CAND-TEC

CANDIDA DETECTION SYSTEM

PACKAGE INSERT

Catalog Number C03/1

 

HISTORY  AND SUMMARY

Disseminated Candiasis associated with the use of broad spectrum antibiotics, indwelling venous catheters, and immunosuppressive therapy continues to become a more serious clinical problem1,2,3,4. The inability of the clinician to detect early dissemination in the face of colonization of multiple body sites often leads to delays in effective treatment, with disastrous results. Although cultures of colonized sites are often positive in patients, blood cultures are frequently negative, or become positive too late to be of diagnostic use5.

Serological techniques for the detection of dissemination or tissue invasion have not proven to be reliable. The commensal nature of Candida species, the lag time required for antibody production in the normal host, and the impaired immune system in the immunosuppressed patient have all rendered antibody detection tests relatively ineffective6,7. Attention has therefore been focused on efforts to detect antigenic components of Candida species in patients with disseminated disease. The detection of either cell wall mannan or cytoplasmic antigens has been recently reported as promising in the detection of disseminated candidiasis in these patients3,8,9,11,12.

CAND-TEC is designed to detect circulating Candida antigen in patients with serious, disseminated infection. Mucocutaneous type infections such as oral or vaginal thrush or esophagitis are not detected by this test. A consistently high (1:4) or rising titer correlates with disseminated disease. Candida antigen titers may change rapidly, for example, rising from 1:2 to 1:8 within 48 hours. As could be predicted, this test is most useful for early diagnosis. Later in the course of disease, some patients may develop antibodies and subsequent immunecomplex formation, giving "false negative" results. In the presence of a positive blood culture and negative antigen titer, I.V. lines should be checked for possible Candida colonization. 

 

TEST PRINCIPLE

CAND-TEC is based on the principle that uniform size latex particles coated with specific anti-Candida antibody will agglutinate in the presence of Candida antigen. The Candida Sensitive Latex is coated with rabbit anti-Candida antibody. Positive and negative controls are performed simultaneously with patient sera to verify that the reagent system is functioning properly. Specimens exhibiting agglutination of the latex are considered positive for Candida and should be serially diluted to determine the highest titer which will cause agglutination.

 

REAGENTS

All reagents supplied with this kit are intended for in vitro diagnostic use only.

 

 

Candida Sensitive Latex

Reactive Ingredients:     Uniform size latex particles coated with rabbit anti-Candida antibody.

Non-reactive Ingredients:     Glycine buffered saline, pH 8.4, containing 0.1% sodium azide.

Precautions:     Exercise normal precautions required for handling all laboratory reagents.

Storage:     Store at 2 - 8C. DO NOT FREEZE. Shake well before each use.

Volume:     2.2ml

 

Sample Diluent

Non-reactive Ingredients:  Glycine buffered saline, pH 9.5, containing 0.1% sodium azide   and a chelating agent.

Precautions:     Exercise normal precautions required for handling all laboratory reagents.

Storage:     Store at 2 - 8C.

Volume:     15.0ml

 

Positive Control

Reactive Ingredients:     Lyophilized rabbit serum containing Candida antigen with sodium azide as a preservative.

Precautions:     Exercise normal precautions required for handling all laboratory reagents.

Storage:     Store at 2 - 8C after reconstitution. May be aliquoted and frozen.

Volume:     Reconstitute with 500ul of distilled or deionized water.

 

Negative Control 

Reactive Ingredients:     Lyophilized normal rabbit serum with sodium azide as a preservative.

Precautions:     Exercise normal precautions required for handling all laboratory reagents.

Storage:     Store at 2 - 8C after reconstitution. May be aliquoted and frozen.

Volume:     Reconstitute with 500ul of distilled or deionized water.

 

PRECAUTIONS

  • Do not use serum that has been heated.
  • Do not use serum that has been treated with any agent which denatures proteins.
  • Do not use kit if the Positive Control fails to agglutinate, or if the Negative Control agglutinates.
  • Do not use latex solution if it has been frozen.
  • Do not mix reagents from different lots or kits.
  • Do not use reagents beyond the expiration date.
  • Do not pipette reagents or patient samples by mouth.
  • Always shake the latex solution prior to use to uniformly resuspend the particles.
  • Do not allow test material to dry on the Slide.
  • Make certain that the Slide is clean and dry prior to use.

The National Institute for Occupational Safety and Health has issued a bulletin citing the potentially explosive hazard due to the reaction of sodium azide with copper, lead, brass, or solder in plumbing systems. Although sodium azide is added at a concentration 0.1%, it is still recommended that copious amounts of water be used to flush the drain pipeline after disposal of these reagents in the plumbing system. Copper-free and lead-free discharge lines should be used wherever possible.

 

SAMPLE COLLECTION AND PRESERVATION

USE ONLY SERUM FOR THIS ASSAY

Collect 5ml of venous blood aseptically in a tube without anticoagulant. Allow blood to coagulate and separate the serum from the clot by centrifugation. Neither moderate hemolysis or lipemia will interfere with the test.

If the assay will be performed within 14 days, store the serum at 2 - 8C. If more than two weeks will elapse before the test is performed, the serum should be frozen. Sera may be stored for 6 months with no change in its antigen content.

No additives, acidifying agents or preservatives should be combined with patient samples either prior to use or in preparation for storage.

 

 

MATERIALS

Materials Provided 

  • One vial of each reagent listed under "Reagents"
  • One glass agglutination Slide

 Materials Required But Not Provided 

  • 100l and 20 ul precision pipettes
  • Laboratory horizontal rotator capable of 140 RPM
  • Timer (separate or as part of rotator)
  • Applicator sticks
  • 12 × 75mm plastic or glass tubes and rack

 

REAGENT PREPARATION

Reconstitute the Positive and Negative Controls using 500 ul of distilled or deionized water for each Control. Allow the reconstituted materials to stand for 60 minutes prior to use. Controls are stable for 10 weeks after reconstitution when stored at 2 - 8C. If longer shelf life is necessary, Controls may be aliquoted and stored frozen.

 

 ASSAY PROCEDURE

Allow all reagents to reach room temperature prior to use. Test patient samples as soon as possible after addition of Sample Diluent.

Note: The Slide should be thoroughly clean and dry prior to use. Wash the Slide using common laboratory detergent and rinse well with tap water. Rinse theSlide again thoroughly with deionized or distilled water. Dry the Slide with paper towel and wipe clean with lint free tissue.

 

1.   Prepare a 1:2 dilution of each patient serum by dispensing 100 ul of the Sample Diluent and 100 ul of the patient serum into a 12 × 75mm test tube. Do not dilute thePositive or Negative Controls.

2.   Dispense 20 ul of the Positive Control inside the ring at sample position 1 on the Slide.

3.   Dispense 20 ul of the Negative Control inside the ring at sample position 2 on the Slide.

4.   Dispense 20 ul of each diluted patient serum inside a separate ring of the remaining sample positions used on the Slide.

5.   Shake the Candida Sensitive Latex solution vigorously before use.

6.   Transfer 20 ul of the Candida Sensitive Latex to the inside of each ring on the Slide that contains a test specimen.

7.   Mix the contents of each ring, using a separate applicator stick for each sample (the mixture should cover roughly ½ to of the surface area of the ring). Do not reuse the tip of the applicator stick to prevent cross contamination of samples.

8.   Place the Slide on a horizontal rotator and rotate at 140 RPM for 10 minutes.

9.   Results should be read immediately after the 10 minute rotation.

A positive reaction is indicated by agglutination of the latex. Agglutination, or lack thereof, should be determined with the unaided eye -- Do Not Use Any Form of Magnification.

NOTE:  Discontinue use of the assay if the Positive Control fails to agglutinate, or if the Negative Control agglutinates.

 

TITRATION OF POSITIVE PATIENT SERA

NOTE:  It is not necessary to run a Positive or Negative Control with these samples or to rerun the patients' positive 1:2 dilution.

  

1.      a. Prepare serial dilutions of the positive sera. Dispense 100 ul of the Sample Diluent into each of three tubes marked 1:4, 1:8, and 1:16, or greater if desired.

b. Remove 100 ul of the initial 1:2 dilution and add it to the tube marked 1:4. Mix well.

c. Remove 100 ul of the 1:4 dilution and add it to the tube marked 1:8. Mix well.

d. Remove 100 ul of the 1:8 dilution and add it to the tube marked 1:16. Mix well.

Further serial dilutions may be prepared by adding 100 ul of Sample Diluent and 100 ul of the previous dilution to a clean tube and mixing.

2.   Clean the Slide as previously described.

3.   Perform steps 4 through 9 of the Assay Procedure on the serial dilutions of the positive sera.

 

  INTERPRETATION OF RESULTS

NOTE:  Do not use any form of magnification when reading the test results.

 

 

 

Negative Test

If no visible latex agglutination is noted after 10 minutes of mixing with the serum diluted 1:2, the result should be reported as negative.

 

Positive Test

Clearly visible agglutination of the latex after 10 minutes mixing with the serum diluted 1:2 should be reported as positive. Further serial 1:2 dilutions of the serum should be made and assayed. The results should be reported as positive and the titer reported as the highest serum dilution causing agglutination of the latex.

 

LIMITATIONS OF THE PROCEDURE

1.   The antigen detected in this test is heat sensitive, but it is stable at room temperature. If, however, the serum has been heated for any reason prior to testing, false negative results will occur.

2.   Sera positive for rheumatoid factor causes an increased incidence of agglutination not specific for Candida antigen.

3.   There is no conclusive evidence that "false positive" results occur in sera from patients with other fungal infections or in patients with high creatinine levels.

4.   This assay detects free Candida antigen. Its principle advantage lies with its ability to detect disseminated candidiasis in the immunosuppressed patient during its early stages or before significant antibody production occurs in those patients capable of mounting a substantial immune response.

5.   A negative reaction does not exclude the possibility of systemic Candida infection since, in immunocompetent patients, the antigen may be present as part of an immunecomplex which does not react.

 

CONTROLS

Sera from one hundred hospitalized patients were randomly selected as controls. Eighty-four (84%) sera were negative for Candida antigen, thirteen (13%) were positive at 1:2, and three (3%) had titers of 1:4. No titer greater than 1:4 was seen. In the three patients with 1:4 titers, one was recovering from a kidney transplant, one was being treated for a pulmonary infiltrate, and the third was undergoing immunosuppressive Chemotherapy. Although a transient infection could not be ruled out, there was no evidence to suggest any of the three patients was systemically infected.

 

PERFORMANCE CHARACTERISTICS

The antibody used to coat the Candida Sensitive Latex is raised in animals immunized with Candida albicans but patients with proven systemic infection due to C. stellatoideae, C. tropicalis and C. parapsilosis have also shown positive test results for Candida antigen.

 

COLONIZATION

Sera from 20 patients with normal hepatic and renal function receiving broad spectrum antibiotic therapy for an established bacterial infection were tested. The presence ofCandida species was established by culturing each patient's nose, mouth, vagina, rectum and urine.

Thirteen patients (65%) were antigen negative, five (25%) had positive undiluted serum only, and two (10%) had an antigen titer of 1:2. No patient in this group had a titer of 1:4 or greater.

 

 

 

SYSTEMIC CANDIDIASIS

The criteria for inclusion into this group was fever plus a positive blood culture or organ culture together with a positive histology. Sera from 33 patients with systemic candidiasis were tested. In this group, the initial titer of antigen was 1:4 or greater in 31 (94%) of the 33 patients. Two patients with positive blood cultures had no detectable antigen in their sera. One of these was on immunosuppressive chemotherapy and daily plasmapheresis for systemic lupus erythematosus. In the 31 patients positive for antigen, eight (26%) had an initial titer that was 1:4, thirteen (42%) had 1:8, eight (26%) had 1:16 and two (6%) had 1:32. Six of the eight patients with a titer of 1:4 survived their infection while two patients died. Three of thirteen patients with titers of 1:8 survived, while nine (69%) died. No follow up data was available for one patient in this group. Two of ten patients with a 1:16 or greater titer survived. No follow up data was available on two patients in this group.

Antigen titers were 1:16 or greater in each of the three patients with Candida endocarditis. Four patients with pulmonary infection had titers of 1:8 or greater, and three patients with endophthalmitis had titers ranging between 1:4 and 1:16. Subsequent antigen determinations were available in nine patients with disseminated disease who responded favorably to amphotericin B therapy. In each case, the antigen disappeared or was present only in undiluted serum upon completion of therapy.

 

 

EXPECTED VALUES

CAND-TEC is designed to detect a heat sensitive circulating antigen associated with the Candida species, and can be expected to give a positive reaction if this antigen is present.

  

CANDIDA ANTIGEN TITER

Negative

Candida species absent or present as a commensal only.

Positive

   =   1:2 An indicator for serial dilution and retesting of the diluted sera.

>/=   1:4 Consistent with systemic candidiasis. Higher titers are likely to reflect more extensive infections.

  

 

BIBLIOGRAPHY

1.  Mazumdar, P.K. and Marks, M.I. 1975. Candida albicans infections in hospitalized children. A survey of predisposing factors. Clin. Pediatr. 14:123-9.

2.  Myervitz, R.L. Razin, G.J. and Allen, C.M. 1977. Disseminated candidiasis; changes in incidence, underlying diseases and pathology. AM.J.Clin.Pathol. 68:29-38.

3.  Odds, F.C. 1979. Candida and Candidosis. University Park Press, Baltimore.

4.  Seelig, M.S. 1966. The role of antibiotics in the pathogenesis of Candida infections. Am.J.Med. 40:887-917.

5.  Kay, J.H., Bernstein, S., Tsuji, H.K. Redington, J.V. Milgram, M., and Brem, T. 1968. Surgical treatment of Candida endocarditis. J. AM Med Assoc. 203:621.

6.  Harding, S.A., Sanford, G.R., and Merz, W.G. 1976. Three serological tests for candidiasis: diagnostic value in distinguishing deep or disseminated infection from superficial infection or colonization. AM. J. Clin. Path. 65:1001-9.

7.  Rosner, F., Gabriel, F.D., Taschdjian, C.L. Cuesta, M.B., and Kosinn, P.J. 1971. Serological diagnosis of systemic candidiasis in patients with acute leukemia. AM. J. Med. 51:54-62.

8.  Greenfield, R.A. and Jones, J.M. 1981. Purification of a major cytoplasmic antigen of Candida albicans. Infect. and Immun. 34:469-477.

9.  Lew, M.A. Siber, G.R. Donahue, D.M., and Maiorca, F. 1982. Enhanced detection with an enzyme-linked immunoabsorbent assay of Candida mannan in antibody-containing serum after heat extraction. J. Infect. Dis. 145:45-56.

10.  Segal, E., Berg, R.A., Pizzo, P.A. and Bennett, J.E. 1979. Detection of Candida antigen in sera of patients with candidiasis by an enzyme-linked immunoabsorbent assay-inhibition technique. J. Clin. Microbiol, 10:116-118.

11.  Weiner, M.H., and Coats-Stephen, M. 1979. Immunodiagnosis of systemic candidiasis: mannan antigenemia detected by radioimmunoassay in experimental and human infections. J. Infect. Dis. 140:989-993.

12.  Gentry, L.O. Wilkinson, I.D., Lea, A.S., & Price, M.F. 1983. Latex agglutination test for detection of Candida antigen in patients with disseminated disease. Eur. J. Clin. Microbiol. 2:122-128. 

 

RAMCO LABORATORIES INC.

 

4100 Greenbriar Drive

Suite 200

Stafford, TX    77477

800-231-6238

 

 

 

 

 

 

 

 

Date Issued: July 1985 Revised: 3/89, 4/92, 10/92, 5/95, 4/04, 1/09