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Current diagnostic tests for tuberculosis (TB) are not able to distinguish active disease from latentMycobacterium tuberculosisinfection, nor are they able to quantify the risk of a latently infected person progressing to active TB. There is interest, however, in adapting antigen-specific gamma interferon (IFN-γ) release assays (IGRAs) to predict disease outcome. In this study, we used the differential susceptibilities of inbred mouse strains toM. tuberculosisinfection to evaluate the prognostic capabilities of IGRAs. Using lung and blood cultures, we determined that CBA/J, DBA/2, and C3H/HeJ mice (models of heightened risk of progression to active TB) produced less antigen-specific IFN-γ in response toM. tuberculosisculture filtrate proteins and early secreted antigenic target-6 than the relatively resistant C57BL/6 mouse strain. Additionally, reduced IFN-γ secretion in supernatants reflected a reduced frequency of IFN-γ-responding cells in the lung and blood and not a specific defect in IFN-γ secretion at the single-cell level. Importantly, detection of antigen-specific IFN-γ from blood cultures accurately reflected lung responses, indicating that blood can be an appropriate test tissue in humans. Furthermore, reduced antigen-specific IFN-γ production and low frequencies of IFN-γ-responding cells from peripheral blood predicted increased risk of TB disease progression across genetically diverse TB disease-susceptible mouse strains, suggesting that similar results may occur in humans. The development of efficacious predictive diagnostic tests for humans would lead to targeted therapy prior to progression to active TB, reducing transmission, incidence, and prevalence rates while maximizing the use of public health resources.

Studies are needed to characterize the reproducibility of QuantiFERON-TB Gold (QFT-G) for targeted U.S. screening populations. Members of northern California households were tested with the QFT-G in-tube assay (QFT-G-IT) at two home visits 3 months apart. Reproducibility and agreement with the tuberculin skin test (TST) were assessed. Monte Carlo simulation was used to evaluate the role of test-related error. Of 63 individuals (49 adults and 14 children) completing QFT-G-IT at both time points, 79% were foreign-born (98% from Latin America) and 68% reported Mycobacterium bovis BCG vaccination. At the baseline visit, 23 (37%) were TST positive and 15 (24%) were QFT-G-IT positive (κ = 0.48 [± 0.11]). At 3 months, 3/48 (6.3%; 95% confidence interval [95CI], 2 to 17) of those initially QFT-G-IT negative converted, and 5/15 (33%; 95CI, 15 to 58) of those initially QFT-G-IT positive reverted. Among the 8 individuals with inconsistent QFT-G-IT results, the maximum gamma interferon response at either visit was 0.68 IU/ml versus means of 4.99 (± 3.74) and 6.95 (± 5.6) for 10 persistent positives at the first and second visits, respectively. Expected false-reversion and -conversion rates were 32% (90CI, 25 to 39%) and 6.95% (90CI, 4.6 to 9.8%) when the sensitivity and specificity were assumed to average 70% and 98%, respectively. Transient responses to QFT-G-IT are common, and low positive results need to be interpreted with caution. Further studies are needed to characterize the predictive value of the test for U.S. foreign-born and other targeted screening populations.

Clostridium difficile causes nosocomial diarrhea and is responsible for complications such as pseudomembranous colitis, megacolon, and perforation. Using 442 stool specimens, we compared the sensitivities and specificities of the Premier toxin A and B (Meridian Bioscience, Inc.) and C. difficile TOX A/B II (TechLab, Inc., Blacksburg, VA) immunoassays in the Virology Department of the Kaiser Permanente Regional Reference Laboratories. The Premier toxin A and B assay demonstrated a higher sensitivity (97.44%) and a higher positive predictive value (79.17%) than the C. difficile TOX A/B II assay (87.18% and 75.56%, respectively), while assay specificities and negative predictive values were similar. We also performed experiments using serially diluted, purified toxin A and B antigens to understand the basis for assay differences. The two assays’ toxin A antibodies detected toxin A at comparable levels. Preliminary results indicated that the toxin B antibody in the Premier toxin A and B assay could detect toxin B at a concentration of 125 pg/100 μl, while the toxin B antibody in the C. difficile TOX A/B II assay could not detect toxin B below a concentration of 250 pg/100 μl. Therefore, the Premier toxin A and B assay provides greater sensitivity than the C. difficile TOX A/B II assay, perhaps due to a superior detection ability of its toxin B antibody.

Tuberculosis (TB) is a serious global disease. The fatality rate attributed to TB is among the highest of infectious diseases, with approximately 2 million deaths occurring per year worldwide. Identification of individuals infected with Mycobacterium tuberculosis and screening of their immediate contacts is crucial for controlling the spread of TB. Current methods for detection of M. tuberculosis infection are not efficient, in particular, for testing large numbers of samples. We report a novel and efficient multiplex microbead immunoassay (MMIA), based on Luminex technology, for profiling antibodies to M. tuberculosis. Microbead sets identifiable by unique fluorescence were individually coated with each of several M. tuberculosis antigens and tested in multiplex format for antibody detection in the experimental nonhuman primate model of TB. Certain M. tuberculosis antigens, e.g., ESAT-6, CFP-10, and HspX, were included to enhance the specificity of the MMIA, because these antigens are absent in nontuberculous mycobacteria and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. The MMIA enabled simultaneous detection of multiple M. tuberculosis plasma antibodies in several cohorts of macaques representing different stages of infection and/or disease. Antibody profiles were defined in early and latent/chronic infection. These proof-of-concept findings demonstrate the potential clinical use of the MMIA. In addition, the MMIA serodetection system has a potential for mining M. tuberculosis open reading frames (about 4,000) to discover novel target proteins for the development of more-comprehensive TB serodiagnostic tests.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

The etiological agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis . Controlling the spread of this disease is hindered by the lack of sensitive, selective, and rapid detection methods for M. avium subsp. paratuberculosis . By using a recently optimized sandwich immunoassay (B. J. Yakes, R. J. Lipert, J. P. Bannantine, and M. D. Porter, Clin. Vaccine Immunol. 15:227-234, 2008), which incorporates a new monoclonal antibody for the selective capture and labeling of M. avium subsp. paratuberculosis and surface-enhanced Raman scattering for sensitive readout, detection limits of ∼630 and ∼740 M. avium subsp. paratuberculosis cells/ml are achieved in phosphate-buffered saline and whole milk samples, respectively, after spiking with heat-treated M. avium subsp. paratuberculosis . Surprisingly, these detection limits are 3 orders of magnitude lower than expected based on theoretical predictions. Experiments designed to determine the origin of the improvement revealed that the major membrane protein targeted by the monoclonal antibody was present in the sample suspensions as shed protein. This finding indicates that the capture and labeling of shed protein function as a facile amplification strategy for lowering the limit of detection for M. avium subsp. paratuberculosis that may also be applicable to the design of a wide range of highly sensitive assays for other cells and viruses.

The first commercial indirect immunofluorescence assay (IFA) using Euroimmun Biochip technology was evaluated for the serodiagnosis of immunoglobulin G (IgG) and IgM antibodies against yellow fever virus (YFV) and was compared with the plaque reduction neutralization test (PRNT), which is currently the gold standard test for YFV. An overall correlation between the tests of 98.7% was established based on the analysis of 150 sera from individuals after vaccination with the 17D yellow fever vaccine. The sensitivity and specificity, calculated using the 150 sera from vaccinees and 150 sera from healthy blood donors, were 95% and 95%, respectively, for the IgG IFA and 94% and 97% for the IgM IFA. Antibody titers found in the PRNT correlated poorly with the IgM and IgG titers detected by IFA. The analysis of preexisting heterologous flaviviral immunity revealed the presence of antibodies reactive with YFV, tick-borne encephalitis virus, West Nile virus, Japanese encephalitis virus, and dengue virus serotypes 1 to 4 in 20 out of the 150 vaccinees. The indirect IFA showed that nine of these individuals with previous flaviviral exposure who received 17D vaccine failed to produce detectable IgM antibodies. Despite this preexisting immunity, all vaccinees developed protective immunity as detected by PRNT and anti-YFV IgG antibodies as detected by IFA. The high specificity and sensitivity of the IFA make it a useful tool for rapid diagnosis of yellow fever during outbreaks, for epidemiological studies, and for serosurveillance after vaccination.

The aim of this study was to develop a highly sensitive human papillomavirus type 31 (HPV31) neutralization assay based on the production of pseudovirions carrying luciferase. Neutralizing antibodies against HPV31 were investigated in a set of HPV31 monoclonal antibodies and in women with evidence of HPV31 infection. Neutralizing antibodies were detected in 78% of subjects with a positive enzyme-linked immunosorbent assay.

Numerous evaluations of the clinical sensitivity and specificity of PCR and serologic assays for Bordetella pertussis have been hampered by the low sensitivity of culture, the gold standard test, which leads to biased accuracy estimates. The bias can be reduced by using statistical approaches such as the composite reference standard (CRS) (e.g., positive if culture or serology positive; negative otherwise) or latent class analysis (LCA), an internal reference standard based on a statistical model. We illustrated the benefits of the CRS and LCA approaches by reanalyzing data from a 1995 to 1996 study of cough illness among 212 patients. The accuracy of PCR in this study was evaluated using three reference standards: culture, CRS, and LCA. Using specimens obtained 0 to 34 days after cough onset, estimates of the sensitivity of PCR obtained using CRS (47%) and LCA (34%) were lower than the culture-based estimate (62%). The CRS and LCA approaches, which utilized more than one diagnostic marker of pertussis, likely produced more accurate reference standards than culture alone. In general, the CRS approach is simple, with a well-defined disease status. LCA requires statistical modeling but incorporates more indicators of disease than CRS. When three or more indicators of pertussis are available, these approaches should be used in evaluations of pertussis diagnostic tests.

We evaluated the T-SPOT.TB and Quantiferon-TB Gold In tube (QFN-G-IT) tests for diagnosing Mycobacterium tuberculosis infection. T-SPOT.TB was more sensitive than QFN-G-IT in diagnosing both active and latent infection. Both gamma interferon tests were unaffected by prior Mycobacterium bovis BCG vaccination. Among children who were not BCG vaccinated but had a positive tuberculin skin test, QFN-G-IT was negative in 53.3% of cases, and T-SPOT.TB was negative in 50% of cases.

Four different recombinant antigens derived from Echinococcus granulosus , designated B1t, B2t, E14t, and C317, were tested with enzyme-linked immunosorbent assays (ELISAs) for the detection of specific immunoglobulin G (IgG) in patients with unilocular hydatid disease (UHD). The results were compared to those obtained with hydatid fluid and were subjected to receiver operator characteristic analysis. The diagnostic performance of the above-listed proteins was defined with respect to their specificity, sensitivity, and predictive values (PV); the influence of cyst location; and usefulness in the follow-up of surgical treatment for UHD and in the determination of whether or not patients have been surgically cured of UHD. The best diagnostic results were obtained with the anti-B2t IgG ELISA, with 91.2% sensitivity, 93% specificity, and high positive and negative PV (89.4 and 94.2, respectively). In addition, this diagnostic tool proved to be useful for the follow-up of surgically treated UHD patients. The anti-B2t IgG ELISA may find an application in the serodiagnosis of UHD in clinical laboratories.

Two hundred seventy-nine serum samples from men attending sexually transmitted disease (STD) clinics in Baltimore, Maryland, were tested for herpes simplex virus type 2 (HSV-2)-specific antibody by three immunosorbent glycoprotein G-2-based assays (the Kalon, Focus, and Biokit assays). The results for all samples with positive results were confirmed by Western blotting (91/279; 32.6% HSV-2 seroprevalence). All patients were also tested for Chlamydia trachomatis , Neisseria gonorrhoeae , Trichomonas vaginalis , Mycoplasma genitalium , human immunodeficiency virus type 1, and hepatitis C virus. The Kalon assay performed very well with samples from this population (90.8% sensitive, 99.4% specific), whereas the Focus assay had a sensitivity (82.6%) much lower than that shown previously. For 19.7% of the samples, the Biokit assay gave an indeterminate result. It was found that the odds of a sample having a Biokit assay indeterminate result compared to that of having a definitive positive or negative results were 3.88 times greater for subjects concurrently infected with N. gonorrhoeae , after the effects of other STDs were controlled for ( P = 0.001; 95% confidence interval, 1.78, 8.45). Unfortunately, we were unable to control for HSV-1 infection status in the regression model, which, on the basis of χ 2 analysis, might also affect the clarity of the Biokit test. The recommended index cutoff value of 1.1 for the Focus and Kalon assays was found to be optimal for this population.

The second-generation Histoplasma antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of Histoplasma antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative Histoplasma antigen immunoassay.

Corroboration of serology results is essential for restricting the risk of inappropriate antileishmanial prescription. A direct agglutination test (DAT) and a recently developed β-mercaptoethanol-modified enzyme-linked immunosorbent assay (β-ME ELISA) based on the use of antigen prepared as described for the DAT were applied to 416 sera from two Sudanese populations with and without clinical evidence of visceral leishmaniasis (VL). Of 285 sera with the lowest antileishmanial DAT titers (≤1:100 to 1:1,600), 270 (94.7%) scored comparable minimum β-ME ELISA absorbance values (≤0.1 to 0.26). In 117 sera that demonstrated the highest DAT titers (1:12,800 to ≥1:25,600), 86 (73.5%) scored maximum (0.81 to ≥1.35) and 30 (25.6%) medium (0.27 to 0.80) β-ME ELISA absorbance values. VL diagnosis was established for 142 (44.1%) patients in the VL-symptomatic group (n= 322), based on positive microscopy forLeishmania donovaniin lymph node aspirates or positive DAT (titer, ≥1:3,200). Of the 125 sera from the symptomatic patients for whom microscopy was positive for VL, 111 (88.8%) had comparable positive DAT and β-ME ELISA readings. In all 17 sera from the symptomatic DAT-positive patients for whom leishmaniasis was not established by microscopy but who responded favorably to antileishmanial therapy, absorbance values (≥0.27) indicative of VL were obtained by β-ME ELISA. Of 197 symptomatic patients for whom microscopy was negative for VL, 172 (87.3%) tested negative in β-ME ELISA and 180 (91.4%) in DAT. Based on the high reliability demonstrated here for VL detection, β-ME ELISA fulfills the requirement of confirming DAT results in patients manifesting suspected VL.

Most persons with rubella virus-specific immunoglobulin M (IgM)- or IgG-positive sera tested positive (98% [ n = 178] and 99% [ n = 221], respectively) using paired filter paper dried blood spot (DBS) samples, provided that DBS indeterminate results were called positive. For persons with IgM- or IgG-negative sera, 97% and 98%, respectively, were negative using DBS.

We describe here a rapid and semiautomated method for the determination of rubella virus immunoglobulin G (IgG) avidity with the VIDAS instrument. A total of 153 serum samples from persons with naturally acquired rubella virus infections ( n = 98), from vaccinated persons ( n = 44), and from patients with autoantibodies ( n = 11) were included in this study. The rubella virus-specific IgG avidity assay we developed for the VIDAS instrument was evaluated by comparison with an in-house method. Results obtained with the VIDAS instrument allow considering this method valuable to help confirm or exclude acute primary infection or recent vaccination.

WOS: 000251125800003, PubMed ID: 17761525, In this study, we aimed to evaluate the validity of the conventional enzyme-linked immunosorbent assay (ELISA) and the Western blotting test for the diagnosis of anthroponotic cutaneous leishmaniasis (ACL) using serum samples obtained from 51 patients with parasitologically proven nontreated CL (NonT-CL patients) and 62 patients under treatment for CL (UT-CL patients). Additionally, 29 serum samples obtained from patients with parasitologically and serologically proven visceral leishmaniasis (VL) were also used as positive controls, and serum samples from 43 blood donors were used as negative controls. All sera were diluted to the same dilution (1/100). Leishmania infantum MON-1 was used as the antigen in the conventional ELISA. The sera of 27 (93.1%) of 29 VL patients were seropositive by ELISA, while the sera of 40 (78.4%) of 51 NonT-CL patients and 43 (69.3%) of 62 UT-CL patients were seropositive by the conventional ELISA. The absorbance values of the CL patients' sera were significantly lower than the absorbance values of the VL patients' sera. Bands between 15 and 118 kDa were detected in two groups of CL patients. Among all bands, the 63-kDa band was found to be more sensitive (88.5%). When we evaluated the Western blotting results for the presence of at least one of the diagnostic antigenic bands, the sensitivity was calculated to be 99.1%. By using serological tests, a measurable antibody response was detected in most of the CL patients in Sanliurfa, Turkey. It is also noted that this response can be changed according to the sizes, types, and numbers of lesions that the patient has. The Western blot test was found to be more sensitive and valid than the conventional ELISA for the serodiagnosis of ACL. In some instances, when it is very difficult to demonstrate the presence of parasites in the smears, immunodiagnosis can be a valuable alternative for the diagnosis of ACL.

Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins.

A microsphere-based immunoassay (MIA) was previously developed that is capable of determining the presence of anti-West Nile (WN) virus or anti-St. Louis encephalitis (SLE) virus immunoglobulin M (IgM) antibodies in human serum or cerebrospinal fluid. The original data set on which the classification rules were based comprised 491 serum specimens obtained from the serum bank at the Division of Vector-Borne Infectious Diseases of the Centers for Disease Control and Prevention (DVBID). The classification rules were used to provide a result and to determine whether confirmatory testing was necessary for a given sample. A validation study was coordinated between the DVBID and five state health laboratories to determine (i) the reproducibility of the test between different laboratories, (ii) the correlation between the IgM-enzyme-linked immunosorbent assay (MAC-ELISA) and the MIA, and (iii) whether the initial nonspecific parameters could be refined to reduce the volume of confirmatory testing. Laboratorians were trained in the method, and reagents and data analysis software developed at the DVBID were shipped to each validating laboratory. Validating laboratories performed tests on approximately 200 samples obtained from their individual states, the collections of which comprised approximately equal numbers of WN virus-positive and -negative samples, as determined by MAC-ELISA. In addition, 377 samples submitted to the DVBID for arbovirus testing were analyzed using the MIA and MAC-ELISA at the DVBID only. For the specimens tested at both the state and the DVBID laboratories, a correlation of results indicated that the technology is readily transferable between laboratories. The detection of IgM antibodies to WN virus was more consistent than detection of IgM antibodies to SLE virus. Some changes were made to the analysis software that resulted in an improved accuracy of diagnosis.

Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzyme-linked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses., Instituto de Biotecnologia y Biologia Molecular

Five commercially available enzyme-linked immunosorbent assays (ELISAs), one in-house ELISA, and two hemagglutination assays were evaluated to determine their diagnostic accuracy for Chagas' disease in two studies. In study 1, ELISA kits showed 100% sensitivity, but specificities ranged from 82.84% to 100% when leishmaniasis cases were included and from 95.57% to 100% when leishmaniasis cases were excluded. Kits using recombinant antigens or synthetic peptides are more specific than those using crude extracts from Trypanosoma cruzi epimastigote forms. Kits evaluated in Panama, in study 2, showed 75% to 100% sensitivity and 97.12% to 100% specificity. These data were obtained by using a Western blot assay with T. cruzi trypomastigote excreted-secreted antigens as a reference test to confirm T. cruzi infection.

Lyme borreliosis (LB) is a disease for which antibody-based detection assays are often required for diagnosis. The variable surface molecule VlsE and IR6, one of its invariable regions, are commonly targeted by the antibody response in infected individuals. A series of enzyme-linked immunosorbent assays was performed to comparatively examine the antibody responses of North American LB patients (n= 37) to VlsE and invariable segments of this molecule. Both immunoglobulin M (IgM) and IgG responses to full-length VlsE and to peptides reproducing invariable regions 2, 4, and 6, as well as the invariable domains at the amino and carboxyl termini of VlsE, were assessed. The proportions and specificities of reactivity to the invariable segments were tested by using cognate peptides as competitors for VlsE binding by patient serum antibodies. IR6 epitopes (by the C6 peptide) were found to dominate the response to invariable segments. IR6 (C6)-specific antibodies were detected in 78% of the serum specimens, whereas n= 8) that contained IgG antibodies reacting with both C6 and VlsE was assessed in competition experiments, using C6 as a competitor. Only half of these specimens contained IgG antibodies whose binding to VlsE could be inhibited >50% by competition with the added C6 peptide. The median percent inhibition was 45.5%. These findings indicate that IR6 epitopes are largely concealed from the VlsE molecular surface and that full-length VlsE-based diagnosis likely detects antibodies to conformational and/or variable region epitopes.

In the absence of erythema migrans, the basis for diagnosis of Lyme disease is the demonstration of an antibody response againstBorrelia burgdorferiin an appropriate clinical setting. The C6 enzyme-linked immunosorbent assay, based on the IR6 region of VlsE, has become widely used in both the United States and Europe. We mapped the antigenic epitopes of IR6 to a shorter sequence that is equivalent in sensitivity and specificity to the full-length IR6 25-residue peptide. In addition, we observed significant differences in sensitivity between serum panels (60 to 100%), indicating that the selection of the serum panels can shape the apparent overall sensitivity of the assay. Contrary to prior reports, the assay sensitivity is greater when the IR6 peptide is derived from the sequence of the same infectingBorreliagenospecies. Using our North American panels and the two panels obtained from European Lyme disease patients, we determined that the IR6 assay that is based on a single genospecies ofBorreliaspp. is not optimal for use as a universal diagnostic assay for Lyme disease.

Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected withPlasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of theP. vivaxDuffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant = 3.62 × 10−8M) and the greatest inhibition activity (50% inhibitory concentration ≈ 2.9 μg/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.

Since the introduction of West Nile virus (WNV) in the United States in 1999, several assays have become commercially available to detect antibodies against WNV. Capture enzyme-linked immunosorbent assays (ELISAs) for the detection of WNV-specific immunoglobulin M (IgM) have been approved by the Food and Drug Administration for clinical testing and are available from Focus Diagnostics and PanBio, Inc. The Focus Diagnostics IgM capture ELISA utilizes a background subtraction protocol in order to detect nonspecific reactivity due to rheumatoid factor, heterophile antibodies, or other interfering substances. A background subtraction procedure is not currently recommended for the PanBio IgM capture ELISA. In previous experiments, we determined the agreement, sensitivity, and specificity of the PanBio first-generation IgM capture ELISA compared to an immunofluorescence assay and the Centers for Disease Control and Prevention's IgM capture ELISA. The PanBio assay has since been reformulated to improve the specificity of the assay. We evaluated the reformulated PanBio assay with and without an antigen subtraction procedure and compared the results to the Focus IgM capture ELISA. Agreement, sensitivity, and specificity of the PanBio assay were, respectively, 85%, 95%, and 76% without the subtraction protocol and 94%, 95%, and 93% with the subtraction protocol. In general, when the subtraction protocol was applied to the PanBio IgM capture ELISA, there was a reduction in some, but not all, false-positive results. We suggest that all WNV IgM assays be standardized with a procedure such as background subtraction to eliminate nonspecific reactivity that may cause false-positive results.

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

The advent of multiplexed bead assays in recent years has introduced a new dimension of testing for complex diseases such as lupus, which can involve multiple autoantibodies. The ability to rapidly identify multiple autoantibodies, with high sensitivity and specificity in an automated fashion, is highly attractive. The aim of this study was to assess the performance and clinical value of multiplexed bead-based (AtheNA Multi-Lyte ANA-II test system) immunoassays both by comparing the results with those achieved by indirect fluorescent-antibody assay (IFA) or conventional enzyme immunoassays (EIAs) and by independent identification of autoantibodies in well-characterized samples. To achieve this goal, 984 samples were tested for seven analytes (SS/A, SS/B, Sm, RNP, Scl-70, double-stranded DNA [dsDNA], and centromere B) in both traditional and bead-based assays. The average concordance for the different analytes was 91%, ranging from 81% (dsDNA) to 97% (centromere B). The average relative specificity and sensitivity for the analytes were also high, 92% and 81%, respectively. An examination of 93 “normal controls” demonstrated a 7% false-positive rate, which was comparable to IFA. Percentages of different autoantibodies found in patients with a variety of disease conditions (34 with calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia; 41 with mixed connective tissue disease; 24 with scleroderma; and 35 with Sjogren's syndrome) were well within the range expected from each group. A scrutiny of results from AtheNA and EIA and Farr results for 185 systemic lupus erythematosus samples revealed comparable results by both methods, with the exception of SS/A and dsDNA, where AtheNA had a higher percentage of SS/A-positive results compared to EIA (51% versus 29%) and a lower percentage of dsDNA-positive results (18% versus 28% at a cutoff of 5 IU/ml).

We previously described a simplified quantitative hemolytic assay for classical pathway (CP) hemolytic function in serum that has been shown to correlate with the 50% hemolytic complement (CH 50 ) assay. In the present study, we used this assay to compare CP functions; plasma levels of C3, C4, and C3dg; and ratios of C3dg to C3 in healthy individuals and patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) with different degrees of complement activation. A significant depression in CP function and levels of C4 and C3 and increased C3dg levels and C3dg/C3 ratios were observed in the SLE patients. In patients with RA, CP function was normal, whereas C3, C4, and C3dg levels and the C3dg/C3 ratio were elevated. The SLE results are compatible with systemic complement consumption, whereas the RA data suggest an acute-phase reaction with a normal C3 catabolic rate. To facilitate the handling of patient samples, we also developed a method to restore the hemolytic function of EDTA-plasma by transferring it to Veronal-buffered saline containing the thrombin inhibitor lepirudin. This process inhibits coagulation and enables complement activation, allowing a longer time lag between sample harvesting and testing. These results, combined with previous correlation studies, suggest that the CP hemolytic assay can effectively replace the CH 50 assay for routine SLE differential diagnosis and monitoring of disease activity.

Because of the occurrence of genotype 3 hepatitis E virus (HEV) in regions of low endemicity, it is important to validate the currently used serological assays for diagnosing infections with viruses belonging to this lineage, since these assays only use antigens derived from genotype 1 and 2 viruses. We evaluated the Genelabs enzyme-linked immunosorbent assay (ELISA) and the RecomBlot from Mikrogen for the detection of HEV-specific immunoglobulin M (IgM) and IgG under conditions of low endemicity. We compared test results of 16 patients with locally acquired genotype 3 HEV, 8 genotype 1 patients, 167 healthy controls from the general population, and 101 cases with hepatitis due to other viral causes. The measured specificities of the ELISA (98%) and the RecomBlot (97%) were comparable to those given by the manufacturer for IgM but were significantly lower for IgG (93% by ELISA and 66% by immunoblotting, versus reported values of 98% for ELISA and 95% for blotting). Antibody levels detected following infections with genotype 3 were lower than those following genotype 1 infections except for those measured in the IgM ELISA. Reactivity to the four antigens used in the immunoblot assay were analyzed and showed differences in the IgM immunoblot reactions between genotype 1 patients and genotype 3 patients. The ORF3 antigen was the most specific antigen. The specificity could be improved by a combined testing regimen with confirmation by immunoblotting of all positive ELISA results and by raising the cutoff of the IgG immunoblot assay without loss of sensitivity. We conclude that a combination of ELISA and immunoblotting is needed for acceptable specificity and sensitivity of HEV assays under conditions of low endemicity.

The diagnosis of chronic Chagas' disease is generally made by detecting antibodies to Trypanosoma cruzi . Most conventional serological tests are based on lysates of whole parasites or semipurified antigen fractions from T. cruzi epimastigotes grown in culture. The occurrence of inconclusive and false-positive results has been a persistent problem with the conventional assays, and there is no universally accepted gold standard for confirmation of positive test results. We describe here an immunoblot assay for detecting antibodies to T. cruzi in which four chimeric recombinant antigens (rAgs), designated FP3, FP6, FP10, and TcF, are used as target antigens. Each of these rAgs is composed of several antigenically distinct regions and includes repetitive as well as nonrepetitive sequences. Each rAg is coated as a discrete line on a nitrocellulose strip. Assay sensitivity was assessed by testing 345 specimens known to be positive for antibodies to T. cruzi . All 345 of these samples showed two to four reactive test bands in addition to the three on-board control bands that are on each strip. Assay specificity was determined by testing 500 specimens from random U.S. blood donors, all of which gave negative results. Based on the results obtained in this study, we propose the following scheme for interpretation of test results: (i) no bands or a single test band = a negative result; (ii) two or more test bands with at least one band showing intensity of 1+ or higher = a positive result; and (iii) multiple faint test bands (±) = indeterminate result. Based on this scheme, the prototype immunoblot assay showed sensitivity of 100% ( n = 345) and specificity of 100% ( n = 500). Additionally, all 269 potentially cross-reacting and T. cruzi antibody-negative specimens tested negative in our immunoblot assay. The rAg-based immunoblot assay has potential as a supplemental test for confirming the presence of antibodies to T. cruzi in blood specimens and for identifying false-positive results obtained with other assays.

The tuberculin skin test (TST) was compared with QuantiFERON-TB Gold in-tube (QFT-GIT) test for the diagnosis of tuberculosis in non- Mycobacterium bovis BCG-vaccinated military personnel. Among subjects positive by TST, 44.4% of recruits were positive by QFT-GIT compared with 11.5% subjects tested after missions abroad, suggesting that most TST conversions in the latter group were caused by nontuberculous mycobacteria.

Cell-mediated immunity, particularly that induced by T cells, is thought to have a key role in controlling infection. The enzyme-linked immunospot (ELISPOT) assay has been successfully adapted to detect T-cell immune response to a variety of pathogens. However, it still remains a challenge to detect antigen-specific T cells when the numbers of circulating cells are low, such as in a local cervical infection caused by genital human papillomavirus (HPV). The goal of this study was to develop a protocol for enhanced detection of HPV-specific CD8 + T cells by examining a number of the variables involved in performing an ELISPOT assay. Since blood samples consistently positive for HPV-specific T cells are difficult to obtain, previously described human papillomavirus type 16 (HPV16) E6 52-61 (FAFRDLCIVY)-specific T-cell clone cells (13) seeded in peripheral blood mononuclear cells from an HLA-B57-positive blood donor were used. The variables examined were the amounts of primary and secondary anti-gamma interferon antibodies, amounts of antigen-presenting monocytes and recombinant vaccinia virus expressing the HPV16 E6 protein, and amounts of exogenous cytokines added (recombinant human interleukin-2 [rhIL-2] and rhIL-7). The amounts of antigen-presenting monocytes, followed by the concentration of exogenous rhIL-2, had the most pronounced and significant effects in enhancing sensitivity of the ELISPOT assay. Blood samples from six patients being monitored for abnormal Pap smear results and from 12 healthy volunteers were examined using the enhanced conditions.

An enzyme-linked immunosorbent assay (ELISA) was developed for specific antibody detection in serum specimens of patients with sporotrichosis. The assay was made with mycelial-phase Sporothrix schenckii exoantigens and was tested against 90 sera from patients with different clinical forms of sporotrichosis. Potential cross-reactions were analyzed with 72 heterologous sera from patients with paracoccidioidomycosis, cryptococcosis, aspergillosis, histoplasmosis, tuberculosis, and American tegumentary leishmaniasis, as well as 76 sera from healthy controls. We found a sensitivity of 97% and a specificity of 89% in this assay. Some cross-reactions were seen, as observed in other immunoassays for the diagnosis of sporotrichosis. The ELISA appears to be especially useful for cutaneous forms of disease, since these are not promptly diagnosed with available immunoprecipitation or agglutination techniques. These results suggest that the ELISA using mycelial-phase S. schenckii exoantigens is a very sensitive diagnostic tool for the serodiagnosis of sporotrichosis and can be used in conjunction with conventional methods of diagnosis, particularly in cases where cross-reactions or false-positive results are experienced with the serodiagnosis.

A single-platform volumetric flow cytometer, the Partec Cyflow SL_3, was evaluated against a BD FACSCalibur/Sysmex XT1800i dual platform for measuring CD4+lymphocytes, total lymphocytes, and the percentage of CD4 lymphocytes in whole-blood samples for monitoring the immune systems of human immunodeficiency virus (HIV)/AIDS patients. Statistical analyses for precision, correlation, and agreement were performed. Coefficients of variation (CV) of 5.8, 4.6, and 3.9% were obtained for low, medium, and high CD4+cell counts, respectively, using the SL_3, and CV of 3.7, 4.0, and 0.94 were obtained for the same categories, using the BD FACSCalibur. Significant correlations (P< 0.005) between the two assays for CD4 counts, total lymphocyte counts, and percentages of CD4 were obtained, with correlation coefficients of 0.99, 0.96, and 0.99, respectively (n= 229). Using the Bland-Altman plot, mean biases of −18 cell/μl (95% confidence interval (CI); −91 to 54 cells/μl), −0.8% (95% CI; −3.6 to 2%), and −36.8 cells/μl (95% CI; −477 to 404 cells/μl) were obtained for comparisons of CD4 counts, percentages of CD4 cells, and total lymphocyte counts, respectively. The effects of the age of the samples on the three parameters were also analyzed by comparing results from the same samples analyzed at 6, 24, and 48 h after collection. The correlation coefficients for comparisons among different time points for the same machine and among all the time points for the two different machines were greater than 0.90. These data showed that the Partec Cyflow SL_3 assay is comparable to the BD FACSCalibur/Sysmex XT1800i dual-platform method for measuring the amount of CD4+cells and total lymphocytes and the percentages of CD4 cells in blood samples for the purpose of monitoring HIV/AIDS patients.

The diagnostic accuracy of an indirect immunofluorescence antibody test (IFAT) for Strongyloides stercoralis at different serum antibody titers was evaluated. To assess diagnostic sensitivity, sera from 156 patients with known strongyloidiasis were collected. Negative control sera were obtained from a composite group of 427 subjects (blood donors and hospitalized patients). With an area under the receiver-operating characteristic plot of 0.98, the IFAT showed a high level of diagnostic accuracy for strongyloidiasis. An antibody titer of ≥1:20, with 97% sensitivity and 98% specificity, was identified as the diagnostic threshold with the best overall performance. Cross-reactions were evaluated with 41 additional samples from patients with other known helminth infections, and the IFAT detected low-titer positivity in only one subject with filariasis. A positive IFAT result at an antibody dilution of ≥1:80 was virtually 100% specific, with 71% sensitivity. To test the usefulness of the IFAT as a monitoring tool, the changes in specific-antibody titers after treatment in a group of 155 patients were evaluated. Seroreversion or a decrease in antibody titer of twofold or more was observed in 60% of the patients. Response to treatment was directly correlated to the initial antibody titer, and a baseline titer of ≥1:80 was identified as the best predictor of response. In conclusion, a positive IFAT result at an antibody dilution of ≥1:20 is the optimal cutoff for screening. A titer of ≥1:80, with virtually no false-positive result, is a reliable cutoff for a serological assessment of treatment efficacy and for inclusion in clinical trials.

We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection.

Detection of antibody to C6, a peptide that reproduces the sequence of the sixth invariable region within the central domain of the VlsE protein of Borrelia burgdorferi, is used currently for the serologic diagnosis of Lyme disease in humans. B. burgdorferi isolates taken from infected humans can be categorized into specific genetic subtypes (designated RST1, -2, and -3) by restriction fragment length polymorphisms in the 16S to 23S rRNA spacer sequence. Many of these, usually categorized as RST2, retain only segments of the linear plasmid lp28-1, which encodes VlsE. The VlsE genetic region is retained, but altered expression of this molecule could affect diagnosis by the C6 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients infected with each of the three genotypes and from mice infected with three RST2 isolates were tested with the C6 ELISA. Such isolates elicited marked C6 responses in infected mice. The sensitivity of C6 antibody detection in patients infected with RST2 spirochetes was statistically indistinguishable from detection of RST1 and RST3 infections. These findings demonstrate that diagnosis by C6 ELISA remains effective for infection with all B. burgdorferi genotypes, including those with incomplete lp28-1 plasmids. The clinical progression of Lyme disease, a tick-borne illness that is caused by the spirochete Borrelia burgdorferi, is divided into early localized, early disseminated, and late stages. During the early localized phase, the disease usually manifests by a characteristic skin lesion, erythema migrans (EM). Several days or weeks later, the spirochetes may spread hematogenously and patients may develop early disseminated disease with dermatologic, cardiac, neurologic, and/or rheumatologic involvement. During the early disseminated phase, some patients exhibit dermatologic signs that appear as multiple EM (24, 27, 29, 33). Late disease presents mainly with arthritis or neurologic manifestations (28). Evidence has been put forward to suggest that a major determinant of the risk of B. burgdorferi dissemination in Lyme disease patients with EM is the genotype of the infecting spirochetal strain. Three genotypes have been identified on the basis of restriction fragment length polymorphisms in the rRNA spacer region and have been designated RST1, -2, and -3 (16). Patients infected with RST1 spirochetes had the highest proportion of blood culture positivity (43%), those infected with RST2 spirochetes were intermediate (20%), and those with RST3 had the lowest proportion (3%). In general, persons with an RST1 infection had more symptoms and symptoms of greater severity than those with either RST2 or RST3 infection (32). Mice that were inoculated with RST1 organisms were significantly more likely to yield cultivable spirochetes from different organs and showed a significantly higher prevalence of both carditis and arthritis than did animals inoculated with RST3 spirochetes (30); RST2 isolates were not assessed. When the plasmid contents of the different human infectious types were assessed, RST2 spirochetes emerged as

Storage of high-quality cryopreserved peripheral blood mononuclear cells (PBMC) is often a requirement for multicenter clinical trials and requires a reproducibly high standard of practice. A quality assurance program (QAP) was established to assess an Australia-wide network of laboratories in the provision of high-quality PBMC (determined by yield, viability, and function), using blood taken from single donors (human immunodeficiency virus [HIV] positive and HIV negative) and shipped to each site for preparation and cryopreservation of PBMC. The aim of the QAP was to provide laboratory accreditation for participation in clinical trials and cohort studies which require preparation and cryopreservation of PBMC and to assist all laboratories to prepare PBMC with a viability of >80% and yield of >50% following thawing. Many laboratories failed to reach this standard on the initial QAP round. Interventions to improve performance included telephone interviews with the staff at each laboratory, two annual wet workshops, and direct access to a senior scientist to discuss performance following each QAP round. Performance improved substantially in the majority of sites that initially failed the QAP ( P = 0.002 and P = 0.001 for viability and yield, respectively). In a minority of laboratories, there was no improvement ( n = 2), while a high standard was retained at the laboratories that commenced with adequate performance ( n = 3). These findings demonstrate that simple interventions and monitoring of PBMC preparation and cryopreservation from multiple laboratories can significantly improve performance and contribute to maintenance of a network of laboratories accredited for quality PBMC fractionation and cryopreservation.

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Chronic hepatosplenic suppurative brucellosis (CHSB) is a local reactivation of a previous brucellosis, coursing with an immunoglobulin G (IgG) and IgA secondary immunological response. The observation of two cases of CHSB with an apparent IgM response gave rise to a detailed serological study of three of our patients. We studied the first sample from all three patients and successive samples from two of them. In cases 1 and 2, we found samples with positive IgM lateral flow and IgM enzyme-linked immunosorbent assay results concomitantly with rheumatoid factor (RF); after absorption with anti-RF serum, these results were rendered negative. In patients 2 and 3 the diagnosis of brucellosis was delayed, because none of the test results were initially very significant. However, a clear seroconversion of IgG antibodies was observed in subsequent months; titers of the Brucellacapt and Coombs tests increased in similar ways, although Brucellacapt decreased more rapidly than Coombs, which persisted at high titers for years. In patient 3 a relapse was observed in the fourth year of follow-up, detected by Coombs and also by IgG lateral flow and counterimmunoelectrophoresis (CIEP), although not by the rose bengal, agglutination, or Brucellacapt tests. Serological changes in CHSB may sometimes be mild and are detected mainly by the Coombs test. Brucellacapt does not offer additional information, although IgG lateral flow and CIEP may be of some use. Careful surveillance of titer changes in the Coombs test is the best marker of infection activity. As the disease progresses, an intense IgG response may develop and RF sometimes appears, simulating an IgM response

The limitations of conventional microbiologic methods (CMM) for etiologic diagnosis of community pneumococcal pneumonia have made faster diagnostic techniques necessary. Our aim was to evaluate the usefulness of the immunochromatography (ICT) technique for detecting urinary Streptococcus pneumoniae antigen in the etiologic diagnosis of community-acquired pneumonias (CAP). This was a prospective study on in-patients with CAP in a tertiary hospital conducted from October 2000 to March 2004. Apart from using CMM to reach an etiologic diagnosis, we determined pneumococcal antigen in concentrated urine by ICT. We also determined the urinary pneumococcal antigen (UPA) content in patients from two control groups to calculate the specificity of the technique. One group was comprised of in-patients diagnosed with chronic obstructive pulmonary disease (COPD) or asthma, with respiratory infection, and without pneumonia; the other group included fractures. We studied 959 pneumonia patients and determined UPA content in 911 (95%) of them. We diagnosed the etiology of 253 cases (28%) using CMM; S. pneumoniae was the most common etiologic agent (57 cases). ICT analysis was positive for 279 patients (31%). Using this technique, the percentage of diagnoses of pneumococcal pneumonias increased by 26%, while the overall etiologic diagnosis increased from 28 to 49%. The technique sensitivity was 81%; the specificity oscillated between 80% in CAP with nonpneumococcal etiology and 99% for patients with fractures without infections. Determination of UPA is a rapid, simple analysis with good sensitivity and specificity, which increased the percentage of etiologic diagnoses. Positive UPA may persist in COPD patients with probable pneumococcal colonization or recent pneumococcal infections.

The diagnostic utility of immunochromatographic (Leptotek) and enzyme-linked immunosorbent assay (ELISA; Panbio) tests for the detection of Leptospira immunoglobulin M antibodies was assessed in febrile adults admitted in Vientiane, Laos. Both tests demonstrated poor diagnostic accuracy using admission serum (Leptotek sensitivity of 47.3% and specificity of 75.5%: ELISA sensitivity of 60.9% and specificity of 65.6%) compared to the Leptospira “gold standard” microscopic agglutination test.

We investigated immunoglobulin G (IgG) subclass antibody responses to Plasmodium falciparum merozoite surface protein 1 (MSP-1) and MSP-2 in 112 malaria-exposed subjects in Brazil. IgG3 polarization was primarily epitope driven, being little affected by cumulative or current exposure to malaria and not affected by a subject's age and Fcγ receptor IIA genotype.

Inflammatory bowel disease (IBD) is the general term used for a heterogeneous group of intestinal disorders, including Crohn's disease (CD) and ulcerative colitis (UC). Serological markers such as anti- Saccharomyces cerevisiae antibodies (ASCA) and atypical perinuclear antineutrophilic cytoplasmic antibody (atypical pANCA) have proven useful in the diagnosis and differentiation of CD and UC. Immunoglobulin A (IgA) antibody directed against the outer membrane protein C (OmpC) of Escherichia coli is said by one group to have clinical utility in diagnosing IBD, specifically in ASCA-negative CD patients. Our objective in this study was to compare the results obtained from two separate laboratories offering similar IBD tests using sera from suspected IBD patients. One hundred ninety-seven sera received for IBD testing were included in the study. The agreement between the two laboratories was 93.4% for ASCA IgA, 90.9% for ASCA IgG, and 87.8% for atypical pANCA IgG. There were 25 sera with ASCA-negative/OmpC-positive results reported by one laboratory. Thirteen of these 25 (52.0%) ASCA-negative/OmpC-positive sera were also atypical pANCA positive (9 as determined by both laboratories, 3 by one, and 1 by the other). Atypical pANCA antibody is found primarily in IBD patients with UC and colon-limited CD (Crohn's colitis). We conclude that the ASCA and atypical pANCA assays showed good agreement between the two laboratories, but the data for ASCA-negative/OmpC-positive sera suggest that many (52.0%) of these patients were more likely to have had UC or Crohn's colitis based on the presence of an atypical pANCA.

The gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay is a reference method for the ex vivo monitoring of antigen-specific T cells and a primary tool for assessing clinical trials of human immunodeficiency virus (HIV) or cancer vaccines. Four experienced laboratories in Paris compared their results with this method by exchanging frozen blood samples from eight HIV-seronegative and eight HIV-seropositive subjects. Each laboratory measured the IFN-γ-producing cells specific for HIV, Epstein-Barr virus, cytomegalovirus, and influenza using the same set of peptides and the same ELISPOT reader but its own ELISPOT technique. The cutoff values for positive responses (50 or 100 spot-forming cells/106peripheral blood mononuclear cells over background) were consistent with the binomial statistic criterion. The global qualitative concordance, as assessed by the kappa index, ranged from 0.38 to 0.92, that is, moderate to excellent, and was better for non-HIV 9-mer peptide pools than for HIV 15-mer peptide pools. The interlaboratory coefficient of variation for the frequency of virus-specific T cells was 18.7% (data are expressed on a log scale). Clustering analysis of HIV-positive subjects showed qualitative agreement for ELISPOT results from all four laboratories. Overall, the good interlaboratory qualitative concordance of IFN-γ ELISPOT assays with only the peptide source and ELISPOT reader in common suggests that a qualitative comparison of interlaboratory findings is feasible. Nonetheless, a single set of standard operating procedures should be used in multicenter trials to improve standardization.

We investigated whether the choice of anticoagulant or the application of density gradient mononuclear cell isolation may account for conflicting published data regarding the levels of the scavenger receptors' expression in healthy individuals. We demonstrate that the detection of CD163, but not CD36, differs dramatically among the methods.

The collection of maternal placental intervillous blood (IVB), without contamination of fetal blood and with an accurate mononuclear cell profile, is essential for immunological studies of placental malaria and other infectious diseases of the placenta. We have compared five documented methods of IVB collection: perfusion, incision, biopsy, tissue grinding, and puncture (prick) for fetal blood contamination and mononuclear cell profiles using flow cytometry. Twenty-five placentas were obtained fromPlasmodium falciparumand human immunodeficiency virus-negative primigravid and secundigravid women delivering at Nyanza Provincial Hospital in Kisumu, western Kenya. Each of the five methods was performed on the same placenta. Fetal red blood cell contamination was significantly lower for the prick and perfusion methods (4.1% and 8.3%, respectively) than for incision (59.5%), biopsy (42.6%), and tissue grinding (19.9%). Significant variation was noted among the five methods in the percentages of monocytes, total T cells, CD4+and CD8+T cells, B cells, and NK cells. Further, a pairwise comparison of prick and perfusion, the two methods with low fetal blood contamination, showed that they were not different for fetal red blood cell contamination levels; however, prick yielded significantly higher percentages of CD4 T cells and CD4 memory T cells than perfusion. Collection by prick was determined to be the best method of intervillous blood collection for immunology studies, and perfusion represented the next best method of choice due to high sample volume yield. Overall, in considering the advantages/disadvantages of the two methods with low fetal cell contamination, we conclude that a combination of prick and perfusion is most suitable for immunology studies.

The determination of functional antipneumococcal capsular polysaccharide antibodies by sequential testing of pre- and postvaccination serum samples one serotype at a time is sample-intensive and time-consuming and has a relatively low throughput. We tested several opsonophagocytic assay (OPA) formats, including the reference killing method, a monovalent bacterium-based flow method, a trivalent bacterium-based flow method, and a tetravalent bead-based flow method using a panel of sera (4 prevaccination and 16 postvaccination, from healthy adults immunized with the 23-valent pneumococcal polysaccharide vaccine). The trivalent and tetravalent methods allow simultaneous measurements of opsonic antibodies to multiple pneumococcal serotypes. The trivalent bacterial-flow OPA had significant correlation to the reference OPA method and to a previously published flow cytometric OPA ( r values ranged from 0.61 to 0.91, P < 0.05) for serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. The tetravalent OPA had significant correlation to all OPA method formats tested ( r values from 0.68 to 0.92, P < 0.05) for all seven serotypes tested. This tetravalent OPA is an alternative to other OPA methods for use during vaccine evaluation and clinical trials. Further, the flow cytometric multiplex OPA format has the potential for expansion beyond the current four serotypes to eight or more serotypes, which would further increase relative sample throughput while reducing reagent and sample volumes used.

Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an “adhesive” state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-μm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3 + CD4 + or CD8 + CD19 − CD16 − CD45RO + CD62L + CD27 + CD57 − . A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.

To increase testing efficiency, a microsphere-based multianalyte immune detection (MAID) system was developed to measure serum immunoglobulin G (IgG) and IgA recognizing two Bordetella pertussis antigens, pertussis toxin (PT) and filamentous hemagglutinin antigen (FHA). The assay was performed as two separate duplexes. One duplex measured IgG to PT and FHA, and the other measured IgA to PT and FHA. The two duplexes were then combined and analyzed as a tetraplex. The MAID system and an in-house enzyme-linked immunosorbent assay (ELISA) system were used to evaluate 100 sera from blood donors and 220 consecutive sera submitted for B. pertussis antibody testing. For both the MAID and ELISA systems, antibody levels were defined as increased if greater than the blood donor group 95th percentile value. The qualitative concordance rates between MAID and ELISA results for the 220 consecutively submitted sera were as follows: PT IgG, 99%; PT IgA, 94%; FHA IgG, 93%; FHA IgA, 94%. The overall concordance rate was 95% (836 of 880 result sets). For 29 of 44 (66%) discordant result sets, the discordant MAID result was supported by the MAID and ELISA results for other B. pertussis antibodies. The MAID and in-house ELISA systems were also used to evaluate 20 sera previously tested for pertussis antibodies at a pertussis vaccine research laboratory; MAID results for all four analytes did not significantly differ from results obtained by the research laboratory. These findings show that antibodies to B. pertussis antigens can be measured easily and accurately using a tetraplex microsphere system.