Predicting Human Immunodeficiency Virus Type 1-Positive Sera by

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1991, p /91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 29, No. 11 Predicting Human Immunodeficiency Virus Type 1-Positive Sera by Using Two Enzyme Immunoassay Kits in a Parallel Testing Format KEVIN FONSECA'* AND CHANDAR M. ANAND" 2 Provincial Laboratory of Public Health for Southern Alberta, 3030 Hospital Drive N. W., P.O. Box 2490, Calgary, Alberta, Canada T2P 2M7,' and Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4NI2 Received 4 March 1991/Accepted 16 August 1991 Two algorithms for screening sera for antibody to human immunodeficiency virus type 1 were compared for their efficiency in identifying a true-positive sample in a population with heterogeneous risk factors, using the criteria of specificity and positive predictive value (PPV). In the first algorithm, all sera were screened by using a single enzyme immunoassay (EIA) kit, and a specificity of 98.6%o and a PPV of 69.3% was calculated for true-positive sera. The second algorithm employed two different EIA kits in parallel to screen each sample. In the first instance, a specificity and a PPV of 100% was calculated if a positive sample was defined as reactive by both EIA kits; in the second, a specificity of 99.97% and a PPV of 99.4% was obtained if this criterion was extended to include a combination of one reactive and one equivocal result obtained with the two EIA kits. Enzyme immunoassays (EIAs) are commonly used as screening tests in detecting antibody to human immunodeficiency virus (HIV) in serum and plasma. In most of these algorithms, samples are tested either singly or in duplicate by using a single EIA kit, and any sera giving reactive results are retested in duplicate to ensure reproducibility of the initial reactivity. Sera giving reactive results on repeat testing are then tested by supplemental procedures to exclude any EIA false-positive reactions. Current supplementary procedures for the verification of EIA-reactive sera include the Western blot (WB) (immunoblot), radioimmunoprecipitation assay (RIPA), and immunofluorescence assay (IFA), each of which have their respective advantages and disadvantages. Despite the commercial availability of the WB kits, the cost of performing an individual test is high, partly because of the need to include the requisite controls. The RIPA requires the propagation and labelling of the HIV with radioactive isotopes, which restricts the availability of this test to laboratories with suitable containment facilities. Consequently, the WB and RIPA are generally referred to larger reference laboratories in which larger volumes of specimens or the appropriate facilities are present; this may lead to some delay in reporting the results to the requesting physician. Although the IFA is technically simple to perform, it requires either exacting methodology for in-house preparation of slides or commercially available kits of good quality, so that the distinction between positive and negative sera is unambiguous. Studies have shown that the distinction between positive and negative tests is occasionally hampered by nonspecific reactions to the propagating cell stock and further testing by WB and RIPA may be required (4). Although EIA kits are generally designed for maximum sensitivity and specificity, in practice, the positive predictive value of the test is directly related to the prevalence of the disease in the population tested. Consequently, a higher proportion of supplementary tests will be used to verify EIA-reactive sera, the majority of which will be false positives, especially when the prevalence of the disease is low * Corresponding author (3). Usually most laboratories will be testing sera collected from such a population, in which relatively few true-positive sera will be identified in relation to the numbers of EIA false-positive sera encountered. We report on the use of an algorithm which uses two different EIA kits in parallel as a presumptive aid in detecting HIV antibody-positive samples in screening a population with different prevalences for this disease. MATERIALS AND METHODS Study population. Sera submitted to the Provincial Laboratory of Public Health for Southern Alberta, Calgary, Alberta, Canada, during the period from December 1986 to December 1989 (inclusive) were included in this survey. During December 1986 to August 1988 (period 1), all sera were tested by using a single EIA kit in the protocol described below. From September 1988 to December 1989 (period 2), sera were tested in parallel by each of two different EIA kits in a protocol defined below. Categories of sera were organized by risk groups of individuals or reason for testing, as ascertained through a history form incorporated as part of the requisition form, and the main categories are shown in Table 1. The "other" category included sera from people with needlestick injuries, hemophiliacs, and organ donors and those samples for which the risk factors of individuals tested were not defined sufficiently for them to be allocated to one of the other categories. Antibody testing protocol. Samples received during period 1 were each tested in duplicate wells in the Genetic Systems LAV EIA (GSC) kit (Genetic Systems, Seattle, Wash.). Sera initially giving absorbance values which corresponded to reactive or equivocal results (see "EIA") were retested to ensure reproducibility, and those which were repeatedly reactive or equivocal were then tested by the IFA. Samples initially giving equivocal results and then two nonreactive results on separate retests were regarded as not reactive. Sera testing nonreactive initially were not retested. Samples which were reactive by both EIA and IFA were reported as reactive. Those samples that gave a reactive or equivocal result by EIA when the IFA result was either nonspecific or

2 2508 FONSECA AND ANAND J. CLIN. MICROBIOL. New Both reactive case IFA Request further sample FIG. 1. Repeat case 1 Screen in both GSC and WEL EIA kits Both non reactive No further testing a - see text fo Simplified algorithm of HIV antibody testing for period 2. The verification tests performed were the IFA and WB. When both tests were performed on the same sample, only the results of the WB are given. IFA. During period 1, a commercially available IFA slide test from Institut Armand-Frappier, Montreal, Quebec, Can- Other ada, was used. In period 2, the laboratory acquired the combinations expertise to produce an in-house IFA which used infected Western Blota r reporting and uninfected MOLT-4-T4 cells, and this IFA replaced the For both periods, a positive result was reported above kit. when the virus-infected cell well showed the typical even fluorescence and the cell control showed none, a negative result was reported when there was no fluorescence in either set of wells, and a nonspecific result was reported when fluorescence was seen in the wells of both infected and uninfected cells. Positive and negative sera were used as controls to monitor the performance of the tests, which were comparable for both periods. WB. During period 1, all WB tests were performed at the FCA. Their criterion for reporting a positive WB result was either the presence of antibody to gag and one env component or the presence of antibodies to env glycoproteins together with an appropriate clinical history. At the beginning of period 2, our laboratory performed the WB tests using a commercially available kit (Dupont de Nemours, Wilmington, Del.). For this study, the criteria of the Association of State and nonreactive were referred to the Federal Centtre for AIDS Public Health Laboratory Directors/Centers for Disease (FCA), Ottawa, Ontario, Canada, for a WB confirmatory Control (CDC) (Atlanta, Ga.) for interpreting the WB band test. profile (8) were adopted. During period 2, ail sera were tested in si ngle wells in All sera giving indeterminate results were sent to the FCA parallel in each of two kits, i.e., GSC and Wellcozyme for verification by RIPA. anti-htlv III EIA (WEL) kits (Wellcome Dartford, England), as shown in a simplified Fig. 1. Sera testing reactive by both kits were retested to the presence of virus-specific precipitable antibodies was Diagnostics, RIPA. The protocol employed by the FCA was as dealgorithm in scribed previously (6). A positive result was reported when ensure reproducibility of the earlier result and then verified detected. by the IFA. A request for a second sample for confirmation Statistics. The statistical measures used were expressed as by the WB test was also made if the IFA gaive a positive percentages. Sensitivity was calculated as [TP/(TP + FN)] x result (2). If the serum sample tested nonspe( cific with the 100, specificity was calculated as [TN/(TN + FP)] x 100, IFA, it was then tested by the WB test. Seraa which gave and positive predictive value was calculated as [TP/(TP + equivocal or reactive results with either EIA kçit and which FP)] x 100, where TP and FP are the numbers of true- and gave similar results after retesting were also 1tested by the false-positive samples, respectively, and where TN and FN WB test, and requests for additional serum samples were are the numbers of true- and false-negative samples, respecresults were lively (5). Confidence intervals and limits were determined made when positive or indeterminate WB obtained. Samples which initially gave equivocal or reactive from standard formulae (1). results with one kit and then gave nonreactive iresults in two A true-positive sample was defined as one which was subsequent separate retests with both kits were reported as positive by IFA, WB, or RIPA. not reactive. Provisional reports were issued f"or indeterminate WB results pending verification by the RIIPA, and these RESULTS samples were referred to the FCA. In the infreqluent instance when the first serum sample was tested by the WB test and Sample distribution. In period 1, a total of 10,387 serum found to be positive, a second sample for confirnation of this samples were tested by using one EIA kit. In period 2, 8,026 result was requested (2). serum samples were tested in parallel by using two EIA kits. Sera testing as nonreactive with both kiits were not The distribution of sera tested in both periods 1 and 2 is retested and were reported as not reactive. shown in Table 1. Generally, the percentages of numbers of The results of all follow-up tests performed on the same tests performed for each of the categories for both periods patient were collated when subsequent seraa from these were comparable, except for the transfusion and emigration individuals were specifically identified. groups. The number of tests performed for the transfusion EIA. All EIAs were performed and interpre-ted in accor- category declined considerably in period 2; in contrast, the dance with the manufacturer's protocol. Categ,ories of reac- requirement for testing for emigration purposes rose. tive, nonreactive, and equivocal results were established. The anxiety category resulted from physicians frequently The equivocal category included those sera ffor which the giving this as a reason for requesting the test. absorbance ranged from the positive cutoffto 2'0% below the The distribution of serologically true-positive individuals cutoff. Likewise, for the WEL kit, which is a competition for both periods is shown in Table 1. After multiple samples reaction assay, the category of equivocal res,ults included from a serologically positive individual were eliminated, a those sera with absorbances ranging from the F>ositive cutoff total of 304 of 10,387 (2.9%) of individuals tested were to 20% above the cutoff. verified as true positives for period 1; this percentage was

3 VOL. 29, 1991 IMPROVED ALGORITHM FOR HIV-1 ANTIBODY TESTING 2509 TABLE 1. Distribution of sera from people tested in periods 1 and 2 Period 1 Period 2 Category No. (%) of No. of true Prevalence No. (%) of No. of true Prevalence individuals positives (%) individuals positives (%) Risk factor Homosexual or bisexual 1,276 (12.3) (11.1) Sexual partner of HIV- 392 (3.8) (3.6) positive patient "AIDS" 20 (0.2) (0.03) Multiple sexual partners 1,664 (16) 0 0 1,380 (17.2) 0 0 Transfusion 1,978 (19) (4.6) Intravenous drug abuser 642 (6.2) (7) Reason for test Emigration 746 (7.2) 0 0 1,202 (15) 0 0 Anxiety 1,241 (12) 0 0 1,244 (15.5) 0 0 Other 2,428 (23.4) ,091 (26.1) Total 10,387 (100) 304 8,026 (100) 214 similar to that for period 2, during which 214 of 8,026 (2.7%) individuals were verified positives. The average number of times an HIV-positive individual was tested in period 1 was 1.03 and this went up to an average of 1.5 in period 2, largely as a result of the follow-up protocol introduced by the laboratory. EIAs. In the analysis of tests for period 1, a total of 453 of 10,387 (4.4%) serum samples reactive by EIA required verification by supplemental tests; of these 453 samples, 182 serum samples were IFA positive, 132 were WB positive, and 139 were WB negative. The 132 WB-positive serum samples were previously tested by IFA and all showed some degree of nonspecific fluorescence, and only 5 were weakly IFA positive. Only 70 of the 139 WB-negative serum samples were unequivocally IFA negative, with the remainder all testing nonspecific by this test. In period 2, a total of 415 of 8,026 (5.2%) serum samples reactive by EIA required verification by supplemental tests (Table 2). Of these 415 samples which were reactive by using both GSC and WEL kits, 321 were verified by IFA (n = 174), WB (n = 145), or RIPA (n = 2). Therefore, al! of the sera testing reactive by both kits were verified as true positives. In contrast, only one of the remaining 94 serum samples which gave a combination of results other than reactive by both EIA kits was verified as a true positive. This serum sample (1 of 3) gave one reactive result and one equivocal result with the EIA kits (Table 2). IFA was performed on 174 of the 321 serum samples giving a reactive result with both EIA kits and also on 24 of TABLE 2. Comparison of combinations of EIA reactive and equivocal results after verification by IFA, WB, or RIPA for period 2 No. verified as positive EIA results No. of serum by supplementary assay samples IFA WB RIPA Both reactive Reactive, equivocal 3 0 O 1 Reactive, nonreactive Both equivocal Equivocal, nonreactive Total the 94 serum samples giving various combinations of EIA results (Table 2). Of the 174 IFA-verified serum samples, all were unequivocally positive and exhibited strong fluorescence. Of these 174 samples, 100 were from individuals who had a second sample collected for the WB test when the first sample was reported as reactive by EIA and IFA as described in the algorithm for period 2. The WB patterns of all these serum samples were consistent with HIV infection. Of the subset tested, namely, the 24 serum samples with combinations of EIA results and WB patterns selected for testing by the IFA, only 1 was positive with some degree of nonspecificity in the cell control and originated from an early seroconverting patient. The remaining 23 serum samples all reacted nonspecifically in the uninfected cell control of the IFA and could not be adequately verified by this test. From these results, only serum samples reactive with both EIA kits could be unequivocally verified by the IFA, and those samples giving other combinations of EIA results were unlikely to be verified by this test. Of the 93 serum samples which were not verified as positive by the supplemental tests (Table 2), 31 were reactive by the GSC kit and 43 were reactive by the WEL kit. An additional 27 serum samples gave equivocal results, 12 for the GSC kit and 15 for the WEL kit. Sera from the intravenous drug user group were more likely to react nonspecifically by using the WEL kit, because the majority of these exhibited no bands by WB testing. No attempt was made to investigate the cause of this nonspecific reaction. Statistical evaluation. In period 1, the results of the GSC kit after verification tests yielded a specificity of 98.6% (9,934 of 10,073) and a positive predictive value of 69.3% (314 of 453) (Table 3). A sensitivity of 100% was assumed for the tests performed during this period. In period 2, the separate and combined performances of the GSC and WEL kits are shown in Table 3. A specificity of 99.4% for the GSC kit in period 2 was similar to that obtained for this kit in period 1, although the positive predictive value calculated was considerably higher in period 2 (69.3 versus 87.96%). In comparison, the WEL kit missed one truepositive sample, which gave an equivocal result with this kit but a reactive result with the GSC kit. Consequently, the sensitivity of the WEL kit relative to that of the GSC kit was determined to be 99.7% (313 of 314) (Table 3), although the positive predictive values for both the WEL and GSC kits were similar. To further assess the sensitivities of both kits

4 2510 FONSECA AND ANAND J. CLIN. MICROBIOL. TABLE 3. Specificity and positive predictive values for EIAS for periods 1 and 2 Period, test, and result True status of sample Total no. Specificity Positive predictive No. positive No. negative of samples (%) value (%) Period 1 GSC kit Positive (9,934/10,073) 69.3 (314/453) Negative 0 9,934 9,934 Total ,073 10,387 Period 2 GSC kit Positive (7,669/7,712) (314/357) Negative 0 7,669 7,669 Total 314 7,712 8,026 WEL kit Positive (7,654/7,712) 84.4 (313/371) Negative 1 7,654 7,655 Total 314 7,712 8,026 GSC and WEL kits in parallel Positive 313 (314) O (2) 313 (316) 100 (7,712/7,712)a 100 (313/313)- Negative 1 (0) 7,712 (7,710) 7,713 (7,710) Total 314 (314) 7,712 (7,712) 8,026 (8,026) [99.97 (7,710/7,712)]b [99-4 (314/316)]b GSC and WEL kits in parallelc Positive (7,611/7,712) 75.7 (314/415) Negative 0 7,611 7,611 Total 314 7,712 8,026 a In calculating the specificity and positive predictive values, these values are the numbers of serum samples in each category for which a positive result is defined as reactive by using both EIA kits. b In calculating the specificity and positive predictive values, the values in the brackets are the values calculated when a positive sample was defined either as a serum sample reactive by using both kits or reactive by using one kit and equivocal by using the other kit. C Positive is defined as reactive or equivocal by at least one EIA kit. and the GSC kit in particular, serum samples collected from six individuals known to have seroconverted were tested by WB and RIPA when appropriate. All of these serum samples testing WB or RIPA positive were also reactive by using the GSC kit. The combinations of results from the GSC and WEL kits in the algorithm shown for period 2 were considered by using two different definitions of a positive result, and the specificity and positive predictive values were calculated for each. With the first definition, a specificity of 100% and a positive predictive value of 100% were obtained if a serum sample was regarded as positive when both the EIA kits gave a reactive result (Table 3). The relative sensitivity of the combined tests was 99.7% (313 of 314). With the second definition, in which a serum sample was considered positive if the above combination was extended to include a positive result and an equivocal result, the specificity and positive predictive values declined marginally to and 99.4%, respectively (Table 3, values in brackets) but the relative sensitivity was 100% (314 of 314). However, as also shown in Table 3, if all combinations of the reactive and equivocal results by EIAs were considered, the positive predictive value declined to 75.7%, which is considerably lower than values obtained for either kit individually, although the specificity for both EIA kits in parallel was calculated to be 98.7%, which is similar to values calculated for each of the kits. Based on 95% confidence limits, the confidence intervals for the specificities and positive predictive values were calculated for the GSC and WEL kits alone and in combination for the algorithm in period 2. Upper limits of 99.6 and 99.4% were calculated for the respective specificities of the GSC and WEL kits alone, compared with a lower limit of 99.93% for the EIA kits in parallel, when a positive was defined as the combination of one equivocal result and one reactive result. Similarly, an upper limit of 91.33% was calculated for the positive predictive value of the GSC kit and an upper limit of 88.1% was calculated for the WEL kit, compared with a lower limit of 98.55% for the parallel kit combination when a positive result is defined as above. Thus, for either the specificity or positive predictive value, the lower limit of the EIA kits used in combination was greater than the upper limit of each EIA kit considered separately. The only exception was when a positive result was defined as any combination of results (Table 3); then an upper limit of 98.95% was derived for the specificity and 79.83% for the positive predictive value, which was below the lower limits of the kits considered either separately or in combination. DISCUSSION Guidelines issued by the CDC for the testing of sera for antibody to HIV-1 cite the use of EIA as a convenient screening test (7) and also stipulate the use of a supplementary test (IFA, WB, or RIPA) for final verification to exclude any EIA false positives. An algorithm using EIA kits which identifies a true-positive sample early in screening clearly has a considerable impact in reducing the number of supplemental tests required, which are laborious and expensive. From our study, the testing scheme in period 1 closely resembles the guidelines issued by the CDC, and our data

5 VOL. 29, 1991 IMPROVED ALGORITHM FOR HIV-1 ANTIBODY TESTING 2511 clearly illustrate that at least one-third of EIA-reactive sera were false positives when these samples were tested by supplemental procedures. In contrast, for period 2, our proposed scheme clearly showed that even the less-stringent combination of an equivocal and a reactive EIA result would yield a specificity and a positive predictive value close to 100% for a true-positive sample. The equivocal category was established by our laboratory to identify any samples from patients in the early stages of the infection for which the absorbance values might, in occasional cases, be below the positive threshold. The serum samples tested at our laboratory likely represent a spectrum of differing risk categories and disease prevalence similar to that encountered by most clinical diagnostic laboratories, and the breakdown into the various risk groups provided serves as a comparison. The value of combination testing for populations with various prevalences of disease has been hypothetically evaluated in studies (3), and the conclusions arrived at showed that in a parallel testing format, such as that described for period 2 of this study, the sensitivity of the combined tests was considerably higher than those obtained for each separate test but the specificity of the combination was lower than those attainable for each. This projection is clearly true if all the combinations of EIA reactive and equivocal results were to be considered in our study (Table 3). However, as the only two combinations of results (i.e., either both reactive or one reactive and one equivocal) were adopted, the evaluation of the data within these parameters was found to yield high specificity and positive predictive values for a population with heterogeneous risk factors. Statistical evaluation of the data would also appear to confirm this finding, in that for period 2, the EIA kits performed better in a parallel format than each kit considered separately for the same sample population. From our observations, it is apparent that a true-positive serum sample reacted in both EIA kits, whereas those samples reacting nonspecifically were reactive or equivocal with one kit and invariably nonreactive or equivocal with the other. Although these kits use two different reaction principles, i.e., the GSC kit is a noncompetitive assay and the WEL kit is a competitive assay, preliminary results with another kit, using a noncompetitive reaction principle, in place of the WEL kit have yielded similar results (unpublished observations). Thus, the parallel testing format of two different EIA kits is in itself highly discriminating for true-positive sera. A scheme employing dual non-eia-based kits in a hierarchical system has been reported for the screening of units of blood for antibody to HIV by using a latex card agglutination and a coated gelatin particle agglutination test (9). Approximately 4,000 specimens were tested in this format, using one test as a screen and a second test as a confirmatory test. In the final analysis, samples positive by both tests were subsequently verified as true positives, and apart from a very few true positives which gave combinations of reactive and equivocal results, the rest were all negative. Our procedure differs from the above by using a parallel format which allows a direct comparison of the results from both EIA kits, thereby reducing the additional time involved in performing the alternate test as described previously. Thus, the status of a reactive sample can be predicted very early and with great confidence in our testing scheme, which can have considerable advantages in selecting these sera for supplemental tests. A further finding was the value of the IFA as a verification procedure compared with the WB assay; of the two tests, the WB test is probably the most frequently performed. The WB test, although commercially available, is both technically exacting and expensive, and depending upon the criteria used to interpret the bands, sera giving indeterminate results require further verification by the RIPA. In period 1 of this study, of 453 serum samples tested by the IFA, only 56% could be unequivocally reported as either negative or positive and the remainder required further verification by WB. In period 2, however, 100 of the 174 IFA-positive serum samples had a WB performed on a paired second sample taken within a short period, and all of these were WB positive. All these sera were reactive by using both EIA kits. Sera giving any other combination of EIA results could not be verified by the IFA, apart from one sample which was taken at early seroconversion. From our results, we conclude that the IFA can be used to verify those sera reactive in both EIA tests as an alternate to the more costly WB kit. Sera giving a combination of results could be verified in the newer recombinant-antigen EIA kits which have been shown to have sensitivities and specificities similar to those of the WB test. In such a study, 2,212 serum samples reactive by using a number of commercial screening EIA kits were tested by a recombinant-protein EIA kit, and a sensitivity of 99.9%, a specificity of 99.7%, and positive and negative predictive values of 99.7% and 99.9%, respectively, were recorded; these results make this type of kit attractive for use as a confirmatory assay (6). An additional advantage of our parallel EIA kit testing scheme is that the cost of performing two EIA tests per serum sample was found to be very similar, if not identical, to that incurred by using one kit alone and testing each sample in duplicate wells. In our laboratory, the cost of reagents for testing each sample in duplicate wells as in period 1 was approximately $3.60, compared with identical costs per sample using a single well in each of the two EIA kits as in period 2. Our experience with using the GSC and WEL EIA kits and with substituting another microwell EIA kit for the WEL kit has shown that they can all be operated interchangeably on different washers and readers without any difficulty. In addition, because the operator has to return to the original sample for each of the two EIAs, this procedure also reduces the chances of an inadvertent sample mix-up going unnoticed. In summary, while the CDC guidelines for HIV-1 antibody testing specify that all EIA-reactive sera be verified by using a different type of test, our study clearly shows that using two EIA kits in parallel will presumptively identify a truepositive sample early in the testing algorithm, which could be of considerable benefit in some settings. On the basis of our data, a sample giving a reactive result in both kits can be regarded as a presumptive true positive. The occasional sample which gives a reactive result and a equivocal result can be verified by using the cheaper recombinant-antigenbased EIA kits in place of the WB test. Any other combinations of results are likely negatives. ACKNOWLEDGMENTS We acknowledge M. V. O'Shaughnessy and his staff at the Federal Centre for AIDS, Ottawa, Ontario, Canada, for performing RIPA on the sera sent for testing and for supplying helpful suggestions; M. Fauvel (Laboratoire de Santé Publique du Québec, Sainte- Anne-de-Bellevue, Canada) and Luc Montagnier (Institut Pasteur, Paris, France) for providing the MOLT-4-T4 cells; and Heather

6 2512 FONSECA AND ANAND Bryant (Department of Community Health Sciences, University of Calgary, Calgary, Canada) for giving valuable statistical advice. REFERENCES 1. Bland, M An introduction to medical statistics. Oxford Medical Publications, Oxford. 2. Burke, D. S., J. F. Brundage, R. R. Redfield, et al Measurement of the false positive rate in a screening program for human immunodeficiency virus infections. N. Engl. J. Med. 319: Galen, R. S Use of predictive value theory in clinical immunology, p In N. R. Rose, H. Friedman, and J. L. Fahey (ed.), Manual of clinical laboratory immunology, 3rd ed. American Society for Microbiology, Washington, D.C. 4. Gallo, D., J. L. Diggs, G. R. Shell, P. J. Dailey, M. N. Hoffman, and J. L. Riggs Comparison of detection of antibody to the acquired immune deficiency syndrome virus by enzyme immunoassay, immunofluorescence, and Western blot methods. J. Clin. Microbiol. 23: J. CLIN. MICROBIOL. 5. Ilstrup, D. M Statistical methods in microbiology. Clin. Microbiol. Rev. 3: Lepine, D. G., P. W. Neumann, S. L. Frenette, and M. V. O'Shaughnessy Evaluation of human immunodeficiency virus test algorithm utilizing a recombinant protein enzyme assay. J. Clin. Microbiol. 28: Morbidity and Mortality Weekly Report Update: serologic testing for antibody to human immunodeficiency virus. Morbid. Mortal. Weekly Rep. 36: , Morbidity and Mortality Weekly Report Interpretation and use of the Western Blot assay for serodiagnosis of human immunodeficiency virus type 1. Morbid. Mortal. Weekly Rep. 38(S-7): Spielberg, F., C. M. Kabeya, T. C. Quinn, R. W. Ryder, N. K. Kifuani, J. Harris, T. R. Bender, W. L. Heyward, M. R. Tam, and K. Auditore-Hargreaves Performance and cost-effectiveness of a dual rapid assay system for screening and confirmation of human immunodeficiency virus type 1 seropositivity. J. Clin. Microbiol. 28: Downloaded from on July 16, 2018 by guest