Direct and indirect diagnostic methods in infectious diseases

1. Direct and indirect diagnostic methods in infectious diseases

2. Learning ObjectivesAt the end of this lecture, the student should be able to: • list the main methods in diagnosis of different type of microorganisms • explain the importance of these methods in diagnosis • List the main advantages and disadvantages of each type of test

3. Case I • 6 year old child presented with a 24 hour history of fever, vomiting and complaint of a sore throat

4. Etiology • Strep throat: S. Pyogenes (A group Beta hemolytic streptococci) • Mainly viruses • Rarely: • Corynebacterium • Arcanobacterium • Neisseria gonorrhoeae

5. Laboratory Diagnosis

6. Diagnosis • Rapid antigen test • Culture and susceptibility test ?

7. Case II • Fever • History

8. Case II • Fever • Has a history to visit to west Africa

9. Ebola • Malaria

10. Case • Peripheral blood smear: • Plasmodium • Babesia • Trypanosome • Microfilaria • Bacteria and fungi

11. Case • 1.5 years old patient presenting to the emergency room with 2 hour of history of vomitting, diarrhea, fever • Stool macroscopy: no mucus no blood

12. Stool microscopy • Fecal leucocyte • Culture: Salmonella, Shigella and camphylobacter

14. Fecal leucocyte

16. Fecal leucocyte absent

17. Season • Winter

18. For rapid diagnosis • Antigen detection • Rotavirus

19. Outbreak! • Norovirus antigen

20. Multiplex PCR

21. case • A child • Maculupapular rash

22. case • Maculupapular rash • Viral(measles, rubella, HHV6,parvovirus B19,EBV, Enterovirus,CMV • Others • HIV

23. Laboratory Diagnosis

24. Maculopapular rash • Specific serology: • IgM • Total antibody • Antigen and antibody • NAT

25. Laboratory diagnosis • Direct • Indirect

26. Laboratory diagnosis • Direct: -Microscopy -Culture -Antigen -Nucleic acid • Indirect: -Specific antibody (Serology)

27. Laboratory diagnosis • Direct: -Microscopy -Culture -Antigen -Nucleic acid • Indirect: -Specific antibody (IgG, IgM, IgA)

28. Laboratory diagnosis • Direct: -Microscopy -Culture -Antigen detection -Nucleic acid detection: Nucleic acid amplification techniques (NAT=NAAT) • Indirect: -Specific antibody (IgG, IgM, IgA)

29. What do you see? Which type of microscopy is this?

30. Microscopic Principles and Applications • In general, microscopy is used in microbiology for two basic purposes: 1-the initial detection of microbes 2-the preliminary or definitive identification of microbes.

31. Microscopic Principles and Applications • The microscopic examination of clinical specimens is used to detect: • bacterial cells, • fungal elements, • parasites (eggs, larvae, or adult forms), and • viral inclusions present in infected cells. • But lacks sensitivity !

32. Stains • Because most organisms are colorless and transparent, various dyes (stains) are used to see the individual cells • A variety of different types of stains are used in the microbiology lab, including: • Contrast stains (e.g., methylene blue, lactophenol cotton blue, India ink, iodine) • Differential stains (e.g., Gram stain, spore stains, acid-fast stains, Giemsa stain, silver stains, Trichrome stain) • Fluorescent stains (e.g., acridine orange, auramine-rhodamine, calcofluor white, antibody-conjugated fluorescent stains)

33. Methylene Blue Stain • ?

34. Methylene Blue Stain • Corynebacterium diphtheriae

35. Direct Examination Thesample: • can be mixedwith alkali todissolve background material (potassiumhydroxide [KOH] method) : fungalelements • mixedwith a combination of alkali and a contrastingdye (e.g., lactophenolcottonblue:fungalelements Lugoliodine :parasitologyspecimen

36. ? Lactophenol Cotton Blue (LCB) Stain

37. primarily for observing the morphology of fungal molds : Aspergillus Lactophenol Cotton Blue (LCB) Stain

38. Enterobius vermicularis • Pinworm eggs: • deposited by adults at night in the perianal area. • Eggs are collected by pressing tape on the anal surface • Eggs appear as an embryo surrounded by a colorless shell that is characteristically flattened on one side.

39. ? Iodine Stain

40. The iodine stain is a contrast stain used primarily for the detection of intestinal parasites (Entamoeba coli in this example). Iodine Stain

41. Direct Examination • India ink method, • in which the ink darkens the background rather than the cell. • This method is used to detect capsules surrounding organisms, such as the yeast Cryptococcus(the dye is excluded by the capsule, creating a clear halo around the yeast cell), and • is a rapid method for the preliminary detection and identification of this important fungus.

42. India Ink Stain • ?

43. India Ink Stain • The India ink stain: • negative contrasting stain • Cryptococcus neoformans. The ink is excluded by the fungal capsule so the fungi (arrows) are unstained and surrounded by a clear halo, while the ink particles provide a background contrast. • But now antigen detection is preferred

44. Differential Stains • Gram stain : -bacteria -Yeasts (yeastsare gram-positive).

48. Gram staining Neisseria gonorrhoae detection in uretral specimen from males

49. Differential Stains Acid-Fast Stains Ziehl-Neelsen stain: Used to stain mycobacteria and other acid-fast organisms. Kinyoun stain: Cold acid-fast stain (does not require heating)

50. Updated Guidelines for the Use of Nucleic Acid Amplification Tests in the Diagnosis of Tuberculosis • Conventional tests for laboratory confirmation of TB include • acid-fast bacilli (AFB) smear microscopy(24 hours) • culture • Although rapid and inexpensive, • AFB smear microscopy is limited by its poor sensitivity • (45%–80% with culture-confirmed pulmonary TB cases)