, 49%; P=0.54) or 7-days (% difference= -6.1%, 95%CI: -31%, 28%; P=0.69) of LP202195 administration, versus placebo (geometric mean concentration=4.3mg Fe/100g dry stool (95%CI:3.3, 5.6); n=53). Inferences were unchanged when groups were disaggregated by FOS co-administration (P>0.05 for all). Similarly, there were no effects of LP202195 on serum ferritin at 2 months of age (P>0.05 for all). Fecal iron quantification by AAS was valid and feasibly implemented in a trial of neonatal administration of Lactiplantibacillus plantarum ATCC 202195. However, the assay is resource-intensive and may not be more informative than conventional measures of iron status when studying the effects of probiotics/synbiotics on iron bioavailability. ClinicalTrials.gov identifier: NCT05180201