7.3 Case Studies

7.3.1 Influenza A virus

This is the only negative sense retrovirus in the A, B, C, and D family of influenza viruses.

Figure 7.7: Schematic on the Influenza A Virus

This is also the most virulent virus and the fastest to mutate. It now infects other animals.

Several serotypes have been based on antibody responses to these viruses. Some of the virus’ surface proteins include:

1. Hemagglutinin (HA)

   For attachment and fusion

2. Neuraminidase (NA)

   For virus release

7.3.2 Diagnosing influenza A

There are several ways:

1. Isolating the virus in a cell culture or a chicken embryo

   This is the gold standard - after culturing the virus, the virus in then verified by the cytopathic effect or a haemagglutination test.

   However, this test cannot tell influenza A and B viruses apart.

2. RT-PCR

   This gives a yes / no answer on the type of virus and can be used to tell influenza A and B viruses apart.

3. Real time RT-PCR

   This is a quantitative, more reliable and sensitive version of RT-PCR as it uses a fluorescent probe.

4. Rapid tests

   This has low sensitivity - lateral flow immunoassay can also differentiate between influenza A and B viruses.

   Some more examples of rapid tests include chromatographic immunoassay; tests can also be as quick as 15 minutes.

It is worth noting that the outcome of the diagnosis also depends on the age of the patient and the sample collection time.

7.3.3 SARS-CoV-2 diagnosis

One can target the E and the N regions and RNA polymerase for viral gene detection via RT-PCR. This is the most reliable technique.

Human antibody detection works for:

1. IgG / IgM
2. Those who have been infected for at least two weeks
3. Possible for uses
4. Late detection and cross reactivity are the main issues.

Viral antigen detection allows for an earlier detection for infection. Some other techniques include an electrical probe and RT-LAMP.

7.3.3.1 USA CDC Real time RT-PCR for SARS-Cov-2

Their approach uses oligonucleotide primers and dual labelled probes that finds virus nucleocapsid genes N1 and N2. An additional probe also detects human RNase P gene; the test also has a positive control, a negative control, and a no template control.

Figure 7.8: Amplification Plot of Test

The sensitivity of this test is around 0.3 - 1 RNA copies / microliter, and its specificity is confirmed by comprehensive testing.

7.3.4 Lab Diagnosis for malaria

Figure 7.9: Summary of Lab Diagnosis Techniques for Malaria

There are currently three ways of finding malaria in a patient:

1. Microscopy

   A thick and thin blood smear study is the gold standard for confirming infection, identifying malaria species, and quantifying parasitic forms.

2. Immunological techniques

   These can either be antibody based or antigen based.

3. Molecular techniques

   PCR techniques can be used for parasite DNA.

7.3.5 Dengue Virus

The dengue virus is a flavivirus and has four different subtypes that have a 60% - 80% homology with one another with major differences in their surface proteins.

One can develop lifelong immunity against a serotype, but it only gives a short time, cross-protecting immunity against other types.

Secondary infections of the dengue virus can cause severe diseases - this is called Antibody-Dependent Enhancement.

7.3.5.1 Tests for diagnosis dengue

Figure 7.10: Flowchart for Confirming Dengue Infections

1. Isolating the virus

   Clinical samples are cultured in a variety of mosquito cell lines or in live mosquitoes before being used in an immunofluorescence analysis.
   This step can take days to weeks.

2. Immunological tests

   These can be used against the viral secretive protein NS1. IgG and IgM capture ELISA and other test (e.g., western blots) can also work.

3. Nucleic acid based tests

   RT-PCR or real time RT-PCR can work. However, the people performing the PCR need to be trained and provided with the appropriate tools.