 Direct  Indirect

 Direct: -Microscopy -Culture -Antigen -Nucleic acid  Indirect: -Specific antibody (Serology)

 Direct: -Microscopy -Culture -Antigen -Nucleic acid amplification technique (NAAT=NAT)  Indirect: -Specific antibody (IgG, IgM, IgA)

 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.

 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.

 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

 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.

 Target molecule ◦ DNA ◦ RNA

The advantages of molecular techniques:  their sensitivity  Specificity  safety..  False positivity !!!!!!!!!!!!  False negativity !!!!!!!!!!

P P C C R R olymerase hain eaction

 Real-time PCR  Multiplex  Rapid  For the time being expensive

 Detect  Identify  Quantitate antigen or antibody Disadvantage: Cross reaction -similar or common epitope

 Detect either  Antigen using a known antibody  Antibody using a known antigen

 Prozone reaction: high antibody causes false negative. The sera should be diluted!!

 Immunofluorescence (IFA)  Enzyme-linked immunosorbant assay (ELISA)-EIA -Western blot

 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)

 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

 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.

 Hepatitis viruses  Epstein-Barr virus  HSV type 2  HIV infection......

 Specific serologic tests ◦ anti-HAV IgM by ELISA

Serologic reactivity Disease state Healthy state EarlyEarly acute AcuteChronicLate acute Resolved vaccinat ed Anti-HBcAnti-HBeAnti-HBsHBeAgHBsAg Infectious virus / /

 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

 Anti-HCV with ELISA ◦ Seroconversion within 7 to 31 weeks of infection  HCV RNA with molecular techniques  HCV Antigen

 Respiratory viruses  Gastroenteritis viruses  Central nervous system infection viruses…

 Quality of the specimen  The way its sent  The method used  The interpretation  The dialogue with the lab!!!!