İ. Çağatay Acuner M.D., Clinical Microbiologist , Associate Professor

1. İ. Çağatay Acuner M.D., Clinical Microbiologist, Associate Professor Department of MicrobiologyFaculty of Medicine, Yeditepe University, Istanbul cagatay.acuner@yeditepe.edu.tr YEDİTEPE SCHOOL OF MEDICINE YEDİTEPE UNIVERSITY Laboratory Diagnosis of Infectious Diseases –II(Examplary Methods and Tests)(Modified from the lecture given by Prof. Gülden Çelik, 2015-2016)

2. Laboratory Methods in Clinical Microbiology • Direct: • -Microscopy • -Culture • -Antigen • -Nucleic acid • Indirect: • -Specific antibody (Serology) (IgG, IgM, IgA)

3. Microscopy Two basic purposes: 1-the initial detection of microbes 2-the preliminary or definitive identification of microbes. • 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. • 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. • But lacks sensitivity !

4. Microscopy • Brightfield (light) microscopy • Darkfield microscopy • Phase-contrast microscopy • Fluorescent microscopy • Electron microscopy

5. Microscopy Treponema pallidum in the direct fluorescent antibody test :more sensitive and specific • Darkfield microscopy (Treponema pallidum) • The microscopic detection of organisms stained with antibodies labeled with fluorescent dyes or other markers: • very useful for the direct, sensitive and specific identification of many organisms.

6. Microscopy • Direct Fluorescent Stains (without antibody marker) • 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. • 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.

8. Microscopy • Other Direct Examination Microscopy Methods • 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 • mixed with 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.

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

10. Microscopy • Other Direct Examination Microscopy Methods • The sample: • mixed with India ink • in which the ink darkens the background rather than the cell. • used to detect capsules surrounding organisms, • the yeast Cryptococcus (the dye is excluded by the capsule, creating a clear halo around the yeast cell), • is a rapid method for the preliminary detection and identification of this important fungus.

11. Microscopy • 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)

12. Microscopy • Differential stains • (e.g., Gram stain, spore stains, acid-fast stains, Giemsa stain, silver stains, Trichrome stain) • Gram stain : • -bacteria (positive, negative) • -yeasts (yeasts are gram-positive). • Iron hematoxylin and trichrome stains: • protozoan parasites • Giemsa stain: • blood parasites and other selected organisms

15. Methylene Blue Stain • Corynebacterium diphtheriae

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

17. 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.

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

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

20. 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.

21. Acid-Fast 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) • Mycobacteria • partially acid-fastorganisms such as Nocardia

22. Microscopy • 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). • Organisms that retain this stain are referred to as partially acid-fast.

23. Panels A and B, Cryptosporidia. Panel C, Cyclospora. Panel D, Isospora.

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

25. Silver Stain • Silver stains are primarily used in anatomic pathology labs and not in microbiology labs. • Fungal elements (hyphae [photo] and cells) are stained with silver particles..

26. Fecal leucocyte negative Fecal leucocyte positive

27. Culture (in vitro; on artificial media or living cells) • Anton van Leeuwenhoek : Microscobic observation (1676 ) • Pasteur: culture of bacteria almost 200 years later • Other rapid tests replaced culture methods • microbial antigen detection • nucleic-acid-based assays • But 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.

28. Culture (in vitro; on artificial media or living cells=cell culture) • The success of culture methods is defined by: • the biology of the organism • the site of the infection • the patient's immune response to the infection • the quality of the culture media • Culture media can be subdivided into four general categories: • enriched nonselective media, • selective media, • differential media, and • specialized media • Certain bacteria need special conditions: • Legionella is an important respiratory pathogen; media should be supplemented with iron and L-cysteine. • Campylobacter, an important enteric pathogen, highly selective media should be incubated at 42° C in a microaerophilic atmosphere. • Chlamydia, an important bacterium responsible for sexually transmitted diseases, is an obligate intracellular pathogen that must be grown in living cells.

29. Culture (in vitro; on artificial media or living cells=cell culture) • Some bacteria and all viruses are strict intracellular microbes • They can only grow in living cells. • In 1949, Enders described a technique for cultivating mammalian cells for the isolation of poliovirus. • This technique has been expanded for the growth of most strict intracellular organisms. • Cell culture is not a routine test! • The cell cultures can either be cells • that grow and divide on a surface (i.e., cell monolayer) or • grow suspended in broth. • Some cell cultures are well established and can be maintained indefinitely. • These cultures are commonly commercially available. • Other cell cultures must be prepared immediately before they are infected with the bacteria or viruses and cannot be maintained in the laboratory for more than a few cycles of division (primary cell cultures).

30. SerologicMethods(Immunologictechniques) • Detect • Identify • Quantitate antigen or antibody Disadvantage: Cross reaction -similar or common epitope

31. Serologic, Serodiagnosis,Serology • Detection of antigen or antibody in serum • The term serologic is used also for searching antigen or antibody in mediums other than serum(saliva,urine) • Serologic assay=immunoassay

32. Immunoassays • Antigen or antibody is detected • In a variety of clinical specimens: • Mostly sera • Body fluids(cerebrospinal fluid) • Tissues • Environmental substances

33. Antibodies Polyclonal: • Heterogeneous antibody preparations • Recognizes many epitopes on a single antigen Monoclonal: • Recognize individual epitoses on an antigen

34. Methods of detection Antibody-antigen complexes can be detected: • Directly • Labelling the antibody or the antigen: -enzyme -radioactive -fluorescent dye

35. Classical serologic methods • Precipitation • Immunodiffusion techniques • Agglutination Other serologic methods • Complement fixation • Hemagglutination inhibition • Neutralization

36. Agglutination tests • Clumping of antigen with its antibody • Flocculation: similar to agglutination; except that agglutinats float rather than sediment • Prozone reaction: high antibody causes false negative. The sera should be diluted!! • Antigens passively absorbed on carriers:passive agglutination

37. Agglutination tests • Antigens passively absorbed on carriers:passive agglutination -Red blood cells: passive hemagglutination -gelatin particles: particle agglutination Classical agglutination in test tubes: -Salmonella:Gruber Widal -Brucella:Wright -Rickettsiae:Weil-Felix reaction

38. Agglutination negative

39. Agglutination positive

40. Precipitation • Tubes:solutions • Gels: • Double diffusion-Quchterlony • Radial immunodiffusion • Countercurrent electrophoresis (pyogenic meningitis and fungal infections)

41. Immunoassays • Immunofluorescence (IFA) • Enzyme-linked immunosorbant assay (ELISA) -Western blot • Radioimmunoassay (RIA)

42. Serology • can be used to identify the infecting agent • evaluate the course of an infection, or determine the nature of the infection-whether it is a primary infection or a reinfection, and whether it is acute or chronic. • Serologic testing is used to identify viruses and other agents that are difficult to isolate and grow in the laboratory or that cause diseases that progress slowly

43. In the diagnosis of infectious diseases by immunoassays • Either spesific antigen: • Directly from specimen • From the culture for identification • Specific antibodies are detected: • IgG • IgM • IgA

44. Specific antibody detection • Seroconversion occurs when antibody is produced in response to a primary infection. • IgM: early in infection (2-3 weeks) transient (3-6 months) *sometimes persists longer • IgG: later highest in 4-6 months usually persists during the whole life • IgG avidity: High: past infection Low: new infection