Module 5 Diagnostic tests

TAS

Global Programme to Eliminate Lymphatic Filariasis (GPELF)

Training in monitoring and epidemiological assessment of mass drug administration for eliminating lymphatic filariasis

Module 5 Diagnostic tests

Module 5 Diagnostic tests

Learning objectives

By the end of this module, you should understand how to:

1. procure diagnostic tests

2. collect blood

3. prepare, conduct and interpret ICTs

4. prepare, conduct and interpret the Brugia RapidTM tests

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Module 5 Diagnostic tests

Overview

Diagnostic tests for TAS

Procurement of diagnostic tests

Blood collection techniques

Standard operating procedures for diagnostic tests:

W. bancrofti – ICT

Brugia spp. – BrugiaRapid™

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Module 5 Diagnostic tests

Diagnostic tests for TAS

Mapping

Mf or Ag≥1% TAS

Surveillance

Baseline

MDA

Follow-up[Eligibility]

Mid-term (optional)

Yes

M&E

Pass

Fail

Assessment tools

Mapping MDA Surveillance

Blood film or ICT Blood film or ICT

ICT or Brugia RapidTM test

Potential for future use:Antibody, xenomonitoring

ICT or Brugia

RapidTM test

TAS

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Module 5 Diagnostic tests

Areas endemic for W. bancrofti: ICT

Areas endemic for Brugia spp.: Brugia RapidTM test

Areas endemic for both W. bancrofti and Brugia spp.: both diagnostic tests, with testing evaluated separately against critical cut-offs

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Diagnostic tests for TAS

Module 5 Diagnostic tests

Field assay Detection target

Blood film Microfilariae

ICT Filarial antigen

Brugia RapidTM test Antifilarial antibody

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Diagnostic tests for TAS

Module 5 Diagnostic tests

Procurement of diagnostic tests

ICT BinaxNow® Filariasis – manufactured by Alere, Inc

(Scarborough, Maine, USA)

A “no objection certificate” is required for importation of the test devices.

Positive control can be obtained from the Filariasis Research Reagent Repository Center (www.filariasiscenter.org)

Brugia RapidTM test Manufactured by Reszon Diagnostics International

(Selangor, Malaysia)

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Module 5 Diagnostic tests

Quality control

Training should be conducted before a TAS to ensure that all protocols are followed properly.

The pouch should be opened just before use.

Diagnostic tests should be tested with a positive control to ensure their validity.

Diagnostic tests should be stored properly to minimize the risk for compromising their quality.

Any indeterminate test result should immediately be read by a second reader or supervisor and the test repeated if necessary.

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Module 5 Diagnostic tests

Blood collection technique

Clean the finger to be pricked with an alcohol swab, and allow finger to dry.

Prick the internal side of the finger with a sterile lancet.

Safely discard the lancet.

Collect the blood (4a) into a calibrated capillary tube coated with an anticoagulant or (b) onto filter paper according to the survey method. If collecting into tubes, collect slightly more than the required volume of blood in case of clotting or spillage.

1 2 3

4 4a 4b

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Module 5 Diagnostic tests

ICT

Sensitive for detecting W. bancrofti antigen.

Do not require laboratory equipment and can be processed quickly.

Positive result indicates the presence of adult worm antigen.

Adequate training is necessary to reduce inter-observer variation and to reduce misreading of cards, which can lead to false-positive results.

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Module 5 Diagnostic tests

ICT: Preparation

Storage – Cards have a limited shelf-life at ambient temperature (3 months at 30 °C) but a longer shelf-life when stored at 4 °C (about 9 months). Cards should not be frozen.

Testing with a positive control – Before a field survey is begun, two cards from each lot should be tested with a weak positive control, which can be obtained from the Filariasis Research Reagent Repository Center (www.filariasiscenter.org). With this control, the test line may be very faint. Do not use cards that give a negative result when tested with the control.

Transport – A cool box is not required for transporting cards for use in the field; however, care should be taken not to expose cards to extreme heat for long periods.

Light – Cards must be read under adequate lighting, as faint lines can be difficult to see. This is especially important when reading cards at night.

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Module 5 Diagnostic tests

Remove card from pouch just before use.

1

2Collect 100 µl of blood by finger prick into a calibrated capillary tube or remove 100 µl of blood from a microcentrifuge tube with a micropipette. Do not add blood directly from the finger to the card.

ICT: Procedure

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Module 5 Diagnostic tests

3

Add blood sample slowly to the white portion of the sample pad.

Do not add blood directly to the pink portion of the sample pad.

Do not close the card before the sample migrates to the pink portion of the sample pad (takes about 30 seconds after addition of blood).

4

Remove adhesive liner and close card. Start timing.

It is helpful to record the starting time on the front of the card.

Do not read cards if the plasma has not flowed all the way down the strip.

If plasma fails to migrate completely past the bottom of the window, a false-positive result may be read.

ICT: Procedure

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Module 5 Diagnostic tests

ICT: Procedure

5 Read test results 10 minutes after closing card.

Do not read cards at any time other than 10 minutes, as the reading may be false-positive.Circle the appropriate

result on the front of the card to create a permanent record.

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Module 5 Diagnostic tests

ICT: Interpretation

T = test

C = control

Positive Negative

InvalidNo lines appear

InvalidNo control line

Positive (weak)

Negative

The test line should be pink. Sometimes, a grey line or shadow appears in the test line position. This should not be misinterpreted as a positive result.

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Module 5 Diagnostic tests

Brugia RapidTM test

Sensitive for detecting antibodies to B. malayi and B. timori.

Does not require laboratory equipment and can be processed quickly.

Positive result indicates the presence of antifilarial antibodies.

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Module 5 Diagnostic tests

Brugia RapidTM test: Preparation

Storage – The test has a shelf-life of 18 months when stored at ambient temperature (20–25 °C); 4 oC (refrigeration) is recommended for long-term storage. The tests should not be frozen.  

Transport – A cool box is not required, although it is desirable, when transporting tests for use in the field. Care should be taken not to expose the tests to extreme heat for long periods.

Lighting – Tests must be read under adequate lighting, as faint lines can be difficult to see. This is especially important when reading tests at night.

The test requires 30 µl of serum or plasma or 35 µl of whole blood.

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Module 5 Diagnostic tests

Brugia RapidTM test: Procedure

2Collect 35 µl of blood by finger prick into a calibrated capillary tube or remove 35 µl of blood from a microcentrifuge tube with a micropipette. Do not add blood directly from the finger to the cassette.

Bring test cassette and chase buffer to room temperature. Remove cassette from foil pouch just before use. Label the cassette with information on the sample.

1

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Module 5 Diagnostic tests

Brugia RapidTM test: Procedure

3

Add blood sample slowly to the square well by touching the capillary tube or pipette tip to the sloping side.

If using serum or plasma, only 30 µl are needed.

The sample will start to flow up the strip. The cassette can be tapped gently on the table to facilitate the flow. Wait until the sample has reached the blue line (A).

If the sample does not reach the blue line (A) after 4 minutes but has reached area B, proceed to the next step.

Add one drop of chase buffer to the same square well.

If using serum or plasma, no chase buffer is required.

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Module 5 Diagnostic tests

Brugia RapidTM test: Procedure

4

Firmly pull the clear tab at the bottom of the cassette until you feel resistance.

When the sample has reached the blue line (A), add three drops of chase buffer to the circle well at the top of the cassette.

Add the buffer drop by drop, and allow each drop to saturate the pad before delivering the next drop.

After pulling the clear tab, add one drop of buffer to the square well.

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Module 5 Diagnostic tests

Brugia RapidTM test: Procedure

5

Start timing. Read test results 25 minutes after adding the final drop of buffer.

Test results for serum and plasma samples should be read after 15 minutes.

Record the start or end time on the front of the cassette.

Write the appropriate result on the front of the cassette to create a permanent record.

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Module 5 Diagnostic tests

Brugia RapidTM test: Interpretation

A = blue line

B = control

C = test line

A

B

C

Positive B and C lines present

Positive (weak) B and C lines present

NegativeB line appears; no C line present

Invalid No B and C lines

present

InvalidNo B line present;

C line appears

Invalid Blood did not clear

The intensity of the blue line does not affect the reading.

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Module 5 Diagnostic tests

Exercise

1. Practise finger-prick blood collection.

2. Observe use of positive controls to ensure the validity of the diagnostic test(s).

3. Practise using the diagnostic test(s) approved for transmission assessment surveys in your country or area.

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