Evaluation of Oral Fluid Enzyme Immunoassay for Confirmation of a Positive Rapid Human Immunodeficiency Virus Test Result

The CDC recommends that a reactive rapid human immunodeficiency virus (HIV) test be confirmed with an approved supplemental test; the performance of an intermediate enzyme immunoassay (EIA) is optional. In support of this recommendation, it was found that of 1,431 reactive rapid HIV test results, 2 (0.1%) had false-negative oral fluid Western blot results and both had false-negative EIA results.Until 2002 in the United States, all tests for human immunodeficiency virus (HIV) infection were conducted in laboratories and the results were reported within several days to 2 weeks. Beginning in 2003, rapid HIV tests waived under the Clinical Laboratory Improvement Amendments of 1988 became available. The rapid tests can be performed with finger-stick whole-blood or oral fluid specimens in nonclinical settings, and the results (negative or preliminary positive) are available within an hour. Negative rapid test results may be reported without further testing. However, preliminary positive rapid test results must be confirmed in a laboratory with a supplemental test (e.g., a Western blot [WB] test) (2, 3). WB results from serum specimens are more accurate than WB results from oral fluid specimens, but because phlebotomy is not always feasible in nonclinical settings, some HIV testing programs use oral fluid for WB confirmation of rapid tests (4) (OraSure HIV type 1 [HIV-1] WB kit package insert; OraSure, Inc., Bethlehem, PA). Before 2007, the CDC recommended that a laboratory-based enzyme immunoassay (EIA) and a WB test be performed when oral fluid specimens were submitted for confirmation of positive rapid tests. In 2007, the CDC changed this recommendation because of the impending withdrawal of the only Food and Drug Administration (FDA)-approved oral fluid EIA and because postmarketing surveillance data identified several instances in which the oral fluid EIA was negative in persons whose rapid tests and oral fluid or serum WB were positive (1, 2). To evaluate the additional diagnostic usefulness of an oral fluid EIA to confirm preliminary positive rapid test results, the CDC examined the EIA and WB results for oral fluid specimens from persons confirmed to be HIV infected by serum WB.Study participants were persons with known HIV infection who had not taken antiretroviral treatment during the past 3 months. During the period from March 2006 to August 2007, 1,436 participants, all confirmed to be HIV infected by serum WB, were enrolled at clinics in six cities (Atlanta, GA; Baltimore, MD; Chicago, IL; Denver, CO; Louisville, KY; and Philadelphia, PA). Participants provided finger-stick whole-blood and oral fluid specimens, which were tested by the OraQuick Advance Rapid HIV-1/2 antibody test (OraSure, Inc., Bethlehem, PA) at the study site. Additional oral fluid specimens were collected using an OraSure HIV-1 oral specimen collection device and tested with the Vironostika HIV-1 Microelisa system EIA (bioMérieux, Marcy-l''Etoile, France) and the OraSure HIV-1 WB. The oral fluid EIA was not conducted for 429 specimens because of the unavailability of test kits due to a manufacturer shortage. Serum specimens were collected using standard BD Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ) and tested with the Genetic Systems HIV-1/HIV-2 Plus O EIA and HIV-1 WB (Bio-Rad, Redmond, WA).Specimens from 5 of the 1,436 participants were excluded from analysis: two specimens were suspected of having an error in identification labeling, and three oral fluid specimens had insufficient volume for confirmatory testing. All whole-blood (n = 1,431) and oral fluid (n = 1,429) specimens tested positive using the OraQuick rapid test. All serum specimens (n = 1,431) tested positive by serum WB. Of the 1,431 oral fluid specimens tested by oral fluid WB, 1,423 (99.4%) were positive, six (0.4%) were indeterminate, and two (0.1%) were negative. Oral fluid EIAs were performed on 994 of the 1,423 specimens that had positive oral fluid WB results, and all 994 oral fluid EIAs were reactive. Of the six oral fluid specimens with indeterminate WB results, five of six (83.3%) were positive by oral fluid EIA. Of the two oral fluid WB-negative specimens, neither was positive by oral fluid EIA.Data from this study indicate that oral fluid WB tests were positive in 99.4% of specimens from persons who were known to be HIV infected. However, specimens from approximately 0.1% of HIV-infected persons had false-negative oral fluid WB results, and both of these specimens also had false-negative oral fluid EIA results. Specimens from six (0.4%) HIV-infected persons had indeterminate oral fluid WB results, and one of these specimens had false-negative results for oral fluid EIA. Current guidelines require additional confirmatory testing using a blood specimen for any persons with a positive rapid test and a negative or indeterminate oral fluid WB (3). For these persons, even if there is a positive oral fluid EIA result, they will still need a follow-up WB.Postmarketing surveillance conducted in 2003 to monitor the performance of the OraQuick test indicated that some HIV-infected persons with preliminary positive rapid test results had false-negative oral fluid EIA results (2). In addition, the only FDA-approved oral fluid EIA was withdrawn from the market (1). The results of this study support current CDC recommendations that all preliminary positive rapid test results be confirmed with an additional approved supplemental test for HIV, such as WB or an immunofluorescence assay, and that performing an intermediate EIA is optional (2, 3). The CDC further recommends that if WB confirmatory testing of an oral fluid specimen produces a negative or an indeterminate result, confirmatory testing should be repeated with a blood specimen because of its greater sensitivity (2, 3).     

New Automated Chemiluminescence Immunoassay for Simultaneous but Separate Detection of Human Immunodeficiency Virus Antigens and Antibodies

The recently launched Liaison XL Murex HIV Ab/Ag assay (DiaSorin S.p.A) uses chemiluminescence immunoassay technology for the combined qualitative determination of p24 antigen of HIV-1 and specific antibodies to both HIV-1 and HIV-2. We studied 571 serum samples from those submitted to our laboratory for HIV screening. The samples were divided into 3 subsets: subset A, 365 samples collected prospectively during 1 week; subset B, 158 samples from confirmed HIV-positive patients; and subset C, 48 samples with a positive screening result but a negative or indeterminate confirmatory test result. Our standard screening/confirmatory algorithm was used as a reference. In subset A (prospective), 5 samples were positive and 360 negative by the standard procedure. Liaison XL Murex HIV Ab/Ag correctly identified all 5 positive samples (100%) and 357 negative samples (99.2%). In subset B (confirmed positive), all 158 positive samples were in total agreement in both procedures. In subset C (screen positive only), Liaison XL Murex HIV Ab/Ag yielded accurate results in 42 out of 48 samples (87.5%). Global sensitivity and specificity for Liaison XL Murex HIV Ab/Ag (all subsets included) were 98.3% and 98.5%, respectively. Considering only nonselected prospective samples and confirmed positive samples (subsets A and B), the corresponding sensitivity and specificity values were 100% and 99.2%, respectively. The new fully automated HIV screening test showed high sensitivity and specificity compared to our standard algorithm. Its added advantage of being able to detect HIV-1 and HIV-2 antibodies and p24 antigen separately could prove useful in the diagnosis of early infections.     

Comparison of a Chemiluminescent Immunoassay with Two Microparticle Enzyme Immunoassays for Detection of Hepatitis B Virus Surface Antigen

A comparative evaluation of the following commercial immunoassays for the detection of hepatitis B virus surface antigen (HBsAg) was performed: the Abbott AxSYM, Abbott IMx, and DPC IMMULITE assays. The specificity was 100% for all assays. Twelve samples were identified and were confirmed to be positive for HBsAg by all three methods. One additional sample was identified as reactive and was confirmed to be positive by the Abbott AxSYM assay only. Prior to confirmation testing the DPC IMMULITE assay produced significantly fewer false-positive results than the Abbott AxSYM assay (P < 0.05).     

Detection of Antibodies to Human Immunodeficiency Virus Type 1 in Oral Fluids: A Large-Scale Evaluation of Immunoassay Performance

Paired serum and oral-fluid (OF) specimens (n = 4,448) were collected from blood donors and patients attending local sexually transmitted disease clinics in Trinidad and Tobago and the Bahamas and were tested for the presence of human immunodeficiency virus type 1 (HIV-1) antibodies. Sera were tested by Abbott AB HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA), and positive specimens were confirmed by Cambridge HIV-1 and HIV-2 Western blotting (WB). OF specimens were collected with the OraSure collection device and were tested by Murex GACELISA and by two EIAs from Organon Teknika (the Oral Fluid Vironostika HIV-1 Microelisa System [OTC-L] and the Vironostika HIV-1 Microelisa System [OTC-M]). EIA-reactive OF specimens were confirmed by miniaturized WB (OFWB). GACELISA detected all 474 HIV-1 seropositive specimens (sensitivity, 100%). OTC-L detected 470 positive specimens (sensitivity, 99.2%), while OTC-M detected 468 positive specimens (sensitivity, 98.8%). Specificities ranged from 99.2 to 100% for the three assays. Concordance of OFWB with serum WB was 99.4%, and banding patterns determined by the two methods were similar. The immunoglobulin G (IgG) concentration of OF specimens ranged from 0.21 to 100 μg/ml, with a mean of 17.1 μg/ml. Significant differences in OF IgG concentrations were observed between HIV antibody-positive and HIV antibody-negative persons (31.94 versus 15.28 μg/ml, respectively [P < 0.0001]). These data further confirm the suitability of OF specimens for detection of HIV-1 antibodies. Currently available HIV-1 antibody assays provide sensitivities and specificities with OF specimens comparable to those achieved with serum specimens.     

Validation of a Modified Commercial Enzyme-Linked Immunoassay for Detection of Human Immunodeficiency Virus Type 1 Immunoglobulin G Antibodies in Saliva

This study was performed to evaluate the performance of a saliva collection device (OmniSal) and an enzyme-linked immunoassay (EIA) designed for use on serum samples (Detect HIV1/2) to detect human immunodeficiency virus type 1 (HIV-1) antibodies in the saliva of high-risk women in Mombasa, Kenya. The results of the saliva assay were compared to a “gold standard” of a double-EIA testing algorithm performed on serum. Individuals were considered HIV-1 seropositive if their serum tested positive for antibodies to HIV-1 by two different EIAs. The commercial serum-based EIA was modified to test the saliva samples by altering the dilution and lowering the cutoff point of the assay. Using the saliva sample, the EIA correctly identified 102 of the 103 seropositive individuals, yielding a sensitivity of 99% (95% confidence interval [CI], 94 to 100%), and 96 of the 96 seronegative individuals, yielding a specificity of 100% (95% CI, 95 to 100%). In this high-risk population, the positive predictive value of the assay was 100% and the negative predictive value was 99%. We conclude that HIV-1 antibody testing of saliva samples collected with this device and tested by this EIA is of sufficient sensitivity and specificity to make this protocol useful in epidemiological studies.     

Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution

A concern during the early AIDS epidemic was the lack of a test to identify individuals who carried the virus. The first HIV antibody test, developed in 1985, was designed to screen blood products, not to diagnose AIDS. The first-generation assays detected IgG antibody and became positive 6 to 12 weeks postinfection. False-positive results occurred; thus, a two-test algorithm was developed using a Western blot or immunofluorescence test as a confirmatory procedure. The second-generation HIV test added recombinant antigens, and the third-generation HIV tests included IgM detection, reducing the test-negative window to approximately 3 weeks postinfection. Fourth- and fifth-generation HIV assays added p24 antigen detection to the screening assay, reducing the test-negative window to 11 to 14 days. A new algorithm addressed the fourth-generation assay''s ability to detect both antibody and antigen and yet not differentiate between them. The fifth-generation HIV assay provides separate antigen and antibody results and will require yet another algorithm. HIV infection may now be detected approximately 2 weeks postexposure, with a reduced number of false-positive results.     

Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection of Mycobacterium tuberculosis Infection

A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.     

Evaluation of a Rapid Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus

A new immunochromatographic rapid test, Determine HIV-1/2, for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 in human whole blood, serum, and plasma was evaluated. Determine HIV-1/2 is a sandwich immunoassay and uses a nitrocellulose strip with a capture site for the patient’s results and a procedural control site to confirm the validity of the assay. The results can be read visually, and a positive result is indicated by the formation of a red line within 15 min after sample application. The test showed 100% sensitivity for HIV-1 with 102 whole-blood, 152 serum, and 144 plasma samples obtained from Ramathibodi Hospital, Bangkok, Thailand. The sensitivity of the test for HIV-2 was 100% with 100 serum or plasma samples obtained from Ivory Coast. The sensitivity of the test with 4 anti-HIV-1 seroconversion panels from Boston Biomedica Inc. was equivalent to or better than those of another agglutination assay with serum or plasma and the enzyme immunoassay licensed by the U.S. Food and Drug Administration. The specificity was 100% with 367 sets of whole-blood, serum, and plasma samples from Ramathibodi Hospital. This method had an analytical sensitivity for the detection of HIV-1 equivalent to or better than that of another agglutination assay with serum or plasma. This test had an analytical sensitivity for the detection of HIV-1 better than that of another immunochromatographic test with whole blood. This evaluation demonstrated the excellent performance of this immunochromatographic test with EDTA-anticoagulated whole-blood, serum, and plasma samples. We conclude that this test is suitable for use in emerging countries and is an excellent alternative to HIV antibody testing at remote sites, as well as in traditional laboratories.     

Diagnosis of Norwalk Virus Infection by Indirect Enzyme Immunoassay Detection of Salivary Antibodies to Recombinant Norwalk Virus Antigen

Simple diagnostic tests are needed for the detection of norovirus (NoV) outbreaks. Salivary antibody assays provide an attractive alternative to collecting and testing serum or stool samples. Antibodies to Norwalk virus (NV) in oral fluid samples were compared with NV antibodies in serum collected from 38 volunteers challenged with NV inoculum. Pre- and postchallenge (day 4, 8, 14, and 21) saliva and serum samples were examined by enzyme immunoassay (EIA) using recombinant NV antigen. Of 18 infected subjects (those who shed NV in stool or who demonstrated immunoglobulin G [IgG] seroconversion), 15 (83%) had ≥4-fold increases in NV-specific salivary IgA and 15 (83%) had ≥4-fold increases in NV-specific salivary IgG when prechallenge and postchallenge saliva samples were compared. When the results of the IgA and IgG assays were combined, all 18 infected subjects showed ≥4-fold increases in NV-specific salivary IgG or IgA postchallenge titers compared to their prechallenge titers. One of 19 uninfected subjects had a ≥4-fold increase in NV-specific salivary IgG. The sensitivity of the combined assay results was 100%, and the specificity was 95%. NV-specific salivary IgA titers peaked around 14 days postchallenge. NV-specific salivary IgG and serum IgG titers continued to rise through 21 days postchallenge. The application of this EIA to an elementary school outbreak indicated that 67% of the subjects with confirmed infections had >4-fold rises in anti-NoV IgA when an antigen in the same genetic cluster as the outbreak virus was used. This is the first documented mucosal antibody response to NoV in children. This EIA provides a useful approach for diagnosing NoV outbreaks.     

Efficacy of a Less-Sensitive Enzyme Immunoassay (3A11-LS) for Early Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Infants

We evaluated a less-sensitive enzyme immunoassay (3A11-LS) for its possible use for early diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in infants. The results were compared with those from the immunoglobulin G-capture enzyme immunoassay. A total of 239 sera from 77 infants were tested. All 25 sera from the 10 infants born to seronegative mothers were found to be negative by both assays. Forty-one seroreverting infants showed a complete decay of maternal antibodies by 4 months by the 3A11-LS assay. However, the assay detected HIV antibodies in only 9 (36%) of 25 sera collected from infected infants between 4 and 6 months and in 27 (63%) of 43 sera collected after 6 months of age. Further analysis with alternative cutoff values indicated that the 3A11-LS had a sensitivity of 12 to 44% and a specificity of 90 to 100% for infants between 4–6 months of age. This data suggest that a diagnosis of HIV infection in some of the infants could be made after 4 months of age by the 3A11-LS assay, although a negative 3A11-LS test result may not rule out infection and may require a further followup.     

Comparison of Human Immunodeficiency Virus Antigens as Stimulants for Lymphocyte Proliferation Assays

CD4 proliferative responses to the human immunodeficiency virus (HIV) type 1 (HIV-1) p24 (gag) antigen inversely correlate with the plasma viral load in HIV-infected subjects who control viral replication without antiretroviral therapy. Use of a single HIV-1 protein to assess CD4 proliferative responses may not reflect the global response to this pathogen. We compared the abilities of HIV p24 and gp120 antigens from two different vendors, an inactivated whole HIV-1 MN virion preparation and an HIV-1E culture supernatant antigen, to elicit proliferative responses in HIV-seropositive and HIV-seronegative donors. Peripheral blood mononuclear cells from 12 HIV-seropositive donors (each with HIV-1 loads <4,000 copies/ml of plasma, >350 CD4 T lymphocytes/mm³, and no antiretroviral therapy) and 15 HIV-seronegative donors were assessed with multiple concentrations of each stimulant by standard lymphocyte proliferation assays. Wide variations in response rates were found, with zero, three, five, and eight individuals demonstrating stimulation indices of >3 for the HIV culture antigen supernatant, gp120, p24, and inactivated whole-virus preparations, respectively. These results suggest that the use of the inactivated whole virus resulted in a more sensitive assay for detection of CD4 T-lymphocyte function in HIV-infected subjects.     

Quantitation of Immunoglobulin to Hepatitis E Virus by Enzyme Immunoassay

We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and intertest coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.     

Impact of Strain Type on Detection of Toxigenic Clostridium difficile: Comparison of Molecular Diagnostic and Enzyme Immunoassay Approaches

A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites'' standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two- and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P = 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P = 0.004, respectively, Fisher''s exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.Clostridium difficile continues to be a significant cause of infectious diarrheal disease among hospitalized patients, particularly in the United States and Europe (2, 11, 13). C. difficile isolates are capable of causing, in addition to diarrheal disease, serious syndromes, such as pseudomembranous colitis and toxic megacolon, which may result in death (11, 13). This organism is also responsible for increasing numbers of community-acquired infections (14, 16). Hypervirulent strains of C. difficile have emerged, including the J strain (9) and 027/NAP1/BI strain (20), the latter of which has been responsible for a series of hospital outbreaks around the world (10, 12, 20, 22). Recent studies indicate that several of the rapid enzyme immunoassay (EIA) methods used to diagnose C. difficile infection (CDI) are less sensitive than previously indicated (1, 29). The sensitivity of EIAs was often assessed initially using direct cytotoxin testing of stool samples, often in high-prevalence or outbreak settings (5). More-recent studies have compared the results of EIA methods against those of toxigenic culture, i.e., culturing C. difficile isolates from stool samples (often using broth enrichment) and testing the organism recovered in culture for cytotoxin production, which has higher sensitivity than direct cytotoxin testing (5, 25). The renewed use of toxigenic culture, particularly in North America, as the reference method has encouraged microbiologists to reassess diagnostic methods for CDI.Another change in the diagnostic landscape has been the introduction of glutamate dehydrogenase (GDH) screening of stool specimens as part of two- or three-step algorithms in an attempt to enhance the sensitivity of C. difficile detection. Data from studies reported by Reller et al. (27) and Ticehurst et al. (36) supported the use of this approach, although a study by Gilligan raised concerns about using less-sensitive toxin EIAs for confirming GDH-positive assays (6).More recently, PCR-based amplification methods for the detection of chromosomal genes encoding C. difficile toxin B (tcdB) or toxin regulatory genes (tcdC) directly in stool samples have been described (7, 15, 24, 29). Evaluations of several commercial amplification methods that target tcdB for detection of C. difficile in stool samples have been reported (8, 23, 30, 31). Three commercial amplification methods have received Food and Drug Administration (FDA) clearance in the United States.The goal of this study was to assess the accuracy of the Cepheid Xpert C. difficile assay in a multilaboratory study using the results of toxigenic culture with broth enrichment (i.e., “enrichment toxigenic culture”) as the reference method. The accuracy of the two commercial EIAs, direct cytotoxin testing, and two algorithms that incorporate GDH screening that were the standard-of-care methods at the study sites was also assessed. We also evaluated the sensitivity of EIA, GDH, and Xpert C. difficile assays for detecting multiple PCR ribotypes of C. difficile, including ribotype 027.     

Analysis of Eight Commercial Enzyme Immunoassay Tests for Detection of Antibodies to Mycoplasma pneumoniae in Human Serum

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.     

合成肽作抗原酶免疫技术检测非甲－戊型肝炎病人血清中抗HGV

本文应用人工合成肽作为抗原建立了检测庚型肝炎病毒（ＨＧＶ）抗体的酶免疫技术（ＥＩＡ），最适抗原包被浓度为４μｇ／ｍｌ。在特异性验证的基础上，检测了３４例非甲－戊５型肝炎病人血清和３２例丙型肝炎感染病人血清。结果表明，在ＨＧＶ感染高危人群中，诸如输血后肝炎－丙型肝炎中有较高的ＨＧＶ的感染率。本方法的建立为临床上难于作出诊断的庚型肝炎提供了敏感、特异、易于应用的新技术。     

Reduction of Diagnostic Window by New Fourth-Generation Human Immunodeficiency Virus Screening Assays

In order to reduce the diagnostic window between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immunosorbent assays (ELISAs) which permit the simultaneous detection of HIV antigen and antibody have been developed. Two fourth-generation assays, HIV DUO (Biomérieux) and HIV Combi (Boehringer Mannheim), for the combined detection of HIV antigen and antibody, were compared with a third-generation assay (HIV-1/HIV-2 3rd Generation Plus enzyme immunoassay [EIA]; Abbott) and a p24 antigen test (HIV-1 Ag monoclonal; Abbott). A total of 17 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 255 potentially cross-reactive serum samples were tested. Ten seroconversions were detected an average of 8.1 days earlier with HIV DUO and 7.5 days earlier with HIV Combi than with the third-generation ELISA. Overall, in the 17 seroconversion panels tested, HIV DUO detected HIV-1 infection an average of 4.8 days and HIV Combi detected infection an average of 4.4 days earlier than HIV-1/HIV-2 3rd Generation Plus EIA. HIV antigen was detected with HIV DUO and HIV Combi in all of the 15 cell culture supernatants infected with different HIV-1 subtypes, including subtype O. With fourth-generation assays, considerably fewer false-positive results (n = 4 to 6) were obtained, in comparison with the third-generation EIA (n = 18). Fourth-generation assays permit an earlier diagnosis of HIV infection than third-generation antibody screening assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion.     

Performance Characteristics of a Rapid New Immunochromatographic Test for Detection of Antibodies to Human Immunodeficiency Virus

A new immunochromatographic rapid test (Rapid Check HIV 1&2; Núcleo de Doenças Infecciosas) for the detection of antibodies to human immunodeficiency virus type 1 and type 2 in human samples (whole blood, serum, and plasma) was evaluated and compared to the commercially available Determine (Abbott Laboratories). When whole-blood samples were evaluated, the specificity and sensitivity of both tests were 100%. However, when plasma samples were used, sensitivity for the Rapid Check HIV 1&2 and the Determine tests were 100 and 98.58%, respectively. The observed specificity for plasma samples was 98.94% for the Rapid Check HIV 1&2 and 96.97% for the Determine test. The results presented here are encouraging and support the adoption of both tests as an alternative to enzyme-lined immunosorbent assay and/or Western blots in regions where laboratorial infrastructure is not available or for use in the management of occupational accidents for healthcare workers.     

微粒子酶联免疫分析法与电化学发光免疫分析法测定甲型肝炎病毒抗体一致性评价

为对微粒子酶联免疫分析法（MEIA）测定甲型肝炎病毒抗体方法学进行验证,用MEIA与电化学发光免疫分析法（ECLIA）同时检测476份血清标本的甲型肝炎病毒抗体,对其测量准确性、特异性及重复性进行评估分析。结果表明,MEIA与ECLIA比较,阳性符合率为98.7%,阴性符合率为97.0%,总符合率为98.3%。类风湿因子（RF）阳性、重度溶血、脂血、黄疸血清对检测无明显影响。MEIA与ECLIA测定甲型肝炎病毒抗体一致性较高。