This document discusses methods for diagnosing viral infections. It describes direct examination methods like antigen detection through immunofluorescence or ELISA, viral genome detection by PCR or hybridization, and electron microscopy. Indirect methods include culturing viruses in cell cultures to detect cytopathic effects or hemadsorption, and performing serology to detect antibodies through techniques like ELISA, complement fixation, or hemagglutination inhibition. Both direct and indirect methods have advantages and limitations for viral detection and diagnosis.
This document discusses methods for diagnosing viral infections. It describes direct examination methods like antigen detection through immunofluorescence or ELISA, viral genome detection by PCR or hybridization, and electron microscopy. Indirect methods include culturing viruses in cell cultures to detect cytopathic effects or hemadsorption, and performing serology to detect antibodies through techniques like ELISA, complement fixation, or hemagglutination inhibition. Both direct and indirect methods have advantages and limitations for viral detection and diagnosis.
This document discusses methods for diagnosing viral infections. It describes direct examination methods like antigen detection through immunofluorescence or ELISA, viral genome detection by PCR or hybridization, and electron microscopy. Indirect methods include culturing viruses in cell cultures to detect cytopathic effects or hemadsorption, and performing serology to detect antibodies through techniques like ELISA, complement fixation, or hemagglutination inhibition. Both direct and indirect methods have advantages and limitations for viral detection and diagnosis.
This document discusses methods for diagnosing viral infections. It describes direct examination methods like antigen detection through immunofluorescence or ELISA, viral genome detection by PCR or hybridization, and electron microscopy. Indirect methods include culturing viruses in cell cultures to detect cytopathic effects or hemadsorption, and performing serology to detect antibodies through techniques like ELISA, complement fixation, or hemagglutination inhibition. Both direct and indirect methods have advantages and limitations for viral detection and diagnosis.
Fluorophore Diagnostics of viral diseases Direct methods 96 well plate
Serology Enzyme Linked Immunosorbent Assay (ELISA). Complement fixation test Procedure Looking for antigens
No hemolysis
Ab , complement, and sRBCs are externally added
Patient serum Positive results
No antigen
Patient serum Negative results
Complement fixation test
No hemolysis Hemolysis
+ _ _ _
+ + + _ Diagnostics of viral diseases
Direct methods Electronmicrographs
Electron Microscopy • 106 virus particles per ml required for visualization. • 50,000 - 60,000 magnification normally used. • Viruses may be detected in the following specimens. • Faeces: Rotavirus, Adenovirus, Norwalk like viruses, Astrovirus, Calicivirus • Vesicle Fluid: HSV, VZV • Skin scrapings: papillomavirus, molluscum contagiosum Diagnostics of viral diseases
Direct methods Electron Microscopy Problems with Electron Microscopy • Expensive equipment • Expensive maintenance Cylindrical (Mumps virus )
• Require experienced observer
Icosahedral (poliovirus) Diagnostics of viral diseases
Direct methods Molecular Methods • Methods based on the detection of viral genome. • By Polymerase Chain Reaction (PCR) • However in practice, although the use of these methods is indeed increasing, the role played by molecular methods in a routine diagnostic virus laboratory is still small compared to conventional methods.
Advantages of PCR: Disadvantages of PCR
• Extremely high sensitivity, may detect – Extremely liable to contamination. down to one viral genome per sample – High degree of operator skill required. volume. – Not easy to set up a quantitative assay. • Easy to set up. • Fast turnaround time Diagnostics of viral diseases
Hemagglutination inhibition test Animals disease or death Diagnostics of viral diseases
Indirect methods Cell Culture Are used for virus isolation. However, they are very expensive and it is often difficult to obtain a reliable supply. Problems with cell culture • Long period (up to 4 weeks) required for result. • Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen. • Susceptible to bacterial contamination. • Susceptible to toxic substances which may be present in the specimen. Hemadsorption • To detect the presence of certain viruses, the hemadsorption test is commonly used. • Influenza and parainfluenza viruses express a viral hemagglutinin on the surface of infected cells. • By the hemadsorption test, the culture medium is removed and replaced with a 0.5% dilute solution of guinea-pig red blood cells. Culture cell
Hemadsorption inhibition
Patient serum with
suspected Influenza Cultured cells Red Blood Cells infection
= No hemadsorption = Positive infection
Adsorbed RBCs on the culture cell Diagnostics of viral diseases
Indirect methods Serology Detection of antibodies against the virus. Criteria for diagnosing primary infection • 4 fold or more increase in titer of IgG or total antibody between acute and convalescent sera • Presence of IgM • Seroconversion Criteria for diagnosing reinfection • fold or more increase in titer of IgG or total antibody between acute and convalescent sera • Absence or slight increase in IgM Diagnostics of viral diseases
Serology
Note that during reinfection, IgM may be absent or present at a low level transiently Diagnostics of viral diseases Indirect methods Serology Direct ELISA
Indirect ELISA Haemagglutination inhibition test Haemagglutination inhibition test Diagnostics of viral diseases
Indirect methods Serology Problems with Serology: • Long period of time required for diagnosis for paired acute and convalescent sera. • Mild local infections such may not produce a detectable Abs. • Immunocompromised patients often give a reduced or absent Abs. • Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result. • Patients given blood or blood products may give a false positive result due to the transfer of antibody
