Nour M. Abd El Kader, Abdel Rahman B. Abdel Ghaffar, Marwa A. Tammam, Jose M.

Rubio, Ahmad Osman, and Nabila El Sheikh

Cryptosporidium is an obligate intracellular protozoan that causes a gastrointestinal disease (cryptosporidiosis) in a wide range of vertebrates, including humans Cryptosporidium exists in the environment as oocyst. Humans and animals are infected by ingesting these oocysts Currently there are 21 valid Cryptosporidium species, at least 8 of them have been reported in humans, Of these, C. hominis and C. parvum are responsible for the majority of human infections The transmission of human cryptosporidiosis has been inferred to occur from human-to-human (anthroponotic transmission) for C. hominis and C. parvum, or animal to- human (zoonotic transmission) for C. parvum

In humans, Cryptosporidium infection can result in severe diarrhoea, which is usually self-limiting in immunocompetent individuals, but may be chronic and life-threatening to those that are immunocompromised Cryptosporidium has emerged as an important enteric pathogen as it has been the cause of multiple waterborne diarrheal outbreaks in the many countries The impact of cryptosporidiosis is compounded by lack of costeffective chemotherapeutic agents or vaccines It is believed that management and control of cryptosporidiosis in human requires knowledge of Cryptosporidium species contributing to human disease

Samples collection Immunochromatographic screening Cryptosporidium detection Microscopic examination

Confirmed Cryptosporidium isolates

Genotyping by PCR - RFLP targeting the SSU rRNA gene

Sub-genotyping targeting the 60-kDa glycoprotein gene (gp60) gene

Sub-genotyping by PCR - RFLP Sequencing of the gp60 amplified product

A total of 391 stool samples were collected from Egyptian patients (228 males, 163 females) with age ranging from 1 month-70 years, in the period between May 2008 and March 2009. Samples were collected from either in-hospital patients suffering from diarrhea or out-patients requesting stool analyses due to gastro-intestinal discomfort associated with diarrhea

Cryptosporidium antigens were detected in 23/391 cases which represents an infection rate (5.88%)
Stick Crypto-Giardia; operon

Higher prevalence was detected for Giardia (22.8%; 89/391) Mixed infections with both Cryptosporidium and Giardia was detected in 5 patients (1.3%)

Modified Ziehl-Neelsen (mZN) stained smear

Cryptosporidium oocysts were conformed in 20 specimens on repeated examination

12 isolates succeeded in amplifying PCR product targeting the SSU rRNA

Species C. hominis C. parvum Prevalence 9/12; 75% 3/12; 25%

Xiao et al., 1999

SspI digestion

VspI digestion

1.

PCR RFLP according to Cohen et al 2006 Sequencing of the amplified PCR products

2.

The conducted gp60 RFLP patterns for the most comparable database sequences

Cohen et al., 2006

AluI digestion
M 2 12 Ia 6 7 Id 11 1 Ie 8 4 5 M 3 9 IIc 10 M M 2 12 Ia 6

RsaI digestion
7 Id 11 1 Ie 8 4 5 M 3 9 IIc 10 M

Ib Ia

Ib Ia

Genotypes (no.) C. hominis (9)

Subtype family Ia Ib Id Ie

No. of cases 3 1 3 2 3

C. parvum (3)

IIc

The BLAST analysis of the sequence data confirmed the PCR- RFLP results. The gp60 genotypic nomenclature was determined according to Jex et al. 2007, based on the tri-nucleotide repeats within the gp60 microsatellite region (which encodes a poly-serine tract within the gene).

Distribution of C. hominis and C. parvum alleles was revealed by a Maximum Likelihood Phylogenic analysis of the DNA sequences

Sequence variations from the data base were observed in 2 C. hominis subtype families 1. Ia 2. Ib The rest of our sequences were similar to the data bases sequences

The analysis of the sequence alignment results revealed the presence of a new allele in the Ia family represented in 3 isolates. The sub-genotype allele was named according to Jex et al., 2007 as IaA7R1which is characterized by shorter poly-serine segment in the microsatellite region The sequence has been submitted to NCBI GenBank under accession number HQ389257

In our study the Ib subtype family was represented by a single isolate, IbA9G3R2, which showed some variations outside the polyserine microsatellite region compared to the database sequence GU214347

DNA sequence alignment

protein sequence alignment

The results of the PCR-RFLP for the gp60 gene were validated by the sequencing results to the level of determining the subtype family Despite the general advise that differentiating gp60 subtypes shouldnt be based on RFLP analysis instead of DNA sequencing. However, RFLP analysis may still be an economical method for primary differentiation followed by sequencing analysis of representative cases. Despite the investigation of limited number of isolates, our study revealed that anthroponotic transmission of Cryptosporidium infection is the predominant mode of transmission in Cairo, Egypt. Finally our results identified a new allele belonging to the Ia subtype family, which require further investigation to determine its characteristics and its geographical distribution in Egypt.

Egyptian team
Prof. Nabila A. El-Sheik 1 Prof. Ahmad Osman 2 Dr. Abdel Rahman B. Abdel Gaffar 2

Spanish team
Prof. Jose M. Rubio 3 Dr. Marwa A. Tammam
1,3

Dr. Nour M. Abd El-Kader 2

1Microbiology

department, Faculty of Medicine for Girls, El-Azhar University,

Cairo, Egypt 2Biochemistry department, Faculty of Science, Ain Shams University, Cairo, Egypt; 3Servicio de Parastologa, Centro Nacional de Microbiologa, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain;