Molecular diagnosis of parasite infection.

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Molecular diagnosis of parasite infection.

• Jo Hamilton
• Parasitology
• BS31820

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Objectives and learning outcomes.

• Recognise limitations of parasitological
  diagnosis.
• Recognise new molecular methods developed.
• Identify 3 main methods biochemical, antibody
  DNA based.
• Give specific examples of each.

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Why identify parasites?

• Treatment.
• Epidemiology.
• Control measures.
• Fundamental research.

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Some definitions

• Diagnostic test sensitive specific.
• Mathematically defined.
• Sensitivity capacity of test to make correct
  diagnosis in known cases.
• Specificity capacity to correctly diagnose
  uninfected individuals.
• Generally a trade off.

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A diagnosis problem?

• Traditionally diagnosis infection
• based on finding parasite.
• Problems
• Some parasites morphologically indistinguishable.
  
• Parasites hidden in host tissue.
• Low sensitivity.

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Skin snips Onchocerciasis
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Traditional diagnosis of Malaria
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Traditional Diagnosis of Chagas' disease.
Cage of triatomine bugs placed on skin for
xenodiagnosis.
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Lumbar puncture for African Sleeping Sickness.
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Faecal smear / urine filtration for
Schistosomiasis.
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The solution?

• Current parasitological diagnostic techniques not
  satisfactory.
• Need trained staff, equipment, slow throughput.
• But gold standard.
• Rapid molecular tests being developed.

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Three types of molecular tests.

• Biochemical (first generation).
• Immunological (antibodies).
• Nucleic acid.

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1. Biochemical molecular tests Enzyme patterns.

• Isoenzymes.
• Perform same functions BUT different movement on
  gels.
• Genetically controlled? parasites with different
  gel patterns genetically distinct.

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Isoenzyme Analysis of Chagas Disease
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Assessment of enzyme pattern based diagnosis

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

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Enzyme pattern based diagnosis.

• Disadvantages
• Significant tissue needed for analysis
• ? visceral leishmaniasis requires spleen,
  liver.
• Technique not rapid ? can take days.
• Sometimes incorrect diagnosis
• ? enzyme labile.
• Technique simple but equipment expensive.

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Iso-enzymes separated by charge Isoelectric
focusing equipment.
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Enzymes separated by size SDS-PAGE.
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2. Antibody based diagnosis.

• Rely on identification of specific antibodies.
• ?Advantages
• Rapid easy field-based tests.
• Both individual mass population screening.
• Ig subclasses to improve specificity /
  sensitivity.

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Antibody based diagnosis.

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

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

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CATT Test for African sleeping sickness.
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Example of antibody based molecular diagnosis

• Chagas Disease
• 1. FATALA kit measures T. cruzi antibodies in
  blood using 2 recombinant proteins
• 2. BIO CHAGAS kit uses cocktail recombinant T.
  cruzi antigens.
• Infection sera produces blue precipitate on
  strip in 60 minutes.
• (25 US dollars per kit).

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Rapid Diagnosis using card kits Malaria.
Results.
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Immuno-diagnosis of Cestode disease.

• Sera from patients with cysticercosis react with
  cysticercosis-specific proteins.
• Sera from patients with echinococcosis do not
  react.

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Future for Ab-based diagnosis?

• Non-invasive sampling? saliva, excreta?
• Good results for saliva abs for African
  trypanosomiasis.
• But will need new tests.
• Saliva abs not detected in CATT or Latex.

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3. DNA based molecular diagnosis.

• DNA probes.
• Polymerase Chain Reaction (PCR).

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PCR in parasite diagnosis.

• Amplifies target sequences increases
  sensitivity.
• Ribosomal DNA/RNA.
• Highly sensitive.
• No good for closely related species.
• Specific sequences of genomic DNA.
• Highly specific for single species - not
  sensitive.
• Random primer amplification (RADP) PCR.
• Very highly sensitive - not specific.

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Nucleic acid based molecular diagnosis.

• 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.

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Nucleic acid based diagnosis.

• Disadvantages
• Expensive - especially PCR .
• Radioactivity needed newer non-radioactive
  probes.
• PCR can fail - Contamination false positives.
• DNA probes do not distinguish between
• dead living parasites

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Parasite nucleic acid based diagnosis.

• Chagas Disease.
• PCR based kit in trials in Brazil.
• Aims to replace Xeno-test.

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Examples of veterinary parasite nucleic acid
diagnosis.

• Eimeria protozoa.
• Southern blot patterns using
• ribsosomal RNA probes
• - requires micrograms of DNA.
• PCR identification of 500 base pair
• sequence in ribosomal RNA
• requires 1pg DNA - lt 10 oocysts.

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Examples of veterinary parasite nucleic acid
diagnosis

• Cryptosporidium parvum protozoa.
• Faecal microscope analysis
• limit 50,000 oocysts per g faeces.
• PCR amplifies 400 base pair
• sequence from faeces
• - sensitivity to 6 oocysts per g faeces.

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PCR diagnostic test for detection of
Cryptosporidium parvum DNA

• C. parvum positive faecal specimen

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Summary.

• Parasitological diagnosis problems.
• New molecular methods - 3 main divisions.
• Biochemical isoenzymes e.g. Chagas.
• Antibody e.g. CATT for sleeping sickness kits
  for Chagas malaria Westerns for cestodes.
• DNA e.g. PCR kit for Chagas PCR for vet
  protozoans.

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Further reading.

• Please see handout for latest parasite diagnosis
  developments.
• The WHO/TDR website is very useful
  http//www.who.int/tdr/
• TDR publications can be found at
• http//www.who.int/tdr/publications/publications/
  default.htm

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Next lecture.

• Parasite vaccines.