Candida Albicans (Ab) IgA (EIA-1744)

Transcription

1 Intended Use The Candiquant-IgA Test measures Candida albicans specific serum IgA antibodies. It is intended for in vitro diagnostic use only. Summary And Explanation Systemic Candidiasis is a fungal infection resulting from the overgrowth and spread of Candida (yeast) into body tissues (1,2). It is an important cause of mortality among patients who are compromised immunologically or undergoing prolonged antibiotic therapy (3,4,5). The passage of C. albicans pathogens through the gastrointestinal (GI) mucosa into the host's bloodstream is believed to be an important mechanism leading to systemic Candidiasis (6,7,8). Symptoms are varied and may include migraine headaches, depression, urogenital dysfunction, intestinal gas and allergies. In more extreme cases, the patient is totally incapacitated (1,2,11,12,13). Studies indicate that a number of fungal pathogens can spread systemically from intestinal lumen to visceral organs (8,9,10). Although there are over 100 species of Candida, only seven species are isolated with any frequency from medical specimens. Two of these, C. albicans and C. tropicalis account for more than 80% of isolated yeast species. C. albicans is a part of the human microflora and it is commonly known that its overgrowth can upset delicate balance and may manifest in the form of oral and/or vaginal thrush. It may release mycotoxins or other products that affect the host's cellular immune system (14,15). Candida cytoplasmic antigens have been detected in patients with systemic Candidiasis, cancer, and those receiving therapies which alter the host s defense mechanisms. Diagnosis of this condition has been limited to medical history, diet diaries and questionnaires (1,14,16). Many have used blood cultures as a diagnostic tool, but have found that it is unreliable with 40-60% false negatives as confirmed by visceral organ infection. Positive blood cultures become evident only at terminal stages. Throat and/or vaginal cultures are commonly found to be positive (14,15), and thus patients with polysystemic Candidiasis cannot be identified easily. The passage of C. albicans and/or its cytoplasmic antigens through the GI mucosa into the host's blood system will stimulate the host's humoral immune system, resulting in the host s production of anti-c. albicans antibodies in their circulatory system. In general, soon after antigen-crossover, the host s immune response will produce IgM antibodies specific to C. albicans, followed by an increase in specific IgG antibodies. Infection of the mucosal membranes may lead to the presence of a high titer of IgA antibodies specific to C. albicans. The presence of an abnormally high titer of any anti-candida antibody is a good indicator of host immune response to C. albicans infection. The Candiquant Test uses ELISA technology and is designed for the measurement of specific antibodies: IgM, IgG and IgA produced by the host against the C. albicans antigen. A high titer of Candida specific IgM may indicate the early onset of Candida infection. A moderate titer of Candida specific IgG may indicate that the patient has experienced an overgrowth of Candida in the past. A high titer of Candida specific IgG may indicate an active or prolonged infection. A high titer of Candida specific IgA may indicate an active infection of the mucosal membranes (17). Three quantitative procedures are available from DRG for the detection of C. albicans: Specific IgG, specific IgM, and specific IgA. These test procedures may also be used to monitor the effectiveness of therapy. The following applies to the detection and quantification of C. albicans specific serum IgA. Principle Of The Test C. albicans cytoplasmic antigens are purified and then immobilized onto microwells. The Calibrators, Controls, and a dilution of the Serum Sample are added into the appropriate wells. The human antibodies in the serum sample are allowed to react immunologically at room temperature with the immobilized C. albicans antigens. After washing off the unbound materials, the bound antibodies are quantified by an enzyme-labelled second antibody specific to human IgA. A substrate is added, and the color generated is read spectrophotometrically. The intensity of the color is directly proportional to the concentration (or titer) of the C. albicans-specific serum IgA. WARNING Potential Biohazardous Material The matrix of the Calibrators and reference sera is human serum. The human serum used has been found negative for HIV antibodies as well as for Hepatitis B surface antigen when tested with FDA licensed reagents. However, as there is no test

2 method that can offer complete assurance that HIV, hepatitis B virus or other infectious agents are absent, these reagents should be handled at the Biosafety Level 2, as recommended for any potentially infectious human serum or blood specimen in the Centers for Disease Control/National Institutes of Health manual, "Biosafety in Microbiological and Biomedical Laboratories," The microwell strips do not contain viable C. albicans organisms. However, the strips should be considered potentially infectious and handled at the Biosafety Level 2. Sodium Azide The reagents contain sodium azide as a preservative. Sodium azide may react with lead, copper or brass to form explosive metal azides. When disposing of these materials, always flush with large volumes of water to prevent azide buildup. Procedural Notes 1. Strict adherence to the specified time and temperature of incubations is essential for accurate results. 2. Do not freeze reagents. 3. Store all components of the test kit at 2-8 C at all times. Do not allow reagents to stand at room temperature for extended periods of time. 4. Positive and Negative Controls must be run each time the test is performed. 5. Do not mix reagents from different lots. 6. Do not use expired reagents. 7. Use only clear sera as test specimens. The samples should not have gross turbidity, hemolysis or any microbial contamination. Additional Materials Required But Not Supplied 1. Micropipet with disposable tips to deliver 25 µl and 100 µl. 2. For washing, use either a microtiter plate washer (automated or manual), or a squeeze wash bottle. 3. Distilled or deionized water ml pipet for sample dilution and 10 ml pipet for substrate buffer delivery. 5. A 500 ml and a 1000 ml graduated container. 6. Microtiter plate reader with 450 nm wavelength absorbance capability. 7. Absorbent paper towels. 8. Test tubes (13 x 100 mm) for serum dilution. 9. Parafilm or plastic wrap. Kit Components x 8 microwell strips coated with inactivated C. albicans antigens. The strips are packaged in a twelve-strip holder, sealed in a ziplock bag. Reseal carefully any unused strips. 2. Candida albicans IgA Calibrators 0 through 4, human serum base. (5 x 1.0 ml vials). 3. Candida IgA Negative Control (1 x 1.0 ml). 4. Candida IgA Positive Control (1 x 1.0 ml). 5. Anti Human IgA-HRP Conjugate (1 x 10.0 ml). 6. Substrate Solution A (1 x 8.0 ml). 7. Substrate Solution B (1 x 8.0 ml). 8. Serum Sample Diluent (concentrate) (1 x 20.0 ml). 9. Washing Buffer (concentrate). Phosphate buffered saline with Tween (1 x 20.0 ml vial). 10. Stopping Solution (1N HCl) (1 x 6.0 ml bottle). Serum Diluent, Calibrators and Controls contain 0.01% sodium azide as a preservative. Store kit at 2-8 C.

3 Specimen Collection Collect venous blood by venipuncture into a glass vaccutainer (red top) tube. Separate the serum by centrifugation. Serum samples may be stored refrigerated (2-8 C) for up to 10 days. If the sample cannot be tested during this period, store it frozen at -20 C. Excess hemolysis, lipemia, the presence of large clots, or visible indications of bacterial growth in the specimen, may interfere with the performance and accuracy of the test. Reagent Preparation And Storage 1. Serum Sample Diluent Buffer (1:25 Dilution): Add the entire contents of the vial into a 500 ml container with 480 ml of distilled water. Mix thoroughly. Label the container as "Working Sample Diluent Buffer", and store at 2-8 C until use. The reconstituted reagent is stable for the shelf-life of the kit when stored at 2-8 C. Use 5 ml for each patient sample. Rinse out any crystals in the vial which may be present. 2. Washing Buffer (1:50 Dilution): Add the entire contents of the vial into 980 ml of distilled or deionized water in a 1000 ml container. Mix thoroughly. Label the container as "Working Washing Buffer" and store at 2-8 C until use. The "Working Washing Buffer" is stable for the shelf-life of the kit when stored at 2-8 C. Rinse out any crystals from the vial which may be present. 3. Substrate Reagent: Mix Substrate Solution A and Substrate Solution B in the ratio of 1:1 and label as Working Substrate. Prepare the Working Substrate solution within one hour prior to use. Prepare only the required volume. For example for six microwell strips mix 2.5 ml of Substrate Solution A with 2.5 ml of Substrate Solution B. 4. Storage of Remaining Kit Components C. albicans antigen coated strips, Calibrators and Controls, Substrate Solutions and Stopping Solution should be stored at 2-8 C and are stable until the kit expiration date. Assay Procedure The test kit contains 12 microwell strips. Assemble the microwell strips (the number depends on the number of specimen samples to be tested) into the left side of the strip holder. Return unused strips to the original ziplock bag and store at 2-8 C. NOTE: Sample Dilution: Accurately pipet 25 µl of each patient serum to 5 ml of Serum Sample Diluent Buffer (see #1 of the above) in a 13 x 100 mm test tube (1:200 dilution). Mix thoroughly by inversion or vortexing. Appropriately label all tubes containing diluted test samples. Well Positions for the Assay: A1,Bl Calibrator 0 C1,D1 Calibrator 1 E1,F1 Calibrator 2 G1,H1 Calibrator 3 A2,B2 Calibrator 4 C2,D2 Negative Control E2,F2 Positive Control G2,H2 Patient Sample #1 A3,B3 Patient Sample #2 1. Serum Incubation Into the appropriate wells, as shown in the above diagram, dispense 100 µl of Calibrators 0 through 4, Negative Control, Positive Control, and diluted patient serum samples in duplicate. There should be 100 µl of solution in each microwell at this point. Cover the plate with parafilm and let it stand for 1 hour at room temperature (22-26 C).

4 2. Wash Procedure After a one hour incubation, discard the contents of all the wells into the sink by quick decantation. Blot the plate dry with a paper towel. If you use an automatic or manual plate washer, wash each well three times with about 300 µl of washing buffer according to the instrument manufacturer's instructions. If you use a squeeze bottle, fill it with washing buffer. Carefully fill the wells up one by one with the washing buffer by squeezing the bottle (avoid air bubbles in the well during washing), discard the washing buffer, blot dry with a paper towel, and then repeat the procedure two more times, making sure that the plate is blotted dry each time between washes. 3. Conjugate Incubation Add 100 µl of Anti-Human IgA-HRP EnzymeConjugate reagent to all the microwells. Cover the plate (as in step # 1c) and let it stand for 30 minutes at room temperature (22-26 C). 4. Wash (same as step #2). 5. Substrate Incubation Add 100 µl of the "Working" Substrate Reagent (see "Reagent Preparation" Section, #3), into all the wells. Cover the plate, as in step #1c and let it stand in the dark at room temperature (22-26 C) for 10 minutes. 6. Stop Reaction/Read Absorbance After exactly 10 minutes, add 50 µl of Stopping Solution to all the wells. Set the microplate reader to read at a wavelength of 450 nm and measure the optical density (O.D.) of each well. The plate should be read within one hour after the addition of the stop solution. Calculaton Of Data 1. Read the O.D. of the Calibrators, Controls and Patient Samples and record the data as shown in Table #1. 2. Calculate the mean O.D. of calibrators, controls and all the samples (see Table #1). 3. Construct a calibration curve on linear graph paper using the mean O.D. on the Y axis and the calibrator value on the X axis. 4. Interpolate the patient values from the calibration curve. TABLE 1 Sample Calculations: I.D. O.D. Duplicates Mean O.D. Value* U/ml Calib Calib Calib Calib Calib Negative Control Positive Control Pat. Spl Pat. Spl Quality Control 1. The O.D. of the Standard 0 must be < Negative Control should read less than 10 units/ml*.

5 3. Positive Control should read units/ml*. Candida Albicans (Ab) IgA (EIA-1744) Interpretation Positive Result: Patient samples with values greater than or equal to 25 units/ml are considered to be positive for the presence of IgA specific antibodies to C. albicans. Negative Result: Patient samples with values less than or equal to 12.5 units/ml should be considered negative for the presence of IgA specific antibodies to C. albicans. Equivocal Result: Patient samples with values falling between 12.5 and 25 units/ml are equivocal. Patients with equivocal results should be redrawn after two weeks, and the second sample run together with the first sample. Expected Values The antibodies to Candida albicans are found in 20-30% of the normal population. A clinical correlation must exist for the test to be useful. The titer of the antibody along with the symptomatic evidence has been taken into consideration in determining the cut-off point in Candiquant-IgA. Some conditions predisposing to candidiasis are: Pregnancy Diabetes mellitus Chronic debilitating diseases Prolonged therapy with broad spectrum antibiotics AIDS (virtually 100% of AIDS patients have very high titers of the antibodies) Limitations 1. The Candiquant-IgA ELISA is a qualitative test to detect the presence of IgA specific antibodies to C. albicans and does not indicate the titer of the antibody. 2. A positive test result should only be used as an aid to the detection of disease along with other clinical tests such as Histology and Culture. 3. A negative test result does not preclude the absence of antibodies to C. albicans. The infection may be there, but may be in its very early stages, or the antibody titer may be too low for the test to detect. 4. If the clinical symptoms and test results are inconclusive, additional testing should be done on a subsequent sample. 5. The Candiquant-IgA ELISA test should be used only for patients reporting clinical signs and symptoms related to candidiasis. The test should not be used for patients who are asymptomatic. Precision 1. Intra-assay precision was determined by measuring the units/ml of 12 replicates of confirmed Low, Medium and High antibody positive C. albicans patient samples. Low Medium High Number of determinations: Mean value: S.D C.V. 8.0% 10.8% 5.8% 2. Inter-assay variability of the test for sample results was calculated from values obtained from confirmed Low, Medium and High antibody positive C. albicans patient samples over a period of five months, using three different operators.

6 Low Medium High Number of determinations: Mean value: S.D C.V. 17.9% 15.1% 13.44% Candida Albicans (Ab) IgA (EIA-1744) Clinical Significance A high IgG antibody titer against cytoplasmic antigens of C. albicans may be indicative of past or ongoing infection. A high IgM titer indicates current infection. High IgG and IgM antibody titers against cytoplasmic antigens of C. albicans is an even better indicator of current infection. High IgA antibody titers against cytoplasmic C. albicans are indicative of mucosal or vaginal Candidiasis. THIS IS A SCREENING TEST ONLY AND LEVELS OF ANTI-CANDIDA SERUM IgA SHOULD BE ASSESSED IN THE LIGHT OF THE PATIENT'S MEDICAL HISTORY. Moreover, in chronically ill patients, assessment for total immune function is recommended. Immuno-suppressive drugs, steroids and fungiostatic medications may alter test results. References 1. Crook, W.G. The Yeast Connection: A Medical Breakthrough. P.O. Box 3494, Jackson, TN Professional Books, Truss, C.O. The Missing Diagnosis. P.O. Box 26508, Birmingham, AL 35226, Meunier-Carpenter, F., T.E. Kiehn, D. Armstrong. Fungaemia in the immunocompromised host: Changing patterns of antigenema high mortality. Amer. J. Med., 71: , Ray, T.L. Fungal infections in the immunocompromised host. Med. Clin. No. Amer., 64: , Wingard, J.R., W.G. Merz, R. Saral. Candida tropicalis: A major pathogen in immunocompromised patients. Ann. Int. Med., 91: , Krause, W., H. Mathesis, K. Wulf. Fungaemia and Funguria after oral administration of Candida albicans, i: , Stone, H.H., C.E. Geheber, L.D. Kolb, W.R. Kitchens. Alimentary tract colonization by Candida albicans. J. Surg. Res,. 14: , Stone, H.H., L.D. Kolb, C.H. Currie, C.E. Geheber, J.F. Cuzzell. Candida sepsis; pathogenesis and principles of treatment. Ann. Surg., 179: , Wingard, J.R., J.D. Dick, W.G. Merz, G.R. Sanford, A. Saral, W.H. Burns. Pathogenicity of Candida tropicalis and Candida albicans after gastrointestinal inoculation in mice. Infect. Immuno.,. 29: , Haupt, H.M., W.G. Merz, W.E. Bochorner, W.P. Vaughan, R. Saral. Colonization and infection with Trichosporon species in the immunosuppressed host. J. Infect. Dis., 147: , Palacios, H.J. Hypersensitivity as a cause of dermatologic and vaginal moniliasis resistant to topical therapy. Ann. Allergy, 37: , Robinet, R.W. Asthma due to Candida albicans. Univ. Mich. Med. Ctr. Bull,. 34: Iwata, K. A review of the literature on drunken symptoms due to yeast in the gastrointestinal tract. In "Proceedings of the Second International Specialized Symposium on Yeast". Univ. of Tokyo Press, pp , Witkins, S.S., I.R. Yu, W.J. Ledger. Inhibition of Candida albicans: Induced lymphocytes proliferation by lympho-cytes and sera from woman with recurrent vaginitis. Amer. J. Obstet. Gynecol., 147: , Witkins, S.S. Defective responses in patients with current Candidiasis infection. Medicine, May, 129, Araj, G.F., R.L. Hopfer, S. Chestnut, V. Fainstein, G.P. Boday. Diagnostic value of ELISA for detection of Candida albicans cytoplasmic antigens in sera of cancer patients. J. Clin. Microbiol., 16:46-59, Wojdani, A., M. Ghoueum, G. Cheung. Measurement of humoral and cellular immunity for the diagnosis of Candidiasis. Clin. Ecol. 4:210, 1986.