1. Laboratory Diagnosis of Infectious Diseases

2. Laboratory diagnosis
Direct
Indirect

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

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

5. 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.

6. 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.

7. 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.
Culture is followed with antibiotic susceptibility test except throat culture

8. Certain bacteria need special conditions:
Legionella is an important respiratory pathogen; however, it was never grown in culture until it was recognized that recovery of the organism required using media supplemented with iron and l-cysteine.
Campylobacter, an important enteric pathogen, was not recovered in stool specimens until highly selective media were 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.

9. Nucleic acid amplification techniques (NAAT)
Target molecule
DNA
RNA

10. Molecular Diagnosis The advantages of molecular techniques:
their sensitivity
Specificity
safety..
False positivity !!!!!!!!!!!!
False negativity !!!!!!!!!!

11. P C R
olymerase
hain
eaction

14. NAAT
Real-time PCR
Multiplex
Rapid
For the time being expensive

15. Serologic Methods (Immunologic techniques)
Detect
Identify
Quantitate antigen or antibody
Disadvantage: Cross reaction
-similar or common epitope

16. Serologic methods Detect either Antigen using a known antibody
Antibody using a known antigen

17. Agglutination tests
Prozone reaction: high antibody causes false negative. The sera should be diluted!!

18. Immunoassays Immunofluorescence (IFA)
Enzyme-linked immunosorbant assay (ELISA)-EIA
-Western blot

19. Examples of Viruses Diagnosed by Serology
Epstein-Barr virus
Rubella virus, Measles,Mumps;Parvovirus
Hepatitis A, B, C, D, and E viruses
Human immunodeficiency virus
Human T-cell leukemia virus
Arboviruses (encephalitis viruses)

20. 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

21. Serology is used to determine the time course of an infection
Serology is used to determine the time course of an infection. Seroconversion occurs when antibody is produced in response to a primary infection.
Specific IgM antibody, found during the first 2 to 3 weeks of a primary infection, is a good indicator of a recent primary infection. Usually lasts for 3-6 months
Specific IgG usually lasts for lifetime. Usually shows immunity except in latent viruses.

22. Diagnosis and course of infection
Hepatitis viruses
Epstein-Barr virus
HSV type 2
HIV infection......

23. Time course of HAV infection

24. Hepatitis A virus/Laboratory diagnosis
Specific serologic tests
anti-HAV IgM by ELISA

28. Disease state Healthy state
Hepatitis B virus/Laboratory diagnosis Interpretation of serologic markers of hepatitis B virus infection
Serologic reactivity
Disease state Healthy state
Early
Early acute
Acute
Chronic
Late acute
Resolved
vaccinated
Anti-HBc
Anti-HBe
Anti-HBs
HBeAg
HBsAg
Infectious virus - +
+/-

29. EBV
Heterophile antibody: results from nonspecific activation of B cells by EBV
IgM antibody recognizes Paul-Bunnell antigen on sheep, horse and bovine erythrocytes not on guinea pig kidney cells
Detected at the end of first week , lasts for several months
Monotest, ELISA: specific antibodies
VCA-IgM, antibody to early antigen (EA): recent infection
VCA-IgG, EBNA: previous infection

30. Viral Kinetics in HIV/AIDS

31. Hepatitis C virus/Laboratory diagnosis
Anti-HCV with ELISA
Seroconversion within 7 to 31 weeks of infection
HCV RNA with molecular techniques
HCV Antigen

32. Rapid Antigen detection

33. Rapid Antigen detection
Respiratory viruses
Gastroenteritis viruses....

34. The success of the Microbiology laboratory
Quality of the specimen
The way its sent
The method used
The interpretation