as it is highly sensitive to denaturation, drying and

microbial contamination

Check the performance of the reagent with positive and negative controls provided with the kit. Avoid exposure of the reagent to light

5. Serum, plasma stored for a long period of time is used for testing Fresh serum or plasma should be used for testing. Samples not

tested immediately may be stored at 2–8°C for up to 48 hours

6. Hemolyzed or lipemic samples may have been used for testing Avoid using hemolyzed or lipemic samples for testing

7. Antigenic suspension has settled down in the vial Shake the Carbogen reagent vial well before use to disperse carbon

particles uniformly and improve test readability

Troubleshooting

Problem: False positive results

Contd...

Serology/Immunology 621

ONE-STEP TEST FOR SYPHILIS: DIPSTICK

SYPHICHECK®

(Courtesy: Tulip Group of Companies) (can also be in

device form)

Introduction

Syphicheck is a one-step; rapid, self-performing, qualitative,

two-site double antigen sandwich immunoassay for the

detection of syphilis.

Summary

Syphilis is a sexually transmitted (venereal) disease caused

by the spirochete Treponema pallidum. The disease can

also be transmitted congenitally thereby attaining its

importance in antenatal screening. After infection, the host

forms non-Treponema antilipoidal antibodies (reagins)

to the lipoidal material released from the damaged host

cells as well as Treponema specific antibodies. Serological

tests for non-Treponema antibodies such as VDRL,

RPR, TRUST, etc. are useful as screening tests. Tests for

Treponema specific antibodies such as TPHA, FTA-ABS,

rapid Treponema antibody tests are gaining importance

as screening as well as confirmatory tests because they

detect the presence of antibodies specific to Treponema

pallidum.

Syphicheck qualitatively detects the presence of IgM

and IgG class of Treponema specific antibodies during

syphilis in serum or plasma specimen within 15 minutes.

Principle

Syphicheck utilizes the principle of immunochromatography, a unique two-site immunoassay on a

membrane. As the test sample flows through the membrane

assembly of the dipstick, the recombinant Treponema

antigen-colloidal gold conjugate forms a complex with

Treponema specific antibodies in the sample. This complex

moves further on the membrane to the test region where

it is immobilized by the recombinant Treponema pallidum

antigen coated on the membrane leading to the formation

of a pink to deep purple colored band at the test region

which confirms a positive test result. Absence of this

colored band in test region indicates a negative test result.

The unreacted conjugate and the unbound complex if

any move further on the membrane and are subsequently

immobilized by the anti-rabbit antibodies coated on the

membrane at the control region, forming a pink to deep

purple colored band. The control band serves to validate

the test results.

Reagents and Materials Supplied

Each individual pouch contains:

1. Dipstick: Membrane assembly predispensed with

recombinant Treponema pallidum antigen-colloidal

gold conjugate, recombinant Treponema pallidum

antigen and anti-rabbit antiserum coated at the

respective regions.

2. Desiccant pouch.

8.  Weak flocculation may be interpreted as negative The carbon antigen should be gently but thoroughly mixed before

testing to disperse the carbon particles uniformly and improve test

readability. The serum and the reagent should be mixed properly.

Small black floccules against a white background indicate a weakly

reactive test result

9. Cold reagents are used for testing Bring all reagents and samples to room temperature before commencing the testing procedure

10. Expired reagents are used for testing Check the expiry date of the reagents before use

11. Error in interpreting the test results Flocculation results should be interpreted carefully after comparing with positive and negative controls. Note: Very slight roughness

should be interpreted as a negative result

12. The reagent used for testing is in a frozen condition The reagent should not be frozen. It should be stored at 2–8°C

13. False negatives are obtained in the tertiary stage of the disease The test should be confirmed with FTA (Fluorescent treponemal

antibody) or TPHA Treponema pallidum hemagglutination) tests

Contd...

622 Concise Book of Medical Laboratory Technology: Methods and Interpretations Additional Material Required

12 × 75 mm test tubes.

Storage and Stability

The sealed pouches in the test kit may be stored between

4 and 30°C for the duration of shelf-life as indicated on the

pouch.

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 specimens as potentially infectious.

5. Follow standard biosafety guidelines for handling and

disposal of potentially infective material.

Specimen Collection and Preparation

No special preparation of patient is necessary prior to

specimen collection by approved techniques. Though fresh

serum/plasma is preferable, serum/plasma specimens may

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

testing. Do not use hemolyzed or contaminated specimens.

Turbid specimens should be centrifuged or allowed to settle

and only the clear supernatant should be used for testing.

Testing Procedure and Interpretation of Results

Bring kit components, specimen to room temperature

prior to testing.

1. Collect serum/plasma in a clean test tube (approximately

0.5–1 mL may be required). Ensure that only sufficient

quantity of the specimen is collected to allow submerging

the red area on the dipstick (about 1 cm high).

2. Bring the sealed pouch to room temperature, open

the pouch and remove the dipstick. Once opened, the

dipstick must be used immediately.

3. Dip the dipstick in serum/plasma specimen

submerging only the red area.

4. The dipstick should be left submerged for the entire

duration of the test ensuring only the red area is

submerged in the specimen.

5. At the end of 15 minutes read the results as follows:

 Negative: Only one pink to deep purple colored band

appears on the dipstick (Fig. 22.25)

 Positive: Two distinct pink to deep purple colored

bands appear on the dipstick

6. The test should be considered invalid if neither the

test band nor the control band appears. Repeat the

test with a new dipstick.

7. Although depending on the concentration of Treponema antibodies in the specimen, positive results may

appear as early as 2 minutes, negative results must be

confirmed only at the end of 15 minutes.

Performance Characteristics

1. Syphicheck detects the presence of Treponema antibodies; thus, a positive result indicates a past or

present infection. Positive results should be evaluated

in correlation with the clinical condition before

arriving at a final diagnosis.

2. Low levels of antibodies to Treponema pallidum

such as those present at a very early primary stage

of infection can give a negative result. But a negative

result does not exclude the possibility of exposure to

or infection with Treponema pallidum. Retesting is

indicated after two weeks if clinically syphilis is still

suspected.

3. In order to assess the clinical response to treatment,