Hello everyone, I’m sure everyone is happy to have completed the lab practical this afternoon! This week we discussed viruses and basic methods for detecting viruses in patient specimens. I will briefly discuss each method of viral detection below.
Cytological and Histological Exam
During a cytological exam, the tissue samples are stained with any of a variety of stains such as PAP, Giemsa, or H & E. The tissue samples are then examined for synctia (multinucleated cells) and nuclear and cytoplasmic inclusions, which are clusters of viruses or viral materials. This method is less sensitive than the culture method but safer and easier than culturing dangerous viruses.
Electron Microscopy
Electron microscopy allows for visualization of the actual viruses. This technique is used more in research laboratories than clinical laboratories. Electron microscopy can be useful in detecting viruses that can’t be grown using culture techniques. This method however is labor intensive and relatively insensitive compared to other methods.
Direct Detection of Viral Ag
Direct detection of viral antigen involves detection of antigens in specimens or after viruses has grown in cell culture. Antigen detection can be accomplished by either direct fluorescence antibody detection methods which utilize fluorescently labeled antibodies that bind directly to viral antigens in the specimen or indirect fluorescence methods that involves direct labeled anti-human globulin against antibodies attached to corresponding viral antigens in a specimen. Fluorescence is then measured and observed using a fluorescence microscope.
NA Probes
Nucleic acid probes can be useful in viral detection when the amount of virus is abundant. This technique can be used when culture is not possible or slow.
Conventional Cell Cultures and Shell Vial Method
The conventional cell culture method is the gold standard for viral detection. Viruses require cells in order to survive and replicate, so culture by conventional methods is not possible. A variety of cell lines including primary, low passage, and continuous cell lines are used to facilitate the growth of viruses. Cultures are then examined for cytopathetic effect (morphological changes to the cells0 using a microscope or tested for hemadsorption/hemagglutination. The Shell Vial technique is a combination of the conventional cell culture method and fluorescent antibody labeling technique.
Serological Testing
When viruses can not be grown using culture techniques and direct antigen detection is not possible, antibody detection can be performed. Detection of IgM antibodies to viral antigens indicates an acute stage of infection while detection of IgG antibodies to viral antigens indicates immunity to the virus.
The following article discusses the discovery of a new Ebola virus species and discusses some of the viral detection techniques we discussed in class. Enjoy!
Liz, I loved what you had to say but wanted to elaborate on what you said about Nucleic Acid Probes with what I found in the text book.
ReplyDelete"Nuclic acid detection can be acomlished using nucleic acid probes, which are short segments of DNA that hybridize with complementary viral DNA or RNA segments. the probe is labeled with a tag (fluorescent, chromogenic, or radioactive) that allows detection if hybridization occurs. Nucleic acid probes are most useful when the amount of virus is relatively abundant, viral culture is slow or not possible, and immunoassays lack sensitivity or specificty. DNA target fragments that are too few in number in the oriinal specimen to be detected by probes can be amplified using molecular techniques such as PCR."
This technique was covered in our molecular diagnostics course in the spring.