Keul-o-test 

Borreliose Complete

Immunochromatographic rapid test for the detection of anti-Borrelia IgG & IgM antibodies in whole blood, plasma or serum samples

KGST199
DIMDI Reg.-Nr.: DE/CA22/1116-130-IVD

I - PRINCIPLE
Lyme disease (1) or borreliosis is caused by the spirochete Borrelia burgdorferi which is transmitted by ticks. The contaminating insects, infected by Borrelia burgdorferi senso layo, are mainly present in North America (Ixodes scapularis or I. pacificus) and in Western temperate European areas(Ixodes ricinus). The European tick species is the vector of Borrelia burgdorferi, B. afzelli and B. garinii (2).
In 30% to 80% of cases, a skin rash, the Erythema Migrans, is the first clinical sign of the infection appearing after 3 to 10 days at the site of the insect bite. 1 to 3 months later, the first neurological signs, headache or severe disorders like myelitis (3), appear even in the absence of the Erythema Migrans. Still later, patients may develop severe complications such as intermittent attacks of articular arthritis as well as myocarditis or acrodermatitis several years after the primary infection.
Keul-o-test Borreliose Complete is a rapid qualitative screening test for detection of human IgG and IgM class antibodies to Borrelia European strains in serum, plasma or whole blood.
The method employs a unique combination of anti-human immunoglobulins dye conjugate and highly purified Borrelia antigens on the solid phase to specifically detect anti Borrelia antibodies.
The test device consists of a plastic housing containing two different sticks for the detection of IgG or IgM class antibodies. After collection of whole blood, plasma or serum, few drops of the sample are added into each well ( =>) of the reaction device.
As the sample flows through the absorbent device of the IgM stick, the anti-human immunoglobulins dye conjugate binds to the human IgM antibodies forming an antibody-antigen complex. In the same way as the sample flows through the absorbent device of the IgG stick, the anti-human immunoglobulins dye conjugate binds to the human IgG antibodies forming an antibody-antigen complex These complex bind to the specific antigens in the positive reaction zone and produces a pink-rose coloured band. In the absence of anti Borrelia antibodies, there is no line in the positive reaction zone. The reaction mixture continues flowing through the absorbent device, past the reaction and control zones.
Unbound conjugate binds to the reagents in the control zone producing a pink-rose colour band, demonstrating that the reagents are functioning correctly.

II- Keul-o-test Borreliose Complete KIT COMPONENTS
Each kit contains everything needed to perform the tests.

• Keul-o-test Borreliose Complete test devices
• Disposable plastic pipettes
• Diluent in a dropper bottle containing saline buffer, detergent and sodium azide (NaN3 < 0.1%)
• Instructions leaflet

III- STORAGE AND STABILITY

1. All Keul-o-test Borreliose Complete kit components should be stored at any temperature between +4°C and +30°C in the sealed pouch.
2. Do not freeze the test kit.
3. The Keul-o-test Borreliose Complete kit is stable until the expiry date stated on the package label.

IV- PRECAUTIONS

1. This test is designed for in vitro diagnostic use and professional use only.
2. Read carefully instructions leaflet before using this test.
3. Do not use beyond the expiration date which appears in the package label.
4. Do not use a test from a damaged protective wrapper.
5. Handle all biological samples as if they contained infectious agents. When the assay procedure is completed, dispose of specimens and other materials used with this kit as biological waste.
6. Wear protective clothing such as laboratory coats and disposable gloves while assaying samples.
7. Do not eat, drink or smoke in the area where specimens and kit reagents are handled.
8. Avoid any contact between hands and eyes or nose during specimen collection and testing.

V- SPECIMEN COLLECTION AND PREPARATION

Serum, plasma (lithium or ammonium heparinate, EDTA) or whole blood

1. The specimen should be collected under the standard laboratory conditions (aseptically in such a way as to avoid hemolysis).
2. When the test is performed with whole blood, fresh samples should be used (< 4 hours).
3. If the test is to be run within 48 hours after collection, the specimen should be stored in the refrigerator (+2°C to +8°C).
   If testing is delayed more than 48 hours, the specimen should be frozen. The frozen specimen must be completely thawed, thoroughly mixed and brought to room temperature prior to testing. Avoid repeated freezing and thawing.
4. In case of cloudiness, high viscosity or presence of particulate matter into the serum specimen, it should be diluted with equal volume (V/V) of diluting buffer (not provided but available upon request) before testing.

VI- ASSAY PROCEDURE

1. Allow samples and Keul-o-test Borreliose Complete test devices to come to room temperature prior to testing.
2. Remove the reaction device from its protective wrapper by tearing along the split.
3. Label device with the patient's name or control number.
4. Fill the serum dropper with specimen (serum or plasma) and by holding it vertically, dispense one drop (25 μL) into each sample wells (=>). If the whole blood is used, dispense two drops (50 μL) into the sample wells (=>).
5. Add exactly 5 or 6 drops of diluent (200 μL) in each sample wells (ð).
6. Read the results after 10 to 15 minutes.
   DO NOT INTERPRET AFTER 15 MINUTES

VII- READING TEST RESULTS

possible results pattern

A. Negative
Only one coloured band appears in the control zone.

B. Positive
In addition of the control band, a clearly distinguishable band also appears in the test zone.

C. Inconclusive
If there is no distinct colour band visible in the test and control areas, the test is inconclusive. In this case, repeat the test.

VIII- PERFORMANCES CHARACTERISTICS

1- LYME IgG

a) Cross-reactions
The LYME IgG rapid test gave negative results in 100 % of the cases with a syphilis positive control sera calibrated against the W.H.O. international standard n° 3-1980.

b) Sensitivity and specificity
Two panels of respectively 9 and 71 sera pre-assayed by EIA methods have been tested with LYME-IgG. The results are summarized in Table I.

Table I

Performances are calculated from data reported in table I as follows:

Sensitivity:                           (44+3) x 100 = 81 %
                                              (55+3)

Specificity:                           20 x 100 = 91 %
                                              22

Overall agreement:              (44+20+3) x 100 = 83 %
                                                       80

2- LYME IgM

a) Cross-reactions
The LYME IgM test gave negative results in 100 % of the cases with a syphilis positive control sera calibrated against the W.H.O. international standard n° 3-1980.

b) Sensitivity and specificity
Two panels of respectively 9 and 30 sera pre-assayed by EIA methods have been tested with LYME IgM test. The results are summarized in Table II.

Table II

Performances are calculated from data reported in table II as follows:

Sensitivity:                           (18+3) x 100 = 91.3 %
                                              (20+3)

Specificity:                           13 x 100 = 81.3 %
                                              16

Overall agreement:              (21+13) x 100 = 87.2 %
                                                 (23+16)

IX- LIMITATIONS

1. As for any diagnostic procedure, the physician should evaluate data obtained by the use of the test in light of other clinical information as patients suffering from borreliosis could exhibit symptoms similar to those of syphilis (4).
2. Antibodies level are variable, depending on the tested patients. Prevalence in the overall population is closed to 3% to 5% while in exposed subjects (foresters, hikers), antibodies could be detected in 25% to 30% of the cases (5).
3. Very early stage of infection could lead to false negative results, due to the low concentration of anti-Borrelia antibodies in the serum, plasma or whole blood samples.
4. A positive result does not exclude the presence of other pathogens. Cross reactions with syphilis or other non venereal treponemal infections. Cross reactions could also be observed in case of autoimmune diseases or infections due to Herpesviridae (5) for the IgM class antibodies.
5. A positive result does not exclude the presence of other pathogens: cross reactions with syphilis or other non venereal treponemal infections. Cross reactions could also be observed in case of autoimmune diseases.

X- BIBLIOGRAPHY

1. Marques, A.R., Hornung, R.L., Dally, L. and Philipp, M.T. 2005. Detection of Immune Complexes is not independent of detection of antibodies in Lyme disease patients and does not confirm active infection with Borrelia burgdorferi. Clinical and Diagnostics Laboratory Immunology, 12/9: 1036-1040.
2. Lyme Disease and Related Tick-Borne infections. 2001. Southwestern Vermont Health Care.
3. Nadelman, R.B. and Wormser, G.P. 1998. Lyme borreliosis. Lancet 352 : 557-565.
4. Duray, P.H. 1989. Clinical pathologic correlations of Lyme disease. Reviews of Infectious Diseases. 11/6 : S1487- S1493.
5. Bertholom, C. 2007. Place et intérêt des méthodes du diagnostic biologique au cours de la borréliose de Lyme. Option Bio. 387 : 20-21.

Manufacturer:

Von-Langen-Weg 10 D-48565 Steinfurt Tel.: 02551/4090  Fax.: 02551/1298 Web: http://www.biogentechnologies.com