Validation of quantitative real-time PCR for the in vitro assessment of antileishmanial drug activity.

In vitro assays play an important role in the discovery and development of new antileishmanial drugs. The classic macrophage-amastigote models using murine peritoneal macrophages or human-monocyte derived macrophages as host cells are useful for drug screening. A major limitation of these models is the dependence on microscopic counting, a time-consuming and subjective method of analysis. The present study describes a detailed protocol for applying quantitative real-time PCR (qPCR) as an accurate and sensitive tool to assess parasite load in an amastigote-macrophage model. This assay can be performed in a standardized medium-to-high throughput procedure, replacing traditional readout of number of amastigote per macrophages by DNA load measurement.

dupA polymorphisms and risk of Helicobacter pylori-associated diseases.

The dupA of Helicobacter pylori has been suggested as a virulence marker associated with the development of duodenal ulcer disease. However, the studies performed in different geographical areas have shown that there are variations in the prevalence of dupA and its association with H. pylori clinical outcomes. Our group did not observe associations between the presence of dupA and H. pylori clinical outcomes in Brazil. On the other hand, we observed 2 mutations in the sequence of dupA that lead to stop codons: a deletion of an adenine at position 1311 and an insertion of an adenine at position 1426 of the gene. Our aim was to evaluate associations of the presence of dupA with duodenal ulcer and gastric cancer, considering dupA-positive only those H. pylori strains that do not have the mutations in the gene sequence. We also evaluated the effect of infection with a strain carrying an intact dupA on the gastric mucosa histology and IL-8 gastric levels. Colonization with strains that had the intact dupA was negatively associated with gastric carcinoma (p=0.001, OR=0.32, 95% CI=0.16-0.66). The presence of dupA was also associated with an increased degree of antral mucosa inflammation (p=0.01) and with decreased corpus atrophy (p<0.01) as well as with increased gastric mucosa IL-8 levels (p=0.04). In conclusion, the infection with a H. pylori strain containing the dupA without the stop codon polymorphisms is associated with a lower risk of development of gastric carcinoma in Brazilian subjects.

Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples.

A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.

Lack of association between Helicobacter pylori infection with dupA-positive strains and gastroduodenal diseases in Brazilian patients.

Duodenal ulcer-promoting gene (dupA) was recently described as a new putative Helicobacter pylori virulence marker associated with an increased risk for duodenal ulcer and reduced risk for gastric carcinoma in Japan and Korea. Since differences regarding the association among H. pylori markers and H. pylori-associated diseases have been demonstrated around the world, we evaluated the presence of the gene in 482 strains from Brazilian children (34 with duodenal ulcer and 97 with gastritis) and adults (126 with duodenal ulcer, 144 with gastritis and 81 with gastric carcinoma) by PCR using the described primers and an additional set of primers based on Brazilian strain sequences. The results were confirmed by sequencing. The presence of cagA was investigated by PCR and also included in the analysis. dupA was present in 445 (92.32%) and absent in 29 (6.02%) strains. All samples from children with and without duodenal ulcer were dupA-positive (p=1.0). No association was observed among the strains from adults with gastritis (92.36%), duodenal ulcer (87.30%, p=0.30) and gastric carcinoma (87.65%, p=0.31). Conversely, cagA-positve status remained independently associated with duodenal ulcer (children: odds ratios (OR)=5.58, 95% confidence intervals (CI)=1.67-18.50; adults: OR=3.33, 95% CI=2.14-5.19) and gastric carcinoma (OR=6.58, 95% CI=3.51-12.30) in multivariate analyses. The presence of dupA was significantly higher in strains from children than in those from adults (p=0.01). In conclusion, dupA is highly frequent and not associated with H. pylori-associated diseases in both Brazilian adults and children, which points to regional differences in the distribution of the gene.

Expression profile of malignant and nonmalignant lesions of esophagus and stomach: differential activity of functional modules related to inflammation and lipid metabolism.

Adenocarcinomas of stomach and esophagus are frequently associated with preceding inflammatory alterations of the normal mucosa. Whereas intestinal metaplasia of the gastric mucosa is associated with higher risk of malignization, Barrett's disease is a risk factor for adenocarcinoma of the esophagus. Barrett's disease is characterized by the substitution of the squamous mucosa of the esophagus by a columnar tissue classified histopathologically as intestinal metaplasia. Using cDNA microarrays, we determined the expression profile of normal gastric and esophageal mucosa as well as intestinal metaplasia and adenocarcinomas from both organs. Data were explored to define functional alterations related to the transformation from squamous to columnar epithelium and the malignant transformation from intestinal metaplasia to adenocarcinomas. Based on their expression profile, adenocarcinomas of the esophagus showed stronger correlation with intestinal metaplasia of the stomach than with Barrett's mucosa. Second, we identified two functional modules, lipid metabolism and cytokine, as being altered with higher statistical significance. Whereas the lipid metabolism module is active in samples representing intestinal metaplasia and inactive in adenocarcinomas, the cytokine module is inactive in samples representing normal esophagus and esophagitis. Using the concept of relevance networks, we determined the changes in linear correlation of genes pertaining to these two functional modules. Exploitation of the data presented herein will help in the precise molecular characterization of adenocarcinoma from the distal esophagus, avoiding the topographical and descriptive classification that is currently adopted, and help with the proper management of patients with Barrett's disease.

Real Time Polymerase Chain Reaction (rt-PCR): A New Patent to Diagnostic Purposes for Paracoccidioidomycosis.

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. OBJECTIVE: The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. METHODS: To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. RESULTS: The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. CONCLUSION: The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological samples in order to validate this method and then generate a diagnostic kit for Paracoccidioidomycosis.

Molecular classifiers for gastric cancer and nonmalignant diseases of the gastric mucosa.

High incidence of gastric cancer-related death is mainly due to diagnosis at an advanced stage in addition to the lack of adequate neoadjuvant therapy. Hence, new tools aimed at early diagnosis would have a positive impact in the outcome of the disease. Using cDNA arrays having 376 genes either identified previously as altered in gastric tumors or known to be altered in human cancer, we determined expression signature of 99 tissue fragments representing normal gastric mucosa, gastritis, intestinal metaplasia, and adenocarcinomas. We first validated the array by identifying molecular markers that are associated with intestinal metaplasia, considered as a transition stage of gastric adenocarcinomas of the intestinal type as well as markers that are associated with diffuse type of gastric adenocarcinomas. Next, we applied Fisher's linear discriminant analysis in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Many classifiers could distinguish between normal and tumor samples, whereas, for the distinction of gastritis from tumor and for metaplasia from tumor, fewer classifiers were identified. Statistical validations showed that trios that discriminate between normal and tumor samples are powerful classifiers to distinguish between tumor and nontumor samples. More relevant, it was possible to identify samples of intestinal metaplasia that have expression signature resembling that of an adenocarcinoma and can now be used for follow-up of patients to determine their potential as a prognostic test for malignant transformation.

IL2-330G polymorphic allele is associated with decreased risk of Helicobacter pylori infection in adulthood.

We evaluated whether polymorphisms in genes coding molecules linked to the innate and adaptive immune response are associated with susceptibility to Helicobacter pylori infection. IL1B-511C-->T, IL1B-31T-->C, IL1RN allele 2, IL2-330T-->G, TNFA-307G-->A, TLR2Arg677Trp, TLR2Arg753Gln, TLR4Asp299Gly, and TLR5(392STOP) polymorphisms were determined in 541 blood donors. IL2-330T-->G allele carriers had a decreased H. pylori infection risk (OR=0.63, 95% CI=0.43-0.93) after adjustment for demographic and environmental factors. Hence, we investigated whether the polymorphism is functional by evaluating IL-2 serum concentration in 150 blood donors and 100 children. IL-2 pro-inflammatory and anti-inflammatory properties were indirectly investigated by determining serum IFN-gamma and IL-10/TGF-beta levels. The polymorphism was associated with increased mean IL-2 levels in H. pylori-positive adults (2.65 pg/mL vs. 7.78 pg/mL) and children (4.19 pg/mL vs. 8.03 pg/mL). Increased IL-2 was associated with pro-inflammatory activity in adults (IFN-gamma=18.61 pg/mL vs. 25.71 pg/mL), and with anti-inflammatory activity in children (IL-10=6.99 vs. 14.17 pg/mL, TGF-beta=45.88 vs. 93.44 pg/mL) (p<10(-3) for all). In conclusion, in the context of H. pylori infection, IL2-330 T-->G polymorphism is functional and is associated with decreased risk of infection in adults.

Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples

A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.

Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.

Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.