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;