Predictive value of two commercial human immunodeficiency virus serological tests in cases with indeterminate Western blot results.

BACKGROUND AND PURPOSE: Serodiagnosis of human immunodeficiency virus (HIV) infection typically requires repeatedly reactive positive screening test followed by Western blot (WB) assay. When WB assay result is indeterminate, the results of follow-up WB assay may remain inconclusive. This study evaluated use of enzyme-linked immunosorbent assay (ELISA) and particle agglutination test (PAT) as sequential screening tests with WB assay for the diagnosis of HIV infection. METHODS: From January 1, 2000 to December 31, 2003, a total of 565 serum samples collected from individuals with a previous positive or borderline positive ELISA test for HIV at regular check-up (281 samples) and a second group of individuals with known risk of HIV exposure or suspected infection based on clinical presentation (284 samples) were tested for HIV infection by ELISA, PAT and WB assay. RESULTS: The result was positive for HIV infection and confirmed by WB assay in 197 samples (22.5%), indeterminate in 127 samples (22.5%) and negative in 241 samples (42.6%). The sensitivity and specificity of ELISA were 100% and 21.6% and of PAT were 99.5% and 95%, respectively. Among the 197 HIV-infected cases, all ELISA and PAT results were concordant with WB assay except 1 (1/197) with negative PAT. Positive ELISA, positive PAT and indeterminate WB assay results were found in 9 of the 284 samples (7 individuals) from at-risk patients. Among these 7 individuals, 5 were later proved to have HIV infection. WB assay in 1 of the 7 individuals remained indeterminate 1 year later, and the remaining case was lost to follow-up. CONCLUSION: We suggest initial ELISA followed by PAT as sequential screening for HIV infection. When both screening tests are concordant but subsequent WB assay is indeterminate, further evaluation (such as nucleic acid amplification test) should be arranged as soon as possible.     

Diagnostic detection of human immunodeficiency virus type 1 antibodies in urine: a brazilian study

We evaluated, for the first time in Latin America, the performance of a commercial enzyme immunoassay (EIA) (Calypte Biomedical Corporation, Berkeley, Calif.) that detects human immunodeficiency virus type 1 (HIV-1)-specific antibodies in urine in comparison to standard serological assays (two commercial EIAs and a commercial Western blot [WB] assay). Paired serum and urine specimens were collected from two different groups of Brazilian patients: 225 drug users with unknown HIV status who attended drug treatment centers in Rio de Janeiro, Brazil, and 135 subjects with known HIV status. Patients showing positive results in the serum EIAs and/or in the urine EIA were serologically confirmed by WB assay. For 135 individuals with known HIV status, the urine EIA showed 100% sensitivity (74 positive samples) and 95.1% specificity (58 of 61 negative specimens). For 225 drug users, the test showed 100% sensitivity (2 positive samples) and 98.7% specificity (220 of 223 negative samples) compared to WB-confirmed serological EIA results. Thus, in a total of 360 samples, the urine EIA correctly identified all 76 HIV-positive samples and 278 of 284 negative samples (100% sensitivity and 97.9% specificity). Detailed analysis of the urine EIA results indicates that an increase of the recommended cutoff value might raise the specificity of the assay without affecting its sensitivity. Our results suggest that the HIV-1 urine EIA is a good screening test suitable for developing countries like Brazil. However, as for all other HIV screening tests on the market, it is not specific enough to be used as a one-step test and therefore requires confirmation.     

Detection of antibody to immunodeficiency viruses by dot immunobinding assay.

The dot immunobinding assay (DIA), a modified enzyme immunoassay (EIA), has been demonstrated to be a highly sensitive and specific assay for the detection of antibody to a number of viruses. Different laboratory procedures are available for detecting antibody to the immunodeficiency viruses; however, these procedures require a certain amount of sophisticated equipment and trained personnel. Further, commercial kits for detecting antibody to human immunodeficiency virus, as now available, are not easy to use in the nonlaboratory setting. The DIA, as described herein, may be formatted to test up to 30 serum samples and is designed to be used in the absence of laboratory equipment. To determine the effectiveness of the DIA as a test kit for the detection of HIV and human T-cell leukemia virus type I (HTLV-I) antibodies, the kit was compared with commercial EIA and Western blot (WB; immunoblot) kits. Testing approximately 1,000 human serum samples for HIV antibody by DIA and EIA revealed a total agreement of 98.1%, a specificity of 99.0%, and a sensitivity of 95.9%. For 804 serum samples tested (200 were tested independently in two laboratories), eight results were discrepant: four DIA negatives which were EIA borderline positive and four DIA positives which were EIA negative. Testing the eight discrepant sera by immunofluorescence assay and WB resulted in their being either negative or indeterminate. The four DIA positives were indeterminate by WB. Close agreement was obtained when the remaining sera were compared by DIA, EIA, and WB. Of interest was finding that the DIA results compared favorably with those obtained by WB. Twenty-six suspect HTLV-I-positive serum samples tested by DIA also gave results comparable to those obtained by EIA and WB.     

Antigen-antibody reaction in solution in capture competition immunoassay for human immunodeficiency virus antibodies.

In the capture competition immunoassay, undiluted serum was reacted in solution with purified human immunodeficiency virus (HIV) antigen in wells of microtest plates coated with anti-HIV immunoglobulin G antibodies (HIV capture antibodies). HIV antibodies present in the serum being tested combined with the HIV antigen and thus blocked (completely or partially) the fixation of the antigen to the capture layer. Unblocked antigenic activity was measured in subsequent steps by the use of biotinylated anti-HIV immunoglobulin G and peroxidase-conjugated avidin. The assay was evaluated in comparison with indirect enzyme-linked immunosorbent assay and Western (immuno-) blot (WB). A total of 180 serum samples which reacted repeatedly as positive in indirect enzyme-linked immunosorbent assay but negative in WB were found to be negative by the capture competition assay. Of 54 serum samples showing dubious reactions (single p24 bands in WB), 53 were clearly separated into positive or negative reactions, whereas 1 serum sample gave a borderline reaction. It was concluded that a characteristic feature of this kind of inhibition assay is a very low frequency of equivocal results.     

Performance of two commercial immunochromatographic assays for rapid detection of antibodies specific to human immunodeficiency virus types 1 and 2 in serum and urine samples in a rural community-based research setting (Rakai, Uganda)

Rapid detection of human immunodeficiency virus (HIV) antibodies is of great importance in developing and developed countries to diagnose HIV infections quickly and at low cost. In this study, two new immunochromatographic rapid tests for the detection of HIV antibodies (Aware HIV-1/2 BSP and Aware HIV-1/2 U; Calypte Biomedical Corporation) were evaluated in rural Africa to determine the tests' performance and comparability to commercially available conventional enzyme immunoassay (EIA) and Western blot (WB) tests. This prospective study was conducted from March 2005 through May 2005 using serum and urine from respondents in the Rakai Community Cohort Survey. Nine hundred sixty-three serum samples were tested with the Aware blood rapid assay (Aware-BSP) and compared to two independent EIAs for HIV plus confirmatory Calypte WB for any positive EIAs. The sensitivity of Aware-BSP was 98.2%, and the specificity was 99.8%. Nine hundred forty-two urine samples were run using the Aware urine assay (Aware-U) and linked to blood sample results for analysis. The sensitivity of Aware-U was 88.7% and specificity was 99.9% compared to blood EIAs confirmed by WB analysis. These results support the adoption of the Aware-BSP rapid test as an alternative to EIA and WB assays for the diagnosis of HIV in resource-limited settings. However, the low sensitivity of the Aware-U assay with its potential for falsely negative HIV results makes the urine assay less satisfactory.     

Detection of antibodies to HIV-1 in serum and saliva

In this study we report the detection of antibodies to HIV-1 in paired serum and saliva collected from 118 HIV-1 infected patients and 80 normal controls in Madras, South India. Saliva was collected using Omnisal (R) collection device. All the reactive samples were confirmed by Western blot test (WB), while all the control serum and saliva were negative for HIV-1 antibodies. 107 (90.6%) HIV individual's serum and saliva contained antibodies to HIV-1. When these reactive samples were tested by WB test for confirmation the following results were obtained; 68% HIV individuals' paired serum and saliva were positive; while 9% of serum samples were positive and the saliva specimens were negative on WB. 3% of paired samples showed indeterminate Western blot pattern in contrast to 10% of serum showed full WB pattern while the saliva result was indeterminate. It is suggested that saliva testing may be appropriate for surveillance and epidemiological studies. However, if used for individual HIV diagnosis it is imperative to use a confirmatory test.     

Detection of antibodies to human immunodeficiency virus in vaginal secretions by immunoglobulin G antibody capture enzyme-linked immunosorbent assay: application to detection of seminal antibodies after sexual intercourse.

In order to evaluate a commercial immunoglobulin G (IgG) antibody capture enzyme-linked immunoassay (ELISA) (Wellcozyme HIV1 + 2 Gacelisa; Murex Diagnostics Limited, Dartford, United Kingdom) for the detection of antibodies to human immunodeficiency virus (HIV) in vaginal secretion samples (VS) from HIV-seropositive and -seronegative women, serum samples (S) and VS were obtained from 129 African women living in the Central African Republic, a country of high HIV prevalence. Sera were tested for HIV by routine second-generation ELISA with confirmatory Western blot (immunoblot) (WB). By the Gacelisa IgG immuno-capture assay, 45 VS were positive and 84 were negative, whereas by WB, 44 VS were confirmed positive and 85 were confirmed negative. Considering WB as a reference, the IgG immunocapture assay in VS was 97.7% sensitive (43 of 44 positive samples) and 97.6% specific (83 of 85 negative samples). Of 42 HIV-seropositive women, 41 (97.6%) had S and VS that both were HIV positive (S+ VS+), and of 87 HIV-seronegative women, 83 (95.4%) had S and VS that both were HIV negative (S- VS-). Five women had discordant results for S and VS. One (S+ VS-) possibly had a false-negative VS result. Two (S- VS+) had similar indeterminate patterns for S and VS in WB. Two (S- VS+) had a typical HIV-positive pattern on WB of VS, whereas S results in WB were indeterminate in one case and negative in the other case; for both women, detection of prostatic acid-phosphatase was positive in VS, strongly suggesting recent sexual intercourse with an HIV-positive man. Because all HIV-infected men have detectable IgG antibodies to HIV in the seminal fluid, an HIV-seronegative rape victim with HIV-positive VS (S- VS+) should receive short-term antiviral therapy to prevent possible HIV transmission.     

Treponema pallidum surface immunofluorescence assay for serologic diagnosis of syphilis

A surface immunofluorescence assay (SIFA) using live spirochetes was analyzed and compared with Western blot (WB), fluorescent treponemal antibody absorption (FTA-ABS), microhemagglutination (MHA-TP), and Treponema pallidum immobilization (TPI) assays for detecting serum antibodies to T. pallidum in patients with syphilis, in disease controls, and in healthy subjects. SIFA and WB were 99% sensitive (99 of 100 positive specimens) and specific (140 of 140 negative specimens); FTA-ABS showed a sensitivity and a specificity of 90 and 89% (90 of 100 positive and 125 of 140 negative specimens), respectively. MHA-TP showed a sensitivity of 84% (84 of 100 positive specimens) and a specificity of 98.5% (138 of 140 negative specimens). Finally, TPI had a sensitivity of 52% (52 of 100 positive specimens) and a specificity of 100% (140 of 140 negative specimens). The T. pallidum SIFA was therefore highly specific, showing no equivocal reactivities with control sera, and sensitive. The results suggest the possible use of SIFA as a confirmatory test in the serologic diagnosis of syphilis.     

Suitability of a Rapid Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus in Ghana, West Africa

In West African countries such as Ghana, efficient human immunodeficiency virus (HIV) testing is a priority in the fight against AIDS. A new immunochromatographic rapid test, Determine HIV-1/2 (Abbott Diagnostics, North Chicago, Ill.), that detects antibodies against HIV type 1 (HIV-1) and/or HIV-2 was evaluated using Ghanaian blood samples. Two hundred four serum and/or plasma specimens were tested. HIV screening was done by a particle agglutination test and confirmed by a Western blot (WB) test as the "gold standard." The results revealed 125 HIV-seropositive AIDS patients, 75 HIV-seronegative healthy individuals, and 4 individuals for whom the HIV-1 result was indeterminate. The results obtained by the Determine HIV-1/2 assay and Diagnostic HIV SPOT (Genelabs), which is currently widely used in many districts in Ghana, were compared with those of the WB test, excluding the four HIV-1-indeterminate samples. The sensitivity of the Determine HIV-1/2 assay was 100%, compared with 98.0% for the HIV SPOT assay. The specificity was 100% for both tests. Determine HIV-1/2 is a single-step assay and was found to be rapid and easy to perform without any special equipment. It was highly sensitive and specific. The kit can be applied without electricity and water supplies, making it suitable for the detection of HIV antibodies especially in the rural areas of Ghana, West Africa.     

A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections.

A simple, specific and economical dipstick immunobinding enzyme-linked immunosorbent assay (DIA) for detecting hepatitis B surface antigen (HBsAg) and antibodies to hepatitis delta virus (anti-HDV), utilizing cellulose nitrate membrane is described. Screening of 815 serum specimens for HBsAg by DIA and micro ELISA revealed a positivity of 22.69% and 22.94% respectively. In the detection of antibodies to delta antigen, DIA was compared with an indirect immunofluorescence technique using A3 cell line as antigen substrate and a commercial macro ELISA. Of the 143 HBsAg positive sera tested for anti-HDV, 59 (41.25%) were positive by both immunofluorescence and macro ELISA and 61 (42.65%) by DIA. While the positive and negative predictive values of DIA for HBsAg were 100% and 99.6%, for anti-HDV by DIA these were 96.7% and 100% respectively. Based on the simplicity of performance and the economical nature of the test system, DIA is recommended as a diagnostic tool for field surveys and small laboratories in developing countries.     

Multiplex analysis of heterophil antibodies in patients with indeterminate HIV immunoassay results

We hypothesized that heterophil antibodies reactive with animal proteins used in blot preparation caused nonspecific staining (NSS) on HIV Western blot (WB) studies, causing indeterminate results. We analyzed samples showing NSS on HIV WB using a multiplexed immunoassay to simultaneously measure IgG antibodies to animal IgG (bovine, goat, sheep, mouse) and bovine serum albumin. Heterophil antibodies reactive with IgG from several animal species were detected in 23 (49%) of 47 samples showing NSS on HIV WB; 15 positive samples demonstrated antibodies to all 5 antigens. Similar IgG heterophil antibodies were detected in only 2 (8%) of 24 control samples. Of the HIV WB samples with a positive HIV-1 enzyme-linked immunosorbent assay (ELISA) result at the time of WB testing (11/47), heterophil antibodies were found in 8 (73%) of 11. Preabsorption with bovine, goat, and sheep IgG removed heterophil antibodies detected by the multiplexed assay and, in some cases, eliminated reactivity in ELISA and WB testing. Heterophil antibodies are associated with indeterminate HIV immunoassay results and are an important cause of false-positive HIV ELISA results. Multiplexed immunoassays provide a powerful tool for screening patients for heterophil antibodies and resolving possible false-positive results.     

Serological survey of human immunodeficiency virus (HIV) in Ethiopia

The presence of anti-human immunodeficiency virus 1 antibodies was tested in 5,565 serum samples from Ethiopia of which 5,265 were collected from military recruits in the framework of a hepatitis B (HBV) seroepidemiological study performed on a national scale in 1985-1986; the remaining were 300 sera from a population of outpatients belonging to the Arsi region. Of the 5,565 sera, 121 (2.1%) were found to be repeatedly reactive by enzyme-linked immunosorbent assay (ELISA) test for HIV-1 antibodies, but these reactivities were confirmed by Western Blot (WB) assay in only four cases (0.07%) and by ENVACOR (confirmatory competitive ELISA) in three samples. Twenty-three sera were positive by WB to one or two bands related to core proteins but were all negative by ENVACOR. However, according to accepted criteria for positivity, these sera must be regarded as indeterminant reactors. A sample of 409 sera, both reactive and nonreactive by HIV-1 ELISA, were further tested for antibodies to HIV-2 by ELISA. Reactive sera were analysed by WB and by radioimmunoprecipitation assay (RIPA) using 35S-cysteine metabolically labelled SIVmac (HTLV-IV) infected cell lysates. Only 11 sera were found to be slightly reactive in ELISA, but this was not confirmed by WB or RIPA. Data indicate that HIV infection was not widespread in the general population of Ethiopia up to 1986.     

A simple semi-rapid HIV-1&2 confirmatory immunoassay using magnetic particles

The Bionor HIV-1&2 Confirmatory Test is a semi-rapid simple immunoassay based on magnetic particles for the confirmation of serological status to human immunodeficiency virus (HIV). The specificity and sensitivity of this assay was evaluated by comparison with the Diagnostic Biotechnology HIV-1 Western blot (WB) 2.2 and the HIV-2/SBL-6669 WB. Bionor's confirmatory test demonstrated 98% specificity when testing sero-negative blood donors and false positive sera in screening tests compared to 81.5 and 71.6%, respectively, using the HIV-1 WB. The sensitivity of this assay for HIV-1 antibody positive sera was 97.9% compared to the WB which was 99.5%. When testing confirmed HIV-2 antibody positive samples, 2/100 scored negative using this confirmatory test similar to other HIV-2 peptide-based line immunoassays available commercially, whilst 8/100 were indeterminate reacting to HIV-2 membrane antigens only. Bionor's confirmatory test detected HIV-1 seropositivity earlier than the WB in longitudinal seroconversion panels and could discriminate between HIV-1 and -2 infection. The number of indeterminate responses was generally reduced significantly using Bionor's confirmatory test compared to the HIV-1 WB. The greater specificity, speed and ease of interpretation of Bionor's confirmatory test renders it an attractive and cost effective alternative to the WB for confirming HIV serological status worldwide.     

Comparative studies of three serologic methods for the measurement of Mycoplasma pneumoniae antibodies

Antibodies against Mycoplasma pneumoniae in the sera of patients and normal adult controls were measured by a standard complement fixation (CF) test, a commercial immunofluorescence (IF) test (CROWNTITRE), and a commercial enzyme-linked immunosorbent assay (ELISA) (MYCOPLASMELISA). The findings showed that, in the control sera, 269 of 277 (97%) had negative results for CF antibodies. Of the 320 controls tested by the IF assay, all (100%) had negative results for IgM antibodies and 314 (98%) had negative results for IgG antibodies. Only 6 of the 201 (3%) controls by the ELISA were classified as negative/equivocal. Among the 450 patient sera, 105 (23%) had positive results for CF antibodies, and 158 (35%) had positive results for IgG and/or IgM membrane antibodies by the IF test; 424 of these patients' sera were also tested by the ELISA, and 397 (94%) of them were found to have positive results for anti-M. pneumoniae IgG antibodies. If the CF test were chosen as the standard for comparison, the IF test would have a sensitivity of 87% and a specificity of 81% and the ELISA would have a sensitivity of 71% and a specificity of 80%, provided an adjustment in the threshold ELISA-positive value was made. A single positive M. pneumoniae membrane IgM antibody titer appeared to be valuable for a presumptive diagnosis of an ongoing infection; 41 of 47 (87%) of the IgM-positive results in the paired sera were supported by a fourfold increase or a stable high level of CF antibody titer.     

Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.     

Evaluation of supplemental testing with the Multispot HIV-1/HIV-2 Rapid Test and APTIMA HIV-1 RNA Qualitative Assay to resolve specimens with indeterminate or negative HIV-1 Western blots

BackgroundThe use of Western blot (WB) as a supplemental test after reactive sensitive initial assays can lead to inconclusive or misclassified HIV test results, delaying diagnosis.ObjectiveTo determine the proportion of specimens reactive by immunoassay (IA) but indeterminate or negative by WB that could be resolved by alternative supplemental tests recommended under a new HIV diagnostic testing algorithm.Study designRemnant HIV diagnostic specimens that were reactive on 3rd generation HIV-1/2 IA and either negative or indeterminate by HIV-1 WB from 11 health departments were tested with the Bio-Rad Multispot HIV-1/HIV-2 Rapid Test (Multispot) and the Gen-Probe APTIMA HIV-1 RNA Qualitative Assay (APTIMA).ResultsAccording to the new testing algorithm, 512 (89.8%) specimens were HIV-negative, 55 (9.6%) were HIV-1 positive (including 19 [3.3%] that were acute HIV-1 and 9 [1.6%] that were positive for HIV-1 by Multispot but APTIMA-negative), 2 (0.4%) were HIV-2 positive, and 1 (0.2%) was HIV-positive, type undifferentiated. 47 (21.4%) of the 220 WB-indeterminate and 8 (2.3%) of the 350 WB-negative specimens were HIV-1 positive.ConclusionApplying the new HIV diagnostic algorithm retrospectively to WB-negative and indeterminate specimens, the HIV infection status could be established for nearly all of the specimens. IA-reactive HIV-infected persons with WB-negative results had been previously misclassified as uninfected, and HIV diagnosis was delayed for those with WB-indeterminate specimens. These findings underscore the limitations of the WB to confirm HIV infection after reactive results from contemporary 3rd or 4th generation IAs that can detect HIV antibodies several weeks sooner than the WB.     

HTLV-I carriers in ATL/L-nonendemic area--with reference to blood transfusions

Sera from 5,705 persons were tested for antibodies to human T-cell leukemia virus type 1 (HTLV-I) by gelatin particle agglutination test (PA) and enzyme immunoassay (EIA) as screening methods, and the antibodies were confirmed by indirect immunofluorescence assay (IF) and Western blotting method (WB). The antibodies were detected in 30 (1.34%) of 2,239 patients who had received blood transfusions, 3 (0.29%) of 1,022 healthy blood donors, all 18 adult T-cell leukemia/lymphoma (ATL/L) patients, 23 (47%) of 49 family members of the ATL/L patients and 10 (0.53%) of the other 1,898 persons. Before the introduction of the mass screening of healthy blood donors by the Japan Red Cross Blood Center, 17 (2.29%) of 743 recipients tested were positive for the antibodies by PA and most of them were confirmed by IF and WB. Ten of these 17 recipients tested were negative for the antibodies before the blood transfusions. After the introduction of mass screening, 13 (0.87%) of 1,496 recipients tested were positive for the antibodies by PA, but the seroconversion of the antibodies by blood transfusions could not be confirmed clearly. In persons with low titers (1:16-1:64) in PA, the coincident rate of positive reaction with EIA, IF and WB was only 9.5%, 0.0% and 23.8%, respectively, and most of the immunoglobulin (Ig) class was IgM. On the other hand, in those with titers of 1:128 or greater in PA, the coincident rate was 72.7%, 81.8% and 90.9%, respectively, and the Ig class was IgG or IgG + IgM.     

Serological detection of Helicobacter pylori by a flow microsphere immunofluorescence assay.

A flow cytometric immunofluorescence assay (FMIA) for the detection of immunoglobulin G antibodies to Helicobacter pylori was developed. A multicomponent antigen was prepared and used to coat carboxylated polystyrene microspheres for reaction with patient sera followed by fluorescein isothiocyanate-labelled goat anti-human immunoglobulin G. The reacted microspheres were collected with a flow cytometer, and fluorescence was quantitated relative to the cutoff value provided by pooled sera from patients in whom H. pylori could not be demonstrated by culture or histology. Serum samples from 28 H. pylori-positive patients and 27 H. pylori-negative patients were tested by FMIA. Additionally, an in-house enzyme-linked immunosorbent assay (ELISA) employing the same antigen preparation and a commercially available ELISA were used to assay the patient population. Both the FMIA and in-house ELISA were 100% sensitive and 89% specific with positive and negative predictive values of 90 and 100% and no equivocal results. The commercial ELISA was 96% sensitive and 89% specific with positive and negative predictive values of 90 and 96% and five equivocal results. FMIA provides a rapid, inexpensive, and easily performed means for serodiagnosis of H. pylori.     

Immunofluorescence assay for detection of antibodies to human immunodeficiency virus type 2.

A total of 215 serum samples were tested for antibodies against human immunodeficiency virus type 2 (HIV-2) with an immunofluorescence assay (IFA). Some samples originated from Denmark and some originated from Guinea-Bissau. The IFA results were compared with enzyme-linked immunosorbent assay (ELISA) and Western (immuno-) blot (WB) results. Twenty-nine serum samples were found to be true positive for HIV-2 antibodies as judged from WB and radioimmunoprecipitation results; all of these were also found to be positive in the HIV-2 IFA. Of 80 serum samples originating from HIV-1-infected persons, 60% showed reactivity in the HIV-2 ELISA, and 51% cross-reacted with at least one band in the HIV-2 WB. None of the sera cross-reacted in the HIV-2 IFA. A total of five serum samples (three African and two Danish) gave unspecific results in the HIV-2 IFA. It is concluded that the HIV-2 IFA is more specific and at least as sensitive as a first-generation ELISA and that IFA is superior to WB in discriminating between HIV-1 and HIV-2 infections.     

Recombinant antigen-based enzyme immunoassay for screening of Treponema pallidum antibodies in blood bank routine.

This work reports a comparison of an enzyme immunoassay (EIA) using two major Treponema pallidum recombinant antigens with a T. pallidum hemagglutination (TPHA) assay and a nontreponemal Venereal Disease Reference Laboratory (VDRL) test. A total of 1,822 normal donor serum samples was tested for cardiolipin and T. pallidum antibodies, respectively, by the VDRL assay and EIA. Among these samples, 440 were further tested by TPHA technology. Four samples were found positive by EIA, while all were reported to be negative by both TPHA and VDRL routine assays. Subsequent testing of EIA-positive samples confirmed 100% (four of four samples) and 25% (one of four samples) positive results, respectively, by immunofluorescence assay and a Western blot (immunoblot) syphilis kit. The sensitivity of the recombinant EIA was estimated at virtually 100% with a reference panel of 50 syphilitic samples. According to this study, the newly developed EIA kit shows 100% sensitivity combined to a specificity greater than 99.8% for detecting treponemal immunoglobulin G antibodies in blood bank syphilis screening.