Specificity & Sensitivity Study KRUUSE Borrelia Quick

Transcription

1 Specificity & Sensitivity Study 2011 KRUUSE Borrelia Quick

2 Abstract Lyme disease (borreliosis) is a tick-borne disease that was first identified in dogs in It is caused by Borrelia burgdorferi, a bacterium of the species spirochete. The KRUUSE Borrelia Quick is a lateral flow immunoassay, using a specific surface protein of Borrelia burgdorferi for the detection of antibodies against borrelia to detect antibodies of a borreliosis in the blood. Within the study of KRUUSE Borrelia Quick, it was determined that the diagnostic sensitivity was 95.83% and the diagnostic specificity was 94.12%. Furthermore, it was shown that the KRUUSE Borrelia Quick, reliably differentiates between field and vaccine antibodies. Introduction The KRUUSE Borrelia Quick is an immunochromatographic rapid test for the qualitative detection of specific antibodies against Borrelia burgdorferi antigens in blood. These specific antibodies against the antigens are exclusively expressed upon an infection with viable Borrelia spp. and are not formed by an infection with other spirochetes, so possible cross reactions can be excluded. Also cross reactions with antibodies that were formed by a previous vaccination against Lyme disease can be excluded. The detection of antibodies in the blood or serum is possible approximately 3-5 weeks postinfection. With the KRUUSE Borrelia Quick, then a field infection can be detected reliably. The causative agent of Lyme disease was first identified in 1981 by Willy Burgdorfer and named after him. Borrelia burgdorferi is a gram-, spiral-shaped and agile bacterium (spirochete). Worldwide at least 13 genospecies belong to the Borrelia burgdorferi sensu lato complex. The three main species B. burgdorferi sensu stricto (Bss), B. garinii (Bg) and B. afzellii (Ba) are also pathogenic for humans. For dogs, the pathogenicity of B. burgdorferi sensu stricto is proven. Its natural reservoir has Borrelia in wild animals. Lyme disease is transmitted by ticks of the genus Ixodes ricinus (vectors). After the bite of an infected tick, the borrelia migrate from the gut of the tick in the salivary gland and then reach the host through the bite. From the injection site the bacteria migrate through the host s tissue mainly into the joints. The clinical picture of the disease in dogs often develops insidiously and is characterized by nonspecific symptoms such as apathy, lymph node swelling and intermittent fevers. Possible clinical manifestations are described in the form of arthropathies with lameness due to infectionrelated arthritis of one or more joints and occasionally glomerulopathies. An infection however may not always and/or maybe only after a long period of time lead to a disease (in experimental studies it was shown that the incubation period can range between 2-5 months). A complete elimination of the pathogens is difficult even under an effective antibiotic therapy, the pathogen persists possibly in the host, it can elude the immune response and disease symptoms can occur at any time again. The safest protection against a Borrelia infection is a vaccination in combination with contactparasiticides and avoiding areas with high infection pressure of Borrelia. A test for antibodies against Borrelia in the blood gives an indication of a possible infection; whereas the serological test result should always be interpreted in conjunction with the clinical picture: - The detection of antibodies against Borrelia is not synonymous with a clinically manifest Lyme infection, but it indicates an infection with viable Borrelia spp (field infection), which can possibly lead to a disease at a later date. - A test result does not exclude an infection, since the titer may be still below the detection limit at the time of testing. In cases of a suspected Lyme infection, a further test should be carried out approximately 4 weeks later. - Antibodies against Borrelia circulate even after successfully treated disease for a longer time in the blood. The KRUUSE Borrelia Quick detects antibodies in the blood by the use of a specific surface protein of Borrelia burgdorferi, thus enabling a distinction between field and vaccination antibodies. Method For this sensitivity and specificity study 41 defined dog sera were tested with laboratory methods for Lyme disease antibodies and subsequently these samples were evaluated with the KRUUSE Borrelia Quick.

3 The dog sera were determined by a commercial laboratory with different tests; depending on the individual sample, using an IgG ELISA, an IgM ELISA and / or an Immunoblot. The results of the laboratory were compared with the test results of the KRUUSE Borrelia Quick for the respective sera. The KRUUSE Borrelia Quick was performed in accordance with the instructions, whereas one drop of serum was put into the sample well of the test cassette, followed by one drop of buffer. The tests were read out and interpreted 10 minutes after the addition of the sample liquid. For the interpretation of the test results of the KRUUSE Borrelia Quick the standardized color scale KRUUSE FF (Q GC 23,092,011) were used (rating scale 1-10, whereas 1 means "no line" and 10 "very intensive test line, see Figure 1). Line intensities greater than / equal to 2 have been interpreted as a positive test result. C-SE positive Field / positive positive C-SE C-SE positive not performed positive positive C-SE positive Field positive positive C-SE not performed C-SE positive Field positive positive C-SE positive Field positive positive C-SE positive C-SE positive Field Positive positive C-SE value value not performed uncertain positive C-SE positive Field Positive positive C-SE positive Field / positive C-SE positive Field positive positive C-SE not performed C-SE C-SE positive Field positive positive C-SE positive not performed positive positive C-SE positive positive Field positive positive C-SE positive Field positive positive C-SE positive Field positive positive Figure 1 C-SE value not performed uncertain Results The test results from the laboratory for the respective samples are compared with the test results of the KRUUSE Borrelia Quick in the following table 1. Sample number Results laboratory IgG ELISA IgM ELISA Immunoblot C-SE positive C-SE positive C-SE C-SE value Interpretation Laboratory Results KRUUSE Borrelia Quick Field / positive positive value not performed uncertain value not performed uncertain C-SE C-SE positive Field positive positive C-SE positive Field positive positive C-SE positive Field positive positive C-SE C-SE positive C-SE positive Field positive positive C-SE not performed C-SE positive Field positive positive C-SE positive Field positive positive C-SE C-SE positive Field / positive positive C-SE positive Field positive positive C-SE C-SE positive Table 1 Test results value not performed uncertain Reason for using different laboratory methods to determine the samples was that with the ELISA positive test results for vaccination antibodies were counted as positive by the laboratory, as well as a positive detection of field antibodies, no differentiation were made. In order to get a reliable differentiation of the antibodies, positive tested samples were then also tested with an Immunoblot. Samples tested with the ELISA giving results are considered as in this study and for the evaluation and were not further tested in an Immunoblot. To illustrate the visual test results of the KRUUSE Borrelia Quick five test cassette of the study are exemplified in Figure 2. C-SE positive positive Field positive positive

4 detection limit of the test strip, which has led to the corresponding outcome. Overall, the results show a significant correlation of the laboratory results compared to the test results of the KRUUSE Borrelia Quick, what is graphically illustrated in figure 3. KRUUSE Quick Figure 2 Test cassette examples Interpretation The results of the sensitivity and specificity study show a highly significant correlation between the results of the laboratory tests and the test results of the KRUUSE Borrelia Quick. The results from the laboratory were first tested by ELISA for present antibodies in the sample (vaccination antibodies, as well as field antibodies). For a correct interpretation of the test results a differentiation between field and vaccination antibodies was necessary and was, when if applicable, determined with an Immunoblot. For the interpretation of the test results of the KRUUSE Borrelia Quick only positive samples were considered as correct positive, which contained field antibodies. Only present vaccination antibodies in the samples should not lead to a positive test result with the KRUUSE Borrelia Quick. The samples are rated as correct if no antibodies against borrelia were in the blood, as well as if clearly only vaccination antibodies were detected by an Immunoblot in the respective sample. Borderline results of the laboratory should lead to test results of the test strip. Equivocal results have not been recognized by the strip test as positive as they were below the detection limit and therefore do not lead to clear test results. Nevertheless, the sample C-SE was detected by the test strip as positive. For an exact interpretation of this test result a followup testing of the corresponding animal about 4 weeks later would have been necessary, to verify the test result. As no follow test was performed, the result is interpreted as false-positive in this study. The sample C-SE showed on the test strip a result, but in the laboratory the sample was defined as positive. Since the sample contained both field and vaccination antibodies, it is highly possible that the concentration of field antibodies was below the Figure 3 Correlation of the test results With the reduction to a comparison of the final results of the laboratory and the test results of KRUUSE Borrelia Quick, the correlation of the results is even more evident, as shown in Figure 4. KRUUSE Quick Figure 4 Correlation of final results Table 2 shows the summary of the cumulative laboratory results compared with the test results of Borrelia Quick KRUUSE. Results laboratory (total) KRUUSE Borrelia Positive Negative Quick Positive 23 1 Negative 1 16 Table 2 Summary of results from the lab and the KRUUSE Borrelia Quick With this result, the following sensitivity and specificity of the KRUUSE Borrelia Quick can be calculated:

5 The diagnostic sensitivity is: 23/(23+1)=95,83 % The diagnostic specificity is: 16/(16+1)=94,12 % Conclusion The study shows a significant correlation of the test results of the laboratory to the test results of the KRUUSE Borrelia Quick. Furthermore it also could be shown that the KRUUSE Borrelia Quick generally can differentiate between field and vaccination antibodies, samples containing field antibodies were detected as positive. According to the results of this sensitivity and specificity study the KRUUSE Borrelia Quick can be assessed as a reliable test for the detection of antibodies against a borreliosis, caused by Borrelia burgdorferi, in the blood of dogs, which offers the veterinarian a simple and reliable onsite solution for a rapid testing in suspected cases. Literature R.K.Straubinger, N. Pantchev (2010): Die Lyme-Borelliose- Impfung beim Hund-kontrovers diskutiert. Kleintier Konkret 5:8-11. R.K.Straubinger (2011): Die Lyme-Borreliose des Hundes- Impfen Sie noch oder schützen Sie schon? Fortbildung des TBv Oberbayern im Pavillon, München, 2011 H.J. Selbitz, U. Tryen, P.V. Weigand (2010):Medizinische Mikrobiologie, Infektions- und Seuchenlehre, 9.Aufl. Enke Verlag, Stuttgart. StIKo VET (2008): Borreliose beim Hund. Leitlinien zur Impfung von Kleintieren, Beilage zum Deutschen Tierärzteblatt 8/2009: G.P. Wormers, I. Schwartz (2009): Antibiotic Treatment of Animals Infected with Borrelia burgdorferi. Clinical microbiology reviews 20/3: Karin Rebel (2007):Die Borreliose des Hundes-ein Update zur Erkrankung und Impfung. Kleintiermedizin 5/6: C. Bauer et.al (2011): Empfehlung zur Bekämpfung von durch Vektoren übertragenen Krankheiten bei Hunden und Katzen; Auszüge der deutschen ESCCAP-Empfehlung Nr. 5: Von Zecken übertragene Erkrankungen. Der praktische Tierarzt 92: Kit Tilly, PhDa, Patricia A. Rosa, PhDb, and Philip E. Stewart,(2008): Biology of Infection with Borrelia burgdorferi. Infect Dis Clin North Am. 22(2): Rev.: st