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Introduction: Approximately 20% - 30% of histological lymph node-negative patients with colorectal cancer relapse at five years after surgical treatment. This recurrence is likely due to occult nodal disease undetected by standard histopathological practice which has implications in terms of the clinical management of patients. Material and Methods: Lymph nodes were collected from colectomy specimens. A central section from each lymph node was histologically examined following haematoxylin-eosin staining and the remaining tissue was subjected to OSNA - one step nucleic acid amplification analysis. Results: A total of 1046 lymph nodes from 59 pN0 patients were assessed. Of these, 753 lymph nodes were examined by both methods. The median number of lymph nodes assessed with OSNA - one step nucleic acid amplification was 12 (IQR: 7;16). Among pN0 patients, 17 had OSNA - one step nucleic acid amplification-positive lymph nodes, resulting in a positive molecular staging rate of 28.8% (95% CI: 17.8 - 42.1). Among these patients, 12 (70.59%) were molecular-staged as pN1 and 5 (29.41%) were molecular staged as pN2. The tumour burden of lymph nodes assessed with OSNA - one step nucleic acid amplification ranged from 270 to 17 000 cytokeratin 19 mRNA copies/μL. Most of these patients (88.2%) were found to have lymph nodes with micrometastases only (250 - 4999 copies/μL). Discussion: We provide the results from the first study of the use of the OSNA - one step nucleic acid amplification assay in colorectal cancer patients in Portugal. Our results are in-line with other international studies, showing the improvement on patients’ staging by molecular examination of lymph nodes. Conclusion: In our study, 28.8% of patients with histologically negative lymph nodes were found to have metastatic lymph nodes using OSNA - one step nucleic acid molecular assessment. OSNA - one step nucleic acid assay allows a more accurate staging of patients with colorectal cancer and standardizes lymph node assessment.

Introduction: Hemotherapy consists of therapeutic treatment using blood transfusion. Because of the vast amount of bloodborne infectious diseases, highly sensitive laboratory tests must be conducted to minimize the transfusion risks for donors and recipients. Thus, the present study aimed to evaluate the prevalence of hepatitis B and C serological markers in samples from a blood bank in Porto Alegre, southern Brazil, as well as the correlation between serology and nucleic acid amplification testing (NAT). Methods: A quantitative, descriptive, retrospective study was conducted to analyze blood donor information obtained from a blood bank database in Porto Alegre, southern Brazil, concerning 2017 and 2018. Serology and NAT results for hepatitis B and C were examined. Results: Of 17,181 blood donations, the total number of serologically reactive samples was 162 (0.94%) for anti-HBc, 20 (0.12%) for HbsAg, and 62 (0.36%) for anti-HCV. Only 4 (0.02%) samples were simultaneously reactive for anti-HBc and HbsAg, being also positive in NAT. The number of samples reactive to hepatitis C in NAT was 18 (0.10%). Conclusions: Biotechnological evolution has contributed to biosafety in blood transfusions. Considering that NAT is relatively recent in blood banks, further studies using different time periods are suggested for yielding satisfactory results for transfusion safety and elucidating the combination of serological testing and NAT at blood banks. [ABSTRACT FROM AUTHOR]

ABSTRACT We estimated the costs of a molecular test for Mycobacterium tuberculosis and resistance to rifampin (Xpert MTB/RIF) and of smear microscopy, within the Brazilian Sistema Único de Saúde (SUS, Unified Health Care System). In SUS laboratories in the cities of Rio de Janeiro and Manaus, we performed activity-based costing and micro-costing. The mean unit costs for Xpert MTB/RIF and smear microscopy were R$35.57 and R$14.16, respectively. The major cost drivers for Xpert MTB/RIF and smear microscopy were consumables/reagents and staff, respectively. These results might facilitate future cost-effectiveness studies and inform the decision-making process regarding the expansion of Xpert MTB/RIF use in Brazil.

Introduction: Chlamydia trachomatis (CT) is the most prevalent sexually transmitted bacterial infection, affecting mainly young, sexually active women. Untreated infection may lead to reproductive complications due to tubal damage. Infections during pregnancy may cause preterm labor, low birth weight, perinatal death, and neonatal conjunctivitis and pneumonia. There are few data on CT infection in Brazil. The aim of this study was to determine CT prevalence in infertile and pregnant women. Methods: A cross-sectional study included 77 infertile and 60 asymptomatic pregnant women. First-void urine was tested for CT using PCR (Polymerase Chain Reaction). Blood samples were collected for CT IgG antibodies testing using indirect immunofluorescence. A questionnaire about medical, gynecological, and sexual history was completed by all participants. Results: We found statistically similar prevalence of PCR and IgG antibodies between the groups. There was a 61% prevalence of CT IgG antibodies in infertile women and 56.7% in pregnant women. PCR was positive in only one (1.3%) infertile woman and in none pregnant women. Conclusion: There is a high prevalence of CT IgG antibody in Brazilian pregnant and infertile women, but we found a low prevalence of positive PCR in the urine samples. CT antibodies were associated with sexual behavior and smoking. Keywords: Chlamydia trachomatis, Chlamydia infections, prevalence, nucleic acid amplification techniques; infertility, female; fluorescent antibody technique

In the last 35 years, major progresses have been accomplished in the prevention, diagnosis and treatment of HIV infection, with reduction of the incidence of infection in Portugal, in the last decade. Nevertheless, there is still a high rate of late diagnosis, with individual and populational adverse outcomes. This paper aims to: present the current situation of HIV infection in Portugal, briefly review the natural course of HIV infection and exhibit the most recent HIV laboratory diagnostic testing algorithm, reviewed in 2014, strengthening the need of maintaining a high grade of clinical suspicion for early diagnosis of infection. [ABSTRACT FROM AUTHOR]

INTRODUÇÃO: A sepse é uma resposta inflamatória sistêmica relacionada com altas taxas de mortalidade no meio hospitalar. O diagnóstico etiológico tardio e terapia antimicrobiana inadequada se associam a falhas do tratamento. Exames moleculares baseados na reação em cadeia da polimerase são considerados métodos mais rápidos e precisos do que técnicas de hemocultura para identificação microbiana, proporcionando uma taxa mais elevada de sucesso terapêutico. OBJETIVO: Desenvolver um painel de seqüências iniciadoras (primers) para fragmentos de DNA de microrganismos associados à sepse. MÉTODOS: Seqüências iniciadoras para amplificação de Enterobacter spp., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus e Candida spp. foram desenvolvidos e testados quanto a sensibilidade e especificidade com base em suas respectivas cepas padrão. RESULTADOS: A especificidade pretendida foi obtida para os primers de P. aeruginosa, S. aureus e Candida spp. O teste de sensibilidade mostrou um limite de detecção de 5 ng a 500 fg em amostras de sangue contaminado com DNA microbiano. CONCLUSÕES: O painel molecular apresentado oferece a vantagem de constituir um sistema flexível "aberto" em comparação a outros métodos de detecção múltipla.INTRODUCTION: Sepsis is a systemic inflammatory response related to high mortality rates in the hospital environment. Delayed etiological diagnosis and inadequate antimicrobial therapy are associated with treatment failures. Molecular tests based on polymerase chain reaction are regarded as faster and more accurate procedures than culture techniques for microbial identification, providing a higher rate of therapeutic success. OBJECTIVE: To develop a panel of primers for DNA fragments of sepsis-related microorganisms. METHODS: Primers for amplification of Enterobacter spp., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp. were designed and tested for sensitivity and specificity on the basis of their respective standard strains. RESULTS: The intended specificity was obtained for P. aeruginosa, S. aureus and Candida spp primers. Sensitivity tests showed a threshold for detection from 5 ng to 500 fg in blood samples contaminated with microbial DNA. CONCLUSIONS: The molecular panel presented offers the advantage of a flexible 'open' system when compared to other multiplex detection methods.

The HCV, agent of hepatitis C, a virus belonging to the genus Hepacivirus of the Flaviviridae family, having a genome comprising a single strand of RNA. HCV transmission occurs via parenteral, such as blood transfusion and blood products, hemophiliacs, intravenous drug users or inhaled, material cutting or piercing can be a vehicle transmitter. The possibility of sexual transmission is still an open question and the transmission is also low, being, however, it may be increased if the patient has strong viremia and/or co-infection with HIV. The diagnosis of hepatitis C is based on serological tests such as ELISA and RIBA and molecular biology techniques. The ELISA detects antibodies against the virus anti-HCV, if the result is indeterminate confirmatory tests must be used such as RIBA. To confirm the diagnosis, we use a qualitative PCR RNA-HCV, and quantitative tests are used to evaluate viral-load, and they are also used to monitor therapeutic response. The nucleic acid amplification technique (NAT) results in a change of conventional laboratory methods, therefore, is quick to identify the "immune gap" period, approximately 50 to 60 days after contamination resulting from 10 to 11 days. The need for incorporation of NAT in blood banks is due to its ability to reduce the immune window period between infection and disease was discovered, thereby increasing transfusion safety. [ABSTRACT FROM AUTHOR]

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Purpose: The epidermal growth factor receptor (EGFR) is overexpressed in the majority of nonsmall- cell lung cancers (NSCLC) and is a major target specific EGFR tyrosine kinase inhibitors (TKIs) developed and used for the treatment of advanced NSCLC. A number of biological factors are also associated with EGFR-TKIs responsiveness. This study was focused on EGFR somatic mutations and amplifications in squamous cell lung cancer., Material and Methods: Representative sections of squamous cell carcinoma were selected from 54 surgical specimens from formalin-fixed paraffin-embedded tissues and submitted to TMA construction. Determination of EGFR protein expression was done by immunohistochemistry( IHC) (Zymed, Laboratories). Fluorescence in situ hybridization (FISH) was performed with LSI EGFR/CEP 7 (Vysis; Abbott Molecular, USA). Genomic DNA was extracted from 48 cases and exon 19 was amplified by polymerase chain reaction (PCR) for search deletions and point mutations for exon 21. All cases expressed high weigh cytokeratin and were observed negativity for CK7, CD56 and chromogranin., Results: EGFR protein overexpression was identified in 49 cases, by the application of Hirsh's scoring system. The chromosome 7 and EGFR gene were analyzed by FISH and scored according to Cappuzzo's method that showed high polysomy in 31 cases and amplification in 7 cases. Deletion in exon 19 of EGFR was detected in 3 cases of 48 samples; the exon 21 of EGFR was expressed in its wild type by RFLP in all cases., Conclusions: Detection of common EGFR deletion and mutation showed to be a rare event in Squamous cell carcinoma of the lung. While EGFR mutation is the most effective molecular predictor or sensitivity in patients with advanced NSCLC submitted to EGFR-TKIs treatment, amplification and polysomy is the most effective molecular predictor for EGFR-TKIs responsiveness in squamous cell carcinoma, when validated isolated from the group of NSCLC.

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.