Confirming Human T-Cell Lymphotropic Virus Type I

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1994, p Vol. 32, No /94/$ Copyright ) 1994, American Society for Microbiology Evaluation of a p2le-spiked Western Blot (Immunoblot) in Confirming Human T-Cell Lymphotropic Virus Type I or II Infection in Volunteer Blood Donors STEVEN H. KLEINMAN, 2* JONATHAN E. KAPLAN,3 RIMA F. KHABBAZ,3 MICHAEL A. CALABRO,4t RUTH THOMSON,5 MICHAEL BUSCH,"7 AND THE RETROVIRUS EPIDEMIOLOGY DONOR STUDY GROUP8 Southern California Region American Red Cross Blood Services, Los Angeles, California 90006'; University of California at Los Angeles, Los Angeles, California ; Retrovirlus Diseases Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia ; SRA Technologies, Inc.,4 and Westat, Inc.,s Rockville, Maryland 20850; Irwin Memorial Blood Centers, San Francisco, California ; University of California at San Francisco, San Francisco, California ; and National Heart, Lung, anid Blood Institute, Bethesda, Maryland Received 26 July 1993/Returned for modification 23 September 1993/Accepted 29 November 1993 Current algorithms for the serologic confirmation of human T-cell lymphotropic virus type I or II (HTLV-I/II) antibody reactivity are complicated. We evaluated the performance of an HTLV-I Western blot (immunoblot) spiked with recombinant p2le protein (p2le WB) as an alternative to current confirmatory methods. These methods include the HTLV-I viral lysate Western blot and either a radioimmunoprecipitation assay or a p2le enzyme-linked immunosorbent assay. Five hundred fifty nine blood donations obtained from five U.S. blood centers and classified as HTLV-I/II seropositive (n = 149) or seroindeterminate (n = 410) by routine testing methods were further evaluated by PCR for proviral DNA and by the p2le WB. On the basis of serologic and PCR testing, 155 donations were classified as HTLV-I/II infected. The sensitivity of the p2le WB was 97.4%, slightly exceeding that of routine confirmatory testing. The specificity of the p2le WB was 97.5%, as determined by testing of 404 seroindeterminate samples that were negative in the PCR. The positive predictive value of the p2le WB was 94%. In contrast, the specificity and positive predictive value of routine confirmatory testing were both 100%. Follow-up sampling of presumptive p2le WB false-positive donors substantiated the absence of HTLV-I/II infection. Although the p2le WB used in this study has high sensitivity and may be useful as a confirmatory assay in epidemiologic research studies, it may not be ideal as a confirmatory test for the notification of blood donors. Human T-cell lymphotropic virus type I (HTLV-I), the causative agent of adult T-cell leukemia or lymphoma and of HTLV-I-associated myelopathy or tropical spastic paraparesis (2), can be transmitted by blood transfusions (12, 13, 16). In November 1988, the Food and Drug Administration issued recommendations to screen all voluntary blood donations in the United States for antibodies to HTLV-I by use of licensed enzyme-linked immunosorbent assays (ELISAs) (14). These tests, which use HTLV-I whole virus lysate as an antigen, can also detect antibodies to closely related human T-cell lymphotropic virus type II (HTLV-II). HTLV-I antibody screening assays produce a significant number of false-positive results and therefore require supplementary tests, such as a Western blot (immunoblot) and a radioimmunoprecipitation assay (RIPA), for confirmation (14). On the basis of an evaluation of a panel of known HTLV-I- and HTLV-II-infected samples, the U.S. Public Health Service recommended that criteria for HTLV-I/II seropositivity include reactivity to gag p24 and env gp46 or gp61/68 in the Western blot and/or the RIPA (14). Antibody to gp46 is directed against the HTLV-I/II envelope * Corresponding author. Mailing address: UCLA Medical Center, LeConte Ave., Room A4-239 CHS, Los Angeles, CA t Present address: Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD. 603 surface glycoprotein, whereas antibody to gp6l/68 is directed against the parent precursor glycoprotein (14). Since HTLV-I Western blots are relatively insensitive for the detection of env reactivity, additional testing with RIPAs is frequently required (6). However, there are significant limitations inherent in RIPA methodology; the test requires the use of radioisotopes, is time-consuming, and can be performed only in the few laboratories that have the capability of growing radiolabelled HTLV-I in tissue culture. In the last few years, many developments have occurred in the field of HTLV-I/II serodiagnostics. Among the most significant has been the use of a highly immunogenic HTLV-I recombinant env p2le antigen which is capable of detecting antibody to the transmembrane portion of the envelope glycoprotein. Such reactivity is usually not detectable in standard Western blots or RIPAs. The p2le antigen has been used in an ELISA format and has also been added to (or spiked onto) HTLV-I whole virus lysate Western blots (7, 11). The use of the p2le antigen has markedly improved the detection of env reactivity and has resulted in a nearly 100% sensitivity for the diagnosis of HTLV-I/II infection, thereby greatly reducing the need for the RIPA (7, 11). However, false-positive p2le reactivity has also been reported (7, 9). The present study was undertaken to evaluate the performance of a p2le-spiked HTLV-I Western blot (p2le WB) in confirming HTLV-I antibody ELISA screening test reactivity for volunteer blood donors. The diagnosis of HTLV-I/II

2 604 KLEINMAN ET AL. infection was made by a combination of routine serology, additional serologic testing for env reactivity, and PCR analysis of peripheral blood mononuclear cells (PBMCs) for HTLV- I/II proviral DNA. (This work was presented in part at the American Association of Blood Banks Annual Meeting in 1992.) MATERLALS AND METHODS J. CLIN. MICROBIOL. Routine blood donor testing. The study population consisted of all blood donors at five regional blood centers in the United States (American Red Cross Blood Services of Greater Chesapeake/Potomac [Baltimore/Washington, D.C.], Southern California [Los Angeles], and Southeastern Michigan [Detroit]; the Irwin Memorial Blood Centers [San Francisco]; and Oklahoma Blood Institute [Oklahoma City]) from October 1990 through December These five blood centers are participants in the National Heart, Lung, and Blood Institute multicenter program entitled the Retrovirus Epidemiology Donor Study (REDS). All blood donations were screened at the respective blood centers for antibody to HTLV-I by an ELISA (Abbott Laboratories, North Chicago, Ill.). Repeatedly reactive samples underwent routine confirmatory testing. For samples donated to the two non-red Cross blood centers, confirmatory testing consisted of an HTLV-I whole virus lysate Western blot; if necessary, a RIPA was performed by standard methods (10). Criteria for seropositivity included p24 and gp46 or gp61/68 reactivity in the Western blot or the RIPA. The three American Red Cross centers used a modified confirmatory testing algorithm consisting of a standard Western blot and a p2le ELISA (Cambridge/Biotech, Worcester, Mass.) performed according to the manufacturer's instructions (7). Samples were classified as HTLV-I/II seropositive when both p24 and gp46 could be identified by the Western blot or when p24 was identified by the Western blot and the p21e ELISA was positive, with a serum/cutoff optical density ratio of greater than 6.0. When, however, gag reactivity was present in the Western blot and the serum/cutoff optical density ratio in the p2le ELISA was between 0.8 and 6.0, the sample was tested further by a RIPA (Abbott Laboratories). This modified confirmatory testing algorithm has been shown to have sensitivity and specificity equal to or superior to those of the standard testing algorithm of a Western blot and a RIPA (5). At all five blood centers, samples that showed no bands on Western blots were classified as confirmatory test negative. Samples with reactivity to HTLV gag antigens (p19 and/or p24) but without env reactivity were classified as seroindeterminate. PCR testing. A 30-ml aliquot obtained from the packed erythrocyte unit corresponding to each repeatedly reactive donation (with the exception of most autologous donations) was shipped to the REDS Central Laboratory (SRA Technologies, Rockville, Md.) within 3 to 7 days of collection. At SRA, PBMCs were isolated within 24 h by Ficoll-Hypaque separation and frozen at - 70 C. Aliquots from these samples were subsequently thawed and tested by PCR for the presence of HTLV-I/II provirus. PCR testing was performed without knowledge of serologic test results. Each PCR was performed on a lysate from 3 x 1(5 PBMCs. Two gene regions were amplified. A TAX/REX region common to HTLV-I/II was amplified for 40 cycles, and products were detected by hybridization to a 32P-labelled oligonucleotide probe, electrophoresis, and autoradiography. HTLV-I was distinguished from HTLV-II by amplification of a POL region and then solution hybridization with HTLV-I- and HTLV-II-specific 32P-labelled probes, electrophoresis, and autoradiography (8). All samples were run in duplicate with one negative control for each sample and with appropriate positive controls. For selected samples, special PCR procedures were used. High-volume PCR was performed with DNA extracted from 9 x 105 PBMCs. Subsequent PCR analysis was performed according to standard procedures. The hot-start PCR modification involves the sequestration of Taq DNA polymerase from other components of the reaction by an impermeable wax layer (3) (Ampliwax; Perkin-Elmer). Since the wax melts at approximately 65 C, nonspecific priming at lower temperatures is avoided. p2le WB. For p21e WB evaluation, 559 samples from HTLV-I/I1-seroindeterminate and -seropositive donors were tested at SRA by the p2l e WB (Cambridge/Biotech). Serologic testing at SRA was performed without knowledge of test results at the blood centers. Seroindeterminate samples that subsequently showed p2le reactivity in the p2le WB were further tested by RIPA, p2le ELISA, and a modified HTLV- I/II Western blot (designated WB 2.3) which incorporates HTLV-I whole virus lysate, p2le, and the specific HTLV-I and HTLV-II recombinant proteins MTA-1 and K-55 (Diagnostics Biotechnology, Singapore, Singapore) (15). Seroindeterminate donors with p21e reactivity in the p21e WB were recontacted by blood center personnel and, after informed consent was obtained, a blood sample was collected for repeat serologic and PCR testing. Samples for PCR testing were shipped to SRA on the day of blood sample collection by overnight carrier; at SRA, PBMCs were harvested within 24 h by centrifugation in Ficoll, frozen with vapor-phase liquid nitrogen, and subsequently thawed for PCR testing. The p2le WB was performed according to the package insert supplied with the kit. The test specimen was serum or occasionally plasma (when serum was not available). Each test run contained three controls: a negative control, an HTLV-Ipositive control, and an HTLV-II-positive control. Runs were considered valid only when the negative control lacked virusspecific bands, the HTLV-I-positive control showed bands at p21e, p19, p24, p28, p38, and gp46, and the HTLV-II-positive control showed bands at p2le and p24. Band strengths were scored as 1+, 2+, or 3+. A 3+ band was equal to or stronger than the corresponding band of the HTLV-I-positive control; 1 + and 2+ bands were weaker and were graded subjectively. A test was considered positive when both p2le and p24 bands were present, each at an intensity of 1 + or greater. RESULTS p2le WB results. Of 1,195,267 blood donations, 939 (0.079%) were repeatedly reactive for HTLV-1/II in ELISA screening. Routine confirmatory test results were available for 915 samples; 199 (22%) were HTLV-1/II routine confirmatory test seropositive, 517 (56%) were routine confirmatory test seroindeterminate, and 199 (22%) were negative. One hundred forty-nine (75%) of the routine confirmatory test seropositive samples and 410 (79%) of the routine confirmatory test seroindeterminate samples were tested further by PCR and the p2le WB. The results are presented in Table 1. One hundred fifty-nine (80%) of the confirmatory test negative samples were tested by PCR; all were found negative. These samples were not tested by the p21e WB. As can be seen from Table 1, samples from 21 routine confirmatory test seroindeterminate donors showed p2le reactivity in the p2le WB; 14 of these also showed p24 reactivity, while 7 did not. These 21 samples were subjected to additional serologic testing, as reported in Table 2. Table 2 can be used to

3 VOL. 32, 1994 TABLE 1. WB reactivities for samples classified as HTLV-I/II seropositive and HTLV-I/II seroindeterminate in routine confirmatory testing in the REDS from October 1990 to December 1991 Routine confirmatory test results" p2le WESTERN BLOT FOR HTLV-I/II INFECTION 605 A B C D E F G H I J K L p2le WB results Seropositive Seroindeterminate (bands) (n = 199) (n = 517) PCR+ PCR- PCR+ PCR- (n = 146) (n = 3) (n = 6) (n = 404) p2le, p24, p19? b 10b p2le, p19, no p " p2le, no p24, no p " lb p24 or p19, no p2le No p19, no p24, no p2le " PCR+, PCR positive; PCR -, PCR negative. b Detailed results are presented in Table 2. compare the serologic results (RIPA and p2le ELISA) for the 15 donors who tested PCR negative (subjects 1 to 15) with those for the 6 donors who tested PCR positive (subjects 16 to 21). Additional testing with WB 2.3 classified 2 of the 6 PCR-positive donors as HTLV-II infected and 1 of the 15 PCR-negative donors as HTLV-I infected. With a freshly obtained sample from this latter donor, PCRs performed by routine, high-volume, and hot-start techniques were all negative and failed to confirm the positive serologic results. Follow-up data were obtained for 10 of the 15 p2le-reactive, PCR-negative donors (subjects 1 to 10 in Table 2) at a median interval of 12 months (range, 1 to 18 months). By using follow-up samples processed into PBMCs within 36 h of collection, we found that all 10 donors still tested PCR negative. RIPA and p2le ELISA results duplicated the findings for the original samples. In contrast, p2le reactivity in the TABLE 2. repeat p2le WB was variable; whereas all 10 donors had shown p2le reactivity upon initial testing, only 4 showed such reacp28 p24 pig - M m p2le FIG. 1. p2le WB strips for study subjects. Lanes A, B, C, D, E, F, G, and H contained initial and follow-up specimens from four HTLV- I/II-seroindeterminate, PCR-negative subjects. Lane I contained a specimen from an HTLV-I/II-seroindeterminate, PCR-positive subject. Lane J contained an HTLV-I-positive control. Lane K contained an HTLV-II-positive control. Lane L contained a negative control. tivity in their follow-up samples. In addition, p19 and p24 reactivities also varied between initial and follow-up samples. To assess whether the loss of p2le reactivity was due to biologic changes in donors or to changes in the performance of different lots of test reagent, we repeated the testing of paired Serologic and PCR results for HTLV-I/II-seroindeterminate donor samples with p2le reactivity in the p2le WB Routine p2le WB result Result in: Donor blot result p2le p19 p24 Interpretation" RIPA p2le ELISA PCR 1 p POS p POS p POS p POS p POS p IND p IND pl9, p IND pl9, p IND p IND p POS p POS p POS p POS pl9, p POS p IND - + HTLV-II 17 p IND - + HTLV-II 18 p POS + + HTLV-II 19 p POS + + HTLV-II 20 p19, p POS + + HTLV-I 21 p19, p POS + + HTLV-II " POS, positive; IND, indeterminate.

4 606 KLEINMAN ET AL. initial and follow-up samples in a single test run. Each of the 10 pairs of donor samples showed identical strengths of p2le reactivity in both initial and follow-up tests. Figure 1 illustrates this result by showing representative paired samples from four donors (lanes A to H). Figure 1 also illustrates the relatively weaker p2le band intensity for PCR-negative samples (lanes A to H) than for a seroindeterminate, PCR-positive sample (lane I). Testing of an additional panel of 20 selected PCR-positive samples showed that 95% had p2le bands of 3+ intensity; in contrast, only 20% of the p2le-reactive, seroindeterminate, PCR-negative samples showed bands of this intensity. To further assess the significance of p2le reactivity in the 15 PCR-negative donors, we compared their age, sex, and racial characteristics with those of HTLV-I/II routine confirmatory test seropositive donors and all other HTLV-I/II routine confirmatory test seroindeterminate donors. We found that 27% of the 15 PCR-negative donors were nonwhite or Hispanic; in comparison, 69% of the routine confirmatory test seropositive donors belonged to this category. This difference was statistically significant (P = 0.002; Fisher's exact text). In contrast, there was no racial difference between the 15 PCRnegative donors and all other routine confirmatory test seroindeterminate donors. Age and sex comparisons showed no statistical differences. In summary, our PCR data established that 6 of the 21 routine confirmatory test seroindeterminate donors with p2le reactivity in the p2le WB were infected with HTLV-I/II; this result was further corroborated by the presence of env reactivity for each of these donors in at least one additional assay. In contrast, the data suggest that all, or nearly all, of the 15 routine confirmatory test seroindeterminate, PCR-negative donors with p2le reactivity in the p2le WB were not infected with HTLV-I/II. Performance characteristics. The sensitivity, specificity, and positive predictive value of the p2le WB as a confirmatory test were established and compared with those of routine confirmatory testing methods. Seropositivity in the p2le WB is defined as the presence of reactivity to both p2le and p24. For these calculations, we assumed that the three HTLV-I/II routine confirmatory test seropositive, PCR-negative donors were indeed HTLV infected, despite our inability to detect HTLV proviral DNA. On the basis of these criteria, the p2le WB detected 151 of 155 HTLV-infected donors (sensitivity, 97.4%); in contrast, HTLV routine confirmatory testing methods classified 149 of these 155 HTLV-infected samples as seropositive (sensitivity, 96.1%). The p2le WB identified 10 false-positive donors. Therefore, the specificity of the test was 97.5% (394 of 404) for PCRnegative, routine confirmatory test seroindeterminate donors. This calculation does not include p2le WB results for the 199 donors whose samples were repeatedly reactive in the ELISA and who had no bands detected in the HTLV-I whole virus lysate Western blot. We believe it is reasonable to assume that, if tested, none of these samples would have been classified as seropositive by the p2le WB, since all lacked p24 reactivity in the standard Western blot. Adding the results expected for these samples to our calculation, we estimated the specificity of the p2le WB for donors whose samples were repeatedly reactive in the HTLV-I antibody ELISA to be 98.3% (593 of 603). In comparison, the specificity determined for routine confirmatory testing methods under our assumptions was 100%. We also calculated the predictive value for a positive test result if the p2le WB were the sole method of confirmatory testing used. We performed this calculation by using two J. CLIN. MICROBIOL. methods: (i) analyzing the data from p2le WB-tested samples and (ii) performing an extrapolation from these results to the entire HTLV-I antibody ELISA-repeatedly reactive population in our study, thereby accounting for the fact that some routine confirmatory test seropositive and routine confirmatory test seroindeterminate donor samples were not tested by the p2le WB. The p2le WB identified 151 true-positive and 10 false-positive samples. When our direct testing data were used, the positive predictive value was 93.8% (151 of 161); when our adjusted calculation was used (data not shown), the positive predictive value was virtually unchanged and was calculated to be 94%. In contrast, the positive predictive value of routine confirmatory testing methods was 100%. DISCUSSION Our study has demonstrated that the Cambridge/Biotech p2le WB, when used as a confirmatory test, has excellent sensitivity, slightly exceeding that of routine confirmatory methods. However, the test appears to detect a small population of donors who are uninfected with HTLV-I/II. We have based this conclusion upon results obtained from PCR testing of optimally collected follow-up samples combined with results obtained from alternative serologic methods used for env detection. We have shown that this optimal PCR technique has a 99% sensitivity for the detection of HTLV-I/II infection in a similar blood donor population (1). Using these data, we calculated the positive predictive value of the Cambridge/Biotech p2le WB in our volunteer blood donor population to be approximately 94%. Therefore, 1 of 20 blood donors classified as seropositive by this test would be inappropriately notified that they are infected with HTLV-I/II. This test may therefore not be ideal for a confirmatory test used in blood donor notification. However, for epidemiologic research studies in which misclassification of an occasional sample would not affect study conclusions, it may be acceptable to confirm HTLV-I/II infection by use of this simple assay. Samples with reactivity to p19 and env but not to p24 are uncommon in HTLV-I/II confirmatory testing but have been shown to be associated with HTLV-I/II infection and with the transmission of infection by blood transfusion (4). In this study, two routine confirmatory test seropositive samples (one of which was PCR negative) demonstrated p19 and p2le but not p24 reactivity in the p2le WB. However, four PCR-negative, routine confirmatory test seroindeterminate samples also demonstrated the same pattern. Therefore, our data indicate that persons demonstrating p19 and p2le reactivity but not p24 reactivity in the p2le WB need to undergo additional testing to determine their HTLV-I/II infection status. We have shown that the p2le band intensity of false-positive samples tends to be weaker than that detected for PCRpositive, HTLV-I/II-infected donors. We have also found that the presence of p2le reactivity in the p2le WB does not correlate with p2le ELISA reactivity. This lack of correlation could be due to slight differences in the amino acid sequences of the two recombinant p2le antigens (9), to different stereochemical presentations of the proteins in the two test systems, or to nonspecificity introduced by steps in the Western blot assay. The observation that p2le reactivity appears to be more specific in the ELISA suggests that there may be ways to improve the specificity of p2le reactivity in the Western blot format. ACKNOWLEDGMENTS This work was supported by contract NO 1 HB from the National Heart, Lung, and Blood Institute.

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