1. Infection and Disease Fungi Parasites Nosocomial infection Diagnosis of infectious disease

2. Fungi

3. Eukaryotes – nucleus / several organelles Non-motile Aerobic Cell wall made of Chitin Need for special antibiotics (antifungals):

4. Fungal diseases –superficial infections Superficial infection– ability to infect intact skin /nail Caused by dermatophytes These fungi use keratin for their growth Eg. Trychophyton spp

5. Fungal diseases –Systemic infections Systemic infections: Lung infections in healthy individuals Not common Eg. Histoplasma spp

6. Fungi opportunistic infection Infection in immunocompromised individuals Eg. Aspergillus spp. and Candida spp. in AIDS patients Can affect any organ

7. Fungal toxins 1960s – “Turkey X disease” – Brazilian peanut feed Linked to fungal toxin from Aspergillus spp. (Aflatoxin) Can affect liver, kidney, heart and brain in humans

8. Parasites Unicellular (Protozoa) – eg. Amoebae Multicellular worms (Helminths) eg. Round worms /flatworms Many parasites require – 2 hosts to complete their life cycle (eg. Plasmodium falciparum) Some parasites require vectors for transmission(eg. Mosquitoes) Entamoeba spp Round worms

9. Protozoa Eg. Entamoeba spp. – amoeba that can cause dysentery / bloody diarrhoea Plasmodium falciparum – causes malaria

10. Helminths Nematodes Unsegmented worms Roundworms Platyhelminthes Segmented worms Flat worms

11. Roundworm (Ascaris spp.) - life cycle < 100µm 500µm 40 cms Abdominal pain Diarrhoea

12. Tapeworm (Taenia spp. ) – life cycle < 50µm 5mm Up to 10 meters Indigestion, loss of appetite, abdominal pain

13. Nosocomial infections or hospital acquired infections

14. Nosocomial or hospital acquired infections infections Hospital acquired infections: new infections acquired in the hospital or immediately after discharge

15. Example of nosocomial infections MRSA – Methicillin Resistant Staphylococcus aureus ; highly antibiotic resistant

16. Diagnosis of infectious diseases

17. Methods in Diagnosis of infectious diseases Direct methods: used to detect the microbe or its components Indirect methods: used to detect antibody response in the host to a microbe Direct methods - Microscopy - Culture - Antigen detection - Detection of genomes Indirect methods - Serology (Ab detection)

18. Choosing the right sample Choosing the appropriate sample is critical Factors: Type of illness Time from appearance of symptoms Typhoid fever Week 1-2 – blood Week 3 - Stool Week 4 - Urine

19. Window period Period between infection and the point when the lab test can identify infection Varies with the test used. (Shorter for direct tests than indirect tests)

20. Choosing the right test Dengue 1-6 days – NS1 antigen 5-15 days – IgM Ab against dengue virus

21. Direct methods

22. (1) Microscopy Light microscopy used for visualization of bacteria, fungi, parasites Staining – enhances contrast Good for rapid presumptive Dx Bacteria - unstained Bacteria stained

23. Microscopy Quick, easy, inexpensive Good for microbes that are difficult to grow – MTB, malarial parasite Viruses – require electron microscopy Bacteria in urine MTB in sputum Malarial parasite in blood

24. (2) Culture Good when the number of microbes in the sample is low Culture media -used for bacteria and fungi Human cell lines – used for some viruses Time – 2 days to 8 weeks Expensive Subsequent tests are required for specific identification Bacteria Poliovirus in human cells Fungi

25. (3) Antigen detection of microbial antigens Detection of microbial proteins (antigens) allows specific identification of microbe Easy, inexpensive and reliable methods available

26. Antigen detection: Immunofluorescence Specific Ab tagged with a fluorochrome Requires fluorescence microscope

27. Antigen detection: ELISA Anti-Target Antibody Enzyme linked immunosorbent assay = ELISA

28. ELISA Principle Anti-Target Antibody Target Protein

29. ELISA Principle Anti-Target Antibody Target Protein Antibody-Enzyme Conjugate

30. Anti-Target Antibody Target Protein Antibody-Enzyme Conjugate Substrate ELISA Principle

31. ELISA-Antigen detection video https://www.youtube.com/watch?v=70TPrfL_8-M

32. (4)Detection of microbial genomes Agarose gel electrophoresis

33. Applications of PCR Highly sensitive (1-10 copies are picked up) Early Dx Can quantitate microbes - virus loads / response to treatment Works on non-cultivable microbes

34. Indirect methods

35. (1)Detection of antibodies produced against microbes Ab detection ELISA Eg. HIV Ab detection ELISA Ab in sample

36. Point-of-care tests

37. Tests performed at the point-of-care (eg. doctor’s office) Can be performed outside a laboratory Does not require special training

38. Point-of-care tests for Ag and Ab detection Principle –similar to ELISA Rapid (< 10 minutes) Available for many infectious agents (eg. HIV, rotavirus..) Antigen Antibody