Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs

This paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT) and hemagglutination test (IHAT) were standardized, and complement fixation test (CFT) and direct agglutination test (DAT) were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1) parasitemic dogs of two localities of endemic area (EA) of Santiago del Estero province in Argentina (n = 134); (2) non-parasitemic dogs of the same area (n = 285); (3) dogs experimentally infected with T. cruzi in the patent period (n = 6); (4) non-infected dogs (n = 56) which were born in the city of Buenos Aires (BA), one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95% of reactive sera. Non parasitemic dogs EA showed 77% of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84% of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61% were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95%, 84%, 97% and 95% respectively.

Efficacy of an enzyme-linked immunosorbent assay as a diagnostic tool for schistosomiasis mansoni in individuals with low worm burden

IgM-ELISA is an immunoenzymatic method useful for detection of IgM antibodies against a fraction of Schistosoma mansoni adult worm antigen (AWA) that is soluble in trichloroacetic acid (AWA-TCA). This method was applied to three groups of individuals with different clinical and epidemiological characteristics, and the results compared with those obtained by other diagnostic methods: immunofluorescence test for detection of IgM antibodies (IgM-IFT) or IgG antibodies (IgG-IFT), ELISA for detection of IgG antibodies (IgG-ELISA), and two parasitological methods, Kato-Katz and miracidium hatching. The IgM-ELISA presented a sensitivity of 98%, when the parasitologic fecal examination was defined as reference diagnostic method, and a specificity of 98 and 97.3%, respectively for the group of clinically healthy individuals and other helminth carriers. A comparative analysis between the results of IgM-ELISA and those obtained by other serologic tests showed a good degree of agreement, with Kappa indices ranging from 0.95 to 0.98. The diagnostic efficacy of 97.8%, as determined with schistosomiasis patients with low parasitic burden, suggests the excellent performance of the IgM-ELISA and its usefulness for the diagnosis of schistosomiasis when applied in low endemic areas.

Polymerase chain reaction with two molecular targets in mucosal leishmaniasis' diagnosis: a validation study

We validated the polymerase chain reaction (PCR) with a composite reference standard in 61 patients clinically suspected of having mucosal leishmaniasis, 36 of which were cases and 25 were non-cases according to this reference standard. Patient classification and test application were carried out independently by two blind observers. One pair of primers was used to amplify a fragment of 120 bp in the conserved region of kDNA and another pair was used to amplify the internal transcript spacers (ITS) rDNA. PCR showed 68.6% (95% CI 59.2-72.6) sensitivity and 92% (95% CI 78.9-97.7) specificity; positive likelihood ratio: 8.6 (95% CI 2.8-31.3) and negative likelihood ratio: 0.3 (95% CI 0.3-0.5), when kDNA molecular target was amplified. The test performed better on sensitivity using this target compared to the ITS rDNA molecular target which showed 40% (95% CI 31.5-42.3) sensitivity and 96% (95% CI 84.1-99.3) specificity; positive likelihood ratio: 10 (95% CI 2.0-58.8) and negative likelihood ratio: 0.6 (95% CI 0.6-0.8). The inter-observer agreement was excellent for both tests. Based upon results obtained and due to low performance of conventional methods for diagnosing mucosal leishmaniasis, we consider PCR with kDNA as molecular target is a useful diagnostic test and the ITS rDNA molecular target is useful when the aim is to identify species.

Comparison of serological responses in two different populations with pulmonary tuberculosis

Observational studies on the humoural immune responses of the Warao indigenous people from Delta Amacuro, an isolated area, were compared with urban residents of the Venezuelan capital. Mycobacterium tuberculosis-specific reactivities (IgM, IgE, sIgA, IgG and IgG subclasses) were measured by ELISA using PPD and 38-kDa M. tuberculosis antigens. A total of 294 individuals were studied, 162 Warao (indigenous people) and 132 Creole (non-indigenous people). The patient group consisted of 87 Warao patients and 58 Creole patients, while the control group consisted of 75 Warao controls and 74 Creole controls. Combinations among the isotypes studied were performed. The findings showed that for the Warao people, sensitivity to the combination including anti-PPD IgG and IgE was 92.0%, while for the Creole people, sensitivity to the combination including anti-PPD IgG but more so anti-PPD IgG1 and IgG2 was 90.0%. Simple tests were able to show higher specificities, which were population-specific; specificities were anti-PPD IgG3, 100.0% and anti-PPD IgM, 97.4% for the Warao and Creole peoples, respectively. In conclusion, while simple tests reached high specificity, the multi-isotype tests improved sensitivity; the latter shows this approach may be useful in diagnostic testing.

Diagnostic reliability of an immunochromatographic test for Chagas disease screening at a primary health care centre in a rural endemic area

Many patients with Chagas disease live in remote communities that lack both equipment and trained personnel to perform a diagnosis by conventional serology (CS). Thus, reliable tests suitable for use under difficult conditions are required. In this study, we evaluated the ability of personnel with and without laboratory skills to perform immunochromatographic (IC) tests to detect Chagas disease at a primary health care centre (PHCC). We examined whole blood samples from 241 patients and serum samples from 238 patients. Then, we calculated the percentage of overall agreement (POA) between the two groups of operators for the sensitivity (S), specificity (Sp) and positive (PPV) and negative (NPV) predictive values of IC tests compared to CS tests. We also evaluated the level of agreement between ELISAs and indirect haemagglutination (IHA) tests. The readings of the IC test results showed 100% agreement (POA = 1). The IC test on whole blood showed the following values: S = 87.3%; Sp = 98.8%; PPV = 96.9% and NPV = 95.9%. Additionally, the IC test on serum displayed the following results: S = 95.7%; Sp = 100%; PPV = 100% and NPV = 98.2%. Using whole blood, the agreement with ELISA was 96.3% and the agreement with IHA was 94.1%. Using serum, the agreement with ELISA was 97.8% and the agreement with IHA was 96.6%. The IC test performance with serum samples was excellent and demonstrated its usefulness in a PHCC with minimal equipment. If the IC test S value and NPV with whole blood are improved, then this test could also be used in areas lacking laboratories or specialised personnel.

Performance of a Brazilian population sample in the Boston Diagnostic Aphasia Examination: A pilot study

Brazilian researchers and health professionals often face the challenge of having to use tests developed in foreign languages and standardized for populations of other countries, especially in the fields of Neuropsychology and Neurolinguistics. This fact promotes a feeling that some scoring systems may be inadequate for our sociocultural reality. In the present study, we describe the performance of a Brazilian population sample submitted to a translated and adapted version of the Boston Diagnostic Aphasia Examination (BDAE). Sixty normal volunteers (21 men and 39 women), all Portuguese native speakers, ranging in age from 15 to 78 years (average 43.7) and with an educational level of 2 to 16 years (average 9.9), were tested using a translated and adapted Portuguese version of the BDAE. Cut-off scores are suggested for our population and the performance of the Brazilian sample is compared to that of American and Colombian samples, with the results being closely similar in all tasks. We also performed a correlation analysis between age, gender and educational level and the influence of these variables on the performance of the subjects. We found no statistically significant differences between genders. Educational level correlated positively with performance, especially in the subtests involving reading and writing. There was a negative correlation between age and performance in two subtests (Visual Confrontation Naming and Sentences to Dictation), but a coexisting effect of educational level could not be ruled out.

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

Diagnostic leaf to evaluate the nutritional status of Jatropha

ABSTRACT The analytical determination of nutrient levels in recently mature leaves in order to diagnose nutritional status is based on the fact that leaves are metabolically active and more sensitive to variation in nutrients of the soil. In most of cases, there is a direct well known between foliar content and the development and yield of the plant. However, for a more accurate interpretation, it is essential to establish the index leaf. There are few published studies about Jatropha with contrasting results. In order to establish the index leaf, in adult plants, the macronutrient levels were evaluated in samples collected in experimental plots, in which doses of nitrogen and phosphorus were applied, in two parts of the floral branches (in the top and in the middle thirds); and in three positions of leaves of the floral branch (between the 1st and 3rd, 6th and 8th, and 13th and 15th leaves below the inflorescence). The location of the leaf on the plant significantly affects nutrient contents. Nitrogen, phosphorus, potassium and sulfur tend to have higher concentration in young tissues. Calcium and magnesium showed higher levels in the basal leaves of floral branches. Samples collected in the top third of plants (between the 6th and 15th leaves of the floral branch) are more sensitive to variations of nitrogen and phosphorus fertilization. Therefore, we indicate the 6th to 15th leaves of the top third plants as index leaves estimate nutritional status of Jatropha.

Schizophrenia in males of cognitive performance: discriminative and diagnostic values

OBJECTIVE: To evaluate the discriminative and diagnostic values of neuropsychological tests for identifying schizophrenia patients. METHODS: A cross-sectional study with 36 male schizophrenia outpatients and 72 healthy matched volunteers was carried out. Participants underwent the following neuropsychological tests: Wisconsin Card Sorting test, Verbal Fluency, Stroop test, Mini Mental State Examination, and Spatial Recognition Span. Sensitivity and specificity estimated the diagnostic value of tests with cutoffs obtained using Receiver Operating Characteristic curves. The latent class model (diagnosis of schizophrenia) was used as gold standard. RESULTS: Although patients presented lower scores in most tests, the highest canonical function for the discriminant analysis was 0.57 (Verbal Fluency M). The best sensitivity and specificity were obtained in the Verbal Fluency M test (75 and 65, respectively). CONCLUSIONS: The neuropsychological tests showed moderate diagnostic value for the identification of schizophrenia patients. These findings suggested that the cognitive impairment measured by these tests might not be homogeneous among schizophrenia patients.

Living conditions and receptive vocabulary of children aged two to five years

OBJECTIVE: To assess the receptive vocabulary of children aged between two years and six months and five years and eleven months who were attending childcare centers and kindergarten schools. METHODS: An analytical cross-sectional study was carried out in the municipality of Embu, Southeastern Brazil. The Peabody Picture Vocabulary Test and analysis of factors associated with children's performance were applied. The sample consisted of 201 children of both genders, aged between two and six years. Statistical analysis was performed using multivariate analysis and logistic regression model. The dependent variable analyzed was test performance and the independent variables were child's age, mother's level of education and family socio-demographic characteristics. RESULTS: It was observed that 44.3% of the children had performances in the test that were below what would be expected for their age. The factors associated with the best performances in the test were child's age (OR=2.4; 95% CI: 1.6-3.5) and mother's education level (OR= 3.2; 95% CI: 1.3-7.4). CONCLUSIONS: Mother's education level is important for child's language development. Settings such as childcare and kindergarten schools are protective factors for child development in families of low income and education.

Factors related to inadequate cervical cancer screening in two Brazilian state capitals

OBJECTIVE:To analyze factors associated with cervical cancer screening failure. METHODS:Population-based cross-sectional study with self-weighted two-stage cluster sampling conducted in the cities of Fortaleza (Northeastern Brazil) and Rio de Janeiro (Southeastern Brazil) in 2002. Subjects were women aged 25-59 years in the last three years prior to the study. Data were analyzed through Poisson regression using a hierarchical model. RESULTS: The proportion of women who did not undergo the Pap smear test in Fortaleza and Rio de Janeiro was 19.1% (95% CI: 16.1;22.1) and 16.5% (95% CI: 14.1;18.9), respectively. Higher prevalence ratios of cervical cancer screening failure in both cities were seen among women with low education and low per capita income, old age, unmarried, who never underwent mammography, clinical breast examination, and blood glucose and cholesterol level testing. Smokers also had lower screening rates compared to non-smoker women and this difference was only statistically significant in Rio de Janeiro. CONCLUSIONS:The study findings point to the need of intervention focusing particularly women in worse socioeconomic conditions and access to healthcare, old-aged and unmarried. Education activities must prioritize screening of asymptomatic women and early diagnosis for symptomatic women and access to adequate diagnostic methods and treatment should be provided.

Seroepidemiology of hepatitis a and b in two urban communities of Rio de Janeiro, Brazil

Hepatitis B markers were determined in 397 individuals from Niterói and 680 from Nova Iguaçu and prevalences of 9.1% (1.0% of HBsAg and 8.1% of anti HBs) and 11.1% (1.8% of HBsAg and 9.3% of antiHBs) were found, respectively. The comparative prevalence of both markers in relation to age showed a higher prevalence of HBsAg in the group 21-50 years old. Considering the antiHBs antibody, it was demostrated a gradual increase with age, reaching 14.9% in Niterói and 29.1% in Nova Iguaçu in individuals over 51 years old. For hepatitis A, in 259 samples from Niterói, equally distributed by age groups, an overall prevalence of 74.5% of anti-HAV antibodies was found. This prevalence increases gradually reaching 90.0% at age over thirty. In 254 samples from Nova Iguaçu analysed, a prevalence of 90.5% of antibodies was encountered when the same criteria of distribution of samples were used. This level of prevalence reached 90.0% already in the age over ten years old. The tests were performed by enzyme immunoassay with reagents prepared in our laboratory.

Predictive value of serologic tests in the diagnosis and follow-up of patients with paracoccidioidomycosis

A serologic study was undertaken in a group of 43 patients with active paracoccidioidomycosis who were treated in the same form (ketoconazole), for identical periods of time (6 months), and folio wed-up for various periods posttherapy. The tests employed were agar gel immunodiffusion (AGID) and complement fixation (FC). Also studied were 50 sera from patients with proven histoplasmosis and pulmonary aspergilloma, 30 patients with culturaly proven tuberculosis as well as 92 specimens from healthy individuals, residents in the endemic area for paracoccidioidomycosis. A single lot of yeast filtrate antigen was used throughout the study. The value of each test was measured according to GALEN and GAMBINO6. Both tests were highly sensitive, 89 and 93% respectively. Regarding their specificity, the AGID was totally specific while the CF exhibited 96.6% and 97% specificity in front of tuberculosis patients and healthy individuals respectively and 82% in comparison with patients with other mycoses. The concept of predictive value, that is, the certainty one has in accepting a positive test as diagnostic of paracoccidioidomycosis, favored the AGID procedure (100%) over the CF test. The latter could sort out with 93% certainty a patient with paracoccidioidomycosis among a group of healthy individuals and with 97.5% in the case of TB patients; when the group in question was composed by individuals with other deep mycoses, such certainty was lower (81%). The above results indicate that both the AGID and the CF tests furnish results of high confidence; one should not relay, however, in the CF alone as a means to establish the specific diagnosis of paracoccidioidomycosis.

Passive haemagglutination test for human neurocysticercosis immunodiagnosis: II - Comparison of two standardized procedures for the passive haemagglutination reagent in the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluids

A comparison of two different standardized reagent procedures for the passive haemagglutination test (PHA) in the detection of specific antibody to Cysticercus cellulosae in cerebrospinal fluid (CSF) was carried out. The formaldehyde-treated group O Rh-human red blood cells (HuRBC) and glutaraldehyde-treated sheep red blood cells (SRBC) were the supplies for the reagents preparation and, in the tests, they were designated as PHA-1 and PHA-2, respectively. For both reagents the cells were coated with the cysticerci total saline extract (TS) antigen. PHA-1 and PHA-2 were assessed in a total of 204 CSF from patients with neurocysticercosis, from non-related infections and from healthy individuals. The positivity and specificity indices obtained were respectively 81.7% and 94.4% for PHA-1 and for PHA-2, 88.7% and 96.6%. Since no significant differences were observed between the results provided by two reagents, at level of significance of 0.05, either processes of cell sensitization can alternatively be used according to the own laboratory convenience.

Isolation of human fungi from soil and identification of two endemic areas of Cryptococcus neoformans and Coccidioides immitis

The present study was carried out in two different areas of Province of Cordoba, Argentina, where there was a suspicious of endemic mycosis. The previous data were the presence of a clinical case of pulmonary cryptococcosis in one area (Alta Gracia) and the previous findings of a high incidence of coccidioidin and cryptococcin reactors in the population of the second one (Villa Dolores). In both areas soil samples for fungi were studied and Cryptococcus neoformans was found in 2/25 samples from Alta Gracia. In Villa Dolores Coccidioides immitis was isolated in 2/40 samples, and C. neoformans in 1/40 samples. Delayed hypersensitivity test with cryptococcin was determined in the population from Alta Gracia and it was found to be 5.3%. Positive cutaneous tests with coccidioidin (33.8%) and cryptococcin (31.9%) in Villa Dolores were obtained. With these findings two endemic areas of systemic mycoses in Cordoba, Argentina were delimited.