prevalence & genetic identification

Upload: mohammed-sarhan

Post on 04-Apr-2018

225 views

Category:

Documents


0 download

TRANSCRIPT

  • 7/31/2019 Prevalence & Genetic Identification

    1/17

    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

  • 7/31/2019 Prevalence & Genetic Identification

    2/17

    Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    3/17

    Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    4/17

    Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    5/17

    Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    6/17

    Recherche et formation

    Study populationin rural area

    70Stool samples

    from calves

  • 7/31/2019 Prevalence & Genetic Identification

    7/17Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    8/17Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    9/17Recherche et formation

    Intraspecific characterization of C. parvumisolates

    GP60 sequencing (Sulaiman et al., 2005)

    Nested PCR

    targeting GP60

    Purification

    Sequencing

    Phylogenic analysis

  • 7/31/2019 Prevalence & Genetic Identification

    10/17Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    11/17Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    12/17Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    13/17Recherche et formation

    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

  • 7/31/2019 Prevalence & Genetic Identification

    14/17Recherche et formation

    In rural area

    70 calves

    C.parvum: 14 (20%)

  • 7/31/2019 Prevalence & Genetic Identification

    15/17Recherche et formation

    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%)

  • 7/31/2019 Prevalence & Genetic Identification

    16/17Recherche et formation

    * 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

  • 7/31/2019 Prevalence & Genetic Identification

    17/17Recherche et formation

    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