Evaluation of Dengue, Zika virus, and Chikungunya virus transmission by blood components in recipients of haematopoietic stem cell transplantation.

BACKGROUND: Brazil has a high prevalence of arboviruses, especially Dengue (DENV), Zika (ZKV), and Chikungunya (CHKV). OBJECTIVES: To study the risk of DENV, ZKV, and CHKV transmission by blood components in the haematopoietic stem cell transplantation (HSCT) population. METHODS: Prospective cohort of HSCT recipients and donors performed at the Hospital das Clinicas da FMUSP, São Paulo-Brazil. Patients were evaluated by serology and RT-PCR for DENV, ZKV, and CHKV pre-transplantation and once a week until neutrophil grafting. In positive cases (positive RT-PCR and/or serology conversion), an investigation was carried out on the blood components that the patient received to evaluate the possibility of it being transfusion transmitted. RESULTS: A total of 93 patients were included during the study period. The mean age was 52 years with a predominance of males (56.9%). We considered five (5.3%) DENV cases positive by seroconversion in our study. One patient had IgM seroconversion and the other four presented IgG seroconversion to DENV. In the investigation of the blood components, 145 individual samples were analysed. None of the investigated blood components showed a positive RT-PCR. CONCLUSION: We observed a low prevalence of DENV, ZKV, and CHKV in HSCT donors and recipients by serology and RT-PCR, and no case of blood transfusion transmission by RT-PCR.

Graphene-based hybrid electrical-electrochemical point-of-care device for serologic COVID-19 diagnosis.

The outbreak of COVID-19 pandemics highlighted the need of sensitive, selective, and easy-to-handle biosensing devices. In the contemporary scenario, point-of-care devices for mass testing and infection mapping within a population have proven themselves as of primordial importance. Here, we introduce a graphene-based Electrical-Electrochemical Vertical Device (EEVD) point-of-care biosensor, strategically engineered for serologic COVID-19 diagnosis. EEVD uses serologic IgG quantifications on SARS-CoV-2 Receptor Binding Domain (RBD) bioconjugate immobilized onto device surface. EEVD combines graphene basal plane with high charge carrier mobility, high conductivity, low intrinsic resistance, and interfacial sensitivity to capacitance alterations. EEVD application was carried out in real human serum samples. Since EEVD is a miniaturized device, it requires just 40 µL of sample for a point-of-care COVID-19 infections detection. When compared to serologic assays such ELISA and other immunochromatographic methods, EEVD presents some advantages such as time of analyses (15 min), sample preparation, and a LOD of 1.0 pg mL-1. We glimpse that EEVD meets the principles of robustness and accuracy, desirable analytic parameters for assays destined to pandemics control strategies.

Polymerase chain reaction targeting 16S ribosomal RNA for the diagnosis of bacterial meningitis after neurosurgery.

OBJECTIVES: Bacterial and aseptic meningitis after neurosurgery can present similar clinical signs and symptoms. The aims of this study were to develop and test a molecular method to diagnose bacterial meningitis (BM) after neurosurgery. METHODS: A 16S ribosomal RNA gene PCR-based strategy was developed using artificially inoculated cerebrospinal fluid (CSF) followed by sequencing. The method was tested using CSF samples from 43 patients who had undergone neurosurgery and were suspected to suffer from meningitis, and from 8 patients without neurosurgery or meningitis. Patients were classified into five groups, confirmed BM, probable BM, possible BM, unlikely BM, and no meningitis. RESULTS: Among the samples from the 51 patients, 21 samples (41%) were culture-negative and PCR-positive. Of these, 3 (14%) were probable BM, 4 (19%) were possible BM, 13 (62%) were unlikely BM, and 1 (5%) was meningitis negative. Enterobacterales, non-fermenters (Pseudomonas aeruginosa and Acinetobacter baumannii), Staphylococcus haemolyticus, Granulicatella, Variovorax, and Enterococcus cecorum could be identified. In the group of patients with meningitis, a good agreement (3 of 4) was observed with the results of cultures, including the identification of species. CONCLUSION: Molecular methods may complement the diagnosis, guide treatment, and identify non-cultivable microorganisms. We suggest the association of methods for suspected cases of BM after neurosurgery, especially for instances in which the culture is negative.

Polymerase chain reaction targeting 16S ribosomal RNA for the diagnosis of bacterial meningitis after neurosurgery

OBJECTIVES: Bacterial and aseptic meningitis after neurosurgery can present similar clinical signs and symptoms. The aims of this study were to develop and test a molecular method to diagnose bacterial meningitis (BM) after neurosurgery. METHODS: A 16S ribosomal RNA gene PCR-based strategy was developed using artificially inoculated cerebrospinal fluid (CSF) followed by sequencing. The method was tested using CSF samples from 43 patients who had undergone neurosurgery and were suspected to suffer from meningitis, and from 8 patients without neurosurgery or meningitis. Patients were classified into five groups, confirmed BM, probable BM, possible BM, unlikely BM, and no meningitis. RESULTS: Among the samples from the 51 patients, 21 samples (41%) were culture-negative and PCR-positive. Of these, 3 (14%) were probable BM, 4 (19%) were possible BM, 13 (62%) were unlikely BM, and 1 (5%) was meningitis negative. Enterobacterales, non-fermenters (Pseudomonas aeruginosa and Acinetobacter baumannii), Staphylococcus haemolyticus, Granulicatella, Variovorax, and Enterococcus cecorum could be identified. In the group of patients with meningitis, a good agreement (3 of 4) was observed with the results of cultures, including the identification of species. CONCLUSION: Molecular methods may complement the diagnosis, guide treatment, and identify non-cultivable microorganisms. We suggest the association of methods for suspected cases of BM after neurosurgery, especially for instances in which the culture is negative.

Detection of HIV-1 infections in blood donors during the pre-seroconversion window period in São Paulo, Brazil.

By decreasing the pre-seroconversion window period, nucleic acid testing (NAT) has improved the safety of blood products and reduced the risk of transfusion-transmitted infections. Between 2011 and 2017, NAT determinations for approximately 898,202 donations were performed at Fundação Pró-Sangue/Hemocentro de São Paulo (FPS-HSP). Three seronegative HIV-viremic donations were detected. The NAT yield rate per million donations was 3.34 for HIV, and the acute HIV-1 infections detected are described, followed by a brief review of the situation in Brazil.

Detection of HIV-1 infections in blood donors during the pre-seroconversion window period in São Paulo, Brazil

Abstract By decreasing the pre-seroconversion window period, nucleic acid testing (NAT) has improved the safety of blood products and reduced the risk of transfusion-transmitted infections. Between 2011 and 2017, NAT determinations for approximately 898,202 donations were performed at Fundação Pró-Sangue/Hemocentro de São Paulo (FPS-HSP). Three seronegative HIV-viremic donations were detected. The NAT yield rate per million donations was 3.34 for HIV, and the acute HIV-1 infections detected are described, followed by a brief review of the situation in Brazil.

Phylogenetic analysis of the emergence of main hepatitis C virus subtypes in São Paulo, Brazil

ABSTRACTBACKGROUND: It is recognized that hepatitis C virus subtypes (1a, 1b, 2a, 2b, 2c and 3a) originated in Africa and Asia and spread worldwide exponentially during the Second World War (1940) through the transfusion of contaminated blood products, invasive medical and dental procedures, and intravenous drug use. The entry of hepatitis C virus subtypes into different regions occurred at distinct times, presenting exponential growth rates of larger or smaller spread. Our study estimated the growth and spread of the most prevalent subtypes currently circulating in São Paulo.METHODS:A total of 465 non-structural region 5B sequences of hepatitis C virus covering a 14-year time-span were used to reconstruct the population history and estimate the population dynamics and Time to Most Recent Common Ancestor of genotypes using the Bayesian Markov Chain Monte Carlo approach implemented in BEAST (Bayesian evolutionary analysis by sampling tree software/program).RESULTS:Evolutionary analysis demonstrated that the different hepatitis C virus subtypes had distinct growth patterns. The introduction of hepatitis C virus-1a and -3a were estimated to be circa 1979 and 1967, respectively, whereas hepatitis C virus-1b appears to have a more ancient entry, circa 1923. Hepatitis C virus-1b phylogenies suggest that different lineages circulate in São Paulo, and four well-supported groups (i.e., G1, G2, G3 and G4) were identified. Hepatitis C virus-1a presented the highest growth rate (r = 0.4), but its spread became less marked after the 2000s. Hepatitis C virus-3a grew exponentially until the 1990s and had an intermediate growth rate (r = 0.32). An evident exponential growth (r = 0.26) was found for hepatitis C virus-1b between 1980 and the mid-1990s.CONCLUSIONS:After an initial period of exponential growth, the expansion of the three main subtypes began to decrease. Hepatitis C virus-1b presented inflated genetic diversity, and its transmission may have been sustained by different generations and transmission routes other than blood transfusion. Hepatitis C virus-1a and -3a showed no group stratification, most likely due to their recent entry.

Phylogenetic analysis of the emergence of main hepatitis C virus subtypes in São Paulo, Brazil.

BACKGROUND: It is recognized that hepatitis C virus subtypes (1a, 1b, 2a, 2b, 2c and 3a) originated in Africa and Asia and spread worldwide exponentially during the Second World War (1940) through the transfusion of contaminated blood products, invasive medical and dental procedures, and intravenous drug use. The entry of hepatitis C virus subtypes into different regions occurred at distinct times, presenting exponential growth rates of larger or smaller spread. Our study estimated the growth and spread of the most prevalent subtypes currently circulating in São Paulo. METHODS: A total of 465 non-structural region 5B sequences of hepatitis C virus covering a 14-year time-span were used to reconstruct the population history and estimate the population dynamics and Time to Most Recent Common Ancestor of genotypes using the Bayesian Markov Chain Monte Carlo approach implemented in BEAST (Bayesian evolutionary analysis by sampling tree software/program). RESULTS: Evolutionary analysis demonstrated that the different hepatitis C virus subtypes had distinct growth patterns. The introduction of hepatitis C virus-1a and -3a were estimated to be circa 1979 and 1967, respectively, whereas hepatitis C virus-1b appears to have a more ancient entry, circa 1923. Hepatitis C virus-1b phylogenies suggest that different lineages circulate in São Paulo, and four well-supported groups (i.e., G1, G2, G3 and G4) were identified. Hepatitis C virus-1a presented the highest growth rate (r=0.4), but its spread became less marked after the 2000s. Hepatitis C virus-3a grew exponentially until the 1990s and had an intermediate growth rate (r=0.32). An evident exponential growth (r=0.26) was found for hepatitis C virus-1b between 1980 and the mid-1990s. CONCLUSIONS: After an initial period of exponential growth, the expansion of the three main subtypes began to decrease. Hepatitis C virus-1b presented inflated genetic diversity, and its transmission may have been sustained by different generations and transmission routes other than blood transfusion. Hepatitis C virus-1a and -3a showed no group stratification, most likely due to their recent entry.

Comparison of two methods of RNA extraction from formalin-fixed paraffin-embedded tissue specimens.

The present study aimed to compare two different methods of extracting RNA from formalin-fixed paraffin-embedded (FFPE) specimens of diffuse large B-cell lymphoma (DLBCL). We further aimed to identify possible influences of variables--such as tissue size, duration of paraffin block storage, fixative type, primers used for cDNA synthesis, and endogenous genes tested--on the success of amplification from the samples. Both tested protocols used the same commercial kit for RNA extraction (the RecoverAll Total Nucleic Acid Isolation Optimized for FFPE Samples from Ambion). However, the second protocol included an additional step of washing with saline buffer just after sample rehydration. Following each protocol, we compared the RNA amount and purity and the amplification success as evaluated by standard PCR and real-time PCR. The results revealed that the extra washing step added to the RNA extraction process resulted in significantly improved RNA quantity and quality and improved success of amplification from paraffin-embedded specimens.

Epstein-Barr Viral Load is Associated to Response in AIDS-Related Lymphomas.

AIDS-related lymphoma (ARL) development is associated to immunodeficiency state with proliferation of B-cells driven by HIV itself and EBV infection. However, Epstein-Barr DNA is not detected in malignant cells of all ARL subtypes. A prospective and controlled study to analyze EBV viral load (VL) in plasma and peripheral blood mononuclear cells (PBMC) of ARL patients was performed to analyze if Epstein-Barr VL could be related to response in these patients. Fifteen patients with ARL were included in this study with measurement of EBV VL at three different periods of time: at lymphoma diagnosis, upon completion of chemotherapy, and 3 months after. Two control groups composed by HIV-negative and HIV-positive patients were also evaluated for EBV VL comparison. In situ hybridization for EBER was performed on diagnostic samples of all ARL patients. Median EBV VL in PBMC and plasma had a significant decrease (p = 0.022 and p = 0.003, respectively) after ARL treatment. EBER was positive in 7 (46.7 %) cases. Median EBV VL in PBMC before lymphoma treatment in patients positive for EBER was significantly higher compared to EBER negative cases (p = 0.041). Reduction of EBV viral load during treatment of lymphoma could be predictive of response. EBER expression was associated to advanced stages of disease and worse immune status. Our study suggests that measurement of EBV VL during ARL treatment could be used as a marker for response, but further studies are needed to validate this association.

Comparação de três protocolos distintos para extração de RNA de amostras fixadas em formalina e emblocadas em parafina / Comparison of three different protocols for extracting RNA from formalin-fixed paraffin-embedded tissues

INTRODUÇÃO: Tecidos fixados em formalina e emblocados em parafina (FFEP) são importantes fontes de amostras para estudos retrospectivos. Apesar de sua capacidade de preservação de proteínas e morfologia celular, a formalina interfere negativamente em testes de biologia molecular por fragmentar e modificar quimicamente os ácidos nucleicos, particularmente o ácido ribonucleico (RNA). OBJETIVO: Comparar a eficiência de três diferentes protocolos de extração de RNA para análise de expressão gênica de tecidos FFEP. MATERIAL E MÉTODOS: Amostras de linfonodo humano FEEP foram submetidas à extração de RNA utilizando-se os kits Ambion e Arcturus Bioscience e o método clássico de Trizol. Após a extração, o RNA foi quantificado e testado quanto à sua capacidade de amplificação pela reação em cadeia da polimerase em tempo real (RT-PCR) utilizando primers do gene endógeno gliceraldeído-3 fosfato desidrogenase (GAPDH). DISCUSSÃO/CONCLUSÃO: Todos os protocolos testados produziram quantidades adequadas e suficientes de RNA total, entretanto, somente os protocolos com uso dos kits Ambion e Arcturus produziram RNA capaz de ser amplificado pela PCR.

INTRODUCTION: Formalin fixed paraffin embedded (FFPE) tissues are an important sample source for retrospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders Molecular Biology tests once it fragments and chemically modifies nucleic acids, particularly RNA. OBJECTIVE: To compare the efficiency of three different RNA extraction protocols for gene expression analysis of FFEP tissues. MATERIAL AND METHODS: RNA was extracted from FFPE samples of human lymph by means of Ambion and Arcturus Bioscience kits and the classical Trizol method. After extraction, RNA was quantified and tested for amplification through real time polymerase chain reaction (RT-PCR) using glyceraldehydes-3 phosphate dehydrogenase (GAPDH) endogenous gene primers. DISCUSSION/CONCLUSION: All the protocols produced sufficient and adequate amounts of total RNA. However, only protocols using Arcturus and Ambion kits generated suitable RNA for PCR amplification.

Distribution of hepatitis B virus genotypes and viral load levels in Brazilian chronically infected patients in São Paulo city.

The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68% samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.

Distribution of hepatitis B virus genotypes and viral load levels in Brazilian chronically infected patients in São Paulo city / Distribuição dos genótipos do vírus da hepatite B e níveis de carga viral em pacientes brasileiros cronicamente infectados na cidade de São Paulo

The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68 percent samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.

O objetivo do presente estudo foi avaliar a carga viral no soro de pacientes cronicamente infectados pelo vírus da Hepatite B (HBV) e investigar a distribuição de genótipos HBV na cidade de São Paulo. PCR quantitativo do HBV e genotipagem ganharam importância para a previsão de progressão da doença, empregada para avaliar a infectividade, para tratamento e acompanhamento e para detectar o aparecimento de resistência aos anti-retrovirais. Vinte e nove pacientes brasileiros com suspeita de hepatite B crônica foram estudados, utilizando PCR em tempo real para a determinação da carga viral e seqüenciamento direto para determinação do genótipo. A sorologia revelou que 22 estavam, de fato, cronicamente infectados pelo HBV. O HBV-DNA foi positivo em 68 por cento das amostras (15/22). Em sete casos, HBV-DNA foi indetectável por PCR quantitativo. A análise filogenética mostra que onze pacientes foram infectados com hepatite B genótipo A, dois com genótipo F e dois com genótipo D. Desta forma, o genótipo A foi o mais prevalente em nosso estudo.