CareStart Malaria PF/VOM Combo

Transcription

1 USING HRP2 FOR P. FALCIPARUM PLDH FOR VIVAX, OVALE, MALARIAE A RAPID TEST FOR THE DETECTION OF MALARIA LDH AND HRP2 IN HUMAN BLOOD PARASITOLOGY SINGLE USE IN VITRO DIAGNOSTIC DEVICE CareStart Malaria PF/VOM Combo

2 CareStart Malaria PF/VOM Combo RAPYDTEST 1 Performance Benefits Distinguishes between a mixed infection and a falciparum Isolates Plasmodium falciparum HRP2 and pldh for P.vivax, P.ovale and P.malariae Combined antigen technology gives you increased accuracy User friendly cartridge format for ease of use and storage Integral vents prevent sample 'back flow' interference Results in 20 minutes Intended Use For the rapid qualitative determination of Malaria Histidine-rich Protein 2 (HRP2) (P. falciparum) and lactate dehydrogenase (pldh) (P. vivax, P. ovale and P. malariae) in human blood as an aid in the diagnosis of Malaria infection. Summary Malaria is a serious parasitic disease characterized by fever, chills, and anemia and is caused by a parasite that is transmitted from one human to another by the bite of infected Anopheles mosquitoes. There are four kinds of malaria that can infect humans: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. In humans, the parasites (called sporozoites) migrate to the liver where they mature and release another form, the merozoites. The disease now occurs in more than 90 countries worldwide, and it is estimated that there are over 500 million clinical cases and 2.7 million malaria-caused deaths per year. The CareStart Malaria PF/VOM Combo contains a membrane strip, which is pre-coated with two monoclonal antibodies as two separate lines across a test strip. One monoclonal antibody (test line VOM) is specific to pldh of Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. The other line (test line Pf) is printed with a monoclonal antibody specific to HRP2 of Plasmodium falciparum. The conjugate pad is dispensed with monoclonal antibodies, which are specific to pldh (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) and P. falciparum specific to HRP2. So, the CareStart Malaria PF/VOM Combo Antigen Test is designed for the differential diagnosis between Plasmodium falciparum and other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae). Precautions In order to obtain reproducible results, the following rules must be observed: For in vitro diagnostic use only. Use disposable gloves while handling potentially infectious material and performing the assay. Wash hands thoroughly afterwards. Do not use it beyond the expiration date. Do not eat or smoke while handling specimens. Clean up spills thoroughly using an appropriate disinfectant. Specimen Collection and Storage Collection by venipuncture 1. Collect the whole blood into the collection tube (containing EDTA, citrate, or heparin) by venipuncture. 2. If specimens are not immediately tested, they should be refrigerated at 2-8 C. For storage periods greater than three days, freezing is recommended. They should be brought to room temperature prior to use. Using the specimen in the long-term keeping more than three days can cause non-specific reaction. 3. When stored at 2-8 C, the whole blood sample should be used within three days. Collection using a lancet 1. Clean area to be lanced with an alcohol swab. 2. Squeeze the end of the fingertip and pierce with a sterile lancet provided. 3. Wipe away the first drop of blood with alcohol swab. 4. Take a sample pipette provided, and while gently squeezing the tube, immerse the open end in the blood drop and then gently release the pressure to draw blood into the sample pipette up to the black line. Sample Pipette 5µl GUIDE LINE VENTS TO PREVENT BACK FLOW OF SAMPLE RESULT WINDOW SAMPLE WINDOW BUFFER WELL

3 Test Procedure 1. Add 5µl of whole blood into the Sample Well (small well). 2. Add two drops (60µl) of Assay Buffer into the Buffer Well. 3. Read the test result in 20min. GENTLY SQUEEZE THE TUBE WHILST SQUEEZED IMMERSE THE END IN BLOOD 1 2 CCON NEGATIVE GENTLY RELEASE TO DRAW THE BLOOD INTO TUBE INVALID Results Interpretation of the test CON 1. Negative reaction The presence of only one band in the CON area within the result window indicates a negative result. 2. Invalid The test is invalid if the line in the CON area does not appear. If this occurs, the test should be repeated using a new strip. 3. Positive reaction - P. falciparum The presence of two bands (one band in the CON area and another band in the Pf area) indicates a positive result for P. falciparum. 4. Mixed infection - P. falciparum and others The presence of three color bands (bands in the CON area, Pf area and VOM area) indicates a positive result of mixed infection for P. falciparum and others (P. vivax, P. ovale or P. malariae). ADD WHOLE BLOOD (5µl) INTO SAMPLE WELL ADD ASSAY BUFFER (2 DROPS OR 60µl) INTO BUFFER WELL CON CON P.falciparum MIXED INFECTION P.falciparum and others READ RESULTS IN 20 MIN CON Other species P.vivax, P.ovale or P.malariae 5. Positive other species- P. vivax, P. ovale, P. malariae The presence of two color bands (one band in the CON area and another band in the VOM area) indicates a positive result for P. vivax, P. ovale or P. malariae. Limitation and interferences The test procedure, precautions and interpretation of results for this test must be followed when testing. Anti-coagulants such as heparin, EDTA, and citrate do not affect the test result. Do not mix reagent of different lots. The test is limited to the detection of antigen to Malaria Plasmodium sp. Although the test is very accurate in detecting pldh and HRP2, a low incidence of false results can occur. Other clinically available tests are required if questionable results are obtained. As with all diagnostic tests, a definitive clinical diagnosis should not be based on the results of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated. CareStart Malaria PF/VOM Combo RAPYDTEST 2

4 CareStart Malaria PF/VOM Combo RAPYDTEST Performance Characteristics The CareStart Malaria PF/VOM Combo kit has tested with positive and negative clinical samples tested by microscopic examination of whole blood. 1. MALARIA P. VIVAX EVALUATION RESULTS References 1. Valecha N., Eapen A., Devi C. Usha, Ravindran J., Aggarwal A., and Subbarao S. K. (2002). Field evaluation of the ICT Malaria P.f./P.v immunochromatographic test in India. Annals of Tropical Medicine & Parasitology. 96: Iqbal J., Hira P. R., Sher A., and AL-Enezi A. A Diagnosis of imported Malaria by Plasmodium Lactate Dehydrogenase (pldh) and Histidine-Rich Protein 2. (PfHRP2)-based immunocapture assays. American Journal of Tropical Medicine and Hygiene. 64: Tjitra E., Suprianto S., Dyer M., Currie B. J. and Anstey N.M. (1999). Detection of Histidine-rich Protein 2 and panmalarial ICT MALARIA P.f./P.v test antigens after chloroquine treatment of uncomplicated falciparum Ordering Information Precision Within-run and between-run precisions have been determined by the testing 10 replicates of three specimens: a negative, a low positive and a strong positive. The agreement between the test results and the expected results were 100%. P.v/P.o/P.m - CONFIRMED SPECIMEN NEGATIVE PRODUCT PACK SIZE CODE SENSITIVITY CARESTART MALARIA AG RAPID /100 X 100% = 96% 2. MALARIA P. FALCIPARUM EVALUATION RESULTS PF - CONFIRMED SPECIMEN NEGATIVE SENSITIVITY CARESTART MALARIA AG RAPID /100 X 100% = 98% 3. MALARIA NEGATIVE NORMAL HUMAN SPECIMEN EVALUATION RESULTS RANDOM NORMAL HUMAN SPECIMEN NEGATIVE SPECIFICITY CARESTART MALARIA AG RAPID /200 X 100% = 97.5% malaria does not reliably predict treatment outcome in eastern Indonesia. Journal of Clinical microbiology. 37: Panton L. J., PcPhie P., Maloy W. L., Wellems T. E., Taylor D. W. and Howard R. J.(1989). Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II. Molecular and Biochemical Parasitology. 35: Leonard K. Basco, Frederique Marquet, Michael M.Makler, and Jacques Le Bras. : Plasmodium falciparum and Plasmodium vivax : Lactate Dehydrogenase Activity and its Application for in vitro Drug Susceptibility Assay. Experimental Parasitology 80, (1995). CareStart Malaria PF/VOM Combo Rapydtest Products can be ordered direct from Apacor or from an appointed distributor Visit our website for all the latest information or on: sales@apacor.com 3 UNIT 5, SAPPHIRE CENTRE, FISHPONDS ROAD, WOKINGHAM, BERKSHIRE, RG41 2QL, ENGLAND TEL: +44 (0) FAX: +44 (0) APA080 - V1 03/2013

5 FOR A RAPID, ONE STEP TEST FOR THE SIMULTANEOUS DETECTION AND DIFFERENTIATION OF IgG AND IgM TO THE SUBSPECIES OF LEISHMANIA DONOVANI IN HUMAN SERUM / PLASMA / WHOLE BLOOD Leishmania IgG/IgM Combo PARASITOLOGY SINGLE USE IN VITRO DIAGNOSTIC DEVICE

6 Leishmania IgG/IgM Combo 1 Intended Use The Apacor Leishmania IgG/IgM Combo Rapydtest is a lateral flow immunoassay for the simultaneous detection and differentiation of IgG and IgM to the subspecies of Leishmania donovani (L. donovani), the visceral leishmaniasis causative protozoans, in human serum, plasma or whole blood. It is intended to be used as a screening test and as an aid in the diagnosis of the disease of visceral leishmaniasis. Any reactive specimen with the Apacor Leishmania IgG/IgM Combo Rapydtest must be confirmed with alternative testing method(s). Summary Visceral leishmaniasis, or Kala-azar, is a disseminated infection caused by several subspecies of L. donovani. The disease is estimated by the World Health Organization (WHO) to affect approximately 12 million people in 88 countries 1. It is transmitted to humans by bites of the Phlebotomus sandflies which acquire infection from feeding on infected animals. Though it is a disease found in poor countries, in Southern Europe, it has become the leading opportunistic infection in AIDS patients 2-3. Identification of L. donovani organisms from the blood, bone marrow, liver, lymph nodes or the spleen provides a definite means of diagnosis. Serological detection of anti-l. donovani IgM is found to be an excellent marker for acute visceral leishmaniasis. Tests used in the clinic include ELISA, fluorescent antibody or direct agglutination tests 4-5. Recently, utilization of L. donovani specific protein in the test has improved the sensitivity and specificity dramatically 6-7. The Apacor Leishmania IgG/IgM Combo Rapydtest is a recombinant rk39 based serological test, which detects IgG and IgM antibodies to L. donovani simultaneously. The test provides a reliable result within 15 minutes without any instruments. Principle The Apacor Leishmania IgG/IgM Combo Rapydtest is a lateral flow chromatographic immunoassay. The test cassette consists of: 1. A burgundy coloured conjugate pad containing recombinant rk39 antigen conjugated with colloidal gold (Leishmania conjugates) and rabbit IgG-gold conjugates, 2. A nitrocellulose membrane strip containing two test lines (M and G lines) and a control line (C line). The M line is pre-coated with monoclonal anti-human IgM for the detection of anti-l. donovani IgM, the G line is pre-coated with reagents for the detection of anti-l. donovani IgG, and the C line is pre-coated with goat anti-rabbit IgG. SAMPLE ID CONTROL LINE TEST LINES SAMPLE WELL When an adequate volume of test specimen is dispensed into the sample well of the cassette, the specimen migrates by capillary action across the cassette. L. donovani IgM, if present in the specimen, will bind to the Leishmania conjugates. The immunocomplex is then captured on the membrane by the pre-coated anti-human IgM antibody forming a burgundy coloured M line, indicating a L. donovani IgM positive test result. L. donovani IgG if present in the specimen will bind to the Leishmania conjugates. The immunocomplex is then captured by the pre-coated reagents on the membrane forming a burgundy coloured G line, indicating a L. donovani IgG positive test result. Absence of any test lines (M and G) suggests a negative result. The test contains an internal control (C line) which should exhibit a burgundy coloured line of the immunocomplex of goat anti-rabbit IgG/rabbit IgG-gold conjugates regardless of colour development on any of the test lines. If the C line does not develop, the test result is invalid and the specimen must be retested with another device. Reagents and Materials Provided 1. Individually sealed foil pouches containing: a. One cassette device b. One desiccant 2. Plastic droppers 3. Sample diluent (1 bottle, 5 ml) 4. One package insert (instruction for use) Materials may be Required and not Provided 1. Positive Control 2. Negative Control Materials Required but not Provided 1. Clock or Timer 2. Lancing device for whole blood test 3. Pipette and tips capable of delivering 20µl volumes with a precision better than 1.5% Warnings and Precautions For in Vitro Diagnostic Use 1. This package insert must be read completely before performing the test. Failure to follow the insert gives inaccurate test results. 2. Do not open the sealed pouch unless ready to conduct the assay. 3. Do not use expired devices. 4. Bring all reagents to room temperature (15-30 C) before use. 5. Do not use components from any other type of test kit as a substitute for the components in this kit. 6. Do not use hemolized blood specimens for testing. 7. Wear protective clothing and disposable gloves while handling the kit reagents and clinical specimens. Wash hands thoroughly after performing the test. 8. Users of this test should follow Good Laboratory Practice for prevention of transmission of HIV, HBV and other blood-borne pathogens. 9. Do not smoke, drink or eat in areas where specimens or kit reagents are being handled. 10. Dispose of all specimens and materials used to perform the test as bio-hazardous waste. 11. Handle the negative and positive controls in the same manner as patient specimens. 12. The test results should be read within 15 minutes after a specimen is applied to the sample well or sample pad of the device. Reading the results after 15 minutes may give erroneous results. 13. Do not perform the test in a room with strong air flow, i.e. an electric fan or strong air-conditioning. Reagent Preparation and Storage Instructions All reagents are ready to use as supplied. Store unused test devices unopened at 2-30 C. If stored at 2-8 C, ensure that the test device is brought to room temperature before opening. The test device is stable through the expiration date printed on the sealed pouch. Do not freeze the kit or expose the kit over 30 C. Specimen Collection and Handling Consider any materials of human origin as infectious and handle them using standard bio-safety procedures. PLASMA Step 1 Collect blood specimen into a lavender, blue or green top collection tube (containing EDTA, citrate or heparin, respectively, in Vacutainer ) by veinpuncture. Step 2 Separate the plasma by centrifugation.

7 Step 3 SERUM Step 1 Step 2 Step 3 Step 4 Carefully withdraw the plasma into new pre-labelled tube. Collect blood specimen into a red top collection tube (containing no anticoagulants in Vacutainer ) by veinpuncture. Allow the blood to clot. Separate the serum by centrifugation. Carefully withdraw the serum into a new pre-labelled tube. Test specimens as soon as possible after collecting. Store specimens at 2-8 C if not tested immediately. Store specimens at 2-8 C for up to 5 days. The specimens should be frozen at -20 C for longer storage. Avoid multiple freezethaw cycles. Prior to testing, bring frozen specimens to room temperature slowly and mix gently. Specimens containing visible particulate matter should be clarified by centrifugation before testing. Do not use samples demonstrating gross lipemia, gross hemolysis or turbidity in order to avoid interference on result interpretation. BLOOD Drops of whole blood can be obtained by either finger tip puncture or veinpuncture. Do not use any hemolized blood for testing. Whole blood specimens should be stored in refrigeration (2-8 C) if not tested immediately. The specimens must be tested within 24 hours of collection. Assay Procedure Step 1 Step 2 Step 3 Step 4 Bring the specimen and test components to room temperature if refrigerated or frozen. Mix the specimen well, prior to assay, once thawed. When ready to test, open the pouch at the notch and remove the device. Place the test device on a clean, flat surface. Be sure to label the device with the specimen s ID number. For whole blood test - Apply 1 drop of whole blood (about µl) into the sample well. Then add 1 drop (about µl) of sample diluent immediately. OR RESULTS 15 mins Quality Control 1. Internal Control: This test contains a built-in control feature, the C line. The C line develops after adding the specimen and the sample diluent. If the C line does not develop, review the whole procedure and repeat the test with a new device. 2. External Control: Good Laboratory Practice recommends using external controls, positive and negative, to assure the proper performance of the assay, particularly under the following circumstances: a. New operator uses the kit, prior to performing the testing of specimens. b. A new lot of test kits is used. c. A new shipment of kits is used. d. The temperature used during storage of the kits fall outside of 2-30 C. e. The temperature of the test area falls outside of C. f. To verify a higher than expected frequency of positive or negative results. g. To investigate the cause of repeated invalid results. Interpretation of the Assay Result 1. Negative Result: If only the C line is present, the absence of any burgundy colour in both test lines (M and G) indicates that no anti-l. donovani antibody is detected in the specimen. The result is non-reactive. 2. Positive Result: 2.1 In addition to the presence of the C line, if only the M line is developed, the test indicates the presence of IgM anti-l. donovani in the specimen. The result is IgM positive or reactive. 2.2 In addition to the presence of the C line, if only the G line is developed, the test indicates the presence of IgG anti-l. donovani in the specimen. The result is IgG positve or reactive. Leishmania IgG/IgM Combo 1 DROP OF WHOLE BLOOD 1 DROP OF SAMPLE DILUENT For serum or plasma test - Dispense 20µl of the specimen into the sample well. Then add 2 drops (about µl) of sample diluent immediately. 2.3 In addition to the presence of the C line, if both the M and the G lines are developed, the test indicates the presence of both IgG and IgM anti-l. donovani in the specimen. The result is also positive or reactive. Step 5 Step 6 15 mins 20µl OF SERUM/PLASMA 2 DROPS OF SAMPLE DILUENT RESULTS Set up timer. Results can be read in 15 minutes. Positive results can be visible in as soon as 1 minute. Do not read the results after 15 minutes. To avoid confusion, discard the test device after interpreting the result. Samples with reactive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made. 2

8 Leishmania IgG/IgM Combo 3. Invalid: If no C line is developed, the assay is invalid regardless of any burgundy colour in the test lines as indicated below. Repeat the assay with a new device. Performance Characteristics 1. Clinical Performance for IgM Test: A total of 234 samples from susceptible subjects were tested by the Apacor Leishmania IgG/IgM Combo Rapydtest and by a commercial L. donovani IgM EIA kit. Comparison for all the subjects is shown in the following table. APACOR LEISHMANIA IgG/ IgM COMBO IgG EIA NEGATIVE TOTAL NEGATIVE TOTAL Relative Sensitivity: 91.2%, Relative Specificity: 99.5%, Overall Agreement: 98.3% 2. Clinical Performance for IgG Test: A total of 214 samples from susceptible subjects were tested by the Apacor Leishmania IgG/IgM Combo Rapydtest and by a commercial L. donovani IgG EIA kit. Comparison for all subjects is shown in the following table. APACOR LEISHMANIA IgG/ IgM COMBO IgG EIA NEGATIVE TOTAL NEGATIVE TOTAL Relative Sensitivity: 92.9%, Relative Specificity: 99.0%, Overall Agreement: 98.6% Limitations of the Test 1. The Assay Procedure and the Interpretation of Assay Result must be followed closely when testing the presence of antibodies to L. donovani in serum, plasma or whole blood from individual subjects. Failure to follow the procedure may give inaccurate results. 2. The Apacor Leishmania IgG/IgM Combo Rapydtest is limited to the qualitative detection of antibodies to L. donovani in human serum, plasma or whole blood. The intensity of the test line does not have a linear correlation with the antibody titer in the specimen. 3. A non-reactive result for an individual subject indicates absence of detectable anti-l. donovani antibodies. However, a non-reactive test result does not preclude the possibility of exposure to the visceral leishmaniasis causative species of L. donovani. 4. A non-reactive result can occur if the quantity of the L.donovani antibodies present in the specimen is below the detection limits of the assay or the antibodies that are detected are not present during the stage of disease in which a sample is collected. 5. Some specimens containing unusually high titers of heterophile antibodies or rheumatoid factor may affect expected results. 6. If the symptoms persist while the result from Apacor Leishmania IgG/IgM Combo Rapydtest is non-reactive, it is recommended to re-sample the patient a few days later or test with an alternative test method such as ELISA. 7. The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings. References 1. WHO. Control of the Leishmaniasis. World Health Organization. Technical Report Series No Rosenthal E, Marty P. Visceral leishmaniases. Rev Prat. 2004;54(20): Molina R, Gradoni L, Alvar J. HIV and the transmission of Leishmania. Ann Trop Med Parasitol ;97 Suppl 1: Allain DS, Kagan IG. A direct agglutination test for leishmaniasis. Am J Trop Med Hyg ;24(2): Badaro R, Reed SG, Carvalho EM. Immunofluorescent antibody test in American visceral leishmaniasis: sensitivity and specificity of different morphological Am J Trop Med Hyg. 1983;32(3): Maalej IA, Chenik M, Louzir H, Ben Salah A, et, al. Comparative evaluation of ELISAs based on ten recombinant or purified Leishmania antigens for the serodiagnosis of Mediterranean visceral leishmaniasis. Am J Trop Med Hyg (3): Burns JM Jr, Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci U S A Jan 15;90(2): Order Information PRODUCT PACK SIZE CODE Apacor Leishmania IgG/IgM Combo Rapydtest Products can be ordered direct from Apacor or from an appointed distributor Visit our website for all the latest information or on: sales@apacor.com 3 UNIT 5, SAPPHIRE CENTRE, FISHPONDS ROAD, WOKINGHAM, BERKSHIRE, RG41 2QL, ENGLAND TEL: +44 (0) FAX: +44 (0) APA139 - V1 06/2013