Prevalence & Genetic Identification of Cryptosporidium Species Infecting Humans & Animals in Tunisia

Rym Essid, Imne Ben Abda, Rym Abdelmalek, Hanne Tiouiri, Ridha Hamza, Karim Aoun & Ada Bouratbine Institut Pasteur de Tunis, LR 11-IPT-06

Cryptosporidium (C.) an emerging enteric protozoan

commonly associated with persistent diarrhea in immunocompromised persons with acquired immunodeficiency syndrome with primary immunodeficiency
a common cause of a self-limited diarrheal disease in immunologically healthy people particularly in children in developing countries in all age groups, associated with water-borne outbreaks in developed countries

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Five species of Cryptosporidium are responsible for most human Cryptosporidium infections
*C. hominis : almost exclusively a parasite of humans *C. parvum: common in calves and other young ruminants *C. meleagridis common in birds *C. felis common in cats *C. canis common in dogs
Microscopic examination of oocysts in clinical specimens does not

enable identification of species

Molecular tools are routinely used for species characterization

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The aim of this study

To identify Cryptosporidium species involved in human infections in Tunisia and to access their respective prevalence in different population groups To characterize C. parvum isolates in humans and animals To discuss the main transmission routes of Cryptosporidium infection in human

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Study population 108

patients hospitalized for immunodeficiency With human immunodeficiency virus infection 27 (25%) with chronic diarrhea 156 living in urban area

642
Human Stool samples

534
apparently immunocompetent children less than 5 years of age

378 living in rural area

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Study population in rural area

70 Stool samples from calves

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Cryptosporidium spp identification

Stool specimens Smears of fecal concentrates (formalin-ethyl acetate concentration) Staining with the modified Ziehl-Neelsen technique

Microscopic examination for Cryptosporidium spp. Oocysts

For all microscopically positive stools DNA extraction (QIAamp DNA Stool MiniKit) For Species identification

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Identification of Cryptosporidium species

Nested- PCR (18S ribosomal RNA gene) + RFLP analysis (Coupe et al, 2005)
Restriction assays. Amplified product initially digested with Taq I The products that were digested by Taq I were digested with Ase I to differentiate C. hominis from C. parvum

In cases of negative results, Mse I, Bst UI, and Ssp I were used to identify other species

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Intraspecific characterization of C. parvum isolates

GP60 sequencing (Sulaiman et al., 2005)

Nested PCR targeting GP60

Purification
Sequencing Phylogenic analysis

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Prevalence (ZNM) and species identification according to immune status

108
patients hospitalized for immunodeficiency

11 positive (10.2%) C. parvum: 6 C. hominis: 4 C. meleagridis: 1

534
apparently immunocompetent children less than 5 years of age

11 positive (2%) C. parvum: 5 C. hominis: 3 C. meleagridis: 3

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Prevalence (ZNM) and species identification according to geographical origin

156 Living in urban area

534
apparently immunocompetent children less than 5 years of age

4 positive (2.5%) C. hominis: 3 C. parvum: 1

378 Living in rural area

7 positive (1.85%) C. parvum: 4 C. meleagridis: 3

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Species identification according to geographical origin

In urban pediatric communities: - the high prevalence of C. hominis indicates that humans are a major source of infection and that person-to-person transmission probably plays a major role in the spread of Cryptosporidium. -The concomitant presence of C. parvum could be the result: > of environmental transmission (contaminated water or food) > of an anthroponotic transmission In fact, there is good evidence that subpopulations of C. parvum, e.g., GP60 type IIc, have a strictly anthroponotic transmission cycle

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Species identification according to geographical origin

In rural pediatric communities:

- C. hominis was absent which suggests that anthroponotic transmission is less common in this population.
- The presence of C. parvum and C. meleagridis is sugestive of zoonotic transmission by > direct contact with farm animals > environmental transmission (contaminated water or food)

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In rural area

70 calves

C. parvum: 14 (20%)

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Intraspecific characterization of C. parvum isolates from calves and humans

The most frequent In calves (85%) Present in human (50%)

Rare in calves Present in human (50%)

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Intraspecific characterization of C. parvum isolates from calves and humans

* Most C. parvum isolates from calves belong to family subtype IIa, also identified in humans This fact suggests that the family subtype IIa can spread easily within cattle populations and can be transmitted to humans too. *In regions where both subtypes IIa and IId are found, (i.e. Spain), family subtype IIa infects preferentially calves, while family subtype IId has a tropism for lambs and kids (Quilez et al., 2008)

This fact, suggests that Sheep and goats can be reservoirs in Tunisian rural areas

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Conclusion

Species identification and intra-specific characterization are valuable tools in the analysis of infection sources and understanding of the dynamics of transmission of cryptosporidiosis. Further studies are required for a deeper understanding of the epidemiology of cryptosporidiosis in men and animals in Tunisia.

Thank you for your attention

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