Diagnostic Virology

In my last post for MICR3350, I wrote about methods used to diagnose bacteria. In this post, I'll be talking about methods used to diagnose viruses!

Background information

This lecture started with some background information, but nothing too complicated. Pretty much all you need to know is that viruses need a host cell in order to grow and replicate. You also need to know about antibodies, which I've described in detail here.

Microscopy

Viruses are small, so they can pretty much only be viewed with the electron microscope. Pretty much the only advantage of using microscopy for viruses is that you get pretty pictures. There are a lot of downsides: specialised, expensive equipment is required, it is not very sensitive (i.e. if you only have a few viral particles, you might not see them on your particular slide), it is not very specific (several viruses look similar and it is difficult to tell them apart), and it is time-consuming.

Culture and susceptibility testing

As viruses can only be grown in host cells, tissue culture is required to culture viruses. Tissue culture can also show something called the "cytopathic effect," or CPE, in which you can see cells that have been infected and killed by viruses. CPE can be used in order to perform phenotypic susceptibility testing (i.e. test the susceptibility of an antiviral by seeing how the number of viral particles decreases etc.).

One good thing about viral culture is that you grow a lot of virus, which is helpful if you intend to study it later. Viral culture is also good when you don't know too much about the virus that you're studying, as some of the later techniques require specific knowledge about your virus of interest. However, tissue culture comes with many disadvantages: it is slow (viruses take longer to replicate than bacteria), labour-intensive (cannot be readily automated), requires special equipment and has low sensitivity and specificity. Additionally, not all viruses can be readily grown in tissue culture.

Direct immunological detection

There are two main methods used in immunological detection. In immunofluorescence, an antibody labelled with a fluorescent tag binds to the protein of interest. In indirect immunohistochemistry, the primary antibody binds to the protein, and a secondary antibody binds to the primary antibody. (Actually, I think that immunofluorescence often uses secondary antibodies too.) The main difference between immunofluorescence and immunohistochemistry is that in the latter, the secondary antibody is connected to some kind of enzyme (rather than a fluorescent tag). The enzyme can catalyse a reaction that forms a coloured product.

Immunological detection has a similar sensitivity to culture, but it is more rapid and specific. Unfortunately, it can be difficult to develop tests, as creating antibodies that bind to the protein of interest is not always easy. Immunological detection also requires access to host cells (which may require a biopsy in some instances), unless the protein of interest is a secreted antigen.

Serology

Serology pretty much involves looking at antibodies in the patient's blood. The main technique used is ELISA, or Enzyme-Linked ImmunoSorbent Assay (also known as "enzyme immunoassay," or EIA). In an indirect ELISA, an antigen is attached to the bottom of a petri dish (or whatever setup you're using). The patient's serum is added, followed by secondary antibodies that bind to any primary antibodies in the patient's serum. The secondary antibodies are attached to an enzyme that can catalyse a reaction forming a coloured product (like in immunohistochemistry). In a sandwich ELISA, a "capture antibody" is attached to the bottom of the petri dish, and then the patient's serum is added. Any antigen present in the patient's serum will bind to the "capture antibody." More primary antibodies are added, as well as secondary antibodies. Sandwich ELISA is more specific than indirect ELISA.

There are many other serological tests that can be performed. The haemagglutination inhibition test tests for antibodies against haemagglutinin (the H spikes on influenza). The neutralisation test, which requires cell culture, is a test for neutralising antibodies.

HIV can be tested by using a Western Blot, where HIV is separated into its component proteins. These proteins can then be reacted with a patient's blood sample. Enzymes can be used to detect antibodies. (See here for more information.)

Several tests can be performed for hepatitis B. Hepatitis B serology may look for surface antigens, surface antibody, total antibody, core IgM, e antigen and e antibody. Both surface antigen and e antigen are secreted antigens. e antigen/antibody concentrations can be used to track progress: e antigen is more common when viral load is high (i.e. the amount of antigen overwhelms the amount of antibody), and e antibody is more common when viral load is low. Vaccinated individuals will have high amounts of surface antibody (but they shouldn't have any other antigen or antibody to a component of Hep B, unless they have been infected).

Serology has its advantages: it is highly sensitive and specific, automatic and fast. IgG antibodies formed against viral antigens are often detectable for long periods of time (often life) after infection, which is good for immunity testing. However, like all of the other tests, it also has its disadvantages. As serology also relies on creating antibodies, it is difficult to create new tests. Also, as many serological tests rely on the detection of antibodies, there are some restrictions with regards to when antibodies are formed: usually it takes a few days for antibodies to form to an illness. Before then, you'll probably get a false negative result.

Nucleic acid tests

Nucleic acid tests, as the name suggests, directly tests for the nucleic acids that make up viruses. There are several different techniques. In FISH (Fluorescence In Situ Hybridisation), a fluorescent probe that is complementary to the sequence of interest is used. Fluorescent antibodies that bind to the probe DNA may also be used. Other techniques include PCR (including real-time PCR) and sequencing (such as Sanger sequencing). Advantages include high sensitivity and specificity, high throughput (i.e. you can do a lot of tests at once), automaticity and speed. Unlike serology, new tests can be easily developed (provided that you know the target nucleic acid sequence) and nucleic acids can be detected at the time of symptom onset. Disadvantages include the need for specialised equipment. Nucleic acid tests also run the risk of being too sensitive- they might pick up on a tiny, clinically insignificant number of particles.