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PubMed1980

Evaluation of the micro enzyme-linked immunosorbent assay for antibodies to Trypanosoma cruzi.

A micro enzyme-linked immunosorbent assay (ELISA) for antibodies to Trypanosoma cruzi was evaluated and the results obtained by ELISA were compared with those obtained by the complement fixation test (CF) and indirect fluorescent antibody test (IFA). Fifty sera collected from residents of the southeastern United States all had reciprocal ELISA titers less than or equal to 320. Similarly, serum samples from 17 patients with T. cruzi infection proven by xenodiagnosis had reciprocal ELISA titers of greater than or equal to 1,280. Specimens from 302 El Salvador Army recruits were tested by ELISA, IFA, and CF. Excellent correlation was observed between results obtained by the three serologic tests; 62.9% of the samples were negative by each of the three tests and 24.5% were positive by all. Overall, 29.5% of the sera were positive for antibodies to T. cruzi by ELISA, 29.5% by IFA, and 31.5% by CF. The data suggest that the micro ELISA is a promising serologic test for measuring antibodies to T. cruzi in individuals and in populations.

Adolescent↗
PubMed1981

The 48-hour exoerythrocytic stage of Plasmodium cynomolgi bastianellii.

Detection and specific identification of the 48-hour exoerythrocytic stage of the primate malaria parasite, Plasmodium cynomolgi bastianellii, was accomplished by means of a highly specific indirect immunofluorescence technique (IFA) applied to hepatic tissue fixed in Carnoy's solution. The 48-hour forms appeared as round-to-slightly-oval bodies of average mean diameter 3.0 micrometers (9 parasites) and lying with the cytoplasm of individual hepatic parenchymal cells; each possessed one to three non-fluorescent nuclei or nuclear sections (mean 1.6) within the brightly fluorescent parasitic cytoplasm. In contrast, 72-hour parasites (6) had an average mean diameter of 4.0 micrometers and a mean of 2.2 nuclei. Restaining of IFA preparations with the Giemsa-colophonium method confirmed the plasmodial nature of fluorescent forms, despite some modification of staining characteristics produced by the prolonged exposure of sections to the aqueous phase of the IFA procedure. Exoerythrocytic forms could not be detected in biopsies obtained 24 hours following sporozoite inoculation.

Animals↗
PubMed1983

Diagnostic criteria for scrub typhus: probability values for immunofluorescent antibody and Proteus OXK agglutinin titers.

The sensitivities and specificities of the indirect microimmunofluorescent antibody (IFA) and Weil-Felix (OXK) tests for scrub typhus were established for a range of titers using groups of diseased and control (other febrile illnesses) patients diagnosed by other methods. At a cut-off point of greater than or equal to 1:400, the IFA test was 0.96 specific, and at greater than or equal to 1:320, the OXK was 0.97 specific. Using either these highly specific levels of antibody or other rigorous diagnostic criteria (isolation or 4-fold rising titers), the prevalence of scrub typhus infection was determined to be 0.22 in an unselected population of febrile patients in a rural Malaysian hospital. Probability values (Pr) for the correct diagnosis of scrub typhus were then calculated from the specificity, sensitivity and prevalence determination for a range of titers. The Pr for an OXK titer of greater than or equal to 1:320 was 0.79, and the Pr for an IFA titer of greater than or equal to 1:400 was 0.78. When both these titers were present in a single specimen, the Pr increased to 0.96.

Agglutination Tests↗
PubMed1983

Anti-sporozoite antibodies induced by natural infection.

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.

Adolescent↗
PubMed1983

Evaluation of promastigote and amastigote antigens in the indirect fluorescent antibody test for American cutaneous leishmaniasis.

Leishmania braziliensis panamensis promastigotes, temperature-induced in vitro-cultivated amastigotes, Vero cell-derived amastigotes, and rodent lesion-derived amastigotes were evaluated as antigens in the indirect immunofluorescent antibody (IFA) test for American cutaneous leishmaniasis. Test sensitivity was determined using sera from 34 U.S. soldiers with leishmaniasis diagnosed by demonstrating parasites in their skin lesions. Sera were collected from 3 to 24 months after exposure to Leishmania. Positive IFA reactions among patient sera were 82% with promastigotes or lesion amastigotes, 79% with in vitro amastigotes, and 76% with Vero cell amastigotes (P = N.S.). Positive titers ranged from 1:8 to 1:128 using all antigens. Test specificity was determined with 30 sera from healthy individuals. False positive reactions ranged from 0-5% depending on the antigen and all titers were less than or equal to 1:8. Test cross-reactivity was assessed with 47 sera from patients with other diseases. Depending on the antigen, cross-reactions occurred with sera from patients with Chagas' disease, lupus erythematosus, malaria, toxoplasmosis and amebiasis. None of the antigens cross-reacted with sera from patients with viral hepatitis, coccidioidomycosis, syphilis, schistosomiasis, and trichinosis. In replicate experiments, 99-100% of the sera varied no more than +/- 1 titer dilution. As sensitivity, specificity, cross-reactivity, and reproducibility of the four antigens were statistically similar, promastigotes, which can be easily and economically cultured in large numbers in vitro are recommended for use in the IFA test for American cutaneous leishmaniasis.

Adult↗
PubMed1986

Epidemiological investigations following an outbreak of hemorrhagic fever with renal syndrome in Greece.

Epidemiological investigations were conducted following an outbreak of hemorrhagic fever with renal syndrome (HFRS) which occurred in the state of Epirus, northwestern Greece, in July and August 1983. A total of 8 patients were hospitalized during the outbreak; 3 were severely ill and 1 died. A serosurvey made in May 1984 sampled 184 of the approximately 400 residents of the village of Tsepelovo, where 4 patients resided, and found 12 (6.5%) persons, including convalescent sera from 4 patients, with antihantaviral antibody by immunofluorescent antibody (IFA) tests. Small mammal collections found house rats common in the village, but none exhibited anti-hantaviral antibody. Collections in nearby fields and mountains found Apodemus flavicollis rodents common, and 2 (6%) of 33 captured had high IFA anti-hantavirus antibody. Virus isolation attempts from rodent tissues were unsuccessful. Testing of convalescent patients' sera by IFA and plaque reduction neutralization tests indicated that the etiological agent was neither Puumala virus nor Seoul virus, but appears to be a strain of Hantaan virus or perhaps a new virus. The rodent host of this virus may be A. flavicollis, and the distribution of this species corresponds with previously reported cases of severe HFRS described elsewhere in central Europe.

Adolescent↗
PubMed1986

The sensitivity of various serologic tests in the diagnosis of Rocky Mountain spotted fever.

Sensitivity indices for various serologic tests in the diagnosis of Rocky Mountain spotted fever (RMSF) were determined from RMSF surveillance data collected at the Centers for Disease Control (CDC) during 1981-1984. During these 4 years, a total of 4,141 cases of RMSF in the U.S. were reported to CDC. Case report forms, which include information on dates and results of serologic testing, were received for 3,567 of these cases; 1,774 were laboratory-confirmed according to criteria published previously by CDC. Sensitivity indices for the Weil-Felix (Proteus OX-19, OX-2), indirect fluorescent antibody (IFA), latex agglutination (LA), complement fixation (CF) and indirect hemagglutination (IHA) tests were examined by analyzing cases confirmed by other means, in which data concerning the test in question were available. Analysis of serum pairs for a 4-fold or greater increase in antibody titer or a minimum diagnostic titer indicated high sensitivity indices for IHA (96%) and IFA (94%), but lower sensitivity indices for OX-19 (70%), OX-2 (47%), CF (63%) and LA (71%). IFA and IHA appear to be the most sensitive serologic tests currently in use for the diagnosis of RMSF.

Agglutination Tests↗
PubMed1987

Suppression of Rickettsia typhi transmission in fleas maintained on murine typhus-immune rats.

The presence of host antibodies against Rickettsia typhi in the hemolymph of Xenopsylla cheopis fed on immune rats was studied using an indirect immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA). The time course experiment revealed that antibody to R. typhi appears in the flea hemolymph after 18 hr exposure to host and persists for 24 hr. IFA tests utilizing specific antisera to rat IgG (Fc) and rat IgG (Fab) fragments, indicate that some unaltered rat IgG to R. typhi were present in the hemolymph of immune fed fleas. Host antibody to R. typhi was detected on the surface of the rickettsiae in the flea hemolymph and gut contents by IFA. Maintenance of infected fleas on immune host had no significant effect on the establishment and subsequent growth of rickettsiae within the fleas. However, 19-22 day infected fleas maintained on immune hosts failed to transmit R. typhi to baby rats.

Animals↗
PubMed1994

Serologic and virologic evidence of a Prospect Hill-like hantavirus in Wisconsin and Minnesota.

The purposes of this study were to determine if hantaviruses were present in the Great Lakes port areas of Wisconsin and Minnesota and if so, to identify which virus and which rodent host species were involved. Rodents were trapped in Duluth, Minnesota, Superior, Green Bay, and Milwaukee, Wisconsin, all ports of call for international maritime shipping. A total of 675 wild rodents were captured and tested, including 310 meadow voles (Microtus pennsylvanicus), 173 Norway rats (Rattus norvegicus), 179 Peromyscus spp., (including white footed mice [P. leucopus] and deer mice [P. maniculatus gracilis and P. maniculatus bairdii]), and 13 house mice (Mus musculus). Twenty percent of the rats, 17% of the meadow voles, 8% of the house mice, and 3% of the Peromyscus spp. had antibody to a hantavirus by immunofluorescent antibody assay (IFA). By the plaque-reduction neutralization test (PRNT), nine of 36 meadow voles, one of 4 P. leucopus, and one of 34 rats had hantavirus antibody, with the highest titers to Prospect Hill (PH) virus. All of the PRNT-seropositive individuals were from the twin cities of Superior and Duluth. Hantavirus antigen was detected in lung tissue by IFA in M. pennsylvanicus and Peromyscus spp., but not in rats. Two hantaviruses, designated SD-1 and SD-2, were isolated from M. pennsylvanicus captured in Duluth and found to be very similar to prototype PH virus by cross-IFA and cross-PRNT. Virus isolation attempts were unsuccessful from tissues of the Peromyscus spp. and the rats.

Animals↗
PubMed1998

Laboratory evaluation of a dot-blot enzyme immunoassay for serologic confirmation of illness due to Rickettsia conorii.

Of the 169 United States Army soldiers who deployed on a field training exercise to a remote area of Botswana for two weeks in January 1992, more than 30% developed a febrile illness within five days of their return. A diagnosis of South African tick typhus was suggested by soldiers' exposure to ticks, as well as the presence of eschars and vesicles at the site of tick bites, and tender regional lymphadenopathies. This high attack rate, experienced during such a short exposure period, emphasizes the hazard of illness due to Rickettsia conorii to persons visiting endemic areas. A rapid, diagnostic, semiquantitative enzyme immunoassay (DS) for detection of IgG and IgM antibodies to R. conorii was performed on 209 acute and convalescent sera from soldiers in the outbreak and on 75 control sera. For the acute sera from soldiers meeting the probable case definition of having both regional lymphadenopathy and tick bite eschar, as judged by an IgG indirect fluorescent antibody (IFA) test, the resulting sensitivity and specificity of the DS test were 100% and 48%, respectively. In the analysis of the acute sera, the DS test identified as reactive more of the probable cases (62%) than either the IgG (16%) or IgM (55%) IFAs. This simple and rapid diagnostic test could be useful in establishing a preliminary diagnosis of R. conorii rickettsiosis in remote settings when immediate confirmation by IFA is impossible.

Antibodies, Bacterial↗
PubMed1999

Detection of specific antibodies to Plasmodium falciparum in blood bank donors from malaria-endemic and non-endemic areas of Venezuela.

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

Adolescent↗
PubMed1998

Serologic survey for antibodies to Borrelia burgdorferi in white-tailed deer in Ontario.

Serum samples collected from 623 white-tailed deer (Odocoileus virginianus) in southern Ontario (Canada) from 1985 to 1989 were tested for antibodies to Borrelia burgdorferi using an indirect fluorescent antibody (IFA) staining method. Samples from 150 of the deer were also tested using an enzyme-linked immunosorbent assay (ELISA). At IFA titers of 1:64 and 1:128 deer with antibodies to B. burgdorferi appeared to be widespread throughout southern Ontario, with an apparent prevalence ranging from 3 to 47%. At IFA titres > or = 1:256 and ELISA titres > or = 1:160 deer with antibodies to B. burgdorferi were only present on Long Point which is the only known endemic focus of Ixodes scapularis, the primary vector for B. burgdorferi, in southern Ontario. At these titres the apparent prevalence of antibodies to B. burgdorferi on Long Point was only 5 to 7%, even though the mean intensity of infestation of adult I. scapularis on deer was > 180, and 60% of the adult ticks are infected with B. burgdorferi. Based on these results, white-tailed deer do not appear to be a good sentinel species for the distribution of B. burgdorferi.

Animals↗
PubMed1999

Antibodies to granulocytic ehrlichiae in white-footed and cotton mice in eastern United States.

Serum samples, collected from Peromyscus leucopus (white-footed mouse) or Peromyscus gossypinus (cotton mouse) during 1987 through 1990 in Florida, Georgia, Maryland, Mississippi, and North Carolina (USA), and in 1997 in southern Connecticut were analyzed by indirect fluorescent antibody (IFA) staining methods or Western blot procedures for antibodies to granulocytic ehrlichiae. Of the 82 sera from white-footed mice in Connecticut tested by IFA methods with either the BDS or NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent, 45 (55%) and 42 (51%) of the samples contained antibodies to these strains, respectively, at concentrations ranging from 1:80 to 1:2560. One (2%) of 43 sera from P. leucopus captured in Assateague Island (Maryland) had a titer of 1:80, while three (20%) of 15 sera from P. gossypinus, captured in Sapelo Island (Georgia) and four (40%) of 10 sera from cotton mice caught in Amelia Island (Florida) had antibodies to the NCH-1 strain at titers of 1:160 to 1:1,280. Fifty-five sera from P. leucopus in Cape Hatteras (North Carolina) and 30 sera from P. gossypinus in Mississippi were negative. Western blot analyses confirmed seropositivity for 19 (95%) of 20 mouse sera positive by IFA staining methods, including samples from both mouse species captured in Connecticut, Maryland, or Florida. There were key banding patterns to proteins having molecular masses of about 44, 80, 105, 110, or 120 kDa. Both serologic assays can be used to determine if mice have been exposed to granulocytic ehrlichiae. These rodents also may be useful in surveillance programs to identify endemic sites for HGE and in performing laboratory studies on immune responses to the etiologic agent.

Animals↗
PubMed1999

Infections of granulocytic ehrlichiae and Borrelia burgdorferi in white-tailed deer in Connecticut.

Serum or whole blood samples, obtained from 141 white-tailed deer (Odocoileus virginianus) in Connecticut (USA) during 1980, 1991, and 1996, were analyzed to detect past or current infections of Ehrlichia phagocytophila genogroup organisms and Borrelia burgdorferi. When the BDS or NCH-1 strains of granulocytic ehrlichiae were used separately in indirect fluorescent antibody (IFA) staining methods, antibody positivity rates varied from 25 to 64% in 1991 and 1996, respectively. All 50 sera tested from 1980 collections were negative. Although percentages of sera with B. burgdorferi antibodies, as detected by an enzyme-linked immunosorbent assay, also differed (23 to 53%), there were coexisting antibodies to both bacteria in 20 (49%) of 41 sera. In tests on specificity, 19 deer sera with ehrlichial antibodies also were tested by IFA staining procedures for Anaplasma marginale antibodies; one serum with a titer of 1:5,120 to ehrlichial antigen reacted to A. marginale antigen at a serum dilution of 1:320. In parallel analyses of 69 sera, results of Western blot analyses for ehrlichial infections in deer were concordant (72% agreement) with those of IFA staining methods containing ehrlichial antigen. All positive immunoblots showed bands to peptides of the NCH-1 strain of the human granulocytic ehrlichiosis (HGE) agent having molecular masses of about 44, 105, or 110 kDa. In polymerase chain reaction (PCR) studies of blood samples from 63 deer, 11 (18%) specimens were positive for 16S ribosomal DNA of an Ehrlichia phagocytophila genogroup organism, whereas 23 (37%) samples were positive for the DNA of the 44 kDa gene of the HGE agent. White-tailed deer are exposed to different tick-borne bacteria in areas where Ixodes scapularis ticks are abundant and may, in some instances, have had concurrent infections.

Animals↗
PubMed1999

Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response.

Traditionally, protein Ags have been injected in CFA (oil with inactivated mycobacteria) to induce immunity and with IFA (oil alone) to induce tolerance. We report here that injection of hen eggwhite lysozyme, a prototypic Ag, in CFA-induced and IFA-induced pools of hen eggwhite lysozyme-specific memory T cells of comparable fine specificity, clonal size, and avidity spectrum, but with type-1 and type-2 cytokine signatures, respectively. This adjuvant-guided induction of virtually unipolar type-1 and type-2 immunity was observed with seven protein Ags and in a total of six mouse strains. Highly polarized type-1 and type-2 immunity are thus readily achievable through the choice of adjuvant, irrespective of the genetic bias of the host and of the nature of the protein Ag. This finding should have far-reaching implications for the development of vaccines against infectious and autoimmune diseases. Furthermore, our demonstration that Ag injected with IFA is as strongly immunogenic for T cells as it is with CFA shows that the presence of the mycobacteria determines not the priming of naive T cells through the second-signal link but the path of downstream differentiation toward CD4 memory cells that express either type-1 or type-2 cytokines.

Alum Compounds↗
PubMed1999

Serologic testing of horses for granulocytic ehrlichiosis, using indirect fluorescent antibody staining and immunoblot analysis.

OBJECTIVE: To diagnose granulocytic ehrlichiosis in horses, compare results of indirect fluorescent antibody (IFA) staining procedures with those of immunoblot analysis, and compare serologic test findings with polymerase chain reaction (PCR) results. ANIMALS: 69 horses with high rectal temperatures (> or = 39 C) and lethargy, anorexia, or limb edema. PROCEDURE: 43 convalescent serum samples obtained from 38 horses 2 to 18 weeks after onset of illness were analyzed by use of immunoblot procedures and IFA staining methods, using the NCH-1 or BDS ehrlichial strains. Blood samples from 69 acutely ill horses were tested by PCR to detect ehrlichial DNA. RESULTS: Antibodies to Erlichial equi were detected in serum samples obtained during all seasons; seropositivity rates ranged from 50 to 93%. In IFA assays using the BDS or NCH-1 strain, seropositivity rates were 70 and 79%, respectively, whereas in immunoblot analyses using the NCH-1 strain, a seropositivity value of 79% was recorded. By immunoblot analysis, all serum samples of all seropositive horses were reactive to a protein having molecular mass of about 44 kd. Blood samples from 29 of 69 (42%) acutely ill horses contained ehrlichial DNA. CONCLUSION AND CLINICAL RELEVANCE: Results of the various serologic testing procedures were in close agreement with each other. All serologic testing methods are suitable for laboratory diagnosis of equine granulocytic ehrlichiosis.

Animals↗
PubMed1998

Antigenic disparity of Plasmodium vivax causing initial symptoms and causing relapse.

Relapse infections are an important obstacle to the successful treatment and control of Plasmodium vivax malaria, but little is known about the nature of the relapse. To provide insight into the antigenic disparity of the parasites causing initial clinical symptoms and causing relapse, a panel of 58 monoclonal antibodies (MAbs) against erythrocytic stages of Plasmodium vivax was tested by indirect fluorescent antibody test in five relapse cases. The initial and relapse strains from three patients (R3, R4, and R5) exhibited similar IFA reactivity with all MAbs tested, whereas the isolates from two relapse cases (R1 and R2) showed different patterns of reactivity and were seen only with 15 MAbs In case R1, different IFA reactivities were observed with 12 MAbs, nine of which reacted with the initial (RPV261) but not the relapse (RPV393) isolates, whereas the other three MAbs reacted only with the relapse isolates. With regards to the second relapse case (R2) in whom two relapses occurred, different IFA reactivities were demonstrated with seven MAbs that reacted only with the initial isolate (RPV 182) and with the isolate from the first relapse (RPV 240) but not with the isolate from the second relapse (RPV 300). The antibody responses from patients who developed primary clinical symptoms and relapse were detected by Western immunoblotting. In cases R3, R4 and R5, there was no difference in the spectrum of antigens from initial and relapse sera recognized by the antibodies. In contrast, in cases R1 and R2, the molecules recognized by antibodies in initial and relapse sera were markedly altered. In case R1, the series of molecules of P. vivax antigens recognized by initial (RPV 261) and relapse (RPV 393) sera were 21, 25, 31, 39, 42, 61, 95, 115, 200, > 200 kDa and 21, 24, 31, 35, 57, 75, 200, > 200 kDa, respectively. In case R2, the initial serum (RPV 182) recognized P. vivax antigens with molecular weights of 23, 30, 52, 57, 68, 75, 85, 95, 115, and 195 kDa while the first relapse (RPV 240) and the second relapse sera recognized P. vivax antigens with molecular weights of 23, 30, 52, 85, 95,115 kDa and 30, 57, 68, 75, 85,195 kDa, respectively.

Animals↗
PubMed1978

Contribution to the serological diagnosis of sarcocystosis.

The importance of the IFA-test (Indirect Fluorescent Antibody Test) in the diagnosis of sarcocystosis in the intermediate host has been confirmed in the present paper. We assessed the time of the first appearance of serum antibodies in the intermediate host in mice inoculated experimentally with the species Sarcocystis dispersa. By means of the IFA-test, the first antibodies were found on day 20 p.i. Cross-reactions among antisera of S. dispersa and a heterologous antigen of S. cernae disclosed that the reaction was not species-specific, but genus-specific. In addition, we confirmed serologically that the antigenic structure of the genus Frenkelia was identical to that of the genus Sarcocystis, because the results of cross reactions obtained with the IFA-test were identical.

Animals↗