False-positive human immunodeficiency virus type 1 western blot tests in noninfected blood donors

Background: The manufacturers' criteria for a positive human immunodeficiency virus type 1 (HIV-1) Western blot (WB) test were recently revised to require reactivity to only two of the following bands: p24, gp41, and gp120/160. In a recent report, low-risk blood donors were identified in whom nonspecific reactivity to multiple env antigens in WB testing resulted in apparently false-positive WBs by these criteria. The present study was conducted to verify the existence of false-positive WBs among noninfected donors and to assess the extent of this problem. Study Design and Methods: Four donors classified as WB- positive on the basis of env-only (3 cases) or p24/env-only (1 case) patterns were investigated. Index and/or follow-up specimens were tested by polymerase chain reaction (PCR), by overlapping recombinant env antigens and synthetic peptides in enzyme immunoassays, and by deglycosylated and denatured antigen WBs. WB records from American Red Cross blood centers were reviewed to determine the frequency of env- only and p24/env-only patterns, relative to all positive WBs, from 1988 through 1993. Results: The four index-case donors denied risk and had stable WB reactivity during follow-up. HIV PCR was negative in all. Env reactivity was restricted to nonglycosylated gp41 epitopes; no gp120- specific reactivity was detected. For three of the four donors, env reactivity was mapped to a 20-amino acid N-terminal epitope of gp41. The rate of detecting WBs with these false-positive patterns increased from 0.6 percent of all positive WBs from 1988 to 1990 (4/776) to 8 percent in 1991 and 1992 (52/683), and then it declined to 6 percent in 1992 and 1993 (47/783). Env-only patterns predominated in 1991 and 1992, whereas p24/env-only patterns were more frequent following implementation of combined anti-HIV-1/HIV type 2 enzyme immunoassays in 1992. Conclusion: Low-risk blood donors can have false-positive results on WB tests. Increased detection of env-only and p24/env-only WBs appears related to the enhanced sensitivity of newer enzyme immunoassays to gp41 and p24 antibodies. Donors with these patterns should undergo follow-up testing to document the presence or absence of HIV infection.     

Contribution of polymerase chain reaction and radioimmunoprecipitation assay in the confirmation of human T-lymphotropic virus infection in French blood donors. Retrovirus Study Group of the French Society of Blood Transfusion

BACKGROUND: To verify the criteria for human T-lymphotropic virus (HTLV) seropositivity in Western blot (WB) proposed by the Retrovirus Study Group of the French Society of Blood Transfusion, 186 blood donations that were repeatedly reactive in HTLV enzyme-linked immunosorbent assay, selected according to their WB pattern, were tested by polymerase chain reaction (PCR) and radioimmunoprecipitation assay (RIPA). STUDY DESIGN AND METHODS: In two commercially available WBs, 12 samples were confirmed as positive (rgp21+p19+p24) and 174 were interpreted as indeterminate (one or two reactivities to these proteins). The primer pairs used for the PCR allowed the amplification of type I (HTLV-I) or type II (HTLV-II) (or both) sequences. The RIPA was performed with two 35S-labeled cell lines: HTLV-I infected HUT 102/B2 and HTLV-II-infected MoT. RESULTS: Of the 12 positive samples, 11 were classified as HTLV-I-positive and one as HTLV-II-positive. Among the 174 indeterminate samples, three (WB pattern: rgp21+, p19+, p24-) were HTLV-I positive in PCR (one of them was positive in RIPA also); the other 171 were HTLV negative. CONCLUSION: In the study of a population in which 97 percent of HTLV infections are due to HTLV-I, these data support the three-protein criteria (rgp21, p19, and p24) for a positive blot reading. No HTLV infection was observed when rgp21 did not react. Consequently, p19 and/or p24 band patterns represent false reactivity and do not require PCR or RIPA confirmation. To discriminate between false- and true-positive results in the absence of MTA-1 or K55 reactivity, PCR and/or RIPA is required only when rgp21 reactivity is associated with one gag band (p19 or p24).     

Transmission of human T-lymphotropic virus type I by blood components from a donor lacking anti-p24: a case report. The Transfusion Safety Study Group

The present criteria for confirmation of human T-lymphotrophic virus types I and II (HTLV-I/II) infection in blood donors are based on seroreactivity to p24 (gag) and gp46 and/or gp61 (env) on Western blot (WB) and radioimmunoprecipitation assays (WB/RIPA). Any single band and other combinations are classified as indeterminate. This case report documents infection in a donor with a repeatedly indeterminate pattern. The blood donor was anti-HTLV-I/II positive on enzyme-linked immunoassay, and two sera taken 5 years apart were WB/RIPA-indeterminate (p19 and gp68 only). His donations in the interval were associated with transmission of HTLV-I to four of the six recipients available for study. Other recipients of blood from donors whose WB/RIPA results were indeterminate by present criteria should be examined to determine if additional patterns are at least occasionally associated with transmission. The likelihood that such donors are infected is important to those who are counseling them and making decisions concerning recipients of their bloody.     

Humoral response in rabbits immunized with calcium phosphate adjuvanted HIV-1 gp160 antigen

Rabbits were immunized with either calcium phosphate adjuvanted purified HIV-1 gp160 or a fluid preparation. Circulating antibodies were detected by ELISA, RIPA and Western Blot tests. Sera of rabbits immunized with the adjuvanted preparation contained high levels of anti-gp160 antibodies, as well as antibodies recognizing p55, p25 and p18. On the contrary, rabbits immunized with the fluid preparation contained only anti-p18 antibodies. Neutralizing antibodies were also detected. It is concluded that the calcium phosphate adjuvant could be used for preparation of candidate anti-HIV vaccines, since it permits one to induce high levels of circulating antibodies, in the absence of untoward reactions as observed when aluminium adjuvants or water in oil emulsions are used.     

HIV (HTLV III/LAV) serology: experiences based on more than 42,000 tests

1,032 sera of diverse origin (AIDS risk groups, prostitutes, inpatients, blood donors) exhibiting a positive HIV (HTLV III/LAV) ELISA result (Organon and/or Abbott) were investigated with different HIV confirmation assays (Western blot, WB; immunofluorescence, IF; competitive enzyme immunoassay against cloned gp41- and p24-antigen). Sera were finally evaluated as positive if at least two confirmation assays turned out positive (IF and WB; IF and p24, gp41; WB and p24, gp41; IF and WB and p24, gp41). Test results were considered false if the respective finding (IF or WB or p24, gp41) differed from two other confirmatory assays. 1,001 out of 1,032 sera (97%) yielded corresponding results in IF and WB. The remaining 3% of the investigated sera showed false positive, false negative, non-interpretable IF results and non-interpretable WB findings. The present study demonstrates that a positive HIV test should be confirmed by at least two confirmatory tests, one of which should be the WB. Non-corresponding results in confirmatory tests necessitate a third test system.     

Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and HSV type 2-specific IgG antibodies

MRL Diagnostics has developed a dual enzyme immunoassay (EIA) system that employs the recombinant Herpes Simplex Virus (HSV) type-specific glycoproteins G1 (HSV1) and G2 (HSV2) to detect HSV type-specific IgG antibodies. This system was evaluated using 155 consecutive sera previously tested in a conventional dual EIA system (Zeus) that employs multiple HSV1 and HSV2 proteins to detect type-common as well as type-specific antibodies. Sera were also analyzed by Western blot to determine the true HSV type-specific IgG reactivity pattern. Of 110 sera giving concordant reactivity patterns in the MRL and Zeus EIA systems, 108 (98%) also displayed concordant Western blot patterns; two sera gave false positive HSV2 reactivity in both EIA systems. Of 45 sera giving discordant MRL and Zeus EIA reactivity patterns, 41 (91%) displayed a Western blot reactivity pattern that matched the MRL reactivity pattern. Both the HSV1 IgG component and the HSV2 IgG component of the MRL EIA system were 100% sensitive and > 95% specific. In contrast, the Zeus HSV1 IgG EIA was 98% sensitive and 79% specific, and the Zeus HSV2 IgG EIA was 85% sensitive and 79% specific. An analysis of the distribution of index values in the MRL EIA system showed that low-positive values (1.0-3.0) were rare, but, when detected, often represented false positive results; only 11 MRL low-positive results were observed, but all 6 MRL false positive results were found within this low-positive subgroup. These findings show that the MRL dual EIA system effectively detects HSV type-specific IgG antibodies.     

Detection of antibodies to Trypanosoma cruzi among blood donors in the southwestern and western United States. II. Evaluation of a supplemental enzyme immunoassay and radioimmunoprecipitation assay for confirmation of seroreactivity

BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is endemic to Latin America and may be transmitted in the United States via blood donated by infected immigrants. Blood- borne pathogens such as T. cruzi require supplemental testing for confirmation of seroreactivity. STUDY DESIGN AND METHODS: A study was undertaken to determine an optimal scheme for confirmation of seroreactivity in repeatedly reactive samples identified by the Chagas antibody enzyme immunoassay (EIA). The procedure for initial confirmation involves three purified antigens coated onto three separate polystyrene beads and uses an EIA format. If the sample is reactive with two of three or three of three antigens, it is confirmed as seroreactive. If none or one of three beads is reactive, the sample is indeterminate and subjected to a radioimmunoprecipitation assay (RIPA). The RIPA must demonstrate characteristic bands at 32, 34, and 90 kDa. RESULTS: When tested with sera from persons with potentially cross-reactive diseases (n = 39) or against a presumed negative population from southeast Wisconsin (n = 289), the confirmatory EIA had a specificity of 100 percent. Sensitivity was 100 percent (28/28) with xenodiagnosis-positive sera and 97.6 percent (80/82) with chagasic sera from Latin America. The RIPA showed a specificity of 100 percent in EIA- nonreactive samples (n = 100) and a sensitivity of 100 percent with both xenodiagnosis-positive (28/28) and chagasic (82/82) sera. CONCLUSION: The confirmatory EIA and the RIPA together provide a highly specific and sensitive means of confirming seroreactivity for antibodies to T. cruzi.     

Evaluation of a commercially developed indirect immunofluorescence system for the detection of antihuman immunodeficiency virus antibodies

A commercially developed IFA system (Electro Nucleonics, Inc.) was compared to EIA (Abbott Laboratories) and WB (Bio-Rad, Inc.) for detection of HIV-1 antibodies. Of 91 EIA negative sera tested, 76 (83.5%) were negative, and 15 (16.5%) were equivocal by IFA on initial testing. Of 156 EIA positive sera tested, 18 were equivocal by either IFA or WB and could not be directly compared. Of 138 EIA positive sera directly comparable, IFA and WB agreed 137 times (99.3% correlation). Based on these findings, the commercially developed IFA system appears to be most useful as a supplementary test, similar to WB, rather than as a screening test.     

Reliable confirmation and quantitation of human immunodeficiency virus type 1 antibody using a recombinant-antigen immunoblot assay

The recombinant DNA-derived, human immunodeficiency virus (HIV) antigen-based immunoblot assay (RIBA-HIV216) is a new supplemental (confirmatory) test developed to detect antibodies to HIV-1. The assay employs four recombinant viral antigens, corresponding to HIV-1 p24, p31, p41 and gp120 proteins, in an immunoblot format. With this assay, HIV-1 antigen reactivity was detected in all 683 infected patient serum or plasma specimens evaluated; 665 (97.6%) of these sera met the criteria for a positive interpretation, and 18 (2.6%) were classified as indeterminate. All 683 samples reacted with the recombinant gp41-equivalent protein. The first sequential enzyme immunoassay (EIA)-reactive samples collected from 33 seroconverting homosexual men reacted on RIBA-HIV216. Eleven (1.1%) of 999 EIA-negative blood donor sera reacted weakly with a single recombinant antigen (p24 or p31), whereas 13 to 48 percent had indeterminate reactions on viral lysate Western blots. One (1.5%) of 66 EIA-positive, Western blot-negative blood donor samples and 19 (29%) of 66 EIA-positive, Western blot-indeterminate blood donor samples scored indeterminate on RIBA-HIV216. Nonspecific reactivity was seen with only 1 (0.8%) of 114 patient sera containing possible interfering antibodies, whereas 33 percent of these samples had indeterminate reactions on Western blot and 35 percent had equivocal reactions on immunofluorescence assay (IFA). We conclude that the RIBA-HIV216 is approximately as sensitive as and significantly more specific than virus-derived Western blot and IFA. The RIBA-HIV216 also allows for semiquantitation of specific antibodies that may be of value in clinical staging and therapeutic monitoring.     

Comparison of enzyme-linked immunosorbent and indirect immunofluorescence assays for the detection of human T-cell lymphotropic virus type-I antibodies in sera from rural Haiti.

Serum samples were obtained from 340 healthy individuals without evidence of neurologic disease living in rural Haiti. Sera were screened for antibodies to human T-cell lymphotropic virus type I (HTLV-I) using two commercially available enzyme immunoassays (EIA) and by an indirect immunofluorescence assay (IFA) using a mixture of uninfected H9 cells and HTLV-I-infected MT-2 cells. Repeatedly positive samples were confirmed by Western blot (WB). Results with the two EIA systems were concordant and detected 13 positive samples, each of which was confirmed by WB. Only 9 (69%) of 13 WB-positive sera were detected by IFA, and four additional samples, positive by IFA, could not be confirmed by WB. The prevalence of HTLV-I seropositivity in this selected rural Haitian population was 3.8% (13 of 340).     

Frequency of human immunodeficiency virus (HIV) infection among contemporary anti-HIV-1 and anti-HIV-1/2 supplemental test- indeterminate blood donors. The Retrovirus Epidemiology Donor Study

Background: Follow-up studies from the mid-1980s showed that 1 to 5 percent of blood donors testing reactive in anti-human immunodeficiency virus type 1 (HIV-1) enzyme immunoassay (EIA) and testing indeterminate in Western blot were infected with HIV-1 and were in the process of seroconverting. The present study was conducted to establish the rate of HIV infection among contemporary anti-HIV-1/HIV type 2 (HIV-2) EIA- reactive, Western blot-indeterminate donors. Study Design and Methods: Donations (n = 607) with indeterminate HIV supplemental test results were identified by screening 3,021,342 donations given from November 1990 through August 1993 at five participating blood centers. Consenting donors were enrolled and samples taken 4 to 8 weeks after donation. Follow-up sera were tested by EIA and Western blot for anti- HIV-1 seroconversion and by type-specific peptide assays for antibodies to HIV-2 and HIV-1 subtype O. Peripheral blood mononuclear cells and/or plasma from the follow-up samples were tested for HIV-1 DNA and/or RNA by polymerase chain reaction. The rate of HIV-1 infection among Western blot-indeterminate donors was also estimated by multiplying the incidence rate of HIV-1 seroconversion in this donor population by the estimated duration of the EIA-reactive and Western blot-indeterminate window during seroconversion (8.5 days). Results: Supplemental test- indeterminate donors (n = 355) enrolled a median of 38 days after donation; 265 (75%) of these donors were identified as indeterminate after an anti-HIV-1/2 EIA-reactive donation. Enrolled and non-enrolled donors had similar distributions of demographic characteristics and band patterns. Follow-up samples from all 355 donors tested negative for HIV-1 in polymerase chain reaction. Follow-up sera tested Western blot-negative in 54 cases (15%) and Western blot-indeterminate in 299 (84%). Two follow-up sera (0.6%) were interpreted, according to manufacturer's package insert criteria, as Western blot positive with p24 and gp41 bands and/or gp120/160 bands; however, paired testing of index and follow-up sera from these two cases showed identical Western blot and EIA reactivity, and polymerase chain reaction was negative for HIV RNA and DNA, which ruled out HIV infection. The absence of HIV infection in 355 Western blot-indeterminate donors was consistent with our incidence-based model analysis, which yielded an estimate of one HIV-1 infection for every 215 Western blot-indeterminate donations (95% CI, 1/39-1/8333). Conclusion: Contemporary blood donors classified as indeterminate in supplemental HIV testing are infrequently infected with HIV. Donors whose follow-up samples test negative in anti-HIV-1/2 EIAs and negative or persistently indeterminate in Western blots should be considered eligible for reinstatement.     

Immunoglobulin (Ig) M antibody against myelin associated glycoprotein (MAG): A comparison of methods

The presence of immunoglobulin (Ig)M antibody against myelin associated glycoprotein (MAG) has been associated with autoimmune demyelinating, sensorimotor neuropathies. Approximately 50% of patients with IgM paraproteinemia and associated peripheral neuropathy possess antibodies against MAG. These autoantibodies are thought to interfere with the process of myelination, myelin maintenance, or axon-Schwann cell interaction. The detection of these autoantibodies is useful to the clinician and is suggestive of active demyelination in a peripheral neuropathy. Our objective in this study was to compare the results obtained using three different methods (dual enzyme immunoassay [EIA], immunofluorescent antibody [IFA] and Western blot [WB]) for detecting IgM antibody against MAG in patients suspected of having autoimmune demyelinating neuropathies. Since the dual EIA utilized two different antigens, results from this assay were separated into two groups: MAG and sulfate-3-glucuronyl paragloboside (SGPG). When compared to WB (gold standard), percent agreement, sensitivity, and specificity for EIA and IFA are as follows: MAG EIA (68.3, 100.0, and 60.6); SGPG EIA (95.1, 100.0, and 93.9); and myelin IFA (97.6, 100.0, and 97.0). The authors conclude that the SGPG EIA and myelin IFA compared well with the standard WB method when detecting IgM antibody against MAG (100 kD). Many sera demonstrated reactivity on the MAG EIA that were negative by WB (100 kD glycoprotein). The authors recommend screening for MAG IgM in suspected patient sera by SGPG EIA or myelin IFA and utilizing these same methods to titer sera confirmed positive by WB.     

Serologic markers for staging human immunodeficiency virus infections

In the present study, 80 serum specimens were tested for human immunodeficiency virus (HIV) antibody by enzyme immunoassay (EIA) and indirect fluorescent antibody tests, HIV (p24) antigen, and Western Blot analyses. A total of 40 specimens were HIV antigen-positive, 35 were antigen-negative and five were indeterminant. Among the 40 antigen-positive sera, 38 had positive antibodies by EIA with confirmation by Western Blot. Two cases were antigen-positive and were thought to be early stages where antibodies had not yet developed. Among the 38 sera, 30 (79%) had decreased or had no reactions by indirect fluorescent antibody tests. Among the 35 antigen-negative cases, all 35 had positive antibodies by EIA and all 35 had bands at gp41 by Western Blot. Among 84 HIV-infected patients, 30 had >400 CD4+ cells per cubic millimeter, 21 patients had 200–400 CD4+ cells and 33 had <200 CD4+ cells. A total of 28 (93%) of the HIV antigen-negative cases with full banding patterns by Western Blot had >400 CD4+ cells. In contrast 18 (55%) of the patients with antigen positivity and incomplete banding on Western Blot had <200 CD4+ cells.     

V3-specific neutralizing antibodies in sera from HIV-1 gp160-immunized volunteers block virus fusion and act synergistically with human monoclonal antibody to the conformation-dependent CD4 binding site of gp120. NIH-NIAID AIDS Vaccine Clinical Trials Network.

Sera from 11 volunteers immunized with a recombinant HIV-1 gp160-expressing vaccinia virus (HIVAC-1e; Oncogen/Bristol-Myers Squibb, Seattle, WA) and boosted with baculovirus-derived rgp160 (VaxSyn; MicroGeneSys, Inc., Meriden, CT) were evaluated for functional serum antibodies and their epitopes. Sera obtained prior to boosting had undetectable HIV-1-specific IgG and neutralizing activity, and did not block HIV-1 from binding or fusing to CD4+ MT-2 cells. 14 d after boosting, sera from each volunteer contained HIV-1-specific IgG titers of 1:40 to 1:1,280. Five of these sera also contained neutralizing antibodies, where most or all neutralizing activity was blocked by a synthetic peptide corresponding to amino acids 307-330 of the V3 loop of gp120, indicating that neutralizing antibodies were mostly V3 loop-specific. All sera obtained after boosting contained HIV-1 binding/fusion-inhibition antibodies, and a significant portion of their activity was blocked by the V3 loop peptide, a result consistent with the presence of antibodies against the region of the V3 loop that participates in fusion. Three sera with V3 loop-specific neutralizing and fusion-inhibition antibodies were studied further. In competitive antibody binding experiments, antibodies reactive with the conformation-dependent, CD4 binding site of gp120 were undetectable in each serum. When evaluated in combination with a monoclonal antibody to the CD4 binding site of gp120, two sera demonstrated synergism in neutralizing assays, and all three sera demonstrated synergism in binding/fusion-inhibition assays, further indicating that the functional antibodies were primarily V3 loop-specific. The synergism also suggests that a vaccine that elicits strong serum antibody responses to both regions of gp120 may improve the potential for inducing protective immunity.     

Causes of false-positive reactions in herpesvirus IgM assays

Antibody class capture assays (ACCA) with detergent-solubilized, peroxidaselabelled membrane antigens of infected and uninfected cells and indirect enzyme immunoassays (EIA) were compared for the detection of IgM antibodies to cytomegalovirus (CMV), herpes simplex virus (HSV) and varicella-zoster virus (VZV). Ninety sera showing a positive reaction against CMV and 105 against HSV and/or VZV on initial testing by indirect EIA were included. Sixteen sera were negative, and 62, 28 and 53 sera were positive for CMV, HSV and VZV, respectively, as determined by both assays. By ACCA, three additional sera contained IgM antibodies to CMV and one to HSV. All twelve sera with a positive result only by indirect EIA contained rheumatoid factor. Owing to the presence of auto antibodies, inconclusive results were obtained from twenty sera. To avoid false-positives, adequate controls are essential. Lack of interference from rheumatoid factor and a higher sensitivity make ACCA a useful test for the detection of IgM antibodies to herpesviruses.     

Effect of 10 minutes heat treatment on HIV antibody testing from alternate testing sites

One hundred and thirty-five sera were tested for the presence of antibody to human immunodeficiency virus (HIV) by enzyme immunoassay (EIA) with the Abbott Diagnostics HTLV-III test before and after heating at 56 degrees C for 10 min. Only one serum specimen was repeatedly reactive after heating when compared with the unheated portion (1/135). The specimen was a low level positive (ratio of test over cutoff = 1.6). Fifty-eight different sera, selected to yield a high percentage of positive results, had both their heated and unheated Western blot (WB) results agree completely with repeated EIA testing. An additional 4,244 sera heated at 56 degrees C for 10 min were tested. Of the 330 repeatable EIA positives, only 38 were not confirmed by WB. HIV infectivity in sera was reduced from 10(3.5) TCID50 to 10(1) TCID50 by the same heat treatment. We conclude that heating sera at 56 degrees C for 10 min significantly reduces HIV infectivity and does not significantly affect the results of HIV antibody testing.     

Comparative evaluation of an immunofluorescent antibody test, enzyme immunoassay and western blot for the detection of HIV-1 antibody

Screening blood and blood products for human immunodeficiency virus type 1 (HIV-1) antibody is predominantly performed by enzyme immunoassay (EIA), and results must be confirmed by the more immunospecific Western blot (WB) assay. This study evaluated an HIV immunofluorescent antibody (IFA) test relative to WB assay for use in confirming EIA designated HIV-1 antibody-positive sera. Specimens from seroconversion and CDC panels as well as clinical specimens obtained for routine EIA HIV-1 antibody screening were evaluated. Results with 209 specimens indicated that sensitivity and specificity of the Fluorognost-HIV assay were equivalent relative to WB. In addition, the Fluorognost-HIV IFA test was faster and easier to perform than the WB assay, and unlike the WB assay was not prone to indeterminate results.     

Longitudinal follow-up of blood donors found to be reactive for antibody to human immunodeficiency virus (anti-HIV) by enzyme-linked immunoassay (EIA+) but negative by western blot (WB-)

Abstract: A cohort of 467 volunteer blood donors who were found to be EIA+/WB- was studied longitudinally for up to two years. EIA screening for anti-HIV and WB testing, regardless of the EIA result, was performed on all 769 subsequent donation events of this cohort to ascertain the consistency of test results over time. The following results were obtained: 1) 8.8% of subsequent donation events were EIA+; 2) Most donors who returned were found to be EIA-/WB-; 3) EIA-/WB? (indeterminate) was 14.5 times more common than EIA+/WB?; 4) EIA and WB results were generally inconsistent from donation to donation; 5) No donor was found to be WB+. These results suggest that, in a volunteer donor population, an EIA+/WB- result may have little value in predicting anti-HIV test results and AIDS infectivity in a future donation. The current practice of not using blood donated subsequently by EIA+/WB- donors unless a re-entry testing scheme is satisfactorily completed should be reconsidered.