Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies

A simplified fluorescence inhibition microtest (SFIMT) was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT) and the fluorescence inhibition microtest (FIMT). The simplified test showed reproductibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL) gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO). The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.

Peroxidase antibody test for mucocutaneous leishmaniasis serology performance indexes and comparison with a fluorescent antibody test: Comparação com o desempenho da reação de imunofluorescência

Performance indexes of the peroxidase antibody test were compared to that of the fluorescent antibody test. The peroxidase antibody test had a statistically higher sensitivity and negative predictive value and a higher efficiency than the fluorescent antibody test but its specificity and positive predictive value were within the 95% confidence limits for the values found for the fluorescent antibody test. Such differences did not change when Chagas' disease and visceral leishmaniasis sera were included in index calculations. Statistical analysis showed that the two tests have a substantial degree of agreement but the immunofluorescent test had a specificity index and a positive predictive value equal to 100.0% when Chagas' disease and visceral leishmaniasis sera were not included in the calculations of the performance index; in this instance, a positive test result equals a disclosure of the disease attribute due to the inexistence of false positive results. The enzyme/ protein ratio of the peroxidase conjugate, resulting in heavy or light-labeled conjugates may pose technical problems to its use in serology tests.

Malaria seroepidemiology: comparison between indirect fluorescent antibody test and enzyme immunoassay using bloodspot eluates

Blood sampling on filter paper is a current practice seroepidemiological studies by indirect fluorescent antibody test (IFAT). There is, however, scant comparative information about the use of bloodspot eluates for detection of malarial IgG antibodies simultaneously by IFAT and enzyme immunoassay (ELISA). Here we report data obtained by both serological methods done on 219 bloodspot eluate samples collected in a rural community in Brazilian Amazon Basin (Alto Paraíso, Ariquemes municipality) where malaria is endemic. Plasmodium falciparum and P. vivax thick smear antigens were used in the IFAT; a detergent-soluble P. falciparum antigen was prepared for ELISA. Substantial agreement of results (Kappa coefficient k = 0.686) was observed when P. falciparum antigen was used in both tests, and IFAT titers were found to be strongly correlated ELISA antibody units (Spearman correlation coeficient rs = 0.818, p < 0.0001). Only moderate agreement (k = 0.467) between IFAT with P. vivax antigen and ELISA with P. falciparum antigen was observed. Spearman correlation coefficient value between quantitative results (IFAT titers and ELISA antibody units) in this case was numerically lowe (rs = 0.540, p < 0.0001). Our results suggest that, with P. falciparum antigen, both IFAT and ELISA performed on bloodspot eluates are equivalent for seropidemiological purposes.

Immunogenicity test of tetanus component in adsorbed vaccines by toxin binding inhibition test

Samples from 20 lots of diphtheria-tetanus (adult use dT) vaccine and from 20 lots of diphtheria-tetanus-pertussis (DTP) vaccine were used to standardize and validate the in vitro toxin binding inhibition (ToBI) test for the immunogenicity test of the tetanus component. The levels of tetanus antitoxin obtained by ToBI test were compared to those obtained using the toxin neutralization (TN) test in mice routinely employed to perform the quality control of the tetanus component in adsorbed vaccines. The results ranged from 1.8 to 3.5 IU/ml for dT and 2 to 4 IU/ml for DTP by ToBI test and 1.4 to 3 IU/ml for dT and 1.8 to 3.5 IU/ml for DTP by TN in mice. These results were significantly correlated. From this study, it is concluded that the ToBI test is an alternative to the in vivo neutralization procedure in the immunogenicity test of the tetanus component in adsorbed vaccines. A substantial refinement and a reduction in use of animals can be achieved.

Evaluation of the indirect fluorescent antibody test and modified agglutination test for detection of antibodies against Toxoplasma gondii in experimentally infected pigs

The study determined the sensitivity and specificity of the indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT) for anti-Toxoplasma gondii antibody detection by analyzing sera from 46 experimentally infected pigs. Values for sensitivity were 95.7% (confidence interval 95%: 84.0-99.2%) and for specificity 97.8% (confidence interval 95%: 87.0-99.9%) in both tests. There was an optimum agreement of results between IFAT and MAT evidenced by a Kappa test of 0.86. These results validate these tests for the detection of T. gondii infection in pigs. IFAT and MAT despite methodologies with different characteristics and readings have similar accuracy in pig serum samples.

Determination of low tetanus or diphtheria antitoxin titers in sera by a toxin neutralization assay and a modified toxin-binding inhibition test

A method for the screening of tetanus and diphtheria antibodies in serum using anatoxin (inactivated toxin) instead of toxin was developed as an alternative to the in vivo toxin neutralization assay based on the toxin-binding inhibition test (TOBI test). In this study, the serum titers (values between 1.0 and 19.5 IU) measured by a modified TOBI test (Modi-TOBI test) and toxin neutralization assays were correlated (P < 0.0001). Titers of tetanus or diphtheria antibodies were evaluated in serum samples from guinea pigs immunized with tetanus toxoid, diphtheria-tetanus or triple vaccine. For the Modi-TOBI test, after blocking the microtiter plates, standard tetanus or diphtheria antitoxin and different concentrations of guinea pig sera were incubated with the respective anatoxin. Twelve hours later, these samples were transferred to a plate previously coated with tetanus or diphtheria antitoxin to bind the remaining anatoxin. The anatoxin was then detected using a peroxidase-labeled tetanus or diphtheria antitoxin. Serum titers were calculated using a linear regression plot of the results for the corresponding standard antitoxin. For the toxin neutralization assay, L+/10/50 doses of either toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus detection, or in guinea pigs for anti-diphtheria detection. Both assays were suitable for determining wide ranges of antitoxin levels. The linear regression plots showed high correlation coefficients for tetanus (r² = 0.95, P < 0.0001) and for diphtheria (r² = 0.93, P < 0.0001) between the in vitro and the in vivo assays. The standardized method is appropriate for evaluating titers of neutralizing antibodies, thus permitting the in vitro control of serum antitoxin levels.

Rabies diagnosis and serology in bats from the State of São Paulo, Brazil

INTRODUCTION: Bats are one of the most important reservoirs and vectors of the rabies virus in the world. METHODS: From 1988 to 2003, the Zoonosis Control Center in São Paulo City performed rabies diagnosis on 5,670 bats by direct immunofluorescent test and mouse inoculation test. Blood samples were collected from 1,618 bats and the sera were analyzed using the rapid fluorescent focus inhibition test to confirm rabies antibodies. RESULTS: Forty-four (0.8%) bats were positive for rabies. The prevalence of rabies antibodies was 5.9% using 0.5IU/ml as a cutoff. Insectivorous bats (69.8%) and bats of the species Molossus molossus (51.8%) constituted the majority of the sample; however, the highest prevalence of antibodies were observed in Glossophaga soricina (14/133), Histiotus velatus (16/60), Desmodus rotundus (8/66), Artibeus lituratus (5/54), Nyctinomops macrotis (3/23), Tadarida brasiliensis (3/48), Carollia perspicillata (3/9), Eumops auripendulus (2/30), Nyctinomops laticaudatus (2/16), Sturnira lilium (2/17) and Eumops perotis (1/13). The prevalence of rabies antibodies was analyzed by species, food preference and sex. CONCLUSIONS: The expressive levels of antibodies associated with the low virus positivity verified in these bats indicate that rabies virus circulates actively among them.

Rabies surveillance in bats in Northwestern State of São Paulo

Introduction Rabies is an important zoonosis that occurs in mammals, with bats acting as Lyssavirus reservoirs in urban, rural and natural areas. Rabies cases in bats have been recorded primarily in urban areas in Northwestern State of São Paulo since 1998. This study investigated the circulation of rabies virus by seeking to identify the virus in the brain in several species of bats in this region and by measuring rabies-virus neutralizing antibody levels in the hematophagous bat Desmodus rotundus. Methods From 2008 to 2012, 1,490 bat brain samples were sent to the Universidade Estadual Paulista (UNESP) Rabies Laboratory in Araçatuba, and 125 serum samples from vampire bats that were captured in this geographical region were analyzed. Results Rabies virus was detected in the brains of 26 (2%) of 1,314 non-hematophagous bats using the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). None of the 176 hematophagous bat samples were positive for rabies virus when a virus detection test was utilized. Out of 125 vampire bat serum samples, 9 (7%) had levels of rabies virus neutralization antibodies (RVNAs) that were higher than 0.5IU/mL; 65% (81/125) had titers between 0.10IU/mL and 0.5IU/mL; and 28% (35/125) were negative for RVNAs using the simplified fluorescent inhibition microtest (SFIMT) in BHK21 cells. The observed positivity rate (1.7%) was higher than the average positivity rate of 1.3% that was previously found in this region. Conclusions The high percentage of vampire bats with neutralizing antibodies suggests that recent rabies virus exposure has occurred, indicating the necessity of surveillance measures in nearby regions that are at risk to avoid diffusion of the rabies virus and possible rabies occurrences.

Toxoplasma gondii Antigenuria in Patients with Acquired Immune Deficiency Syndrome

A longitudinal study was performed with sera and urine of patients with acquired immune deficiency syndrome (AIDS), taken before, during and after clinically Toxoplasma infection. The tested patients were followed for an average of two years. The titres of the specific IgG and IgM antibodies were measured by an indirect fluorescent antibody test (IFAT), and the appearance of circulating antigens of T. gondii was determined in 36 urine samples of 13 patients with neurotoxoplasmosis by means of the coagglutination test. The presence of T. gondii antigens in the urine of AIDS patients by this test was correlated with the immunoblot technique, with clinical symptoms and also with pathological findings. Our results indicate that the detection of T. gondii antigens in the urine of AIDS patients can be regarded as a rapid and efficient method for the diagnosis of acute toxoplasmosis

Genetic and Antigenic Analysis of Adenovirus Type 3 Strains Showing Intermediate Behavior in Standard Seroneutralization Test

During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

The measurement by serological means of the impact of the Hong Kong/68 influenza virus on a population

In order to obtain evidence on the size of the impact of the Hong Kong/68 variant of influenza A2 virus on the population of São Paulo, Brazil, serum samples taken in 1967 before this variant appeared and during successive years after it appeared were examined for their antibody content. Haemagglutination-inhibition tests performed on a total of 2726 serum samples from adults showed a sharp decrease in 1969 of the proportion of sera without antibody to the Hong Kong/68 variant and a corresponding mercase in the proportion with high titres. It was concluded that about three-quarters of the adult population became infected at some time after the variant appeared, the majority in the first year of prevalence.

Serological analysis reveals circulation of influenza C viruses, Brazil

The circulation of influenza C viruses in Rio de Janeiro, Brazil, was studied when significant levels of antibodies were detected (56.7%) with hemagglutination inhibition test, used as a standard methodology for influenza virus studies.

Bat rabies in the north-northwestern regions of the state of São Paulo, Brazil: 1997-2002

OBJECTIVE: Reports on bat rabies in Brazil are sporadic and isolated. This study aimed at describing the detection of rabies virus in bats in the state of São Paulo. METHODS: A total of 7,393 bats from 235 municipalities of the north and northwestern areas of the state of São Paulo, Southeastern Brazil, were assessed according to their morphological and morphometric characteristics from 1997 to 2002. Fluorescent antibody test and mice inoculation were used for viral identification. RESULTS: Of all samples examined, 1.3% was rabies virus positive, ranging from 0.2% in 1997 to 1.6% in 2001. There were found 98 bats infected, 87 in the urban area. Fluorescent antibody test was detected in 77 positive samples, whereas 92 produced rabies signs in mice; incubation period ranging from 4 to 23 days. In 43 cities at least one rabid bat was observed. The highest proportion (33.7%) of rabies virus was found in Artibeus lituratus. Eptesicus and Myotis were the most frequent positive species (24.5%) of the Vespertilionidae family. The species Molossus molossus and Molossus rufus showed 14.3% positive bats. There were no differences in the distribution of positive rabies between females (33; 48.5%) and males (35; 51.5%). CONCLUSIONS: Rabies-infected bats were found in environments that pose a risk to both human and domestic animal population and there is a need for actions aiming at the control of these species and public education.