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Abstract Amoebae belonging to the genus Acanthamoeba are frequently found in soil, freshwater, and other habitats. They can cause diseases and can also carry other harmful microorganisms. When these amoebae contaminate healthcare facilities, they pose a microbiological risk to medical staff and immunocompromised patients. The purpose of the current study is to evaluate the extent to which Acanthamoeba species contaminate water in different parts of the hemodialysis unit in MRI and to assess the possible risk of transmission to hemodialysis patients. A total of 52 water samples were collected from these systems, including two pre- treatment and two post–treatment water samples of the main water treatment station, one input and one output sample of each of the 24 dialysis machines in the Dialysis Unit. The collected samples were cultivated on non-nutrient agar medium streaked with living E. coli for detection of Acanthamoeba spp., followed by morphological confirmation of Acanthamoeba using direct smear, iodine and Giemsa stained smears. Blood samples were collected from 70 hemodialysis patients attending the hemodialysis Unit of Medical Research Institute (MRI), Alexandria University for receiving three dialysis sessions/week and 22 healthy subjects as a control group. Sera were separated and examined by an ELISA assay for the detection of anti-Acanthamoeba IgG antibodies. Participating patients were interviewed using a structured questionnaire to collect demographic data and medical history. This study revealed that Acanthamoeba spp. were not detected in the pre-treatment water but were detected in the two post-treatment samples of the main water treatment station of the dialysis unit. Acanthamoeba was detected in 19 out of 24 input samples (79.2%) and 4 out of 24 output samples (16.7%) with a removal percentage of 78.9%. The difference in Acanthamoeba spp. detection rates between input and output water was statistically significant (p<0.001). Anti-Acanthamoeba IgG antibodies were detected in 23 out of 70 hemodialysis patients (32.9%) and in none of healthy control (p=0.002) Acanthamoeba IgG antibodies were detected in nearly equal percentages of males and females (32.5% and 33.3% respectively) with a non-significant difference (P=0.941) Also, there was no significant difference between the mean age of seropositive and seronegative patients (p=0.699). The duration of maintenance hemodialysis was longer in seropositive patients (median: 13 years, range: 3 months-28 years) compared to seronegative patients (median: 4 years, range; one month to 22 years) with a statistically significant difference (p=0.008) Regarding comorbidity, there was no significant association between Acanthamoeba seropositivity and the presence of diabetes mellitus, hypertension, or HCV infection among hemodialysis patients. Acanthamoeba antibodies were not detected in SL patients but 17 % of seronegative patients had SL (p=0.05) Acanthamoeba antibodies in serum samples of hemodialysis patients were not significantly associated with anemia, abdominal pain or recurrent headache. Summary, Conclusion and Recommendations 45 Regarding the relation between contamination of input water and seropositivity, antibodies were detected in 16 out of 55 patients (29.1%) performing dialysis on Acanthamoeba-positive machines and in seven out of 15 patients (46.7%) using negative machines The difference was not statistically significant (p= 0.204; OR: 0.469; 95% CI: 0.146 – 1.510). Regarding output water, antibodies were detected in one out of 13 patients (7.7%) performing dialysis on Acanthamoeba-positive machines and in 22 out of 57 patients (38.6%) using negative machines. The difference was not statistically significant (p= 0.06; OR |