Detection and Molecular Identification of Cryptosporidium Species Among Children with Malignancies

Abstract

Cryptosporidiosis represents a major health problem worldwide particularly among children. Its diagnosis is still difficult and demands sensitive methods. In Egypt, there is little documentation of infection among children with malignancies. This work was designed to study the infection rate of Cryptosporidium among children with malignancies, compare the performance of modified Ziehl-Neelsen (MZN) stain with nested polymerase chain reaction (PCR) and identify the species subtypes of positive cases. The study was conducted on 100 children with malignancies (leukemia, lymphoma and solid tumors), below 10 years of age, from El-Shatby hospital, Alexandria University. After obtaining the informed consent, their stool samples were collected and examined microscopically following MZN stain for the diagnosis of Cryptosporidium spp. All samples were then subjected to nested PCR. Restriction fragment length polymorphism (RFLP) targeting the Cryptosporidium oocyst wall protein (COWP) gene was applied to positive cases, using restriction enzyme RsaI for digestion of nested PCR products. Out of the 100 examined children, MZN detected higher positive cases compared to nested PCR. Six cases (6%) were diagnosed positive by MZN stain, three of which (3%) were concordantly positive by nested PCR. All positives were among children with acute lymphoblastic leukemia (ALL). Fair agreement was found between the two tests (K = 0.36). Genotyping results revealed that positive samples were of Cryptosporidium parvum (C. parvum) type II. Low Cryptosporidium infection rate was detected among children with malignancies. MZN diagnosed more positive cases compared to nested PCR. C. parvum type II was the identified species among the examined children. Further optimization of PCR steps is needed.