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PubMed1988

Imported fire ant allergy in young children: skin reactivity and serum IgE antibodies to venom and whole body extract.

We studied 19 young children (aged 15 to 39 months) who had had a systemic allergic reaction after imported fire ant (IFA) sting. Skin testing was performed with whole body extract (WBE) of both Solenopsis species; positive reactions to S. invicta was noted in 94% and to S. richteri in 100%. Serum was tested for total IgE level and for specific IgE antibodies by RAST with WBE and venom (V) of S. invicta. In vitro results were compared with those of 19 age and sex-matched atopic control subjects. The range of total serum IgE level was 31 to 1720 IU/ml (geometric mean 160 IU/ml), compared with 7.5 to 6756 IU/ml (geometric mean 85 IU/ml) in the control subjects. The difference between the two groups, however, was not statistically significant (p greater than 0.05). In the IFA-allergic group, the IFA WBE RAST mean binding was 13.5% (range 2.0% to 30.2%), and the IFA V RAST mean was 29.2% (range 7.8% to 46.7%). The control sera demonstrated low levels of activity to both IFA WBE RAST (0.7% to 4.8%) and IFA V RAST (0.7% to 5.9%). IFA WBE RAST demonstrated that 26% of the IFA-allergic patients had counts within the range of the atopic control subjects. In contrast, the IFA V RAST clearly separated all IFA-allergic patients from their age and sex-matched atopic control subjects. A scoring system for IFA V RAST is proposed. These results demonstrate superiority of IFA V over IFA WBE RAST in the diagnosis of allergy to S. invicta.

Animals↗
PubMed1990

12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoic acid: biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase.

Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-[(5-iodo-4-azido-2-hydroxybenzoyl)amino]dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl [14C]oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent Ki of 150 microM. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid (1 mM) provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions (apparent Ki = 20 microM), and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of 125IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.

Acyl Coenzyme A↗
PubMed2003

Fire ants represent an important risk for anaphylaxis among residents of an endemic region.

BACKGROUND: Imported fire ants (IFA) represent a potential anaphylactic risk to IFA-sensitized individuals. OBJECTIVE: We examined the prevalence of allergic sensitization to IFA, yellow jacket venom (YJV), and peanut in an adult population from an IFA-infested region, Augusta, Georgia. METHODS: Specific IgE to IFA, YJV, and peanut were determined by using the Pharmacia UniCAP assay in 200 random blood donors from an Augusta blood bank. These results were compared with specific IgE to identical allergens in a random sample of blood donors from Oklahoma City, Oklahoma (OKC), a nonendemic region for IFA. RESULTS: Prevalence of IFA-specific IgE (17%) in the Augusta population was significantly higher than to YJV (10%, P =.04) or peanut (7.5%, P =.004). The majority of individuals who had significant IgE to IFA (> or =0.35 kIU/L) did not have IgE to YJV (24/34 = 71%). YJV caused significantly more inhibition of IgE binding to a YJV solid phase than to an IFA solid phase when the 10 dual-positive sera were analyzed (58% vs 11%, P =.005). The prevalence of IFA-specific IgE in adults from Augusta was higher than in OKC (17% vs 2%, P =.0002). YJV-specific IgE was also more prevalent in Augusta compared with OKC (10% vs 6.0%, P =.04), whereas no difference was demonstrated for peanut-specific IgE (7.5% vs 6.5%, P =.6). CONCLUSIONS: Allergic specific IgE to IFA is 1.7 times more common in adults living in an endemic area than specific IgE for other allergens associated with potentially fatal anaphylaxis. This suggests that IFA may pose the greatest risk of anaphylaxis for adults residing in IFA-endemic regions.

Adolescent↗
PubMed1999

Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and -negative human sera by using native and recombinant E. chaffeensis and E. canis antigens.

Human monocytic ehrlichiosis is an emerging infectious disease caused by Ehrlichia chaffeensis, a gram-negative obligatory intracellular bacterium closely related to E. canis. The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes of E. chaffeensis and E. canis, respectively. Western immunoblotting was performed to analyze the antibody responses of the 37 E. chaffeensis indirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30. All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity). Of 37 IFA-positive sera, 34 sera reacted with any native proteins of E. chaffeensis ranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa native E. canis antigens. The 28-kDa E. chaffeensis and 30-kDa E. canis native proteins were recognized by 25 IFA-positive sera. Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera. These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera. Except for three rP30-negative but IFA-positive specimens that instead showed an E. ewingii infection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results. Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer. These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.

Animals↗
PubMed1993

Relationship between collagen-induced and adjuvant arthritis in the Lewis rat.

Adjuvant arthritis (AA) and type II collagen (CII)-induced arthritis (CIA) in the rat serve as models of chronic human arthritis. Adoptive transfer of AA was observed in 21 of 25 Lewis rats given concanavalin A (Con A)-treated spleen cells prepared from animals immunized with Mycobacterium butyricum in mineral oil (complete Freund's adjuvant, CFA). No arthritic changes were noted in rats given spleen cells obtained from donors that had received incomplete Freund's adjuvant (IFA, 0/22), type I collagen in IFA (CI-IFA, 0/6) or CII-IFA (0/28). Administration of spleen cells from IFA, CI-IFA or CII-IFA-injected animals did not modify the development of CIA when these rats were subsequently challenged with CII-IFA. However, partial protection against induction of AA was provided by the transfer of spleen cells prepared from rats immunized with CII-IFA (6/11) but not by those obtained from rats injected with IFA (1/15) or CI-IFA (0/3). Rats that did not develop clinically evident arthritis following the administration of spleen cells prepared from CFA-injected rats were also resistant to AA induction by CFA. Pre-treatment of rats with a synthetic peptide, corresponding to amino acids 180-188 of the Mycobacterium 65 kD heat shock protein (65 kD HSP), significantly delayed the onset of AA, but not that of CIA. Disease-specific resistance to AA, provided by spleen cells prepared from rats injected with CII-IFA and by pre-treatment with the 65 kD HSP 180-188 peptide, may result from the induction of protective tolerance to arthritogenic epitopes present in the Mycobacterium and CII preparations.

Amino Acid Sequence↗
PubMed1999

[Serum antibodies to rubella virus in healthy individuals with indirect immunofluorescent method].

To investigate sensitivity to rubella virus (RV) in healthy individuals, we examined levels of antibodies to RV in sera by an indirect immunofluorescence assay (IFA) and compared levels of antibodies by IFA with those by a hemagglutination inhibition (HI) assay. Of 114 healthy individuals, we detected antibodies to RV in serum specimens from 103 (90.3%) by IFA and in those from 109 (95.6%) by HI assay. The peak value of levels of antibodies by HI assay was 4 fold higher than that by IFA. When levels of antibodies by IFA were less than 32, levels of antibodies by HI assay ranged from < 8 to 1024. We did not detect anti-rubella antibodies of IgM class in all serum specimens and detected anti-rubella antibodies of IgA class in serum from only 1 individual by IFA. We detected antibodies to rubella in sera from 51 (94.4%) by IFA and in sera from 52 (96.3%) by HI assay of 54 individuals who reported having had rubella, and in sera from 23 (88.5%), by IFA and in sera from 26 (100%) by HI assay of 26 individuals reported having been vaccinated. Also, we detected anti-rubella antibodies in sera from 13 (76.5%) by IFA and in sera from 15 (88.2%) by HI of 17 individuals who reported having had neither rubella nor vaccination. In serum from 1 individual who reported having had rubella, we detected antibodies to rubella by IFA but not by HI assay. In serum specimens from 2 individuals who reported having had rubella vaccination, from 3 having had vaccination, from 2 having had neither rubella nor vaccination, we detected anti-rubella antibodies by HI assay but not by IFA. On the other hand, by both assays, we detected antibodies to RV in all sera of individuals who reported having had rubella and been vaccinated. The serodiagnosis, at least, by two methods is necessary to prevent individuals from rubella virus infection, because of following results: 1) influence of an inhibitor in serum specimens was thought to be variable. 2) The results measured by IFA were differed from those by HI assay in some individuals. 3) It is difficult in diagnosis of rubella from clinical symptoms alone. Also, it might be required to use vaccine to the individual who lacks detectable antibodies to rubella in serum by any method to prevent rubella infection.

Adult↗
PubMed2000

New immunofluorescence assays for detection of Human herpesvirus 8-specific antibodies.

Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a "gold standard." Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (kappa > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV(+)) Kaposi's sarcoma (KS) patients, HIV(+) KS(-) patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.

Animals↗
PubMed2006

Enantiomer-enantiomer interaction of ifosfamide in the rat.

The objective of this study was to study the enantiomer-enantiomer interaction of ifosfamide (IFA) in a rat model. Following intravenous administration of individual IFA enantiomers or pseudo-racemates to male Sprague-Dawley rats, two enantiomers and their metabolites, 4-hydroxyIF (HOIF), N2-dechloroethylIF (N2D), N3-dechloroethylIF (N3D), and isophosphoramide (IPM), were quantified using gas chromatography/mass spectrometry (GC/MS) and isotope dilution techniques. In addition, the mutual inhibition in the metabolism between two stereoisomers was also investigated in vitro using rat liver microsomes. Pharmacokinetic parameters were similar between (R)-IFA and (S)-IFA when individual enantiomers were intravenously administered to rats separately. However, in the rats administered with the IFA racemate, half-life, mean residence time (MRT), and area under the concentration-time curve (AUC) values of (S)-IFA were significantly increased with total body clearance (CLT) being decreased. No significant difference in volumes of distribution (Vss), and renal clearance (CLr) and blood cell partition was observed between two enantiomers regardless of (R)-IFA and (S)-IFA being administered separately or in combination as a racemate. The results from the in vitro metabolism and inhibition experiment suggested that each IFA enantiomer inhibited the metabolism of its antipode in a competitive manner. It is concluded that the enantiomeric interaction of IFA mainly occurred in the process of metabolism with (S)-IFA being affected to a larger extent.

Animals↗
PubMed1996

Novel indirect fluorescent antibody test for Lyme disease.

An indirect fluorescent antibody (IFA) test was developed using a novel format of Borrelia burgdorferi organisms adhered to a monolayer of cultured endothelial cells derived from an equine tumor. Sensitivity and specificity of the new IFA test for detecting anti-B, burgdorferi antibodies were evaluated using sera from dogs inoculated with live B. burgdorferi or vaccinated with B. burgdorferi bacterin or leptobacterins and from unvaccinated specific-pathogen-free (SPF) dogs. To compare the new IFA test with existing tests, serum samples were submitted to independent laboratories to be tested by enzyme-linked immunosorbent assay (ELISA) and a traditional IFA test. Samples were also tested with 2 commercially available membrane-bound ELISA kits. Both Borrelia-inoculated dogs and dogs vaccinated with B. burgdorferi bacterin developed levels of antibody detectable by the new IFA test. Dogs vaccinated with a combination canine vaccine or leptobacterin for food animal use developed detectable levels of antibody against Leptospira but remained seronegative for Borrelia by the new IFA test, as did the unvaccinated SPF dogs. The new IFA test was sensitive, detecting antibodies against B. burgdorferi as early as 7 days postinoculation. It was also specific, showing no cross-reactivity with anti-Leptospira antibodies induced by vaccination with leptobacterins. The new IFA test compared favorably with both the standardized traditional IFA test and ELISA. Results from both membrane-bound ELISA kits were not consistent when compared with each other or with the new IFA test. The new IFA test had low nonspecific fluorescence, which made it easier to evaluate and reduced the human error and variability of test results.

Animals↗
PubMed2004

New indirect immunofluorescence assay as a confirmatory test for human immunodeficiency virus type 1.

BACKGROUND: Screening of blood and blood products for human immunodeficiency virus (HIV) is routinely performed using the enzyme-linked immunosorbent assay (ELISA), and the results confirmed by Western blot (WB). However, western blot is expensive and mostly performed in developed countries. A technique more superior or comparable to WB and adaptable to developing countries must be sought. In an effort to identify such a technique, this study determined the efficiency of indirect immunofluorescence assay (IFA) to detect antibodies to HIV-1. OBJECTIVE: To determine the accuracy and sensitivity of an in-house immunofluorescence assay (IFA) to detect antibodies to HIV-1 in plasma. DESIGN: A comparative study to evaluate the performance of indirect immunofluorescence assay (IFA) and western blot (WB) techniques in the detection of antibodies to HIV-1. SETTING: Kenya Medical Research Institute, Centre for Virus Research. The study was conducted between June and December 2001. METHODS: The evaluation of IFA as a technique for detecting antibodies to HIV-1 utilized a total of 400 samples. For these samples, IFA was compared with ELISA and particle agglutination (PA) (manuscript under preparation). Of the 400 samples, there were discrepant results in the three assays in only 36 samples. IFA was compared with Western blot (WB) to confirm the true HIV-1 serostatus in these 36 plasma specimens. The IFA technique used acetone-fixed HIV-1 infected MOLT-4 cells in one spot on a Teflon coated slide and uninfected MOLT-4 cells alone in a second spot to asses non-specific fluorescence. Western blot was performed according to the instructions of the manufacturer. RESULTS: The sensitivity and specificity of IFA based on 36 plasma specimens tested was 71.4% and 100% respectively. All samples that were HIV seronegative by WB were also HIV seronegative by IFA. However, two (5.6%) samples were HIV seronegative by IFA but seropositive by WB. CONCLUSION: The data obtained show that IFA can be used as a primary confirmatory test in Kenya.

AIDS Serodiagnosis↗
PubMed1999

Use of ELISA to measure antinuclear antibodies in children with juvenile rheumatoid arthritis.

OBJECTIVE: To compare a series of commercial ELISA tests with an indirect immunofluorescent antibody (IFA) test for the detection of antinuclear antibodies (ANA) in children with juvenile rheumatoid arthritis (JRA). METHODS: Sera from 178 patients with JRA (88 pauciarticular, 68 polyarticular, 22 systemic) were compared with 26 healthy pediatric subjects. Twenty-one samples from patients with systemic lupus erythematosus (SLE) were also tested. All samples were analyzed by IFA and by 3 commercial ELISA methods. Concordance of ELISA results with IFA results (selected standard) were used as a measure of performance. Sensitivity and specificity were calculated for each test and likelihood ratios (LR) were established for IFA and ELISA in pauciarticular and polyarticular JRA sera. The increment in pretest probability was then obtained for each test as an additional measure of test performance. RESULTS: IFA rendered positive results on 18-77% of the JRA sera depending upon the subset, 100% of SLE sera, and 15% of normal patient sera. Using IFA as the standard, correspondence with positive results among patients with JRA ranged from 0 to 74% for the 3 ELISA tests, while it ranged from 5 to 73% in IFA negative sera. IFA tests showed intermediate range likelihood ratios (0.3, 0.5, 3.5, and 5) and increments in pretest probability ranging from 25 to 45%. While one of the ELISA tests attained 50% of increment in pretest probability for the positive test, it showed 0% increment as a negative test. The other 2 ELISA tests incremented the pretest probability from 0 to 25%. CONCLUSION: Our findings indicate that in JRA, the lack of correspondence with the historic standard IFA precludes the use of ELISA tests for detection of ANA. In addition, IFA out-performs ELISA by a substantial degree when "clinical utility" analysis of test performance is utilized. Detection of ANA in children with JRA should either continue to rely on IFA or be based on a different set of antigens if an ELISA format is chosen.

Antibodies, Antinuclear↗
PubMed1987

Comparison of four serotests for the detection of Dirofilaria immitis infection in dogs.

Three hundred two dogs were tested with 4 serotests for heartworm antigen (AG) or antibody (AB) and with the Knott test. The 4 serotests evaluated were an enzyme-linked immunosorbent assay (ELISA) for adult heartworm-specific AB (AB-ELISA), a quantitative, indirect immunofluorescent assay (IFA) for adult heartworm-specific AB (AB-IFA), an IFA test for microfilaria (MF)-specific AB (MF-IFA), and an ELISA for adult heartworm AG (AG-ELISA). The presence of heartworms was ascertained in all dogs by necropsy examination. Of 302 dogs, 20 (6.6%) had heartworms in the heart at necropsy. Of infected dogs, 9 (45%) had occult infections. Test sensitivities were 75%, 95%, 70%, and 75% for the AB-ELISA, AB-IFA, MF-IFA, and AG-ELISA, respectively. Test specificities were 85% (AB-ELISA), 77% (AB-IFA), 87% (MF-IFA), and 99% (AG-ELISA). The best agreement between serotest results and necropsy findings was obtained with the AG-ELISA (97%). The 4 serotests detected 86% (AB-ELISA), 100% (AB-IFA), 67% (MF-IFA), and 78% (AG-ELISA) of the dogs with occult heartworm infection. A significant (P less than 0.05) association between intestinal parasitism and positive heartworm test results was found with only AB-IFA. Seemingly, the Knott test, or some other concentration method for detecting circulating MF should be the first heartworm test performed. If the examination for MF is negative, the dog has clinical signs, and radiographic findings are suggestive of occult heartworm infection, then a serotest for adult heartworm AG is recommended.

Animals↗
PubMed2001

IgA antibodies against endomysium and transglutaminase: a comparison of methods.

Recently, the endomysial antigen has been identified as the protein cross-linking enzyme known as tissue transglutaminase (tTG). Our objective was to compare a novel enzyme immunoassay (EIA) that detects IgA antibody against tTG to two standard IFA methods utilizing thin tissue sections of rat kidney/rat stomach (KS) and distal primate esophagus (PE) as substrates to detect IgA antibody against endomysium (EMA). Sera from 100 patients suspected of having gluten-sensitive enteropathy (GSE) and 23 sera possessing various antibodies used for EIA cross-reactivity studies were included. Additional tests, performed routinely in our laboratory, were utilized to further assess sera from patients suspected having GSE. These tests include anti-gliadin IgA antibody (AGA) and anti-reticulin IgA antibody (ARA) and are part of the European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) revised criteria for diagnosing GSE. When compared to IFA using KS, the tTG EIA had a sensitivity of 87.5%, was 97.1% specific, and had an overall agreement of 94.0%. When compared to IFA using PE, the tTG EIA had a sensitivity of 92.6%, was 93.2% specific, and had an overall agreement of 93.0%. When the KS IFA was compared to the PE IFA for EMA, the KS IFA had a sensitivity of 96.3%, was 91.8% specific, and had an overall agreement of 93.0%. The majority of sera that were positive for tTG but were negative by IFA (KS, n = 2/PE, n = 5) possessed IgA antibodies against gliadin and/or reticulin. Five of six sera with negative results by PE IFA were positive by the KS IFA and possessed one or more antibodies to tTG and/or gliadin and/or reticulin. We conclude that the tTG EIA compares well to both KS and PE IFAs when detecting IgA antibody against endomysium. We do not recommend the use of PE to detect EMA primarily because of the inconsistencies (i.e., tissue selection, quality, and preparation) and limited availability of commercially prepared PE tissue.

Animals↗
PubMed1995

Isolation, properties and chromosomal localization of four closely linked hamster interferon-alpha-encoding genes.

Three recombinant phages containing hamster interferon-alpha-encoding genes (Ha Ifa) were isolated from a Ha genomic library, using a murine (Mu) Ifa probe. The phage inserts contained overlapping genomic fragments which span a total length of approx. 30 kb, on which four Ha Ifa genes are localized. The Ifa gene cluster could be assigned to hamster chromosome 2q. The nt sequences of the four Ifa genes were determined. Two of the genes are functional (Ha Ifa-1 and Ifa-3) and two are pseudogenes (Ifa-ps2 and Ifa-ps4). Ha Ifa-1 and -3 were transiently expressed in COS cells and they gave rise to protein products (A1 and A3, respectively) with antiviral properties on hamster CHO cells. In addition, Ha A1 revealed high antiviral activity on murine L929 cells.

Amino Acid Sequence↗
PubMedDate not supplied

Two-dimensional color Doppler estimation of the severity of atrioventricular valve regurgitation: important effects of instrument gain setting, pulse repetition frequency, and carrier frequency.

Literature reports of good correlation between two-dimensional color Doppler and ventriculography for estimation of the severity of atrioventricular valve regurgitation have appeared. Local experience has been variable. The purpose of this study was to evaluate three factors for their effect on two-dimensional color Doppler display of the apparent area of regurgitant flow. The factors studied were instrument gain setting, pulse repetition frequency (PRF), and carrier frequency. Sixteen regurgitant valves were examined with high quality commercial instrumentation from two manufacturers. Electrocardiographic-triggered image gating was used for recording and planimetry of maximal imaged flow area (IFA). Examinations were performed at two gain settings with constant carrier frequency and PRF, at three carrier frequencies (2.5, 3.5, and 5 MHz) at constant gain, and at differing PRF (3.0 to 12 kHz). Reduction in gain setting made little difference in asthetic quality of regurgitant jets, but their areas decreased by 29%. Carrier frequency was found to greatly affect IFA, with each higher carrier frequency giving significantly smaller IFAs. PRF also affected IFA consistently, with progressively smaller IFAs at each higher PRF. There was no useful correlation between IFA of a single jet evaluated on the two instruments at generally similar settings. It is concluded that instrument gain setting, PRF, and carrier frequency each significantly affects the size of IFA within a single instrument and between different instruments. Given the variety of instruments and settings employed within the cardiology community, it is unlikely that even moderately accurate grading of severity of regurgitation based on IFA can be done unless instrument settings are specified and standardized. There is a clear need for annotation of instrument settings during color Doppler examinations and a need for concensus regarding "optimal" settings. Finally, the consistency of reduction in IFA with increasing carrier frequency and PRF within each of the instruments tested suggests investigation of "normalization" of IFA displays.

Adolescent↗
PubMed2006

Comparison of an indirect immunofluorescence assay, western blot analysis, and a commercially available ELISA for detection of Ehrlichia canis antibodies in canine sera.

OBJECTIVE: To examine the correlation between results for an indirect immunofluorescence assay (IFA) that uses Ehrlichia canis antigen as a substrate (ie, E canis-IFA), 2 western blot (WB) analyses, and a commercially available ELISA in the detection of E canis antibody in dog sera. SAMPLE POPULATION: 54 canine serum samples that were reactive on E canis-IFA and 16 canine serum samples that were E canis-IFA nonreactive. PROCEDURE: Serum samples were evaluated by use of 2 WB analyses and a commercially available ELISA. Correlation between results of the 3 testing modalities (ie, IFA, WB analyses, and the ELISA) was examined by use of nonreactive (E canis-IFA reciprocal titer, < 20), low-titer (reciprocal titer, 80 to 160), medium-titer (reciprocal titer, 320 to 2,560), and high-titer (reciprocal titer, 5,120 to > 20,480) serum samples. RESULTS: For all serum samples in the nonreactive (n = 16), medium-titer (17), and high-titer (18) groups, correlation of results among IFA, WB analyses, and the commercially available ELISA was excellent. A poor correlation was found between IFA results and those of WB analyses and the ELISA for serum samples in the low-titer group (19), with only 4 of the 19 serum samples having positive results on both WB analyses and the commercially available ELISA. CONCLUSIONS AND CLINICAL RELEVANCE: The discrepancy between E canis-IFA, WB analyses, and the commercially available ELISA results for the low-titer serum samples may be related to a high IFA sensitivity or, more likely, a lack of specificity associated with cross-reactivity among Ehrlichia spp.

Animals↗
PubMed1996

Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (FA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum.

A commercial enzyme immunoassay (EIA) (ProSpect Rapid Assay), a direct immunofluorescence antibody (IFA) test for stool testing (MERIFLUOR Cryptosporidium/Giardia), and an indirect IFA test for environmental testing (Hydrofluor-Combo Cryptosporidium/Giardia) were evaluated for detection of low public health risk Cryptosporidium oocyst isolates, and for C. parvum oocyst isolates from human and bovine feces that represent a high public health risk. There was no cross-reactivity of EIA with ova of eight medically important helminths, three Eimeria species oocysts, Sarcocystis cruzi sporocysts, and two Candida sp. isolates. All nine snake oocyst isolates (C. serpentis), two of seven lizard oocyst isolates, one turtle oocyst isolate, two avian oocyst isolates (turkey, C. meleagridis), one C. wrairi oocyst isolate from guinea pigs, one C. muris oocyst isolate from hyrax, one heifer C. muris isolate, and two C. muris-like oocyst isolates from a camel were positive by both IFA tests; six of these 19 oocyst isolates were EIA-positive. There was no difference in the sensitivity and specificity between direct and indirect IFA tests. The sensitivity of the EIA and both IFA tests to the C. parvum oocysts was 100%. The EIA showed less cross-reactivity with the non-C. parvum oocysts (24%) than direct or indirect IFA (76%), and was less sensitive to those isolates (20%) than both IFA tests (63%). A simulated sampling model for high and low public health risk Cryptosporidium oocysts showed that the low risk oocyst isolates may constitute up to 35% of all positive environmental samples by direct or indirect IFA determination, and up to 12% of all EIA positive samples. This study indicates a superiority of direct and indirect IFA and EIA for screening of human-or-bovine-origin fecal specimens, whereas testing of environmental samples may lead to misidentification of medically important isolates. The results demonstrated that the EIA kit can more accurately identify environmental samples containing oocytes pathogenic for humans than both IFA tests. The specificity of commercially available diagnostic kits to C. parvum should be critically examined for cross-species identification before they are recommended or adopted for use in testing environmental samples.

Animals↗
PubMedDate not supplied

Human immunodeficiency virus (HIV)-specific immune responses are generated with the simultaneous vaccination of a gp120-depleted, whole-killed HIV-1 immunogen with cytosine-phosphorothioate-guanine dinucleotide immunostimulatory sequences of DNA.

OBJECTIVES: We compared the effect of priming with a synthetic oligodeoxynucleotide (ODN) immunostimulatory DNA sequence followed by vaccination with human immunodeficiency virus type 1 (HIV-1) in incomplete Freund's adjuvant (IFA) or HIV-1 antigen alone to the simultaneous administration of immunostimulatory sequences (ISS) with HIV-1 in IFA. METHODS: We examined immune function involving interferon-gamma (IFN-gamma) production, RANTES (regulated upon activation, normal T cell expressed and secreted) production, and lymphocyte proliferation, all of which appear to be augmented in HIV-1-exposed, but uninfected, individuals. RESULTS: We demonstrate that similar levels of antigen-specific IFN-gamma were produced from lymph node cells of the animals immunized with HIV-1 antigen in IFA containing the CpG ODN 1826 (ISS; mean +/- SE = 450.8 +/- 224.3 pg/mL) and the group of animals primed with the ODN before injection with the HIV-1 in IFA (mean +/- SE = 377.7 +/- 294.8 pg/mL) or HIV-1 antigen alone (IFN-gamma = 0 pg/mL). However, the group that received the HIV-1 in IFA plus ISS mounted a stronger lymphocyte proliferation (mean net +/- SE = 29,180 +/- 1,932 cpm) compared to the group primed with the ODN before injection with HIV-1 in IFA (mean net +/- SE = 8,575 +/- 2,978 cpm). Furthermore, the group that received the HIV-1 in IFA plus ISS also mounted stronger beta-chemokine production measured as RANTES (mean +/- SE = 1,217 +/- 267.4 pg/mL) compared to the group that received the ODN before injection with HIV-1 in IFA (mean +/- SE = 129.1 +/- 48.5 pg/mL). Antibody responses from the group that received the HIV-1 in IFA plus ISS also showed a higher p24-specific response that was predominantly of the immunoglobulin G IgG2b isotype. CONCLUSION: These results suggest that the simultaneous administration of the ISS in the HIV-1 in IFA emulsion may be a condidate for testing in non-human primates and in human studies as a therapeutic and preventative vaccine.

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