The study included a total of 25 cases of colour or structure changing of Eucharistic Host (in the form of white bread) after the consecration. They come from churches and other places in Poland, Germany, the USA, and South Africa, where Communion was distributed. All examinations were ordered by the Church’s officials. In most cases, the hosts fell to the ground shortly after the consecration, mostly during distribution of the Holy Sacrament. They were then picked up and placed in the water-filled containers (vascula) to dissolve, a procedure according to the ‘Liturgica instaurationes’ (Kozłowski 2018). However, after several days of storage and observation, some of the hosts did not dissolve. Instead, they exhibited the signs of red and purple colour stains (Fig. 1a–h), with corresponding liquid turning red, rusty, yellowish, or dark brown (Fig. 1a, e–h). The stains were usually either jelly-like (Fig. 1a, e–h) or dried (Fig. 1b–d), sometimes with corresponding signs resembling the mould colonies (Fig. 1a, c), while the liquids (if present) were mostly opaque.

Examples of the materials found on the liturgical hosts: a case no.13; b case no.14; c case no. 2; d case no.10; e case no. 20; f case no. 16; g case no. 17; h case no. 15

In one instance, the material came from the hosts stored in a chalice and stolen from a chapel, which was then found in a forest area nearby. Each case was assigned a sequential number (Tables 1 and 2). The cases were analysed in the laboratories of the Collegium Medicum in Bydgoszcz, or in the laboratories of the Medical University of Wrocław and the University of Warsaw. Documentation of cases 1–15 is deposited in the Pontifical Faculty of Theology in Wrocław, whereas the documentation of cases 16–25 is deposited at the Department of Forensic Medicine, Ludwik Rydygier Collegium Medicum of the Nicolaus Copernicus University, Bydgoszcz, Poland.

The overview of the analysed specimens is given in Tables 1 and 2. In most cases, both the isolated fragments of stains (typically, of a 1-cm diameter) and 5–10 uL of liquid substance were assigned to different laboratories (genetic, microbiological, mycological, histological) and subjected to further analysis. The scheme of the procedure undertaken is presented in Fig. 2.

The procedural framework adopted for this study. The sample undergoes division to facilitate microbiological and mycological surveys, alongside forensic examination. Notably, both forensic and microbiological analyses encompass genetic assessments

In the following subchapters, the methods used during investigations are introduced.

The samples 1–15 were analysed in Wrocław or Warsaw, whereas the samples 16–25, in Bydgoszcz. All samples were inoculated on the microbiological media to stimulate fungal and bacterial growth.

In Bydgoszcz, the samples were inoculated on BD Columbia Agar with 5% sheep blood, BD MacConeky Agar, BD Pseudosel Agar (Cetrimide Agar), BD Enterococcosel Agar for a 5-day incubation at 37 °C in air atmosphere, and on BD Sabouraud agar with gentamicin and chloramphenicol (Becton Dickinson) for a 10-day incubation at 30 °C in air atmosphere. Samples were also incubated in brain–heart broth. The identification of isolated strains was performed with MALDI Biotyper (Bruker) according to the manufacturer’s instructions. The application of the MALDI Biotyper identified the isolates to species with an identification score of 2.000 and category A—a reliable identification at the species level.

The samples 2–15 analysed in mycological laboratories at the University of Warsaw were inoculated to Maltose Agar and/or Potato Glucose Agar and/or Sabouraud Glucose Agar to stimulate fungal growth from the inoculum. The incubation for fungal growth was performed at the temperature of 18 °C. The samples were observed and documented using the dissecting and optical microscopes (Nikon SMZ 800, Nikon Eclipse E200 and Nikon Eclipse E600 using Nikon DX-1200 or Nikon DS—Ri 2 cameras) with NIS Elements software. Slides were stained using lactophenol blue or material was suspended in lactic acid.

Mycelium fragments with a thin layer of the medium (0.9–1.0 μm thick) were prepared for LM and FM. For general observation, they were immersed in a lactic acid. They were observed with white light, and 300 nm UVB light. They were examined using a Nikon Eclipse 90i microscope. A UV2B filter (Nikon) was used to check both hyphae autofluorescence and fluorescence with Calcofluor White. Micrometry and photomicrography were accomplished by means of a Nikon Eclipse 90i (NIS-Elements AR software).

The DNA isolation was performed from the original sample (if homogenous), or from 7 days culture (if heterogenous). The DNA isolation was performed using ExtractMe Genomic DNA Kit (Blirt S.A, Gdańsk, Poland) following the manufacturer’s instructions. The PCR with the primers ITS1 and ITS4f (White et al. 1990) was performed. ITS region was used as the universal fungal barcode (Schoch et al. 2012). The amplification was performed using the ‘TaqNova-RED’ PCR mix, containing Taq DNA polymerase. The PCR reaction was conducted using the BioRad T100TM Thermal Cycler, with the programme described in Okrasińska et al. (2021). Products were visualised by electrophoresis in 1% agarose gel with Midori Green DNA stain (Nippon Genetics). PCR products were purified with the ExtractMe DNA Clean-Up & Gel-Out kit (Blirt S.A.). Sequencing was outsourced to an external company, Genomed S.A. (Warsaw, Poland). The sequences, forward and reverse, if both were obtained, were assembled using the Chromas (https://technelysium.com.au/wp/chromas/) or DNA Subway software (Williams et al. 2014) and compared against NCBI nucleotide databases using the BlastN search (Altschul et al. 1990). The nucleotide sequences have been deposited in the GenBank under accession numbers PQ470030, PQ475905, PQ481993, PQ481994, PQ482317, and PQ489325. To check the presence of Serratia marcescens the CHROMagar™ Serratia (distribution Biomaxima) were used.

Specimens prepared for histopathology examinations taken form solid material were fixed with formalin, paraffin-embedded, and then stained with haematoxylin and eosin (H&E) staining, and additionally, the Grocott’s method was used for the visualisation of fungi. The results were interpreted by a specialised pathologist.

Initially, the samples from cases no. 16–25 (Table 1) were examined for the presence of blood and specifically, for human blood (haemoglobin), using HemoPhan (Erba Lachema) or Bluestar OBTI (Bluestar Forensic) and HemCheck (Hydrex) tests, respectively. In one instance of an inconclusive result in HemCheck test (case no. 23), the sample was subjected to alternative RNA analysis with the use of High-Capacity cDNA Reverse Transcription Kit and human haemoglobin (HBB) TaqMan™ Gene Expression Assay (Thermo Fisher Scientific). The samples from cases no. 1–15 (Table 1) were not subjected to initial biochemical tests for the presence of blood and human blood; instead, they were directly examined for the presence of human and male DNA, on demand of the officials ordering the research.

Both stains and liquids were subjected to DNA extraction using GeneMATRIX Bio-Trace DNA Purification Kit (EurX). In one instance (case no. 23. Table 1), RNA was extracted with the use of organic method employing TRIzol™ Reagent (Invitrogen). Total amounts of human DNA and human male DNA in the samples were quantified using the Quantifiler® Duo DNA Quantification Kit (Thermo Fisher Scientific) and ViiA 7 Real-Time PCR System (Applied Biosystems). The latter was also used for performing HBB TaqMan™ Gene Expression Assay (case no. 23, Table 1).

Depending on a result of DNA quantitation, microsatellite markers were tested using the PowerPlex ESI Pro System (Promega), or AmpFℓSTR® NGM Select™ PCR Amplification Kit (Life Technologies), including 16 STRs (D3S1358, D19S433, D2S1338, D22S1045, D16S569, D18S51, D1S1656, D10S1248, D2S441, TH01, vWA, D21S11, D12S391, D8S1179, FGA, ACTB2), and amelogenin (X,Y) locus. In one instance, the AmpFLSTR ™ Yfiler ™ Amplification Kit (Thermo Fisher Scientific) was used to analyse 17 Y-STRs (DYS385, DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y GATA H4). PCR reactions were performed in the GeneAmp PCR System 9700 thermal cycler (Applied Biosystems), while the products were analysed on the ABI PRISM® 3130xl Genetic Analyzer and the GeneMapper® ID v. 3.2 software (Applied Biosystems). For autosomal STR probability estimates with corresponding values of likelihood ratio (LRs), both STR frequency database from Polish population and global STRidER (STRs for Identity ENFSI Reference Database, available online at https://strider.online) were used. Y-STR haplotype frequency was estimated with the use of YHRD database (Willuweit and Roewer 2015) for both minimal (DYS385, DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393 loci) and complete haplotype. The Y-chromosome haplogroup prediction based on Y-STR haplotype was performed using the Y-DNA Haplogroup Predictor NEVGEN available online at http://nevgen.org.