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 different types of infections caused by microorganisms • explain the importance of these methods in diagnosis • List the main advantages and disadvantages of each type of test

3. Methods in Laboratory diagnosis of infectious diseases • Direct • Indirect

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

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

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

7. Determinating the value of tests • Sensitivity • Specificity • Positive predictive values • Negative predictive values

8. Sensitivity • Analytical or epidemiologic • Analytical sensitivity refers to the ability of a test to detect very small quantities of antibodies as occur during seroconversion • Epidemiologic sensitivity(clinical sensitivity): refers to the ability of a test to detect persons with established infection.

9. Sensitivity True positives • Sensitivity = X 100 True positives + False negatives

10. Specificity True negatives • Specificity = X 100 True negatives+ False positives

11. Positive predictive value True positives • PPV = X 100 True positives + False positives

12. Negative predictive value True negatives • NPV = X 100 True negatives+ False negatives

13. Presence of disease Number of people with + test Number of people with - test Total Sick TP FN TP+FN Healthy FP TN FP+TN Total TP+FP FN + TN TP + FP + FN + TN DiagnosticSensitivity= [TP/(TP+FN)] x 100 (Positivityin sickpeople) Diagnosticspesifity= [TN/(TN+FP)] x 100 (Negativity in healtypeople) Positive predictive value= [TP/(TP+FP)] x 100 Negative predictive value= [TN/(FN+TN)] x 100

14. PREVALANCE : % 0.02 Presence of disease Number of people with + test Number of people with - test Total Sick 199 1 200 Healthy 2000 997.800 999.800 Total 2199 997.801 1.000.000 DiagnosticSensitivity= % 99.8 (Positivityin sickpeople) Diagnosticspesifity= = % 99.8 (Negativity in healtypeople) Positive predictive value= [199/(199+2000)] x 100 = % 9 Negative predictive value= [997.800/(1+997.801)] x 100 = %100

16. 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.

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

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

19. 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.

20. 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

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

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

23. B. burgdorferi • present in low numbers in the skin So: • culture of the organism from skin lesions • detection of bacterial nucleic acids by polymerase chain reaction (PCR) amplification • is not prefered

24. 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

25. In every infectious disease • The tests to be used • The clinical material • should be properly selected !

26. 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.

27. 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 !

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

29. 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)

30. Methylene Blue Stain • ?

31. Methylene Blue Stain • Corynebacterium diphtheriae

32. 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

33. ? Lactophenol Cotton Blue (LCB) Stain

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

35. 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.

36. ? Iodine Stain

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

38. 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.

39. India Ink Stain • ?

40. 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

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

45. Gram staining • Neisseria gonorrhoae detection in uretral specimen from males

46. 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)

47. 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)

48. Acid-Fast Stains

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

50. 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.