update on clinical parasitology developments
TRANSCRIPT
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!