In-house ELISA method to analyze anti-Trypanosoma cruzi IgG reactivity for differential diagnosis and evaluation of Chagas disease morbidity

INTRODUCTION: The goal was to develop an in-house serological method with high specificity and sensitivity for diagnosis and monitoring of Chagas disease morbidity. METHODS: With this purpose, the reactivities of anti-T. cruzi IgG and subclasses were tested in successive serum dilutions of patients from Berilo municipality, Jequitinhonha Valley, Minas Gerais, Brazil. The performance of the in-house ELISA was also evaluated in samples from other relevant infectious diseases, including HIV, hepatitis C (HCV), syphilis (SYP), visceral leishmaniasis (VL), and American tegumentary leishmaniasis (ATL), and noninfected controls (NI). Further analysis was performed to evaluate the applicability of this in-house methodology for monitoring Chagas disease morbidity into three groups of patients: indeterminate (IND), cardiac (CARD), and digestive/mixed (DIG/Mix), based on their clinical status. RESULTS: The analysis of total IgG reactivity at serum dilution 1:40 was an excellent approach to Chagas disease diagnosis (100% sensitivity and specificity). The analysis of IgG subclasses showed cross-reactivity, mainly with NI, VL, and ATL, at all selected serum dilutions. Based on the data analysis, the IND group displayed higher IgG3 levels and the DIG/Mix group presented higher levels of total IgG as compared with the IND and CARD groups. CONCLUSIONS: These findings demonstrated that methodology presents promising applicability in the analysis of anti-T. cruzi IgG reactivity for the differential diagnosis and evaluation of Chagas disease morbidity.

A Bayesian approach to estimate the accuracy of in-house ELISA assay to measure rabies antibodies from compulsory vaccinated dogs and cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioMérieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

A simple and cheaper in house varicella zoster virus antibody indirect ELISA

We have developed a cheaper an simple in house indirect ELISA that uses the live attenuated VZV vaccine as a coating antigen. The alternative ELISA had an agreement of 94% when compared with a commercial VZV ELISA kit. Moreover, our ELISA proved to be more reliable than the kit when assessing true negative samples. By adding a standard serum, we were able to produce results in international units per millilitre. Also, the addition of an extra step with 8M urea allowed the assessment of VZV IgG avidity without excessive costs. The cost per sample to test VZV IgG was 2.7 times cheaper with our ELISA, allowing the testing of many samples without the burden of production of VZV antigen in the laboratory.

Evaluation of the diagnostic value of the ELISA tests developed by using EgHF, Em2 and EmII/3-10 antigens in the serological diagnosis of alveolar echinococcosis

Alveolar echinococcosis (AE), caused by larva stage of Echinococcus multilocularis, is one of the lethal parasitic diseases of man and a major public health problem in many countries in the northern hemisphere. When the living conditions and habits in Turkey were considered in terms of relation with the life cycle of the parasite, it was suggested that AE has been much more common than reported mainly from the Eastern Anatolia region of Turkey. Since in vitro serologic diagnosis tests with high specificity for AE have not been used in our country, most of the cases with liver lesions were misdiagnosed by radiological investigations as malignancies. The aim of this study was to evaluate the diagnostic value of the in-house ELISA methods developed by using three different antigens (EgHF, Em2, EmII/3-10) in the serological diagnosis of AE. The study samples included a total of 100 sera provided by Bern University Parasitology Institute where samples were obtained from patients with helminthiasis and all were confirmed by clinical, parasitological and/or histopathological means. Ten samples from each of the cases infected by E.multilocularis, E.granulosus, Taenia solium, Wuchereria bancrofti, Strongyloides stercolaris, Ascaris lumbricoides, Toxocara canis, Trichinella spiralis, Fasciola hepatica and Schistosoma haematobium were studied. In the study, EgHF (E.granulosus hydatid fluid) antigens were prepared in our laboratory from the liver cyst fluids of sheeps with cystic echinococcosis, however Em2 (E.multilocularis metacestode-purified laminated layer) and EmII/3-10 (E.multilocularis recombinant protoscolex tegument) antigens were provided by Bern University Parasitology Institute. Flat bottom ELISA plates were covered with EgHF, Em2 and EmII/3-10 antigens in the concentrations of 2.5 µg, 1 µg and 0.18 µg per well, respectively, and all sera were tested by EgHF-ELISA, Em2-ELISA and EmII/3-10-ELISA methods. For each tests, the samples which were reactive above the cut-off value (mean OD of negative controls+2 SD) were accepted as positive. The sensitivity of the ELISA tests performed with EgHF, Em2 and Em2II/3-10 antigens were estimated as 100%, 90% and 90%, respectively, whereas the specificity were 63%, 91% and 91%, respectively. When Em2-ELISA and EmII/3-10-ELISA tests were evaluated together, the specificity increased to 96%. Our data indicated that the highest sensitivity (100% with EgHF-ELISA) and specificity (96% with Em2-ELISA + EmII/3-10-ELISA) for the serodiagnosis of AE can be achieved by the combined use of the ELISA tests with three different antigens. It was concluded that the early and accurate diagnosis of AE in our country which is endemic for that disease, could be supported by the use of highly specific serological tests such as Em2-ELISA ve EmII/3-10-ELISA contributing radiological data.

Synthetic peptides as an antigenic base in an ELISA for laboratory diagnosts of schistosomiasis mansoni

A pool of five synthetic peptides was used as an antigenic base in an ELISA (ELISA-Pp) for laboratory diagnosis of Schistosoma mansoni. Serum samples were obtained from individuals with acute (n = 23) and chronic (n = 30) schistosomiasis, with other parasitoses (n = 39) or without parasitic infections (n = 100). ELISA-Pp was compared with other immunoenzymatic methods for detection of IgM (IgM-ELISA) or IgG (IgG-ELISA) as well as an immunofluorescence test for detection of IgM antibodies (IgM-IFT). The sensitivity and specificity of ELISA-Pp was 86.8% and 94.2% when tested on the schistosomiasis group and the non-schistosomiasis group, respectively. Comparison of ELISA-Pp with other serological methods resulted in K concordance indices varying from 0.59 to 0.75. Evaluation of anti-peptide IgG antibodies showed higher Levels in patients with acute compared with chronic schistosomiasis (P = 0.001). ELISA-Pp showed satisfactory sensitivity and high specificity and may constitute a potentially useful method for laboratory diagnosis of schistosomiasis mansoni. (c) 2007 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

The origin of a Robertsonian chromosomal translocation in house mice inferred from linked microsatellite markers

The Western European house mouse, Mus domesticus, includes many distinct Robertsonian (Rb) chromosomal races. Two competing hypotheses may explain the distribution of Rb translocations found in different populations: they may have arisen independently multiple times or they may have arisen once and been spread through long distance dispersal. We investigated the origin of the Rb 5.15 translocation using 6 microsatellite loci linked to the centromeres of chromosomes 5 and 15 in 84 individuals from 3 Rb populations and 4 neighboring standard-karyotype populations. Microsatellite variation on the 5.15 metacentric chromosomes was significantly reduced relative to the amount of variation found on acrocentric chromosomes 5 and 15, suggesting that linked microsatellite loci can track specific mutational events. Phylogenetic analyses resulted in trees which are consistent with multiple origins of the 5.15 metacentric found in the three Rb populations. These results suggest that cytologically indistinguishable mutations have arisen independently in natural populations of house mice.

A contratação "in house" nas relações contratuais entres os municípios e as empresas municipais e intermunicipais

A possibilidade de escolha de empresas municipais e intermunicipais como entidades cocontratantes é uma "tentação" para os municípios que têm a seu cargo um conjunto de tarefas através das quais visam satisfazer o interesse público local. Terão estas empresas características especiais que as tornam entidades preferenciais para a derrogação das regras da contratação pública? Existe um mercado concorrencial quando estamos perante serviços públicos locais? Justifica-se a aplicação das regras gerais da contratação pública às adjudicações de serviços públicos locais? O mercado em que se desenvolvem as empresas municipais apresenta uma relevância tal que permita haver concorrência? Estas questões têm vindo a ser especialmente tratadas no quadro da jurisprudência comunitária, dentro da qual, é de fazer sobressair o Acórdão Stad Halle, que veio limitar a possibilidade de recurso à exceção "in house providing", ampliando em consequência o âmbito de aplicação das regras gerais da contratação pública, nomeadamente os princípios comunitários da transparência e igualdade de tratamento dos concorrentes. A empresa municipal está mais próxima das necessidades dos cidadãos e, em princípio, estará mais apta para desenvolver um projeto à medida do serviço pretendido. Poderemos questionar se a proximidade da empresa municipal às necessidades reais de certos cidadãos tem uma expressão tal que coloque a empresa numa posição especial relativamente às outras, para permitir a adjudicação direta.

CALIBRATION AND USE OF AN IN-HOUSE ANTI-MEASLES IgG STANDARD SERUM

For the purpose of research a large quantity of anti-measles IgG working reference serum was needed. A pool of sera from five teenagers was prepared and named Alexandre Herculano (AH). In order to calibrate the AH serum, 18 EIA assays were performed testing in parallel AH and the 2nd International Standard 1990, Anti-Measles Antibody, 66/202 (IS) in a range of dilutions (from 1/50 to 1/25600). A method which compared parallel lines resulting from the graphic representation of the results of laboratory tests was used to estimate the power of AH relative to IS. A computer programme written by one of the authors was used to analyze the data and make potency estimates. Another method of analysis was used, comparing logistic curves relating serum concentrations with optical density by EIA. For that purpose an existing computer programme (WRANL) was used. The potency of AH relative to IS, by either method, was estimated to be 2.4. As IS has 5000 milli international units (mIU) of anti-measles IgG per millilitre (ml), we concluded that AH has 12000 mIU/ml.

Some ultrastructural superficial changes in house fly (Diptera: Muscidae) and blow fly (Diptera: Calliphoridae) larvae induced by eucalyptol oil

The ultrastructural superficial changes in third instar house fly (Musca domestica) and blow fly (Chrysomya megacephala) induced by eucalyptol oil were observed using scanning electron microscopy. Dipped in 0.902 g/ml eucalyptol for 30 sec, the larvae integument of both species showed significant aberrant appearance of the body surface, particularly swelling integument, bleb formation, partial breach and deformation of spines.

Primary screening of blood donors by nat testing for HCV-RNA: development of an "in-house" method and results

An "in-house" RT-PCR method was developed that allows the simultaneous detection of the RNA of the Hepatitis C Virus (HCV) and an artificial RNA employed as an external control. Samples were analyzed in pools of 6-12 donations, each donation included in two pools, one horizontal and one vertical, permitting the immediate identification of a reactive donation, obviating the need for pool dismembering. The whole process took 6-8 hours per day and results were issued in parallel to serology. The method was shown to detect all six HCV genotypes and a sensitivity of 500 IU/mL was achieved (95% hit rate). Until July 2005, 139,678 donations were tested and 315 (0.23%) were found reactive for HCV-RNA. Except for five false-positives, all 310 presented the corresponding antibody as well, so the yield of NAT-only donations was zero, presenting a specificity of 99.83%. Detection of a window period donation, in the population studied, will probably demand testing of a larger number of donations. International experience is showing a rate of 1:200,000 - 1:500,000 of isolated HCV-RNA reactive donations.

Otimização de processos de logística in-house

A presente dissertação foi realizada no âmbito do Mestrado de Engenharia e Gestão Industrial da Escola Superior de Estudos Industriais e de Gestão, de Vila do Conde. O projeto desenvolvido tem como tema principal a Otimização de processos de Logística in-house baseado num projeto, em contexto empresarial da empresa cliente, Continental Mabor S.A., da Rangel Distribuição e Logística, S.A. Este projeto tem como objetivo a “aglomeração” de dois armazéns do cliente, devido à necessidade de ocupação do armazém de produto acabado interno, para aumento da área de produção. Inicialmente foi feita uma revisão de literatura sobre os temas mais relevantes de suporte para o projeto, nomeadamente na otimização e melhoria contínua. Seguidamente é apresentado o Grupo Rangel, bem como a Rangel Distribuição e Logística, S.A., onde se enquadra o projeto e para se perceber o enquadramento e objetivo. A metodologia usada, caso de estudo, permitiu a aplicação de conceitos e ferramentas usados na literatura neste contexto, como ferramentas de otimização e melhoria continua como as melhores práticas de Kaizen-Lean. Na fase de diagnóstico do atual sistema, foi realizado um mapeamento de fluxo de processos e uma descrição detalhada do layout dos dois armazéns: Armazém de Produto Acabado (APA) e Armazém de Produto Acabado Externo (APAE), bem como todos os recursos, quer técnicos quer humanos necessários. Verificamos ao longo deste projeto várias limitações, inclusive limitações impostas pelo cliente, tal como não aprovar um estudo para um novo layout do armazém. Foi aprovado apenas a replicação do já existente. Com isto, depararam-se constrangimentos na gestão deste projeto. Os custos aumentaram significativamente, embora estes não sejam apresentados por questões de confidencialidade, principalmente com a necessidade de aquisição de novos equipamentos retráteis, e mais baterias para os mesmos, devido às grandes distâncias que irão ser percorridas. Finalmente foi projetado o sistema futuro, de acordo com as necessidades reais do cliente tendo em consideração a otimização de recursos e uma gestão magra (Lean Management). Foi desenvolvida a implementação da metodologia “Kaizen diário”, a dar início em 2016 juntamente com o novo projeto APAE. Com esta projeção foram identificados problemas e implicações no projeto, bem como possíveis melhorias.

The performance of an in-house nested-PCR technique for pleural tuberculosis diagnoses

Introduction This study evaluated the performance of an in-house nested-PCR system for the detection of the Mycobacterium tuberculosis complex in pleural fluid, blood and urine samples from pleural effusion tuberculosis patients by health services physicians in Pernambuco, Brazil. Methods A prospective double-blind study with 37 hospitalized patients of both sexes, aged over 15, was used to investigate the diagnosis of pleural effusion. The criteria used to define the cases included the demonstration of bacillus in biological samples by smear or culture or by a granulomatous finding in the histopathological examination, associated with an evident response to specific treatments to each clinical situation. Pleural fluid, blood and urine samples were collected and subjected to routine tests and the nested PCR technique to assess for M. tuberculosis amplification. Results In total, 37 pleural effusion patients took part in the study, of whom 19 (51.3%) had tubercular etiologies and 18 (48.7%) had etiologies from other causes. When the pleural fluid, blood and/or urine sample in-house nested-PCR sensitivities were evaluated simultaneously, the results were positive regardless of the biological specimen (the sensitivity was 84.2%); however, when the blood and/or urine samples were analyzed together, the sensitivity was 72.2%. When the pleural fluid samples were evaluated alone, the sensitivity was only 33.3%. Conclusions The performance of the diagnostic pleural tuberculosis nested-PCR was directly related to the diversity of the samples collected from the same patient. Additionally, this study may identify a need to prioritize non-invasive blood and urine collection for this diagnosis.

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.