VZV. Hepatitis B virus (HBV) screening and employee immunization are also commonly offered. The rou- tine screening of blood donors for HIV-1 antibody now places this test in the category of a high volume test. In addition, the test is used to diagnose patients with AIDS and is part of the infection control program for certain needle-stick injuries in health-care workers.

A large proportion of the population has been infected with cytomegalovirus (CMV), and the virus remains latent in blood and tissue cells. Screening for CMV antibody is important in institu- tions that perform organ transplanta- tion. Both donors and recipients of organ transplants should be screened for CMV antibody. Published evidence suggests that seronegative patients who receive organs from seropositive donors may suffer significant infections due to CMV (6). Some donors and recipients of organs such as bone marrow are matched for CMV antibody status if possible.

Use of EIAs for Antigen Detection An important advantage of EIA

systems is that they can be adapted to detect microbial antigens in human specimens. Therefore, they may be- come a substitute for culture in cases where the etiologic agents of disease grow slowly or are difficult to culture. HBsAG assays for blood and blood products were developed for detecting active HBV carriers, but the tests can also be used to diagnose hepatitis B in patients. Other infectious diseases for which the presence of an antigen is tested include respiratory syncytial virus (RSV) in respiratory sections (7), group A streptococci in throat swabs, Chlamydia trachomatis in genital spec- imens (8), and rotavirus (9) and Clos-

tridium difficile toxin A in stool specimens. These tests can be done in minutes to hours, allowing the highly contagious RSV and rotavirus agents to be detected and controlled before hos- pital outbreaks occur. In addition, de- tection of RSV offers the possibility of intervention with antiviral therapy. In the near future, antigen detection tests for Chlamydia pneumoniae (TWAR agent), herpes simplex virus (HSV), and HIV-1 p24 antigen may be diag- nostically or prognostically useful.

Convenient disposable units for rapid antigen detection have been developed by several companies. These flow- through systems use EIA technology to detect infectious agents such as group A streptococci, RSV, HSV (10), and C. trachomatis. However, the cost of these units is high (7), and some of the systems lack sensitivity, and may be no more sensitive than the less expensive agglutination procedures (11).

Automation of EIA Assays Manufacturers of immunoassay re-

agents are currently involved in the de- velopment and marketing of new EIAs. The 1990-1991 edition of Clinical Laboratory Reference (supple- ment to Medical Laboratory Observer) lists 74 companies offering EIAs or re- lated products. These products gener- ally consist of kits containing reagents, washing solutions, and antigen-coated wells in a 96-microwell plate format. The wells may be washed manually or by using a programmable microwell plate washer. All systems require a spectrophotometer for final assay readout.

Most of the established manufac- turers in the immunoassay field (Ab- bott, Clinical Sciences, Inc., Diamedix, Ortho, Sigma, and Whittaker) provide a broad range of test kits and products, especially for the high volume tests

such as rubella, CMV, HIV-1, HTLV-1, toxoplasma, and HSV. Sev- eral companies have also expanded their test kit offerings to include many useful but low volume antibody assays such as for hepatitis A virus, Enta- moeba histolytica, Epstein-Barr virus, Lyme disease, measles, and mumps. Other manufacturers are concentrating exclusively on the HIV-1/HTLV-1 an- tibody market (Coulter, DuPont).

Some manufacturers sell spectropho- tometers and automated washers with their kits (Behring, Diamedix, Ortho, Sigma, and Whittaker). Other compa- nies specialize only in the plate reader or washer equipment (Flow Laborato- ries, Dynatech, Molecular Devices). The ultimate in automation are robotic workstations (Diagnostic Products, Beckman, Diamedix) that can be pro- grammed to perform automatically al- most all steps of the assay.

Data management software is avail- able from some companies for use with an interfaced personal computer (PC). Although its use may not be justified by the workload of some laboratories, such a PC-based system can be inter- faced with the microwell plate reader and with the laboratory information system. The PC can then provide a worksheet instructing the technologist which patients' and control sera to add to each well, calculate a calibration curve for the assay from the control readings, blank and calibrate the patient specimens, and produce a patient- chartable report after completing the assay.

Some obvious and subtle differences among the EIA products should be mentioned. Some kits may be used only with a dedicated reader supplied by the same manufacturer. Some com- panies provide IgG, but not IgM, assays. Some package their antigen wells in 8-well strips and some in a 12-well format. Not all strip-holders fit

NOTE. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. No suggested test or procedure should he carried out unless, in the reader's judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug dosages should he made. Discussions, views and recommendations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors.

Clinical Microbiology Newsletter (1SSN 0196-4399) is issued twice monthly in one indexed volume by Elsevier Science Publishing Co., 655 Avenue of the Americas, New York, NY 10010. Subscription prices per year: $110.00 including postage and handling in the United States, Canada, and Mexico. Add $48.00 for postage in the rest of the world. Second-class postage paid at New York, NY, and at additional mailing offices. Postmaster: send address changes to Clinical Microbiology Newsletter, Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 113010.

74 0196-4399/91/$0.00 + 02.20 © 1991 Elsevier Science Publishing Co., Inc. Clinical Microbiology Newsletter 13:10,1991

equally well into all readers and washers. Some systems require ro- tating or shaking of the plates during incubation. Most manufacturers use wash volumes and protocols unique to their line of products. The spectropho- tometer wavelengths required by some kits are unusual. One advantage of using a family of kits from the same manufacturer is that these differences are minimized, and the time-saving benefits of automation are realized.

A departure from the microwell format is the VIDAS system (Vitek, Hazelwood, Mo.). In this highly auto- mated system, patient specimens are added to predispensed reagent strips and the test is then completely pro- cessed by the instrument. VIDAS has been adapted for both antibody and an- tigen detection assays. This assay is not compatible with conventional EIA systems, and the FDA-approved tests include only rubella, RSV, and toxo- plasma. Tests for Clostridium difficile toxin, HSV, and chlamydia will be ap- proved soon.

Assays Currently Being Adapted to EIA

The identification and cloning of the virus that causes non-A, non-B hepa- titis, now called hepatitis C virus (HCV), has recently been reported (12). A viral antigen that can be used to screen blood donors for antibody to this virus is likely to be available soon. Individuals who have antibody to the virus are assumed to be chronic carriers, and will be excluded from do- nating blood. All blood donors will soon need to be screened for HCV an- tibody as well as HBV and HIV-1. Laboratories involved in blood screening will need a rapid, sensitive, and inexpensive HCV test, most likely an EIA.

Human herpes virus 6 has been iden- tified recently as the cause of exanthem subitum. This virus may also be an important factor in the pathogenesis of AIDS and possibly in bone-marrow transplant recipients. Since culture of the virus is difficult at this time, sero- logic testing may become a commonly used procedure for diagnosing human herpes virus 6 infection (13).

Lyme disease is another recently characterized syndrome that appears to be Spreading into areas of the United States where it was previously not found. The first tests for Lyme disease measured antibodies to the spirochete, Borrelia burgdorferii, by IFA. Re- cently, EIA methods appear to be sup- planting IFA as the preferred tests.

Problems Associated with Automated EIA Systems

Several issues must be addressed when conversion from IFA to EIA methods is being considered. IFA tests use whole organisms or cells such as Toxoplasma gondii tachyzoites or CMV-infected human fibroblast cells as solid-phase antigens. In contrast, EIAs use purified proteins that may be ex- tracted from the infecting organism, but may also be produced in vitro by mo- lecular cloning (14). Since the target antigens of the two tests may be dif- ferent, the two methods may measure slightly different types of antibodies in patient sera.

Potential disadvantages of EIAs in- clude their dependence on the selection and purification of antigen substrates to provide optimum sensitivity and speci- ficity. Another problem common to both methods is the presence of inter- feting antibodies (IgG in IgM assays, rheumatoid factor, antinuclear anti- bodies, etc.) that can give rise to false- positive or false-negative results (15, 16).

The reporting method for the two techniques is not the same. IFA results are expressed in a series of discrete serum dilutions (1:16, etc.), whereas EIA systems give continuous numerical readouts based on spectrophotometric absorbance readings. Most physicians are familiar with serial two-fold dilu- tion results obtained by IFA, but may not understand EIA units. EIA manu- facturers have provided verbal interpre- tations for some tests (low, medium, high) and some provide tables relating EIA units to equivalent IFA titers.

A widely-accepted rule for serologic testing is that a 4-fold rise in IgG titer between acute and convalescent sera is usually significant. EIAs do not use

serial dilutions of patient sera, and do not use the 4-fold rule. The product literature for most EIA systems ex- plains interpretation criteria for parallel acute and convalescent sera testing. Considerable education efforts may be needed to avoid confusing physicians during the transition period from one type of test to another.

The spectrophotometer readouts of EIAs make these tests much more ac- curate, precise, and discriminating than IFA readings by the human eye. The continuous nature of EIA data produces a spectrum of patient values, many of which are in the lower ranges, and called equivocal readings. IFAs do not use an equivocal zone because interme- diate dilutions are not used, and the eye cannot pick up subtle differences in fluorescence. Each laboratory may have to determine how such equivocal readings are to be interpreted (negative, equivocal, low positive, etc.). Our data have shown that equivocal EIA re- sults of some kits are consistently posi- tive by IFA while those of other kits are negative (17).

Cost of EIAs EIAs have been criticized for being

more expensive than IFAs. Both test methods require heavy initial invest- ment in expensive equipment; IFA re- quires a fluorescence microscope, EIA requires at least a microplate reader, and perhaps a washer, a reagent dis- penser, and a PC-based data manage- ment system to realize the full benefit of automation.

To compare costs of EIA systems versus IFA, we calculated the cost of performing the CMV IgG assay with a set of four specimens and controls using three EIA kits. EIA prices per run were determined by dividing the list price of the kit by the number of wells included (generally 96) to arrive at a per well test cost. This cost included diluents, wash solution, and controls, but did not include the labor cost or cost of the reader. Cost of the IFA assay included only the list price of the slides needed to titer (but not initially screen) the four specimens. Our data showed that the EIA costs per run,

Clinical Microbiology Newsletter 13:10,1991 © 1991 Elsevier Science Publishing Co., Inc. 0196-4399/91/$0.00 + 02.20 75

using kits from three different manu- facturers, were $23.24, $20.40, and $19.25 (17). In contrast, the IFA cost for the same tests was $30.24. In both EIA and IFA tests, batching specimens into larger runs would slightly reduce per patient test cost due to reduction in cost of controls. EIA methods would show much more economy of scale than IFA, because the labor cost to run a few specimens or a full plate (96 wells minus the control wells) is ap- proximately the same. These data in- dicate that most EIAs are likely to be more cost-effective than IFA assays.

Choosing an EIA EIAs are intrinsically easier, simpler,

and less subjective than IFA assays. The two systems use somewhat dif- ferent methods of antibody detection, however, and one should not expect perfect correlation between them. Ad- ditionally, studies have shown that the performances of different commercial EIA kits vary from good to unaccept- able. Laboratories should always con- duct parallel tests of new systems to discover those positive and negative characteristics that may not be immedi- ately evident in the salesperson's dem- onstration.

Once a laboratory decides to invest in an automated EIA system, it is probably advantageous to adapt as many high volume tests to it as pos- sible. The system should be flexible to allow the use of multiple tests from different vendors. Purchasing several tests from the same vendor can mini- mize the number of washer and reader protocols that must be programmed into the equipment. Also, the addition of a microcomputer for data analysis, either initially or in the future, should be a strong consideration when purchasing an automated EIA system.

Summary EIA methodology is rapidly super-

seding IFA as the technique of choice

for serologic testing. Advantages in- clude lower cost, economy of scale for large volume tests, ease of use, non- subjective results, and greater accuracy and precision. Disadvantages include the requirement for new instrument purchase, lack of complete correlation with IFA, and lack of user familiarity with the results data if IFA titers are currently being used. Challenges to la- boratorians interested in converting to EIA methodology include choosing equipment and test kits among the large and increasing number available, edu- cating physicians in the new data ex- pression, establishing the degree of correlation with IFA, and determining the significance of borderline or equiv- ocal EIA results.

References 1. Seppanen, H. 1990. Development of a

highly specific and sensitive rubella immunoglobulin M antibody capture enzyme immunoassay that uses en- zyme-labeled antigen. J. Clin Micro- biol. 28:719-723.

2. James, K. 1990. Immunoserology of infectious diseases. Clin. Microbiol. Rev. 3:132-152.

3. Filice, G. A., A. S. Yeager, and J. S. Remington. 1980. Diagnostic signifi- cance of immunoglobulin M antibodies to Toxoplasma gondii detected after separation of immunoglobulin M from immunoglobulin G antibodies. J. Clin. Microbiol. 12:336-342.

4. Stagno, S. et al. 1985. Immunoglobulin M antibodies detected by enzyme- linked immunosorbent assay and radio- immunoassay in the diagnosis of cytomegalovirus infections in pregnant women and newborn infants. J. Clin. Microbiol. 21:930-935.

5. Sluiter, J. F. et al. 1989. Indirect en- zyme-linked immunosorbent assay for immunoglobulin G and four immuno- assays for immunoglobulin M to Toxo- plasma gondii in a series of heart transplant recipients. J. Clin. Micro- biol. 27:529-535.

6. Pass, R. F., P. D. Griffiths, and A. M. August. 1983. Antibody response to cytomegalovirus after renal transplanta- tion: Comparison of patients with pri-

mary and recurrent infections. J. Infect. Dis. 147:40-46.

7. Halstead, D. C., S. Todd, and G. Fritch. 1990. Evaluation of five methods for respiratory syncytial virus detection. J. Clin. Microbiol. 28:1021 - 1025.

8. Gaydos, C. A. et al. 1990. Evaluation of Syva enzyme immunoassay for de- tection of Chlamydia trachomatis in genital specimens. J. Clin. Microbiol. 28:1541-1544.

9. Christy, C., D. Vosefski, and H. P. Madore. 1990. Comparison of three enzyme immunoassays to tissue culture for the diagnosis of rotavirus gastroen- teritis in infants and young children. J. Clin. Microbiol. 28:1428-1430.

10. Dorian, K. J., E. Beatty, and K. E. Atterbury. 1990. Detection of herpes simplex virus by the Kodak SureCell herpes test. J. Clin. Microbiol. 28:2117-2119.

11. DeBates, M. J. et al. 1988. Comparing direct detection kits for group A strep. Lab. Management 26:26-32.

12. Choo, Q. et al. 1989. Isolation of a eDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 244:359-364.

13. Chou, S. and K. M. Scott. 1990. Rise in antibody to human herpesvirus 6 de- tected by enzyme immunoassay in transplant recipients with primary cyto- megalovirus infection. J. Clin. Micro- biol. 28:851-854.

14. Gorgievski-Hrisoho, M. et al. 1990. Serodiagnosis of infectious mononucle- osis by using recombinant Epstein-Ban" virus antigen and enzyme-linked im- munosorbent assay technology. J. Clin. Microbiol. 28:2305-231 I.

15. Champsaur, H., M. Fattal-German, and R. Arranhado. 1988. Sensitivity and specificity of viral immunoglobulin M determination by indirect enzyme- linked immunosorbent assay. J. Clin. Microbiol. 26:328-332.

16. Tsue-Ming, L. et al. 1986. An enzyme immunoassay for immunoglobulin M antibodies to Toxoplasma gondii which is not affected by rheumatoid factor or immunoglobulin G antibodies. J. Clin. Microbiol. 23:77-82.

17. Van Enk, R. A., K. K. James, and K. D. Thompson. 1991. Evaluation of three enzyme immunoassays for Toxo- plasma and cytomegalovirus antibodies. Am. J. Clin. Pathol. 95:428-434.

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