Immunology and Serology MLS115 LABORATORY

MODES OF TRANSMISSION
OUTLINE 1. Direct contact with a syphilis sore
1. Syphilis  e.g. Kissing a person with active oral
a. Modes of Transmission lesion
b. Stages of Disease
i. Primary Syphilis 2. Sexual transmission – the primary mode of
ii. Secondary Syphilis dissemination
iii. Latent Syphilis vaginal, anal, or oral sex
iv. Chronic Syphilis
• Gummas 3. Pregnant women with the disease can pass
• Cardiovascular complications it to the babies they are carrying.
• Neurosyphilis (Congenital infections)
v. Congenital Syphilis  Transmission to the fetus is possible in
c. Diagnosis mothers with clinically LATENT disease.
i. Direct Detection of Spirochetes
• Darkfield microscopy 4. Other potential means of transmission:
• Fluorescent antibody testing ֍ parenteral exposure through
ii. Serological Tests
contaminated needles or blood
• Nontreponemal Tests
a. VDRL  extremely rare
b. RPR
2007 Guidelines by the American Red Cross
c. TRUST
for blood donation:
• Treponemal Tests
a. FTA-ABS Wait for 12 months after treatment
b. MHA-TP or TP-PA for syphilis before donating
d. Nontreponemal vs. Treponemal Tests
e. Treatment for Syphilis
* Because syphilis can only be transmitted by means
of fresh blood products, the use of stored blood
components has virtually eliminated the possibility of
1. SYPHILIS transfusion-associated syphilis.
‐
A sexually transmitted disease (STD) Pregnancy – most infectious during LATENT disease of
‐
Caused by Treponema pallidum
o a member of the family the mother  congenital infection
Spirochaetaceae Parental exposure – extremely rare, this is why excluded
o have no natural reservoir in the na sya sa screening sa blood banks
environment - But you have to wait 12 mos after treatment
o must multiply within a living host before ka makadonate if may ara syphilis
- Length: 6 to 2- mm - Need sang syphilis fresh blood to be transmitted,
- Width: 0.1 to 0.2 mm since ang bb nga blood ginastore, di na sya
- Number of coils: 6 to 14 coils
katransmit
- its outer membrane is a phospholipid bilayer
with very few exposed proteins - Place blood in room temp for 30 mins to 1 hr to
o identified proteins: TROMPS allow the bacteria to die before iplace sa
(Treponemal rare outer membrane refrigerator.
proteins)
* The scarcity of such proteins delays the host
immune response. STAGES OF THE DISEASE
1. Primary stage
2. Secondary stage
TROMPS – very few exposed proteins, cannot be easily
3. Latent stage
recognized by the immune system  delayed immune a. Early latent
response b. Late latent
4. Tertiary stage
5. Neurosyphilis Congenital syphilis
Immunology and Serology MLS115 LABORATORY
1. Primary Syphilis
• Once contact has been made with a
susceptible skin site, endothelial cell
thickening occurs with aggregation of
lymphocytes, plasma cells, and
macrophages.
• Initial lesion = CHANCRE
- develops between 10 and 90 days
after infection (average 21 days)
- a painless, solitary lesion
- characterized by raised and well-
defined borders
- in men: usually occur on the outside
of the penis
- in women: may appear in the vagina
or on the cervix (may go undetected)
• lasts from 1-6 weeks, during which time the lesion
heals spontaneously.
‐ after 3 weeks, chancre develops
‐ Chancre is more noticeable in men than women
‐ They are undetected most of the time cause they
are painless
2. Secondary Syphilis
• If the initial chancre is untreated, about 25%
of such cases progress to the secondary
stage.
• systemic dissemination of the organism
occurs
• usually observed about 1 to 2 months after
the primary chancre disappears
(15% of reported cases, primary
lesion may still be present)
Symptoms:
1. generalized lymphadenopathy (enlargement
of the lymph nodes)
2. malaise
3. fever
4. pharyngitis
5. RASH on the skin and mucous membranes
- rough, red, or reddish brown spots both on
the palms of the hands and the soles of
the feet
- rashes with a different appearance may
occur on other parts of the body
*The rash usually does not cause itching.
6. Involvement of the central nervous system
- may occur earlier than previously suspected
because viable organisms have been found in
the CSF of several patients with 1 0 or 20
syphilis.
7. neurological signs
a) visual disturbances
b) hearing loss
c) tinnitus
d) facial weakness
Lesions persist from a few days up to 8
weeks. After this time, spontaneous healing occurs.
The signs and symptoms of secondary
syphilis will resolve with or without treatment, but
without treatment, the infection will progress to the
LATENT and possibly LATE STAGES of disease.
‐ Systemic na
‐ Di na kinanglan patapuson ang primary stage,
kis-a gaoverlap sila
‐ If you don’t treat primary stage, maprogress sa
next stage
‐ Rashes occur not only on the skin but on other
parts as well  no itching
‐ Tinnitus  ringing in the ear, echoing of sound
pero wala man actually
‐ Pwede lang kaheal on their own, if indi galling
matreat, it will progress to latent or late stage
3. Latent syphilis
• Follows the disappearance of secondary
syphilis
• Characterized by a lack of clinical symptoms
• Divided into:
1. Early latent
- less than 1 year’s duration
2. Late latent
- the primary infection has
occurred more than 1 year
previously
• Patients are noninfectious at this time.
• Pregnant women can pass the disease on to
the fetus even if they exhibit no symptoms.
‐ Abi sang patients, healed na sila
‐ Tanan non-infectious, EXCEPT SA PREGNANT!!
‐ HIGH RISK OF CONGENITAL INFECTION
Immunology and Serology MLS115 LABORATORY
4. Tertiary Syphilis
• About 1/3 of the individuals who remain
untreated develop tertiary syphilis.
• Appears anywhere from months to years
after secondary infection.
• This occurs most often between 10 and 30
years following the secondary stage.
• Has 3 major manifestations:
1. gummatous syphilis
2. cardiovascular disease
3. neurosyphilis
a) GUMMAS
- localized areas of granulomatous
inflammation that are most often
found on bones, skin, or
subcutaneous tissue.
- can reach up to several
centimeters in diameter
- they contain lymphocytes,
epithelioid cells, and fibroblastic
cells.
- may heal spontaneously with
scarring, or they may remain
destructive areas of chronic
inflammation.
- they represent the host response
to infection.
- may scarring gd
b) CARDIOVASCULAR
COMPLICATIONS
- usually involve the ascending
aorta
- symptoms are due to destruction
of elastic tissue, especially in the
ascending and transverse
segments of the aortic arch.
- may result in:
• aortic aneurysm
• thickening of the valve
leaflets causing aortic
regurgitation
• narrowing of the ostia,
producing angina pectoris
- guba si ascending aorta
- angina pectoris  chest pain
c) NEUROSYPHILIS
- the complication most often
associated with the tertiary stage
- can occur anytime after the primary
stage and can span all stages of the
disease
- In immunodeficient individuals, there
has been a large rise in the incidence
of early neurosyphilis.
- During the first 2 years following
infection, central nervous system
involvement often takes the form of
ACUTE MENINGITIS.
- Late manifestation of neurosyphilis include:
1. tabes dorsalis – a degeneration of
the lower spinal cord
2. general paresis – chronic
progressive dementia
*It usually takes more than 10 years for these to
occur, but both are the results of structural central
nervous system damage and cannot be reversed.
*Fortunately, these symptoms are now very RARE
because of early detection and treatment with
penicillin.
- can happen even sa primary stage pa lang, pero
most often, it is observed in tertiary syphilis
- dementia  memory loss
- if tabes dorsalis and general paresis occur, indi na
mareverse back ang imo condition. ;-; sad
5. Congenital Syphilis
• Occurs when a woman who has early
syphilis or early latent syphilis transmits
treponemes to the fetus.
• Typically the fetus is most affected during the
2nd or 3rd trimester
• Fetal or perinatal death occurs in
approximately 10% of the cases.
• Infants who are liveborn often have no
clinical signs of disease during the first few
weeks of life.
• Some remain asymptomatic.
 But between 60-90% of such infants
develop later symptoms if not treated
at birth.
• Necrotizing funisitis
 inflammation of the umbilical cord
 the first indication of the disease
Immunology and Serology MLS115 LABORATORY
- Pregnant women need to be screened gd if may 1. Darkfield Microscopy
ara or wala syphilis - primary and secondary syphilis can be
- biskan normal nabata ang baby, maggrow sya, diagnosed by demonstrating the presence of
eventually madevelop ang symptoms T. pallidum in exudates from skin lesions
- A darkfield condenser is used to keep all
incident light out of the field except for that
Necrotizing funisitis
captured by the organisms themselves.
1. clear or hemorrhagic rhinitis or runny nose - it is essential to a good specimen in the form
2. skin eruptions (macropapular rash) of serous fluid from a lesion.
 prominent around the mouth, the  usually obtained by cleaning the lesion
palms of the hands, and soles of the with sterile saline and rubbing it with
feet clean gauze.
3. generalized lymphadenopathy - pathogenic treponemes are identified on the
4. hepatosplenomegaly basis of characteristic corkscrew
5. jaundice morphology and flexing motility.
6. anemia - because observation of motility is the key to
7. painful limbs identification, specimens must be examined as
8. bone abnormalities (saddle nose, saber shins) quickly as possible, before they become dried
9. neurosyphilis – 60% of infants with congenital out.
syphilis - False negative results can occur due to:
a. delay in evaluating the slides
DIAGNOSIS b. an insufficient specimen
Traditional laboratory tests for syphilis can be c. pretreatment of the patient with
classified into 3 main types: antibiotics
1. Direct detection of spirochetes *A negative test does not exclude a diagnosis of
2. Non-treponemal serological tests syphilis.
3. Treponemal serological tests * If a specimen is obtained from the mouth or the
rectal area, morphologically identical non-
pathogens can be found, so these must be
1. Direct Detection of Spirochetes
differentiated from the true pathogens.
 DIRECT MICROSCOPY
- primary and secondary stages
METHOD ANTIGEN ANTIBODY COMMENTS - need gd buhi ang specimen kay para
Darkfield T. pallidum None Requires active Makita ang motility
microscopy from lesion. Must
patient have good 2. Fluorescent Antibody Testing
specimen, - sensitive and highly specific alternative to
experienced darkfield microscopy
technologist; - can be performed by:
inexpensive a. Direct method – uses fluorescent-labeled
antibody conjugate to T. pallidum
Fluorescent T. pallidum Anti- Requires active
a. Indirect method – using antibody specific
antibody from treponemal lesion; more
for T. pallidum and a 2nd labeled anti-
testing patient antibody specific than
immunoglobulin Ab
with darkfield;
- advantage: Live specimens are not
fluorescent specimen does
required
tag not have to be
*A specimen can be brought to the laboratory in a
live.
capillary tube, and fixed slides can be prepared for
later viewing.
Immunology and Serology MLS115 LABORATORY
*Even after fixing, treponemes can be washed off - prone to PROZONE (Ab excess)
the slide, so each slide must be handled individually,
and rinsing must be carefully done. - reagin titers tend to decline in later
- more advantageous kay di na need live stages of the disease, even if the patient
specimen  less dangerous remains untreated.
- examples:
2. Serological Tests a. VDRL - Venereal Disease
- if a patient does not have active lesions, as Research
may be the case in 20 or 30 syphilis, then Laboratory
serological testing for antibodies is the key a. RPR – Rapid Plasma Reagin
to diagnosis. b. TRUST – Toluidine Red Unheated
Serum Test
 biskan wala na active lesions (needed if direct c. USR – Unheated Serum Reagin
detection), pwede lang japon serum or plasma d. RST – Reagin Screen Test

1. NONTREPONEMAL TESTS VDRL – commonly used

- determine the presence of REAGIN RPR – more common, kag mas dasig ubrahon
reagin – an Ab that forms against  black ang color due to charcoal
cardiolipin particles (carbon)
cardiolipin – a lipid
material from damaged cells
TEST ANTIGEN ANTIBODY COMMENTS
- found in the sera of patients with
syphilis and several other disease VDRL Cardiolipin Reagin Flocculation; good for
states screening tests,
- An antigen that is a combination of treatment monitoring,
cholesterol, lecithin, and cardiolipin spinal fluid testing; false
is used in the reaction to detect the positives
nontreponemal reaginic antibodies, RPR Cardiolipin Reagin Modified VDRL with
which are either of the IgG or IgM class. charcoal particles, More
- Ab to cadiolipin sensitive than VDRL in
- SCREENING ONLY primary syphilis
- determine presence of REAGIN (ab-like TRUST Cardiolipin Reagin Uses red particles to
substance nga Makita sa mga patients with visualize the
syphilis) reaction; Similar to RPR
- since antibody imo ginapangita, antigen ang
gamiton nga reagent 2. TREPONEMAL TESTS
-detect antibody directed against the T.
Flocculation – traditional method pallidum organism or against specific
- a specific type of precipitation that occurs treponemal antigens
over a narrow range of antigen concentrations -2 main types:
Flocculation – more on sa secondary stage, pero be a. FTA-ABS = Fluorescent
careful kay damo na ang antibody formation Treponemal Antibody-
(PROZONE) Absorption Test
- POSITIVE within 1 to 4 weeks after the - one of the most used
appearance of the primary chancre. confirmatory tests
- titers usually peak during the secondary - an indirect fluorescent
or early latent stages. antibody test
Primary – 13%-41% non reactive
Secondary - all patients are reactive
Immunology and Serology MLS115 LABORATORY
- a dilution of heat-inactivated - time consuming to perform
patient serum is incubated - usually positive before reagin
with a sorbent consisting of an tests (20% of primary syphilis
extract of nonpathogenic cases are NR)
treponemes (Reiter strain). - in 20 and latent syphilis =
 this removes cross- 100% reactive
reactivity with *Once a patient is reactive, that individual remains so
treponemes other than for life.
T. pallidum
a. FTA-ABS
- Slides used for this test have
the Nichols strain of T. - sa latent period
pallidum fixed to them. They - once reactive, maremain for life
are kept frozen until use and
then are equilibrated at room
temperature for 30 minutes. b. Agglutination tests
- Diluted patient samples and 1. passive hemagglutination
controls are measured and b.2. particle agglutination
applied to individual wells on b.3. microhemagglutination tests
the test slide. Slides are then
incubated in a covered moist  Particle agglutination: TP-PA
chamber at 37 C for 30 - uses colored gelatin particles
minutes. They are rinsed with coated with treponemal antigens
deionized water and placed in - more sensitive in detecting primary
a Coplin jar with phosphate- syphilis
buffered saline for 5 minutes.
a. Patient serum or plasma is
- After a 2nd rinsing, the slides
diluted in microtiter plates.
are air-dried, and Ab
conjugate (AHG conjugated b. Incubated with either T.
with fluorescein) is added to pallidum- sensitized gel
each well. particles or unsensitized gel
- Slides are reincubated as particles as a control.
before, and a similar washing - Presence of T. pallidum Abs = Agglutination
procedure is followed. - negative = gel particles settle to the well’s
- Mounting medium is applied, bottom and form a compact button
and coverslips are placed on
the slides.
TEST ANTIGEN ANTIBODY COMMENTS
- They are examined under a
fluorescence microscope as FTA-ABS Nichols strain Anti-treponemal Confirmatory;
soon as possible. of T. pallidum Specific,
- If specific Ab is present sensitive; May
 it will bind to the T. be negative in
pallidum antigens primary stage
- When slides are read under
MHA-TP or Sheep RBCs Antitreponemal Not as
a fluorescence microscope,
TP-PA or gel sensitive as
the intensity of the green
particles FTA-ABS
color is reported on a scale sensitized
of 0 to 4+ with T.
- negative result = No fluorescence pallidum
- reactive result = 2+ or above sonicate
- minimally reactive = 1+ - MHA-TP– microheme agglutination-
 test must be repeated
Treponema pallidum
with a 2nd specimen
drawn in 1 to 2 weeks b. Agglutination test (Treponema pallidum –
- highly sensitive and specific Particle agglutination TP-PA)
Immunology and Serology MLS115 LABORATORY
SENSITIVITY OF COMMONLY USED SEROLOGICAL - usually reactive before reagin tests in
TESTS FOR SYPHILIS primary syphilis
- suffer from a lack of sensitivity in
TEST PRIMARY SECONDARY LATENT LATE
congenital syphilis and neurosyphilis
(%) (%) (%) (%)
Non-Treponemal (Reagin) Tests
TREATMENT FOR SYPHILIS
VDRL 78 100 95 71
• Penicillin – Drug of Choice for treating
RPR 86 100 98 73 primary or secondary syphilis
Specific Treponemal Tests • a single intramuscular injection is
FTA- 84 100 100 96 usually effective.
ABS • Doxycyline – can be used as an alternative
MHA- 76 100 97 94 if the patient is allergic to penicillin
TP
• Azithromycin – can also be used, but
resistant strains are arising

Nontreponemal vs. Treponemal Tests

• Nontreponemal tests For NEUROSYPHILIS:
- inexpensive 1. Crystalline penicillin
- simple to perform 2. Procaine penicillin
- can yield quantitative results
- uses: 3. Probenecid
1. as a screening tool For CONGENITAL SYPHILIS
2. in monitoring the progress of
1. Crystalline penicillin – administered for 10
the disease days
3. in determining the outcome of
treatment – sa baby ihatag indi sa mama, okay?
- disadvantage:
¤ they are subject to false positives
Transient false positives occur in:
a. Hepatitis
b. Infectious mononucleosis
c. Varicella
d. Herpes
e. Measles
f. Malaria
g. Tuberculosis
h. During pregnancy
- if reactive
 should be confirmed by a
more specific treponemal test
• Treponemal tests
- more difficult to perform
- traditionally used as a confirmatory
tests to distinguish false-positive
from true-positive reagin results
- help establish a diagnosis in late
latent syphilis or late syphilis,
because they are more sensitive than
nontreponemal tests in these stages
 SUPPLEMENTARY NOTES
IMMUNOLOGY

SYPHILIS
- a contagious disease caused by a spirochete Treponema pallidum
- Treponema pallidum – extremely susceptible to heat and drying, so that direct transfer by
intimate contact, preferably in the presence of moisture, is essential for its survival.
- Sexual contact – an ideal mode of transfer of syphilis

Classification of Venereal Syphilis:

1. ACQUIRED
Stages:
a. Early
1. Primary – chancre develops 3-4 weeks after initial entrance of the treponemes
into the host.
2. Secondary – skin eruptions appear about 6 weeks later
- Treponema pallidum can be demonstrated in these lesions
- diagnosis is dependent upon the observation of the characterized
skin lesions, darkfield detection of T. pallidum in the lesions of
increasing serologic test for syphilis
3. Early latent – infectious
- with duration of more than 4 years

b. Late
- not infectious except for the fetus
- Treponema pallidum cannot be demonstrated from the lesion, serologic test will
remain reactive indefinitely or will descend over a period of years.
1. Late latent
2. Tertiary Benign
3. Gummatous
4. Quaternary

2. CONGENITAL SYPHILIS – affects the developing fetus

Stages:
a. Early
b. Late

PREPARATIONS OF SPECIMEN FOR SEROLOGICAL TEST FOR SYPHILIS

1. SERUM
a. Inactivate clear serum at 56 0C for 30 minutes or 60-62 0C for 5 minutes. If delayed,
reheat serum at 56 0C for 10 minutes.
b. Allow specimens to return to room temperature before testing
c. Specimens that are excessively hemolyzed, grossly contaminated with bacteria or
extremely turbid are unsatisfactory for testing.
d. Serum specimens not tested more than 4 hours are reheated at 56 0C for 10
minutes.

2. SPINAL FLUID
a. Centrifuge and decant the fluid.
b. Tested without preliminary testing.
c. Spinal fluids which are visibly contaminated or certain gross blood are
unsatisfactory for testing.

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 2 MAJOR GROUPS OF SEROLOGIC TESTS FOR SYPHILIS
1. Non-Treponemal Test or Reagin Test
2. Treponemal Test

NON-TREPONEMAL TEST
Cardiolipin-lecithin – antigen employed
- obtained by chemical extraction
= Cardiolipin  contains 4 phosphorus
 no nitrogen
 alcohol soluble
 acetone insoluble
 requires the addition of lecithin and cholesterol or other sensitizers
to flocculate or fix complement in the presence of syphilitic reagin
- Used to detect REAGIN – antibody-like substance
- [in theory] = the result of the interaction of Treponema with
body tissues
- Non-specific for syphilitic antibodies
- Inexpensive to carry out and easy to perform

Tests commonly employed to detect and measure reagin Ab:

1. Flocculation Method
a. VDRL
b. Kahn
c. Kline
d. Eagle
e. Manzzini
f. Hinton
g. Rapid Reagin Test
1. RPR – Rapid Plasma Reagin
a. RPR (Teardrop) card
b. RPR (circle) card
2. Plasmacrit
3. Unheated Serum Reagin (USR)

2. Complement Fixation
a. Kolmer Complement Fixation
b. Wasserman Method

VDRL Test
= Venereal Disease Research Laboratory

Reagents:
1. VDRL Antigen – stable for 1 day only
- addition of 0.05 of 1% benzoic acid to each single volume (5 mL)
will stabilize the antigen at 6-10 0C.
- consist of a proper balance of cardiolipin, purified lecithin,
cholesterol and alcohol which is standardized by adjustment of
the lecithin content to give reproduction qualitative and
quantitative results
Cholesterol – provides absorption centers so that
agglutinated particles can be visualized.

2. VDRL Buffered Saline – containing 1% NaCl

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 - pH 6.0 + 0.1-1% NaCl solution with phosphate buffer is
important for proper agglutination of antigen in the
presence of Ab, which in this system is called “REAGIN”
 not to be
confused with the term
that was applied to allergic
persons in the older
literature.

2 Ways of VDRL Testing

1. Slide Test – Qualitative Test
- Graded as:
Non-Reactive = No agglutination
Weakly Reactive = Slight agglutination
Reactive = Definite agglutination
2. Quantitative Test

 All REACTIVE sera are then diluted 1:2, 1:4 and so forth and each serum giving
reaction is reported.
 Rarely, a patient with a high titer of regain will have a negative VDRL with
undiluted serum (PROZONE PHENOMENON).
Prozone phenomenon – usually observed in secondary syphilis
Quantitative test results – obtained by diluting the serum in geometrical
progression until a non-reactive result is obtained
- the report is the endpoint titer.
- this type of result is often used as a therapeutic
index in
the treatment of the disease

Important Notes to Remember in VDRL Testing

1. SLIDE FLOCCULATION TEST for syphilis is affected by room temperature.
= should be performed within 73-85 0F or 23-29 0C
 to have reliable and reproductive results
At lower temperature
 test reactivity is decreased.
AT higher temperature
 test reactivity is increased

2. It is of primary importance that the proper amounts of reagents be used.

= needles used each day should be checked.

3. SLIDE QUALITATIVE TEST on serum, dispense antigen suspension from a

syringe fitted with an 18-gauge needle without bevel which will deliver 60 drops
+/- 2 drops of antigen suspension per mL when the syringe and needle are held
vertically.

4. SLIDE QUANTITATIVE TEST on serum, dispense antigen suspension from a

syringe fitted with a 19-gauge needle (with or without bevel) which will deliver
100 drops +/-2 drops on antigen suspension per mL when the syringe and needle
are held vertically.

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 5. SLIDE QUANTITATIVE TEST on serum, dispense 0.9% saline from syringe fitted
with a 23-gauge needle (with or without bevel) which will deliver 100 drops +/-2
drops of saline per mL when the syringe and needle are held vertically.

RPR (Rapid Plasma Reagin)

- antigen: Modified VDRL antigen suspension with choline chloride and EDTA
added.
- does not require inactivation because of the choline chloride that has been added to
the reagents to inactivate the substances present in the unheated serum.
- mostly used as a screening procedure in field situations where equipment may be
limited.
- more sensitive
- less specific than VDRL
- any positive test must be followed by a VDRL or any other more specific procedures.

1. RPR (Teardrop) Card Test

- uses plasma from a fingerstick blood specimen
- reaction is carried out on a plastic-coated card that is rotated by hand.
2. RPR (Circle) Card Test
- uses unheated serum and a plastic-coated card
- requires mechanical rotator
- comparable to the VDRL qualitatively but not quantitatively
- now accepted as an alternative to VDRL as a general screening test for syphilis

TREPONEMAL TESTS
- Use Treponema pallidum antigens to detect specific antibodies developed in response to
treponemal infections
- Not designed for routine use but are reserved as verification procedures to be performed
on diagnostic problem cases.
- Highly specific and sensitive
- Once a diagnosis of syphilis is made, is not useful in the evaluation of a therapeutic
response
- Expensive

Tests employing T. pallidum or extracts of the treponemes as antigen:

1. COMPLEMENT FIXATION METHOD
a. RPCF = Reiter Protein Complement Fixation
b. KRP = Kolmer Reiter Protein
c. TPCF = Treponema pallidum Complement Fixation

2. Immobilization Test
a. TPI = Treponema pallidum Immobilization

3. Agglutination Test
a. TPA = Treponema pallidum Agglutination
b. TPHA = Treponema pallidum Hemagglutination
c. MHA-TP = Microhemagglutination-Treponema pallidum

4. Immunofluorescence
a. FTA-200 – Fluorescent Treponemal Antibody 200
b. FTA-ABS – Fluorescent Treponemal Antibody-Absorption Test

5. ELISA
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 Complement Fixation Methods
RPCF & KRP
- Antigen: protein extract from the Reiter strain of T.pallidum (non-pathogenic)
- require bacteriolytic and haemolytic amboceptor to complete the system

TPCF
- Antigen: extract of virile T. pallidum
- require both bacteriolytic and haemolytic amboceptors to complete the system

Immobilization
TPI
- the serum of syphilitic patients contains specific antibodies against T. pallidum
- these antibodies are capable of immobilizing the actively motile spirochetes
- serial dilutions of the patient’s serum (a normal serum, T. pallidum obtained from
the
testicular chancre of an infected rabbit).
- the serum antigen mixtures are examined under darkfield illumination and the
highest
dilution of serum which readily immobilizes the spirochetes reported as the
immobilizing antibody titer of the patient’s serum.
- the test, in which the antigen is the actual causative agent of syphilis and not lipoidal
extract of mammalian tissue, represents a true antigen-antibody reaction and is not
subject to the biological false positive reactions which plague the reagin test.
- considered the most reliable
- has been the standard test against which all other treponemal tests were evaluated
- has certain limitations:
1. It does not distinguish the various treponematoses.
2. It cannot distinguish between active and latent infection.
3. It cannot be used as an index of therapeutic response.
4. It fails to detect early syphilis.
5. It is ineffective when the patient is on antibiotics.

Immunofluorescence
FTA-ABS
- antigen: Nichols strain of T. pallidum (growth in rabbit testes) fixed to a slide, and
overlaid with the test serum.
- a 1:5 dilution of the patient’s serum was used, but this gave too many false (+)
reactions owing to non-specific antibodies in patient sera.
- continued dilution of serum to 1:200 (FTA-200) removed the non-specific reactions,
but many true (+) reactions were also diluted out.
- the patient’s serum is diluted 1:5 in sorbent to remove the non-specific antibodies
before reacting the serum with the Nichol’s strain of T. pallidum.
- fluorescent-labeled anti-human globulin then demonstrates the presence of the
patient’s Ig on the surface of the treponemes.
- has the first 3 limitations of the TPI test
- able to detect early cases
- not affected by antibiotics
- simply and quickly performed
- highly specific and sensitive

MTEC 205 – IMMUNOLOGY & SEROLOGY Supplementary Notes in Syphilis Page 5 of

6
False (+) Serologic Test for Syphilis
1. Technical False (+)
a. Spontaneous deterioration of complement
b. Anti-complementary antigen
c. Cell suspensions are too thick – red blood cells will overcome the complement and
produce no hemolysis
d. Amboceptor too weak

2. Biologic False (+) – may be obtained in:

a. Leprosy
b. Malaria
c. Infectious mononucleosis
d. Rat-bite fever
e. Relapsing fever
f. Leptospirosis

MTEC 205 – IMMUNOLOGY & SEROLOGY Supplementary Notes in Syphilis Page 6 of

6
Immunology and Serology MLS115
2. Toxoplasmosis
 Toxoplasmosis is a widespread disease in
humans and many species of animals.
 This infection is caused by Toxoplasma
gondii
 recently recognized as a tissue coccidian
that belongs to the Phylum Apicomplexa
 this is a parasite
Epidemiology
 Toxoplasma gondii was first discovered in a
North African rodent and has been observed
in numerous birds and mammals worldwide,
including humans.
 a parasite of cosmopolitan distribution
able to develop in a wide variety of
vertebrate hosts.
Definitive Host: House Cat and certain other
Felidae
 definitive host is where the parasite
lasts/end
Mode of Transmission
 Accidental ingestion of mature oocyst in
undercooked meats
 Infective stage of T. gondii is mature
oocyst
 by accidental ingestion of undercooked
meat with mature oocyst
 if the meat was cooked properly, it will not
transmit T. gondii
 Food and water contaminated with
household cat feces
 The cat plays a vital role in the life cycle
of the T. gondii
 If you accidentally ingested infected
food/water, definitely you will have T.
gondii
 Transfusion – if leukocyte concentrates are
used
 It has been identified that it can only be
found in leukocyte concentrates
 Transplacental – takes place in the course of
an ACUTE but inapparent or undiagnosed
maternal infection
 This means to say that the mother is
pregnant and while being pregnant,
nakuha nya si T. gondii.
 The baby/fetus will be affected
 ACUTE= active; apparent = indi
halata/kitaon; undiagnosed= not able to
do some tests.
 It is a protocol that pregnant mothers will
undergo Toxoplasmosis testing
Major Sources of Human infection:
 Fecal contamination of food and water
 Fecal contamination because of the
feces coming from the cats
 Soiled hands
 This happens when you’re handling
meat(with mature oocyst) and you were
not able to wash your hands.
 Inadequately cooked or infected meat
 Undercooked meat (medium rare)
 Raw milk
 It is recommended that ALL PREGNANT
women BE TESTED for toxoplasmosis
immunity.
 Because there are cases of T. gondii that
are undiagnosed, inapparent, yet active
ang infection
 If a patient is susceptible, screening should
be repeated during pregnancy and at
delivery.
 Patient is pregnant.
 Prevention of infection in pregnant women
should be practiced to avert congenital
toxoplasmosis.
 It is important that testing should be
practiced to rule out the chance of having
Toxoplasmosis
 To further prevent infection of the fetus,
women at risk should be identified by
serologic testing, and pregnant women with
primary infection should receive drug
therapy.
Signs and Symptoms
 Adults and children  Asymptomatic
(other than newborns) as long as the immune
system of the patient
is functioning well
- A generalized infection probably occurs.
- Although spontaneous recovery follows
acute febrile disease.  The organism can
localize and multiply in any organ of the body
or the circulatory system.
 Generalized infection, exhibit nonspecific
signs and symptoms like fever and
myalgia
 Toxoplasmic encephalitis in AIDS patients
may result in death and even when treated.
 Another complication of toxoplasmosis
because not only that the baby is affected
if the mother is pregnant but, likewise
Immunology and Serology MLS115
there is a tendency that anybody who has
Toxoplasmosis can develop to have
encephalitis if their immune system is
really down such as in cases of AIDS
patients.
 Once it has already affected the CNS,
even if you have treated it, it is already
irreversible.
 persons at risk can be identified by
screening patients positive for
HIV(high-risk px) for antibody to T.
gondii.
ACQUIRED INFECTION
- symptoms are frequently MILD. - (5) variable coinfections with other
Toxoplasmosis can stimulate infectious
mononucleosis, with chills, fever, headache,
lymphadenopathy and extreme fatigue
 if you have ACUTE Toxoplasmosis,
pwede sya mka tulod nga mag gwa ang
infectious mononucleosis.
- Primary infection- may be promoted by
immunosuppression.
- A chronic form of toxoplasmic
lymphadenopathy exists.
- T. gondii presents a special problem in
immunosuppressed or otherwise
compromised hosts.
(Hodgkin’s and on-Hodgkin’s
lymphoma, recipients of organ
transplant)
 some of these patients have experienced
REACTIVATION of a latent
toxoplasmosis.
 Stages: Primary Toxoplasmosis, Chronis
form of Toxoplasmosis, also consider
that there is a tendency of reactivation of
latent Toxoplasmosis. Latent= it doesn’t
mean that you don’t have the dse.
nagpahuway ang dse. And if you are
immunocompromised there is a tendency
of reactivation
REACTIVATION OF CEREBRAL
TOXOPLASMOSIS
- common in patients with AIDS
- often occurring when the total CD4 count
falls below 100 X 109/L
 100 is nubo because you should at least
have a 1000 esp in AIDS px, manubo pgd
depending on how the body can fight
against the virus(?)
T. gondii-positive HIV-infected patients may develop
toxoplasmic encephalitis because of:
(1) genetic susceptibility in the human
immune response to T. gondii
(2) subtle differences in patients’
immunocompromised status
 not all have the same level of activity in
the immune system. May hina immune system, may
kusog immune system esp/ AIDS px
(3) differences in the virulence of individual
strains of T. gondii
(4) possible recurrent infections with
different strains
opportunistic pathogens
CONGENITAL INFECTION
 When the mother has Toxoplasmosis,
there is a possibility of passing it to the
baby (Transplacental)
- can result in CNS malformation or prenatal
mortality.
 Wala pa nabata ang bata napatay na sya
- infants who are serologically positive at birth,
many fail to display neurologic, ophthalmic,
or generalized illness at birth.
 baby positive with T. gondii can be born
normally/naturally but do not present the
neurologic, ophthalmic, or generalized
illness
- acquired in utero can result in:
a. Blindness
b. Encephalomyelitis neonates
c. mental retardation
d. convulsions
e. death in infected
- all these are called (TORCH syndrome)
IMMUNOLOGIC MANIFESTATIONS
 Clinical & Lab findings  resemble IM
(infectious
mononucleosis)
 increased number of variant
lymphocytes in peripheral blood smear.
 variant lymphocytes are not normal
lymphocytes aka reactive lymphocytes.
 The diagnosis can be established
serologically by detecting a marked of
elevation of Toxoplasma antibodies.
 Antibodies are demonstrable within the first
2 weeks after infection, rising to high levels
early in the infection, then falling slightly, but
persisting at an elevated level for many
months before declining to low levels after
Immunology and Serology MLS115
many years. Diagnostic Evaluation of Toxoplasmosis
 That’s why it can produce chronic TEST Recommended Use
infection Toxoplasma gondii First-line test in
antibodies, IgG and IgM endemic areas for
presence of IgM to T. gondii  INFECTION identifying T. gondii
 If there is the presence of IgM (primary infection in pregnant
response antibody), that means to say, women Diagnostic of
na-produce sya because of an existing opportunistic infections
infection. But if IgG ang ara sa imo, that in
means to say ayo kna because IgG is the immunocompromised
second response antibody and hosts.
sometimes IgG persists throughout life. T. gondii by polymerase Confirmation of
chain reaction (PCR) toxoplasmosis infection
low levels of Abs that may persist for more than 12 in
months after infection immunocompromised
hosts.
DIAGNOSTIC EVALUATION
T. gondii antibody, IgM When results from
The diagnosis of toxoplasmosis can be established initial antibody from
by the following: testing are equivocal.
A. Serological tests T. gondii antibody, IgG When results from
B. Polymerase chain reaction (PCR) initial antibody from
C. Indirect Fluorescent antibody (IFA) testing are equivocal.
D. Isolation of the organism IgM Antibodies
Serologic Tests  IgM assay - is not recommended for routine
 serological testing - the mainstay of use in adults because it may yield frequent
diagnosis of T. gondii infection. false-positive or false negative results to:
a. Immunocompromised patients
 ELISA - the method of choice for detection of
b. pregnant women
IgM antibodies in toxoplasmosis.
c. patients exposed to places where
 For the detection of IgM antibodies to T.
Toxoplasma infection is highly
gondii:
endemic.
o indirect immunofluorescence
 Appears for few months and some persists
antibody (iIFA)
up to 12 years in patients with recently
o ELISA tests
acquired infection.
 In direct qualitative enzyme immunoassay for
 • IgM-positive sera undergo confirmatory
IgM, or IgG, IgM (capture):
testing
o microparticle enzyme immunoassay
results are interpreted as:
using the automated AxSYM System
1. a recently acquired infection
o chemiluminescent immunoassay
2. an infection acquired in the past
 A panel of tests performed by specialized
3. a false-positive result
reference laboratories can determine
whether an infection is consistent with a new
IgA Antibodies
or past infection.
 detected in: acutely infected adults
The T. gondii serologic profile (TSP) consists of the congenitally infected infants
ff:  detected using ELISA or Immunosorbent
o IgM, IgA, and IgE ELISAs agglutination Assay (ISAGA) methods
o IgE immunosorbent agglutination assay  persist for many months to more than 1 year
o Sabin-Feldman dye test (IgG) with acute infections
o Differential agglutination test (AC/HS  IgA can be detected in first trimester of
test, IgG antibody) pregnancy in determining if there is an
infection.
 Increased sensitivity of IgA assays over IgM
assays for diagnosis of congenital
toxoplasmosis represents an advance in
Immunology and Serology MLS115
diagnosis of the infection in the fetus and
newborn.
IgE Antibodies
 detectable by ELISA in:
acutely infected adults
congenitally infected infants
children with congenital toxoplasmic
chorioretinitis
 The duration of IgE seropositivity is less than
with IgM or IgA and appears useful as an
adjunctive method for identifying recently
acquired infections.
IgG Antibodies
 appear 1 to 2 weeks after the initial infection,
peak after about 6 to 8 weeks, decline
gradually over the next 1 to 2 years and in
some cases, persists for life.
 The most widely used tests to measure IgG
responses to T. gondii are:
1. Sabin-Feldman dye test (DT),
2. ELISA
3. IFA test
4. modified (differential) agglutination
test
Sabin Feldman Dye Test
 Considered as the GOLD STANDARD
 IgG are primarily measured - a sensitive and
specific neutralization test
 live organisms are lysed in the presence
of complement and the patient’s IgG T.
gondii specific antibody.
 IgG Abs usually appear within 1-2 weeks of
the infection, peak within 1-2 months, fall at
variable rates, and usually persist for life.
 available mainly in reference laboratories
ELISA
 detects IgG antibodies to T. gondii.
 No single IgG titer can differentiate a recent
infection from a previously acquired infection.
IFA
 uses killed organisms as a substrate, with
patient serum assayed for activity against
them.
 used widely because it measures the same
antibodies as Sabin-Fieldman Dye Test.
False (+) = sera that contain antinuclear
antibodies
False (-) = when using sera from patients
with low titers of IgG antibody
Differential Agglutination Test
 also known as AC/HS test
 uses two antigen preparations that express
antigenic determinants:
AC antigen  found early after acute
infection
HS antigen  found in later stages of
infection
 Ratios of titers using AC versus HS antigen
are interpreted as ACUTE, EQUIVOCAL,
NON-ACUTE patterns of reactivity, or
NONREACTIVE.
 This test helps in differentiating a recent
(acute) from a remote (chronic) infection in
adults and older children, also in pregnant
women.
Avidity Test
 Can be used as an additional confirmatory
diagnostic tool in patients with a positive or
equivocal IgM test or with an acute or
equivocal pattern in the AC/HS test.
 Performed kung indi klaro ang result sng
AC/HS test
 The avidity result is determined using the
ratios of antibody titration curves of urea-
treated and untreated serum.
Polymerase Chain Reaction
 Mainly used to detect T. gondii DNA in the
body fluids and tissues.
 Can also be used to detect the presence or
absence of T. gondii DNA in fresh or frozen
biopsy tissue, CSF, Amniotic fluid, serum, or
plasma.
Lateral Blot Rapid Tests
- A special test
 Lateral blot Toxo IgG card tests (BioQuant,
San diego) is a chromatographic
immunoassay in which a lateral flow
immunoblot assay designed for qualitative
detection of IgM or IgG Abs to T. gondii in
human serum or plasma.
 Histologic diagnosis is the demonstration of
tachyzoites in tissue sections or smears of
body fluids establishes the diagnosis of the
acute infection.
Treatment:
 Sulfadiazine and Pyrimethamine can be
used to treat patients with acute
toxoplasmosis.
Immunology and Serology MLS115
OUTLINE 2. blood transfusion
1. INFECTIOUS MONONUCLEOSIS 3. bone marrow and solid organ transplants
I. Epstein-Barr Virus
4. sexual contact
a. MOT
II. Epstein-Barr Virus Antigens 5. perinatal transmission
a. Antigens identified in EBV-
infected cells  In developing nations:
b. Summary - EBV infections usually occur during early
III. Clinical Manifestations
IV. Laboratory Findings childhood
V. Serological Responses of Patients with EBV-Associated  In industrialized nations:
Diseases - infections are typically delayed until
VI. Supplementary Notes adolescence or adulthood
2. CYTOMEGALOVIRUS INFECTION by adulthood  more than 90% of
I. Epidemiology
II. Transmission individuals have been
III. Latent infection infected
a. True Viral Latency - Selectively infects host cells that are positive
b. Latency (operational definition) for the CD21 molecule  receptor for the
IV. Congenital Infection
V. Signs and Symptoms virus
VI. 3 types of CMV infections possible in o CD = cluster designation
blood transfusion recipients - The virus initially infects epithelial cells in the
VII. Immunological Manifestations oropharynx, where it enters a lytic cycle (viral
VIII. Laboratory Evaluation
IX. Supplementary Notes
replication, lysis of host cells, release of
3. HEPATITIS SEROLOGY infectious virions) until the acute infection is
I. Hepatitis virus resolved.
II. Fulminant Hepatitis - The virions also infect B lymphocytes, which
III. Signs and Symptoms spread the virus throughout the
IV. Hepatitis A
V. Hepatitis B
lymphoreticular system.
VI. Hepatitis C - The virus-infected B cells become
VII. Hepatitis D polyclonally activated, proliferating and
VIII. Hepatitis E secreting a number of antibodies, including
EBV-specific antibodies, heterophile
1. INFECTIOUS antibodies; and autoantibodies such as cold
agglutinins, rheumatoid factor, antinuclear
MONONUCLEOSIS antibodies.
- Caused by a virus known as Epstein-Barr - Just by mere infection, ga release na sa mga
Virus antibodies
EPSTEIN-BARR VIRUS
- Causes a wide spectrum of diseases: EPSTEIN-BARR VIRUS ANTIGENS
1. Infectious mononucleosis Antigens identified in EBV-infected cells:
2. Burkitt’s lymphoma Antigens produced:
3. nasopharyngeal carcinoma
4. neoplasms of the thymus, parotid 1. EA = Early Antigens
gland, and supraglottic larynx - during the initial stages of viral replication in
- not only causing one disease (IM) but may the lytic cycle
also be an agent of cancers. The one we call - classified based on their location within the
oncogenic viruses. cell
- Oncogenic viruses are the viruses that can
cause cancers a. EA-D = has a diffuse distribution in the
MODE OF TRANSMISSION nucleus and cytoplasm
1. intimate contact with salivary secretions from b. EA-R = restricted to the cytoplasm only
an infected individual 2. Late Antigens
- in the form of kissing [alangan naman
mag duplaanay kmo da (de la Rama, - those that appear during the period of the
lytic cycle following viral DNA synthesis
2021) 😭 ]
a. VCAs = Viral Capsid Antigens - in the
- Infectious mononucleosis aka “kissing
protein capsid (of the virus)
disease”
Immunology and Serology MLS115
b. MAs = Membrane Antigens - in the viral LABORATORY FINDINGS
envelope - Characteristic finding:
3. LATENT PHASE ABSOLUTE LYMPHOCYTOSIS
a. EBNA = EBV Nuclear Antigen (>50% of the total WBCs and
(EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, at least 10% atypical
EBNA3C, EBNA-LP) lymphocytes)
atypical lymphocytes (or reactive
b. LMP = Latent Membrane Proteins lymphocytes) – activated cytotoxic T cells
(LMP-1, LMP-2A, LMP-2B) - Serological findings: presence of a
SUMMARY heterophile antibody
antibodies to certain
EARLY EBV antigens
ACUTE LATE PHASE LATENT PHASE
PHASE - the presence of the heterophile
antibody, mka pa-conclude nga may
EA-R (early VCA (viral EBNA (EBV IM
antigen capsid nuclear antigens)
restricted) antigen) - pag positive, automatic that is IM
supported by other laboratory testing
EA-D (early MA EBNA-1, EBNA-2, such as CBC
antigen diffuse) (membrane EBNA-3A, EBNA-
antigen) 3B, 3BNA-3C, SUMMARY
EBNA-LP
Classic Laboratory Findings in Acute Infectious
Mononucleosis
Latent membrane ASSAY RESULT
proteins (LMP-1,
LMP-2A, LMP-2B) Heterophile antibody test Positive
Anti-VCA IgM Elevated titer
CLINICAL MANIFESTATIONS -kung elevated ang
- Clinical manifestations vary with the host’s IgM meaning bag-o
age and immune status. pa nga infection

- In infants and young children: Liver enzymes Elevated

ASYMPTOMATIC or MILD -SGPT
- In adolescents or adults: INFECTIOUS
MONONUCLEOSIS (IM) Leukocytes differential Increased number of
- 3 classic symptoms: variant (atypical)
1. fever lymphocytes
2. lymphadenopathy
3. sore throat
- non-specific symptoms Heterophile antibodies – are antibodies that are
- Other symptoms: capable of reacting with
1. splenomegaly similar antigens from 2 or
2. hepatomegaly more unrelated species
3. periorbital edema - sometimes, these unrelated species comes
- Symptoms usually last for 2 to 4 weeks, but from animals like horses, sheep, even pigs.
fatigue, myalgias, and need for sleep can
persist for months. - Heterophile antibodies associated with IM
are IgM antibodies produced as a result of
- Although these symptoms are essential in polyclonal B-cell activation (of EBV) and are
diagnosing IM, they can also be caused by capable of reacting with horse RBCs, sheep
many other infectious agents, so RBCs, and bovine RBCs.
LABORATORY TESTING plays an
important role in differentiating IM from other ‐ these Abs are produced by 40%
infections. of patients with IM during the first
week of clinical illness and by up
Immunology and Serology MLS115
to 90% of patients by the 4th further testing for EBV-specific antibodies is
week. indicated.
- Kung ga lawig nga ga lawig ang infection nga - you have to do further testing
wala treatment, naga taas ang
level/percentage/concentration sng EBV-specific antibodies are detected by:
heterophile antibodies.
1. IFA = indirect immunofluorescence assays
‐ they disappear in most patients
by 3 months after the onset of - using EBV-infected cells
symptoms but can be detected in
2. ELISA techniques
some patients for up to 1 year.
- using recombinant or synthetic EBV proteins
- Because heterophile antibody is present in *Both methods have a high level of sensitivity (95-
most patients during the acute phase of 99%),
illness, testing for this antibody is typically IFA – “gold standard” = higher level of
performed as a SCREENING TEST FOR IM specificity
in patients who present with symptoms of the
disease. Many laboratories prefer ELISA tests, because:
֍ less time-consuming
֍ easier to interpret
“Monospot”
- a rapid slide agglutination method that More recent method:
detects the heterophile antibody of IM CHEMILUMINESCENCE-based detection of the
- tests the ability of serum absorbed with antibodies
guinea pig kidney or beef erythrocyte IgM to VCA (Viral Capsid Antigen)
antigens to agglutinate horse RBCs - the most useful marker for acute IM
 usually appears at the onset of clinical
- the antibody could then be titered by symptoms
incubating serial dilutions of the patient’s  disappears by 3 months
serum with sheep RBCs in the Paul-Bunnell IgG anti-VCA
test - present at the onset of IM but persists for life
- Paul-Bunnell test – differential test  indicate past infection
Abs to EA-D (Early Antigen – Diffused)
*These methods (Monospot and Paul-Bunnell test)
- seen during acute IM
have been replaced today by: Anti-EBNA
1. RAPID LATEX AGGLUTINATION TESTS or - EBNA one of the antigens that are seen in
latent phase
2. SOLID PHASE IMMUNOASSAYS
- appears during convalescence
using purified bovine RBC extract as the antigen.
sensitivity: 71-95% in adults Serological Responses of Patients with
25-50% in children under 12 years EBV-Associated Diseases
old ANTI-VCA
specificity: 82-99%
CONDITION IgM IgG IgA
False positive – can occur in patients with: Uninfected Negative Negative Negative
a. Lymphoma IM + ++ ±
b. Viral hepatitis
c. Malaria Convalescent IM Negative + Negative
d. Autoimmune disease Past infection IM Negative + Negative
Negative heterophile antibody results occur in:
 about 10% of adult patients with IM Chronic active Negative + + + ±
 up to 50% of children less than 12 years infection IM
old Post-transplant Negative + + ±
* In these patients, who demonstrate symptoms of lymphoproliferative
IM but are negative for the heterophile antibody, disease
Immunology and Serology MLS115
Burkitt’s lymphoma Negative + + + Negative HETEROPHILE AB
CONDITION
- chronic (IgM)
form of
cancer Uninfected Negative
Nasopharyngeal Negative + + + + IM +
carcinoma
Convalescent IM ±
Past infection IM Negative
ANTI-EA
Chronic active infection Negative
CONDITION EA-D EA-R IM
Uninfected Negative Negative Post-transplant Negative
IM + Negative lymphoproliferative
disease
Convalescent IM Negative ±
Burkitt’s lymphoma Negative
Past infection IM Negative Negative
Nasopharyngeal Negative
Chronic active infection + ++
carcinoma
IM
Post-transplant + +
lymphoproliferative - Molecular tests may be more reliable than
disease serology in immunocompromised patients
who may not demonstrate a good humoral
Burkitt’s lymphoma ± ++ response, and they are also useful in
monitoring viral load in patients with EBV-
Nasopharyngeal ++ ± related malignancies who are undergoing
carcinoma therapy.
- It has been established that you do not rely
solely on serological tests but you also have
CONDITION ANTI-EBNA to look for other alternative methods esp if
Uninfected Negative the patients are immunocompromised
because if immunocompromised sila, some if
IM Negative them could not react to the presence of
Convalescent IM + antibodies/ could not produce antibodies.

Past infection IM +
Chronic active infection ±
IM
Post-transplant ±
lymphoproliferative
disease
Burkitt’s lymphoma +
Nasopharyngeal +
carcinoma
 SUPPLEMENTARY NOTES
Infectious Mononucleosis
Infectious Mononucleosis
- A.k.a. Glandular Fever
- A.k.a. Kissing Disease – transmitted through saliva
- An acute/subacute benign infectious viral disease of the reticuloendothelial system
- Onset is insidious
- Main clinical symptoms:
a. Fever d. Malaise
b. Chills e. Sore throat
c. Headache f. Enlargement of lymph nodes, liver, spleen
- Hematologic picture:
a. Mild leukocytosis
b. Lymphocytosis
c. Presence of atypical lymphocytes (DOWNEY CELLS) after a short while
d. Follows presence of heterophil antibodies

1911 – Forssman = noted that when rabbits were injected with tissues from guinea pigs, cats, or horses,
they produced antibodies against sheep RBCs.

Forssman heterophile antigens – agents producing this response.

Forssman heterophil antibodies – the corresponding antibodies
- one of a family of heterophile antibodies which have
the ability to react with antigens that were not
responsible for their production

heterophil antibodies – present in normal individuals, in patients with infectious

mononucleosis and in patients with serum sickness
- produced by one species and can infect other species

SEROLOGIC TESTS
Methods:
1. Paul-Bunnell Test  a presumptive hemagglutination test for heterophile antibodies
2. Davidson’s Test
a. Davidson’s Presumptive Test  routinely performed to determine the titer of total
heterophil antibody contents of the serum
1:224 or higher – considered as a (+) routine presumptive test in the presence of clinical
and/or cytological findings suggestive of IM.

b. Davidson’s Differential Test

- Differential because you have to identify nga ang ina nga heterophile antibody is
really the heterophile antibody that is produced by IM because probably there are
heterophile antibodies nga gina produce sng other diseases. The heterophile antibody
may react with other diseases.
Principle:
Absorption with a suspension of a Forssman antigen (Horse/Guinea pig kidney)
removes anti-sheep agglutinins in the serum of patients with serum sickness but
not from patients with infectious mononucleosis.
 Absorption with a suspension of beef cells removes anti-sheep agglutinin in
infectious mononucleosis.

Type of Heterophile Absorbed by Guinea Absorbed by Absorbed by Beef

Antibodies Pig Kidney Antigen Sheep’s RBC Erythrocyte Antigen
1. Forssman Positive Positive Negative
2. Serum sickness Positive Positive Positive
3. IM Negative Positive Positive

3. Rapid Slide Test / Monospot  a qualitative screening test

 horse erythrocytes are used instead of sheep cells
 Contribute higher specificity

4. Ox Cell Hemolysin Test

5. Immunoblot – confirmatory test for heterophile antibodies to IM
Immunology and Serology MLS115
- IM can be mistaken from CMV 4. breastmilk
- the same sila manifestations, except that 5. vaginal fluid
CMV is negative for heterophile antibodies 6. blood
7. cervical secretions
1. CYTOMEGALOVIRUS INFECTION 8. virus-infected grafts from a donor
‐ A ubiquitous human viral pathogen 9. semen
‐ Classified as a member of the herpes family 10. respiratory droplets
of viruses (HERPESVIRUSES)
TRANSMISSION
5 Recognized human herpesviruses: - appears to require intimate contact with
1. herpes simplex 1 secretions or excretions
2. herpes simplex 2 are strongly
3. Varicella-zoster virus (VZV) peripheral blood leukocytes
transplanted tissues incriminated as
4. Epstein-Barr virus (EBV) sources of CMV
5. Cytomegalovirus (CMV)
4 & 5 belong to the same family of viruses ‐ Transmission of CMV by transfusion of blood
or blood components containing WBCs is
All the herpesvirurses: assuming increased importance in patients
1. relatively large with severely impaired immunity who require
2. enveloped DNA viruses that undergo a supportive therapy.
replicative cycle involving DNA expression o ara si CMV sa leukocytes
and nucleocapsid assembly within the ‐ Low-birth-weight neonates are also at high
nucleus risk for CMV infection through transfusion of
3. the viral structure gains an envelope hen the CMV-infected blood products.
virus buds through the nuclear membrane,  Preventive methods in these patients:
which in turn is altered to contain specific 1. effective donor screening
viral proteins 2. leukocyte-depleted blood products
4. share the basic characteristic of being cell- 3. immune globulin containing
associated. passively acquired CMV antibodies

- Once in a person’s body, CMV stays there for

EPIDEMIOLOGY
- infection is endemic worldwide
- the prevalence of seropositivity increases
steadily with age.
- ↑age = ↑dasig mainfect
- found in all geographic and socioeconomic
groups, but in general it is more widespread
in developing countries and areas of lower
socioeconomic conditions.
- CMV is the most common virus transmitted
to the fetus
life.
- Most CMV infections are “silent”
causing no signs or symptoms
- Individuals who are CMV positive (infected
with CMV in the past) usually do not have
virus in urine or saliva, so the risk of acquiring
a CMV infection from casual contact is
negligible.
- Women who are pregnant or planning a
pregnancy should follow hygienic practices
to avoid CMV infection.  HANDWASHING

TRANSMISSION LATENT INFECTION

1. oral route - persistent infections characterized by
2. respiratory route periods of reactivation
3. venereal route - CMV can persist in a latent state, and active
4. transfusion of fresh blood infections may develop under a variety of
5. vertical transmission/transplacental conditions:
transmission 1. pregnancy
2. immunosuppression
The virus has been isolated in: 3. after organ, bone, or stem cell
1. urine transplantation
2. saliva 4. Only seronegative patients appear to
3. feces be at a significant risk of developing
Immunology and Serology MLS115
CMV infection. 6. myalgia
7. Lymphadenopathy
Patients at the highest risk of mortality from CMV 8. Splenomegaly
infections: ‐ persons experiencing acquired infection,
1. allograft transplant reinfection with the same or different strains of
2. seronegative patients who receive tissue CMV, or reactivation of a latent infection can
from a seropositive donor excrete the virus in titers as high as 106 infective
‐ great majority of infections in units/mL in the urine or saliva for weeks or
allograft recipients are months.
transmitted by a donated organ or
arise from the reactivation of the ‐ infrequent complications of CMV infection in
recipient’s latent virus previously healthy individuals include:
1. interstitial pneumonitis
a. True Viral Latency 2. hepatitis
3. Guillian-Barre syndrome
- defined by the presence of the genetic
4. meningoencephalitis
information in an unexpressed state in
5. myocarditis
the host cell.
6. thrombocytopenia
b. Latency (operational definition) 7. hemolytic anemia
- include the conditions of a dynamic
relationship between the virus and the host - CMV infection can be life threatening in
immunosuppressed patients.
*CMV reactivation is possible at any time, but rarely  Infections in these patients may result in
manifests in immunocompetent individuals. disseminated multisystem involvement:
1. pneumonitis
CONGENITAL INFECTION 2. hepatitis
3. GI ulceration
- the most common intrauterine infection 4. arthralgias
- the presence of maternal antibody to 5. meningoencephalitis
CMV before conception provides a 6. retinitis
substantial protection against damaging
congenital CMV infection in the newborn. Retinitis
- Infected infants can become severely ill, Common manifestations of
encephalitis disseminated CMV
and premature infants may die.
- Most newborns infected with CMV survive, - ulcerative damage of tissues (esophagus) –
but they may be mentally impaired or may another demonstration of the cytopathic effect
develop other health problems. of CMV
- interstitial pneumonitis – the major cause of
SIGNS AND SYMPTOMS death after allogeneic bone marrow
Acquired Infection transplantation
- usually ASYMPTOMATIC - in premature infants – CMV infection can result
- self-limited, heterophile-negative, in:
mononucleosis-like syndrome results 1. atypical lymphocytosis
 IM is heterophile-positive 2. hepatosplenomegaly
- can persist in the host as a chronic or latent 3. pneumonia
infection. 4. death
- incubation period: 3 to 12 weeks 3 types of CMV infections possible in
- hepatitis can occur as well (same kay IM)
blood transfusion recipients:
- symptoms: (same kay IM) 1. PRIMARY INFECTION
1. sore throat - occurs when a previously unexposed
2. fever (seronegative) recipient is transfused with
3. swollen glands blood from an actively or latently infected
4. chills donor
5. profound malaise - recipient NEGATIVE; donor POSITIVE
Immunology and Serology MLS115
- accompanied by the presence of virus in the
blood and urine, an immediate antibody
response, and eventual seroconversion.
- SYMPTOMATIC, but the great majority is
asymptomatic
2. REACTIVATED INFECTION
- can occur when a seropositive recipient is
transfused with blood from either a CMV Ab-
positive or -negative donor
- recipient POSITIVE;
- donor POSITIVE/NEGATIVE
- donor leukocytes are thought to trigger an
allograft reaction, which in turn reactivates the
recipient’s latent infection
- accompanied by significant increases in CMV-
specific Ab
- exhibit viral shedding as their only
manifestation
3. REINFECTION
- can occur by a CMV strain in the donor’s blood
that differs from the strain originally infecting
the recipient
- recipient POSITIVE; donor POSITIVE
but different strain
- a significant Ab response is observed, and
viral shedding occurs.
Congenital Infection
- the classic congenital CMV syndrome is
manifested by:
1. a high incidence of neurologic
symptoms
2. neuromuscular disorders
3. jaundice
4. hepatomegaly
5. splenomegaly
6. Petechiae – the most common
clinical sign, seen in about 50% of
CMV-infected infants
- Congenitally infected newborns, especially
those who acquire CMV during a maternal
primary infection, are more prone to develop
severe cytomegalic inclusion disease
(CID)
- severe form of CID – may be fatal or can
cause permanent neurologic sequelae:
1. Intracranial calcifications
2. Mental retardation
3. Deafness
4. Vision defects
5. Microencephaly
6. Motor dysfunction
7. Psychomotor impairment
- we do not rely only on clinical manifestation,
need serologic testing
IMMUNOLOGIC MANIFESTATIONS
Serologic Markers
1. Immediate-early antigens
- appear within 1 hour of cellular
infection
- present in the nuclei of infected cells
2. Early antigens
- present within 24 hours
- present in the nuclei of infected cells
3. Late antigens
- about 72 hours after infection or the
end of the viral replication cycle
- present in the nucleus and
cytoplasm of infected cells
Immune Ab response:
Abs against Immediate-early and Early antigens
‐ ACTIVE INFECTION (Primary or
Reactivated)
*Ab to early antigen undergoes a relatively rapid
decline after recovery but can persist for up to 250
days, and it may identify patients with recent, as well
as active, infections.
*Ab to early antigen is strongly associated with viral
shedding.
The characteristic antibody responses associated
with infection:
1. Primary infection
‐ demonstrated by a transient virus-
specific IgM Ab response and eventual
seroconversion to produce IgG Abs to
the virus
‐ IgM, IgG
2. Reactivation of latent infection in seropositive
individuals
- accompanied by significant increases in
IgG Abs to the virus
- elicits no detectable IgM response
- IgG, no IgM
3. Reinfection by a strain of CMV different from
the original infecting strain
- a significant IgG Ab response is
demonstrated.
Immunology and Serology MLS115
- not known whether an IgM response Laboratory Diagnosis of CMV Infection
occurs.
Target Test Method Recommende
- IgG; ? IgM
d Use
CMV cell Culture, Diagnose CMV
* There is no available vaccine for
Immunofluores infection Gold
preventing congenital CMV disease (present at
cence standard test for
birth).
tissue
hematologic examinations: CMV PCR Rapid test for
- leukocytosis diagnosing
- slight lymphocytosis (> 20% variant CMV in
lymphocytes) immunocompro
sa IM, almost 50% lymphocytes lang, taas RBC mised patients
count, may ara man variant lymphocytes same kay or organ donors
CMV CMV DNA PCR Diagnose CMV
quantitation infection
CMV infection is possible in the ff. situations: Monitor disease
1. The patient has mononucleosis-like state in organ
symptoms but exhibits a negative transplant and
EBV test result. HIV patients
2. The patient manifests hepatitis CMV: whole PCR Diagnose CMV
symptoms but does not demonstrate blood or infection
any positive results when tested for bone marrow
common hepatitis viruses CMV Latex Screen
 if negative sya sa hepatitis, CMV sya antibodies: agglutination pregnant
IgG and IgM women and
in affected infants: infants possibly
- low platelet count (thrombocytopenia) infected with
- abnormal liver function tests CMV
- presence of infection demonstrated by CMV Solid-phase Screen organ
inclusion bodies in leukocytes in urine antibodies: agglutination donors
sediment Total
CMV ELISA Confirm
antibody: equivocal CMV
LABORATORY EVALUATION IgM IgM results
- In immunocompromised patients, CMV CMV Chemilumines Confirm
serology is not recommended. antibody: cent equivocal CMV
IgG immunoassay IgG results
- Culture of the virus Preferred method for
- PCR diagnosis CULTURE  gold standard

- The standard reference method for detecting

congenital CMV infection is to isolate the virus
from the urine or saliva of the neonate within 3
weeks of birth.
 SUPPLEMENTARY NOTES IN CMV

SIGNS AND SYMPTOMS

The following 3 types of CMV infections are possible in blood transfusion recipients:
1. Primary infection
- occurs when a previously unexposed (seronegative) recipient is transfused with blood from an
actively or latently infected donor.
- this type of infection is accompanied by the presence of virus in the blood and urine, an
immediate antibody response, and eventual seroconversion.
- Patients may be symptomatic, but the great majority are asymptomatic.

Summary: Recipient = Negative

Donor’s blood = Positive

2. Reactivated infection
- can occur when a seropositive recipient is transfused with blood from either a CMV antibody-
positive or –negative donor.
- donor leukocytes are thought to trigger an allograft reaction, which in turn reactivates the
recipient’s latent infection
- such infections may be accompanied by significant increases in CMV-specific antibody.
- some reactivated infections exhibit viral shedding as their only manifestation.
- largely asymptomatic.

Summary: Recipient = Positive

Donor’s blood = Positive

3. Reinfection
- can occur by a CMV strain in the donor’s blood that differs from the strain originally infecting
the recipient.
- a significant antibody response is observed, and viral shedding occurs
- Although it is difficult to differentiate a reactivated infection if both the patient and the donor
are CMV antibody positive before transfusion, reinfections can be documented if isolates can be
obtained from both donor and recipient.

Summary: Recipient = Positive different

Donor’s blood = Positive strains

IMMUNOLOGIC MANIFESTATIONS

The characteristic antibody responses associated with infections are as follows:

1. Primary Infection
- demonstrated by a transient virus-specific IgM antibody response and eventual seroconversion
to produce IgG antibodies to the virus

SUMMARY: IgM, IgG present

2. Reactivation of latent infection in seropositive (IgG) individuals, which may be accompanied by significant
increases in IgG antibodies to the virus, but which elicits no detectable IgM response.

SUMMARY: IgG present

No IgM

3. Reinfection by a strain of CMV different from the original infecting strain.

- A significant IgG antibody response is demonstrated.
- It is not known whether an IgM response occurs

SUMMARY: IgG present

? IgM
Immunology and Serology MLS115

Hepatotropic viruses  viruses that have

3. HEPATITIS SEROLOGY predilection toward the liver

All of these viruses produce acute inflammation of

HEPATITIS VIRUS the liver.
- at least 7 recognized hepatitis viruses:
1. Hepatitis A
SIGNS AND SYMPTOMS
2. Hepatitis B
3. Hepatitis C 1. fever
4. Hepatitis D 2. nausea
5. Hepatitis E 3. vomiting
6. Hepatitis F 4. jaundice  maglala na
7. Hepatitis G 5. dark urine
- these are unrelated viruses that are 6. fatigue
biologically and morphologically disparate 7. headache
*Many of the clinical symptoms caused by the 8. anorexia
different hepatitis viruses are similar, so 9. abdominal pain
differentiation on the basis of clinical findings is not (Right Upper Quadrant [RUQ] / diffuse)
reliable.
- they are called hepatitis viruses altogether 1. Hepatitis A
because they have a common symptom: - small, non-enveloped
inflammation of liver - icosahedral
- but if you look at their structure and families, - ss RNA
lainay sila - family: PicoRNAviridae
- localizes primarily in the cytoplasm of
- An inflammation of the liver the liver, where it multiplies easily
- Results from the damage to liver cells - does not produce a coat protein
(hepatocytes) Causes: - is not detectable in serum
a. viruses, bacteria, fungi, parasites, - stable (despite non-enveloped) in
drugs, toxins ether and to a pH of 3.0
b. Heat, hyperthermia, radiation - an ENTEROVIRUS (Enterovirus 72)
c. Excessive alcohol intake  gastrointestinal tract sya gainfect
- can eventually lead to - a human pathogen
cirrhosis
d. Others – idiopathic  we don’t know CLINICAL INFECTION
the cause HEPATITIS A infection
Fulminant hepatitis - a.k.a. Infectious Hepatitis
– term applied when the number of - the type seen in most epidemic outbreaks of
hepatocytes destroyed is so great that hepatitis in the normal population (3rd world
too few remain to maintain basic liver countries)  related to POOR SANITATION
function. - IP: 2-6 weeks (ave. 25 days)
- abrupt onset
Terms: Viral hepatitis & Acute viral hepatitis - s/s: fever, chills, fatigue, malaise, aches,
 generally used only to refer to cases caused pains anorexia, nausea, vomiting RUQ
by specific hepatotropic viruses: pain dark urine, clay-colored stools jaundice
a. Hepatitis A Virus (HAV) in the sclera and skin
b. Hepatitis B Virus (HBV) - once the jaundice appears, there is a rapid
c. Hepatitis C Virus (Non A-Non B improvement in symptoms  liver is
Hepatitis) regenerating, it is a sign that the liver is
d. Hepatitis D Virus (HDV) recovering
Immunology and Serology MLS115
- convalescence can last weeks and complete
recovery can take months
- low mortality, no persistence, and no
evidence of chronic liver damage by HAV
infection  very nice ! :>
- MOT: ORAL – FECAL route
- more common in countries with low
standards of living
 “POOR MAN’S HEPATITIS”
 even if you are a rich man, you
can still get HAV
SEROLOGY
At the onset of apparent Hepa A,
antibodies to HAV appear in the plasma.
Ab: initially IgM (Anti-HAV IgM)
: replaced by IgG – persists for years
probably for life
There is a vaccine for Hepatitis A infection.
HAV MARKERS  confirmation of HA infection
1. HAV Ag – not usually detected
- excretion of virus first before the
appearance of jaundice
2. Anti-HAV IgM
- early infection = appears 1st
3. Anti-HAV IgG
- late infection = appears later
To detect HAV Ag?Specimen: STOOL (not blood)
- nagkadto na sa GIT ang virus
To detect Anti-HAV IgM & Anti-HAV IgG
 ? Specimen: SERUM
2. Hepatitis B Virus
- most controversial virus, prone si
MEDTEK ;-;
- microbiologically unrelated to HAV
- pero related si B and D
- partially ds DNA that exists in 3
forms:
a. Spherical particle
predominant form seen in the
blood
a. Filamentous form – slightly
less common
a. Dane particle – represents the
virion - least common in blood
- Family: HepaDNAviridae
- MOT: blood transfusion, sexual
contact, perinatal means,
parenteral means
- High-risk groups:
a. IV drug abusers
(intravenous)
b. Homosexuals
c. Individuals from endemic
areas
d. Household & sexual
contacts of HBV carriers
e. Healthcare personnel
f. Newborns of carrier mothers
Hepatitis B other names:
- Serum Hepatitis
- Long Incubation Hepatitis
- incubation period: 4 – 26 weeks
- s/s: chronic hepatitis, cirrhosis, liver
cancer
 lain kay HAV, kay longer ang incubation,
rapid si HAV
SEROLOGY: HBV MARKERS
1. HBsAg (surface) – earliest to be detected
- early stage of infection
2. HBeAg (envelope) – high infectivity
- high vertical transmission risk
3. HBcAg (core) – not found in the blood
- only found in infected hepatocytes
 LIVER FNAB (fine needle
aspiration biopsy)
4. Anti-HBc IgM – Acute Phase Marker
Anti-HBc IgG – Chronic Phase Marker
Anti-HBc = life long marker
= the only Ab detected in the
window phase
 negative/normal ang results sa
ibn, pero sya lang gataas
5. Anti-HBe – low infectivity marker
- low transmission risk
- CONVALESCENCE PERIOD
6. Anti-HBs – last to appear
- immune state marker
–
Prevention: IMMUNIZATION!!!
• Prior to Immunization: Do Screening Tests
• Vaccine was prepared from the plasma of
chronic carriers of HBsAg
Immunology and Serology MLS115

HBsAg Pt1 Pt2 Pt3 Pt4 Pt5 Pt6 3. Hepatitis C

(-) (+) (+) (-) (-) (-) - Family: Flaviviridae
Anti-HBc (-) (-) (+) (+) (+) (-) - major cause of Non-A, Non-B
Anti-HBs (-) (-) (-) (-) (+) (+) Hepatitis (NANBH)  old name nya
- no universally accepted serum
Pt 1 = No Exposure  Candidate for Immunization marker yet
Pt 2 = INFECTED!!!  No need to immunize (time - You can do molecular test, HCV RNA
will come for antibodies to appear)  - HCV RNA  more complicated
SEROCONVERSION method since you are dealing with
Pt 3 = Acute / Chronic  No need to immunize molecular level na
Pt 4 = Window phase period
Pt 5 = Immune  No need to immunize Non A – Non B Hepatitis
Pt 6 = Immunize (pwede ka kadonate blood - a.k.a. Post transfusion hepatitis
huehuehue)  depends on the titer: - MOT: Blood transfusion
>10U = immune - s/s: minimal clinical manifestations
<10U = no infection - incubation period: 6 - 10 wks

Tests Results Interpretation 4. Hepatitis D

HBsAg Negative Susceptible (wala - a.k.a. Hepatitis Delta Virus
Anti-HBc Negative masakit, pero wala - ss RNA virus ( a.k.a. Delta Antigen )
Anti-HBs Negative protection) - spherical in shape
HBsAg Negative IMMUNE because of - a defective virus
Anti-HBc Positive natural infection - needs HBV
Anti-HBs Positive  it requires obligatory helper functions
HBsAg Negative IMMUNE because of from HBV in order to ensure its
Anti-HBc Negative Hepatitis B replication and infectivity.
Anti-HBs Positive vaccination  HBV provides HDV w/ a protein coat of
HBsAg.
Acutely infected
HBsAg Positive Chronically infected Antibodies to HDV
Anti-HBc Positive HDV Ag – the first marker to appear in HDV infection
IgM Anti- Negative - but transient in serum (1– 4 days )
HBc Negative ↓ HDV Ag – Anti-HDV IgM appear
Anti-HBs  seroconversion followed by low levels
HBsAg Negative 4 interpretations are of IgG Ab in acute infection
possible:
Anti-HBc Positive
1. Might be recovering The progression of high levels of IgG anti-HDV in
Anti-HBs Negative from acute HBV infection
2. Might be distantly HBsAg (+) indicates the switch to chronic HDV
immune and test not infection.  may help na ni HBV
sensitive enough to detect
Anti-HBs in serum
3. Might be susceptible Hepatitis D infection
with a false-positive anti- - MOT: Parenteral & Transmucosal routes
HBc Horizontal transmission
4. Might be undetectable
level of HBsAg present in - Co-infection
the serum, and the person = HDV, HBV w/o Chronic Infection
is actually chronically - Superinfection
infected
= HDV, HBV w/ Chronic infection
- Prevention: Vaccine
Immunology and Serology MLS115

Tx: No effective antiviral drugs or

immunosuppressive agents for treatment of
chronic HDV infections :<

5. Hepatitis E
- Enterically Transmitted through
sewage contaminated drinking
water
- waterborne enteric agent believed to
believed to be enterically transmitted
via the fecal-oral route, similar to
HAV  “puno kag punta”
Immunology and Serology MLS115 LECTURE
OUTLINE
1. MUMPS Gahabok sa neck area, because the salivary glands that
a. Vaccine are mostly infected are the ones within the tongue.
b. Passive Immunization against Mumps
c. Diagnosis
Bilateral – both sides of neck/jaw
d. Serological Testing Unilateral – either left or right side
e. Prevention
2. MEASLES
Blue color = “aniel”?  cold feeling, alleviates the pain
a. Pathogenesis and Clinical Features Ice pack = ginabutang man sa neck area para mabuhinan
b. Vaccine
c. Diagnosis
ang pain
3. RUBELLA
a. Vaccine
b. Diagnosis
4. VARICELLA-ZOSTER VIRUS
a. Vaccine
b. Diagnosis

1. MMRV
- Mumps, Measles, Rubella, Varicella

MUMPS
- a.k.a Acute Parotitis
- Inflamed parotid glands
only one of the many manifestations
- Single-stranded RNA
Complications of Mumps:
- Family: Paramyxoviridae
1. asymptomatic meningitis – 50-60% of
- Genus: Rubulavirus
cases
- Transmitted from person to person by infected
2. symptomatic meningitis – 10-30% of
respiratory droplets and possibly fomites
cases
NOT AIRBORNE, need direct contact with
3. testicular inflammation (ORCHITIS) – 20-
respiratory droplets
50% of post-pubertal males
Fomites – non-living material [doorknobs,
4. ovarian inflammation - 5% of post-
mouse of computer]
pubertal females
- Replicates initially in the nasopharynx and
5. deafness – 1 case per 20, 000
regional lymph nodes
• post-pubertal males = adults na gd
- Incubation period:14-18 days
• orchitis  dangerous since it may lead to
- The virus spreads from the blood to various
sterility(?)
tissues, including:
o meninges of the brain
Prior to routine immunization, mumps was one of the
o salivary glands
most common causes of:
o pancreas
1. aseptic meningitis
o Testes
2. sensorineural deafness in children
o Ovaries
- Most common clinical manifestation: PAROTITIS
Infrequent but important complications of mumps:
o inflammation of the parotid glands
3. pancreatitis
o occurs in 30% to 40% of cases
4. encephalitis
o results in earache and tenderness of the
5. myocarditis
jaw
6. polyarthritis
 can be bilateral or unilateral
7. thrombocytopenia
 resolve in 7 to 10 days
Immunology and Serology MLS115 LECTURE
‐ Mumps infection in pregnant women results in
increased risk for fetal death when it occurs in
the first trimester of pregnancy, but it is not
associated with congenital abnormalities.
if nagkamumps ang nanay esp sa 1st
trimester  increased risk of FETAL DEATH, but
doesn’t mean nga may congenital abnormalities
Once maovercome nya ang death, normal sya
japon
VACCINE
• Combined with the vaccines for:
MMR  Mumps, Measles, Rubella
MMRV  Mumps, Measles, Rubella, Varicella
Passive Immunization Against Mumps
Passive immunization – ginahatag na
ang antibodies, no need for production
‐ Immune globulin ineffective for post-exposure
prophylaxis
o does not prevent disease or reduce
complications
‐ Transplacental maternal antibody appears to
protect infants for first year of life
DIAGNOSIS
‐ Diagnosis is usually made on the basis of clinical
symptoms, especially PAROTITIS, and does not
require laboratory confirmation.
 however, laboratory testing is very
useful in cases in which parotitis is
ABSENT or when differentiation from
other causes of parotitis is required.
DOES NOT REQUIRE LAB CONFIRMATION –
clinical symptoms lang ang iobserve, commonly
esp if may PAROTITIS
• Within the 1st few days of illness, mumps virus
can be isolated from:
a. Saliva
b. Urine
c. CSF
d. Swabs from the area around the
excretory duct of the parotid gland
• Can then be grown in shell vial cultures of rhesus
monkey kidney cells or human embryonic lung
fibroblasts and identified by staining with
fluorescein-labeled monoclonal Abs.
Viruses – grown in cell cultures, need
special requirements
• Culture methods require experienced personnel
and specialized reagents and may not be
performed in the routine clinical laboratory.
Reverse transcriptase polymerase chain reaction (RT-
PCR) methods
a. have been developed to detect viral RNA in
specimens collected from the buccal cavity,
throat, cerebral spinal fluid, or urine of patients
with a suspected mumps infection
b. have not been standardized but may be useful in
confirming infection in cases where viral isolation
is not successful.
RT-PCR  gamiton mo if wala ka may makita sa culture
 more sensitive
Serological testing
- provides the most simple and practical means of
confirming a mumps diagnosis
1. complement fixation
2. hemagglutination inhibition
3. hemolysis in gel
4. neutralization assays
5. immunofluorescence assay most commonly
6. ELISA used
They are:
a. Sensitive
b. Specific
c. Cost-effective
d. Readily performed by the routine clinical
laboratory
Serological testing
Use of solid-phase IgM capture assays reduces the
incidence of false-positive results due to rheumatoid
factor.
Current or recent infection is indicated by:
1. the presence of mumps specific IgM Ab in a single
serum sample, or
2. by at least a 4-fold rise in specific IgG Ab between 2
specimens collected during the acute and
convalescent phases of illness  PAIRED SERA
• IgM Abs can be detected within 3-4 days of illness
and can persist for at least 8-12 weeks.
Immunology and Serology MLS115 LECTURE
• IgG Abs become detectable within 7-10 days and ‐ During the prodromal period, Koplik spots
persist for years. appear on the mucous membranes of the inner
• The presence of specific IgG antibodies indicates cheeks or lips.
immunity to mumps, either as a result of natural  appear as gray-to-white lesions against
infection or immunization. a bright red background and persist for
If nagkamumps ka na sang-una, there is a several days.
lesser chance nga magka-mumps ka liwat [presence Koplik spots inner part of the mouth
of IgG ab] ‐ Rash – appears about 14 days after exposure to
the virus
PREVENTION - is characterized by an erythematous,
• Minimize close contact with other people, especially maculopapular eruption that begins on the
babies and people with weakened immune systems face and head and spreads to the trunk and
who cannot be vaccinated. extremities
• Stay home from work or school for 5 days after your - lasts 5-6 days
glands begin to swell, and try not to have close
contact with other people who live in your house.
• Cover your mouth and nose with a tissue when you
cough or sneeze, and put your used tissue in the trash
can. If you don’t have a tissue, cough or sneeze into
your upper sleeve or elbow, not your hands.
• Wash hands well and often with soap, and teach
children to wash their hands too.
Measles Pathogenesis and Clinical Features
• Don’t share drinks or eating utensils.
• Koplik spots
• Regularly clean surfaces that are frequently touched
(such as toys, doorknobs, tables, counters) with soap • 2-4 days after prodrome, 14 days after exposure
• Maculopapular, becomes confluent
and water or with cleaning wipes.
• Begins on face and head
• Persists 5-6 days
MEASLES • Fades in order of appearance
- a.k.a. Rubeola [one L = one word]
– kung diin una pakita, didto man una dula
- not associated with fomites
- highly contagious
‐ Single-stranded RNA virus
‐ Genus: Morbillivirus
‐ Family: Paramyxoviridae
‐ Spread by direct contact with aerosolized
droplets from the respiratory secretions of
infected individuals
if may nagsneeze sa room, there is 100%
chance of infecting others in the room
• Incubation period: 10-12 days
• The virus produces prodromal symptoms of:
1. fever
2. cough
3. coryza (runny nose)
4. conjunctivitis – last 2-4 days
Immunology and Serology MLS115 LECTURE
A systemic infection that can result in complications.
 most common in adults, children less than 5 years
of age, and immunocompromised persons
1. diarrhea
2. otitis media
3. croup
4. bronchitis
5. pneumonia
6. encephalitis
Subacute sclerosing panencephalitis (SSPE)
- a fatal degenerative disease of the CNS
- can result from persistent replication of measles
virus in the brain, with onset of symptoms
typically appearing 7-10 years after primary
measles infection.
reactivated nga measles infection, can
be FATAL
Measles infection during pregnancy results in:
- a higher risk of premature labor
- spontaneous abortion
- low birth weight
but it is not associated with a defined pattern of
congenital malformations in the newborn.
same sa mumps, wala gadepict nga
makacause sya congenital abnormalities
VACCINE
A vaccine consisting of killed rubeola virus was
originally licensed in 1963 but was ultimately ineffective.
A more effective vaccine consisting of live,
attenuated rubeola virus was licensed in 1968 and is used
in the routine immunization schedule of infants and
children:
• either in combination with rubella and mumps
(MMR)
• or in combination with rubella, mumps, and
varicella (MMRV)
Recommended administration of the vaccine is in 2
doses:
1st dose: between the ages of 12 and 15 months
2nd dose: between the ages 4-6
• Administration of the first dose prior to the age of
12 months may result in vaccine failure.
 because the presence of maternal
antibodies can interfere with the infant’s
immune response.
DIAGNOSIS
• Diagnosis has typically been based on clinical
presentation of the patient.
same japon kay mumps, clinical presentation
lang ang ginaobserve para sa diagnosis
• Laboratory tests are therefore of value in ensuring
rapid, accurate diagnosis of sporadic cases; they are
also important for epidemiological surveillance and
control of community outbreaks.
• Isolation of rubeola virus in conventional cell cultures
is technically difficult and slow and is not generally
performed in the routine diagnosis of measles, but it
may be useful in epidemiological surveillance of
measles virus strains.
• The optimal time to recover measles virus:
from nasopharyngeal aspirates, throat swabs, or
blood
 from the prodrome period up to 3 days after
the rash onset
from urine
 1 week after appearance of the rash
• Serological testing provides the most practical and
reliable means of confirming a measles diagnosis.
Confirm diagnosis of measles
• Clinical symptoms + presence of rubeola-
specific IgM Abs
• 4-fold rise in the rubeola-specific IgG Ab titer
between serum samples collected soon after the
onset of rash and 10-30 days later
• SSPE is associated with extremely high titers of
rubeola Abs.
• IgM Abs are preferentially detected by an IgM
capture ELISA method
 highly sensitive
 has a low incidence of false-positive
results
• IgM Abs become detectable 3-4 days after
appearance of symptoms and persist for 8-12
weeks.
Immunology and Serology MLS115 LECTURE
• Samples collected before 72 hours may yield
false-negative results, and repeat testing on a
later sample is recommended in that situation.
• Methods to detect IgG rubeola Abs:
• hemagglutination inhibition
• microneutralization
• plaque reduction neutralization
• complement fixation
• indirect fluorescent Ab tests
• ELISA – most commonly used
• IgG Abs become detectable 7-10 days after the
onset of symptoms and persist for life.
• Presence of rubeola-specific IgG Abs indicates
immunity to measles due to past infection or
immunization.
• Testing for IgG Abs is therefore routinely
performed by clinical laboratories in order to
detect immune status of individuals such as
healthcare workers to the virus.
• Molecular methods to detect rubeola RNA can
be used in cases in which serological tests are
inconclusive or inconsistent and can be used to
genotype the virus in epidemiological studies.
RT-PCR = preferred molecular technique
-sensitive
-can be performed on a variety of clinical
samples or in infected cell cultures
-can detect viral RNA within 3 days or rash
appearance
Cell culture  not commonly done, difficult and slow
RUBELLA
- a.k.a. German Measles [two L’s = two words]
- 3-day measles  3 days lang gaappear ang
rashes, healed ka na
- Single-stranded
- Enveloped RNA virus
- Genus: Rubivirus
- Family: Togaviridae
- Transmitted through:
1. respiratory droplets
2. transplacental infection of the fetus
during pregnancy
‐ Causes German measles
 benign, self-limited disease
 mainly a disease of young children, prior to
widespread use of the rubella vaccine
 today, occurs most often in young,
unvaccinated adults
‐ Incubation period: 12-23 days
‐ The virus replicates in the upper respiratory tract
and cervical lymph nodes, then travels to the
bloodstream.
‐ 50% of infections are ASYMPTOMATIC
‐ The infection usually resolves without
complications
same kay Ruboela  Fades in order of
appearance – kung diin una pakita, didto man
una dula
‐ Infected adult women experience arthralgias
and arthritis, but chronic arthritis is rare.
‐ Other clinical manifestations: (INFREQUENT)
• encephalitis
• thrombocytopenia with hemorrhage
• neuritis
‐ Rubella infection during pregnancy may have
severe consequences:
1. miscarriage
2. stillbirth
3. Congenital Rubella Syndrome (CRS)
‐ The likelihood of severe consequences increases
when infection occurs earlier in the pregnancy,
especially during the first trimester.
‐ Infants with CRS may present:
1. deafness
2. eye defects (cataracts, glaucoma)
3. cardiac abnormalities
4. mental retardation
5. motor disabilities
Immunology and Serology MLS115 LECTURE
VACCINE
• Consisting of live, attenuated rubella virus
- primary goal: preventing infection of
pregnant women by preventing dissemination
of the virus in the population as a whole
• Given in combination with vaccines for measles
and mumps (MMR)
• Possibly with varicella (MMRV)
DIAGNOSIS
‐ Laboratory testing is helpful in confirming
suspected cases of German measles, whose
symptoms may mimic those of other viral
infections.
‐ It is essential in the diagnosis of CRS and in the
determination of immune status in other
individuals.
‐ Virus can be grown in cultures inoculated with
respiratory secretions or other clinical
specimens.
 however:
a. growth is slow
b. may not produce characteristic
cytopathic effects upon primary
isolation
Serological testing = the method of choice
• To detect rubella Abs:
1. hemagglutination inhibition (HI)
– once the standard technique for
measuring rubella Abs
2. passive hemagglutination
3. complement fixation
4. latex agglutination
5. immunoassays, ELISA
– most commonly used method today
 because of its sensitivity, specificity, ease
of performance, and adaptability to
automation
Serological testing - can be used in both the diagnosis of
rubella infections and in screening for rubella immunity
IgM and IgG – appear as the rash of German measles
begins to fade.
IgM – decline by 4 or 5 weeks but may persist in low
levels for a year or more in some cases
IgG – provide immunity and persist for life
Primary rubella infection
= presence of rubella-specific IgM
OR
= 4-fold rise in rubella-specific IgG Ab titers
between acute and convalescent-phase
samples
10-15 IU/mL  Ab level considered to be protective
To enhance the reliability of a CRS diagnosis, any positive
IgM results should be confirmed by:
a. Viral culture
b. Demonstration of persistently high titers of
rubella IgG Abs after 3-6 months of age
c. RT-PCR
– a highly sensitive and specific aid in
prenatal or postnatal diagnosis and can be used
to detect rubella RNA in a variety of clinical
samples (chorionic villi, placenta, amniotic fluid,
fetal blood, lens tissue, products of conception,
pharyngeal swabs, spinal fluid, or brain tissue).
VARICELLA-ZOSTER VIRUS (VSV)
- a.k.a Chickenpox or Shingles
- Cause of 2 distinct diseases:
Varicella – Chickenpox
Herpes zoster – Shingles
- Transmitted primarily by:
o inhalation of infected respiratory
secretions or aerosols from skinlesions
associated with the infection
o transplacental transmission to the fetus
Rubella & Varicella  may
transplacental transmission (?)
1. PRIMARY INFECTIONS IN ADULTS, NEONATES, OR
PREGNANT WOMEN tend to be MORE SEVERE, with
a larger number of lesions and a greater chance of
developing other complications such as pneumonia.
2. VARICELLA INFECTION IN PREGNANT WOMEN may
also cause premature labor or congenital
malformations if the infection is acquired during the
first trimester of pregnancy or may cause severe
neonatal infection if transmission of the virus occurs
around the time of delivery.
 Immunology and Serology MLS115 LECTURE
3. INFECTIONS IN IMMUNOCOMPROMISED PATIENTS
are more likely to result in disseminated diseased,
with extensive skin rash, neurological conditions (e.g.,
encephalitis), pneumonia, or hepatitis.
‐ During the course of primary infection, VZV is thought
to travel form the skin to the sensory nerve endings
to the dorsal ganglion cells, whre it establishes a
latent state.
‐ Reactivation of the virus occurs in 15-30% of persons
with a history of varicella infection, probably as a
result of a decrease in cell-mediated immunity.
‐ Reactivation results in the virus moving down the
sensory nerve to the dermatome supplied by that
nerve, resulting in eruption of a painful vesicular rash
known as HERPES ZOSTER / SHINGLES, in the affected
area.
‐ The rash may persist for weeks to months and is more
severe in immunocompromised and elderly
individuals.
Complications of shingles:
1. post-herpetic neuralgia – characterized by
debilitating pain that persists weeks, months, or
even years after resolution of the infection.
2. herpes ophthalmicus – leading to blindness
3. pneumonia
4. visceral involvement
VACCINE
• 1995 – VZV vaccine
 for use in children aged 12 months or
older, adolescents, and adults
• Although most patients experience mild illness,
they are still contagious, and some individuals
may experience more severe disease.
 this prompted CDC to recommend adding
a booster dose of the vaccine to routine
childhood immunization schedules.
• 2005 – a vaccine was licensed for use in healthy
children that combines the varicella vaccine with
that for measles, mumps, and rubella (MMRV)
• 2006 –a single-agent VZV vaccine was licensed
for prevention of herpes zoster in persons aged
60 or older, presumably by boosting the immune
response.
• Because these vaccines all contain a live agent,
they are not recommended for use in
immunocompromised persons.
 these patients should receive an antiviral
drug or injections of varicella immune
globulin within 96 hours after exposure to
the virus.
DIAGNOSIS
‐ Diagnosis of the disease was based primarily on
clinical findings, and laboratory testing was usually
unnecessary.
‐ Definitive diagnosis is based on identifying VZV or
one of its products in skin lesions, tissue, or vesicular
fluids.
‐ Rapid identification of the virus can be performed
by microscopic examination of smears made from
the base of the vesicles and stained with H & E,
Wright- Giemsa, toluidine blue, or Papnicolaou’s
stain to reveal multinucleated giant cells called
TZANCK CELLS.
 this procedure cannot distinguish between
VZV and herpes simplex virus (HSV)
Tzank cells –only be seen using microscopic
examination
 definitive diagnosis
PCR – the most accurate and sensitive method of
detecting the infection
- detects the VZV DNA from clinical specimens
• Serology testing is of limited use in detecting
current infections, because accurate detection
requires demonstration of a 4-fold rise in Ab titer
between acute and convalescent samples, a
process that take 2 to 4 weeks to perform.
IgM Abs – may not be detectable until the convalescent
stage of illness
- cannot distinguish between primary and
reactivated infection
- may not be free of IgG Abs when serum is
processed for testing.
-
Serology is most useful in determining immunity to VZV
in individuals such as healthcare workers and in
identifying VZV-susceptible persons in outbreak settings
who may benefit from prophylactic treatment.
Immunology and Serology MLS115 LECTURE
• Most serology tests detect TOTAL VZV ANTIBODY
primarily IgG
 Methods:
1. Point-of-care testing – latex agglutination
and membrane-based EIAs
2. FAMA (Fluorescent antibody to membrane
antigen)
- most sensitive and reliable method of
detecting VZV antibody
- detects antibody to the envelope
glycoproteins of the virus
- REFERENCE METHOD
- requires live, virus-infected cells and not
suitable for large-scale routine testing
Immunology and Serology MLS115 (LECTURE)
OUTLINE
Human Immunodeficiency Virus
I. History
II. HIV Structure
III. HIV Genome
a. Regulatory
Proteins
IV. HIV Infection
a. Epidemiology
b. Transmission
c. Cell Infection
d. Pathogenesis
e. CD4 Count
Human Immunodeficiency Virus
Human Retrovirus
History
- When HIV was discovered, they had so
many controversies about the virus
 By mid-1970’s, retroviruses had been
discovered in many vertebrate species,
including apes
 That is why they always associate HIV with
apes
 Hypothesized that humans may also be
infected with retroviruses
 True enough, humans really are infected
 Searched (Researches) that eventually led
to the isolation of a retrovirus from the cell
lines and blood of patients with adult T-cell
leukemia.
 There has been a strong association
between HIV and people who have had
adult T-cell leukemia. That is why dira nag
gwa ang HTLV, which in some studies
would be almost synonymous to HIV
 Kung mag hambal ka nga may HTLV ka,
daw halos may HIV na sya. And they have
actually identified that there are 2 tyopes of
HTLVs: HTLV-I and HTLV-II
 HTLV – I = Human T-cell Leukemia
Virus - now linked to a paralytic disease
that occurs in the tropics (Carribean
islands) called tropical spastic
paraparesis.
 A 2nd human retrovirus was isolated from
T-cells of patients with a T-cell variant of
hairy cell leukemia
 They were able to identify that aside from
the hairy cell leukemia or aside from the
appearance of the hairy cells sa blood, na
identify nga may variant of hairy cell
leukemia nga associated with another T-cell.
That is why dira nag gwa ang HTLV-II
 HTLV – II – has no known role in
producing disease
 Daw nag sprout lng sa pero indi nila makita
ang association or mechanism of HTLV-II in
producing the leukemia
 Subong ang ma associate/synonymous sa
HTLV-I is ang HIV-I and HTLV-II is
synonymous to HIV-II. Amu na sng una
kung may ara ka both HTLV-I and HTLV-II
it’s as good as you already have HIV-I and
HIV-II
 That is why in our lab, sa antibody assay,
gina check if HIV-I or HIV-II. Kay dira nag
ugat kung ngaa may I and II pero HIV mna
sya japun, virus mna sya japun
 In early 1980 a new epidemic was first
noted that we now call the Acquired
Immuodeficiency Syndrome (AIDS).
 This is controversial, because of the name
gay-associated(?). damo sya name. (indi
ma identify ni miss. Lol)
 There was one child who had blood
transfusion, after a few months after na
transfuse sya, nakitaan nga nag + sya sa
virsus. Kay before indi pa HIV indi pa AIDS
ang ngalan nya. HIV= gay-related virus
something. Nag protest ang mga gay nga
ilisan ang ngalan becoz bata ang patient kg
never have been associated w/ gay people
pero ngaa nagka HIV or AIDS sya. Until gn
island ang ngalan into AIDS. CDC gave the
official name nga AIDS nlg gd sa
 Factors suggested that this was caused by
a retrovirus:
1. The infectious agent was present in
filtered blood products (concentrated
Factor VIII) given to patients with
hemophilia.
 Esp. that hemophilia patients really need
blood transfusion, dira nila na associated
nga pwede ma transmit ang AIDS thru
blood transfusion
 suggested a viral etiology
 something small would be
necessary to pass the filters
Immunology and Serology MLS115 (LECTURE)
2. There was a delayed onset b/w
exposure (sexual/blood products)
and the development of disease.
 delayed onset had been
observed in the other known
retroviral diseases
3. Immunodeficiency occurs with other
animal retroviruses (feline leukemia
virus)
 even HTLV-I can cause
immunosuppression
4. AIDS patients have destruction of the
T-helper lymphocytes.
 HTLV-I & II were both T-cell
tropic.
 T-cell tropic= they have a predilection
towards the t-cells. Kung diin sila na
attract nga cell didto sila gapang guba
bcoz it’s a virus it has to enter a living
cell in order for them to replicate
 Investigators stimulated T-cell culture
growth (T-cells from patients infected with
AIDS) with interleukin-2 and were able to
find RNA & DNA, suggesting a retroviral
etiology.
 virus: Human Immunodeficiency
Virus (HIV)
 A 2nd retrovirus, called HIV-2, causes a
disease similar to AIDS is Western Africa.
 Even if you say it’s HIV-I or HIV-2, HIV mna
japun it only depends on the ethnicity or
group of population kung diin sya common.
Bcoz HIV-2 is common in western Africa,
didto na identify nga probably different
strain of virus pero HIV man japun
 a distantly related virus with 40%
sequence homology with HIV-1
 Simian Immunodeficiency virus (SIV)
- a virus that causes an AIDS-like disease
in primates
- shares a close sequence homology with
HIV-2
HIV Structure
 Spherical
 Enveloped
 central cylindrical nucleocapsid
1. Virion core:
- 2 identical ss RNA pieces (dimer)
- nucleocapsid (NC) proteins bound to the
RNA
- nucleocapsid encloses the genetic material.
In HIV, the genetic material is RNA
- 3 essential retroviral enzymes:
a. Protease
b. Reverse transcriptase
- in the central dogma, DNA is
converted into RNA to produce proteins. In
case of the RNA virus, convert sya danay
from DNA  ma copy to produce RNA and
then mka produce a sya sng protein.
c. Integrase
2. Capsid shell
- surrounds the RNA dimer
- icosahedral symmetry
- icosahedron or icosahedral may have more
or less 20 sides or facets
- capsid proteins (CA) – the proteins that
constitute this shell
o p24 = the major capsid protein =
measured in the serum to detect
early HIV infection
o even in serology, we have to identify
this nga dapat ara sya para mka
confirm kita nga HIV ang virus nga
ara sa blood sng patient
3. The rest of the virus has the same structure
described for the influenza virus:
a. Matrix proteins – proteins under the
envelope
- serve to hold the
glycoprotein spikes that
traverse the lipid bilayer
membrane (envelope)
b. Surface glycoproteins  gp (gp120,
gp41)
- along with p24, u need to
identify the gp120 and gp41 to strengthen the
diagnosis nga HIV kay kung p24 kis-a ma duwa2
kpa. GP=glycoprotein
Immunology and Serology MLS115 (LECTURE)
HIV Genome – sa RNA ngd
1. LTRs (Long Terminal Repeat sequences)
- flank the whole viral genome
- serve 2 functions:
a. Sticky ends
- the sequences, recognized by
integrase
- involved in the insertion into the
host DNA
* Transposons – have similar
flanking DNA pieces
b. Promoter/Enhancer function
- once incorporated into the host
DNA, proteins bind to the LTRs
that can modify viral DNA
transcription
- always remember the central
dogma
2. gag (Group antigen) sequences
- code for the proteins inside the envelope:
a. Nucleocapsid (NC)
b. Capsid (CA) – p24
c. Matrix (MA) proteins
- codes for the virion’s major structural
proteins that are antigenic
- dapat ara ang gag para ma produce ang
mga proteins mentioned. Bcos these the
proteins that are basic sa structure sng HIV
3. pol
- ara sa genetic material; responsible for the coding
of the enzymes to be produced
- encodes the vital protease, integrase, and
reverse transcriptase enzymes
protease - cleaves gag and pol proteins from
their larger precursor molecules
(post-translational modification)
protease-deficient HIV virions – cannot form
their viral core and are non-
infectious
4. env
- codes for the ENVelope proteins that, once
glycosylated, form the glycoproten spikes
gp 120 and gp 41
gp 120 – forms the head
+ gp 41 – forms the stalk
--------------------------------
gp 160 – bind to CD4 receptors on
T-cells
- indi pag e literal nga add ang gp 120 and gp
41. Gp 160 is the overall structure nga gka
kilala ni T-cell. If may gp 160 ka, it will be
easier for the virus to attach to the t-cells
bcos ang gp 160 is ang gina tapikan ni CD4
receptors
Regulatory proteins
- encoded by the regulatory genes:
a. tat
b. rev
c. nef
- major regulatory genes
d. vif have poorly
e. vpr understood
f. vpu actions
- all of these are found in the HIV genome (genetic
material)
a. tat – encodes the viral TrAnsacTivator protein
Transactivator protein
- binds to the viral genome
- activates transcription (thus
transactivates)
- a potent promoter of viral activity
b. rev - another promoter that REVs up viral activity:
* HIV has multiple reading frames,
producing different mRNAs depending on
where splicing occurs.
* It can be spliced into many pieces,
producing the regulatory proteins such as
tat, rev, nef (and others: vif, vpr, and vpu).
* Alternately, it can be spliced only a few
times to produce the major gag, pol, and
env products that form the virion.
* The rev protein binds to the env gene to
decrease splicing.
* So it REVs up the reading of gag, pol, and env to
produce virions.
c. nef - its function is uncertain -
- experiments have demonstrated that it can
both positively and negatively regulate HIV
expression
* Recent studies have shown that persons
infected with nef deficient HIV-1 do not develop
AIDS and do not suffer T cell destruction.
Immunology and Serology MLS115 (LECTURE)
HIV Infection
Epidemiology
- global pandemic
- 47 million persons worldwide – infected
with HIV
- In the last 5 years, medyo nagtaas naman
ang cases of HIV
- ~14 million – died
- Countries in Sub-Saharan Africa = ¼ - 1/3
of all adults
are infected!
- Rapidly spreading in South & Southeast
Asia
- In the Philippines, manila followed by cebu,
3rd after cebu is neg. occ. ang may
pinakadamo cases
- Declining number in Western Europe,
Australia, U.S.
- U.S. = CDC: ~650-900 thousand persons
are currently infected with HIV
Transmission
2 patterns of transmission:
1. Americas & Europe: 90% of cases are
among homosexuals and IV drug users
 Men > Women
- Greater in men
2. In developing areas (Sub-Saharan Africa):
spread is heterosexual
 Men = Women
- Spread by the parenteral route (like
Hepatitis B virus)
- If may Hepa B virus ka, u are also being
treated with HIV virus bcoz same ang MOT
nila
Detailed transmission:
1. Sexual Activity (heterosexual & - to reduce the risk of transmission via blood
homosexual)
- most common mechanism of transmission
- HIV is present in seminal fluid as well as
vaginal and cervical secretions
- Most likely in contact with mucous
membrane to mucous membrane
- During or following intercourse, the viral
particles penetrate tiny ulcerations in the
vaginal, rectal, penile, or urethral mucosa
- Women – 20% more likely than men to get
HIV with vaginal intercourse
 ‘coz of the prolonged exposure
of the vagina, cervix, and uterus,
to seminal fluid
- Receptive anal intercourse – increase the
risk of transmission
 secondary to mucosal trauma
of the thin rectal wall
- receptive, sya ang nag baton, sya ang
nasudlan
- since wala natural lubricant ang anus, mas
difficult compared sa vagina that is why may
tendency nga mapilas and the irsk of
transmission is higher
- STDs also increase the risk of transmission:
- Organisms:
a. Treponema pallidum
b. Herpes simplex virus
c. Chlamydia trachomatis
d. Neisseria gonorrhoeae
 cause mucosal erosions and may
even increase the concentration of
HIV in semen and vaginal fluids
 inflammation of the epididymis,
urethra, and vaginal mucosa results
in an increase in HIV laden
macrophages and lymphocytes.
- oral sex – much less likely to result in
transmission
- unless may pilas2 baba mo kay once may
ulceration, dira ma penetrate ang virus. But
less likely ang oral sex kay ang saliva has a
protective mechanism kay may antibodies
so mka help prevent pero may possibility
japun
2. Blood Product Transmission
- HIV can be transmitted in whole blood,
concentrated RBCs, platelets, WBCs,
concentrated clotting factors, and plasma
- γ globulin – not associated with
transmission
- that’s why kung positive ka, basi forever ka
di ka donate dugo. Indefinite ang deferral
products: blood donors are screened
self reported risk factors
and serologic markers of
HIV infection.
- pinaka dako nga problem sa screening kung
mag butig if nka contact sa mga sexual
workers. Boo!
- serologic markers:
a. Abs to HIV-1 & HIV-2
(by ELISA)
b. p24 antigen
3. Intravenous drug use with needle
sharing
- led to growing numbers of infected
Immunology and Serology MLS115 (LECTURE)
persons in US urban centers
4. Transplacental viral spread from mother
to fetus
- rate of transmission = ~ 30%
- not a 100% chance nga ma transmit sa
fetus may tendency nga msg normal
ang bata kay 30% lng ang chance nga
ma transmit
- infection occurs transplacentally, during
delivery, and perinatally
5. Students & Healthcare Providers
- mga interns wala gina pa collect blood
from HIV px.
- 3 out of 1000 (0.3%) – the risk of
contracting HIV from a stick
with a needle, contaminated
HIV infected blood
- much lower – for accidental body fluid
contact with broken skin
- no risk in touching an HIV infected
patient, unless there is contact with
blood or body fluid
- the risk goes up if:
♥ the injury is deep
♥ the needle was in a patient’s
artery or vein
♥ had blood visible on it
♥ if the patient has a high viral
load
0.3% - risk of transmission of HIV by needle
stick
30% - the risk of transmission of HBV after
a needle stick from a patient who is
HBeAg (+)
3% - the risk of transmission of Hepatitis C
virus
6. NOT spread by mosquito bites or casual
contact (kissing, sharing food)
- there is NO evidence that saliva, urine,
tears, or sweat, can transmit the virus
Cell Infection
1. Once the HIV virion is in the bloodstream,
its gp160 (gp 120 + gp 41) bind to the CD4
receptor on target cells.
 CD4 receptor is present in
high concentration on T-
helper lymphocytes.
CD4+ T-helper cells
2. Other cells that possess CD4 receptors in
lower concentrations and which can
become infected:
a. Macrophages
b. Monocytes
c. CNS dendritic cells
3. Following HIV binding to the CD4 receptor,
the viral envelope fuses with the infected
host cell, allowing capsid entry.
- Sulod si capsid, sulod si
virus
How does HIV bind to the CD4 receptor?
2 cell surface proteins:
1. Fusin - produced by T-lymphocytes
2. CKR5 - produced by macrophages
- serve as co-factors with the CD4 molecule
for binding of HIV to lymphocytes and
macrophages
- low levels of CKR5  patients appear to be
resistant to HIV
infection
4. Following viral replication, the new capsids
form around the new RNA dimers. The
virion buds through the host cell membrane,
stealing portions of the membrane to use as
an envelope, leaving Tcell dead.
- Diri makita ngaa ang tcells ganubo ang
count kung may HIV kay gina guba sng
virus ang tcells
Pathogenesis
1. Acute viral illness
- fever
- malaise
- lymphadenopathy
- pharyngitis
* viremia – high levels of blood-borne
HIV at this stage
- the viruses spread to infect lymph nodes
and macrophages
- an HIV-specific immune response arises
 resulting in decreased
viremia and resolution of
the symptoms
- HIV replication continues in lymph
nodes and PB (peripheral blood)
2. Clinical latency
- follows for a median of 8 years
- pwede sobra 8 years pwede less
- no symptoms of AIDS
- some patients develop a dramatic
generalized lymphadenopathy (20 to an
Immunology and Serology MLS115 (LECTURE)
aggressive immune attack against HIV common as CD4 counts drop
harbored in the lymph nodes) below 400
- NOT a true viral latency without viral  kung HIV px gina test for TB to
replication confirm if may TB amu na ma
 HIV continues to replicate in send xray
the lymphoid tissue; and
 there is a steady gradual 3. CD4+ T-cell count < 200 (about 8 years):
destruction of CD4 T-  serious opportunistic killers set in
lymphocytes a. Pneumocystis carinii
- ang drug is ARV (antiretroviral) drugs pneumonia
nga gina provide sng gov’t sa HIV px b. Cryptococcus neoformans
- toward the end of the 8 years, patients c. Toxoplasma gondii
are more susceptible to bacterial and
skin infections and can develop 4. CD4+ T-cell count <50:
constitutional/systemic symptoms:  immune system is almost
a. Fever completely down
b. Weight loss Mycobacterium avium-
c. Night sweats intracellulare
d. Adenopathy - most common nga naga
cause sng opportunistic
3. AIDS infection sa HIV
- If u are HIV positive, it doesn’t mean that - normally only causing
u are already an AIDS px bcoz ang infection in birds
AIDS sa pinaka end na sng course of - causes disseminated
infection disease in the AIDS
- develops for a median of 2 years patients
followed by death Cytomegalovirus
- sa AIDS nga stage - infections also rise as the
immunocompromised kna count moves from 50 to 0
- having a CD4 T-lymphocyte count of
<200 and/or one of many AIDS-defining
opportunistic infections
- opportunistic infections:
a. Candida esophagitis
b. Pneumocystis carinii
pneumonia
c. Kaposi sarcoma
- skin infection

CD4 Count
1. Normal CD4 + T-cell counts = 1000
cells/uL blood
 HIV-infected: count declines by
about 60 cells/mL blood/year
2. CD4 + T-cell count of 400-200 (about 7
years):
 constitutional symptoms + skin
infections
skin infections:
a. severe athlete’s foot
b. Oral thrush (Candida
albicans)
c. Herpes zoster infection
 bacterial infections (M.
tuberculosis) become more