antibodies (IgM and IgG) to Dengue virus, if present in the

sample. This complex moves further on the membrane

to the test region where it is immobilized by the specific

human IgM antibody and/or human IgG antibody binding

proteins coated on the membrane leading to formation of a

colored band which confirms a positive test result. Absence

of these colored bands in the test window “T” indicates a

negative test result. A built-in control band in the control

window “C” appears when the test has been performed

correctly, regardless of the presence or absence of antiDengue virus antibodies in the specimen and serves to

validate the test performance.

Reagents and Materials Supplied

Each kit contains:

A. lndividual pouches, each containing:

1. Denguecheck-WB (Device) Membrane test assembly predispensed with recombinant Dengue

virus specific antigen colloidal gold conjugate,

streptavidin gold conjugate, anti-human IgM at

test region ‘M’ Protein A at the test region ‘G’ and

Biotin at the control region ‘C’.

2. Desiccant pouch

3. 5 µL sample loop.

B. Sample running buffer.

C. Package insert.

Storage and Stability

The sealed pouches in the test kit and the kit components

may be stored between 4 and 30°C for the duration of the

shelf-life as indicated on the pouch.

Optional Material Required: 5 µL precision micropipettes.

Note

1. Forin vitro diagnostic use only. Not for medicinal use.

2. Do not use beyond expiry date.

3. Read the instructions carefully before performing the

test.

4. Handle all specimen as potentially infectious.

5. Follow standard biosafety guidelines for handling and

disposal of potentially infective material.

Specimen Collection and Preparation

1. No special preparation of the patient is necessary prior

to specimen collection by approved techniques.

2. Though fresh serum/plasma is preferable, specimen

may be stored at 2–8° C for up to 24 hours, in case of

delay in testing.

3. Whole blood samples collected with a suitable

anticoagulant such as EDTA or Heparin or Oxalate

can also be used.

4. Do not use turbid, lipemic, icteric and hemolyzed

specimen.

5. Repeated freezing, thawing of the specimen should be

avoided.

6. Specimen containing precipitates or particulate matter

must be centrifuged and the clear supernatant only

should be used for testing.

Testing Procedure and Interpretation of Results

1. Bring the kit components to room temperature before

testing.

2. Open the pouch and retrieve the test device. Once

opened, the device must be used immediately.

3. Label the test device with patient identity.

4. Add 5 µL of serum/plasma/whole blood, with the

micropipette into the sample port ‘A’, or using the

5 µL sample loop provided with the-kit, dip the loop

Serology/Immunology 641

into the sample and then blot into the sample port ‘A’.

Ensure that the loop does not retrieve clots or debris

from the sample.

5. Add five drops of sample running buffer in the reagent

port ‘B’.

6. Exactly at the end of 15 minutes, read the test results.

Interpretation of Results

Negative Test Result

The presence of only the single red/purple colored band

in the control window “C” indicates the absence of specific

antibodies against dengue virus or that the amount of

antibodies is below the detection limit of the test.

Positive Test Result

1. In addition to the band in the control window ‘C’,

appearance of two red/purple colored bands in the

test window at region ‘M’ and region ‘G’ indicates

the presence of Dengue virus specific IgM and IgG

antibodies (acute secondary infection).

2. In addition to the control band in the control window

‘C’, appearance of a red/purple colored band in the test

window at region ‘M’ indicates the presence of Dengue

virus specific IgM antibodies (acute primary infection).

3. In addition to the control band in the control window

‘C’, the appearance of a red/purple colored band in

the test window at region ‘G’ indicates the presence of

Dengue virus specific IgG antibodies (acute secondary

infection).

Invalid Result: If, after 15 minutes, no band is visible

either in the test or control window, the result is

considered invalid. The test should be repeated with a

new device.

Remarks

1. Do not use test kit beyond expiration date.

2. While sample should be collected as soon as possible

after onset of illness, it is recommended that followup of testing should be done on day 10 after the first

sample to allow seroconversion, especially when the

test is negative and Dengue virus infection is clinically

suspected.

3. Though Denguecheck-WB does provide evidence

to distinguish the past (secondary) infection from

current (primary) ongoing infection, a negative result

does not preclude the possibility of infection with

Dengue virus.

4. As with all diagnostic tests, a definitive clinical

diagnosis should not be based on the results of a single

test but should rather be made by a clinician after all

clinical findings have been evaluated.

5. The DHF is primarily the disease of children

under 15 years in hyperendemic areas. Impending

DSS symptoms include suspected abdominal

pain, persistent vomiting, change in the level of

consciousness, hypothermia and sudden decrease in

platelet counts.

6. Eighty percent of the patients may have detectable

levels of IgM antibody by day 5 of illness and 99% by

day 10.

7. IgM levels rise quickly and peak by two weeks

after onset of symptoms and then fall to become

undetectable over 2 to 3 months. IgG antibodies rise

quickly and peak at about two weeks postinfection and

then decline slowly over 3 to 6 months.

642 Concise Book of Medical Laboratory Technology: Methods and Interpretations Troubleshooting

Dengue Check WB and Leptocheck-WB

Problem: False positive results

Possible causes Solutions

1.  The flow properties of the nitrocellulose membrance are partially

affected leading to the movement of partially aggregated gold-sol

particles

Check the pouch for pinholes and also observe the desiccant for any

color change. The results of the test should be correlated with clinical

findings

Problem: Faint lines observed in control and test region

Possible causes Solutions

1. Hemolyzed blood samples were used for testing Do not use hemolyzed blood samples for testing

2. Reading taken after 15 min Read results exactly at 15 minutes

Problem: Delayed results and altered flow

Possible causes Solutions

1. Whole blood samples having microclots or fibrin Ensure that the whole blood collected directly from fingerprick (without anticoagulant) should be free from microclots to avoid altered

flow and delayed reaction time

Problem: False negative results

Possible causes Solutions

1. Inadequate quantity of sample used for performing the test The exact number of drops of the sample as mentioned in the pack

insert should be dispensed for performing the test using the dropper

provided with the kit

2. The kit is exposed to very high temperatures leading to deterioration of the antibodies coated on the device

Store at recommended temperature when not in use

3. Turbid or contaminated serum samples were used for testing Do not use turbid or contaminated serum samples for testing

4. Sample tested early after infection Specific antibodies reach detectable levels about one week after onset

of disease; hence follow-up testing after day 10 is recommended to

allow seroconversion

Problem: Invalid results

Possible causes Solutions

1. Pinholes/defect in the pouch.