Virology/Serology/Immunology

Viruses are some of the most interesting and sophisticated things on the planet! The HIV virus consists of less than 10,000 base pairs of DNA. If you think
about all the damage and trouble it has caused our population, it is truly remarkable.
Of the vast amount of viruses in the world, there are only a few which are critical to understand for the clinical laboratory scientist.
Hepatitis B:
Hepatitis B antigens and antibodies are frequently tested on and important to understand. There are two antigens to be aware of, hepatitis B surface antigen,
and hepatitis e antigen. There are three antibodies to be aware of, hepatitis B surface antibody, hepatitis B core antibody, and hepatitis B e antibody. Each
of these has an abbreviation which can be seen in the table below.

Name Abbreviation Alternative Name

Hepatitis B surface antigen HBsAg
Hepatitis B e antigen HBeAg
Hepatitis B surface antibody Anti-HBs HBsAb
Hepatitis B e antibody Anti-HBe HBeAb
Hepatitis B core antibody (IgM) Anti-HBc (IgM) HBcAb (IgM)
Hepatitis B core antibody (IgG + IgM) Anti-HBc HBcAb

Watch out for alternative antibody names! They only differ from the name of the antigen by one letter and can cause confusion or mistakes.
You’ll notice there is an IgM and an IgG core antibody. The presence of IgM anti-HBc generally indicates an acute HBV infection. IgM anti-HBc is the
first antibody to appear when fighting a HBV infection. The presence of IgG anti-HBc generally indicates a chronic HBV infection. Once infected with
HBV, IgG anti-HBc will generally persist for life. Oftentimes total hepatitis B core antibody will be referenced because the two tests commonly run in the
clinical lab are total hepatitis B core antibody and IgM anti-HBc. Total hepatitis B core antibody (anti-HBc) simply means IgM and IgG anti-HBc.

With all of these different antigens and antibodies there are a number of different scenarios possible. The presence/absence of individual antigens or
antibodies indicate what is going on with the patient. The table below outlines commonly seen scenarios.

Result Interpretation
HBsAg (-)
Anti-HBs (-) Patient is susceptible to HBV
Anti-HBc (IgG and IgM) (-)
HBsAg (-)
Anti-HBs (+) Immunity from a natural infection
Anti-HBc (IgG and IgM) (+)
HBsAg (-)
Anti-HBs (+) Immunity from a hepatitis B vaccination
Anti-HBc (IgG and IgM) (-)
HBsAg (+)
Anti-HBs (-)
Acute HBV infection
Anti-HBc (IgG and IgM) (+)
Anti-HBc (IgM) (+)
HBsAg (+)
Anti-HBs (-)
Chronic HBV infection
Anti-HBc IgG (+)
Anti-HBc IgM (-)
The important thing with HBV is being able to think your way through results. There are cases where results may appear discrepant but let’s start with
general rules and the most common cases and then we’ll break down results that are less frequent.

General rules:

1. If a person is negative for HBsAg, they have either not been exposed to the virus or have immunity (natural or from a vaccination).
2. If a person is negative for HBsAg and positive for anti-HBc, the immunity is from a natural infection.
3. If a person is negative for HBsAg and positive for anti-HBs but negative for anti-HBc the immunity is from a vaccination.
4. One type of HBV vaccines are proteins of the HBsAg produced by yeast cells, so building an antibody to the “core” of HBV can only come from natural
infection. The vaccine will cause you to build an antibody to HBsAg.
5. In order for a person to have a current infection (acute or chronic) they must be positive for hepatitis B surface antigen (HBsAg).
6. If a person is positive for HBsAg and positive for anti-HBc (IgM) the HBV infection is acute.
7. If a person is positive for HBsAg and negative for anti-HBc (IgM) the HBV infection is chronic.
These rules are just general, and as always there are exceptions to the rules. For example take a look at the results in the table below.

Result
HBsAg (-)
Anti-HBs (-)
Anti-HBc (+)

If we followed the above rules we would assume the immunity is from a natural infection, however, in the larger table above, natural immunity will also be
anti-HBs (+). So what could be happening here?
There are four possible scenarios:
1. A person who had HBV in the past and resolved the infection may have very low levels or decreasing levels of anti-HBs. HBsAg is negative because the
infection was cleared and anti-HBc (most likely IgG) remains for life.
2. The person may have a low level chronic infection and HBsAg levels cannot be detected by the test.
3. The anti-HBc could be a false positive meaning the person is actually susceptible.
4. A person may be recovering from an HBV infection. During the recovery period anti-HBs will not appear for a few weeks after HBsAg has been
cleared. It is possible for both HBsAg and anti-HBs to be negative during recovery. Some will refer to this as the window period in acute infection.

HBeAg and anti-HBe:

In acute infections HBeAg appears slightly after HBsAg and disappears shortly before HBsAg in recovering patients. HBeAg can also be present in
chronic HBV infections and its presence is indicative of an active infection.
Anti-HBe appear after clearance of HBV and indicate recovery. Anti-HBe can persist and provide immunity for years.

HBV DNA:
HBV DNA is commonly measured using rt-PCR and other methods. It’s another useful tool for elucidating what is happening in a patient. HBV DNA can
be detected before HBsAg which can be valuable for blood banking to detect HBV in the blood supply; the HBV DNA viral load can be measured to
monitor chronically infected patients or determine how well a patient is responding to therapy; HBV DNA can also be used to detect variants and mutations
where serologic tests come back negative.

Other rare cases of HBV:

Due to genetic complexity there are all kinds of HBV types with all sorts of properties. Some HBV can evade the HBV vaccine and are referred to as
“escape mutants.” HBV is also known to colonize the liver and be generally undetected by current clinical lab tests. This is referred to as “occult HBV.” If
you are interested in learning more, I encourage you to take a deeper dive into the world of HBV because there isn’t a cure yet, more research needs to be
done!

HIV:
HIV is screened for using an enzyme immunoassay (EIA) using an antibody to HIV-1 and an antibody to HIV-2. HIV-1 and HIV-2 are the two main types
of HIV. They are frequently screened for together, however confirmation is done using a Western blot. Two out of the following three antigens (p24, gp41,
or gp120/gp160) must be present to call a test positive.
Hypersensitivity
Hypersensitivity
The body’s immune system is not a perfect system. Oftentimes, it will attack itself. There are many different scenarios and events that can cause a person’s
immune system to attack its own cells or tissue. The term hypersensitivity is used to describe heightened states of immune responsiveness, and it breaks
down into four main types (types 1-4).
Type 1:
Type 1 hypersensitivity is generally caused by cell bound antibody reacting with free antigen. Good examples are pollen and peanuts. Although very
complex, in its simplest form, a small amount of antigen causes a large amount of IgE to be produced. That IgE will then bind to mast cells, and when re-
exposed to the same antigen, the mast cells degranulate and release large amounts of histamine and other products. Conditions most commonly associated
with type 1 hypersensitivity are: anaphylaxis, food allergies, pollen or plant allergies, and asthma.
An old test to be aware of, radioallergosorbent test (RAST) was the original test used to detect specific IgE types. RAST used a radioactive label which has
been replaced by newer methods although the principles remain the same.

Type 2:
Type 2 hypersensitivity involves free antibody (IgG and IgM) binding to antigens on cell surfaces leading to cell death via opsonization or the complement
system. The best example of this are transfusion reactions. Hemolytic disease of the newborn (HDN) and autoimmune hemolytic anemia will also fall into
this category.

Type 3:
Type 3 hypersensitivity also involves antigen-antibody complexes. The antigens in type 3 are soluble, meaning when they bind to antibody they can
precipitate out of the serum and sometimes end up in tissue. The complexes can activate complement and other processes and cause tissue damage.
Examples of type 3 hypersensitivity are systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and serum sickness.

Type 4:
Type 4 hypersensitivity is also known as delayed hypersensitivity because of the involvement of T-cells. In type 4, contact with an antigen will induce a
sensitization phase (1-2 weeks) where T-cells are presented with the antigen. Upon re-exposure to the same antigen, T-cells activate and create an
inflammatory response 4-6 hours after contact and peaking 2-3 days after contact. Example of type 4 hypersensitivity are tuberculosis, leprosy, graph vs
host disease, and poison oak.

Nerdy Note
The rash from poison oak and poison ivy is caused by the oil urishiol. Urishiol is a small molecule that binds to skin cell protein and changes its shape. The
shape change causes it to be engulfed by an antigen presenting cell called a Langerhans cell. This causes a T-cell activation which leads to an upregulation
in cytokines and other products that cause the rash.

Study Tip
Using the phrase “All Dogs Can Dance” is an easy way to remember the different types of hypersensitivity. Using the first letter from each word (ADCD),
can help you remember properties of the different types of hypersensitivity.

A = Anaphylactic
D = Cell Death
C = Complexes
D = Delayed
Anti-nuclear antibodies (ANA)
Anti-nuclear antibodies (ANA):

Anti-nuclear antibodies are autoimmune antibodies that attack a person’s own system. In biology it always seems as if every iteration exists somewhere,
and in the case of antinuclear antibodies, no part of the nucleus is off limits for an attack.

The most common condition associated with anti-nuclear antibody testing is systemic lupus erythematosus (SLE). Different conditions associate with
specific autoantibodies, and SLE is associated with anti-double stranded DNA (anti-ds DNA).

The screening test of choice for SLE and many other autoimmune conditions is fluorescent nuclear antibody testing (FANA). In FANA, patient serum is
reacted with a reagent epithelial cell line (HEp-2 is a common cell line used). After washing away excess unbound antibody from the patient serum,
antihuman globulin with a fluorescent tag is added. The sample is then viewed under the fluorescent scope. FANA testing is very sensitive but not highly
specific. For example, FANA testing will miss cases of SLE as well as call about 2% of people with no SLE positive.

There is another less frequently used SLE test to be aware of that involves the parasite Crithidia luciliae. Crithidia luciliae contains a structure called a
kinetoplast which is a mitochondrial structure that houses DNA. In the case of Crithidia luciliae it houses a lot of circular ds-DNA. A similar fluorescent
test as the one outlined above can be used with Crithidia luciliae. This test is more specific but less sensitive than FANA.

Nerdy note
The most common host of Crithidia luciliae is the house fly, so next time you’re swinging at one with your fly swatter, remember there may be Crithidia
luciliae inside!

There are a few specific ANA patterns and associations the clinical lab scientist needs to be aware of, they are outlined in the table below.

ANA Pattern Autoantibody Disease Association

Homogenous/Peripheral Rim Anti-ds-DNA SLE
Homogenous Anti-histone Drug induced lupus
Speckled Anti-Smith (Anti-Sm) SLE
Speckled Anti-SS-A, Anti-SS-B SLE and Sjogren’s Syndrome
Speckled Anti-Scl-70 Scleroderma
Speckled Anti-centromere CREST syndrome

A few quick notes. Anti-Sm antibodies are antibodies to extractable nuclear antigens associated with uridine-rich RNA. Anti-Sm antibodies are specific for
SLE but are only found in a small percentage of people with SLE.

SS-A and SS-B are also extractable nuclear antigens. SS-A is RNA complexed to one of two proteins, and SS-B is a phosphoprotein bound to an RNA
polymerase.

Scl-70 is a DNA topoisomerase 1, which is involved with DNA assembly and disassembly.
Syphilis
Syphilis:
Good old syphilis! Syphilis is caused by the spirochete Treponema pallidum. It is transferred by person to person contact and is a well-known sexually
transmitted disease.
Syphilis broadly breaks down into primary, secondary, latent, and tertiary syphilis. Let’s break down each one:

Primary syphilis:
This occurs about 2 weeks to 3 months after infection and presents as a localized sore or chancre where the bacteria came in contact with the person. This
stage usually lasts between 1-6 weeks if left untreated.

Secondary syphilis:
If primary syphilis is left untreated, new symptoms will arise in about 25% of people about 1-2 months after the primary sore or chancre has disappeared
(the sore or chancre does sometimes persist). The most notable symptom of secondary syphilis is a rash on the palms of the hands and/or soles of the feet.
This rash is indicative that the organism has begun to spread through the person’s body. This stage usually lasts between 1-8 weeks.

Latent syphilis:
This stage occurs after secondary syphilis and is characterized by a lack of symptoms but the person is still carrying the bacteria. Latent syphilis is not
contagious but pregnant women can still pass the bacteria to their child.

Tertiary syphilis:
If still untreated, about 33% of people will develop tertiary syphilis. Symptoms here are severe and include neurological and cardiovascular issues that can
result in death. This stage occurs 10-30 years after initial infection.

Screening and testing for syphilis in the clinical lab is generally done using serology tests because of efficiency and cost. There are seven syphilis tests to
be aware of and know the basics of:

1. Rapid plasma reagin test (RPR)

     a. There are two syphilis tests, RPR and VDRL, that involve testing for “nontreponemal” antigens. Nontreponemal antigens are nonspecific for the
causative bacteria Treponema pallidum but are associated with syphilis. The RPR reagent antigen is cardiolipin attached to charcoal particles (the charcoal
particles are for visualization). The term reagin generally means antibody, so the name just indicates it’s a quick antibody test. The RPR test also utilizes a
silicon needle for dispensing the antigen onto the testing card.
     b. Malaria, rheumatoid arthritis, infectious mononucleosis, leprosy, hepatitis, and systemic lupus erythematosus (SLE) can all cause a false positive
RPR or VDRL test.

2. Venereal disease research laboratory (VDRL)

     a. The VDRL test is very similar to RPR except it uses the reagent antigen cardiolipin and lecithin (lecithin enhances sensitivity). VDRL serum must
also be heat inactivated to inactivate complement. VDRL is less sensitive than the RPR test. Both RPR and VDRL are associated with false positives so
any positive in either of these tests must be confirmed by a specific Treponema pallidum test. A modified VDRL test can be used in cases of syphilis in the
cerebral spinal fluid (CSF) and is generally diagnostic.

3. Fluorescent treponemal antibody absorption test (FT-ABS)

     a. The FT-ABS test is an indirect immunofluorescence test. The test utilizes a specific Treponema pallidum strain called the Nichols strain. FT-ABS is
more specific than RPR and VDRL but is more time consuming.

4. Microhemagglutination (MHA-TP)
     a. The MHA-TP uses a sheep red cell line that is sensitized with Treponema pallidum. It is about equal strength to the FT-ABS test. This test has been
replaced by the TP-PA test.

5. Treponema pallidum immobilization test (TPI)

     a. The TPI test uses live Treponema pallidum. Patient serum is added to the live treponemes and if there is antibody in the patient’s serum, the
treponemes will be immobilized. Dark field microscopy is used to visualize the organisms. The test is expensive and seldom used.

6. Dark field microscopy

     a. Primary and secondary syphilis can be diagnosed by looking at skin lesions under dark field microscopy. The presence of the corkscrew shaped
Treponema pallidum is diagnostic.

7. Treponema pallidum particle agglutination test (TP-PA)

     a. The TP-PA test is very similar to the MHA-TP test; however, the TP-PA test uses gelatin particles sensitized with Treponema pallidum instead of
sheep red cells. TPPA is generally positive for life.

Study Tip
It is important to know which tests are more sensitive than others in primary, secondary, and tertiary syphilis. Use the image below to keep them straight.