RIDA QUICK Clostridium difficile Toxin A/B

Transcription

1 RIDA QUICK Clostridium difficile Toxin A/B Art. No.: N0803 R-Biopharm AG, An der neuen Bergstraße 17, D Darmstadt, Germany Tel.: +49 (0) / Telefax: +49 (0)

2 1. Intended use For in vitro diagnostic use. RIDA QUICK Clostridium difficile Toxin A/B is an immunochromatographic rapid assay for the qualitative determination of the toxins A and B of Clostridium difficile in stool samples and in culture supernatants. 2. Summary and explanation of the test Diarrhoea illnesses are a relatively frequent side effect of antibiotic therapies. However, more severe forms of the illness have been occurring more frequently, particularly since the introduction of clindamycin at the beginning of the 70's, and can even produce a severe clinical picture of the pseudo membranous colitis (PMC). This antibiotic-associated diarrhoea (AAD) is essentially caused by Clostridium difficile and therefore also called Clostridium difficile associated diarrhoea (CDAD). It is one of the most common nosocomial infections in the industrial nations. The carrier rate of hospitalised patients has now risen to %. Strains with an increasing virulence due to particular pathogenicity mechanisms have now become known and have made a Clostridium difficile infection (CDI) a major cost factor in health care. The production of Toxin A and B by toxinogenic strains of Clostridium difficile is of primary aetiological importance for the clinical picture of the illness. These toxin proteins, each of which has high molar masses of around 300 kda can be distinguished by their immunology and functionality. Toxin A is an enterotoxin whereas Toxin B is a cytotoxin. Both toxins can operate on their own or synergistically. Since not all strains of Clostridium difficile form toxins and approx. 2-8 % of healthy adults and up to 80 % of children under the age of 2 years may be parasitised with Clostridium difficile, the determination of Toxins A and B in stool samples in conjunction with the appearance of a CDAD is primarily of crucial importance for the diagnosis and decision about treatment. The RIDA QUICK Clostridium difficile Toxin A/B is a rapid assay for determining Toxin A and Toxin B, specifically and simultaneously, in the stool samples of patients using monoclonal and polyclonal antibodies. The test produces a reliable result after only 15 minutes and enables effective therapeutic measures to be taken promptly. 3. Test principle This rapid assay is a single-step, immunochromatographic lateral-flow test in which both biotinylated and gold-labelled Anti-Toxin A and Anti-Toxin B antibodies are used. As soon as the Clostridium difficile Toxins A and/or B are present in a positive sample, immune complexes with the labelled Anti-Toxin A and Anti-Toxin B antibodies form which then pass through the membrane. The Streptavidin on the test line T bonds the inflowing immune complexes via the Biotin coupled to the Anti-Toxin A and Anti-Toxin B antibodies, thus causing a red-violet coloration of the T line. Percolating, noncomplexed, gold-labelled antibodies attach themselves to the subsequent control line C. Therefore, in the case of negative samples there is no RIDA QUICK Clostridium difficile Toxin A/B

3 attachment of gold-labelled immune complexes to the T line but only to the C line. The red C line always indicates whether the test is valid. 4. Reagents provided There are enough reagents in the pack for 25 determinations Cassette 25 det. 25 individually packed test cassettes Reagent A 13.5 ml Specific Anti-Toxin A and Anti-Toxin B antibodies; contains 0.05 % azide, ready for use, blue coloured Reagent B 13.5 ml Specific Anti-Toxin A and Anti-Toxin B antibodies; contains 0.05 % azide, ready to use, yellow coloured Pipet 25 pieces Bag containing 25 disposable pipettes Reagent vial 25 pieces Bag containing 25 reagent vials Pipet Tip 25 pieces Bag containing 25 pipette tips Microlit Pipet 1 piece Pipette for 150 µl volume 5. Storage instructions The pack can be stored at 2-25 C and can be used until the printed expiry date. After the expiry date, the quality guarantee is no longer valid. Likewise, the usability of the cassettes cannot be guaranteed once the packaging of the cassette has been damaged. 6. Additional necessary reagents and necessary equipment - Vortex mixer (optional) - Waste container containing 0.5% sodium hypochlorite solution 7. Precautions for users For in vitro diagnostic use only. This test must only be carried out by trained laboratory personnel. The guidelines for working in medical laboratories must be followed. The instructions for carrying out the test must be strictly adhered to. The reagents contain sodium azide as a preservative. These substances must not be allowed to come into contact with the skin or mucous membrane. RIDA QUICK Clostridium difficile Toxin A/B

4 Do NOT pipette samples or reagents by mouth. Avoid contact with injured skin or mucous membranes. When handling the samples, wear disposable gloves and when the test is finished, wash your hands. Do not smoke, eat or drink in areas where samples are being used. All reagents and materials which come into contact with potentially infectious samples must be treated with suitable disinfectants (e.g. sodium hypochlorite) in exactly the same way as the samples themselves or autoclaved for at least one hour at 121 C. 8. Sample collection and storage Stool samples must be collected in clean containers without any additives and stored at 2-8 C before beginning the test. If stored for more than 3 days, the sample must be frozen at -20 C. In this case, the sample must be completely thawed out and brought to room temperature before testing begins. Avoid repeatedly freezing and thawing the sample. If rectal swabs have to be used, make sure that sufficient stool material (approx. 50 mg) is collected to carry out the test. 9. Test procedure 9.1. General information The samples, reagents and test cassettes must be brought to room temperature (20 25 C) before use. The test cassettes must only be removed from the external packaging shortly before they are used. Once used, the cassettes must not be used again. The test must not be carried out in direct sunlight. Do not pour reagents back into vials as this may cause reagent contamination Preparing the sample tests In a labelled reagent vial Reagent vial you should place 0.5 ml (approx drops) each of reagent A Reagent A and Reagent B Reagent B. The 0.5 ml and 1.0 ml graduation on the reagent vial takes priority over the number of drops of reagents A and B. Reagents A and B must have a ratio of Using the stool samples With a liquid stool sample pipette Pipet 50 µl (up to the second thickening) of the sample and suspend it in the reagent mix placed in the tube beforehand. With solid stool samples, suspend 50 mg of the sample in there same way in the buffer. The reagent vial is then sealed tight and the sample homogenised by thorough mixing (optionally by vortexing). After this, allow the homogenized suspension to precipitate for 5 minutes until a RIDA QUICK Clostridium difficile Toxin A/B

5 largely clear supernatant is formed. The reagent vial can be placed in one of the three middle openings of the reagent insert for sedimentation Using liquid and solid cultures of Clostridium difficile 50 µl of a culture medium (e.g. thioglycolate broth) are pipetted into 1.0 ml of the mixture of reagent A (0.5 ml) and reagent B (0.5 ml) produced beforehand and mixed. 150 µl of the mixture are used for the sample test (Section 9.3.). If using solid culture media as many colonies as possible should be taken from the base of the culture medium and initially suspended completely in 1 ml of distilled water or 0.9% saline solution (NaCl). 50 µl of this suspension are pipetted into 1.0 ml of the mixture of reagent A (0.5 ml) and reagent B (0.5 ml) produced beforehand in the reagent vial and mixed. 150 µl of the mixture are used for the sample test (Section 9.3.) Testing the sample When removed from the external packing, first lay the test cassette Cassette on a level mat. An unused pipette tip Pipet Tip is then placed on the microlit pipette Microlit Pipet and 150 µl supernatant taken from each reagent vial and pipetted in the application field of the test cassette. Make sure that the liquid flows through the membrane unimpeded. If this is done correctly the control band appears on control line C after around 3 minutes. If the control line does not appear after 3 minutes a newly produced sample has to be sedimented more strongly (optionally by centrifuging for 2 minutes at 2000 g) and pipetted into the application field of a new test cassette. The test result should always be read off after 15 minutes. The colour of the bands and their intensity may change during the overall development time and after the band dries from redviolet to a blue to grey-violet. 10. Quality control indications of reagent expiry The test must only be evaluated if the test cassette is intact before the sample suspension is pipetted in and no colour changes or bands are visible on the membrane. In addition to this, at least the red-violet control band must be visible after the 15 minute incubation period. If this does not appear, the following must be checked before repeating the test: - Expiry date of the test cassettes and the reagents being used - Has the correct test procedure been used? - Are the reagents contaminated? If the control band is still not visible after repeating the test with a new test strip, please contact the manufacturer or your local R-Biopharm distributor: RIDA QUICK Clostridium difficile Toxin A/B

6 11. Evaluation and interpretation A maximum of two bands should appear in the following order, as seen from the sample application field: One red-violet test band at test line T and one red-violet control band at control line C. If the control band is missing, the test is invalid and cannot be evaluated! The following interpretations are possible: - Clostridium difficile toxin positive : both bands are visible. - Clostridium difficile toxin negative : only the control band is visible. - Not valid : no band is visible or a constellation different to that described above. Likewise, changes in band colour which appear long after 15 minutes are also without any diagnostic value and must not be used for evaluation. 12. Limitations of the method The RIDA QUICK Clostridium difficile Toxin A/B detects the toxins A and/or B of Clostridium difficile in stool samples or, after their enrichment, in C.difficile cultures. The test cannot be used to derive a relationship between the intensity of the specific visible band and the occurrence or severity of clinical symptoms. The results obtained must always be interpreted in combination with the clinical picture. A positive result does not rule out the presence of another infectious pathogen or cause. A negative result does not necessarily mean that there is no Clostridium difficile infection. This can be due to intermittent excretion of the pathogenic toxins or to the quantity of toxins in the sample being too small. If an infection with the pathogen being sought is suspected on the grounds of the case history, another of the patient's stool samples should be tested. An excess of stool sample can cause a brownish discoloration of the test strip that masks the red-violet colour of the specific test bands. In such cases, it will be necessary to repeat the test with a smaller stool quantity or a stool suspension that has been diluted even further bycentrifuging in order to clarify whether the Clostridium difficile toxins being sought are in fact present in the sample but have been masked by too much stool matrix. 13. Performance characteristics 13.1 Clinical sensitivity and specificity In a validation study with 181 deep-frozen and PCR previously characterised stool samples (116 negative and 65 positive) the RIDA QUICK Clostridium difficile Toxin A/B rapid assay was compared with 5 commercially available ELISA kits. For this purpose, the stool samples were thawed, homogenised and used in the 5 different tests according to the manufacturer's instructions. The relative sensitivities and specificities were determined from the results for all test kits used and compiled in Table 1. RIDA QUICK Clostridium difficile Toxin A/B

7 Table1. Sensitivity and specificity of the RIDA QUICK Clostridium difficile Toxin A/B compared to 5 commercially available Elisa test kits to detect toxins A and B of Clostridium difficile in stool samples. [ % ] RIDA QUICK ELISA 1 ELISA 2 ELISA 3 ELISA 4 ELISA 5 Sensitivity Specificity Analytical sensitivity In order to determine the analytical sensitivity of the RIDA QUICK Clostridium difficile Toxin A/B rapid assay a defined concentration of the pure toxins A and B were each mixed into a negative stool sample and serially diluted. The reaction of the individual dilution levels was tested in both RIDA QUICK Clostridium difficile Toxin A/B as well as in two further commercially available immunochromatographic rapid assays. The results are compiled in Table 2. Table 2. Analytical sensitivity of the RIDA QUICK Clostridium difficile Toxin A/B compared to 2 further commercially available rapid assays to detect toxins A and B of Clostridium difficile. Toxin concentration Toxin A and B [ ng/ml ] RIDA QUICK C.difficileToxin A/B Rapid Immunoassay 1 Rapid Immunoassay 2 A B A B A B (+) - - (+) - 4 (+) (+) (+) The analytical sensitivity of the RIDA QUICK Clostridium difficile Test was determined with 4,2 ng/ml for toxin A and 2,1 ng/ml for toxin B using purified toxins. RIDA QUICK Clostridium difficile Toxin A/B

8 13.3 Precision To determine the precision of the RIDA QUICK Clostridium difficile Test, the intra-assay reproducibility, the inter-day reproducibility (10 days), the inter-operator reproducibility (3 operators) and the inter-lot reproducibility (3 lots) were investigated. For each analysis, 5 references were measured in replicate: one negative, one medium positive with toxin A and one with toxin B, one low positive with toxin A and one with toxin B. The RIDA QUICK Clostridium difficile Test showed the expected result in 100% of the measurements Cross reactivity Different pathogenic organisms of the intestinal tract have been tested using the RIDA QUICK Clostridium difficile Test and have shown no cross reactivity. The tests were carried out with bacterial suspensions (10 7 to 10 9 cfu/ml), parasite cultures (10 7 to 10 9 organisms/ml) and the cell-culture supernatants of cells infected with viruses. The results are listed in Table 2. Test germ Origin Name / source Result Adenovirus Cell culture supernatant Micromun GmbH negative Aeromonas hydrophila Culture DSM negative Astrovirus Cell culture supernatant Micromun GmbH negative Bacillus cereus Culture DSM negative Candida albicans Culture DSM negative Citrobacter freundii Culture DSM negative Cryptosporidium muris Culture Waterborne Inc. negative Cryptosporidium parvum Culture Waterborne Inc. negative E. coli EPEC Culture DSM 8695 negative E. coli ETEC Culture DSM negative E. coli STEC Culture DSM 8579 negative Enterobacter cloacae Culture DSM negative Enterococcus faecalis Culture DSM 2570 negative Giardia lamblia Culture Waterborne Inc. negative Klebsiella oxytoca Culture DSM 6673 negative Proteus vulgaris Culture DSM negative Pseudomonas aeruginosa Culture DSM 288 negative Rotavirus Cell culture supernatant Micromun GmbH negative Serratia liquefaciens Culture DSM negative RIDA QUICK Clostridium difficile Toxin A/B

9 Shigella flexneri Culture DSM 4782 negative Staphylococcus aureus Culture DSM negative Staphylococcus epidermidis Culture DSM 1798 negative Vibrio parahaemolyticus Culture DSM 2172 negative Yersinia enterocolitica Culture DSM negative 14. Interfering substances When mixed with the Clostridium difficile Toxin A/B positive and negative stool samples in the given concentrations, the following substances had no effect on the test results: Barium sulphate (5% w/w), loperamide (antidiarrhoeal agent; 5% w/w), Peptobismol (antidiarrhoeal agent; 5% v/w), mucin (5% w/w), Cyclamate (artificial sweetener 5% v/w), human blood (5% v/w), stearic acid/palmitic acid (mixed 1:1, 40% w/w), metronidazole (0.5) (antibiotic 5% v/w), diclofenac ( % v/w). RIDA QUICK Clostridium difficile Toxin A/B

10 References 1. Lyerly, D.M. et al.: Clostridium difficile: Its disease and toxins. Clin. Microbiol. Rev. (1988); 1: Knoop, F.C. et al.: Clostridium difficile: Clinical disease and diagnosis. Clin. Microb. Rev. (1993); 6: Kelly, C.P. et al.: Clostridium difficile Colitis. New Engl. J. Med. (1994); 330: Sullivan, N.M. et al.: Purification and characterization of toxins A and B of Clostridium difficile. Infect. Immun. (1982); 35: Thomas, D.R. et al.: Postantibiotic colonization with Clostridium difficile in nursing home patients. J. Am Geriatr. Soc. 38, (1990). 6. Bartlett, J.G.: Clostridium difficile: Clinical considerations. Rev. Infect. Dis. (1990); 12: Loeschke,K., Ruckdeschel, G.: Antibiotikaassoziierte Kolitis - aktualisiert. Internist (1989); 30: Enzensberger, R. et al.: Clostridium difficile-induzierte Enterokolitis. DMW(1986); 111: Cefai, C. et al.: Gastrointestinal carriage rate of Clostridium difficile in elderly, chronic care hospital patients. J. Hosp. Infect. (1988); 11: Samore, M.H. et al.: Wide diversity of Clostridium difficile types at a tertiary referral hospital. J. Infect. Dis. (1994);170: Lipsett, P.A. et al.:pseudomembranous colitis: A surgical disease? Surgery (1994);116: Asha, N.J. et al.: iology of Antibiotic associated diarrhea due to Clostridium difficile, Clostridium perfringens, and Staphylococcus aureus. J.Clin. Microbiol. (2006); 44: Voth, D.E., Ballard, J.: Clostridium difficile toxins: Mechanism of action and role in disease. J.Clin. Microbiol. (2005); 18: Borgmann, S. et al.: Increased number of Clostridium difficile infections and prevalence of Clostridium difficile PCR ribotype 001 in southern Germany. Eurosurveillance (2008); Vol 13: Bartlett 15. Mc.Donald, L.C. et al.: An epidemic, toxin gene-variant strain of Clostridium difficile. N.Engl.J.Med. (2005); 353: Loo,V.G. et al.: A predominantly clonal multi-institutional outbreak of Clostridium difficileassociated diarrhea with high morbidity and mortality. N.Engl.J.Med. (2005); Bartlett,J.G., Gerding,D.N.: Clinical recognition and diagnosis of Clostridium difficile infection. CID (2008); 46(Suppl. 1): RIDA QUICK Clostridium difficile Toxin A/B