CYTOMEGALOVIRUS procedure

8/7/2019 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.

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CYTOMEGALOVIRUS procedure