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.

Evaluation of a rapid dipstick test, Malar-CheckTM, for the diagnosis of Plasmodium falciparum malaria in Brazil

The present study was carried out to evaluate the Malar-CheckTM Pf test, an immunochromatographic assay that detects Plasmodium falciparum Histidine Rich Protein II, does not require equipment, and is easy and rapid to perform. In dilution assays performed to test sensitivity against known parasite density, Malar-CheckTMwere compared with thick blood smear (TBS), the gold standard for diagnosis. Palo Alto isolate or P. falciparum blood from patients with different parasitemias was used. The average cut-off points for each technique in three independent experiments were 12 and 71 parasites/mm³ (TBS and Malar-CheckTM, respectively). In the field assays, samples were collected from patients with fever who visited endemic regions. Compared to TBS, Malar-CheckTMyielded true-positive results in 38 patients, false-positive results in 3, true-negative results in 23, and false-negative result in 1. Malar-CheckTMperformed with samples from falciparum-infected patients after treatment showed persistence of antigen up to 30 days. Malar-CheckTM should aid the diagnosis of P. falciparum in remote areas and improve routine diagnosis even when microscopy is available. Previous P. falciparum infection, which can determine a false-positive test in cured individuals, should be considered. The prompt results obtained with the Malar-CheckTM for early diagnosis could avoid disease evolution to severe cases.

Field evaluation of the whole blood immunochromatographic test for rapid bancroftian filariasis diagnosis in the northeast of Brazil

This study evaluated the whole blood immunochromatographic card test (ICT card test) in a survey performed in Northeastern Brazil. 625 people were examined by the thick blood film (TBF) and ICT card test. Residents of a non-endemic area were also tested by the whole blood card test and Og4C3. The sensitivity of the ICT card test was 94.7% overall, but lower in females than males, based on the reasonable assumption that TBF is 100% specific. However, since TBF and other methods have unknown sensitivity, the true specificity of the card test is unknown. Nevertheless, it is possible to estimate upper and lower limits for the specificity, and relate it to the prevalence of the disease. In the endemic area, the possible range of the specificity was from 72.4% to 100%. 29.6% of the card tests performed in the non-endemic area exhibited faint lines that were interpreted as positives. Characteristics of the method including high sensitivity, promptness and simplicity justify its use for screening of filariasis. However, detailed information about the correct interpretation in case of extremely faint lines is essential. Further studies designed to consider problems arising from imperfect standards are necessary, as is a sounder diagnostic definition for the card test.

Prevalence of IgG antibodies specific to Toxoplasma gondii among blood donors in Recife, Northeast Brazil

A serological survey of Toxoplasma gondii infection in blood donors was carried out in order to identify seroprevalence in Recife, Brazil. Sera from 160 individuals (119 male and 41 female) were evaluated by using a Toxoplasma IgG-antibody enzyme immunoassay (Denka Seiken Co., LTD., Tokyo, Japan). The seropositive percentual for males (79.0%) showed to be higher (p < 0.05) than for females (63.4%). This percentage increases with age, ranging from 18.2% to 92.6% for individuals aging under 20 and 40-50 years old, respectively. For women of childbearing age (18-40 years) it was found a prevalence of 51.6%.

Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies

A rapid test based on an immunochromatography assay - Determine™ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine™ test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine™ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

Prevalence of IgG varicella zoster virus antibodies in the Kuikuro and Kaiabi indigenous communities in Xingu National Park, Brazil, before varicella vaccination

The purpose of the study was to estimate the prevalence of IgG antibodies against varicella zoster virus (VZV) in the two most populated indigenous ethnic groups from Xingu Indigenous National Park, in Brazil, prior to the introduction of vaccination against the disease, and to determine the positive and the negative predictive values of a history of varicella infection. In 2001, 589 inhabitants of two Kuikuro villages and three Kaiabi villages were evaluated and provided information concerning previous varicella infection. An indirect immunosorbent assay (ELISA) to detect IgG anti-VZV antibodies was performed in 224 blood samples - volunteer selection had no interference of anamnesis. IgG prevalence was 80.8% (95% Confidence Interval: 76% - 86%). The seroepidemiology of varicella in Xingu National Park prior to varicella vaccine introduction was comparable to the Brazilian national seroprevalence described in the literature, and so were the positive (98%) and the negative predictive value (41%) of the referred history.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

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.

Anti-Taenia solium metacestode IgG antibodies in serum samples from inhabitants of a central-western region of Brazil

A total of 354 serum samples from inhabitants who frequent the Clinical Laboratory in Catalão, Goiás, in the central-western region of Brazil, were collected from June to August, 2002. The samples were evaluated by indirect immunofluorescence antibody tests and an enzyme linked immunosorbent assay in order to detect anti-Taenia solium metacestode IgG antibodies. Reactive and inconclusive samples were tested by Western blotting (WB). Considering WB as a confirmation, the frequency of antibodies in the serum samples of the above population was 11.3% (CI 5.09 - 17.51). The immunodominant bands most frequently recognized in WB were 64-68 kDa (97.5%) and 47-52 kDa (80%). The percentage of seropositivity to cysticercosis was significantly higher for individuals residing in areas without sewage systems (p < 0.0001). In conclusion, the results indicate a probable endemic situation of cysticercosis in this population. These results reinforce the urgent need for control and prevention measures to be taken by the local public health services.