SEROLOGY OF VIRAL

INFECTIONS

VH REVIEW CENTER
DELFIN, RMT
 Hepatitis
General term meaning inflammation of the liver, usually
accompanied with fever, nausea, vomiting and jaundice

can be caused by radiation, chemicals, disease processes such as

autoimmune disease, viruses and cancer

5 distinct viruses A, B, C, D and E

all of these are RNA viruses except hepatitis B which is a DNA

virus

initial infection may be clinically silent

chronic carrier state may develop and may result to liver failure
due to cirrhosis, hepatocellular carcinoma, or fulminant hepatitis
 Hepatitis

Type Route of transmission Acute/Chronic Vaccine

A Fecal-oral Acute 2 doses, first at
12 mo, then 6
mo later

B Contact with infected blood, Chronic, can lead to 3 doses anytime

seminal fuid, vaginal cirrhosis and cancer with 6 mo
secretions between

C Infected blood Chronic, can lead to No

cirrhosis and cancer

D Infected blood, sexual contact Only infects people HBV vaccine

with HBV
E Fecal-oral Acute none
 Hepatitis A virus (HAV)

Transmitted by fecal oral route

Occurs worldwide
Most hepatitis epidemics are due to HAV
Progress of infection:
Incubation of 2-7 weeks, may be asymptomatic or may
include jaundice
Clinical illness develop abruptly and include fever, anorexia,
vomiting, fatigue and malaise
Increase in serum transaminases
dark urine and pale stool
Recovery 2-4 weeks, no carrier state
Mortality 0-1%
 Antibody and antigen markers

First and most clinically useful is IgM antibody to HAV

IgM indicates acute infection, appears 4-5 weeks after

exposure

IgM disappears in 3-6 months, replaced by IgG anti-

HAV

IgG peaks during convalescence and may remain

detectable for life
Time course of Hepatitis A virus (HAV)
infection
 Hepatitis B virus (HBV)

Old term serum hepatitis, incubation period of 4-26 weeks

Route of infection is usually parenteral, direct inoculation

Incidence of infection is 140,000-320,000 cases per year

resulting in 5-6,000 deaths per year.
Duration of acute infection ranges from 4-8 weeks with
symptoms similar to HAV.
10% progress to chronic

One-third of chronic at risk of developing chronic active

hepatitis, cirrhosis and/or hepatocellular carcinoma
Spread of Hepatitis B virus (HBV) in the
body
Laboratory Diagnosis

Involve the detection of three marker system

Hepatitis B surface antigen (HBsAg) is the first to appear, appears 2-

4 weeks during late incubation, marker of choice for recent infection

Anti-Hepatitis B surface antigen (anti-HBs) is the last antibody to

appear, may persist for life

Between disappearance of HBsAg and appearance of anti-HBs is

known as the core window
 Hepatitis B virus (HBV): Laboratory Diagnosis

IgM antibody to Hepatitis B core antigen (anti-HBc) may be the

only detectable marker during the core window, differentiates
recent infection from chronic carrier state

Third marker is Hepatitis Be antigen (HBeAg), appearance of

HBeAg and anti-HBe, closely coincide with HBsAg
Hepatitis B status HBsAg Anti-HBc Anti- Anti-HBs
(total) HBc
Hepatitis B Demystified
Never infected with the virus (consider getting the
(IgM)

vaccine). Negative Negative Negative Negative

Infection likely took place over the last six months and
is still active. Positive Positive Positive Negative

Infection likely took place over the past six months

and is in the process of clearing. A false-positive is
another possibility (HIV-positive people with this Negative Negative Positive Negative
particular test result should have their HBV viral load
checked).
Infection likely took place more than six months ago
and has been successfully controlled by the immune Negative Positive Negative Positive
system.
The vaccine was successfully given to prevent HBV
infection. Negative Negative Negative Positive

Chronic HBV infection.

Positive Negative Positive Negative
 Serodiagnosis of Acute Viral Hepatitis
Test Result:

HBsAg HBsAb HBcAb HAV-I Interpretation

- - - + Recent acute hepatitis A infection

+ - - - Early acute type B hepatitis
+ - + - Acute of chronic type B hepatitis
+ + + - Chronic or acute type B hepatitis
- + + - Recovery from or past infection of type B hepatitis
- - + - Remote past infection or immediate recovery phase or low
level carrier of type B hepatitis
- + - - Remote past infection or immunization with HBsAg
+ - + + Recent probable hepatitis A infection and probable chronic
type B hepatitis
- - - - Possible non-A, non-B hepatitis infection, other viral
infection or liver or liver toxin
 Serological Tests for HBSAg

Third generation
Radioimmunoassay
Reverse passive agglutination
Enzyme-linked Immunosorbent assay (ELISA)
Reverse passive latex agglutination

Second generation
Counterelectrophoresis
Rheophoresis
Complement Fixation

First generation
Ouchterlony double diffusion (agar gel diffusion)
 Hepatitis D virus (HDV)

Requires infection with Hepatitis B

Route of transmission the same as HBV
Can occur as coinfection or superinfection
Consequences of delta virus infection
 Serological markers

HDAg found early, disappears rapidly, not very useful

IgM anti-D and total anti-HD (IgM and IgG) detected during
acute phase

Presence of IgM anti-D and HBsAg together with IgM anti-HBc

indicates co-infection

Absence of IgM anti-HBc indicates superinfection

Presence of anti-HD indicates chronic infection

 Hepatitis C virus (HCV)

Clinically and epidemiologically similar to HBV

60-70% of HCV patients will develop chronic hepatitis, 10-20%

cirrhosis and 15% hepatocellular carcinoma.

HCV and HBV may be present as co-infections

 Hepatitis C virus (HCV)

Serological Markers

Serological profile not fully developed

Present of HCV antibodies only indicates present or past
infection
Can have false negative in some patients
 Hepatitis E virus (HEV)

Similar to HAV in transmission and clinical course.

Found primarily in developing countries, Africa and Asia

Results in acute hepatitis, no risk of chronic hepatitis

Pregnant women with HEV may develop fulminant liver failure

and death

No distinctive markers, diagnosis based on symptoms for

exposed individuals in endemic countries
 Hepatitis G virus

Independently discovered 1995-1996 by 2 separate

research groups
RNA virus
Transmissible by blood-borne route
Found in patients with acute or chronic liver dse.
Exact clinical significance needs to be further defined
ELISA and Western Blot methods have been developed
 HERPES VIRUS GROUP

Includes EBV, CMV, Herpes simplex virus type I and II,

Varicella-zoster virus

DNA viruses that remain within nucleus while completing

life cycle

most infections are subclinical and result in latent stage

 Epstein-Barr Virus (EBV)

Spread through oral transmission of infective saliva and is the

cause of infectious mononucleosis

Other diseases :
Burkitts lymphoma, nasopharyngeal carcinoma, B-cell lymphoma

Virus may become reactivated and is the suggested cause of

chronic fatigue syndrome
 Characteristics of infection

4-7 week incubation, acute self limiting

Enlarged LN in the neck, sore throat, fever, rash
Malaise, lethargy, extreme tiredness
Liver and spleen involvement and enlargement
Hematology : high WBC, over 20% atypical reactive lymphocytes
Serological Findings:

Increased titer of Heterophile antibodies

Heterophile antibodies have the ability to react with antigens that are
not responsible for their production.
Example of cross reactivity

It is noted that when rabbits were injected with tissues from guinea
pigs, cats or horses, they produce antibodies against sheep rbc.
The agents producing the response are known as Forssman (discovered
by forssman, 1911) or heterophile antigens and the corresponding antigen
are called heterophile antibodies.
Types of Heterophile Antibodies

1. Forssman Heterophile Antibodies

Produced in response to incidental contact with heterophile antigens
such as:
Salmonella
Shigella
Other bacterial species
Other antigens of plants and animal origin
May enter thru minor lesions of the gastrointestinal wall
Could be found in low titers in nearly all normal adults.
Have no clinical significance
2. Serum Sickness Heterophile Antibodies

Produced in response to parenteral injection of horse serum or

products from horse during immunization.

This type of antibody is rarely seen because of the more modern

preparations of vaccines.
3. Infectious Mononucleosis Heterophile Antibodies:

Produced in response to virus capsid antigen found in EBV.

IgM antibodies
Appear on the first week of the illness and peak about the third
to fourth week.
The antibody level declines after the fourth week and become
undetectable thereafter.
 Serological Tests for IM heterophile Antibodies

1. Routine Presumptive Test (Paul and Bunnel Test)

Principle:

Test is based on an increase heterophile antibodies (anti-

sheep agglutinins)

This does not distinguish among Forssman, serum sickness

and heterophile antibodies.

Note that a positive Paul and Bunnel test indicates the

presence of heterophile antibodies but does not identify the
particular antibody
 Method
Employs simple dilution of inactivated serum in saline to which
small amounts of sheep rbc are added.
1:7, 1:14, 1:28, 1:56, 1:112, 1:224, 1:448, 1:896, 1:792

Inactivation is done by heating the serum at 56C for 30 min.

Clinical Significance:
A titer of 1:112 or higher may be considerd as a positive routine
presumptive test in the presence of clinical and hematologic
findings suggestive of IM.
2. Differential Test (Davidson)
Done if Presumptive Test is Positive
This test is carried out to differentiate among 3 different
antibodies using
GPK: guinea pig kidney
BEA: beef erythrocyte antigen

Types of Heterophile Absorbe Absorbed

antibodies d by by BEA
GPK
Forssman Antibody + -
Serum Sickness + +
Infectious Mononucleosis - +
Types of Heterophile Agglutination RXN Agglutination
antibodies After Reaction After
Absorbed by GPK Absorbed by BEA
Forssman Antibody - +
Serum Sickness - -
Infectious + -
Mononucleosis
 Principle

The patient sera are absorbed by GPK and BEA. Absorbed

agglutinins are removed by simple centrifugation and aspirating
the resulting supernatant liquid.

The presence of non-absorbed or absorbed agglutinins maybe

detected by retesting the supernatant fluid with sheep rbc.

The highest dilution of serum that gives agglutination is the titer.

 Interpretation:

The differential test results must be correlated with presumptive

test results.
After GPK absorption, The test is positive for IM if
Agglutination occurs at the same titer as that of routine
presumptive test
Agglutination occurs not more that 3 tubes lower that the
routine presumptive test
The test is negative for IM if the titer after GPK absorption is more
than 3 tubes lower than that of routine presumptive test.
 After BEA Absorption: the test is positive for IM if:

BEA completely removes heterophile antibodies.

GPK does not completely remove heterophile antibodies.
 Presumptive DIFFERENTIAL TEST AFTER INTERPRETAT
Test titer ABSORPTION WITH ION
GPK BEA
1:224 1:112 1:7 Positive
1:224 1:56 0 Positive
1:224 1:28 0 Positive
1:224 1:14 1:112 Negative
1:224 1:7 1:112 Negative
1:56 1:28 0 Positive
1:56 1:14 0 Positive
1:56 1:7 0 Positive
1:28 1:28 0 Positive
1:28 1:14 0 Positive
3. Miscellaneous Heterophile Tests:
Monospot
Rapid slide test 20% horse rbc are used instead of rbc
contribute higher specificity. The patient is pre-absorbed by
GPK and BEA

Monotest
Stabilized horse rbc agglutinate in the presence of IM
heterophile antibodies but not Forssman and serum sickness
heterophile antibodies
 Ox cell hemolysin test

Patients with IM produce high titers of ox hemolysins that normal

individuals
In this test ox rbc and complement are added to serially diluted
serum.
The titer of the serum is the reciprocal of the highest dilution
showing 50% hemolysis.
 EBV specific antibodies

Must know pattern of appearance of EBV antigens

Most valuable is IgM antibody to viral capsid antigen (VCA),
indicates a current infection (best marker), lasts about 12
weeks
Can also detect anti-early antigen (EA), recent infection and
anti-EB nuclear antigen (EBNA), older infection
ELISA and immunofluorescence techniques most commonly
used
4. EBV-specific antibodies
IgM anti VCA (Viral Capsid Antigen)
This is the most useful marker for acute IM. It usually
appears at the onset of clinical symptoms and disappears by 3
months.
IgM anti VCA
This is present at the onset of IM. It persists for life
Indicates past infection
 Anti-EA-D (Early Antigen Diffuse)
This appears during acute IM

Anti-EBA
This appears during convalescence
 Cytomegalovirus

Transmission occurs from person to person

Symptoms resemble IM but has negative test for EBV
In babies may cause life-threatening illness resulting in CNS
involvement, hearing loss, and mental retardation
Seen in patients with deficient immune system, AIDS,
transplantation
Immunologic Response:

For best diagnostic results, lab tests for CMV antibody should be
performed by using paired serum samples.

One blood sample should be taken upon suspicion of CMV, and

another one taken within 2 weeks. A virus culture can be performed
at any time the pt. is symptomatic.

IgM antibodies produced against early and intermediate-early (IE)

CMV antigens, last for 3 to 4 months

IgG appear shortly after and peak at 2 to 3 months

Laboratory Diagnosis

Range from culture and cytologic techniques to DNA probes,

PCR and serologic techniques

Detection of antibodies indicator of recent infection

Viral culture lack sensitivity and are time consuming and

expensive

Microscopic examination of biopsy specimens, urine sediment or

peripheral blood may reveal the typical cytomegalic cell with
owls eye inclusion
 Detection of CMV Ag in cells more appropriately detected by
immunofluorescent techniques using monoclonal antibodies
ELISA is the most commonly available serologic test for measuring
antibody to CMV
The result can be used to determine if acute infection, prior
infection, or passively acquired maternal antibody in an infant is
present
Other tests include various fluorescence assays, indirect
hemagglutination, and latex agglutination
Screening tests using coated latex particles compare favorably to
more complex tests for antibody detection
False positives can occur = RA and Ebstein-Barr antibodies
Laboratory Testing

Recovery of the virus in cell culture is considered the gold

standard for detection of this virus from sources other than CSF,
culture helpful in differentiating types of HSV

Direct examination using immunofluorescence or

immunoperoxidase staining of cells from lesion

DNA probes, ELISA, latex agglutination, RIA and indirect

immunofluorescence

Serology is not very useful because there is a high prevalence of

antibody in the normal population
 Varicella-Zoster Virus

Laboratory testing

important to distinguish VZV from other infections, selection of

antiviral drugs, or determining immune status of individuals

PCR is now the routine testing method for VZV

Direct fluorescent antibody staining and viral culture techniques

may be used for the detection of VZV in most specimen types

IgG and IgM antibody tests by ELISA may be used

 Rubella Virus
GERMAN MEASLES

Laboratory testing

Performed primarily for diagnosis of acquired infections and to determine

immune status of pregnant patients.

Some tests detect IgG antibodies, other IgM

Methods include : hemagglutination inhibition, passive hemagglutination,

neutralization, hemolysis in gel, complement fixation, fluorescent
immunoassay, RIA, ELISA and latex agglutination

Method depends on volume of testing, turn around time, complexity,

expense and whether a qualitative or quantitative test is needed
 Rubeola
MEASLES

Serology testing provides best means of confirming a measles

diagnosis

Methods to detect Rubeola antibodies include : hemagglutination

inhibition, endpoint neutralization, complement fixation, IFA and
ELISA
In addition to signs and symptoms, diagnosis confirmed by presence
of Rubeola specific IgM antibodies or four-fold rise in IgG antibody
titer in paired samples taken after rash to 10 to 30 days later

IgM test highly depended on time of sample collection with 3-11

days after rash being optimal
 MUMPS

Methods to detect mump antibodies include : complement fixation,

hemagglutination inhibition, hemolysis-in-gel, neutralization assays,
IFA and ELISA

Current or recent infections indicated by presence of specific IgM

antibody in single sample which can be detected within 5 days of
illness

Fourfold rise in specific IgG antibody in 2 samples collected during

acute and convalescent phases

Fluorescent antibody staining for mumps antigens developed but not

widely used

Cross-reactivity between antibodies to mumps and parainfluenza

viruses has been reported in test for IgG
 Human Immunodeficiency Virus (HIV)

Etiologic agent of AIDS

Discovered independently by Luc Montagnier of France and
Robert Gallo of the US in 1983-1984
Former names of the virus include :
Human T cell Lymphotrophic virus (HTLV-III)
Lymphadenopathy associated virus (LAV)
AIDS associated retrovirus (ARV)
HIV-2 discovered in 1986, antigenically distinct virus endemic in
West Africa
One million people infected in US, 30 Million worldwide are
infected
Leading cause of death of men aged 25-44 and 4th leading cause
of death of women in this age group in the US
 Structural genes

Gag is p55 from which three

core proteins (p15, p17 and
p24) are formed

Env gene codes for envelope

proteins gp160, gp120 and
gp41

Pol codes for p66 and p51

subunits of reverse
transcriptase and p31 an
endonuclease
Immunologic Manifestations

Early stage slight depression of CD4 count, few symptoms,

temporary
Window of up to 6 weeks before antibody is detected, by 6
months 95% positive
During window p24 antigen present, acute viremia and
antigenemia
Antibodies produced to all major antigens
First antibodies detected produced against gag proteins p24 and
p55
Followed by antibody to p51, p120 and gp41
As disease progresses, antibody levels decreases
 Immune abnormalities associated with increased viral
replication
Decrease in CD4 cells
B cells have decreased response to antigen
CD8 cells initially increase and may remain elevated
As HIV infection progresses, CD4 T cells drop resulting in
immunosuppression and susceptibility of patient to
opportunistic infections
Death comes due to immuno-incompetence
 Laboratory diagnosis of HIV infection

Methods utilized to detect

Antibody
Antigen
Viral nucleic acid
Virus in culture
ELISA Testing

first serological test developed to detect HIV infection

antibodies detected include those directed against p24,

gp120, gp160 and gp41, detected first in infection and
appear in most individuals

used for screening only, false positives do occur

 Western Blot Testing

most popular confirmatory test

antibodies to p24 and p55 appear earliest but decrease or
become undetectable
antibodies to gp31, gp41, gp120, and gp160 appear later but
are present throughout all stages of the disease

INTERPRETATION OF RESULT
no bands: negative
in order to be interpreted as positive a minimun of 3 bands
directed against the following antigens must be present : p24, p31,
gp41 or gp120/160
CDC criteria require 2 bands of the following : p24, gp41 or
gp120/160
 indeterminate results are those samples that produce bands but
not enough to be positive, may be due to the following:

1. prior blood transfusions, even with non-HIV-1 infected blood

2. prior or current infection with syphilis
3. prior or current infection with malaria
4. autoimmune diseases
5. infection with other human retroviruses
6. second or subsequent pregnancies in women
*** run an alternate HIV confirmatory assay
 Indirect immunofluorescence assay

Can be used to detect both virus and antibody

Antibody is detected by testing patient serum
against antigen applied to a slide, incubated,
washed and a fluorescent antibody added
Virus is detected by fixing patient cells to slide,
incubating with antibody
 Detection of p24 HIV antigen
p24 antigen only present for short time, disappears when antibody to p24
appears
anti-HIV-1 bound to membrane, incubated with patient serum,
second anti-HIV-1 antibody attached to enzyme label is added
(sandwich technique), color change occurs

optical density measured, standard curve prepared to

quantitate results

Positive result is confirmed by neutralizing reaction

preincubate patient sample with anti-HIV, retest
if p24 present immune complexes form that will prevent binding to
HIV antibody on membrane added
SEROLOGICAL DIAGNOSIS OF INFECTIOUS
DISEASES = HIV

test not recommended for routine screening as

appearance and rate of rise are unpredictable
sensitivity lower than ELISA
most useful for the following
early infection suspected in seronegative patient
Newborns
CSF
monitoring disease progress
 Polymerase Chain Reaction (PCR)
Looks for HIV DNA in the WBCs of a person

Amplifies tiny quantities of the HIV DNA present, each cycle of

PCR results in doubling of the DNA sequences present

The DNA is detected by using radioactive or biotinylated probes

Once DNA is amplified it is placed on nitrocellulose paper and

allowed to react with a radio-labeled probe, a single stranded
DNA fragment unique to HIV, which will hybridize with the
patients HIV DNA if present

Radioactivity is determined
 Virus isolation

definitively diagnose HIV

best sample is peripheral blood, but can use CSF,

saliva, cervical secretions, semen, tears or material
from organ biopsy

cell growth in culture is stimulated, amplifies

number of cells releasing virus

cultures incubated one month, infection confirmed

by detecting reverse transcriptase or p24 antigen in
supernatant
Viral Load Tests
viral load or viral burden is the quantity of HIV-RNA that is in the
blood
measures the amount of HIV-RNA in one milliliter of blood

take 2 measurements 2-3 weeks apart to

determine baseline

repeat every 3-6 months in conjunction with CD4

counts to monitor viral load and T-cell count

repeat 4-6 weeks after starting or changing

antiretroviral therapy to determine effect on viral load
 Testing of neonates

difficult due to presence of maternal IgG antibodies

use tests to detect IgM or IgA antibodies, IgM lacks
sensitivity, IgA more promising
measurement of p24 antigen
PCR testing maybe helpful but still not detecting
antigen soon enough : 38 days to 6 months to be
positive
Algorithm for Serologic Testing for AIDS
Summary:

ELISA screening test.

Western blot is confirmatory test.

Molecular testing used to follow HIV load and determine success

of treatment.
CD4+ counts are used to determine if the individual has progressed to
AIDS.
The Centers for Disease Control and Prevention considers HIV-infected
persons who have CD4 counts below 200 cells/mm3 to have AIDS
 DENGUE FEVER

Transmitted by mosquitoes
There are 4 known distinct serotypes ( dengue virus 1, 2, 3 and
4)
In children , infection is often sub-clinical or causes a self-
limited febrile disease
Secondarily infected with a different serotype, dengue
hemorrhagic fever or dengue shock syndrome
Serological Test

Dengue IgG/IgM Rapid Test

solid phase immunochromatographic assay for the rapid

qualitative and differential detection of IgG and IgM antibodies
to dengue virus in human serum, plasma or whole blood.

This test can also detect all 4 Dengue serotypes by using a

mixture of recombinant Dengue envelop proteins
Rapid Test for Dengue
 Interpretation of the test
IgG and IgM positive
indicative of a late primary or early secondary dengue infection
IgM positive
indicative of primary Dengue infection
IgG positive
indicative of secondary or past dengue infection
Negative
retest in 3-5 days if Dengue infection is suspected
Invalid
insufficient specimen volume or incorrect procedural technique.
Repeat the test using a new test device