Vol. 7, No. 3

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1978, p. 255-260 0095-1137/78/0007-0255$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

Premarket Evaluation of Commercial Toxoplasmosis Indirect Fluorescent-Antibody Reagents THENA M. DURHAM* AND HENRY M. COLVIN Center for Disease Control, Atlanta, Georgia 30333

Received for publication 17 August 1977

The quality of commercial toxoplasmosis indirect fluorescent-antibody reagents evaluated over a 6-year period. Seven manufacturers voluntarily submitted their products for evaluation in the Center for Disease Control Premarket Evaluation Program. Each product was tested in accordance with the Center for Disease Control performance and labeling specifications and evaluation methods. Only 49% of all of the products tested met the Center for Disease Control requirements. Performance criteria are outlined, and suggestions are offered to assist laboratorians in obtaining toxoplasmosis indirect fluorescent-antibody reagents of high quality.

was

To be consistently useful as an in vitro diagnostic product, a reagent or a multiple-component system (kit) must meet standardized performance and labeling specifications for microbiological reagents. As part of its comprehensive laboratory improvement program, the Bureau of Laboratories, Center for Disease Control (CDC) has compiled certain performance and labeling specifications and reference laboratory methods in Specifications and Evaluation Methods for Immunological and Microbiological Reagents, vol. I: Bacterial, Fungal, and Parasitic Reagents (4). This publication, compiled in cooperation with representatives of state public health laboratories, industry, universities, and other federal agencies, provides the reference guidelines used in the CDC Premarket Evaluation Program. For many years, the CDC has operated the voluntary Premarket Evaluation Program, inviting manufacturers and suppliers of commercial in vitro diagnostic products to submit certain products for testing before marketing. The present program includes many of the serological reagents most commonly used by public health and private diagnostic microbiology laboratories. After

a premarket reagent is evaluated, the results are reported to the submitter. If the evaluated lot of the reagent meets the CDC specifications, the producer (or distributor) is allowed to use the following statement on the package labels or in the product brochures accompanying that product lot: "Samples of this lot were tested by the Center for Disease Control and were found to meet CDC specifications." Also, at the end of each month, a list of all the reagents that met the CDC specifications during

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that month is compiled. Approximately 5,000 copies of this Premarket Evaluation Program Monthly Report ultimately reach state laboratory directors and other interested consumers in federal, foreign, commercial, clinical, and private laboratories. Since early 1971, the CDC has invited manufacturers to voluntarily submit toxoplasmosis IFA reagents for premarket evaluation. The response of manufacturers has varied from excellent to very poor. Some manufacturers' products are seen only through follow-ups on consumer complaints. Reagents of poor quality are obviously being marketed, and this practice could seriously affect the validity of IFA results provided by clinical laboratories for the serological diagnosis of toxoplasmosis. MATERIALS AND METHODS Control sera. Positive and negative control sera were obtained from selected human donors. Two positive sera, one with a high titer and the other with a low titer, were prepared by appropriately diluting the high-titered serum with nonreactive human serum. Each control serum was evaluated in the IFA test with previously established reference reagents as described in the CDC specifications (4). Each serum was also evaluated in the Sabin-Feldman dye test as modified by Beverly and Beattie (3) to confirm the IFA test titers. In subsequent IFA tests, a set of three control sera was used: one negative, one with a low titer, and the third with a high titer. This set of control sera met the performance requirements stated in Table 1. Control antigen. The Rh strain of Toxoplasma gondii was obtained from the Parasitic Serology Branch, CDC, and was cultured in the peritoneum of 3-week-old Institute of Cancer Research mice (6). The peritoneal exudate was harvested and processed according to the method of Fletcher (5) as modified by Harrell et al. (6). The formalinized, whole-cell suspen-

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TABLE 1. CDC performance requirements for toxoplasmosis IFA reagents Requirements

Reagent

Human control seraa Negative

Low-titer positive'

High-titer positiveb T. gondii antigen slides

Potency: Must demonstrate no peripheral fluorescence, or peripheral fluorescence of less than 1+ intensity at a dilution of 1:16 or 1:32. Polar staining acceptable. Potency: Must demonstrate complete peripheral fluorescence of 1+ intensity within a dilution range of 1:32 to 1:128, but less than 1+ at 1:256. Potency: Must demonstrate complete peripheral fluorescence of 1+ intensity within a dilution range of 1:512 to 1:2,048, but less than 1+ at 1:4,096.

Reactivity of trophozoites: Must demonstrate well-defined serum titration end points within the specified dilution ranges for toxoplasmosis human control sera. Smears should contain 50 to 100 well-distributed organisms per highpower microscopic field (x400 to x648). Morphology of trophozoites: Should be comma-like to ovoid in shape with cellular membranes essentially intact. Specificity: Smears prepared from mouse peritoneal exudate must not contain more than 10 leukocytes per high-power field. Smears must not contain microbial contaminants such as bacterial or fungal elements.

Reactivity: When used at the working dilution recommended by the producer, the conjugate must demonstrate well-defined serum titration end points within the specified dilution ranges for toxoplasmosis human control sera. At the recommended working dilution, the conjugate must demonstrate reactivity to human immunoglobulin G (reactivity to immunoglobulin M is not required). Specificity: At the recommended working dilution, the conjugate must not react directly with T. gondii trophozoites. The conjugate may react with toxoplasmosis-negative sera that contain antinuclear antibodies (2). a No specificity requirements for toxoplasmosis human control sera. b When used as a pair, low-titer and high-titer positive sera must demonstrate at least an eightfold difference in titer. c FITC, Fluorescein isothiocyanate.

Anti-human FITC' conjugate

sions were lyophilized and stored at 4°C (6, 16). Slides were prepared by reconstituting the suspensions and dispensing droplets onto the reaction sites of microscope slides (25 by 76 mm; Cel-Line Associates, Inc.). Each slide contained eight reaction sites (6mm wells formed by an epoxy paint coating. The antigen droplets were allowed to air dry, and the slides were stored at -20°C. The antigen slides were evaluated in the IFA test with previously established reference reagents as described in the CDC specifications (4). The antigen slides met the performance requirements listed in Table 1. Control anti-human conjugate. The anti-human conjugate was prepared from immune goat serum produced against human immunoglobulins G, M, and A. The antiserum was fractionated with ammonium sulfate, and globulins obtained were conjugated with fluorescein isothiocyanate according to the direct method of Hebert et al. (7). The conjugate was dialyzed to remove the unreacted fluorescent material, and the fluorescein-to-protein ratio was determined to be 15 jig of fluorescein isothiocyanate per mg of protein (7). The conjugate was titrated in the IFA test with previously established reference reagents as described in the CDC specifications (4). The titration diluent was 0.01 M phosphate-buffered saline (pH 7.6 to 7.8) containing 0.2% Evans blue dye as a counterstain. After the optimal working dilution was determined,

the conjugate was appropriately diluted with a stabilizing medium that contained normal goat serum. It was then lyophilized and stored at 4°C. When reconstituted to the working dilution with 0.2% Evans blue diluent prepared in phosphate-buffered saline, the conjugate used in subsequent evaluations met the performance requirements presented in Table 1. Commercial products evaluated. The products tested were either multiple-unit products (kits) or individual reagents, and were voluntarily submitted by the following seven manufacturers: Clinical Sciences, Inc.; Cooke Laboratory Products (Div. Dynatech Laboratories, Inc.); Virgo Reagents (Electro-Nucleonics Laboratories, Inc.); International Biological Laboratories, Inc.; ICN Medical Diagnostic Products; Microbiological Research Corp.; and Wellcome Reagents Ltd. (For the sake of anonymity, the manufacturers' names will henceforth be referred to by letters assigned on a random basis). A total of 112 products were evaluated: 66 kits, 22 T. gondii antigens, 11 anti-human conjugates, and 13 human sera. Each product was evaluated in accordance with the CDC specifications, with established reference reagents as described (4). IFA test procedure. The same lyophilized serum pools and antigen suspension were used to prepare the CDC control reagents throughout the entire study. The 66 toxoplasmosis kits were evaluated according to

COMMERCIAL TOXOPLASMOSIS IFA REAGENTS

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Reading of results. The slides were examined on Leitz SM fluorescence microscope (monocular) equipped with an Osram HBO 200 mercury arc lamp, a cardioid dark-field condenser, Schott BG-38 and BG12 primary filters, a Leitz blue-absorbing secondary filter, a x10 dry objective and a X54 oil objective, and a X10 eyepiece. For viewing the reactions, the central portion of the antigen well was critically focused under low power, and the entire well was scanned under high power. Numerical notations as defined below were used to describe the peripheral fluorescence observed on the T. gondii trophozoites: 4+, brilliant yellow-green fluorescence around the entire cell periphery, internal red counterstain (Evans blue) fluorescence partially or completely masked; 3+, brilliant yellow-green fluorescence around the entire cell periphery, internal red counterstain exposed; 2+, less brilliant, thin band of

the manufacturers' instructions. All kit components were tested together as a system, and each antigen, conjugate, and serum component was also tested in reciprocal combinations with CDC reference reagents. The IFA test protocol outlined in the CDC specifications was used to evaluate the 46 individual toxoplasmosis reagents (4). This procedure was essentially as follows. T. gondii antigen slides were removed from the freezer and allowed to equilibrate to room temperature. The slides were then rinsed in a light stream of distilled water directed down the center of each slide to achieve an outward flushing action. (This rinse was necessary to remove residual salts from the antigen reaction sites.) The slides were blotted dry with bibulous paper, appropriately labeled, and placed face up on wet blotting pads in a shallow tray. Toxoplasmosis sera (negative, low-titer positive, and high-titer positive) were diluted with 0.01 M phosphate-buffered saline (pH 7.6 to 7.8) in serial twofold dilutions beginning at 1:16 (or at 1:32 for those test sera reconstituted to an initial dilution of 1:16). Dilutions prepared for each serum were as follows: negative, 1:16 and 1:32; low-titer positive, 1:16 through 1:256; and high-titer positive, 1:16 through 1:4,096. The dilutions tested were 1:16 and 1:32 of the negative serum, 1:32 through 1:256 of the low-titer serum, and 1:512 through 1:4,096 of the high-titer serum. A drop of phosphate-buffered saline was applied to one antigen well to serve as a conjugate staining control. The tray of slides was covered with a lid and placed in a 37°C incubator for 30 min. After incubation, each slide was individually washed with a light stream of distilled water (directed down the center of the slide), and all of the slides were placed in a slide carrier and taken through three phosphate-buffered saline rinses and one distilled-water rinse. (Each rinse lasted for 30 s, during which the slides were gently agitated up and down.) The slides were blotted dry and again placed on the wet blotting pads in the tray. The working dilution of anti-human conjugate (as previously determined by titration with reference sera, or as specified by the manufacturer) was then applied to the antigen wells. The slides were incubated, rinsed, and blotted as described above. Cover slips (no. 1, or as provided by the manufacturer) were mounted with buffered glycerol (pH 8.5 to 9.0, or as provided by the manufacturer).

a

fluorescence around the entire cell periphery, internal red counterstain very apparent; 1+, very thin band of yellow-green fluorescence around the entire cell periphery, internal red counterstain dominant; ±, barely visible band of yellow-green fluorescence around the entire cell periphery, cells exhibit mostly red counterstain fluorescence; negative, cells exhibit red fluorescence only with no yellowgreen fluorescence extending around the cell periphery, or only the anterior end of the cells fluoresces bright yellow-green, with no extension of yellow-green fluorescence around the cell periphery (polar staining). The preparation was considered to be positive if at least 50.0% of the T. gondii trophozoites displayed 1+ or greater peripheral fluorescence. The serum titer was judged to be the highest (final) serum dilution that demonstrated a 1+ level of fluorescence. yellow-green

RESULTS

The CDC performance requirements outlined in Table 1 were used to assess the 112 products tested (Table 2). Only 49.1% of the products met the CDC requirements: 36.4% of the kits, 45.5% of the conjugates, 72.7% of the antigens, and 76.9% of the sera. Any product that was unsatisfactory in its initial evaluation was reevaluated to confirm the accuracy of the first results.

TABLE 2. Premarket evaluation results for commercial toxoplasmosis IFA reagents listed by manufacturera Kits Manufacturer

No. of reagents evaluated

Individual reagents

Conjugates Sat

Sat

A B C D E F G Total

Antigens

Sera

Unsat Unsat

Sat

Unsat

Sat

Unsat

NR NR NR NR 4 1 1 0 0 NR 2 0 1 NR 5 10 NR 2 0 NR 6 10 5 4 8 3 8 2 3 6 NR 2 1 NR 0 4 NR 1 1 NR 24 42 5 6 16 6 10 3 a Sat, Satisfactory performance as judged by the performance requirements in the CDC specifications. Unsat, Unsatisfactory performance as judged by the performance requirements in the CDC specifications. NR, No reagents of the specified type evaluated. 4 24 3 17 46 12 6 112

1 9

3 9

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No test was considered valid unless all CDC control reagents also met the performance requirements. Most of the products were received after 1973, with kits becoming more prevalent with each ensuing year (Table 3). The quality of the individual reagents improved slightly over the years; however, the quality of the kits showed a definite

decline, as indicated by the decrease in the satisfactory incidence rate from 83.3% in 1973 to 31.6% in 1976. During the first 6 months of 1977, only 16.6% of the kits were satisfactory. The performance problems encountered and the frequency of their occurrence are given in Table 4. When individual reagents were combined into kits, problems occurred not only with product

TABLE 3. Chronological distribution ofpremarket evaluation results for toxoplasmosis IFA reagentsa No. of manufacturers

Yearb

participating

No. of reagents evaluated

Kits

Individual reagents Antigens Unsat Sat Unsat

Conjugates St Sat

U Unsat

Sat

Sera Sat

Unsat

4 2 1 3 1971 NR NR 0 1 4 12 2 7 1 1972 0 0 1 1 0 2 8 1 5 1 1973 0 1 NR 0 21 3 2 6 4 4 5 1974 0 2 1 5 11 5 26 4 0 5 1 NR 1975 27 5 6 13 1 0 1976 3 4 NR 2 14 1 2 1 5 3 1 1977 1 0 112 24 42 5 6 16 6 Total 10 3 a Sat, Satisfactory; Unsat, unsatisfactory. NR, No reagents available of specified type evaluated. See Table 2, footnote a. " From May 1971 to June 1977.

TABLE 4. Frequency distribution ofperformance problems encountered with commercial toxoplasmosis IFA reagents Reagenta

Reagent"

T. gondii antigenc

Unacceptable characteristic

of times ~~~~~~~~~~~~~~~~~~~~~~No

Too few trophozoites per microscopic field (usually accompanied by excessive clumping of cells) Bacterial contamination

Under-reactivity Over-reactivity Atypical morphology (sickling or rounding of cells; degradation of cell membranes) Excessive leukocytes and tissue debris from mouse peritoneal exudate Buckling or excessive hydrophobic properties of slide coatings Uneven distribution of trophozoites in antigen wells (accumulation of cells at the well periphery, etc.) Too many trophozoites per microscopic field Human control serad

Over-reactivity of positive sera (exceeding maximum allowable titer) Under-reactivity of positive sera (less than minimum required titer) "Positive" reactivity of serum labeled as "negative" Less than an eightfold titer difference between low- and high-titer positive

encounteredb

10 10 6 4 4 4 2 1 1

9 8 7 3

serum end points

Anti-human

FITCe

Over-sensitivity

14

conjugates Ancillary

Under-sensitivity Inclusion in kits of insufficient Evans blue diluent to reconstitute the companion conjugate accurately

5 2

Supplied as individual reagents or as kit components. b Among 57 unsatisfactory reagents. Supplied as prepared slides or as lyophilized, standardized suspensions from which slides were prepared according to the manufacturer's instructions. d Usually supplied in sets of three standardized sera; a negative, a low-titer positive, and a high-titer positive. Positive sera in a given set had to demonstrate an eightfold difference in titer. a

e

FITC, Fluorescein isothiocyanate.

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COMMERCIAL TOXOPLASMOSIS IFA REAGENTS

performance, but also with product labeling, as foilows: (i) kit expiration dates did not match those of the earliest-expiring kit component; (ii) kits were not assigned identifying kit (or master) lot numbers; (iii) labeling of positive control sera was incomplete in that they were designated only as "positive," and not as "low titer" and "high titer"; and (iv) storage instructions on the outer kit containers and those on component containers often disagreed. When these labeling problems were brought to the attention of the producers, most of the products were appropriately relabeled; however, some kits are still being sold with inconsistent storage instructions. Even more importantly, some products (kits and individual reagents) were being sold with the statement in their labeling that the product was tested and approved by the CDC, when in fact the product lots so labeled were never submitted for evaluation. This was brought to the attention of the producer on several occasions before the problem was eventually corrected. DISCUSSION Often active toxoplasmosis is not easy to diagnose, and usually the diagnosis is based on the critical assessment of certain serological tests (8-10, 13, 14). The IFA test has become an important tool in the serological diagnosis of this disease because this test is safer and more adaptable than the standard Sabin-Feldman dye test, and the reagents used in the IFA test are relatively stable. Several workers have shown that the IFA test is sensitive and reliable (15, 16, 19) and is specific under most circumstances (2); however, the quality of the commercial reagents must be improved for the results obtained with this test to be reliable. Obviously, quality reagents can be produced, as demonstrated by those products that did meet the CDC specifications. Ironically, kits are the most widely distributed commercial product used in toxoplasmosis IFA serology, even though problems with performance and labeling are compounded when individual reagents are combined into a unit. The greatest difficulty with kits is that the two most important components, the antigen and the conjugate, often do not perform satisfactorily in the test (see Table 4). An unsatisfactory antigen or conjugate component cannot accurately reflect the titers of control serum components, even though the sera may have been properly standardized. Likewise, the titer of the patient's serum is inaccurately reflected when determined with such reagents. In small laboratories, the IFA test may be the sole serological procedure used for the routine

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diagnosis of toxoplasmosis; therefore, the availability of high-quality reagents, particularly in kits, is imperative. Proper standardization of reagents for use in this test is very important because this type of staining procedure is extremely sensitive (11). False-positive results could lead to unnecessary treatment with toxic drugs and to needless anxiety in pregnant and nonpregnant women (12). False-negative results could lead to a lack of necessary treatment, which could have dire consequences, particularly when immunologically compromised individuals are involved (12). Until the quality of the toxoplasmosis IFA reagents is substantially improved, the following suggestions are offered to laboratorians. (i) Laboratories may request and accept only those products tested at CDC and found to meet CDC performance specifications. The CDC's Premarket Evaluation Program Monthly Reports identify products that have been tested and found satisfactory. (ii) Before using them in routine diagnostic tests, laboratories should check-test individual products with known control materials and should check kits with control human sera of known titer. Kit components should work together as a system to produce the expected results with these control sera. (iii) Laboratories should notify the producer (or supplier) and the Food and Drug Administration of any unsatisfactory products. A Problem Product Report may be sent to the United States Pharmacopeia (forms obtainable from USP, 12601 Twinbrook Parkway, Rockville, MD 20852), which forwards copies to the Food and Drug Administration and the producer. All reports should be coordinated with quality control/assurance procedures required by your laboratory. ACKNOWLEDGMENT We thank Ruth M. Dodge, State Laboratory of Hygiene, University of Wisconsin Medical Center, for determining the Sabin-Feldman dye test titers of our control human sera.

L1TERATURE CITED 1.

Ambroise-Thomas, P., J.-P. Garin, T. K. Truong, A. Cornet, M. A. Fournis, and M. J. Despeignes. 1971.

Our experience of immunofluorescence in the serological diagnosis of toxoplasmosis-comparative evaluation with Sabin and Feldman test in more than 7500 human sera, p. 61-66. In D. Hentsch (ed.), Toxoplasmosis. Hans Huber, Bern. 2. Araujo, F. G., E. V. Barnett, L. 0. Gentry, and J. S. Remington. 1971. False-positive anti-Toxoplasma fluorescent-antibody tests in patients with antinuclear antibodies. Appl. Microbiol. 22:270-275. 3. Beverly, J. K. A., and C. P. Beattie. 1952. Standardization of the dye test for toxoplasmosis. J. Clin. Pathol. 5:350-353. 4. Center for Disease Control. 1975. Specifications and

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5. 6.

7.

8. 9.

10. 11. 12. 13.

DURHAM AND COLVIN

evaluation methods for immunological and microbiological reagents, vol. 1: Bacterial, fungal, and parasitic, 4th ed. Center for Disease Control, Atlanta, Ga. Fletcher, S. 1965. Indirect fluorescent antibody technique in the serology of Toxoplasma gondii. J. Clin. Pathol. 18:193-199. Harrell, W. K., H. Ashworth, L E. Britt, J. R. George, S. B. Gray, J. H. Green, H. Gross, and J. E. Johnson. 1973. Procedural manual for production of bacterial, fungal, and parasitic reagents, 3rd ed. Center for Disease Control, Atlanta, Ga. Hebert, G. A., B. Pittman, R. M. McKinney, and W. B. Cherry. 1972. The preparation and physicochemical characterization of fluorescent antibody reagents. Center for Disease Control, Atlanta, Ga. Jacobs, L., and M. N. Lunde. 1957. Haemagglutination test for toxoplasmosis. J. Parasitol. 43:308-314. Karim, K. A., and G. B. Ludlam. 1975. The relationship and significance of antibody titers as determined by various serological methods in glandular and ocular toxoplasmosis. J. Clin. Pathol. 28:42-49. Kelen, A. E., L. Ayllon-Leindl, and N. A. Labzoffsky. 1962. Indirect fluorescent antibody method in serodiagnosis of toxoplasmosis. Can. J. Microbiol. 8:545-554. Nairn, R. C. (ed.). 1969. Fluorescent protein tracing, 3rd ed. The Williams and Wilkins Co., Baltimore. Remington, J. S. 1974. Toxoplasmosis in the adult. Bull. N.Y. Acad. Med. 50:211-227. Sabin, A. B. 1949. Complement fixation test in toxoplas-

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14.

15.

16. 17.

18.

19.

mosis and persistence of the antibody in human beings. Pediatrics 4:443-453. Sabin, A. B., and H. B. Feldman. 1948. Dyes as microchemical indicators of a new immunity phenomenon affecting a protozoan parasite (Toxoplasma). Science 108:660-663. Sulzer, A. J., and E. C. Hall. 1967. Indirect fluorescent antibody tests for parasitic diseases. IV. Statistical study of variation in the indirect fluorescent antibody (IFA) test for toxoplasmosis. Am. J. Epidemiol. 86:401-407. Sulzer, A. J., M. Wilson, and E. C. Hall. 1971. Toxoplasma gondii: polar staining in fluorescent antibody test. Exp. Parasitol. 29:197-200. Takumi, K., I. Takebayashi, H. Takeuchi, H. Ikeda, and N. Toshioka. 1966. The use of lyophilized parasites in indirect fluorescent antibody technique for detection of Toxoplasma antibody. Jpn. J. Microbiol. 10:189-191. Van Renterghem, L., and L Van Nimmer. 1976. Indirect immunofluorescence in toxoplasmosis: frequency, nature and specificity of polar staining. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 235:559-565. Walton, B. C., M. Benchoff, and W. H. Brooks. 1966. Comparison of the indirect fluorescent antibody test and methylene blue dye test for detection of antibodies to Toxoplasma gondii. Am. J. Trop. Med. Hyg. 15:149-152.

Premarket evaluation of commercial toxoplasmosis indirect fluorescent-antibody reagents.

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