Laboratory Diagnosis of Infectious Diseases

Laboratory diagnosis Direct Indirect

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

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

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.

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.

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

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.

Nucleic acid amplification techniques (NAAT) Target molecule DNA RNA

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

P C R olymerase hain eaction

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

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

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

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

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

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)

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

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.

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

Time course of HAV infection

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

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 - + +/-

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

Viral Kinetics in HIV/AIDS

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

Rapid Antigen detection

Rapid Antigen detection Respiratory viruses Gastroenteritis viruses....

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