Sample population and study design

The Hospital San Juan de Dios in Armenia (Quindío Department, Andean Western Region of Colombia) is a third-level departmental referral hospital that treated 1,533 children with diarrhoea symptoms in 2020. This study was a descriptive, observational, cross-sectional investigation. To assess potential sources of Cryptosporidium spp. and Cyclospora spp. infection beyond the hospital setting, information on the residential addresses of study participants was recorded. The children resided in the municipalities of Armenia, La Tebaida, Montenegro, Quimbaya, and Circasia, all within the Quindío Department. A geographical map was generated to illustrate the distribution of the study participants’ residences across the region (Fig. 1).

Fig. 1

Geographic distribution of study area locations in Quindío (map from Google Earth)—The map displays the municipalities of Armenia, La Tebaida, Montenegro, Quimbaya, and Circasia, where the enrolled children resided. Yellow labels indicate the housing locations of all the children included in the study. Red labels mark the residences of children who tested positive for Cyclospora spp., whereas green labels indicate the residences of children who tested positive for Cryptosporidium spp. Although all stool samples were collected at the hospital, this spatial representation provided insight into the geographic distribution of the study participants within Quindío

A total of 150 stool samples were collected from paediatric patients under 15 years of age presented with acute diarrheal disease at the emergency department of the Hospital San Juan de Dios between March and May 2022. The sample size was determined based on the resources available to conduct the project. Additionally, 24 stool samples from children hospitalized for non-diarrheal conditions were analysed as controls.

Ethical aspects

This study was approved by the Bioethics Committee of the Faculty of Health Sciences at the Universidad del Quindío, as documented in Act No. 17 on June 17, 2022. The study was subsequently endorsed by the Bioethics and Research Committees of the Hospital Universitario San Juan de Dios, as per their communication dated December 22, 2021.The parents or guardians of the children completed an informed consent form. All measures were respected in accordance with Ministry of Health Resolution No. 8430 of 1993. The results were provided to the parents or guardians of the children, and the indicated treatment was provided in cases of positive results.

Collection of information

The emergency department of the Hospital San Juan de Dios served as the site for collecting medical history information and stool specimens. For the purposes of this study, diarrhoea was characterized by the occurrence of three or more watery stools within a 24-hour period. The Colombian Guidelines were employed to categorize dehydration severity: Level I (mild) involved no hemodynamic alterations and approximately 3–5% body weight loss; Level II (moderate) was marked by tachycardia and roughly 6–8% body weight loss; Level III (severe) presented with hypotension, compromised perfusion, and an estimated ≥ 10% body weight loss. An axillary temperature surpassing 37.8 °C was considered indicative of fever. Participants submitted a single stool sample on the day of hospital admission. These specimens were subsequently transported under suitable conditions to the Centro de Investigaciones Biomédicas de la Universidad del Quindío, where they were preserved at -20 °C for future parasitological and molecular examinations. A uniform data collection instrument was employed to document the sociodemographic and clinical details for each case, drawing upon information extracted from the hospital’s medical records.

Parasitological analysis

Stool samples were collected in sterile containers with saline or phosphate-buffered saline (PBS), pH 7.4, on the day of consultation. They were transported to the laboratory in a Styrofoam cooler to maintain the appropriate conditions for subsequent parasitological and molecular analyses. Each sample was anonymized using unique codes to ensure blind evaluation by the bioanalyst, who was unaware of the patients’ identities.

Diagnosis was performed by a trained professional, whose competence was externally validated by the National Institute of Health of Colombia, achieving 100% concordance in quality control tests. Stool samples were stained using the modified Ziehl-Neelsen (ZN) stain and examined under a light microscope with a 40X objective. ZN-stained plate samples of Cryptosporidium spp.-positive stool samples that were verified by PCR amplification of the 16s ribosomal subunit and sequenced were obtained from previous studies in our laboratory and used as positive controls in microscopy analysis. Reference stool samples containing C. cayetanensis were kindly donated by Dr. Ynes Ortega of the University of Georgia (USA).

Each sample was subjected to three plate reading mounts (0,83% saline, 1% Lugol, and 0,83% saline-eosin solution). Additionally, two concentration techniques were applied to enhance the detection of parasitic forms: the 0,7% formalin-ether concentration technique and the 0,8% zinc sulfate flotation technique. Morphological descriptions of the parasitic forms were conducted using fresh preparations stained with 1% parasitological Lugol solution and observed under a light microscope with a 40X objective. Positive samples for protozoa were preserved in a 10% formalin solution.

DNA extraction

A combination of chemical and mechanical lysis was used to extract DNA from stool samples. First, 300 µL of each sample was concentrated using the formalin-ether method [17, 18]. Then, 300 µL of each sample was shaken in a Mini Bead-Beater (Stratech, UK) machine for one minute and incubated on ice for another minute. This process was repeated ten times. Subsequently, 100 µl of protein precipitate was added, vortexed for 20 s, allowed to stand on ice for 5 min, and centrifuged at 13,500 rpm for 1 min. The supernatant was transferred to a clean tube and added and DNA purification using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI), the tube was allowed to dry and 80 µl of rehydrating DNA was added.

PCR detection

Detection of Cryptosporidium spp. DNA, nested PCR was initially used to amplify the gene encoding the membrane glycoprotein GP60 [19]. The primers used for the first amplification were Crsp601F 5´-ATAGTCTCCGCTGTATTC-3´ and Crsp601R 5´-GCCGAGGAACCAGCATC-3 × (863 bp). The following primers were used for the second PCR: Crsp602F 5´-TCCGCTGTATTCTCAGAC-3´ and Crsp602R 5´-GAGATATATCTTGGTGCGGG-3 × (443 bp). The PCR mix included GoTaq Green Master Mix (Promega, Madison, WI), primers, molecular water, BSA and DNA in a final volume of 25 µl. For the nested PCR the concentrations of the reagents were the same as for the first PCR and 1 µl of the already amplified product was added The parameters of the amplification cycles were an initial denaturation at 94°C for 5 min, followed by 35 cycles starting with denaturation at 94°C for 1 min, annealing at 46°C for 1 min, followed by an extension at 72°C for 1 min, and a final extension step for 10 min. For the second PCR, denaturation of 94°C for 10 min followed by 14 cycles with an initial denaturation at 94°C for 1 min, annealing at 54°C for 30 s, and extension at 72°C for 30 s, and an additional final extension step for 5 min at 72°C. The presence of 443 bp amplified product was visualized by 1.5% agarose gel electrophoresis, performed in a horizontal chamber with 1 × TBE running buffer at 100 V. The amplified product was visualized by 1,5% agarose gel electrophoresis. Amplification of C. cayetanensis was carried out by conventional PCR using conventional PCR with the 5’-GCAGTCAGAGGAGGAGGCATATATATCC-3’ and 5’-ATGAGAGACCTCACAGCCAAAC-3’ primers, which amplify a 116 bp fragment of the 5,8 S subunit of ribosomal RNA (SSUrRNA) [20] The PCR mix consisted of 12,5 µl of GoTaq Green Master Mix (Promega), 1.5 µl of primers (10 µM), and 3 µl of DNA in a final volume of 25 µl. The amplification cycle for the first cycle consisted of denaturation at 95 °C for 2 min, followed by 40 cycles consisting of 30 s at 95 °C, annealing at 59 °C for 30 s, and 30 s at 72 °C, followed by a final extension of 5 min at 72 °C. PCR amplifications were carried out on a Veriti thermocycler (Thermo Fisher Scientific), and the resulting products were analysed by 1.5% agarose gel electrophoresis to confirm positive samples.

Positive and negative controls for PCR

DNA was obtained from C. parvum oocysts purchased from the University of Arizona (https://acbs.cals.arizona.edu/crypto/purchasing). The method of obtention consisted of propagation in neonatal Holstein calves, approximately 1–2 times per month. The oocysts were purified using discontinuous sucrose and caesium chloride centrifugation gradients. The purified oocysts were stored at 4 °C in an antibiotic solution containing 0,01% Tween 20, 100U of penicillin, 100 µg of gentamicin per ml. These oocysts were used as positive amplification controls for the Cryptosporidium spp.

Additionally, a plasmid (pUC57) containing a 116 bp insert of the 5.8 SSU rRNA subunit of C. cayetanensis (GenScript HK Limited, Hong Kong) served as an additional positive control.

DNA-free samples (300 µL PBS) were used as negative controls at every stage of the process, from DNA extraction to sequence analysis, to monitor potential contamination from reagents or consumables and to detect any cross-contamination between samples during handling.

To minimize the risk of contamination, strict laboratory protocols were followed, including the use of separate pre-PCR workspaces, filter tips, and dedicated pipettes. To check for the presence of potential inhibitors, negative samples were spiked with C. parvum and C. cayetanensis DNA and reanalysed.

DNA sequencing and phylogenetic analysis

Samples that were positive after PCR amplification were sent to Psomagen Inc. software (Rockville, MD, United States) for Sanger sequencing. The nucleotide sequences obtained for the C, parvum gp60 marker, and C. cayetanensis SSUr-RNA were subjected to purification and quality control. Sequences with a QV score > 20 (values above 20 generally indicate that a sequence is reliable) were curated and aligned. In addition, nBLAST was performed for both chains (Forward and Reverse) using the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm whether the sequences obtained corresponded to the amplification target under study. Subsequently, the obtained sequences were aligned with Clustal W and trees for similarity of sequences analysis were derived in Molecular Evolutionary Genetics Analysis (MEGA) software, version 6 (available at: http://www.megasoftware.net/). All sequences were aligned with the MUSCLE algorithm and phylogeny was established using MUSCLE software in the MEGA X program to establish phylogenetic inferences (http://www.megasoftware.net/).

Immunofluorescence for Cryptosporidium spp

Stool samples that tested positive for Cryptosporidium spp. using the ZN method were further analyzed using immunofluorescence. Each ZN-positive sample was homogenized for one minute, and 100 µL of the homogenate was processed for staining. Immunofluorescence staining was performed with monoclonal antibodies specific to C. parvum using the Easy Stain kit (Biopoint Advancing Microbiology).

Statistical analysis

The data were processed using EpiInfo version 7.2.5.0 (Centers for Disease Control and Prevention, Atlanta, USA), available at https://www.cdc.gov/epiinfo/. A descriptive analysis of the total sample was performed, followed by bivariate analysis considering as dependent variable the presence of the parasite in the faeces of the children included in the sample. Point prevalence of parasitism and odds ratios (ORs) were calculated for each analysed factor. Chi-square tests were performed to assess statistical significance, and Fisher’s test was applied when required. For quantitative variables, normality was evaluated using the Kolmogorov-Smirnov test. Depending on the results, parametric tests (Student’s t-test) or nonparametric tests (Mann-Whitney U test) were used to compare the means. Statistical significance was defined as an alpha value of ≤ 0.05.