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PubMedDate not supplied

Chagas' disease: an algorithm for donor screening and positive donor counseling.

Classical serological screening assays for Chagas' disease are time consuming and subjective. The objective of the present work is to evaluate the enzyme immuno-assay (ELISA) methodology and to propose an algorithm for blood banks to be applied to Chagas' disease. Seven thousand, nine hundred and ninety nine blood donor samples were screened by both reverse passive hemagglutination (RPHA) and indirect immunofluorescence assay (IFA). Samples reactive on RPHA and/or IFA were submitted to supplementary RPHA, IFA and complement fixation (CFA) tests. This strategy allowed us to create a panel of 60 samples to evaluate the ELISA methodology from 3 different manufacturers. The sensitivity of the screening by IFA and the 3 different ELISA's was 100%. The specificity was better on ELISA methodology. For Chagas disease, ELISA seems to be the best test for blood donor screening, because it showed high sensitivity and specificity, it is not subjective and can be automated. Therefore, it was possible to propose an algorithm to screen samples and confirm donor results at the blood bank.

Algorithms↗
PubMed2002

Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

OBJECTIVE: To determine apparent seroprevalence of antibodies against Sarcocystis neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. DESIGN: Cross-sectional study. SAMPLE POPULATION: Serum from 196 domestic cats. PROCEDURE: Banked serum samples submitted to the Michigan State University Animal Health Diagnostic Laboratory for T. gondii diagnostic testing were tested for antibodies against S. neurona by use of an indirect fluorescent antibody (IFA) test and a western blot test. Submission records were analyzed to determine descriptive statistics and test for associations between positive results of a test for S. neurona and other variables in the data set. RESULTS: 10 of 196 (5%) samples yielded positive results for antibodies against S. neurona by use of western blot analysis, whereas 27 samples yielded positive results by use of the IFA. No association was found between S. neurona western blot test results and T. gondii test results, age, sex, or the reason for T. gondii testing. The S. neurona IFA titer was positively and significantly associated with positive results of western blot analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Domestic cats are not likely to play a substantial role as intermediate hosts in the natural life cycle of S. neurona. Results indicate that natural infection of domestic cats may occur, and small animal practitioners should be aware of this fact when evaluating cats with neurologic disease. The S. neurona IFA test had lower specificity than western blot analysis.

Animals↗
PubMed1997

Evaluation of serological assays for diagnosis of onchocercosis.

Microscopic analysis of skin snips is the most widely used diagnostic technique for onchocercosis in endemic countries. The invasive nature and low sensitivity of that procedure has called for alternative diagnostic methods for this disease. Presently, serological assays detecting filariosis in general are available for routine analysis. However, serological assays specific for onchocercosis are still not universally available. We have evaluated the performance of a dot blot assay (DBA) as a potential method for specific detection of onchocercosis. The DBA, which detects IgG, as compared with 2 IgG4-immunoblot assays, all employing Onchocerca volvulus antigen. Furthermore, an enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA) based on antigens from Acanthocheilonema viteae and Brugia malayi, respectively, were included in the comparison. Samples from microfilariae-positive patients and negative controls from the onchocercosis-endemic country Ghana were analysed. The DBA was significantly more sensitive and specific than the IgG4-assays and the ELISA, respectively. Furthermore, the anti-filarial Ig was increased in patients 1 month post-ivermectin treatment. Sera from patients with suspected filariosis from different parts of the world were analysed using DBA, ELISA and IFA. Patients responding positively in the DBA (12%) had clinical symptoms compatible with onchocercosis whereas those positive in ELISA and IFA (53% and 48%, respectively) had various clinical symptoms. These results indicate that the DBA is more specific than and as sensitive as the ELISA and the IFA presently used for the diagnosis of onchocercosis.

Animals↗
PubMed1993

The adjuvant effect of silicone-gel on antibody formation in rats.

The extent of immunological adjuvancy of silicone-gel, from mammary implants, up to now, has not been determined definitively. This study compares the immune potentiation effects of silicone-gel with that of Freund's adjuvant, using bovine serum albumin (BSA) as the test antigen in rats. Sixty, 250 gr., male Sprague Dawley rats were divided into six groups: I- phosphate buffered saline (PBS) only, II- silicone oil (Dow Corning Medical Grade 360 liquid silicone), III- 50% silicone-gel (McGhan Medical Corp.- mammary implant) in silicone oil, IV- complete Freund's adjuvant (CFA), V- incomplete Freund's adjuvant (IFA), and VI- 50% silicone oil in IFA. Each adjuvant was mixed or emulsified with an equal volume of 50 micrograms of BSA in 150 microliters of PBS. Each immunization was given intramuscularly in a single injection. Cardiac puncture test bleeds were taken at 12, 22, 40 and 56 days post immunization and the serum anti-BSA-antibody was measured by ELISA. The results indicate that silicone-gel is a potent immunological adjuvant, compared to both CFA and IFA. Silicone oil alone is not as potent as adjuvant and seems to inhibit the immune response when mixed with IFA. There thus appears to be a distinct possibility that silicone-gel may also be able to mediate an auto-immune reaction.

Animals↗
PubMed2002

CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.

Adjuvants, Immunologic↗
PubMed1997

Clinical comparison of borreliacidal-antibody test with indirect immunofluorescence and enzyme-linked immunosorbent assays for diagnosis of Lyme disease.

OBJECTIVE: To compare the clinical results with the borreliacidal-antibody test (BAT) and two standard screening serologic tests for Lyme disease (LD)-the indirect immunofluorescence assay (IFA) and the enzyme-linked immunosorbent assay (ELISA). DESIGN: The medical records of patients from an endemic LD area, who had been serologically tested during the summer of 1992, were retrospectively categorized by clinical diagnoses without results of serologic tests. Serologic testing, which included control serum samples from patients from a nonendemic LD area, was performed in a blinded fashion, and the results were compared with the clinical categories. MATERIAL AND METHODS: Medical records of 307 patients who had been serologically tested for LD were reviewed. We found untreated, active LD in 43 patients (early-localized LD, 21; early-disseminated LD, 14; and late-disseminated LD, 8) and treated LD in 33. Non-LD cases were categorized into acute or chronic conditions of unknown or known cause. RESULTS: Overall, the BAT had a sensitivity of 11% in active LD and did not correlate with results of other conventional surface antibody assays. The IFA and ELISA were more sensitive (67 to 93%), but false-positive results frequently were noted (20 to 40%) in acute and chronic non-LD inflammatory conditions. The specificity of the BAT, IFA, and ELISA in the control group was 96%, 93%, and 97%, respectively. CONCLUSION: Until the sensitivity, as measured by prospective clinical studies, is improved without loss of specificity, the BAT should not be used clinically for the diagnosis of LD. Suspected cases of LD with atypical clinical manifestations should have positive ELISA and IFA results confirmed with a standardized immunoblot assay.

Antibodies, Bacterial↗
PubMed1976

The seroepidemiology of malaria in Middle America. III. Serologic assessment of localized Plasmodium falciparum epidemics.

Epidemiologic studies were done in two localities, one in Panama and one in El Salvador, where outbreaks of Plasmodium falciparum malaria occurred. The indirect fluorescent antibody (IFA) method was helpful in defining the intensity of the outbreaks, the species of parasite involved, and, through parallel surveys of adjacent localities, the localized character of outbreaks. In Escobal, Panama 71 (34%) of 211 persons examined were IFA-positive for P. falciparum, although only 1 positive blood film was seen in this survey. In Jocomontique, El Salvador, 15 (28%) of 53 persons examined were IFA-positive for P. falciparum; 6 persons were parasite-positive. In the Panama study a number of children with positive IFA titers at the time of the epidemic had reverted to negative 6 months later. This lability of titers, in children, probably resulting from very early treatment of their first infection and, thus, a very brief exposure to the parasite antigen, suggests the need for caution in interpreting such negative serologic findings. Serologic studies provide valuable information in malaria epidemics, but must be interpreted in association with other known epidemiologic factors in the area concerned.

Adolescent↗
PubMed1979

A comparison of the complement fixation, indirect fluorescent antibody, and microagglutination tests for the serological diagnosis of rickettsial diseases.

Three techniques for the serological diagnosis of Rocky Mountain spotted fever were compared by testing 417 sera from 178 patients who very probably did not have rickettsial infections and 88 sera from 41 patients who very probably had Rocky Mountain spotted fever (SF). The techniques were complement fixation (CF), indirect fluorescent antibody (IFA), and microagglutination (MA). To avoid possible degradation during unnecessary purification, the antigens were prepared by methods that were as simple as possible. In the CF tests of 417 sera from patients with nonrickettsial diseases there was only one titer of 8 and none at higher dilutions, whereas with the IFA and MA tests 4-8% of the sera reacted with SF antigens and 4-20% reacted with murine typhus (MT) antigens; the evidence indicated that these reactions were not caused by specific rickettsial antibody. With the SF sera, it could be seen that the IFA test was the most sensitive and the MA test was the least sensitive at each interval after infection. Moreover, the IFA results showed the least number of confusing cross-reactions with MT antigens and the MA test showed the most. The relative advantages of the three tests in serodiagnosis of rickettsial diseases are discussed.

Agglutination Tests↗
PubMed1980

Serologic studies on onchocerciasis in Guatemala using fixed-tissue sections of adult Onchocerca volvulus.

Sera from 458 residents of a coffee plantation in an area of Guatemala endemic for onchocerciasis were examined for the presence of antibodies to Onchocerca volvulus using a fluorescent antibody (IFA) test with sections of adult worms as the antigen. Antibody response increased with age of the residents and microfilarial density. A total of 70.0% of the residents were serologically positive and 65.1% were positive for microfilariae. Males had higher microfilarial densities and higher IFA responses. Sixty-three individuals were positive for microfilariae and IFA-negative indicating a lack of sensitivity on the part of the test antigen. Seventy-four individuals were negative for microfilariae and IFA-positive, suggesting the possible usefulness of the test in the determination of present or past infection in the absence of microfilariae.

Adolescent↗
PubMed1983

Identification and partial characterization of exoantigens derived from medium used to culture Plasmodium falciparum.

Rabbits were immunized with exoantigens from the spent medium of Plasmodium falciparum cultures, and the resultant immunologic responses were studied by indirect fluorescent antibody (IFA), hemagglutination (HA), and two-dimensional crossed immunoelectrophoretic (IEP) techniques. By crossed IEP, three parasite antigens, identified and characterized as proteins lacking lipid and carbohydrate moieties, reacted with rabbit antiserum and human immune serum. Pre-immunization sera of the rabbits used in these experiments had 1:80 IFA titers against P. falciparum schizonts which were then boosted eightfold by immunization with parasite exoantigens, in contrast to IFA titers of less than 1:2 for coccidia- and Pasteurella-free rabbits. Experimental infections of coccidia- and Pasteurella-free rabbits with rabbit Eimeria spp. resulted in 1:80 anti-P. falciparum IFA titers, suggesting cross-reactivity of coccidial and plasmodial antigens. Post-immunization sera demonstrated extremely high HA titers against human erythrocytes, underscoring the potency of human blood components containing parasite antigens prepared from cultures. These results suggest that coccidia- and Pasteurella-free rabbits may be useful in the analysis of antigen of cultured P. falciparum.

Animals↗
PubMed1990

Canine visceral leishmaniasis in northeast Brazil: assessment of serodiagnostic methods.

Domestic dogs are considered to be a major reservoir of Leishmania donovani chagasi in northeast Brazil, and the elimination of infected dogs is an important part of the control program. We assessed 2 serological methods, IFA and ELISA. Of 405 dogs, 8% were positive by IFA obtained from blood collected by drying onto filter paper followed by elution, 17% were positive by IFA performed using sera, and 38% were positive by ELISA on the same sera. Thirty-five dogs, seropositive by 1 or more of the above tests, were killed and touch preparations were made of liver, spleen, and mesenteric lymph nodes. Samples were cultured in enriched NNN media. The ELISA recognized all dogs with proven infection; IFA detected 10 of 12. Eleven dogs were positive by touch preparations and 7 by culture. In addition, kDNA hybridization was undertaken with probes to L. donovani chagasi, L. braziliensis ssp., and L. mexicana amazonensis. Positive results were obtained from tissue in 19 instances, but 10 culture positive specimens were not recognized.

Animals↗
PubMed2002

Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand.

We compared the performance of 2 commercially available dipstick assays, 2 enzyme-linked immunosorbent assays (ELISAs), and an indirect immunofluorescent antibody (IFA) assay for the diagnosis of scrub typhus, using the indirect immunoperoxidase (IIP) test as the reference standard. The dipstick assays were the Integrated Diagnostics (Baltimore, MD) Dip-S-Ticks Scrub Recombinant (r56) dipstick test (INDX assay) and the PanBio (Brisbane, Australia) Scrub Typhus IgM and IgG Rapid Immunochromatographic test (PanBio assay). One of the ELISAs used pooled cell lysates of Karp, Kato, and Gilliam strain Orientia tsutsugamushi as antigen (pooled-antigen ELISA), and the other used a recombinant r56 protein as the antigen (recombinant ELISA). With a panel of 123 positive and 227 negative sera, sensitivity and specificity of the assays were as follows: INDX assay, IgG, 60% and 95%, IgM, 60% and 97%; PanBio assay, IgG, 94% and 96%, IgM, 83% and 93%; IFA (1:400 cutoff), IgG, 91% and 96%, IgM, 85% and 98%; pooled-antigen ELISA, IgG (1:1600 cutoff), 97% and 89%, IgM (1:400 cutoff), 94% and 91%; recombinant ELISA, IgG (1:1600 cutoff), 97% and 92%, IgM (1:400 cutoff), 93% and 94%. Because of its excellent performance and use of a standardized, commercially available antigen, the recombinant ELISA is suitable for use in a diagnostic laboratory, where it may be able to replace the IFA and IIP assays. In contrast, the PanBio dipstick assay was easy to perform and did not require sophisticated equipment, making it suitable for use in rural areas where more sophisticated diagnostic tests such as the ELISA and IFA may not be available.

Antibodies, Bacterial↗
PubMed1993

Babesiosis in Washington State: a new species of Babesia?

OBJECTIVE: To characterize the etiologic agent (WA1) of the first reported case of babesiosis acquired in Washington State. DESIGN: Case report, and serologic, molecular, and epizootiologic studies. SETTING: South-central Washington State. PATIENT: A 41-year-old immunocompetent man with an intact spleen who developed a moderately severe case of babesiosis. MEASUREMENTS: Serum specimens from the patient were assayed by indirect immunofluorescent antibody (IFA) testing for reactivity with seven Babesia species and with WA1, which was propagated in hamsters inoculated with his blood. A Babesia-specific, ribosomal-DNA (rDNA) probe was hybridized to Southern blots of restriction-endonuclease-digested preparations of DNA from WA1, Babesia microti, and Babesia gibsoni. Serum specimens from 83 family members and neighbors were assayed for IFA reactivity with WA1 and B. microti. Small mammals and ticks were examined for Babesia infection. RESULTS: The patient's serum had very strong IFA reactivity with WA1, strong reactivity with B. gibsoni (which infects dogs), but only weak reactivity with B. microti. DNA hybridization patterns with the rDNA probe clearly differentiated WA1 from B. gibsoni and B. microti. Four of the patient's neighbors had IFA titers to WA1 of 256. The tick vector and animal reservoir of WA1 have not yet been identified, despite trapping 83 mammals and collecting 235 ticks. CONCLUSIONS: WA1 is morphologically indistinguishable but antigenically and genotypically distinct from B. microti. Some patients elsewhere who were assumed to have been infected with B. microti may have been infected with WA1. Improved serodiagnostic and molecular techniques are needed for characterizing Babesia species and elucidating the epidemiology of babesiosis, an emergent zoonosis.

Adolescent↗
PubMed1994

Response of the meadow vole (Microtus pennsylvanicus) to experimental inoculation with Borrelia burgdorferi.

The response of the meadow vole (Microtus pennsylvanicus) to infection by experimental inoculation with Borrelia burgdorferi was evaluated. Forty-two adult voles were inoculated subcutaneously with 0.5 x 10(6) spirochetes. Sera taken during the 196 day trial were tested by indirect fluorescent antibody (IFA) assay for antibodies to B. burgdorferi. Tissues from animals which died during the trial, and from animals killed at 28, 112 and 196 days post-inoculation (DPI), respectively, were cultured in BSK-II medium for < or = 6 weeks. They also were examined histologically for lesions and the presence of spirochetes. All inoculated animals developed antibodies by 14 DPI and maintained titers > or = 1:10 for the duration of the trial. Spirochetes were isolated from ears, bladder, and spleen. Spirochetes also were identified by Bosma-Steiner silver stain or tissue IFA assay in sections of ears, bladder, kidney and heart. Infection as confirmed by re-isolation persisted for < or = 111 days. No lesions were identified in association with the presence of spirochetes. No increase in mortality was observed in inoculated animals compared with controls. Sensitivity of the IFA test at a cut-off titer of 1:10 was 100% from > or = 14 DPI, but at 1:20 reached a maximum of 97%. Specificity at 1:10 was 84% and at 1:20 was 97%. Use of antiserum to Microtus immunoglobulin (Ig) in a double-layered test provided no significant advantages over use of a commercial fluorescein-conjugated anti-mouse Ig in a single-layered IFA test.

Animals↗
PubMedDate not supplied

Serosurveillance for Pseudomonas pseudomallei infection in Thailand.

A nation-wide survey was conducted to see the prevalence of serosensitivity to Pseudomonas pseudomallei antigens by indirect hemagglutination (IHA) and indirect immunofluorescent assay (IFA) for IgG and IgM. Serum samples were collected from blood donors in eight selected areas and bacteriologically confirmed melioidosis patients in Ubon Ratchathani province. The distribution patterns of antibody titers were compared among the survey areas with cut-off points set at 1:160 for IHA, 1:4 for IFA-IgM and 1:32 for IFA-IgG. These cut-off points were decided by ROC (Receiver Operating Characteristics) analysis. The specificity (% true negative reactions) of each serological test in the general population differed significantly among survey areas, possibly reflecting the extent of inapparent infection in each community. IFA was more successful than IHA in differentiating between negative from positive reactions. The survey classified the areas into endemic (Khon Kaen, Ubon Ratchathani), transported (Bangkok), and non-endemic (other provinces) types.

Antibodies, Bacterial↗
PubMed1999

Strain variation in autoimmunity: attempted tolerization of DA rats results in the induction of experimental autoimmune encephalomyelitis.

This paper reports that DA rats develop experimental autoimmune encephalomyelitis (EAE) when immunized with encephalitogenic myelin basic protein (MBP) peptide (MBP63-81) in IFA. In contrast, most rodent strains are tolerized by this procedure. Doses as low as 5 micrograms peptide + IFA induced EAE in DA rats. Lewis (LEW) rats did not develop EAE, even after immunization with 100 micrograms encephalitogenic peptide (MBP68-86) + IFA, but were rendered tolerant to EAE. DA rat T cells proliferated to peptide, and proliferation was inhibited by CTLA4Ig, and by anti-B7.1 and anti-B7. 2 mAbs. This indicates that the ease of induction of EAE in this strain does not reflect a decreased requirement for T cell costimulation through the B7/CD28 costimulatory pathway. The inhibitory effect of CTLA4Ig was abrogated in the presence of anti-TGF-beta-neutralizing Ab. An encephalitogenic DA T cell line expressed mRNA for the Th1 cytokines IFN-gamma and TNF-alpha, as well as IL-10, and secreted these cytokines. In contrast, a T cell line from peptide + IFA-immunized LEW rats (which did not develop EAE) failed to secrete these cytokines. Although this line did not express TNF-alpha or IL-10 mRNA, IFN-gamma mRNA was detected, suggesting posttranscriptional regulation of IFN-gamma expression. Attempts to induce unresponsiveness in DA rats with encephalitogenic peptide-coupled splenocytes were also unsuccessful.

Amino Acid Sequence↗
PubMedDate not supplied

[Norms for authors of Mexican medical journals in 1994 and 1995].

OBJECTIVE: To find out how much information of the Vancouver Norms (VANNOR) are given to prospective authors in the Instructions for Authors (IFA) in a sample of Mexican medical journals (MMJ) published in 1994 and 1995. DESIGN: Prospective, cross sectional, descriptive, observational. MEASUREMENTS: The IFA in the last issue of the MMJ that were published in 1994 and 1995, were reviewed and 121 characteristics in the VANNOR were scored in nine categories (characteristics, presentation, title page, summary and key words, text order and contents, references, tables, illustrations and other instructions). A one point score was given to any characteristic present in order to obtain a score for each category and a total score for each journal. The total scores in the VANNOR affiliated journals (N = 33) versus the non-affiliated ones (N = 27) were evaluated by the Mann-Whitney and exact Fisher test with alpha = 0.05 (two-tailed). RESULTS: A statistically significant higher score was found in the IFA of VANNOR journals in 22 of the 121 characteristics, in six of the nine categories, as well as in the total score. CONCLUSION: VANNOR affiliated journals offer a significantly higher amount of information regarding the IFA than those not affiliated.

Bibliometrics↗
PubMed1975

Effect of prolonged administration of homologous and heterologous intrinsic factor antibodies on the parietal and peptic cell masses and the secretory function of the rat gastric mucosa.

In order to determine the possible effects of the circulating intrinsic factor antibodies (IFA) on gastric morphology and secretory function, four groups of 12 rats each received intravenous injection daily for 8 to 12 weeks of immunoglobulin G (IgG) fractions separated on DEAE-cellulose columns from various sources: (1) sera of patients with pernicious anemia, containing both IFA and parietal cell antibodies (PCA), (2) sera from patients with atrophic gastritis, containing parietal cell antibody only, and (3) and (4) sera of rabbits immunized with semipurified human or rat intrinsic factor (IF). In addition three control groups of 12 rats each received intravenous injections daily for 8 to 12 weeks of either (5) saline or (6) and (7) IgG processed from human or rabbit normal sera. Still another group of 12 rats (8) did not receive any injections whatsoever for the same duration of time. One-third of the rats were intubated biweekly after histamine stimulation and the hourly outputs of CHl pepsin, and IF were determined. At conclusion of the experiments, rats were killed, the mucosal surface and thickness of the mucosa were measured, and parietal cell and peptic cell masses were counted. The control groups showed either progressive growth of the cellular mass in gastric mucosa and increase of the HCl, pepsin, and IF outputs, or no significant changes. In contrast, rats injected with IgG containing IFA to human or rat IF showed a statistically significant thinning of the gastric mucosa, reduction of peptic cells, which are known to secret IF in this species, and corresponding decreases in the ouputs of pepsin and IF. These became reduced by about 50% from initial values, and by 62 or 75%, respectively, when compared to rats injected with normal human or rabbit IgG's. In rats injected with IgG's from pernicious anemia sera, which contained both IFA and PCA, the outputs of IF, pepsin, and HCl decreased signigicantly, as well as the peptic and parietal cell masses. The rats injected with PCA only demonstrated thinning of the gastric mucosa, reduction of parietal cell mass, and a significant decrease of the HCl output. These findings imply an active role of the circulating gastric.

Anemia, Pernicious↗