1. ZOO405 by Rania Baleela is licensed under a Creative Commons Attribution- NonCommercial-ShareAlike 3.0 Unported LicenseRania BaleelaCreative Commons Attribution- NonCommercial-ShareAlike 3.0 Unported License ZOO405, Week 7

2. This week A case of molecular epidemiology Methods of diagnosis Human genome project Recombination Genetic mapping

3. MOLECULAR EPIDEMIOLOGY How useful are molecular tools in disease control

4. (Baleela et al., 2014) Maximum likelihood (ML) tree based on phased and concatenated L. donovani haplotypes across eight coding loci (10.9 kb). Country code: SD – Sudan, IN-India, KE-Kenya, ET-Ethiopia, PT- Portugal, FR-France, ES-Spain.

5. Genetic clustering among Sudanese and geographically representative L. donovani strains: discriminant analysis of principal components based on 14 microsatellite loci. The optimal number of populations (10) is defined in the Bayesian information criterion (BIC) curve on the right hand side, by the ‘elbow’ of the BIC (y axis) vs population number (x axis) curve. 7 principal components were retained, explaining 80% of the total variation (Baleela et al., 2014)

6. Microsatellite-based neighbor-joining tree reveals genetic diversity among L. donovani from Sudan (Baleela et al., 2014) Key observation = dogs and humans in the region share similar strains.

7. “Man has tamed many creatures, but has himself become the most domestic animal of all” Steve Jones, Almost like a whale…the origin of species updated, ed. 2001

8. Malaria kills 2000000 people a year Malaria appear as a single illness BUT it is actually 4! Caused by parasites not much related to each other Malarias native continent is Africa Malaria parasites feed on haemoglobin… significance???? Certain people resist infection for reasons unknown… e.g. Fulani vs Rimaibe. Both tribes of Sudan

9. Diagnosis Traditionally => find a parasite. Problems:  Some parasites are morphologically indistinguishable.  Some hide in host tissue.  Low sensitivity.

10. Traditional diagnosis of Malaria

11. Skin snips: diagnosis of onchocerciasis

12. Lumbar puncture for African Sleeping Sickness

13. Faecal smear/urine filtration for Schistosomiasis.

14. Traditional parasitological diagnostic techniques = not satisfactory:  Need trained staff => local problems???  Need equipment => local problems???  slow throughput.  But “gold standard”.

15. Rapid tests development 3 types of “molecular” tests: 1.Biochemical (1 st generation). 2.Immunological (antibodies). 3. Nucleic acid. The solution?

16. 1. Biochemical Molecular Tests MLEE

17. Enzyme patterns: Isoenzymes (MLEE)  Perform same functions BUT have different movement on gels.  Genetically controlled  parasites with different gel patterns genetically distinct.

18. Chagas’ Disease MLEE profile of T. cruzi isolated by starch-gel electrophoresis

19. Assessment of enzyme pattern based diagnosis Advantages: Simple technique. Large number of typing enzymes available. Many samples typed at the same time. Power to distinguish morphologically similar parasites.

20. Enzyme pattern based diagnosis. Disadvantages:  Significant tissue needed for analysis  Technique not rapid  can take days.  Sometimes incorrect diagnosis  Technique simple but equipment expensive.  Silent mutations that does not affect the protein structure can not be detected.  Results can not be compared across labs.

21. 2. ANTIBODY BASED DIAGNOSIS immunological

22. Rely on identification of specific antibodies.  Advantages: Rapid easy field-based tests. Both individual & mass population screening. Ig subclasses – to improve specificity/sensitivity.

23. Antibody based diagnosis. Disadvantages:  Cannot distinguish past / present infections.  Cannot distinguish morphologically similar parasites.  Expensive to develop – significant research prior to commercialization.

24. Basic principles of Enzyme-Linked Immunosorbant Assay (ELISA) Ag Microtitre plate well Antigen Block unbound sites Primary antibody Secondary antibody with label Positive Negative

25. 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

26. CATT Test for African sleeping sickness.

27. Rapid Diagnosis using card kits – Malaria. NegativePositive 1.2.3. Results.