CYTOMEGALOVIRUS procedure
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Transcript of CYTOMEGALOVIRUS procedure
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CYTOMEGALOVIRUS
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Is most prevalent among very young children,
particularly those live in crowded conditions.
Transmitted by: oral, respiratory or venereal routes,through organ transplant, or fresh transfused blood.
Without any detectable signs or symptoms.
Some of them develop a fever-like syndrome, with
prolonged fever, and a mild hepatitis.
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Major areas of risk of infection include pre-
natal or postnatal infants
and immunocompromised individuals, such as organtransplant recipients, persons with leukemia, or those
infected with human immunodeficiency virus (HIV). In
HIV infected persons, HCMV is considered an AIDS-
defining infection, indicating that the T-cell count has
dropped to low levels.
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LABORATORY DIAGNOSIS Urine, saliva, blood and biopsy samples can be used for
virus isolation.
Urine should be collected a sterile container without
additives.
Saliva samples should first be soaked on to a swabwhich 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
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SEROLOGIC TEST FOR CMV
Passive latex agglutination
y Method for the detection of antibodies to CMV
involves the mixing of latex particles with the
patients serum..
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METHOD 1 : PASSIVE LATEX
AGGLUTINATION FOR THEDETECTION OF
ANTIBODIES TO CYTOMEGALOVIRUS IN
HUMAN SERUM
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MATERIALS
The kit contains :
1. CMV antigen-coated latex participles prepared fromdisrupted CMV that has been judged to be inactivated bybioassay procedures.
2. Phosphate-buffered saline , PH 7.4, containing bovineserum albumin with 0.02 percent sodium azide.
3. Test cards, which must be flat for proper reactions .4. Plastic stirrers
5. Dispensing needle , 2-1 gauge, green hub .
6. High-reactive control serum (human) with 0.1 percentsodium azide .
7. Low-reactive control-serum (human) with 0.1 percentsodium azide .
8. Nonreactive control serum (human) with 0.1 percentsodium azide .
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Additonal material needed (not provided by the kit ):
1. Centrifuge
2. Rotator with humidifying cover .
3. High-intensity incandescent lamp .
4. Micropipettors, 25 microliter delivery .
5. Vortex mixer .
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SPECIMEN REQUIREMENTS
A minimum of 2 ml of clotted blood or
anticolagulated promptly and an aliquot of serum
or plasma removed plasma specimens containing
EDTA or heparin as an anticoagulant can be
used for qualitative or quantitative testing using
the same technique as for serum samples .
Specimens may be stored for up to 1 week in the
refrigerator.18 degrees C if longer freezing
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PROCEDURE (QUALITATIVE) 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. Using a micropipettor , place 25 microliter of each
specimen
2. Using 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.
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4. Dispense 1 free-falling drop (approximately 15
microliter )
5. Hand rotate the card back and forth 3 or 4 times todistribute the latex antigen throughout the circle.
6. Place the card on the rotator and mix for 8 mins.
Under a moistened humidify cover.
7. Immediately after rotation, read the cardmacroscopically in the wet state. Test should be read
under a high intensity incandescent lamp.
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INTERPRETATION
Any agglutination of the latex reagent is
regarded as a positive. If the suspension remains
evenly dispersed with no agglutination, report as
negative.
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DISCUSSION
Antibodies may not be detectable in early stage.
The presence of CMV antibodies in qualitative
testing on a single acute-phase is a n indication
of previous exposure to the virus but does notindicate immunity to subsequent reinfection.
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THE FF SHOULD BE NOTED WITH THE RESPECT
TO THIS PROCEDURE THAT INVOLVE CMV
ANTIBODYDETECTION:
1. Pt. with acute infection may not be have detectable
antibody.
2. Seroconversion may indicate recent infection, but an
increase in antibody titer by this method does notdifferentiate bet. A primary and secondary antibody
response.
3. The timing of antibody response during a primary
infection may differ slightly.
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The Cytomegalovirus IgMAssay
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This test, which is an indirect enzyme-labeled
immunosorbent assay, detects IgM antivodies to CMV,
using antigen-coated microwells as a solid phase. IgMantbodies ma persist as long as 9 mos. In
immunocompetent pt.
IbM responses vary bet. Diff indivuals. For example,
10-30% of infants cond=genitally infected with CMV
fail to develop IgM antibody
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METHOD 2: CYTOMEGALOVIRUS IgMASSAY
(QUANTITATIVE)
The technique describe here is intended for usewith the kit provided by Sigma chemical Co. St
Louis, MO
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MATERIALS
1. Microplate walls coated with CMV antigen.
Store at 2-6 degrees C with desiccant in the
reusable plastic bag bag and reseal after
opening.
2. Holder or wells
3. Diluent, a buffered protein solution,containing
surfactant and blue dye,pH 7.5
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4. Calibrator. This is the human serum containing IgM
antibodies to CMV at 100 arbitrary units.
5. Conjugate. Contains goat antibodies to human IgMlabeled with calf alkaline phosphatase.
6. Substrate. This contains p-nitrophenyl phosphate,
disodium, and hexahydrate 1mg/ml, pH 9.6 .
7. Wash concentrate. This is buffer solution concentratewith surfactant.
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8. Stop solution. An alkaline solution pH 12.0. Store at
room temp.
9. Positive control. This is human serum containing
IgM antibodies to CMV and .1 percent sodium azide
as a preservative.
10. Negative control. This is human serum containing no
detectable antibodies to CMV and .1 percent sodiumazide as a preservative.
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ADDITIONAL MATERIAL REQUIRED
1. Spectrophotmeter
2. Pipettes
3. Timer
4. 1 liter measuring cylinder5. Squeeze bottle for dispensing wash solution
6. Dilution plates
7. Test tubes or cuvettes
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SPECIMEN REQUIREMENT
Like the first method.
If the test cannot be performed immediately, the
specimen should be refrigerated or frozen.
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PROCEDURE
1. Dilute 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. Using a pipette tip, mix the samples and the
diluent by drawing up and expelling two or
three times.
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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. Shake out or aspirate the contents of the wells.
7.Place 2 drops (100 UL) conjugate in each well,
including the reagent blank well.8. Allow to stand at room temp 3o mins.
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9. Wash wells by repeating step 6.
10. Place 2 drops (100 UL) substrate in each well,
including the reagent blank well.11. Allow to stand at room temp 3o mins.
12. Place 2 drops (100 UL) stop solution in each well,
including the reagent blank well.
13. Read and record absorbance of each test at 405 nmwithin 2 hours after the reaction has been stopped.
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DISCUSSION
False negative results can be due to a low level of
specific IgM antibodies or interference by
competitive antigen specific CMV-specific
IgG(Especially in congenitally infected newborns
because of the presence of maternal Ig)
False positive results can be due to interference
by IgM rheumatoid factor.