Update onClinical Parasitology Developments

Dr. Claire AlexanderConsultant Clinical Scientist

Scottish Parasite Diagnostic and Reference LaboratoryStobhill Hospital

•Infective oocysts in faeces

Transmission routes;•Water/food to human •human to human •animal to human

•Can infect over 150 species of mammals

•11 Cryptosporidium species known to infect humans

Cryptosporidium

andersoni

hominis

canis

meleagridis

felis

parvum

suis

muris

Cryptosporidium Species

tyzzeri

cuniculus

ubiquitum

Cryptosporidium genotypes (60+)

pig horse sheepcattlemarsupial opossum skunkmonkeyferretfox muskrat squirrelchipmunkbear deer mouse

• Diarrhoea (98%)• Abdominal pain (95%) • Low grade fever (59%)• Nausea & vomiting (65%)

• Incubation period is 2-14 days

• Length of symptoms is 3-21 days

• Need as few as 9 oocysts for infection

• Single bowel movement can contain 10000000000 oocysts!!

Clinical Symptoms

What happens when a Cryptosporidium positive stool arrives at SPDRL?

1 5 1 2 0 01 9 8 C A C C T C C C T C A G C T C C T C C G A C T A C A C C T C C C T C A G C T C C T C C A G T T G C A4 8 9 C A C C T C C C T C A G C T C C T C C G A C T A C A C C T C C C T C A G C T C C T C C A G T T G C A9 2 5 C A C C T C C C T C A G C T C C T C C G A C T A C A C C T C C C T C A G C T C C T C C A G T T G C A2 0 7 C A C C T T C C T C A G C T C C T C C G A C T G C A C C T T C C T C A G C T C C T C C A G T T G C A2 5 0 C A C C T T C C T C A G C T C C T C C G A C T G C A C C T T C C T C A G C T C C T C C A G T T G C A2 9 3 C A C C T C C C T C A G C T C C T C C G A C T G C A C C T T C C T C A G C T C C T C C G A C T G C A8 0 2 C A C C T C C C T C A G C T C C T C C G A C T G C A C C T C C C T C A G C T C C T C C G A C T G C A9 2 2 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ A C G C C c C a g c T C C C T C A G C T C C T C C G A C T G C A

2 0 1 2 5 01 9 8 C C T C C C C C A G C T C C T C C A C T C G C T C C A G C T C C T C C A C C T A C T C C T G C T C C4 8 9 C C T C C C C C A G C T C C T C C A C T C G C T C C A G C T C C T C C A C C T A C T C C T G C T C C9 2 5 C C T C C C C C A G C T C C T C C A C T C G C T C C A . . . . . . . . . . . . . . . . . . G C T C C2 0 7 C C T C C C T C A G C T C C C C C A G T T G C A C C T C C C T C A G C T C C C C C A G T T G C A C C2 5 0 C C T C C C T C A G C T C C C C C A G T T G C A C C T C C C T C A G C T C C C C C A G T T G C A C C2 9 3 C C T T C C T C A G C T C C T C C A G T T G C A C C T C C C T C A G C T C C C C C A G T T G C A C C8 0 2 C C T T C C T C A G C T C C T C C A G N T G C A C C T C C C T C A G C T C C C C C A G T T G C A C C9 2 2 C C T T C C T C A G C T C C T C C G A C T G C A C C T C C C T C A G C T C C C C C A G T T G C A C C

2 5 1 3 0 01 9 8 T C C A C C T A C T C C T G C T C C T C C A C T T G C A C C T C C C T C A G C T C C A C C A C C T G4 8 9 T C C A C C T A C T C C T G C T C C T C C A C T T G C A C C T C C C T C A G C T C C A C C A C C T G9 2 5 T C C A C C T A C T C C T G C T C C T C C A C T T G C A C C T C C C T C A G C T C C A C C A C C T G2 0 7 T C C C T C A G C T C C T C C A G T T G C A C . . . C T C C C T C A G C T C C T C C A C C A C C T G2 5 0 T C C C T C A G C T C C T C C A G T T G C A C . . . C T C C C T C A G C T C C T C C A C C A C C T G2 9 3 T C C C T C A G C T C C C C C A G T T G C A C . . . C T C C C T C A G C T C C T C C A C C A N C T G8 0 2 T C C C T C A G C T C C C C C A G T T G C A C . . . C T C C C T C A G C T C C T C C A C C A C C T G9 2 2 T C C C T C A G C T C C C C C A G T T G C A C . . . C T C C C T C A G C T C C T C C A C C A C C T G

ssrRNA PCR-RFLP Sequencing

Multiplex PCR

Concentrated DNA Extraction

+ Internal Control DNA

Subtyping Assay

C.hominisC.parvum

Other Crypto species

1) Observe oocysts2) Speciate3) GP60 Gene Amplification

• Families - Extensive sequence differences in the non-repeat regions

C.hominis families = Ia, Ib, Id-IgC.parvum families = IIa-IIii, IIk, IIl

• Tandem repeats - trinucleotides TCA, TCG or TCT at the 5’end of the gene

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• Subtypes differ in the number of tandem repeats

Subtyping of Cryptosporidium

The subtype begins with the family, followed by the number of trinucleotide repeats

e.g. C.hominis IbA10G2

Reporting of Cryptosporidium Subtypes

Subtype family Ib; 10 copies of the TCA repeat; 2 copies of the TCG repeat

Subtype family IIa;

C parvum has repeat sequences (usually one or two, ACATCA) after the serine repeats labelled R;e.g. C.parvum IIaA15G2R1

15 copies of TCA; 2 copy of TCG;1 copy of ACATCA Repeat

Scottish Crypto Ref Lab Data April 2012 –present2012 – present 563 Scottish cases

176 samples referred to SPDRL

C.parvum n=114 (110 between April – July 96%)

C.hominis n=50 (37 between Aug-Nov 74%)Mixed (C.parvum & C.hominis) n=1

Others (C.ubiquitum n=5 (Scottish Water & Grampian; C.meleagridis n=1 (Glasgow); C.cuniculus n=1 Lanarkshire)

Insufficient material n=3; Unamplifiable sample n=1

Less common subtypes isolated; C.parvum IIgA9R1 (Glasgow) - rare C.parvum IIdA16G1 (Fife) C.parvum IIcA5G3 (Lothian)

5 potential outbreaks;2 Lothian (?petting farm, predominant subtype C.parvum IIaA17G1R1; ?swimming pool C.hominisIbA10G2)

1 Tayside (?swimming pool C.parvum IIaA15G2R1; IIaA19G1R1)

1 UK wide (?foodborne C.parvum IIaA15G2R1)

1 Borders (All C.hominis but 2 groups - IbA10G2; IaA14R3).

Future Challenges

Prevailing Cryptosporidium subtypes in Scottish human cases?

Prevailing Cryptosporidium subtypes in farm animals in Scotland – Moredun Institute

Optimum procedures for water sampling – Scottish Water, Drinking Water Inspectorate

Cryptosporidium whole genome sequencing – Sanger Institute

Metabolomics

Other Developments

Entamoeba species;•E.histolytica•E.dispar•E.moshkovskiiPCR assays for detection in stools & liver abscesses; real-time multiplex; PCR-RFLP approach

Molecular sequencing-based assays for detection of common and unusual malaria species

Molecular testing of Ancathamoeba species

Rapid antigen detection of Schistosome species

Thanks!