Laboratory Diagnosis of Infectious Diseases

1. Laboratory Diagnosis of Infectious Diseases Prof Dr Gülden Çelik gulden.yilmaz@yeditepe.edu.tr

2. Learning ObjectivesAt the end of this lecture, the student should be able to: • list the main methods in diagnosis of infectious diseases caused by different type of microorganisms • explain the importance of these methods in diagnosis • List the main advantages and disadvantages of each type of test • Ensure the type, storage and transportation of specimen in appropriate tests

3. Laboratory diagnosis • Direct • Indirect

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

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

6. Boy with fever and rash In early June a 15-year old boy comes to your practice with his mother. He had been fine until about five days ago when he developed a fever. He has a stiff neck and a rash on his back. His mother reports that he was playing in the woods with some friends recently.

7. Which of the following bacteria may be the agent Pseudomonas aeruginosa Clostridium perfringens Borrelia burgdorferi Streptococcus pyogenes

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

9. Tick-born disease Borrelia burgdorferi is the causative agent of Lyme disease. This bacterium, just like Treponema pallidum, is a member of the spirochetes, the family of spiral-shaped bacteria.

10. Boy with fever and rash After an incubation period of 3 to 30 days, develop at the site of the tick bite. The lesion (erythema migrans) begins as a small macule or papule and then enlarges over the next few weeks, ultimately covering an area ranging from 5 cm to more than 50 cm in diameter

11. Definition of Lyme Disease • Lyme disease begins as an early localized infection, progresses to an early disseminated stage, and if untreated, can progress to a late manifestation stage.

12. B. burgdorferi organisms are present in low numbers in the skin when erythema migrans develops. This has been shown by culture of the organism from skin lesions or detection of bacterial nucleic acids by polymerase chain reaction (PCR) amplification.

13. Microscopic examination of blood or tissues from patients with Lyme disease is not recommended, because B. burgdorferi is rarely seen in clinical specimens.

14. Lyme Disease • Microscopy • Culture • Nucleic-Acid-Based Tests :65% to 75% with skin biopsies, 50% to 85% with synovial fluid • Antibody Detection : • Spesific IgM: IgM antibodies appear 2 to 4 weeks after the onset of erythema migrans in untreated patients; the levels peak after 6 to 8 weeks of illness and then decline to a normal range after 4 to 6 months. • Spesific IgG

15. 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.

16. 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.

17. Microscopic Principles and Applications • Characteristic morphologic properties can be used for the preliminary identification of -most bacteria and -are used for the definitive identification of many fungi and parasites.

18. Microscopic Methods • Brightfield (light) microscopy • Darkfield microscopy • Phase-contrast microscopy • Fluorescent microscopy • Electron microscopy

19. Darkfield Microscopy • Treponema pallidum (syphilis) • Leptospira spp. (leptospirosis)

20. Direct Examination • The sample: • can be suspended in water or saline (wet mount), • mixed with alkali to dissolve background material (potassium hydroxide [KOH] method) : fungal elements • mixed with a combination of alkali and a contrasting dye (e.g., lactophenol cotton blue: fungal elements Lugol iodine : Iodine is added to wet preparations of parasitology specimens to enhance contrast of internal structures. Facilitates differentiation of protozoa and host white blood cells. • The dyes nonspecifically stain the cellular material, increasing the contrast with the background, and permit examination of the detailed structures.

21. Direct Examination • A variation is the 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.

22. Differential Stains • Differential stains: • Gram stain : -bacteria -Yeasts (yeasts are gram-Ipositive). • Iron hematoxylin and trichromestains:protozoan parasites • Giemsastain: blood parasites and other selected organisms

23. 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)

24. Auramine-rhodamine: Same principle as other acid-fast stains, except that fluorescent dyes (auramine and rhodamine) are used for primary stain • Modified acid-fast stain: Weak decolorizing agent is used with any of three acid-fast stains listed. Whereas mycobacteria are strongly acid-fast, other organisms stain weaker (e.g., Nocardia, Rhodococcus, Tsukamurella, Gordonia, Cryptosporidium, Isospora, Sarcocystis, and Cyclospora). • These organisms can be stained more efficiently by using weak decolorizing agent. Organisms that retain this stain are referred to as partially acid-fast.

25. Microscopic Principles and Applications • The microscopic detection of organisms stained with antibodies labeled with fluorescent dyes or other markers has proved to be very useful for the specific identification of many organisms.

26. Fluorescent Stains • Acridine orange stain: Used for detection of bacteria and fungi in clinical specimens. • Auramine-rhodamine stain: Same as acid-fast stains. • Calcofluor white stain: Used to detect fungal elements and Pneumocystis spp.

27. Direct fluorescent antibody stain • Antibodies (monoclonal or polyclonal) are complexed with fluorescent molecules. Specific binding to an organism is detected by presence of microbial fluorescence. Technique has proved useful for detecting or identifying many organisms (e.g., Streptococcus pyogenes, Bordetella, Francisella, Legionella, Chlamydia, Pneumocystis, Cryptosporidium, Giardia, influenza virus, herpes simplex virus). • Sensitivity and specificity of the test are determined by the number of organisms present in the test sample and quality of antibodies used in reagents.

28. 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)

29. Methylene Blue Stain • ?

30. Methylene Blue Stain • Corynebacterium diphtheriae

31. ? Lactophenol Cotton Blue (LCB) Stain

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

33. India Ink Stain • ?

34. 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.

35. ? Iodine Stain

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

37. Gram Stain • ?

38. Gram Stain • gram-positive (purple) • from gram-negative (red) bacteria.

39. ?

40. Staphylococcus aureus and Candida albicans • S. aureus(black arrow) and • yeasts, in this case Candida albicans(red arrow). • Yeast can appear as gram-positive, although they tend to decolorize readily.

41. ?

42. Candida Species • Candida albicans: yeast cells and pseudohyphae. • Candida glabrata. These are smaller than other yeasts; they also do not form pseudohyphae. This is a common cause of urinary tract infections and is the second most common cause of fungemia.

43. Acid-Fast Stains

44. Acid-Fast Stains • Mycobacteria • If a weak decolorizing solution is used to remove the primary stain, then partially acid-fast organisms such as Nocardia

45. Giemsa Stain • differential stain used for detection of parasites in blood smears

46. Giemsa Stain • Plasmodium

47. Silver Stain • Silver stains are primarily used in anatomic pathology labs and not in microbiology labs.

48. Silver Stain • Fungal elements (hyphae [photo] and cells) are stained with silver particles..

49. In Vitro Culture: Principles and Applications • Anton van Leeuwenhoek : Microscobic observation (1676 ) • Pasteur: culture of bacteria almost 200 years later • Over the years, microbiologists and cooks have returned to the kitchen to create hundreds of culture media that are now routinely used in all clinical microbiology laboratories.

50. In Vitro Culture: Principles and Applications • Although tests that rapidly detect microbial antigens and nucleic-acid-based molecular assays have replaced culture methods for the detection of many organisms, • the ability to grow microbes in the laboratory remains an important procedure in all clinical labs. • For many diseases, the ability to grow a specific organism from the site of infection is the definitive method to identify the cause of the infection.