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Prevalence & Genetic Identificationof CryptosporidiumSpeciesInfecting 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
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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
Cryptosporidium(C.) an emerging enteric protozoan
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Five species of Cryptosporidiumare responsiblefor most human Cryptosporidiuminfections
*C. hominis: almost exclusively a parasite of humans*C.parvum: common in calves and other young ruminants
*C. meleagridiscommon in birds*C. feliscommon in cats*C. caniscommon 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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To identify Cryptosporidiumspecies involved in human infections inTunisia and to access their respective prevalence in different
population groups
To characterize C. parvumisolates in humans and animals
To discuss the main transmission routes ofCryptosporidiuminfectionin human
The aim of this study
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642Human
Stoolsamples
534apparently
immunocompetent
children less than5 years of age
108
patients hospitalizedfor
immunodeficiency
With human
immunodeficiency
virus infection
27 (25%) with
chronic diarrhea
156living in urban
area
378living in rural
area
Study population
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Study populationin rural area
70Stool samples
from calves
7/31/2019 Prevalence & Genetic Identification
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Stool specimens
Smears of fecal concentrates
(formalin-ethyl acetate concentration)
Staining with the modified
Ziehl-Neelsen technique
Microscopic examination
forCryptosporidiumspp. Oocysts
For all microscopically positive stools
DNA extraction (QIAamp DNA Stool MiniKit)
For Species identification
Cryptosporidium sppidentification
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Identification of Cryptosporidiumspecies
Nested- PCR (18S ribosomal RNA gene) + RFLP analysis (Coupe et al, 2005)
Restriction assays.
Amplified product initially digested
with TaqI
The products that were digested by TaqIwere digested with AseIto differentiate C. hominisfrom C. parvum
In cases of negative results,MseI, BstUI, and SspIwere used to identify other species
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Intraspecific characterization of C. parvumisolates
GP60 sequencing (Sulaiman et al., 2005)
Nested PCR
targeting GP60
Purification
Sequencing
Phylogenic analysis
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108patients hospitalized
for
immunodeficiency
11 positive (10.2%)
C. parvum: 6
C. hominis: 4
C. meleagridis: 1
Prevalence (ZNM) and species identificationaccording to immune status
534apparently
immunocompetent
children less than5 years of age
11 positive (2%)
C.parvum: 5
C. hominis: 3
C. meleagridis: 3
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534apparently
immunocompetentchildren less than
5 years of age
156Living in urban area
378Living in rural area
Prevalence (ZNM) and species identificationaccording to geographical origin
4 positive (2.5%)
C. hominis: 3
C.parvum: 1
7 positive (1.85%)
C.parvum: 4
C. meleagridis: 3
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In urban pediatric communities:
- the high prevalence ofC. hominisindicates that humans are a majorsource of infection and that person-to-person transmission probably
plays a major role in the spread ofCryptosporidium.
-The concomitant presence ofC. parvumcould be the result:> of environmental transmission (contaminated water or food)
> of an anthroponotic transmission
In fact, there is good evidence that subpopulations ofC. parvum,
e.g., GP60type IIc, have a strictly anthroponotic transmission cycle
Species identification according togeographical origin
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In rural pediatric communities:
- C. hominis wasabsent which suggests that anthroponotictransmission is less common in this population.
- The presence ofC. parvum and C. meleagridisis sugestive ofzoonotic transmission by
> direct contact with farm animals
> environmental transmission (contaminated water or food)
Species identification according togeographical origin
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In rural area
70 calves
C.parvum: 14 (20%)
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Intraspecific characterization of C. parvumisolates from calves and humans
The most frequentIn calves (85%)
Present in human
(50%)
Rare in calves
Present in human
(50%)
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* Most C. parvum isolatesfrom calves belong to family subtype IIa, alsoidentified 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
Intraspecific characterization of C. parvumisolates from calves and humans
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Species identification and intra-specific characterizationare 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 theepidemiology of cryptosporidiosis in men and animals in Tunisia.
Conclusion
Thank you for your attention