A cross-sectional study was conducted among residents of Thi-Qar Province, Iraq. Participant enrollment, questionnaire interviews, stool sample collection, microscopic examination and molecular confirmation were conducted between January 28 and April 30, 2026 following approval by the Thi-Qar Health Directorate. Data verification and coding were performed progressively as participant recruitment and laboratory results became available. Following completion of participant enrollment and laboratory investigations on April 30, 2026, final data validation, statistical analyses, interpretation of the findings and manuscript preparation were completed during early May, 2026. A total of 420 participants presenting with gastrointestinal symptoms and attending primary healthcare centers were enrolled. Participants were classified into two groups according to the presence of Cryptosporidium infection in their stool samples, as determined by modified Ziehl-Neelsen staining and nested polymerase chain reaction (PCR): Cryptosporidium-positive cases (n=296) and Cryptosporidium-negative controls (n=124).

The study protocol was approved by the Research Ethics Committee of Thi-Qar Health Directorate, Ministry of Health (Approval no. 2026/27, approved on January 27, 2026), following the official institutional authorization issued by the College of Science, University of Thi-Qar (Reference no. 97, dated January 20, 2026), which supported the ethical review and approval process by the Health Directorate. Written informed consent was obtained from all adult participants and from the parents or legal guardians of minors before enrollment. Participant recruitment, questionnaire interviews, and stool sample collection commenced only after all required institutional and ethical approvals had been obtained.

Data were collected through direct face-to-face interviews using a structured questionnaire between January 28 and April 30, 2026. Questionnaire interviews and stool sample collection were performed during the same participant visit, thereby ensuring direct linkage between the demographic and epidemiological information of the participants, and their corresponding laboratory findings. Information obtained included age, sex, education level (illiterate/educated), residence (rural/urban), the presence of domestic animals (animal contact: Yes/no) and the source of drinking water. Water sources were categorized as verified reverse osmosis (RO)-treated water or non-RO water. Commercial bottled water and filtered tap water were grouped together as information regarding RO treatment was not available for all products, and their level of protection against Cryptosporidium oocysts could not be assumed to be equivalent to that provided by verified RO-treated water.

Fresh stool samples were collected in sterile wide-mouthed containers between January 28 and April 30, 2026 during the same visit in which participants completed the study questionnaire.

Thin fecal smears were prepared on clean glass slides by mixing a small amount of stool with a few drops of water and allowed to air dry. Smears were stained using a modified Ziehl-Neelsen stain kit (REF BS0243250; Bio Research for Medical Diagnostics). Slides were covered with carbol fuchsin for 5 min with gentle heating until steam appeared, rinsed with tap water, decolorized with acid alcohol for 10-15 sec, rinsed again with water, counterstained with methylene blue for 1 min, washed with water, and air dried. Slides were examined using an light microscope (Olympus Corporation) at x40 magnification and subsequently under 100X oil immersion objective. Cryptosporidium oocysts appeared as bright pink to red spherical bodies (4-6 µm) against a blue-green background (9).

Nested PCR was performed only on microscopy-positive samples to confirm the presence of Cryptosporidium DNA (this was a limitation to the present study, as discussed below).

Genomic DNA was extracted from 200 mg stool samples using the Presto™ Stool DNA Extraction kit (cat. no. STLD100; Geneaid Biotech Ltd.). Nested PCR targeting the SSU rRNA gene was performed using outer primers (Crypto Outer F/R, 608 bp) and inner primers (Crypto Inner F/R, 500 bp) based on the reference sequence GenBank accession number LC844811.1. Reactions were performed using GoTaq^® Green Master Mix (Promega Corporation) in a T100 Thermal Cycler (Bio-Rad Laboratories, Inc.). The cycling conditions consisted of an initial denaturation 95˚C/5 min; 35 cycles of 95˚C/30 sec, annealing 58˚°C (first round) or 60˚C (second round)/30 sec, extension 72˚C/60 sec (first) or 45 sec (nested); final extension 72˚C/5 min. PCR products were analyzed on 2% agarose gel, stained with ethidium bromide and visualized under ultraviolet illumination.

A representative subset of nested PCR-positive amplicons was submitted to Macrogen Inc. (Seoul, Korea) for bidirectional Sanger sequencing to confirm species identification. The obtained nucleotide sequences were compared with reference sequences available in the NCBI GenBank database using the BLAST program. Multiple sequence alignment was performed using ClustalW implemented in MEGA version 11.0.13, and phylogenetic associations were inferred using the UPGMA method. The representative sequences were deposited in the NCBI GenBank database under accession nos. PZ245405, PZ245406, PZ245407, PZ245408, PZ245409, PZ245410, PZ245411, PZ245412 and PZ245413.

Data were analyzed using SPSS software version 26. Categorical variables are summarized as frequencies and percentages. Associations between variables were evaluated using Pearson's Chi-squared test. Independent predictors of Cryptosporidium infection were identified using multivariable logistic regression analysis, including animal contact (yes/no), drinking water source (non-RO vs. RO), age category (1-5), residence (rural vs. urban), sex (female vs. male) and education level (educated vs. illiterate). Adjusted odds ratios (AORs) and their corresponding 95% confidence intervals (CIs) were calculated. Model fit was assessed using the Hosmer-Lemeshow goodness-of-fit test, with a value of P>0.05 indicating adequate model fit. In addition, a P-value <0.05 was considered to indicate a statistically significant difference.

Cryptosporidium infection was detected in 296 participants, whereas 124 participants tested negative, yielding an overall prevalence of 70.5%. The sociodemographic characteristics and potential risk factors of the study participants are presented in Table I. The frequency of exposure to animals and the ingestion of non-RO water was higher among the infected participants compared with the non-infected participants. Age distribution differed significantly between the two groups (P<0.001), indicating a significant univariate association. However, following multivariable adjustment, age was not independently associated with Cryptosporidium infection (P=0.097; Table II).

Microscopy-positive samples subjected to nested PCR yielded the expected 500-bp amplicon. Representative microscopic and electrophoretic findings are presented in Fig. 1 (microscopy) and Fig. 2 (gel electrophoresis), respectively. Sanger sequencing of selected isolates confirmed species-level identification of Cryptosporidium hominis and Cryptosporidium parvum among study samples. Among the sequenced isolates, 77.8% were identified as Cryptosporidium parvum and 22.2% as Cryptosporidium hominis. Representative Sanger sequencing chromatograms with sequence alignment and variant annotation are presented in Fig. 3. Sequence data were deposited in the NCBI GenBank database under accession nos. PZ245405, PZ245406, PZ245407, PZ245408, PZ245409, PZ245410, PZ245411, PZ245412 and PZ245413.

The results of the multivariable logistic regression analysis are presented in Table II. Following adjustment for all covariates, two variables remained significantly associated with Cryptosporidium infection. Participants with animal contact had significantly higher odds of acquiring the infection compared with those without animal contact (AOR=2.98; 95% CI, 1.75-5.02; P<0.001). Similarly, the consumption of non-RO water was associated with an ~a 2-fold increase in the odds of infection (AOR=2.05; 95% CI, 1.10-3.82; P=0.024). Age exhibited a positive, yet statistically non-significant association with infection (AOR=1.18 per category increase; 95% CI, 0.97-1.42; P=0.097). Residence, sex and educational status were not significantly associated with infection (P>0.05 for all variables). The Hosmer-Lemeshow goodness-of-fit test indicated that the final model demonstrated an adequate fit to the observed data (χ²=10.89, df=8; P=0.208).