SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

Double coverage and demand for health care: Evidence from quantile regression

An individual experiences double coverage when he bene ts from more than one health insurance plan at the same time. This paper examines the impact of such supplementary insurance on the demand for health care services. Its novelty is that within the context of count data modelling and without imposing restrictive parametric assumptions, the analysis is carried out for di¤erent points of the conditional distribution, not only for its mean location. Results indicate that moral hazard is present across the whole outcome distribution for both public and private second layers of health insurance coverage but with greater magnitude in the latter group. By looking at di¤erent points we unveil that stronger double coverage e¤ects are smaller for high levels of usage. We use data for Portugal, taking advantage of particular features of the public and private protection schemes on top of the statutory National Health Service. By exploring the last Portuguese Health Survey, we were able to evaluate their impacts on the consumption of doctor visi

COMPARISON OF A COMMERCIAL ENZYME IMMUNOASSAY WITH PLAQUE REDUCTION NEUTRALIZATION FOR MATERNAL AND INFANT MEASLES ANTIBODY MEASUREMENT

The most practicable assay for measurement of measles IgG (mIgG) in large numbers of sera is an enzyme immunoassay (EIA). To assess how EIA results would agree with those by the gold standard method of plaque reduction neutralization (PRN) we compared the results from the two methods in 43 pairs of maternal and umbilical cord sera, and sera from the corresponding infants when aged 11 - 14 months. In maternal-cord sera, the differences between mean antibody levels by EIA or PRN were not statistically significant, though in individual sera, differences could be large. However, agreement was less good for infants sera, in which levels of mIgG were very low. The conclusions of a study of transplacental transport of mIgG would not be affected by the use of either technique. When studying waning immunity in infants, PRN should be the method of choice, while results from studies using EIA should be interpreted with caution.

A survey of congenital Chagas’ disease, carried out at three Health Institutions in São Paulo City, Brazil

The congenital transmission of Chagas’ disease was evaluated in 57 pregnant women with Chagas’ disease and their 58 offspring. The patients were selected from three Health Institutions in São Paulo City. The maternal clinical forms of Chagas’ disease were: indeterminate (47.4%), cardiac (43.8%) and digestive (8.8%); 55 were born in endemic areas and two in São Paulo City. The transmission of Chagas’ disease at fetal level was confirmed in three (5.17%) of the 58 cases studied and one probably case of congenital Chagas’ disease. Two infected infants were born to chagasic women with HIV infection and were diagnosed by parasitolological assays (microhematocrit, quantitative buffy coat-QBC or artificial xenodiagnosis). In both cases the placenta revealed T. cruzi and HIV p24 antigens detected by immunohistochemistry. In one case, a 14-week old abortus, the diagnosis of congenital T. cruzi infection was confirmed by immunohistochemistry. The other probable infection, a 30-week old stillborn, the parasites were found in the placenta and umbilical cord. The Western blot method using trypomastigote excreted/secreted antigens of T. cruzi (TESA) was positive for IgG antibodies in 54/55 newborns and for IgM in 1/55 newborns. One of the two newborns with circulating parasites had no detectable IgG or IgM antibodies. The assessment of IgG antibodies in the sera of pregnant women and their newborns was performed by ELISA using two different T. cruzi antigens: an alkaline extract of epimastigotes (EAE) and trypomastigote excreted/secreted antigens (TESA). The analysis showed a linear correlation between maternal and newborn IgG antibody titers at birth.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Randomized, double-blind trial comparing indinavir alone, zidovudine alone and indinavir plus zidovudine in antiretroviral therapy-naive hiv-infected individuals with CD4 cell counts between 50 and 250/mm3

Treatment with indinavir has been shown to result in marked decreases in viral load and increases in CD4 cell counts in HIV-infected individuals. A randomized double-blind study to evaluate the efficacy of indinavir alone (800 mg q8h), zidovidine alone (200 mg q8h) or the combination was performed to evaluate progression to AIDS. 996 antiretroviral therapy-naive patients with CD4 cell counts of 50-250/mm3 were allocated to treatment. During the trial the protocol was amended to add lamivudine to the zidovudine-containing arms. The primary endpoint was time to development of an AIDS-defining illness or death. The study was terminated after a protocol-defined interim analysis demonstrated highly significant reductions in progression to a clinical event in the indinavir-containing arms, compared to the zidovudine arm (p<0.0001). Over a median follow-up of 52 weeks (up to 99 weeks), percent reductions in hazards for the indinavir plus zidovudine and indinavir groups compared to the zidovudine group were 70% and 61%, respectively. Significant reductions in HIV RNA and increases in CD4 cell counts were also seen in the indinavir-containing groups compared to the zidovudine group. Improvement in both CD4 cell count and HIV RNA were associated with reduced risk of disease progression. All three regimens were generally well tolerated.

Parasitological and immunological diagnoses of strongyloidiasis in immunocompromised and non-immunocompromised children at Uberlândia, state of Minas Gerais, Brazil

Parasitological and immunological diagnoses were part of a study conducted among 151 children, 83 immunocompromised (IC) and 68 non-immunocompromised (non-IC) aged from zero to 12, seen at the University Hospital, Universidade Federal de Uberlândia, State of Minas Gerais, Brazil, from February, 1996, to June, 1998. Three fecal samples from each child were analyzed for the parasitological diagnosis by Baermann-Moraes and Lutz methods. The immunological diagnosis to detect IgG and IgM antibodies was carried out by the indirect immunofluorescence antibody test (IFAT) with cryo-microtome sections of Strongyloides stercoralis and Strongyloides ratti larvae as antigens and by the ELISA test with an alkaline extract of S. ratti as the antigens. Of the 151 children 5 (3.31%) were infected with larvae of S. stercoralis (2 cases IC, 2.41%, and 3 cases non-IC, 4.41%). The IFAT-IgG detected 7 (8.43%) serum samples positive among IC, and 2 (2.94%) cases among non-IC. The ELISA-IgG test detected 10 (12.05%) serum samples positive among IC, and 1 (1.47%) case among non-IC. The IFAT-IgM detected 6 (7.22%) positive cases among IC, and 3 (4.41%) cases among non-IC. ELISA-IgM test detected 10 (12.05%) positive cases among IC, and 3 (4.41%) cases among non-IC. It was concluded that the immunological tests can help in the diagnosis of strongyloidiasis in immunocompromised children.

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

Prevalence of hepatitis B and C in the sera of patients with HIV infection in São Paulo, Brazil

The objective of this study was to evaluate the prevalence of hepatitis B and C viruses in a group of HIV infected patients, followed at a single institution since 1996. 1,693 HIV positive patients (1,162 male, 531 female) were tested for HBV infection. Virological markers for HBV included HBsAg and total anti-HBc by ELISA. 1,457 patients (1,009 male, 448 female) were tested for HCV infection. Detection of HCV antibodies was carried out by ELISA. A sample of HCV antibody positive patients was tested for HCV by PCR to confirm infection. Of 1,693 patients tested for HBV, 654 (38.6%) and 96 (5.7%) were anti-HBc and HBsAg positive, respectively. Of 1,457 patients tested for HCV, 258 (17.7%) were anti-HCV positive. 82 of these patients were also tested by PCR and 81 were positive (98%). Of 1,411 patients tested for HBV and HCV 26 (1.8%) were positive for both viruses.

Detection of hepatitis A antibodies by ELISA using saliva as clinical samples

The possibility of detecting acute infection and immunity using body fluids that are easier to collect than blood, mainly in children, would facilitate the investigation and follow-up of outbreaks of hepatitis A (HAV). Our study was carried out to evaluate the detection of anti-HAV IgM, IgA and total antibodies in saliva using serum samples as reference. Forty three paired serum and saliva samples were analyzed. From this total, 24 samples were obtained from children and 1 from one adult during the course of acute hepatitis A; an additional 18 samples were obtained from health professionals from Adolfo Lutz Institute. The sensitivity to detect anti-HAV IgM was 100% (95%CI: 79.1 to 100.0%), employing saliva as clinical samples. In detecting anti-HAV IgA, the sensitivity was 80.8% (95%CI: 60.0 to 92.7%) and for the total antibodies was 82.1% (95%CI: 62.4 to 93.2%). The specificity was 100% for each. The rate of agreement was high comparing the results of serum and saliva samples for detecting HAV antibodies. We conclude that saliva is an acceptable alternative specimen for diagnosing acute hepatitis A infection, and for screening individuals to receive hepatitis A vaccine or immunoglobulin.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.

Imunossensores Eletroquímicos para o Diagnóstico Precoce e Descentralizado do Cancro da Mama

O cancro é uma das principais causas de morte em todo o mundo. Entre as mulheres, o cancro da mama é o mais frequente. A deteção precoce do cancro é de extrema importância na medida em que pode aumentar as possibilidades de cura dos pacientes e contribuir para a diminuição da taxa de mortalidade desta doença. Um método que tem contribuído para a deteção precoce do cancro é a análise de biomarcadores. Biomarcadores associados ao cancro da mama, como o Recetor 2 do Fator de Crescimento Epidérmico Humano (HER2) e o Antigénio Carbohidratado 15-3 (CA 15-3), podem ser detetados através de dispositivos como os biossensores. Neste trabalho foram desenvolvidos dois imunossensores eletroquímicos para a análise de HER2 e CA 15-3. Para ambos os sensores foram utilizados, como transdutores, elétrodos serigrafados de carbono. A superfície destes transdutores foi nanoestruturada com nanopartículas de ouro. Foram realizados imunoensaios não-competitivos (do tipo sandwich) em ambos os imunossensores, cuja estratégia consistiu na (i) imobilização do respetivo anticorpo de captura na superfície nanoestruturada dos elétrodos, (ii) bloqueio da superfície com caseína, (iii) incubação com uma mistura do analito (HER2 ou CA 15-3) e o respetivo anticorpo de deteção biotinilado, (iv) adição de estreptavidina conjugada com fosfatase alcalina (S-AP; a AP foi utilizada como marcador enzimático), (v) adição de uma mistura do substrato enzimático (3-indoxil fosfato) e nitrato de prata, e (vi) deteção do sinal analítico através da redissolução anódica, por voltametria de varrimento linear, da prata depositada enzimaticamente. Com as condições experimentais otimizadas, foi estabelecida a curva de calibração para a análise de HER2 em soro, entre 15 e 100 ng/mL, obtendo-se um limite de deteção de 4,4 ng/mL. Para o CA 15-3 a curva de calibração (em solução aquosa) foi estabelecida entre 15 e 250 U/mL, obtendo-se um limite de deteção de 37,5 U/mL. Tendo em conta o valor limite (cutoff value) estabelecido para o HER2 (15 ng/mL) pode-se comprovar a possível utilidade do imunossensor desenvolvido para o diagnóstico precoce e descentralizado do cancro da mama. No caso do CA 15-3 serão necessários estudos adicionais para se poder avaliar a utilidade do imunossensor para o diagnóstico do cancro da mama.

Hepatitis B vaccine in infants: a randomized controlled trial comparing gluteal versus anterolateral thigh muscle administration

A significantly diminished antibody response to hepatitis B vaccine has been demonstrated in adults when the buttock is used as the injection site. However, in Brazil, the buttock continues to be recommended as site of injection for intramuscular administration of vaccines in infants. In this age group, there are no controlled studies evaluating the immunogenicity of the hepatitis B vaccine when administered at this site. In the present study, 258 infants were randomized to receive the hepatitis B vaccine either in the buttock (n = 123) or in the anterolateral thigh muscle (n = 135). The immunization schedule consisted of three doses of hepatitis B vaccine (Engerix Bâ, 10 mug) at 2, 4 and 9 months of age. There were no significant differences in the proportion of seroconversion (99.3% x 99.2%), or in the geometric mean titer of ELISA anti-HBs (1,862.1 x 1,229.0 mIU/mL) between the two groups. This study demonstrates that a satisfactory serological response can be obtained when the hepatitis B vaccine is administered intramuscularly into the buttock.

Significance of isolated hepatitis B core antibody in blood donors from São Paulo

The clinical significance of isolated anti-HBc is still a challenge. To elucidate the real importance of this finding in our blood donors, an investigation algorithm was tested. One hundred and twelve isolated anti-HBc seropositive blood donors underwent clinical evaluation and retesting of HBV markers. Those who presented repeatedly reactive isolated anti-HBc, received a single dose of hepatitis B recombinant vaccine to verify anti-HBs early response. A HBV-DNA determination by PCR was done for those who did not test positive to anti-HBs after vaccine. The level of anti-HBc was recorded as a ratio of the sample-to-cut-off values (S:C ratio) in 57 candidates at donation. Comparing true and false-positive anti-HBc results, the different S:C ratios of them were statistically significant and when less than 2, implying in a false-positive result probability over 80%. A high percent of false-positive results (16.07%) was verified after anti-HBc retesting. HBV immunity was characterized in 49.11%, either by anti-HBs detection in retesting (15.18%), or after a single dose HBV vaccination (33.93%). HBV-DNA was negative in all tested donors. In conclusion, this algorithm was useful to clarify the meaning of isolated anti-HBc in most of our blood donors.

Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens

Considering the impact of cysticercosis on public health, especially the neurologic form of the disease, neurocysticercosis (NC), we studied the frequency of positivity of anti-Taenia solium cysticercus antibodies in serum samples from 1,863 inhabitants of Cássia dos Coqueiros, SP, a municipal district located 80 km from Ribeirão Preto, an area considered endemic for cysticercosis. The 1,863 samples were tested by enzyme linked immunosorbent assay (ELISA) using an antigenic extract from Taenia crassiceps vesicular fluid (Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Of the 459 samples submitted to immunoblotting, 40 were strongly immunoreactive to the immunodominant 18 and 14 kD peptides. Considering the use of immunoblotting as confirmatory due to its high specificity, the anti-cysticercus serum prevalence in this population was 2.1%.