Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.

Valoração de contingente pelas modelagens logit e análise multivariada: um estudo de caso da disposição a aceitar compensação dos cafeicultores vinculados ao PRO-CAFÉ de Viçosa - MG

O objetivo deste estudo foi, justamente, identificar, entre os métodos LOGIT e a análise multivariada, qual a mais eficaz para estimar a Disposição a Aceitar Compensação (DAC) dos cafeicultores quando o viés da utilidade marginal é passível de ocorrência. Para tal, foi elaborado um formulário com 33 perguntas envolvendo informações sobre características socioeconômicas dos cafeicultores, o uso da metodologia de valoração de contingente (MVC) e do veículo de pagamento dos "Jogos de Lances", que revelou a Disposição a Aceitar uma Compensação (DAC) na troca de um hectare de café por um hectare de mata. Como esperado, por causa do viés da utilidade marginal o método LOGIT foi incapaz de produzir resultados consistentes. Já a estimação da DAC pela análise multivariada mostrou que, caso o governo estivesse disposto a aumentar a provisão de mata em 70 ha, ele deveria despender 254.200 reais por ano, tratando apenas dos cafeicultores vinculados ao programa do PRO-CAFÉ.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Um protocolo de "nested-PCR" para detecção do vírus da anemia das galinhas

Este trabalho descreve o estabelecimento de um pro!tocolo de "nested-PCR" para a detecção do vírus da anemia das galinhas (CAV, chicken anemia virus), agente causador da anemia infecciosa das galinhas. Para a extração de DNA a partir de amostras clínicas um método baseado no uso de tiocianato de guanidina mostrou-se mais sensível e prático, do que os demais avaliados. Para a PCR inicial foi selecionado um par de primers que amplifica uma região de 664 pares de bases (pb) do gene VP1. Para a "nested-PCR" propriamente dita, foi selecionado um segundo par que amplifica uma região interna de 520 pb. A especificidade dos primers foi avaliada utilizando amostras de lotes controlados para CAV. Outras trinta amostras vírus e bactérias, causadoras de doenças em aves, não geraram produto de amplificação. A sensibilidade do teste foi determinada a partir de diluições seriadas de uma amostra vacinal de CAV. A "nested-PCR" mostrou ser mais sensível do que a PCR e foi capaz de detectar pelo menos 0,16 TCID50% da cepa vacinal. Além disso, detectou DNA viral em tecidos, soro e cama aviária de lotes com e sem sinais clínicos. Conclui-se que, como técnica para a detecção do CAV, o protocolo de "nested-PCR" aqui descrito, é mais sensível, rápido e menos trabalhoso do que o isolamento viral em cultivo celular.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2%) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2% of the patients with CMV infection were symptomatic.

Typing class I HLA-A gene using a nested PCR-RFLP procedure

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A®, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Dinâmica da estratégia logística em empresas brasileiras

O objetivo do artigo é analisar a estratégia logística das empresas brasileiras a partir da mensuração do impacto de diferentes características do negócio sobre as principais decisões de produção e distribuição física de produtos acabados. Foi conduzida uma pesquisa de campo em empresas listadas no Ranking Exame para confrontar as quatro políticas propostas por Pagh e Cooper (1998), derivadas das combinações entre centralizar/descentralizar os estoques e produzir para estoque/produzir contra-pedido, com diversas características do produto, da operação e da demanda. Técnicas de análise multivariada, como as análises de regressão logística simples e multinomial, foram empregadas, permitindo o desenvolvimento de diversos modelos quantitativos. Seus resultados apontam para avanços acadêmicos e gerenciais, como a quantificação e hierarquização de relações empíricas descritas na literatura e o direcionamento da atenção gerencial para as variáveis relevantes à decisão. Os modelos também permitem avaliar a adequação dessas políticas para diferentes valores do tempo de entrega, da variabilidade das vendas e do grau de obsolescência dos produtos.

Detecção do gerenciamento de resultados pela análise do diferimento tributário

O objetivo deste trabalho é verificar se a análise do diferimento tributário aumenta signifi cativamente a detecção do gerenciamento de resultados nas companhias abertas brasileiras. Desenvolveu-se uma pesquisa empírica nos setores de mineração, siderurgia e metalurgia, e têxtil, durante o período de 2000 a 2004, utilizando-se as regressões probit e logit baseadas nos modelos Jones Modificado e Kang e Sivaramakrishnan (KS). Este estudo apresenta evidências de que a análise do diferimento tributário não aumenta o poder preditivo dos modelos analisados. Essa divergência pode ser explicada pelo fato de que a maioria dos trabalhos realizados foi direcionada ao contexto norte-americano, que supostamente apresenta diferentes fatores motivadores para o gerenciamento de resultados. Além disso, as diferenças das evidências empíricas no âmbito brasileiro podem ser originadas pelo uso da flexibilidade do método parcial de reconhecimento do diferimento dos tributos.

Análisis del credit scoring

El problema de la morosidad está cobrándose una gran importancia en los países desarrollados. En este trabajo realizamos un análisis de la capacidad predictiva de dos modelos paramétricos y uno no paramétrico abordando, en este último, el problema del sobreaprendizaje mediante la validación cruzada que, muy habitualmente, se obvia en este tipo de estudios. Además proponemos la distinción de tres tipos de solicitudes dependiendo de su probabilidad cumplimiento: conceder, no conceder (de forma automática), y dudoso y, por consiguiente, proceder a su estudio manual por parte del personal bancario.

O uso do tabaco por estudantes de primeiro e segundo graus em dez capitais brasileiras: possíveis contribuições da estatística multivariada para a compreensão do fenômeno

Analisou-se o uso de tabaco por estudantes de primeiro e segundo graus de dez capitais brasileiras, utilizando técnicas estatísticas bi e multivariadas (modelo logit). Pela análise bi-variada detectou-se que o uso de tabaco está positivamente associado ao fato de o estudante trabalhar, freqüentar o curso noturno, estar atrasado nos estudos e ter pais fumantes. Pela aplicação de técnicas multivariadas e do modelo logit, verificou-se que as variáveis de maior peso são a defasagem escolar (no curso noturno) e o fato de os pais fumarem (diurno). Discutiu-se a importância da utilização de técnicas multivariadas para a compreensão de fenômenos complexos como o do uso de tabaco e outros psicotrópicos.