BackgroundWe report the detailed development of biomarkers to predict the clinical outcome under dengue infection. Transcriptional signatures from purified peripheral blood mononuclear cells were derived from whole-genome gene-expression microarray data, validated by quantitative PCR and tested in independent samples.Methodology/Principal FindingsThe study was performed on patients of a well-characterized dengue cohort from Recife, Brazil. The samples analyzed were collected prospectively from acute febrile dengue patients who evolved with different degrees of disease severity: classic dengue fever or dengue hemorrhagic fever (DHF) samples were compared with similar samples from other non-dengue febrile illnesses. The DHF samples were collected 2–3 days before the presentation of the plasma leakage symptoms. Differentially-expressed genes were selected by univariate statistical tests as well as multivariate classification techniques. The results showed that at early stages of dengue infection, the genes involved in effector mechanisms of innate immune response presented a weaker activation on patients who later developed hemorrhagic fever, whereas the genes involved in apoptosis were expressed in higher levels.Conclusions/SignificanceSome of the gene expression signatures displayed estimated accuracy rates of more than 95%, indicating that expression profiling with these signatures may provide a useful means of DHF prognosis at early stages of infection.

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Primers targeting the gene encoding the small subunit rRNA were designed to amplify DNA from Schistosoma mansoni with high specificity. Three PCR systems were developed: conventional PCR, two-step nested PCR (NPCR) and single-tube nested PCR (STNPCR). The limits of detection of parasite DNA for the conventional PCR, NPCR and STNPCR were 10 pg, 0.1 fg and 1 fg, respectively. The assays were highly specific for S. mansoni and did not recognise DNA from closely related non-schistosome trematodes. Using pools of Biomphalaria molluscs, PCR, NPCR and STNPCR were positive in 6/16 (37.5%), 15/16 (93.8%) and 13/16 (81.3%) of the tested samples, respectively, whereas the observation of cercariae shedding after exposure to light was able to detect S. mansoni infection in 6/16 (37.5%) of the pools. Thus, the molecular detection systems had a higher level of sensitivity than standard screening of intermediate hosts by cercarial shedding when DNA was purified from pools of snails collected from endemic areas. These PCR protocols have potential to be used as tools for monitoring of schistosome transmission.

The detection of specific DNA sequences by polymerase chain reaction (PCR) Key words: polymerase chain reaction -molecular diagnosis -schistosomiasis -transmission -snail -Schistosoma Schistosomiasis is a disease transmitted by fresh water snails. It affects more than 200 million people worldwide and is endemic in 74 countries, with more than 80% of infected persons living in Africa. In Brazil, Schistosoma mansoni is the only causative species of schistosomiasis, and there are three species of intermediate hosts: Biomphalaria glabrata, B. straminea, and B. tenagophila. Adult S. mansoni live in the bloodstream. Their eggs pass out of the host with the faeces. In contact with water, free-swimming miracidia emerge from the egg and penetrate the soft tissues of the snail host. Within the snails, miracidia immediately differentiate into sporocysts and, as they migrate through the snail tissues, give rise to cercariae. Cercariae are shed from the snails and are infective to humans by direct penetration of the skin. Thus, molecular approaches can be used to detect DNA of different life cycle stages of S. mansoni by analyzing different biological samples: feces, snail tissues, and infested water bodies, resulting in diagnosis of vertebrate and invertebrate host infections, and identification of transmission sites.The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens (Leal et al.

BackgroundSymptomatic infection by dengue virus (DENV) can range from dengue fever (DF) to dengue haemorrhagic fever (DHF), however, the determinants of DF or DHF progression are not completely understood. It is hypothesised that host innate immune response factors are involved in modulating the disease outcome and the expression levels of genes involved in this response could be used as early prognostic markers for disease severity.Methodology/Principal FindingsmRNA expression levels of genes involved in DENV innate immune responses were measured using quantitative real time PCR (qPCR). Here, we present a novel application of the support vector machines (SVM) algorithm to analyze the expression pattern of 12 genes in peripheral blood mononuclear cells (PBMCs) of 28 dengue patients (13 DHF and 15 DF) during acute viral infection. The SVM model was trained using gene expression data of these genes and achieved the highest accuracy of ∼85% with leave-one-out cross-validation. Through selective removal of gene expression data from the SVM model, we have identified seven genes (MYD88, TLR7, TLR3, MDA5, IRF3, IFN-α and CLEC5A) that may be central in differentiating DF patients from DHF, with MYD88 and TLR7 observed to be the most important. Though the individual removal of expression data of five other genes had no impact on the overall accuracy, a significant combined role was observed when the SVM model of the two main genes (MYD88 and TLR7) was re-trained to include the five genes, increasing the overall accuracy to ∼96%.Conclusions/SignificanceHere, we present a novel use of the SVM algorithm to classify DF and DHF patients, as well as to elucidate the significance of the various genes involved. It was observed that seven genes are critical in classifying DF and DHF patients: TLR3, MDA5, IRF3, IFN-α, CLEC5A, and the two most important MYD88 and TLR7. While these preliminary results are promising, further experimental investigation is necessary to validate their specific roles in dengue disease.

Four genetic polymorphisms located at the promoter (C-257T) and coding regions of CFH gene (exon 2 G257A, exon 14 A2089G and exon 19 G2881T) were investigated in 121 dengue patients (DENV-3) in order to assess the relationship between allele/haplotypes variants and clinical outcomes. A statistical value was found between the CFH-257T allele (TT/TC genotypes) and reduced susceptibility to severe dengue (SD). Statistical associations indicate that individuals bearing a T allele presented significantly higher protein levels in plasma. The –257T variant is located within a NF-κB binding site, suggesting that this variant might have effect on the ability of the CFH gene to respond to signals via the NF-κB pathway. The G257A allelic variant showed significant protection against severe dengue. When CFH haplotypes effect was considered, the ancestral CG/CG promoter-exon 2 SNP genotype showed significant risk to SD either in a general comparison (ancestral × all variant genotypes), as well as in individual genotypes comparison (ancestral × each variant genotype), where the most prevalent effect was observed in the CG/CG × CA/TG comparison. These findings support the involvement of –257T, 257A allele variants and haplotypes on severe dengue phenotype protection, related with high basal CFH expression.

The aims of this study were to determine the seroprevalence of infection by ruminants Lentivirus in dairy goats in the semiarid of the Paraiba State, Northeastern Brazil, to identify risk factors associated with the herd-level prevalence and to perform molecular detection of the agent. A total of 1,047 dairy goats from 110 herds were randomly selected from the county of Monteiro, Paraiba State, and serum samples were collected from March 2009 to December 2011. For the diagnosis of Lentivirus infection, the agar gel immunodiffusion test (AGID) was used. One year after that a new serology was performed and the real-time PCR assay was applied in blood and milk samples from 48 goats from four herds with seropositive animals. Prevalence of positive herds and seropositive animals at AGID were 44.6% (95% CI=35.1-54.3%) and 8.1% (95% CI =5.6-16.8%), respectively. Umbilical cord cutting and disinfection (odds ratio = 2.44; p = 0.048) and conditions of animal agglomeration (odds ratio=3.45; p=0.048) were associated with herd-level prevalence. One year after the serological profile, the permanence of infected animals detected by real-time PCR in blood and milk samples was verified. Real-time PCR using white blood cells had a good performance, with sensitivity of 100%, specificity of 92.86%, concordance of 93.75% and Kappa index of 0.765. It was suggested to teach sanitary measures to the herd owners in order to encourage them to adopt prevention measures aiming to reduce the spread of the infection in the herds.