IMMUNOLOGY

SEROLOGY
Part 4

MTAP2
BE “CLINICALLY
SIGNIFICANT”.
Syphilis
o a.k.a.: Great Pox, Evil Pox, French disease, Italian disease, , Spanish disease,
Lues Venereal
o Causative agent:
Treponema pallidum subsp. pallidum
(Spirochaeta pallida)
o Modes of transmission:
o Sexual Contact
o Direct Blood Transfusion
Very low risk of transmission via transfusion of blood:
o
Treponema pallidum dies within THREE DAYS at
refrigeration temp (2-6C)
o Transplacental Route
o In the treatment of syphilis, heavy metals such as arsenic were replaced by
PENICILLIN in the 1940s
**Arsenic-derived drug: ARSPHENAMINE(aka. SALVARSAN 606)

 Penicillin-allergic: Tetracycline

 Pregnant women: Erythromycin

Stages of Syphilis
1. PRIMARY STAGE
Demonstrated by the presence of dry, non-tender
lesion on the usual sites of inoculation

Primary lesion develops within 10-90 days

“Hard chancre ”/”Hunterian chancre”
-painless and firm nodule

Lab diagnosis:
Darkfield microscopy: YES
Serology: NO
No antibody production yet, serology not
practical
Stages of Syphilis
2. SECONDARY STAGE
o O c curs within 2-10 weeks following appearance of 1o
lesion
o Rash occurs on palms and soles
o on mucous membranes, the lesions may appear as
white mucous patches
o “Condyloma” / “Condylomata lata”: wart-like lesions in
the moist areas of the body
o 1/3 of cases will be treated, 1/3 will proceed to latent
stage
o Lab diagnosis:
o Darkfield microscopy: YES
o Serology:YES
Stages of Syphilis
3. LATENT STAGE
o ABSENCE of clinical symptoms
o Lab diagnosis:
o recognized ONLY by a serological test
o Darkfield microscopy:NO
o Serology:YES
Stages of Syphilis
4. TERTIARY STAGE
o Deep involvement of the organs of the body
o “Gummas”/”Gummata”
o Untreated cases may develop:
o Benign Tertiary Syphilis : granulomatous
lesions in skin, bones and liver
o Neurosyphilis: degenerative changes inn

CNS
o Syphilitic cardiovascular lesions
o Lab diagnosis:
o Serology: YES
o Darkfield microscopy: NOT practical
 DARKFIELD
STAGE SEROLOGY DISTINGUISHING FEATURE
MICROSCOPY

HARD CHANCRE
Primary YES NO (painless and
firm nodule)
CONDYLOMATA LATA
(wart-like lesions in the
Secondary YES YES moist areas of the body)

ABSENCE of clinical
Latent NO YES symptoms

GUMMAS
Tertiary NO YES Onset of neurosyphilis
Congenital Syphilis
o Caused by maternal spirochetemia and
transplacental transmission of the
microorganism to the child

o Characterized by the presence of a

condition known as HUTCHINSON's TRIAD
o Notched teeth
o Keratitis
o Eight nerve deafness
CONGENITAL SYPHILIS
Lab Detection of Syphilis
1. Direct Detection of T. pallidum
○ Darkfield Microscopy
○ **Fluorescent Antibody Test
2. Serologic Tests
○ Nontreponemal
○ Treponemal
○ Hemagglutination tests
Direct Detection of Syphilis

1. DARKFIELD MICROSCOPY
○ Used for primary and secondary syphilis
○ (+) coiled organisms with corkscrew motility
○ TEST OF CHOICE FOR SYMPTOMATIC
PATIENTS WITH PRIMARY SYPHILIS

2. FLUORESCENT ANTIBODY TESTING

OF SPECIMEN
Serologic Tests for Syphilis
● WASSERMAN TEST
○ Principle: COMPLEMENT FIXATION
○ (+) result: NO HEMOLYSIS
○ FIRST DIAGNOSTIC TEST FOR
SYPHILIS
○ Classified as treponemal or
nontreponemal serologic tests
Serologic Tests for Syphilis
1. NONTREPONEMAL SEROLOGIC TESTS
● Determine the presence of REAGIN
○ antibody to cardiolipin
● Used as a SCREENING TEST for syphilis
○ False (+): measles, chickenpox, hepatitis, infectious
mononucleosis, leprosy, tuberculosis, autoimmune
disorders, pregnancy, etc.
● Examples:
▪ Venereal Disease Research Laboratory Test (VDRL)
▪ Rapid Plasma Reagin (RPR)
▪ Toluidine Red Unheated Serum Test (TRUST)
▪ Unheated Serum Reagin (USR)
▪ Reagin Screen Test (RST)
Nontreponemal Serologic Test
1. VDRL
○ Principle: FLOCCULATION
■ special type of precipitation reactions involving fine
particles
○ The only nontreponemal test that can be performed on
CSF; beneficial for neurosyphilis diagnosis
⦁ Specimen: 50uL (0.05ml) serum HEATED at 56°C for 30 mins
● Reagents
○ VDRL antigen consist of
❑ 0.03% cardiolipin - main reacting component
❑0.9% cholesterol - enhances the reacting surface or
cardiolipin
❑ 0.21% lecithin - removes anticomplementary activity
of cardiolipin
● Both a qualitative and quantitative
VDRL
● Uses a SLIDE with ring having 1.75 mm deep concavity
● ANTIGEN DELIVERY NEEDLES:
o Qualitative serum VDRL
o 18G NEEDLE without bevel that will deliver 60 DROPS
of antigen suspension per ml (ring: 14mm in diameter)
o Quantitative serum VDRL
o 19G NEEDLE without bevel that will deliver 75 DROPS of
antigen suspension per ml
o23G NEEDLE with or without bevel that will deliver
100 DROPS of saline per ml (ring: 14mm in diameter)
o CSF VDRL
o 21 OR 22G NEEDLE that will deliver 100 DROPS per ml
(ring: 16mm in diameter)
o ROTATIONS:
o Serum: 180 RPM for 4 minutes
o CSF: 180 RPM for 8 minutes
o Examine microscopically using L PO
VDRL SLIDES
Nontreponemal Serologic Test
2. RPR
● Principle: AGGLUTINATION
○ Agglutination can be observed easily due to the
addition of charcoal in the reagents
● Used to screen for blood donors
● Specimen: 50ul (0.05ml) UNHEATED SERUM
● Reagents:
○ Modified VDRL antigen:
■ 0.03% cardiolipin

■ 0.9% cholesterol

■ 0.21% lecithin

■ Charcoal - makes the test easier to read

■ EDTA - prevents oxidation of lipid

■ Thimerosal - preservative

■ Choline chloride - inactivates the complement

RPR
● Uses PLASTIC CARD
● Antigen delivery needles:
○ 20G DISPOSABLE NEEDLE without
bevel, 60 DROPS are obtained in 1 ml
○ Ring: 18 mm in diameter
○ Rotation: 100 RPM for 8 minutes
○ Examine macroscopically
RPR CARD
Nontreponemal Serologic Tests

REPORTING FOR VDRL AND RPR

● Nonreactive: No Clumps
● Weakly reactive: Small Clumps
● Reactive: Medium to Large Clumps
Treponemal Serologic Tests
● Detect TREPONEMAL ANTIBODIES (specific)
● Used to CONFIRM results for those REACTIVE
on screening tests.
● Examples of treponemal tests:
1. Treponema pallidum Immobilization (TPI) Test
✔ REFERENCE METHOD
2. Flourescent Treponemal Antibody Absorption
Test (FTA-ABS)

1. Hemagglutination Test
■ Hemagglutination Treponema test for Syphilis
(HATTS)
■ T. pallidum Heagglutination Assay (TPHA)
■ Microhemagglutination Assay for Antibodies to T.
pallidum (MHA-TP)
Treponemal Serologic Tests
1. TREPONEMA PALLIDUM IMMOBILIZATION
TEST
○ Reference/Standard test to which other
treponemal test are evaluated
○ Involves mixing of patient serum with live actively
motile T. pallidum extracted from testicular
chancre of a rabbit and complement.

○ Live T. pallidum + patient’s serum

(with anti-treponemal antibody)
■ POSITIVE: > 50% organism is IMMOBILIZED
■ NEGATIVE: < 20% immobilized
■ DOUBTFUL: 20-50% immobilized
Treponemal Serologic Tests
2.FLUORESCENT TREPONEMAL
ANTIBODY ABSORPTION TEST (FTA-ABS)

○ Detects treponemal antibodies

○ Patient serum is heat inactivated and

reacted with a sorbent consisting of
nonpathogenic treponemes (Reiter strain)
which removes cross-reactivity with
Treponemes other than T. pallidum)

○ Remains POSITIVE even AFTER

TREATMENT
Fluorescent Treponemal Antibody
Absorption Test (FTA-ABS)
○ Consist of two parts
1. Adsorption Phase
● Patient serum heated at 56°C for 30mins +
nonpathagenic treponemes (Reiter's strain)
○ Reiter’s strain acts as a SORBENT which
removes cross-reacting antibodies

2. Indirect Immunofluorescence
● Nichol's strain (VIRULENT strain of T.pallidum,
affixed to the slide) + patient serum (anti-trep
ab) + FITC-labeled AHG reagent
(+) FLUORESCENCE
Treponemal Serologic Tests
3. HEMAGGLUTINATION TESTS
○ Specific
○ Uses RBC's sensitized with Nichol's strain
○ Examples:
✔Hemagglutination Treponema Test
for Syphilis (HATTS) – glutaraldehyde-stabilized turkeyRBCs
✔T. pallidum Hemagglutination Assay (TPHA)- tanned
sheeprbc coated with Nichol’s strain antigens
✔Microhemagglutination Assay for Antibodies to T.
pallidum (MHA-TP)-formalinized tanned sheeprbc coated with
Nichol’s strain antigens in microtiter plates

✔TPPA-gelatin particles instead of red cells

Nonvenereal Syphilis
Yaws
○ a.k.a. :frambesia, pian, buba, bouba,
tropical syphilis, framboesia tropica,
○ chronic nonvenereal disease of skin and bones
○ Caused by:
Treponema pallidum subsp. pertenue
○ Transmission:
Traumatized skin comes in contact with infected
lesion
○ Primary lesion =Mother yaw/Framboise
Nonvenereal Syphilis

Bejel
○ a.k.a. :endemic syphilis, siti,
dichuchwa
○ lesions on the oral cavity, oral
mucose, skin, bones and nasopharynx
○ Caused by:
Treponema pallidum subsp.
endemicum
○ Transmission:
Mouth to mouth by utensils
Nonvenereal Syphilis

Pinta
○ a.k.a. carate, mal de pinto
○ Caused by: Treponema carateum
○ ulcerative skin disease
○ Transmission:
Traumatized skin comes in contact
with infected lesion
Streptococcal
Serology
Streptococcus pyogenes
● Group A beta-hemolytic streptococcus
● Gram (+) catalase (-) cocci in chains

● White PINPOINT colonies on BAP

⮚ MOST COMMON PATHOGEN OF THE

UPPER RESPIRATORY TRACT

● Considered universally susceptible to

penicillin, hence does not require antibiotic
susceptibility testing.
Virulence Factors
○M protein – major virulence factor
● Antibodies against the specific M protein confer lifelong type-specific immunity
● Inhibits p h a g o c y t o si s

○ Lipoteichoic acid - permits bacterial adherence to

the respiratory epithelium

○ Pyrogenic (erythrogenic toxins)

-responsible for scarlet fever a.k.a. s c a r l a t in a

○ Superantigens – with high mitogenic capabilities

-Associated with cases of necrotizing fasciitis or toxic
shock syndrome
Virulence Factors
● Enzymes
○ Streptolysins O
○ Streptolysins S

○ Hyaluronidase
- a.k.a. "spreading or Duran-Reynal factor“

○ Streptokinase - promotes fibrinolytic activity by

converting plasminogen to plasmin

○ Deoxyribonucleases (DNases)
○ Nicotinamide adenine dinucleotidase (NADase)
 Number Other names for the disease Etiology(ies)

Rubeola, Measles, Hard measles, 14-day

First disease Measles virus
measles, Morbilli

Second disease Scarlet Fever, Scarlatina Streptococcus pyogenes

Third disease Rubella, German measles, 3-day measles Rubella virus

Some say the disease does

not exist1. Others believe it
Filatow-Dukes' Disease, Staphylococcal is due to Staphylococcus
Fourth disease
Scalded Skin Syndrome, Ritter's disease aureus strains that make
epidermolytic (exfoliative)
toxin2,3

Erythrovirus (Parvovirus)
Fifth disease Erythema infectiosum
B19

Exanthem subitum, Roseola infantum,

Human Herpes Virus 6B or
Sixth disease "Sudden Rash", rose rash of infants, 3-day
fever Human Herpes Virus 7
Erythema
● Erythematous Rash: SLE
● Erythema infectiosum: Parvovirus B19
● Erythema chronicum migrans, or erythema
migrans: Lyme Disease
o Erythrasma: caused by Corynebacterium
minutissimum
Clinical Manifestations
● Pharyngitis -MOST COMMON CLINICAL MANIFESTATION
○ may be ac c ompanied by scarlet fever (2nd disease)
● Acute rheumatic fever
○ M protein is homologous to myosin light chains of the
heart, thus antibodies produced against M protein cross-
react with myocardial tissues, damaging it in the
process.
● Acute post-streptococcal glomerulonephritis
○ Caused by deposits of circulating streptococcal–
antistreptococcal immune complexes in the glomeruli and
subsequent activation of complement~>lysis
● Impetigo
● Erysipelas
● Necrotizing fasciitis- “flesh-eating disease”
● Toxic shock–like syndrome
Immunologic Manifestation
● Infection with S. pyogenes induces production of
several different antibodies:

● Anti-streptolysin O (ASO) antibody

○ begins to rise about 7 days post-infection
○ not a reliable indicator of recent infection because
it remains elevated for 1 year

● Anti-DNase B (ADN-B) antibody

○ MOST RELIABLE measure of recent
S. pyogenes skin infection
Diagnostic Evaluation
● Throat culture
○ Swab must be Dacron or rayon (synthetic) tipped on a plastic
shaft
○ Specimen must be transported using modified Stuart's liquid
or semisolid medium or Amie’s liquid or semisolid medium

● Serologic tests (Antigen and antibody screening)

○ Should compare acute and convalescent sera collected 3
weeks apart
■ Acute and convalescent phase in parallel testing -> a
FOURFOLD RISE titer is considered SIGNIFICANT
● Molecular tests
○ Rapid-Cycle Real Time Polymerase Chain Reaction
○ Group A strep Direct Probe Test
Detection of
Streptococcal Antigens
1.Optical Immunoassay for Direct
Detection of Group A Streptococcal Ag
○ allows for the direct visual detection of the physical
change in thickness of thin films resulting from
binding reactions between antigens and antibodies.

○ Polyclonal anti-Group A streptococcal

antigen (anti-GAS) antibody is attached to
a thin silicon wafer.
○ (+) purple color
○ (-) no color change
Detection of
Streptococcal Antibodies
1. Antistreptolysin O Titer
○ First test developed to measure streptococcal abs
○ Based on neutralization of the hemolytic activity of
streptolysin O antigen reagent by the ASO antibodies
present in the unknown serum

○ dilution of unknown serum + streptolysin O antigen +

5% suspension rabbit or human RBCs

○ The reciprocal of the highest dilution (tube containing

the least amount of serum) which demonstrate no
hemolysis is the ASO titer
○ RESULTS:
■ Test tube 13(RBC control) should show NO

hemolysis
■ Test tube 14 (ASO control) should show

COMPLETE hemolysis

○ ASO titer = Todd unit or IU

○ INTERPRETATION:
■ <166 Todd units = normal

■ 240 Todd units= moderately elevated (adult)

■ 320 Todd units = moderately elevated (children)

■ 5-125 Todd units = usual ASO titer in children

2. Anti-DNAse B Testing
○ Anti-DNAse B sometimes appear earlier than
streptolysin O in streptococcal pharyngitis

○ Principle: Neutralization

○ If Anti-DNase B antibodies are present, they will

neutralize the reagent DNase B, preventing it
from depolymerizing DNA. Presence of DNase is
measured by its effect on DNA-methyl
conjugate. This complex is green in its intact
form, but when hydrolyzed by DNase, the methyl
green is reduced and become colorless.
3. Streptozyme Testing
○ A slide hemagglutination screening test for the
detection of antibodies to several streptococ cal
antigens

○ Sheep RBCs coated with streptolysin,

streptokinase, hyaluronidase, DNase and NADase
+patient’s serum (for antibody detection)

○+result: hemagglutination

○ Highest dilution to show a positive agglutination is

taken as STZtiter and reported as STZunits
Hepatitis
Hepatitis
■ Inflammation of the liver
■ most common liver disease worldwide

◻ Non-viral causes
■ Alcoholism, ingestion of drugs and toxins,
autoimmunity, non-alcoholic fatty liver disease,
ischemia, etc.

◻ Viral causes
■ MOST COMMON CAUSE OF HEPATITIS
WORLDWIDE
 Forms of Hepatitis
FORM CHARACTERISTICS
Acute hepatitis • Typical form with associated jaundice.
•Has 4 Phases: Incubation, Pre-
icteric, Icteric, & Convalescence
Fulminant acute Rare form of hepatitis associated with
hepatitis hepatic failure
Subclinical •Probably accounts for persons with
hepatitis without demonstrable antibodies in their serum but
jaundice no reported history of hepatitis

Chronic • Accompanied by hepatic inflammation

hepatitis and necrosis that lasts for at least 6
months
•Occurs in about 10% of patients with
HBV infection
 Primary Hepatitis Viruses
VIRUS FAMILY/ TYPE MOT
Hepatitis A Picornaviridae Fecal-oral route
Hepatitis B Hepadnaviridae Sexual contact,
parenteral and
perinatal
transmission
Hepatitis C Flaviviridae Parenteral, vertical,
via blood
transfusion, sexual
contact
Hepatitis D Hepadnaviridae **MOSTLY
Parenteral(sexual
and perinatal)
Hepatitis A
● Causes “infectious hepatitis” or “short-incubation hepatitis”
● Genus Hepatovirus
● MOT: Fecal-oral transmission or Person-person
● Incubation period: 14-28 days
● The only hepatitis virus that has been successfully grown in
culture
● Treatment: there is NO specific treatment for HAV
● **may be transmitted via clotting factor concentrates

○ HEPATITIS A ANTIGENS:
■ Shed in the feces of infected px during the incubation period
and early acute stage of infection
■ When symptoms start to appear, antigen level decreases
■ Not clinically useful
⦁ HEPATITIS A ANTIBODIES:
1. IgM Anti-HAV
⮚ MARKER FOR ACUTE HEPATITIS A
● Peak during the first month of illness

● Decline to undetectable levels within 6-12 mos

● Detected using solid-phase antibody-capture ELISA

2. IgG Anti-HAV
● Produced after a natural infection or immunization
● Detected
using competitive inhibiton ELISA
⮚INDICATES IMMUNITY AND PAST INFECTION W/
HAV
Hepatitis B
● DANE PARTICLE:
○ complete HBV that causes infection

● "Long-incubation hepatitis“/**SERUM
HEPATITIS
● Incubation period: 60-90 days

● MOT:
○ Sexual contact
○ Parenteral transmission
○ Perinatal transmission
● HBV ANTIGENS:
1. Hepatitis B Surface Ag (HBsAG)
■ Viral envelope
■ Formerly known as the “Australian antigen”
■ INDICATOR OF ACTIVE INFECTION
■ Important marker in screening blood donors
2. Hepatitis B Envelope Ag (HBeAg)
■ Soluble nucleocapsid protein
■ present during periods of active viral replication
■ INDICATES A HIGH DEGREE OF INFECTIVITY
WHEN PRESENT
■ High vertical transmission risk
3. Hepatitis B Core Ag (HBcAg)
■ Structural nucleocapsip core protein
■ NOT detectable in serum because of the

viral envelope that mask it

■ Detected through LIVER BIOPSY ONLY

■ Not considered as a serologic marker

because it is not detectable in serum
● HEPATITIS B ANTIBODIES:
1. IgM Anti-HBc
■ FIRST ANTIBODY TO APPEAR DURING INFECTION
■ MARKER FOR ACUTE HBV INFECTION
■ INDICATOR OF CURRENT OR RECENT INFECTION
■ ONLY USEFUL/ DETECTABLE MARKER DURING

THE WINDOW PERIOD

2. IgG Anti-HBc
■ PERSIST FOR THE INDIVIDUAL’S LIFETIME
■ LIFELONG MARKER OF HBV INFECTION
○ NOTE: Total Anti- HBC measures IgG and
IgM antibodies against HBV and is helpful for
the diagnosis of acute or chronic hepatitis

3. Anti-HBe
■ Indicates that the patient is recovering from HBV
infection
■ Marker for CONVALESCENCE and favorable
prognosis
3. Anti-HBs
■ Persists for years, appears during the recovery
period of acute hepatitis B, weeks to months after
HBsAg disappears
■ Marker of PAST infection and immune state
■ PROVIDE PROTECTIVE IMMUNITY

■ Produced also after immunization with the hepatitis

B vaccine
● consists of recombinant HBsAg produced from genetically
engineered yeast
● Protective titer > 10mIU.mL of serum
■ Tested for vaccination screening and follow up
Clinical Course
● Acute HBV infection:
-HBsAg and IgM anti-HBcAg +
*Initial phase of infection,: HBeAg is also +

● Chronic HBV infection:

-persistence (>6 months) of HBsAg (with or
without concurrent HBeAg)
-persistence of HBsAg (principal marker of risk
for developing chronic liver disease and
hepatocellullar carcinoma)
Clinical Course

● Window Phase
○ the period of time between the
disappearance of HBsAg and appearance of
Anti-HBs secondary to complexing of HBsAg
with Anti-HBs.

○ IgM Anti-HBc is the only indicator of

infection during the window phase.
Interpretation of HBV Serologic Test Results
HBsAg Anti-HBc Anti-HBs

No HBV infection - - -
Early HBV infection + - -
Acute HBV + + -
Window period - + -
Past infection - + +
Immunization - - +
Tests for HBsAg Detection
● First Gen
○ Ouchterlony double diffusion
● Second gen
○ Counterelectrophoresis
○ Rheophoresis
○ Complement fixation

● Third gen
○ RIA
○ ELISA
○ Reverse passive Agglutination
○ Reverse passive latex agglutination
Must know!
● The hepatitis B virus can survive outside the
body for at least 7 days.

● During this time, the virus can still cause

infection if it enters the body of a person who is
not protected by the vaccine.
HEPATITIS C
o Previously classified as "nonA-nonB" hepatitis
o Genus Hepacivirus
o Incubation period: 7 to 8 weeks
o ACCOUNTS FOR THE MAJORITY OF CASES OF POST-
TRANSFUSION HEPATITIS
o 10%of chronic hepatitis C is attributed to blood
transfusion
o 80%of post-transfusion related hepatitis is caused by
HC V, 10%is caused by HBV
o MOT:
o Parenteral or via contaminated needles and
hospital equipments
o Blood transfusion
o Vertical transmission from mother to c hild
o Sexual contact
 Laboratory Assays for HCV
● Serology
○ Screening test:
Chemiluminescence and enzyme immunoassays
○ Confirmatory test:
Western Blot Radioimmunoblot assay[RIBA]
○ **Surrogate tests for HCV: ALT & (+)Anti-HBc

● Molecular Tests
○ HCV PCR (nucleic acid amplification test)
- Detects HCV RNA
- BEST CONFIRMATORY ASSAY

● Positive screening test with negative confirmatory test may indicate

○ False-positive reaction
○ Recovery from hepatitis C
○ Viral infection with viral load too low to be detected
Hepatitis D
● Genus Deltavirus
● Single-stranded, circular RNA coated in HBsAg
● A defective RNA viroid that requires HBV
surface antigen (HBsAg) for full expression,
replication, and transmission

● MOT: Parenteral transmission

○ can only occur in the presence of Hepatitis B

● No therapy has proved to be effective

● Infection with the two viruses occurs in 2 ways:
○ COINFECTION = both agents (HBV and HDV) are
acquired simultaneously, rarely leads to chronic
infection
○ SUPERINFECTION = there is an established HBV
infection after which HDV is acquired, can transform
mild infection into persistent infection *(sequentially)

● Anti-HDV IgG and IgM testing is available when HDV coinfection is

suspected in HBsAg-positive individuals

● Serologic markers: HDV Ag, Anti-HDV antibody

● Molecular markers: HDV RNA
Hepatitis E
● Reclassified as Hepeviridae
● Associated with HIGH MORTALITY RATE IN
PREGNANT WOMEN
● **FULMINANT LIVER FAILURE

● MOT:
○ Fecal-oral transmission
○ Ingestion of contaminated water

● Usually presents as an acute, self-limiting hepatitis

without progression to a chronic carrier state
● HEV ANTIBODY:
○ IgM Anti-HEV
■ Marker of ACUTE INFECTION but rapidly

declines in the early recovery period

■ May be detected by serologic tests

● HEV RNA
○ Detected in feces of most patients for about 2
weeks after the onset of illness
○ May persist longer in some cases
○ Identified using PCR
Other Hepatitis Viruses
● Hepatitis GB virus-C (GBV-C)
○ Formerly known as Hepatitis G (HGV)
○ Member of the family Flaviviridae
○ Genus Pegivirus
○ Considered as an ORPHAN VIRUS.
■ Has been identified in post-transfusion hepatitis
■ Does not appear to be hepatotropic, does not
replicate efficiently in hepatocytes, and does not
cause acute or chronic hepatitis

○ Coinfection with GBV-C and HIV appeared to induce

host antiviral activity against HIV and prolong survival.
☺
Other Hepatitis Viruses
● Hepatitis F
○ Hypothetical virus
○ Discovered in1994 by Deka et al.
○ novel viral particle in the stool of post-transfusion,
non-hepatitis A, non-hepatitis B, non-hepatitis C,
non-hepatitis E patients
○ injection of these particles into the bloodstream of
Indian rhesus monkeys caused hepatitis
○ named hepatitis F or Toga virus.
○ Further investigations failed to confirm the existence
of the virus, and it was delisted as a cause for
infectious hepatitis
Human
Immunodeficiency
Virus
Human Immunodeficiency Virus
● Etiologic agent of the Acquired Immunodeficiency
Syndrome (AIDS)
● from the Retroviridae family, Genus Lenti virus
● ssRNA, icosahedral, enveloped
● Has reverse transcriptase
○ enzyme that transcribesRNA to DNA
● 2 Types:
○ HIV-1: Common causative agent of HIV in the United
States and Europe
○ HIV-2: Less pathogenic and endemic in West
Africa
Main Structural Genes of HIV
1. Env (envelope)
○ Involved in the attachment and fusion of HIV to CD4+ cells
■ Gp160
● Gp120: knobs and spikes on the envelope(SU)
● Gp 41: spans the inner and outer membrane(TM)
2. Gag (group antigen gene)
○ Found in the nucleocapsid and nucleus
■ p55
● p15
● P17(MA)
● P24(CA)
Main Structural Genes of HIV
3. Pol (polymerase)
○ Reverse transcriptase: transcribes RNA to
DNA
○ Integrase : inserts viral DNA to host DNA
○ RNAse: located in the core near the nucleic
acid
○ Protease: cleaves structural proteins
Review!
● CXCR4(alpha chemokine) coreceptor on T cells
● CCR5(beta chemokine) coreceptor on macrophages

● 3 MAJOR ROUTES!!!
○ Intimate sexual contact
○ Parenterally from infected blood/body fluids
○ Perinatally (in vivo, breastmilk, during delivery)

○ NOT INFECTIOUS unless they contain VISIBLE INFECTED

BLOOD:
■ SALIVA, SPUTUM, SWEAT, NASAL SECRETIONS, TEARS,
URINE, VOMITUS
AIDS
● MOST ADVANCED STAGE of HIV infection
● Development takes about 2-15 years

● Diagnosis: <200/uL CD4 (<14%) + cell count AND presence

of an opportunistic infection or an “AIDS-defining illness”

● CD4+ T-CELL CATEGORIES:

○ Category 1: > 500/uL
○ Category 2: 200-499/uL
○ Category 3: <200/uL

● FULL BLOWN AIDS: characterized by Kaposi’s sarcoma, P.

carinii pneumonia, CMV and
AIDS-related dementia and illnesses
Examples of AIDS-defining
Illnesses ▪ M. avium or M. kansasii
▪ MTB infections infections
▪ Candidiasis ▪ Salmonella septicemia
▪ Coccidioidomycosis ▪ Kaposi’s Sarcoma
▪ Cryptococcosis
▪ Lymphomas
▪ Cryptosporidiosis
▪ CMV infection ▪ Recurrent pneumonia
▪ HSV infection ▪ Wasting syndrome
▪ Histoplasmosis ▪ Encephalopathy
▪ Isosporiasis ▪ Progressive multifocal
▪ Toxoplasmosis leukoencephalopathy
▪ P. carinii pneumonia ▪ Invasive cervical CA
● CLINICAL CATEGORIES
○ Category A
■ Asymptomatic HIV infection, generalized
lymphadenopathy and acute HIV infection with
accompanying illness
○ Category B
■ Symptomatic HIV infection with conditions
attributed to an impaired cell-mediated
immunity and conditions complicated by HIV
infection
○ Category C
■ Acquired Immunodeficiency Syndrome
● STAGES
○ Stage 1: Primary
■ Time during which people are MOST infective
○ Stage 2: Asymptomatic
■ HIV antibodies are detectable
■ Lasts for an average of 10 years
○ Stage 3: Symptomatic
■ Symptoms are mild, immune system
deteriorates, opportunistic infections and
cancers emerge
○ Stage 4: AIDS
Nice to Know!
Among HIV patients,
✔ TUBERCULOSIS: Most common cause of death

✔ CRYPTOCOCCOSIS: Most common fungal opportunistic infection

✔ PENICILLIUM MARNEFEII INFECTION: 3rd most common

opportunistic infection (after MTB and cryptococcus infection) among HIV patients

✔ CRYPTOSPORIDIOSIS: Most common parasitic opportunistic

infection
HIV Serology
A. SCREENING TESTS
a) Antibody Assay:
ELISA - STANDARD SCREENING TEST
■ Anti-p24 antibody
● FIRST ANTIBODY TO APPEAR

■ Anti-GP41 antibody

b) Antigen Assay:
■ p24 antigen detection- **identify infection during
WINDOW before abs appear
c) Agglutination tests
d) Dot-Blot testing
ELISA
FALSE POSITIVE
RESULT
● Heat inactivation of serum
prior to testing
● Repeated freezing/thawing
● Autoreactive antibodies
● Multiple pregnancies
● Severe hepatic disease
● Passive Ig administration
● Recent exposure to vaccines
● Malignancies
FALSE NEGATIVE
RESULT
● Collection of test serum prior
to SEROCONVERSION
● Immunosuppresive
therapy/replacement
transfusion
● Hypogammaglobulinemia
● Technical error
● Patient harbors a genetically
diverse, recombinant strain of
HIV
**LAB TESTS

● CD4 T-cell enumeration- GOLD

STANDARD: FLOW CYTOMETRY

● HIV NUCLEIC ACID TEST – to determine viral

load and dev’t of drug-resistant strains

● PCR – preferred mtd. for infants and children

<18 yrs. Old
○ UNRELIABLE ELISA
ACCORDING TO CDC: 

● ELISA yields (+) result --- RETEST IN

DUPLICATE with same ELISA test.

● IF 2 OUT OF 3 SPECIMENS (REACTIVE)

------- CONFIRM WITH MORE
SPECIFIC TEST, ex. WESTERN BLOT
HIV Serology
B. CONFIRMATORY TESTS
● Western Blot Testing
○ STANDARD CONFIRMATORY TEST
● Immunofluorescence Assay
● Line Immunoassay

● PCR - can be used for diagnosis of very early, post-

exposure HIV infection in the window period

NOTE: A REPEAT screening test is required before

proceeding to confirmatory tests
PCR
● Denaturation – dsDNA heated to 96 degrees C
● Annealing – cooled to 56 degrees C to allow primers to
bind/anneal to complimentary sequences on the separate DNA
strands
● Elongation/Extension- 72 degrees C, heat stable
polymerase(Taq polymerase) binds to the 3’ end of each primer
and synthesizes a new strand of DNA
CDC Criteria for Western Blot
● Presence of at least 2 out of possible 3 antibody
bands to p24, GP41 and GP160/120
✔p24: must marker
✔ Gp41: must marker (Transmembrane GP/TM)
✔ Gp120: good marker (Surface GP/SU/KNOBS)
✔ Gp160: good marker

● RULE:
✔ HIV (+): p24 and gp41 are BOTH PRESENT
✔ HIV (-): p24 and gp41 are ABSENT
✔ Indeterminate: Only one must marker is present (either p24
or gp41) repeat the test after 6 MONTHS
HIV confirmation of seropositive blood

● PATIENT
● DONORS
HERPESVIRUSES
● “Herpein” – meaning “ creep”
○ referring to the latent or recurrent infection
caused by herpes viruses

● Common factors among Herpes viruses:

○ Common virion morphology

○ Basic mode of replication
○ May establish latent and recurrent infection
○ Cell-associated
● Herpes Simplex
○ Incubation: 2-12 days
○ Most common manifestation: COLD SORES
○ 2 Types:
■ HSV-1: found in the oral cavity and parts of the body
ABOVE the waist
■ HSV-2: found in genital tract and skin lesions
BELOW the waist
● Associated with human cervical cancer
○ Herpetic whitlow- infection on the finger which
is painful but heals without treatment
● Diagnosis
1. Serology
2. Cytology
■ TZANCK TEST- examination of scraped-cells

obtained from herpetic lesions to look for Tzanck

cells (multinucleated giant cells)
■ Tzanck test uses Giemsa stain to visualize cells
3. Virus isolation
■ MOST DEFINITIVE ASSAY
■ Employs a tissue culture medium for viral
culture
● Varizella-Zoster Virus
○ Incubation: 14-17 days
○ MOT: Respiratory route/ Droplet transmission
○ Causes CHICKENPOX (common during childhood)
and its sequelae, SHINGLES (adulthood)

○ Diagnosis:
■ TZANCK test

■ Virus isolation-BEST CONFIRMATORY

METHOD;
uses Human Diploid Fibroblast cells
■ Serology-FAMA (Fluorescent Antibody to

Membrane Antigen) test

● Varizella-Zoster Virus

○ Active immunization
-uses live attenuated VZV
-recipient is deferred from donation for 4
weeks ☺

○ Passive immunization
-uses VZV immunoglobulin
-given within 4 days from exposure
● Epstein Barr Virus
○ MOST INTENSIVELY STUDIED HUMAN CANCER VIRUS
○ MOST UBIQUITOUS VIRUS KNOWN TO MAN
**transmitted through SALIVA, BLOOD TRANSFUSION,
TRANSPLACENTAL and possibly MOSQUITOES
• IM Postperfusion Syndrome-infxn from blood
transfusion
○ Causative agent of:
■ Infectious Mononucleosis/
Kissing’s Disease/ Glandular Fever and Burkitt’s Lymphoma
● Incubation is 10-50days
○ Receptor : CD21 in B cells
■ T cytotoxic cells respond by becoming Reactive/Atypical/Variant
Lymphocytes
■ Enlarged lymphocytes with Atypical Nuclei -> DOWNEY CELL
(ballerina skirt)
● Epstein Barr Virus

○ Antigens:
❑ EBNA: Epstein Barr Nuclear antigen
❑ LYDNA: Lymphocyte Detected Membrane Ag
❑ MA: Membrane Antigen
❑ VCA: Viral Capsid Antigen
❑ EA: Early Antigen
● Diagnosis:
1. Paul-Bunnel Test
■ Screening test for heterophil antibodies
■ Principle: HEMAGGLUTINATION
● Based on ability oh heterophil antibodies to agglutinate
sheep RBCs
● Px serum containing antibodies+Sheep RBCs
■ Positive result: Agglutination **(NORMAL TITER≤ 56)

1. Davidsohn Differential Test

■ Confirmatory/Verification Test for heterophile antibodies
■ Principle: HEMADSORPTION-HEMAGGLUTINATION
■ Uses Guinea pig kidney cells and Beef RBC for
adsorption phase
■ Employs Sheep RBCs for agglutination phase
 Heterophi Adsorption Agglutination w/
l Sheep RBcs
Antibody
GPK BE GPK BE
IM - + + -
Forssman + - - +
Serum + + - -
Sicknes
s !!!!
GPK: RICH IN FORSSMAN ANTIGENS

BEA: LITTLE TO NO FORSSMAN

3. Monospot Test
○ Spot test for IM based on the principle that horse RBCs are moree sensitive than sheep
RBCs in testing for IM
○ Uses 20% horse RBCs

● Serologic Markers of EBV

○ Viral Capsid Ag

■ Anti-VCA IgM (early stages)

■ Anti-VCA IgG (4-7 days)

○ Early Antigen

■ Anti-EA-D IgG (highly indicative of active infection)

○ Epstein Barr Nuclear Antigen

■ Found in nucleus of all infected cells

■ Seen in convalescent period
o Cytomegalovirus
o MOST COMMON VIRUS TRANSMITTED TO FETUS
o MOST COMMON CAUSE OF HETEROPHIL-
NEGATIVE MONONUCLEOSIS
o Diagnosis:
o Cytology-SHELL VIAL CULTURE: rapid detection of
CMV
o Cytologic finding: OWL’S EYE CPE
o Virus isolation-uses Human Diploid Fibroblast Cells
o Serology
o Hematology (w/ atypical lymphocytes)
o Molecular Methods-PCR (preferred method together with
cell culture)
● HSV-6
○ Causes sixth disease/ roseola infantum/
exanthem subitum
○ MOT: Respiratory route

● HSV-7
○ No disease association
○ “ORPHAN VIRUS”

● HSV-8
○ Causes Kaposi’s Sarcoma
WIDAL TEST
● Febrile antibody test
● detection of antibodies in typhoid fever, brucellosis,
tularemia
● Clinically significant titer: greater than or equal to 160

WEIL-FELIX TEST
● Uses cross-reacting Proteus vulgaris & Proteus mirabilis

antigens to diagnose rickettsial infection.

● Clinically significant titer: greater than or equal to 320

● Antigens used: OX-2, OX-19 – P. vulgaris

OX-K – P. mirabilis
WEIL FELIX
ORGANISM DISEASE OX-2 OK-19 OX-K

R. prowazekii
+ + 0
R. typhi
+ + 0
R. rickettsia
+ + 0
R. akari
0 0 0
R.
tsusugamushi 0 0 +
B. quintana
0 0 0
C. burnetii
0 0 0
MALARIA

● OptiMAL
• Detects pLDH produced by viable parasites in which
different isoforms of LDH are present in diff. species

• pLDH can be detected when there are 100-200

parasites/uL blood

MalaQUICK Standby Malaria Test

○ Detects plasmodium
species