Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Anti-Taenia solium metacestodes antibodies in serum from blood donors from four cities of Triângulo Mineiro area, Minas Gerais, Brazil, 1995

Serological survey was performed to detect IgG antibodies anti-Taenia solium metacestodes in blood donors of Hemocentro Regional de Uberlândia, Minas Gerais, Brazil. A total of 1133 sera from blood donors coming from four cities of Triângulo Mineiro area were analyzed by the indirect fluorescence antibody test (IFAT) and the enzyme linked immunosorbent assay (ELISA). Specific IgG antibodies were found in 5.6% of the studied population, showing differences in the positive rates according to their origin: Araguari (13.5%), Tupaciguara (5.0%), Monte Alegre de Minas (4.8%) and Uberlândia (4.7%). The results indicate the probable endemicity of cysticercosis in this population.

Heterologous antigen extract in ELISA for the detection of human IgE anti-Strongyloides stercoralis

Strongyloides ratti larval extract was used for the standardization of ELISA to detect genus-specific IgE in human strongyloidiasis. Forty serum samples from monoinfected patients shedding S. stercoralis larvae (Group I), 40 from patients with other intestinal parasites (Group II), and 40 from copronegative healthy subjects (Group III) were analyzed. Genus-specific IgE levels (ELISA Index: EI) were significantly higher in the group I (EI = 1.43) than groups II (EI = 0.70) and III (EI = 0.71), showing positivity rates of 55%, 2.5% and 0%, respectively. Similarly, sera from copropositive patients had significantly higher levels of total IgE (866 IU/mL) as compared to those from group II (302 IU/mL) and III (143 IU/mL). A significant positive correlation was found between levels of Strongyloides specific-IgE and total IgE in sera from patients with strongyloidiasis. In conclusion, S. ratti heterologous extract showed to be a useful tool for detecting genus-specific IgE by ELISA, contributing for a better characterization of the immune response profile in human strongyloidiasis.

Evidence of active herpesvirus 6 (variant-A) infection in patients with lymphadenopathy in Belém, Pará, Brazil

A total of 323 patients with lymphadenopathy were selected in Belém, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates are found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across < 5 through 41-50-year age-groups for patients whose serum samples were IgM+, with rates ranging from 7.7% (female subjects aged < 5 years) to 80.0% (male, 11-20 years). Among patients whose serological status was IgG+, HHV-6 DNA was detected in < 5, 6-10, 21-30 and > 50 age-groups at rates that ranged from 15.4% (male, < 5 years of age) to 100.0% (female aged 11-20 years). Swelling cervical lymph nodes were the most common sign, accounting for 9 (32.0%) cases in each gender group. Among patients (n = 28) with active infection by HHV-6A variant, duration of symptoms lasted 1-5 days in 35.7% of subjects, whereas in 64.3% of them the disease lasted 6-20 days. Our data suggest that it is worth seeking for HHV-6 infection whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.

Human toxocariasis: frequency of anti-Toxocara antibodies in children and adolescents from an outpatient clinic for lymphatic filariasis in Recife, Northeast Brazil

In a transversal study on a sample of 386 children and adolescents from an outpatient clinic for filariasis in Recife, Northeast Brazil, the frequency of anti-Toxocara antibodies and its relation to age, gender, number of peripheral eosinophils, Wuchereria bancrofti microfilariae and intestinal helminths was determined. The total anti-Toxocara IgG antibody frequency was 39.4%, by ELISA technique. The difference in frequency between males (40.1%) and females (37.6%) was not statistically significant. The 6 to 10-year-old subset presented the highest frequency of anti-Toxocara antibodies (60%), and within this age group there was a statistically significant male bias. There was also a significant association between the number of eosinophils and the presence of anti-Toxocara antibodies. Intestinal parasite frequency was 52.1%, but no association was found between this data and the presence of anti-Toxocara antibodies. In the present sample, 42.2% of the patients were Wuchereria bancrofti carriers, however, again this was not associated with the presence of anti-Toxocara antibodies. In conclusion, anti-Toxocara antibodies were highly prevalent in this sample. The present data show that there is no cross correlation between anti-Toxocara IgG antibody and the presence of intestinal helminths and filariasis.

Dot-ELISA for the diagnosis of neurocysticercosis

The aim of the present study was to standardize and evaluate dot-Enzyme linked immunosorbent assay (Dot-ELISA), a simple and rapid test for the detection of cysticercus antibodies in the serum for the diagnosis of neurocysticercosis (NCC). The antigen used in the study was a complete homogenate of Cysticercus cellulosae cysts obtained from infected pigs and dotted on to nitrocellulose membrane. Test sera were collected from the patients of NCC, and control sera from patients with other diseases and healthy students and blood donors of the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, during a study period from 2001 to 2003. Dot-ELISA detected antibodies in 14 of 25 (56%) in clinically suspected cases of NCC, 13 of 23 (56.5%) in CT/MRI proven cases of NCC and 2 of 25 (8%) each in non-cysticercal CNS infection controls and healthy controls. The test showed a sensitivity of 56.25%, specificity of 92%, positive predictive value of 87.09%, and negative predictive value of 70.76%. Results of the present study shows that the Dot-ELISA as a simple test can be used in the field or poorly equipped laboratories for diagnosis of NCC .

Cysticercus antibodies and antigens in serum from blood donors from Pondicherry, India

The aim of the present study was to screen the serum of blood donors, which are apparently healthy and residing in Pondicherry or its neighboring districts of Tamil Nadu State, for specific detection of Cysticercus antigens and antibodies. A total of 216 blood samples were collected from blood donors at the Central Blood Bank, JIPMER Hospital, Pondicherry, India during January and February 2004. Enzyme-linked immunosorbent assay (ELISA) was used to demonstrate anti-Cysticercus antibodies and the Co-agglutination (CoA) was used to detect antigen in sera. 14 (6.48 %) males were positive for either anti-Cysticercus antibodies or antigens. Of these eight sera were positive for anti-Cysticercus antibodies and six were positive for antigens. Results of the present study show that serum Cysticercus antigen detection may be a useful adjunct to antibody testing for seroprevalence studies of cysticercosis in the community. The present study is the first kind of study, carried out to determine both cysticercal antibodies as well as antigens in the serum samples collected from the healthy blood donors.

St. Louis encephalitis vírus: first isolation from a human in São Paulo state, Brasil

This paper reports the isolation of St. Louis encephalitis virus (SLEV) from a febrile human case suspected to be dengue, in São Pedro, São Paulo State. A MAC-ELISA done on the patient's acute and convalescent sera was inconclusive and hemagglutination inhibition test detected IgG antibody for flaviviruses. An indirect immunofluorescent assay done on the C6/36 cell culture inoculated with the acute serum was positive for flaviviruses but negative when tested with dengue monoclonal antibodies. RNA extracted from the infected cell culture supernatant was amplified by RT-PCR in the presence of NS5 universal flavivirus primers and directly sequenced. Results of BLAST search indicated that this sequence shares 93% nucleotide similarity with the sequence of SLEV (strain-MSI.7), confirmed by RT-PCR performed with SLEV specific primers. Since SLEV was identified as the cause of human disease, it is necessary to improve surveillance in order to achieve early detection of this agent in the state of São Paulo and in Brazil. This finding is also an alert to health professionals about the need for more complete clinical and epidemiological investigations of febrile illnesses as in the reported case. SLEV infections can be unrecognized or confused with other ones caused by an arbovirus, such as dengue.

Serodiagnosis of neurocysticercosis in patients with epileptic seizure using ELISA and immunoblot assay

Sera from 88 patients from Santa Catarina and São Paulo states of Brazil, with epileptic seizures who underwent cerebral computed tomography (CT) were analyzed for the detection of antibodies to T. solium cysticercus by ELISA and Immunoblot (IB) with the following antigens: Taenia solium cysticercus total saline (Tso), Taenia crassiceps cysticercus vesicular fluid (Tcra-vf) and T. crassiceps cysticercus glycoproteins (Tcra-gp). ELISA carried out with Tso, Tcra-vf and Tcra-gp antigens showed 95%, 90% and 80% sensitivities, respectively, and 68%, 85% and 93% specificities, respectively. In the epileptic patients group, ELISA positivity was 30%, 51% and 35% with Tso, Tcra-vf and Tcra-gp antigens respectively. Considering the IB as the confirmatory test, the positivity was 16% (14/88) in the epileptic patients total group and 22% (12/54) in the epileptic patients with positive CT and signals of cysticercosis. We found a significant statistical correlation among ELISA or IB results and the phase of the disease when any antigens were used (p < 0.05). We emphasize the need to introduce in the laboratory routine the search for neurocysticercosis (NC) in patients presenting with epileptic seizures because of the high risk of acquiring NC in our region and its potential cause of epilepsy.

Dot-Elisa for evaluation of hydatid cyst wall, protoscoleces and hydatid cyst fluid antigens in the serodiagnosis of cystic echinococcosis

The aim of the present study is to evaluate cyst wall and protoscolex as an alternate source of antigen in serodiagnosis of cystic echinococcosis (CE). A total of 90 blood samples, 30 each of confirmed CE cases, disease controls and healthy controls were collected. Dot-ELISA using cyst wall, protoscolex and cyst fluid were used to demonstrate anti-hydatid antibodies. The sensitivity of Dot-ELISA using cyst wall, protoscolex and cyst fluid was 96.66%, 86.66% and 93.33% respectively and the specificity of the assay was 70% for Dot-ELISA using cyst fluid, protoscolex and cyst wall antigens. Results of the present study show that cyst wall and protoscolex can also be an useful source of antigen in detection of hydatid antibodies in the serodiagnosis of CE.

Correlation between specific IgM levels and percentage IgG-class antibody avidity to Toxoplasma gondii

Toxoplasmosis is an usually asymptomatic worldwide disseminated infection. In its congenital presentation it may lead to abortion or fetal malformations. Antenatal evaluation is considered of paramount importance to identify seronegative women and allow for prophylaxis. Recent improvements in sensitivity of IgM tests has made IgM detection an extremely protracted acute phase marker, and IgG avidity evaluation test became necessary. Observation has shown that a correlation can be established between IgM levels and avidity percentages, suggesting that frequently the avidity test may not be necessary. In this study we analyzed Toxoplasma gondii IgM levels of 202 samples and their IgG avidity percentages, in order to define specific levels whose IgM quantification could by itself define serodiagnosis and therefore make the avidity evaluation unnecessary. We showed that for IgM levels bellow 2.0 and above 6.0 serodiagnosis of toxoplasmosis could be established without need of IgG avidity test. IgM levels between these two parameters are associated with varying avidity indexes highlighting the importance of its evaluation as a means to confirm toxoplasmosis. Following this demonstration it was possible to avoid the avidity test for 75% of the cases, to reduce the turnaround time and to reduce costs.

Seroepidemiology of toxoplasmosis in women of childbearing age from a marginal community of Maracaibo, Venezuela

The aim of this study was to determine the prevalence of anti-T. gondii total and IgM antibodies in women of childbearing age. One hundred serum samples of women were studied with age range from 11 to 45 years old. Samples were chosen by random. The determination of total antibodies was carried out through the indirect hemagglutination technique and IgM antibodies by ELISA's technique. The statistical analysis was carried out through the Chi square and the Spearman correlation tests. The theoretical estimated incidence of congenital toxoplasmosis was calculated, according to the annual increment of antibody prevalence among the age groups. The overall prevalence of toxoplasmosis was 33%, while only six individuals (18.2%) were positive to IgM. The highest prevalence was observed in the 11-35 year-old age group. The theoretical estimated incidence was 1.5 for 100 pregnancies in women of 21-25 year-old group; it decreased until 0.1% in the 41-45 year-old age group. The findings show a high prevalence of toxoplasmosis in this community with a high infection risk in women of the studied age group and the high cat population observed, suggesting that the transmission way by contaminated soils may play a main role in the spreading of toxoplasmosis in this community.