2. RECAP

3. Housekeeping POSTINGS PCR results gel (report)
Next lecture paper discussion
Vaccine; why, possible?
What knowledge required?
Different approaches, failure, success?
ALSO ELISA LAB-HANDOUT READ, ANSWER PRE-LAB QUESTIONS

4. Diagnosis

5. Why identify parasites?

6. Why identify parasites?
Treatment, Epidemiology, Control
Research

7. Sensitivity and Specificity
An optimal test has
Sensitivity: capacity to make correct diagnosis in known cases (proportion of correctly identified subjects with infection)
Specificity: capacity to correctly identify uninfected subjects (proportion: number of true negative results divided by the sum of the numbers of true negative plus false positive results)
Usually one limits the other.

10. Sensitivity: capacity to make correct diagnosis in known cases.
Specificity: capacity to correctly diagnose uninfected individuals.

11. Diagnosis difficulties
Find/see the parasite.
But:
Some parasites morphologically indistinguishable.
Parasites reside in organs/tissues.
Low sensitivity.

12. Xenodiagnosis
(Toxoplasma/mice)

13. Better methods needed?
Traditional parasitological diagnosis experienced person
equipment
slow throughput
Visual detection IS positive ID
Alternative molecular tests being developed.

14. Antibody Antigen
DNA (RNA)

15. Antibody based diagnosis.
Detection of specific anti-parasite antibodies.
Advantages:
Rapid easy field-based tests.
Both individual & mass population screening.
Ig subclasses – to improve specificity / sensitivity (e.g. IgE – helminths).

16. Classical Ab test for diagnosis Enzyme-Linked Immunosorbant Assay (ELISA).
Positive
Negative

17. Basic principles of ELISA.
Secondary antibody
with label
Primary antibody
Block
unbound
sites
Antigen Ag Ag Ag
Microtitre plate well

18. Antibody based diagnosis.
Disadvantages:
Cannot distinguish past / present
infections.
False positives/negatives, cross reactions
Expensive to develop – significant
research prior to commercialization.

19. Antigen-based diagnosis.
Antigen is more directly associated with active infection

20. TO THE FIELD?

21. Example of antibody-based molecular diagnosis.
African Sleeping Sickness
Anti-trypanosomal IgM detected by simple / rapid
CATT (Card Agglutination Test for Trypanosomiasis)
Drop of blood 
Mixed with fixed parasites on plastic card
Blue granular deposits = infection
25 US cents per test

22. CATT Test for African sleeping sickness.

23. Test principle. The lateral flow assay involves the use of nitrocellulose strips through the capillaries of which a mixture of the urine sample and a detection conjugate (monoclonal antibody labeled with carbon particles) flows. The presence of the analyte (CCA) is made visible by capture of the immune complex of antigen and carbon-labeled antibody by the anti-CCA monoclonal antibody that is immobilized on the strip as a test line. In addition, a line of immobilized polyclonal anti-mouse antibodies is used to capture the excess carbon-labeled antibodies to act as a positive control

26. DNA-based diagnosis PCR (Alberts)

27. PCR in parasite diagnosis.
Amplifies target sequences & increases sensitivity.
MUST KNOW TARGET
Ribosomal DNA/RNA.
Sensitive (multiple targets/cell).
not as specific with closely related species.
Specific sequences from mitochondrial DNA.
Specific for groups of species
Specific sequences from genomic DNA.
Not as sensitive (few targets/cell).
Highly specific for single species

30. RT-PCR
qPCR

31. DNA-based diagnosis. - Specific - detect single parasite species.
Advantages:
Genomic DNA constant -parasite & hosts unique DNA sequences .
Very sensitive - small biopsy.
Probes can be designed with flexibility:
- Specific - detect single parasite species.
- Less specific - detect group of parasites.

32. PCR based diagnosis. Disadvantages: PCR expensive, requires equipment
PCR can fail: - Contamination & false positives.
DNA probes do not always distinguish dead from living parasites

33. Increased sensitivity PCR detection
Cryptosporidium parvum protozoa
Faecal microscope analysis:
- 50,000 oocysts per g faeces.
PCR: amplifies ~400 base pair
sequence from faeces
- sensitivity to 6 oocysts per g faeces.

34. PCR diagnostic test for detection of Cryptosporidium parvum DNA
C. parvum positive faecal specimen

40. Gauci C, Jenkins D, Lightowlers MW. 1.
Strategies for Optimal Expression of Vaccine Antigens from Taeniid Cestode Parasites in Escherichia coli.
Gauci C, Jenkins D, Lightowlers MW.
Mol Biotechnol Jan 11. [Epub ahead of print]
PMID:
[PubMed - as supplied by publisher]
Related citations 2.
Vaccines to combat the neglected tropical diseases.
Bethony JM, Cole RN, Guo X, Kamhawi S, Lightowlers MW, Loukas A, Petri W, Reed S, Valenzuela JG, Hotez PJ.
Immunol Rev Jan;239(1): doi: /j X x.
[PubMed - in process] 3.
Fact or hypothesis: Taenia crassiceps as a model for Taenia solium, and the S3Pvac vaccine.
Lightowlers MW.
Parasite Immunol Nov-Dec;32(11-12): doi: /j x. Review.
[PubMed - indexed for MEDLINE]
Free PMC Article
Free full text Related citations