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

Yellow fever vaccine viremia following ablative BM suppression in AML

Univ São Paulo, Sch Med, Dept Infect & Parasit Dis, São Paulo, BrazilHosp Sirio Libanes, São Paulo, BrazilUniv São Paulo, Sch Med, Div Clin Immunol & Allergy, São Paulo, BrazilFundacao Prosangue Hemoctr São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Infect Dis Div DIPA, São Paulo, BrazilFundacao Oswaldo Cruz, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Infect Dis Div DIPA, São Paulo, BrazilWeb of Scienc

Plasma Cytokine Profile in Tropical Endomyocardial Fibrosis: Predominance of TNF-a, IL-4 and IL-10

Background: the participation of immune/inflammatory mechanisms in the pathogenesis of tropical endomyocardial fibrosis (EMF) has been suggested by the finding of early blood and myocardial eosinophilia. However, the inflammatory activation status of late-stage EMF patients is still unknown.Methodology/Principal findings: We evaluated pro- and anti-inflammatory cytokine levels in plasma samples from late stage EMF patients. Cytokine levels of Tumor Necrosis Factor (TNF)-alpha, Interferon (IFN)-gamma, Interleukin (IL)-2, IL-4, IL-6, and IL-10 were assayed in plasma samples from 27 EMF patients and compared with those of healthy control subjects. All EMF patients displayed detectable plasma levels of at least one of the cytokines tested. We found that TNF-alpha, IL-6, IL-4, and IL-10 were each detected in at least 74% of tested sera, and plasma levels of IL-10, IL-4, and TNF-alpha were significantly higher than those of controls. Plasma levels of such cytokines positively correlated with each other.Conclusions/Significance: the mixed pro-and anti-inflammatory/Th2circulating cytokine profile in EMF is consistent with the presence of a persistent inflammatory stimulus. On the other hand, the detection of increased levels of TNF-alpha may be secondary to the cardiovascular involvement observed in these patients, whereas IL-4 and IL-10 may have been upregulated as a homeostatic mechanism to buffer both production and deleterious cardiovascular effects of pro-inflammatory cytokines. Further studies might establish whether these findings play a role in disease pathogenesis.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Sch Med, Inst Heart InCor, Immunol Lab, São Paulo, BrazilUniv São Paulo, Sch Med, Div Clin Immunol & Allergy, São Paulo, BrazilUniv São Paulo, Sch Med, Inst Heart InCor, Cardiomyopathy Unit, São Paulo, BrazilProSangue Fdn, São Paulo, BrazilInst Investigat Immunol, INCT, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Div Immunol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Div Immunol, São Paulo, BrazilWeb of Scienc

Hcv genotypes, characterization of mutations conferring drug resistance to protease inhibitors, and risk factors among blood donors in São Paulo, Brazil

Nishiya, AS. HCV Genotypes, Characterization of Mutations Conferring Drug Resistance to Protease Inhibitors, and Risk Factors among Blood Donors in São Paulo, Brazil. São Paulo: Universidade Federal de São Paulo, Escola Paulista de Medicina. Background: Infection with Hepatitis C Virus (HCV) is a major cause of chronic hepatitis, affects 150 million people worldwide and is the cause of 350,000 deaths per year due cirrhosis or hepatocellular carcinoma. The aim of this study is to analyze the subtypes and existence of variants resistant to protease inhibitors and their association with potential HCV risk factors among blood donors in Brazil. Methods: Repeat anti-HCV reactive blood donors are systematically asked to return for retest, notification, and counseling in which they are interviewed for risk factors for transfusion-transmitted diseases. We analyzed 202 donors who returned for counseling from 2007 to 2010 and presented enzyme immunoassay- and immunoblot-reactive results. The HCV genotypes and resistance mutation analyses were determined by the direct sequencing of the NS5b and NS3 regions, respectively. The HCV viral load was determined using an in-house real-time PCR assay targeting the 5'-NCR. Results: HCV subtypes 1b, 1a, 3a and others genotypes (2b, 2c , 4 e 5) were found in 45.5%, 32.0%, 18.0% and 4.5% of the donors, respectively. The mean viral load of genotype 1 was significantly higher than that of genotype 3 isolates. Subtype 1a was more frequent among young donors and 3a among older donors. Protease inhibitor-resistant variants were detected in 12.8% of the sequenced samples belonging to genotype 1, and a higher frequency was observed among subtype 1a (20%) in comparison to 1b (8%). There was no difference in the prevalence of HCV risk factors among genotypes or drug-resistant variants. Conclusions: We found a predominance of subtype 1b, with an increase in the frequency of subtype 1a in young subjects. Mutations conferring resistance to NS3 inhibitors were frequent in treatment-naïve blood donors, particularly those infected with subtype 1a. These variants were detected in the major viral population of HCV quasispecies, have replicative capacities comparable to nonresistant strains, and could be important for predicting the response to antiviral triple therapy.Introdução: A infecção pelo Vírus da Hepatite C (HCV) é uma das principais causas de hepatite crônica, atinge 150 milhões de pessoas no mundo e é a causa de 350.000 mortes por ano devido cirrose ou carcinoma hepatocelular. O objetivo deste estudo é verificar os subtipos, a existência de variantes associadas à resistência aos inibidores da protease e os possíveis fatores de risco associados à transmissão do HCV na população de doadores de sangue de São Paulo. Métodos: Doadores de sangue repetidamente reativos ao EIA anti-HCV são rotineiramente convocados a retornar para re-teste, notificação e aconselhamento, quando responderam às questões relacionadas aos potenciais fatores de risco envolvidos na transmissão deste vírus. Foram analisados 202 doadores que apresentaram EIA e immunoblot reativos para anti-HCV de 2007 a 2010. Os genótipos e a análise das mutações associadas à resistência foram determinados pelo sequenciamento direto de parte das regiões NS3 e NS5B, respectivamente. A carga viral foi determinada utilizando um ensaio próprio de PCR em tempo real com primers da região 5'-NCR. Resultados: Os subtipos 1b, 1a, 3a e outros genótipos (2b, 2c, 4 e 5) foram encontrados em 45,5%, 32,0%, 18,0% e 4,5% dos doadores, respectivamente. A carga viral média de genótipo 1 foi significativamente mais elevada do que a do genótipo 3. Subtipo 1a foi mais frequente entre os doadores jovens e 3a foi mais frequente entre os doadores mais velhos. Variantes associadas à resistência aos inibidores de protease foram detectadas em 12,8% das amostras sequenciadas do genótipo 1, e observou-se uma frequência mais elevada entre os subtipos 1a (20%) em comparação com o 1b (8%). Não houve diferença significante na prevalência dos fatores de risco entre os genótipos ou entre as variantes associadas à resistência aos medicamentos. Conclusões: Houve predominância do subtipo 1b, com um aumento na frequência do subtipo 1a, em indivíduos jovens. As mutações de resistência aos inibidores da NS3 foram frequentes em doadores de sangue virgens de tratamento, principalmente aqueles infectados pelo subtipo 1a. Apesar das variantes encontradas apresentarem mutações de baixo nível de resistência, elas foram detectadas na população viral principal dentre as quasispecies do HCV e apresentaram capacidade replicativa comparável às cepas selvagens, que pode ser importante para predizer a resposta à terapia antiviral tripla.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Investigación de Torque teno virus (TTV) en Suero y Sangre de Primates No Humanos Brasileños y en Plasma de Pollo (Gallus gallus domesticus) por PCR en la Region N22

Torque teno virus (TTV) is a recently discovered DNA virus that was originally isolated from a Japanese patient ( initials, TT) with post-transfusion hepatitis of unknown aetiology. TTV is an circular DNA virus classified recently together with related Torque teno minivirus, into a new genus called Anellovirus. Infection TTV has been detected in a range of non-human primates as well as domestic animals. The purpose of this study was to search TTV in the serum and total blood of Brazilian monkeys and in plasma of domestic chickens by seminested PCR of coding region (N22), followed by a genomic sequence and phylogenetic analysis. No serum sample was amplified. TTV DNA was detected in total blood from 3 (4%) out of 75 brown-capuchin ( Cebus apella) and from 1 (25%) out of 4 golden-headed lion-tamarin ( Leontopithecus chrysomelas). Phylogenetic analysis revealed that one sample showed similarity with one sequence of the cotton top tamarin ( Saguinus oedipus) (So-TTV2) and with one of the douroucoulis (Aotes trivirgatus) (At-TTV3). Two samples showed similarity with a human Torque Teno Mini Virus (TLMV). The other sample clustered with one sequence of the chimpanzee (Pt-TTV6) and with the human TTV strain TA278. The plasma chicken samples tested were all negative. The amino acid sequences reported in this study are the first obtained in Brazil from total blood of non-human primates naturally infected by TTV.Biol Inst Sao Paulo, Electron Microscopy Lab, BR-04014002 Sao Paulo, BrazilUniv Fed Sao Paulo, Lab Retrovirol, Sao Paulo, BrazilFundaCao Problood Homectr Sao Paulo, Sao Paulo, BrazilUniv Sao Paulo, Inst Biomed Sci, BR-05508 Sao Paulo, BrazilInst Adolfo Lutz Registro, Sao Paulo, BrazilEcol Pk Tiete, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Surg Tech, Sao Paulo, BrazilUniv Fed Sao Paulo, Lab Retrovirol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Surg Tech, Sao Paulo, BrazilWeb of Scienc

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. Keywords: Growth rate, HCV, Phylogenetic analysis, Subtype

Cytokine plasma levels of endomyocardial fibrosis patients and healthy subjects.

<p>Dot plot represents cytokine levels (interleukin 2, 4, 6, 10, TNF-α and IFN-γ) from endomyocardial fibrosis patients (EMF) and healthy subjects (HS), evaluated by CBA. Horizontal lines indicate the median values. *Differences where <i>P</i>≤0.05 are indicated. (•Healthy Subjects;▪EMF patients).</p

Demographic and echocardiographic data from EMF patients and healthy subjects.

<p>NA – Not applicable; AF – Atrial Fibrillation; (Percentage of EMF patients with the conditionregistered in the medical records); Systolic dysfunction: Ejection Fraction <55%; *Valvar regurgitation level: mild, moderate, and severe, respectively; Diastolic dysfunction: grades mild, moderate, and severe ** It was not possible to evaluate diastolic function in 4 patients, due to the presence of pacemaker or bioprosthetic heart valve.</p><p>Demographic and echocardiographic data from EMF patients and healthy subjects.</p

Correlation among cytokine plasma levels of endomyocardial fibrosis patients.

<p>Dot plots represent correlation between cytokine levels (interleukin 2, 4, 6, 10, TNF-α and IFN-γ) from endomyocardial fibrosis patients, evaluated by CBA.</p