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

This week content Diagnosis Classical Molecular Prenatal

ScienceSociety Epidemiology Medicine Revolution in Molecular Biology has Impacted Molecular Epidemiology Challenge for the 21 st Century

“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

~3.2 billion people (~1/2 world’s population) are at risk of malaria 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 Parasites feed on haemoglobin… significance???? Certain people resist infection for reasons unknown… e.g. Fulani vs Rimaibe. Both tribes of Sudan Malaria kills people a year

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

e.g. To diagnose malaria 1.Clinical Diagnosis: symptoms and physical findings at examination. 2.Microscopic Diagnosis 3.Antigen Detection: detect antigens (ICT) 4.Molecular Diagnosis: PCR. 5.Serology: detects antibodies (IFA) 6.Drug Resistance Test

Traditional diagnosis of Malaria

IFA: the fluorescence indicates that the presence of antibodies that are reacting with the antigen preparation (Plasmodium falciparum ). a positive test for Plasmodium falciparum.

Diagnosis of malaria can be difficult: Where malaria is not endemic any more In some malaria-endemic areas, malaria transmission is so intense that a large proportion of the population is infected but not made ill by the parasites.

Skin snips: diagnosis of onchocerciasis

Lumbar puncture for African Sleeping Sickness

Faecal smear/urine filtration for Schistosomiasis.

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

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

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

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

MLEE Advantages: Simple technique. Large number of typing enzymes available. Many samples typed at the same time. Power to distinguish morphologically similar parasites.

MLEE 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.

B. Antibody based diagnosis (immunological) Rely on identification of specific antibodies. 

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

2. Rapid CATT 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

CATT Test for African sleeping sickness.

3. Rapid Diagnosis using card kits – Malaria. NegativePositive Results.

Advantages Rapid easy field-based tests. Both individual & mass population screening. Ig subclasses – to improve specificity/sensitivity.

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