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GENOTYPING AND SUBGENOTYPING ANALYSIS OF CRYPTOSPORIDIOSIS
INFECTION IN EGYPT
Nour M. Abd El Kader, Abdel Rahman B. Abdel Ghaffar, Marwa A. Tammam, Jose M. Rubio, Ahmad
Osman, and Nabila El Sheikh
What is Cryptosporidium
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
Cryptosporidium is an obligate intracellular protozoan that causes a gastrointestinal disease (cryptosporidiosis) in a wide range of vertebrates, including humans
Human Cryptosporidiosis 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 cost-effective 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
Microscopic examination
Genotyping by PCR - RFLP targeting the SSU rRNA
gene
Sub-genotyping targeting the 60-kDa glycoprotein gene (gp60) gene
Sequencing of the gp60 amplified product
Experimental procedures
Cryptosporidium detection
Confirmed Cryptosporidium isolates
Sub-genotyping by PCR - RFLP
Cases 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
Immunochromatogrphic Screening
Cryptosporidium antigens were detected in 23/391 cases which represents an infection rate (5.88%)
Higher prevalence was detected for Giardia (22.8%; 89/391)
Mixed infections with both Cryptosporidium and Giardia was detected in 5 patients (1.3%)
“Stick Crypto-Giardia; operon”
Microscopic Examination
Cryptosporidium oocysts were conformed in 20 specimens on repeated examination
Modified Ziehl-Neelsen (mZN) stained smear
Genotyping targeting the SSU rRNA gene
12 isolates succeeded in amplifying PCR product targeting the SSU rRNA
SspI digestion
VspI digestion
Species Prevalence
C. hominis 9/12; 75%
C. parvum 3/12; 25%
Xiao et al., 1999
Subgenotyping targeting the gp60 gene
1. PCR –RFLP according to Cohen et al 2006
2. Sequencing of the amplified PCR products
The conducted gp60 RFLP patterns for the most comparable database sequences
Sub-genotyping by PCR –RFLP
Cohen et al., 2006
AluI digestion
RsaI digestion
Ia Id Ie Ib Ia IIc
M 2 12 6 7 11 1 8 4 5 M 3 9 10 M
Ia Id Ie Ib Ia IIc
M 2 12 6 7 11 1 8 4 5 M 3 9 10 M
Genotypes (no.) Subtype family No. of
cases
C. hominis (9) Ia 3
Ib 1
Id 3
Ie 2
C. parvum (3) IIc 3
Sequencing data 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).
Phylogenic analysis
Distribution of C. hominis and C. parvum alleles was revealed by a Maximum Likelihood Phylogenic analysis of the DNA sequences
Sequencing diversities
Sequence variations from the data base were observed in 2 C. hominis subtype families
1. Ia2. Ib
The rest of our sequences were similar to the data bases sequences
Sequence variation in the Ia subtype family
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 “IaA7R1”which is characterized by shorter poly-serine segment in the microsatellite region
The sequence has been submitted to NCBI GenBank under accession number HQ389257
Sequence variation in the Ib subtype family
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
Conclusion
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 shouldn’t 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 it’s characteristics and it’s geographical distribution in Egypt.
Collaborators
Egyptian team Spanish team
Prof. Nabila A. El-Sheik 1
Prof. Ahmad Osman 2
Dr. Abdel Rahman B. Abdel Gaffar 2
Dr. Nour M. Abd El-Kader 2
Prof. Jose M. Rubio 3
Dr. Marwa A. Tammam 1,3
1Microbiology department, Faculty of Medicine for Girls, El-Azhar University, Cairo, Egypt
2Biochemistry department, Faculty of Science, Ain Shams University, Cairo, Egypt;
3Servicio de Parastología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain;