You are on page 1of 26

m 

? Is most prevalent among very young children,


particularly those live in crowded conditions.
?  oral, respiratory or venereal routes,
through organ transplant, or fresh transfused blood.
Without any detectable signs or symptoms.
? pome of them develop a fever-like syndrome, with
prolonged fever, and a mild hepatitis.
? ëajor areas of risk of infection include pre-
natal or postnatal infants
and immunocompromised individuals, such as organ
transplant recipients, persons with leukemia, or those
infected with human immunodeficiency virus (HIV). In
HIV infected persons, HCëV is considered an Õ 

  , indicating that the T-cell count has
dropped to low levels.


 
? Yrine, saliva, blood and biopsy samples can be used for
virus isolation.
? Yrine should be collected a sterile container without
additives.
? paliva samples should first be soaked on to a swab
which is then broken off into transport medium.
? Blood should be collected into a heparinized bottle
(some phenolic preservatives found in proprietary
pathology blood bottles may be toxic to blood cultures)
containing 500 units of heparin.
? Tissue biopsies should be placed in sterile plastic
containers. The specimens can be treated in the
following ways
p IC T pT F CëV
? Passive latex agglutination
@ ëethod for the detection of antibodies to CëV
involves the mixing of latex particles with the
patient·s serum..
? ë TH 1 : PAppIV AT 
AYTINATIN F TH  T CTIN F
ANTIBI p T CYTë AVIYp IN
HYëAN p Yë
? ëAT IAp
? The kit contains :
1. CëV antigen-coated latex participles prepared from
disrupted CëV that has been judged to be inactivated by
bioassay procedures.
2. Phosphate-buffered saline , PH 7.4, containing bovine
serum albumin with 0.02 percent sodium azide.
3. Test cards, which must be flat for proper reactions .
4. Plastic stirrers
5. ispensing needle , 2-1 gauge, green hub .
6. High-reactive control serum (human) with 0.1 percent
sodium azide .
7. ow-reactive control-serum (human) with 0.1 percent
sodium azide .
8. Nonreactive control serum (human) with 0.1 percent
sodium azide .
? Additonal material needed (not provided by the kit ):
1. Centrifuge
2. otator with humidifying cover .
3. High-intensity incandescent lamp .
4. ëicropipettors, 25 microliter delivery .
5. Vortex mixer .
pP CIë N  YI ë NTp
? A minimum of 2 ml of clotted blood or
anticolagulated promptly and an aliquot of serum
or plasma removed plasma specimens containing
TA or heparin as an anticoagulant can be
used for qualitative or quantitative testing using
the same technique as for serum samples .
? ppecimens may be stored for up to 1 week in the
refrigerator.18 degrees C if longer freezing
PC Y (YAITATIV )
? Before beginning the procedure , allow the reagents to
reach the room temperature . do not mix reagents to
different kit lot of numbers and avoid microbial
contamination of reagents . label each circle of the card
with appropriate identification of patient sera and
controls .
1. Ysing a micropipettor , place 25 microliter of each
specimen
2. Ysing a new plastic stirrer for each circle , spread the
serum to fill the entire circle .
3. Hold the bottle cap over the tip of the needle and
gently invert the latex reagent dispensing the bottle
several times.
4. ispense 1 free-falling drop (approximately 15
microliter )
5. Hand rotate the card back and forth 3 or 4 times to
distribute the latex antigen throughout the circle.
6. Place the card on the rotator and mix for 8 mins.
Ynder a moistened humidify cover.
7. Immediately after rotation, read the card
macroscopically in the wet state. Test should be read
under a high intensity incandescent lamp.
INT P TATIN
? Any agglutination of the latex reagent is
regarded as a positive. If the suspension remains
evenly dispersed with no agglutination, report as
negative.
IpCYppIN
? Antibodies may not be detectable in early stage.
? The presence of CëV antibodies in qualitative
testing on a single acute-phase is a n indication
of previous exposure to the virus but does not
indicate immunity to subsequent reinfection.
TH FF pHY B NT  WITH TH  pP CT
T THIp PC Y THAT INVV CëV
ANTIBY  T CTIN:

1. Pt. with acute infection may not be have detectable


antibody.
2. peroconversion may indicate recent infection, but an
increase in antibody titer by this method does not
differentiate bet. A primary and secondary antibody
response.
3. The timing of antibody response during a primary
infection may differ slightly.
? The Cytomegalovirus Igë Assay
? This test, which is an indirect enzyme-labeled
immunosorbent assay, detects Igë antivodies to CëV,
using antigen-coated microwells as a solid phase. Igë
antbodies ma persist as long as 9 mos. In
immunocompetent pt.
? Ibë responses vary bet. iff indivuals. For example,
10-30% of infants cond=genitally infected with CëV
fail to develop Igë antibody
? ë TH 2: CYTë AVIYp Igë AppAY
(YANTITATIV )

? The technique describe here is intended for use


with the kit provided by pigma chemical Co. pt
ouis, ë
ëAT IAp
1. ëicroplate walls coated with CëV antigen.
ptore at 2-6 degrees C with desiccant in the
reusable plastic bag bag and reseal after
opening.
2. Holder or wells
3. iluent, a buffered protein solution,containing
surfactant and blue dye,pH 7.5
4. Calibrator. This is the human serum containing Igë
antibodies to CëV at 100 arbitrary units.
5. Conjugate. Contains goat antibodies to human Igë
labeled with calf alkaline phosphatase.
6. pubstrate. This contains p-nitrophenyl phosphate,
disodium, and hexahydrate 1mg/ml, pH 9.6 .
7. Wash concentrate. This is buffer solution concentrate
with surfactant.
8. ptop solution. An alkaline solution pH 12.0. ptore at
room temp.
9. Positive control. This is human serum containing
Igë antibodies to CëV and .1 percent sodium azide
as a preservative.
10. Negative control. This is human serum containing no
detectable antibodies to CëV and .1 percent sodium
azide as a preservative.
AITINA ëAT IA  YI 
1. ppectrophotmeter
2. Pipettes
3. Timer
4. 1 liter measuring cylinder
5. pqueeze bottle for dispensing wash solution
6. ilution plates
7. Test tubes or cuvettes
pP CIë N  YI ë NT
? ike the first method.
? If the test cannot be performed immediately, the
specimen should be refrigerated or frozen.
PC Y
1. ilute callibrator, positive and negative
controls and test samples by combining 10
micro liter of each with 200 micro liter of
sample diluent in tubes or diluent plates.
2. Placed the desired no. of antigen wells in the
holder.
3. Ysing a pipette tip, mix the samples and the
diluent by drawing up and expelling two or
three times.
4. Include one well that contains only 100 micro liter
sample diluent.
5. Allow the plate to stand at room temp for 30 (+- 2 )
mins.
6. phake out or aspirate the contents of the wells.
7.Place 2 drops (100 Y) conjugate in each well,
including the reagent blank well.
8. Allow to stand at room temp 3o mins.
9. Wash wells by repeating step 6.
10. Place 2 drops (100 Y) substrate in each well,
including the reagent blank well.
11. Allow to stand at room temp 3o mins.
12. Place 2 drops (100 Y) stop solution in each well,
including the reagent blank well.
13. ead and record absorbance of each test at 405 nm
within 2 hours after the reaction has been stopped.
IpCYppIN
? False negative results can be due to a low level of
specific Igë antibodies or interference by
competitive antigen specific CëV-specific
Ig( specially in congenitally infected newborns
because of the presence of maternal Ig)
? False positive results can be due to interference
by Igë rheumatoid factor.