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PubMed2005

New criteria for immunofluorescence assay for Q fever diagnosis in Japan.

A study was made to evaluate the cutoff value of indirect immunofluorescent-antibody (IFA) test for Q fever diagnosis in Japan. We used 346 sera, including 16 from confirmed Q fever cases, 304 from Japanese pneumonia patients, and 26 from negative cases. Thirteen sera from the confirmed Q fever cases with an immunoglobulin M (IgM) titer of > or =1:128 and/or IgG titer of > or =1:256 by the IFA test were positive by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assay (WBA), whereas 298 sera from pneumonia patients and 26 negative sera with an IgM titer of < or =1:16 and an IgG titer of < or =1:32 by the IFA test were negative by both ELISA and WBA. In the proposed "equivocal area," with an IgM titer of > or =1:32 and < or =1:64 and/or an IgG titer of > or =1:64 and < or =1:128, we found 9 sera, 3 from confirmed Q fever cases and 6 from Japanese pneumonia patients, by the IFA test. Three sera from the confirmed Q fever cases and one of the sera from pneumonia patients were IgM and/or IgG positive by both ELISA and WBA. These results suggest that a single cutoff value for the IFA test may cause false-positive and false-negative results. In conclusion, this study showed that an "equivocal area" should be used for the IFA test rather than a single cutoff value and that sera in the equivocal area should be tested by additional serological assays for confirmation.

Antibodies, Bacterial↗
PubMed1978

Immune response in urinary tract infection determined by radioimmunoassay and immunofluorescence: serum antibody levels against infecting bacterium and Enterobacteriaceae common antigen.

A solid-phase radioimmunoassay (RIA) procedure was compared with the indirect fluorescent antibody (IFA) test in a serological study of 76 female adults with urinary tract infections. Relative serum antibody activity was determined against patients' homologous infecting enterobacteria by RIA and IFA and against heterologous enterobacterial common antigen (Escherichia coli O14) by RIA. There was marked correlation between results of the IFA and RIA methods using the homologous system; 22 of 51 patients (43%) with pyelonephritis had significantly elevated serum antibody activity by both IFA (titers greater than or equal to 512) and RIA (binding ratio greater than or equal to 2.0) when compared with normal serum controls; three had significant antibody activity detectable by RIA only. Eighteen (72%) of 25 patients with pyelonephritis had RIA binding ratios of greater than or equal to 2.0 against their homologous bacterial isolates and the enterobacterial common antigen; an additional 6 patients had binding ratios of greater than or equal to 2.0 against the antigen only. All 25 patients with cystitis had low serum antibody levels by IFA and RIA when tested against their own isolate as well as enterobacterial common antigen. The RIA procedure was objective, quantitative, and less tedious to perform than IFA.

Adult↗
PubMed1979

Sensitive microplate enzyme-linked immunosorbent assay for detection of antibodies against the scrub typhus rickettsia, Rickettsia tsutsugamushi.

A microtiter enzyme-linked immunosorbent assay (ELISA) has been developed for the titration of antibodies against scrub typhus in human and animal sera. Scrub typhus rickettsiae were grown in monolayers of irradiated mouse LM3 cells and separated from host cell materials by differential centrifugation, filtration through a glass filter (AP-20, Millipore Corp.), and isopycnic banding in Renografin density gradients. The scrub typhus ELISA antigens were obtained from the purified viable rickettsiae by French pressure cell disruption and addition of 0.2% Formalin to the soluble extract. Antisera prepared in rabbits against the prototype Karp, the Kato, and the Gilliam strains of scrub typhus were used to standardize the ELISA and to compare its sensitivity and specificity to that of the indirect fluorescent antibody test (IFA). ELISA titers were measured as the greatest serum dilution showing an optical density 0.25 above controls or by the optical density achieved at a fixed serum dilution. The IFA and ELISA end point titers were quite similar, and all three measures of titer had comparable specificity for the strains of scrub typhus. No cross-reactions between the typhus and scrub typhus wera were observed by ELISA. Both the immunoglobulin M (IgM) and IgG antibody titers of 12 sequential sera from four patients with scrub typhus were obtained by IFA and ELISA. The IFA and ELISA end point titers for IgM and IgG had correlation coefficients of 0.91 and 0.97, respectively, whereas the ELISA optical density values at a serum dilution of 1:100 had slightly lower correlations with IFA titers (0.80 and 0.94). Early rising IgM titers followed by rising IgG titers were demonstrated by ELISA in three patients with primary scrub typhus infections, whereas the IgG response predominated in a patient with a reinfection. It is concluded that the ELISA for scrub typhus is a very satisfactory alternative to the IFA test.

Animals↗
PubMed1996

Iris indocyanine green videoangiography in diabetic iridopathy.

AIMS/BACKGROUND: Iris fluorescein angiography (IFA) is not commonly used in clinical practice, although its value has been demonstrated especially in cases of diabetic disease. IFA is able to show neovascular tufts in order to guide the laser treatment, and it is highly recommended in diabetic patients who need cataract surgery or vitrectomy. Nevertheless, IFA fails to demonstrate the iris vascular pattern in heavily pigmented iris and conspicuous leakage cases. The aim of the study was to evaluate the feasibility of iris indocyanine green videoangiography (IICGV) and to correlate its findings with those of IFA in diabetic iridopathy. METHODS: Thirty six patients affected in varying degrees by diabetic retinopathy underwent an ophthalmic examination including retinal fluorescein angiography, IFA, and IICGV. IICGV was performed using IMAGEnet System H1024. RESULTS: The results demonstrated that IICGV allows precise visualisation of the iris vascular pattern, also in cases of heavily pigmented iris. CONCLUSIONS: Three main findings seemed to be evident: firstly, iris neovascularisations are detected with IFA far more easily than IICGV; secondly, capillary dilatations and iris hypoperfusion are identified far more clearly using IICGV; thirdly, there is no evident relation between capillary dilatation or iris hypoperfusion, and degree of diabetic retinopathy.

Aged↗
PubMed2003

Improved detection of dengue-1 virus from IgM-positive serum samples using C6/36 cell cultures in association with RT-PCR.

Dengue is the most important arboviral disease in the world, and its diagnosis is primarily made by serology. Virus isolation has been successful mainly in clinical samples obtained during the acute phase of illness, and is carried out through inoculation of clinical samples into C6/36 cell monolayers followed by the detection of infection by indirect immunofluorescence assay (IFA). We compared the efficiency of RT-PCR and IFA in the detection of dengue-1 virus after inoculation of C6/36 cells with samples obtained in the convalescent period of dengue infection. Out of 75 IgM-positive samples inoculated into C6/36 cells, 2 were positive by IFA while 17 were positive by RT-PCR. The 2 IFA-positive samples were collected during the acute phase of the illness; 17 positive samples were found by RT-PCR, including the 2 detected by IFA. For both methods, we also investigated the time necessary for viral detection using a fixed dose of 1 x 10(4) viruses/ml. RT-PCR and IFA detected the dengue virus 1 and 4 days after virus inoculation, respectively. The results obtained here indicate that RT-PCR is the most sensitive method in the detection of dengue viruses using C6/36 cells for viral isolation.

Animals↗
PubMed1999

Independent factor analysis.

We introduce the independent factor analysis (IFA) method for recovering independent hidden sources from their observed mixtures. IFA generalizes and unifies ordinary factor analysis (FA), principal component analysis (PCA), and independent component analysis (ICA), and can handle not only square noiseless mixing but also the general case where the number of mixtures differs from the number of sources and the data are noisy. IFA is a two-step procedure. In the first step, the source densities, mixing matrix, and noise covariance are estimated from the observed data by maximum likelihood. For this purpose we present an expectation-maximization (EM) algorithm, which performs unsupervised learning of an associated probabilistic model of the mixing situation. Each source in our model is described by a mixture of gaussians; thus, all the probabilistic calculations can be performed analytically. In the second step, the sources are reconstructed from the observed data by an optimal nonlinear estimator. A variational approximation of this algorithm is derived for cases with a large number of sources, where the exact algorithm becomes intractable. Our IFA algorithm reduces to the one for ordinary FA when the sources become gaussian, and to an EM algorithm for PCA in the zero-noise limit. We derive an additional EM algorithm specifically for noiseless IFA. This algorithm is shown to be superior to ICA since it can learn arbitrary source densities from the data. Beyond blind separation, IFA can be used for modeling multidimensional data by a highly constrained mixture of gaussians and as a tool for nonlinear signal encoding.

Algorithms↗
PubMed1996

A comparative study of histological conditions suitable for both immunofluorescence and in situ hybridization in the detection of Herpesvirus and its antigens in chicken tissues.

Our objective was to identify an optimal single set of conditions for use in both indirect immunofluorescence assays (IFA) and in situ hybridization (ISH) to detect viral proteins and nucleic acids in avian lymphoid and neural tissues. Various fixatives were evaluated for use with IFA to detect turkey Herpesvirus (HVT) glycoprotein B (gB) and ISH to identify HVT mRNA in chicken tissues. A precipitating fixative (acetone) was compared to crosslinking fixatives [buffered glutaraldehyde-picric acid (BGPA), 10% formalin, and 4% paraformaldehyde] for both IFA and ISH using spleen, thymus, bursa, sciatic plexus, and brachial plexus of 28-day-old chickens. Four percent paraformaldehyde was found to be the optimal fixative for preservation of all chicken tissues examined with both IFA and ISH. Glass slide preparation, incubation temperatures, and tissue processing were each individually evaluated for ISH and IFA. Silylated slides provided the best retention of tissue sections for both procedures. For IFA, 37 degrees C was the ideal incubation temperature tested, whereas the optimal incubation temperature tested for ISH was 47 degrees C. Of the blocking agents compared, Evans blue dye prevented background fluorescence to a greater extent than either calf serum or bovine serum albumin. These findings provide a technical basis for investigations into various aspects of the molecular pathology of avian diseases.

Animals↗
PubMed2005

Soy isoflavone aglycone modulates a hematopoietic response in combination with soluble beta-glucan: SCG.

Soy isoflavone aglycones (IFAs) have a wide range of biological actions that suggest they may be of use in cancer prevention. On the other hand, a branched beta-glucan from Sparassis crispa (SCG) is a major 6-branched 1,3-beta-D-glucan in an edible/medicinal mushroom: Sparassis crispa showing antitumor activity. We have previously reported that both oral and intraperitoneal administration of SCG enhanced the hematopoietic response in cyclophosphamide (CY)-induced leukopenic mice. In this study, we investigated the hematopoietic response due to IFA in combination with SCG in CY-induced leukopenic mice. The oral administration of IFA in combination with SCG synergistically enhanced the number of white blood cells, and increased spleen weight. Analyzing the leukocyte population by flow cytometry, the combination of IFA and SCG increased the number of monocytes and granulocytes in the spleen. Taken together, the combination of IFA and SCG synergistically provides the hematopoietic responses that are enhanced over IFA or SCG alone.

Administration, Oral↗
PubMed2000

Revisiting tolerance induced by autoantigen in incomplete Freund's adjuvant.

Injection of autoantigens in IFA has been one of the most effective ways of preventing experimental, T cell-mediated, autoimmune disease in mice. The mechanism that underlies this protection has, however, remained controversial, with clonal deletion, induction of suppressor cells or of type 2 immunity being implicated at one time or another. Using high resolution enzyme-linked immunospot (ELISPOT) analysis, we have revisited this paradigm. As models of autoimmunity against sequestered and readily accessible autoantigens, we studied experimental allergic encephalomyelitis, induced by myelin oligodendrocyte glycoprotein, proteolipid protein, myelin basic protein, and renal tubular Ag-induced interstitial nephritis. We showed that the injection of each of these Ags in IFA was immunogenic and CD4 memory cells producing IL-2, IL-4, and IL-5, but essentially no IFN-gamma. IgG1, but not IgG2a, autoantibodies were produced. The engaged T cells were not classic Th2 cells in that IL-4 and IL-5 were produced by different cells. The IFA-induced violation of self tolerance, including the deposition of specific autoantibodies in the respective target organs, occurred in the absence of detectable pathology. Exhaustion of the pool of naive precursor cells was shown to be one mechanism of the IFA-induced tolerance. In addition, while the IFA-primed T cells acted as suppressor cells, in that they adoptively transferred disease protection, they did not interfere with the emergence of a type 1 T cell response in the adoptive host. Both active and passive tolerance mechanisms, therefore, contribute to autoantigen:IFA-induced protection from autoimmune disease.

Adoptive Transfer↗
PubMed1980

Seroepidemiological studies of malaria in pregnant women and newborns from coastal El Salvador.

A cohort of 113 women and their newborns from the coastal area of El Salvador were studied longitudinally to estimate malaria incidence and indirect fluorescent antibody (IFA) response to malaria infection. The district in which the study was conducted had an estimated annual parasite index of 600/1,000 inhabitants, and all malaria infections were treated immediately with a 4-aminoquinoline. In the third trimester of pregnancy, the IFA response to Plasmodium falciparum was significantly depressed. As a result of antimalarial therapy and depressed immune responsiveness, 49% (P. vivax) and 53% (P. falciparum) of the pregnant subjects had a malaria IFA titer less than 1:20 at the time of delivery. Malaria IFA crossed the placenta to the fetus with a step-down of approximately a 4-fold dilution, except for the step-up noted in the P. falciparum titer for 17 of 116 newborns. Due to the overall low prevalence and intensity of maternal IFA, a titer of at least 1:20 was passed to only 23% (P. vivax) and 45% (P. falciparum) of newborns. Passively-acquired malaria IFA degraded with a half-life estimated between 43 and 52 days. During follow-up of infants to 6 months of age, no protection from malaria resulting from passively-acquired antibody could be demonstrated. Because of the limited transplacental immunization of these newborns with antimalarial antibody, it appears that passive immunity can exert little effect on the incidence of infant malaria in coastal El Salvador.

Antibodies↗
PubMed1986

Application of indirect hemagglutination test and indirect fluorescent antibody test for IgM antibody for diagnosis of melioidosis in Thailand.

In hyperendemic areas such as Thailand, rapid diagnosis of melioidosis depends upon both bacteriological culture and serological methods. However, interpretation of indirect hemagglutination (IHA) for melioidosis which is the only test available, is seriously hampered by increased IHA titers present in one-third to one-half of the population. In order to get the best results from the available tests, IHA and indirect fluorescent antibody for IgM (IFA-IgM) were evaluated in controls and patients in Thailand. IHA titers of greater than or equal to 1:40 were considered remote or recent exposure to P. pseudomallei. IHA titers of this level were found in 47.1% of 227 blood donors and 29.5% of 210 sera submitted for other tests, while IFA-IgM was positive in only one donor who had an IHA titer of 1:1,280. IHA was positive in eight out of nine patients with melioidosis with IHA titers of less than 1:20 to 1:2,560. IFA-IgM was positive in six out of seven melioidosis patients whose sera were available for this test including a serum with IHA titer of less than 1:20. Six patients were predisposed by diabetes mellitus. Among sera serologically tested for melioidosis, 33 had IHA titers of 1:80-1:1,280, 10 of which were positive for IFA-IgM. This study demonstrates high background IHA titers among IHA titers among Thai people which greatly limits its use for serodiagnosis of melioidosis. In sharp contrast, serodiagnosis by IFA-IgM was more successful. Positive IFA-IgM among healthy Thais did exist indicating that serologic tests for melioidosis at best are only supplementary to bacteriological culture and clinical awareness.

Animals↗
PubMed1993

Rickettsia conorii in Greece: comparison of a microimmunofluorescence assay and western blotting for seroepidemiology.

Since 1972, there have been no reports of Mediterranean spotted fever (MSF) in Greece. In 1991, a seroepidemiologic survey was conducted in three rural villages in the Province of Fokida in central Greece, using both an immunofluorescence assay (IFA) and Western blot to evaluate the prevalence of specific IgG and IgM antibodies to Rickettsia conorii, and to compare these two techniques. Of 254 sera tested by IFA, 148 (58.3%) were positive for IgG at a titer > or = 32 and 117 (46.1%) at a titer > or = 64. Two sera were positive for IgM at a titer > or = 32. Among the IFA-positive sera, 115 reacted against the specific protein antigen (SPA) using Western blotting and were considered as confirmed positive results, indicating a seroprevalence of 45.3%. Using the SPA reaction as a reference, the IFA diagnostic value of a single serum is poor in such an endemic area, suggesting the need for more specific tests. The results of this study have led us to conclude that for seroepidemiologic use, the IFA is not specific for MSF. Thus, the IFA is useful for sero-epidemiologic analysis only in areas where the true prevalence of MSF has been estimated using Western blotting.

Adult↗
PubMed1995

Evaluation of the immunofluorescent antibody test for the diagnosis of human leptospirosis.

Sera from 175 patients with clinically suspected leptospirosis were tested with the immunofluorescent antibody (IFA) assay and compared with the microscopic agglutination (MA) method. Overall, the IFA test recognized all 58 MA-positive patients with leptospirosis. On acute sera testing, the IFA titer > or = 1:100 was 0.97 specific and more sensitive than the conventional MA method (sensitivities = 0.48 versus 0.17, respectively). None of the 117 MA-negative patients, 101 healthy blood donors, and 93 patients with five diseases commonly confused with leptospirosis had IFA titers > or = 1:400. However, cross-reactivity was seen with sera from patients with syphilis. On serial testing, the IFA antibody first appeared during the first week of illness, peaked by the fourth week, and generally decreased below 1:400 after the fourth month. The IFA test appears to be moderately sensitive and specific for the initial diagnosis of leptospirosis. It could replace the more complicated and less sensitive MA assay.

Adult↗
PubMed1995

Hantavirus infection among Rattus norvegicus in Japan.

Seroepizootiological study of hantavirus infection among 393 urban rats (Rattus norvegicus) captured in six regions in Japan during the period from 1990 to 1994 was carried out by the indirect fluorescent antibody (IFA) test and Western blot (WB). Fifteen out of 393 (3.8%) rat sera were antibody-positive by IFA, i.e., Tokyo Port (12.8%, 6/47), Shimizu Port (5.7%, 2/35), Otaru Port (1.5%, 1/65) and Nagoya City (3.6%, 6/167). In two other regions, i.e., Kasai Seaside Park and Haneda Airport, rat sera were antibody-negative. One serum with a lower IFA titer, 1:64, from Otaru Port was confirmed to be antibody-positive by WB, while two sera from Shimizu Port (IFA titer, 1:32 and 1:64) were not. In Nagoya City, one out of four sera (IFA titer, 1:32) and one of two sera (IFA titer, 1:64) were also confirmed to be antibody-positive by WB. Continuous hantavirus infection among rats in Tokyo Port, Shimizu Port and Nagoya City and the existence of hantavirus among rats in Otaru Port were demonstrated.

Animals↗
PubMed1999

Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles.

The contributions of specific human liver cytochrome P-450 (CYP) enzymes to the activation, via 4-hydroxylation, of the oxazaphosphorine anticancer prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) were investigated. Analysis of a panel of 15 human P-450 cDNAs expressed in human lymphoblasts and/or baculovirus-infected insect cells (Supersomes) demonstrated that CYPs 2A6, 2B6, 3A4, 3A5, and three CYP2C enzymes (2C9, 2C18, 2C19) exhibited significant oxazaphosphorine 4-hydroxylase activity, with 2B6 and 3A4 displaying the highest activity toward CPA and IFA, respectively. CYP2B6 metabolized CPA at a approximately 16-fold higher in vitro intrinsic clearance (apparent Vmax/Km) than IFA, whereas 3A4 demonstrated approximately 2-fold higher Vmax/Km toward IFA. A relative substrate-activity factor (RSF)-based method was developed to calculate the contributions of individual P-450s to total human liver microsomal metabolism based on cDNA-expressed P-450 activity data and measurements of the liver microsomal activity of each P-450 form. Using this method, excellent correlations were obtained when comparing measured versus predicted (calculated) microsomal 4-hydroxylase activities for both CPA (r = 0. 96, p <.001) and IFA (r = 0.90, p <.001) in a panel of 17 livers. The RSF method identified CYP2B6 as a major CPA 4-hydroxylase and CYP3A4 as the dominant IFA 4-hydroxylase in the majority of livers, with CYPs 2C9 and 2A6 making more minor contributions. These predicted P-450 enzyme contributions were verified using an inhibitory monoclonal antibody for 2B6 and the P-450 form-specific chemical inhibitors troleandomycin for 3A4 and sulfaphenazole for 2C9, thus validating the RSF approach. Finally, Western blot analysis using anti-2B6 monoclonal antibody demonstrated the presence of 2B6 protein at a readily detectable level in all but one of 17 livers. These data further establish the significance of human liver CYP2B6 for the activation of the clinically important cancer chemotherapeutic prodrug CPA.

Antibodies, Monoclonal↗
PubMed1999

Fire ants: a continuing community health threat in South Carolina.

Imported fire ants are now firmly established in all 46 counties of South Carolina. In 1998 there were an estimated 660,000 cases in the state of which approximately 33,000 sought medical treatment at an estimated cost of 2.4 million dollars. Residents and visitors are at risk for IFA attacks that may occur indoors as well as outdoors. While IFA sting victims in endemic areas of the state may be less likely to seek medical treatment, patients in recently infested areas seem to be more likely to seek treatment since they are relatively unfamiliar with the multiple, painful IFA stings and pustules. Citizens need to control IFA infestations around and within their homes according to guidelines recommended by the Clemson University Cooperative Extension Service. Regional IFA control programs were discontinued in the past because of costs and environmental chemical concerns. Obviously, we need to support ongoing research aimed at developing improved and safe strategies for the local and regional control of IFA.

Animals↗
PubMedDate not supplied

Detection of antibodies to avian pneumovirus by a micro-indirect immunofluorescent antibody test.

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.

Animals↗
PubMedDate not supplied

[Clinico-morphological and molecular-biological characteristics of idiopathic fibrous alveolitis and lung sarcoidosis].

Lung biopsies of 16 patients with classic idiopathic fibrosing alveolitis (IFA) and 6 patients with sarcoidosis were studied immunohistochemically. It was found that adenomatous hyperplasia (AH) and fibrosing in IFA are potentiated by high expression of thrombocytic growth factor (TGF) and insulin-like growth factor type 1 (IGF-1). Tumor necrosis factor alpha (TNFa) in IFA is actively produced at early stages of the disease. TNFa is drastically reduced in IFA with atypical AH. High level of TNFa produced by alveolar macrophage, pneumocytes type 2 and by other cells is observed in the lungs in sarcoidosis. The level of transforming growth factor (B1-TGF) is rather low in sarcoidosis this being combined with weak interstitial fibrosis (3.75 against 5.06 in IFA). Sarcoidosis granuloma contains proliferating epithelioid cells (as judged by Ki-67), produces a high amount of FTN (4.66), moderate amount of cell fibronectin (2.7), fibroblast growth factor, beta-TGF and IGF-system (1.7; 1.1; 0.8, respectively). Thus, IFA and sarcoidosis are distinguished not only morphologically but by the cytokine profile as well. This implies different approaches to their diagnosis and treatment.

Adult↗