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PubMed1997

Detection of Pneumocystis carinii in induced sputa from immunocompromised patients using a repetitive DNA probe.

A hybridization assay for the detection of Pneumocystis carinii was developed using a repetitive DNA fragment of P.c. hominis. The assay was specific as different micro-organisms typically found in the respiratory tract, normal human lung DNA (A 549 cell line) and normal rat lung DNA did not react with the repetitive probe. In a slot blot (SB) hybridization assay, the repetitive probe was able to detect as few as 100 P.c. hominis organisms with no false-positives. The results of the SB hybridization assay were compared with an immunofluorescence (IFA) assay for the detection of P.c. hominis in 84 induced sputum (IS) samples obtained from 52 human immunodeficiency virus (HIV)-seropositive patients, 22 HIV-seronegative patients and 10 healthy individuals. Samples from 24 patients clinically diagnosed with P. carinii pneumonia (PCP) were positive for P.c. hominis by both assays. In addition, the SB assay detected P.c. hominis in 14 patients (10 HIV-positive and four HIV-negative) who were negative by IFA. All 14 samples showed a positive PCR signal for the P.c. hominis dihydrofolate reductase gene, further confirming the presence of P.c. hominis in these specimens. Twelve of these patients had a clinical course highly suggestive of PCP and were either on P. carinii prophylaxis or P. carinii chemotherapy. The other two samples were from HIV-positive patients who had respiratory illness due to causes other than P.c. hominis (disseminated histoplasmosis and fatal Bordetella pneumonia). Detection of P.c. hominis in these samples suggests that these patients may have subclinical colonization by P.c. hominis. Furthermore, P.c. hominis was detected in all 12 sequential IS samples from six AIDS patients who had primary episodes of PCR using the SB assay, while P.c. hominis was detected only in eight samples by IFA (66.6%). All six patients developed recurrent PCP within 6 months from the time the assays were performed, further illustrating the potential of the SB hybridization assay in monitoring PCP recurrence. Thus, the ability of the SB hybridization assay to detect a low parasite load suggests that this assay may become an important supplemental tool, along with current cytological methods, for detecting P.c. hominis in patient populations with lower burdens of the organism and in identifying asymptomatic carriers of the parasite in healthy and immunosuppressed individuals.

AIDS-Related Opportunistic Infections↗

PubMed1996

Induction of type II collagen arthritis in the DA rat using silicone gel as adjuvant.

The Dark Agouti (DA) rat has been shown recently to have a high susceptibility for developing arthritis when challenged with either heterologous or homologous collagen II mixed with mineral oil, or with mineral oil challenge alone. This study determined the arthritogenic potential of silicone gel by either mixing it with bovine collagen II (BII) or by injecting silicone gel alone in DA rats. The incidence of collagen induced arthritis was as follows: PBS group- 0/10, silicone gel group- 4/10, and IFA group- 8/9. Anti-BII antibodies were formed in most of the rats treated with either silicone gel or IFA and these groups of rats showed a positive DTH reaction. The PBS treated rats were negative for both anti-BII antibodies and DTH reaction. The incidence of arthritis formation in rats injected with silicone gel alone was 0/10, while the IFA injected rats showed an incidence of 8/10. Silicone gel taken from a commercial breast implant thus is capable of mediating collagen induced arthritis in the DA rat. However, silicone gel alone does not appear to be arthritogenic.

Adjuvants, Immunologic↗

PubMed1985

Do iodinated fatty acids undergo a nonspecific deiodination in the myocardium?

The intracellular and subcellular distribution of 16-(123I)-iodo-9-hexadecenoic acid were studied in isolated rat hearts, perfused with or without glucose. At various time intervals after injection, cardiac lipids were extracted and the activity was determined for all fractions and all lipid classes. The total cardiac activity was maximal within 1 min postinjection and most of the activity was in the aqueous phase. The presence of glucose in the perfusion medium induced an increase of total cardiac and organic fraction activities. In the latter fraction, activity was very low for FFA, but high for triglycerides (TG), and especially polar lipids. The presence of an exogenous substrate, led to a more active esterification of fatty acids. Coronary effluent analysis showed, in the hydrophilic phase, a lower activity spike in the presence than in the absence of glucose. In the mitochondrial fraction most activity occurred in the organic phase, especially as polar lipids. In the nonmitochondrial fraction, activity was much higher in the aqueous phase. At 90 s postinjection of 1-14C-palmitic acid, over 80% of the myocardial activity was found in the hydrophilic fraction, which indicates, as for the iodo-fatty acid (IFA), an immediate and important oxidation, especially without glucose. These data seem to prove that IFA is taken up by the myocardial cell, subsequently enters the mitochondria and, without an early deiodination, is oxidized with iodide release. Changes in IFA metabolism, consecutive to modifications of glucose concentration in the perfusion medium can be observed by external detection of the myocardial activity curve. Omega-Iodinated fatty acids do not undergo a nonspecific deiodination and are therefore well suited for an external study of myocardial metabolism.

Animals↗

PubMed1988

Association of HLA-DR2 antigen with serum IgG antibodies against Borrelia burgdorferi in Bannwarth's syndrome.

The frequency of the HLA-DR2 antigen in 33 patients with clinical symptoms and signs of Bannwarth's syndrome was 33%, which was not significantly different from the 29% occurrence in 505 control subjects. However, all 11 HLA-DR2-positive patients had elevated serum levels of IgG antibodies against Borrelia burgdorferi, and these were present in 45% of 22 HLA-DR2-negative patients (P = 0.004). In the 21 patients with anti-B. burgdorferi antibodies the frequency of HLA-DR2 was 52%, which is significantly higher than the frequency in the control group (P = 0.04). The diagnosis of Bannwarth's syndrome was serologically confirmed by a positive indirect immunofluorescence assay (IFA). A negative test result does not exclude the diagnosis, as has recently been demonstrated with more sensitive techniques. The association between HLA-DR2 and a positive IFA suggests that the IFA selects a subgroup of patients with Bannwarth's syndrome and a different immune response. We could not demonstrate differences in the clinical spectrum and outcome between the two groups.

Antibodies, Bacterial↗

PubMed1975

Immunoprecipitation and class-specific immunofluorescence titration of human serum antibodies to Schistosoma mansoni antigens.

Twenty sera of individuals with Schistosoma mansoni infections were examined by means of immunoelectrophoresis, Indirect Flourescent Antibody (IFA) technique and the Defined Antigen Substrate Spheres (DASS) system. Immunoprecipitins against S. mansoni antigen and against antigen of the intermediate host, Biomphalaria glabrata, were demonstrated. The presence of specific anti-S. mansoni IgA, IgM, IgG and IgE antibodies were demonstrated. The presence of specific anti-S. MANSONI IgA, IgM, IgG and IgE antibodies was identified with the IFA-technique on frozen sections of adult parasites. The DASS-system proved to be more sensitive than the IFA-technique.

Adolescent↗

PubMed1978

Iris fluorescein angiography in diabetic vitrectomy patients.

Preoperative iris fluorescein angiograms (IFA) and ophthalmic records of 34 diabetic patients who underwent vitrectomy were reviewed. Of 2 patients without clinically apparent rubeosis, 23 showed evidence of iris microvascular abnormalities or rubeosis on IFA. Patients with rubeosis (Grades III and IV) on IFA had a 63% incidence of postoperative neovascular glaucoma, thus isolating them as a high risk group. The visual outcome of patients developing neovascular glaucoma was poor.

Diabetic Retinopathy↗

PubMed1977

Characterization of immunoglobulin classes of human antibodies to cryptococcus neoformans.

Immunoglobulin G (IgG), immunoglobulin M (IgM) and immunoglobulin A (IgA) levels were determined by radial immunodiffusion techniques in sera from 11 patients with cryptococcosis. Most specimens showed increased levels of IgM. Studies with fluorescein-labeled monospecific antihuman IgG and IgM, however, indicated that IgG was the immunoglobulin reactive in the indirect fluorescent antibody (IFA) test. In addition, cross-reacting sera from mycotic infections other than cryptococcosis were also shown to contain IFA antibodies of the IgG class. Sera treated with 2-mercaptoethanol continued to react in both the IFA test and the tube agglutination test. No correlation could be established between IgG AND IgM concentrations and serological reactivity in the sera evaluated in this study.

Antibodies, Fungal↗

PubMed1991

A comparison of the sensitivity and specificity of sialodacryoadenitis virus, Parker's rat coronavirus, and mouse hepatitis virus-infected cells as a source of antigen for the detection of antibody to rat coronaviruses.

Sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC) are two recognized viral strains which cause spontaneous disease in the laboratory rat. Currently there is no recognized practical procedure which will accurately differentiate infections with these strains. Using SDAV- and PRC-infected L-2 cells as the source of antigen, and sera from rats collected post inoculation with either of these viral strains, the indirect fluorescent antibody (IFA) procedure was used to determine whether antibody titers could be used to differentiate infections from the homologous and heterologous virus. There was no detectable difference in the sensitivity or specificity of these systems in detecting antibody to the homologous or heterologous virus. Thus there was no evidence that SDAV- and PRC-infected cells would serve to differentiate antibody to the homologous virus using the IFA technique. In addition, antibody titers were similar when mouse hepatitis virus (MHV)-infected cells were used as the source of antigen for the IFA technique. However, using MHV or SDAV-infected cells as the source of antigen, there was a significant difference in antibody titers to the homologous virus detected using the immunoenzyme technique.

Animals↗

PubMedDate not supplied

Capsular and 'O' serotype determinants of bacteroides fragilis.

Forty-one strains of Bacteroides fragilis, 20 strains of other Bacteroides species and 14 strains of other genera were examined by the indirect immunofluorescent assay (IFA) using anticapsular serum. The sixty-one Bacteroides strains were 'O' serotyped by direct agglutination tests using absorbed antisera raised against 23 strains, each with a different 'O' antigenic determinant. All 41 B. fragilis strains tested were positive by IFA with the anticapsular serum, but apart from one strain of B. distasonis, none of the remaining 19 strains of other bacteroides, i.e. B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. melaninogenicus group and B. ureolyticus, and none of the 14 other bacterial species examined were positive. The majority of strains of saccharolytic bacteroides tested reacted with one of the 23 'O' antisera and were designated as a specific 'O' serotype; a few Bacteroides strains had multiple agglutination reactions indicating the presence of multiple antigenic determinants. All 'O' serotypes gave positive IFA tests with their homologous 'O' antisera. Common capsular determinants and 'O' antigenic determinants appear to exist on the same strains of B. fragilis. Serological typing of B. fragilis and related species would be useful in epidemiological studies.

Antigens, Bacterial↗

PubMed1996

Seroprevalence of Lyme borreliosis in forestry workers from Brandenburg, Germany.

In 1992 blood samples were taken from 630 forestry workers in the state of Brandenburg, Germany, and an inquiry about tick bites and possible symptoms of Lyme borreliosis carried out in order to determine the seroprevalence of the disease. To estimate the rate of seroconversion within six months, 406 of the individuals were investigated a second time. IgG and IgM antibodies against Borrelia burgdorferi were detected in serum using an indirect immunofluorescence assay (IFA) and an immunoblot assay (IBA). Fifty-three percent of the forestry workers reported suffering a tick bite, 8% of whom recalled an erythema after the bite. Positive results were found more frequently in the forestry workers than in a control group of 200 healthy blood donors in both the IgG-IFA (8% vs. 4%, p < 0.05) and the IgG-IBA (18% vs. 5%, p < 0.05). The detection of IgG antibodies correlated with a tick bite and erythema history. There was a tendency of lower seropositivity by the IgG-IBA in individuals who treated the ticks before removal with chemicals or other agents compared to those without such treatment (16.8% vs. 23.9%, 0.05 > p < 0.1). Likewise, there was a tendency of lower seropositivity by the IgG-IFA in individuals being treated with antibiotics for other reasons compared to untreated individuals (3.15% vs. 8.9%, p < 0.05), although the two groups did not differ in the IgG-IBA (13.8% vs. 18.5%, p > 0.1). The rate of seroconversion within six months ranged from 5 to 7%. It is concluded that forestry workers in Brandenburg, Germany, are at risk for infection with Borrelia burgdorferi, but clinical signs of infection are rare.

Adolescent↗

PubMed1995

Enzyme immunoassay for detection of antibodies against isolated flagella of Legionella pneumophila.

Flagella of Legionella pneumophila serogroup 1, strain Philadelphia (ATCC 33152), were isolated and used as antigen to detect antibodies by enzyme immunoassay (EIA) in sera of patients with significant titers against Legionella as determined by the indirect immunofluorescence assay (IFA). Healthy blood donors served as a control group. Whereas the sera of blood donors were negative in both assays, sera of patients showed two patterns of reactivity. Sera with elevated titers against Legionella pneumophila serogroup 1 or Legionella gormanii in IFA usually demonstrated high titers in EIA. However, high IFA titers against Legionella bozemanii were associated with low titers in EIA. The data show that flagella protein of Legionella pneumophila serogroup 1 strain Philadelphia is of limited value for the detection of antibodies against Legionella other than Legionella pneumophila serogroup 1, although in vitro studies revealed genus-specific epitopes. This might be due to unpredictable variabilities in the expression of flagella in vivo.

Antibodies, Bacterial↗

PubMed1986

Use of monoclonal antibodies for rapid detection of influenza A virus in nasopharyngeal secretions.

Two monoclonal antibodies against influenza A virus were assessed for use as diagnostic reagents in an indirect immunofluorescence assay (IFA) of nasopharyngeal secretions. Monoclonal antibody IA-52, directed at an internal antigen, reacted with all influenza A tested. The high stability of this epitope permitted its use in a rapid IFA test, which gave results comparable to those obtained with polyclonal antibodies and viral isolation. The second monoclonal antibody, IA-279 was directed at a surface epitope (hemagglutinin); it reacted with almost all H3 subtype strains. Positive IFA using these monoclonal antibodies permitted rapid preliminary differentiation between the current two major subtypes of influenza A virus (H1N1, H3N2).

Antibodies, Monoclonal↗

PubMed1983

[Biliary excretion of choleretically active cinnamic acid derivatives in the rat].

Concerning the well-known choleretic effect of ferulic acid, we made further investigations on biliary excretion, metabolism and bile flow after application of ferulic acid and related compounds. In anesthesized bile duct cannulated male Wistar-rats, we studied the biliary excretion of the following 14-C labelled cinnamic acid compounds: caffeic acid (CA), ferulic acid (FA), m-coumaric acid (mCA), p-coumaric acid (pCA), isoferulic acid (IFA), p-methoxycinnamic acid (pMCA), 3,4-dimethoxycinnamic acid (DMCA), and 3,4,5-trimethoxycinnamic acid (TMCA). These compounds were given intraduodenally (128-384 mumoles/kg b. wt.). All of the tested compounds were excreted to some extent in bile, giving a bile to the serum conc. ratio greater than 1. The radioactivity excreted by bile within 2 hours (as percent of the given dose) was about 20% in the case of FA, IFA, DMCA, and TMCA, about 8% for pMCA, and less than 5% for CA, mCA and pCA. The biliary excretion increased in a dose-dependent manner only after FA and IFA, but not after application of the other compounds.

Animals↗

PubMed1990

Comparison of an indirect fluorescent antibody test and stool examination for the diagnosis of cryptosporidiosis.

A total of 342 patients, 70 with AIDS and 272 without AIDS, who had gastro-intestinal illness were selected for tests for cryptosporidiosis. Stool and serum specimens were obtained from each patient. The stool specimens were examined for cryptosporidial oocysts by acid-fast stain and the serum specimens were tested for specific antibodies against oocysts by an indirect fluorescent antibody (IFA) test. The oocysts were detected in stool specimens of 68 patients (19.8%), whereas specific antibodies were demonstrated in 102 patients (29.8%). The incidence of cryptosporidiosis was 47.1% in AIDS and 12.9% in non-AIDS patients as shown by stool examination, compared to 50% and 24.6% as shown by the IFA test. Based upon the finding of oocysts in stool specimens, the sensitivity and specificity of the IFA test in the diagnosis of cryptosporidiosis was 92.6% and 85.8%, respectively.

Acquired Immunodeficiency Syndrome↗

PubMed1980

Quantitative epidemiological studies on the prevalence of babesiosis in horses in Kuwait.

Racehorses imported into Kuwait were tested for serum antibodies to Babesia equi and B. caballi by complement fixation (CF) and indirect fluorescent antibody (IFA) tests. The prevalence of B. equi was high (77.1% by IFA) whereas that of B. caballi was much lower (11.4%). Data for B. equi showed that infection was acquired after about 6 months at risk and that in the following 12 months animals had antibody levels detectable by both CF and IFA tests by 24 months the CF reaction was no longer detectable. Estimates of incidence and inoculation rates were calculated and found to be consistent with a considerable infection pressure.

Animals↗

PubMed1988

Prevalence of piroplasmosis in equines in the Colombian province of Cordoba.

Eighty-two equine sera from 13 farms in northern Colombia were examined for antibodies to Babesia caballi and B. equi using the complement fixation (CF) and the indirect fluorescent antibody (IFA) test. Seroreactors to both piroplasms were present on all farms. The IFA test indicated a prevalence of 90% for B. caballi and 94% for B. equi. The CF test detected antibodies to B. caballi in 41% and to B. equi in 65% of the animals. The prevalence of seroreactors in different age groups revealed a significant decline in CF antibodies to B. caballi in animals older than three years. IFA titres for both Babesia spp. gradually declined with increasing age of the animals but were still present in most animals of the oldest age group (over nine years old). Anocentor nitens was found on all farms whereas Amblyomma cajennese was found only on two farms located on the coast.

Age Factors↗

PubMed1985

Seroepidemiology of bovine anaplasmosis and babesiosis in Venezuela.

The serological prevalence of bovine anaplasmosis and babesiosis in the Centro-Occidental region of Venezuela was determined using the indirect fluorescent antibody (IFA) and latex agglutination (LAT) tests. Anaplasma marginale IFA and LAT prevalence rates were 57.7 and 48.6% respectively for cattle of all ages and breeds. The respective prevalence of IFA activity to B. bigemina and B. bovis was 78.2 and 38.8%. The LAT test specific only for Babesia spp. revealed that 61.4% of the animals tested were serologically positive for Babesia parasites. Anaplasma marginale and B. bigemina organisms were detected in peripheral blood smears of 21.1 and 7.5% respectively of cattle surveyed. A longitudinal survey of antibody activity and incidence of parasitaemia for Anaplasma and Babesia infections was conducted in two groups of calves from their birth to seven months of age. The animals generally experienced infections between three and four months of age the period subsequent to decline in colostral antibody levels. Seroepidemiological data are considered in the formulation of proposed vaccination regimens for anaplasmosis and babesiosis.

Anaplasmosis↗

PubMed1998

Parameters for using mannan-MUC1 fusion protein to induce cellular immunity.

We have previously reported preclinical studies in mice of the human mucin 1 (MUC1) antigen covalently linked to the yeast cell-wall mannan polysaccharide (MFP), and shown strong cellular responses of the T1 type using mice. We now describe the optimum parameters for administration of MFP to obtain cellular immunity [as measured by the cytotoxic T cell precursor (CTLp) frequency]. In dose/response studies, in which 1 microg-150 microg was given by the i.p. route, it was clear that doses of 1-7 microg led to cellular and not humoral immunity; at doses above 7 microg humoral immunity prevailed with little cellular immunity increasing doses giving greater amounts of antibody. The most favoured routes of administration were intraperitoneal or intradermal immunisation, which were substantially better than i.m., i.v.; s.c. administration was the worst. Three immunisations were necessary for a maximum cellular response, further immunisation decreasing the CTLp frequency. Six different adjuvants were used with MFP [complete and incomplete Freund's adjuvant (CFA, IFA) Alum, Adjuprime, muramyl dipeptide (MDP) and glutaminyl-muramyl dipeptide (GMDP)]; Alum, GMDP, MDP and IFA moderately increased the CTLp frequency, IFA being the best. Even though preclinical studies of the immunogen in mice may not necessarily mirror the behaviour of the immunogen in humans, these studies demonstrate the factors to be taken into account for phase I/II clinical trials.

Animals↗