Susceptibility of Aedes aegypti populations to pyriproxyfen in the Federal District of Brazil.

INTRODUCTION: In Brasilia, pyriproxyfen (PPF; 0.01 mg/L) has been used for the larval control of Aedes aegypti mosquitoes since 2016. Information on the susceptibility of Ae. aegypti to PPF, and the development of resistance in populations from the Federal District of Brazil (FD) is limited. It is essential to monitor the susceptibility of Ae. aegypti to insecticides in order to improve vector control strategies. This study aimed to evaluate the susceptibility of Ae. aegypti populations from five areas of Brasilia to PPF. METHODS: We performed dose-response tests to estimate the emergence inhibition and resistance ratio of each field population, including the Rockefeller reference population. We also analyzed egg positivity, and the density and mortality of larvae and pupae. RESULTS: Populations from Vila Planalto (RR50=1.7), Regiment Guards Cavalry (RR50=2.5), and Sub-secretary of Justice Complex (RR50=3.7) presented high susceptibility to PPF, while the RR values of populations from Lago Norte (RR50=7.7) and Varjão (RR50=5.9) were moderately high, suggesting the emergence of insipient resistance to PPF in Brasilia. At 30 ng/mL, the highest larvae mortality rate was 2.7% for the population from Lago Norte, while that of pupae was 92.1% for Varjão and Vila Planalto. CONCLUSIONS: The five populations of Ae. aegypti from the FD are susceptible to PPF and there is a need to monitor the susceptibility of Ae. aegypti in new areas of the FD.

Susceptibility of Aedes aegypti populations to pyriproxyfen in the Federal District of Brazil

Abstract INTRODUCTION: In Brasilia, pyriproxyfen (PPF; 0.01 mg/L) has been used for the larval control of Aedes aegypti mosquitoes since 2016. Information on the susceptibility of Ae. aegypti to PPF, and the development of resistance in populations from the Federal District of Brazil (FD) is limited. It is essential to monitor the susceptibility of Ae. aegypti to insecticides in order to improve vector control strategies. This study aimed to evaluate the susceptibility of Ae. aegypti populations from five areas of Brasilia to PPF. METHODS: We performed dose-response tests to estimate the emergence inhibition and resistance ratio of each field population, including the Rockefeller reference population. We also analyzed egg positivity, and the density and mortality of larvae and pupae. RESULTS: Populations from Vila Planalto (RR50=1.7), Regiment Guards Cavalry (RR50=2.5), and Sub-secretary of Justice Complex (RR50=3.7) presented high susceptibility to PPF, while the RR values of populations from Lago Norte (RR50=7.7) and Varjão (RR50=5.9) were moderately high, suggesting the emergence of insipient resistance to PPF in Brasilia. At 30 ng/mL, the highest larvae mortality rate was 2.7% for the population from Lago Norte, while that of pupae was 92.1% for Varjão and Vila Planalto. CONCLUSIONS: The five populations of Ae. aegypti from the FD are susceptible to PPF and there is a need to monitor the susceptibility of Ae. aegypti in new areas of the FD.

Efficacy and Safety of Ombitasvir/Paritaprevir/Ritonavir and Dasabuvir ± Ribavirin for HCV in Brazilian Adults with Advanced Fibrosis.

INTRODUCTION AND AIM: Approximately 650,000 people in Brazil have chronic hepatitis C virus (HCV) infection. We evaluated the safety and efficacy of ombitasvir (OBV)/paritaprevir (PTV)/ritonavir (r) plus dasabuvir (DSV) with/without ribavirin (RBV) in an openlabel multicenter phase 3b trial in treatment-naive or interferon (IFN) treatment-experienced Brazilian patients with advanced hepatic fibrosis (METAVIR F3/4) and HCV genotype (GT) 1 infection. MATERIAL AND METHODS: All patients received coformulated OBV/PTV/r daily + DSV twice daily (3-DAA). GT1a-infected patients received 3-DAA plus RBV for 12 weeks, except for prior pegIFN/RBV nonresponders with cirrhosis who were treated for 24 weeks. GT1b-infected patients received 3-DAA alone (F3) or in combination with RBV (F4) for 12 weeks. The primary endpoint was sustained virologic response (HCV RNA < 15 IU/mL) at post-treatment Week 12 (SVR12). RESULTS: The study enrolled 222 patients, 214 achieved an SVR12 (96.4%; 95% CI, 93.1-98.2%), one GT1a-infected patient experienced virologic breakthrough, six (5 GT1a) relapsed, and one was lost to follow-up. SVR12 was achieved in 111/ 112 (99.1%) GT1b-infected patients, including 42/43 (97.7%) noncirrhotic, and 69/69 (100%) cirrhotic patients; and in 103/110 (93.6%) GT1a-infected patients, including 44/46 (95.7%) noncirrhotic and 59/64 (92.2%) cirrhotic patients. Overall there was a low rate of serious adverse events (n = 6, 2.7%). One patient experienced a treatment-related serious adverse event and one patient discontinued treatment because of an adverse event. DISCUSSION: The results confirm that the 3-DAA regimen with/without RBV is well tolerated and had a favorable safety profile and is efficacious in GT1-infected patients with advanced fibrosis (METAVIR F3/4).

HCV kinetic and modeling analyses indicate similar time to cure among sofosbuvir combination regimens with daclatasvir, simeprevir or ledipasvir.

BACKGROUND & AIMS: Recent clinical trials of direct-acting-antiviral agents (DAAs) against hepatitis C virus (HCV) achieved >90% sustained virological response (SVR) rates, suggesting that cure often took place before the end of treatment (EOT). We sought to evaluate retrospectively whether early response kinetics can provide the basis to individualize therapy to achieve optimal results while reducing duration and cost. METHODS: 58 chronic HCV patients were treated with 12-week sofosbuvir+simeprevir (n=19), sofosbuvir+daclatasvir (n=19), or sofosbuvir+ledipasvir in three French referral centers. HCV was measured at baseline, day 2, every other week, EOT and 12weeks post EOT. Mathematical modeling was used to predict the time to cure, i.e., <1 virus copy in the entire extracellular body fluid. RESULTS: All but one patient who relapsed achieved SVR. Mean age was 60±11years, 53% were male, 86% HCV genotype-1, 9% HIV coinfected, 43% advanced fibrosis (F3), and 57% had cirrhosis. At weeks 2, 4 and 6, 48%, 88% and 100% of patients had HCV<15IU/ml, with 27%, 74% and 91% of observations having target not detected, respectively. Modeling results predicted that 23 (43%), 16 (30%), 7 (13%), 5 (9%) and 3 (5%) subjects were predicted to reach cure within 6, 8, 10, 12 and 13weeks of therapy, respectively. The modeling suggested that the patient who relapsed would have benefitted from an additional week of sofosbuvir+ledipasvir. Adjusting duration of treatment according to the modeling predicts reduced medication costs of 43-45% and 17-30% in subjects who had HCV<15IU/ml at weeks 2 and 4, respectively. CONCLUSIONS: The use of early viral kinetic analysis has the potential to individualize duration of DAA therapy with a projected average cost saving of 16-20% per 100-treated persons.

CD8+ T lymphocyte expansion, proliferation and activation in dengue fever.

Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population.

Simeprevir with pegylated interferon alfa 2a or 2b plus ribavirin in treatment-naive patients with chronic hepatitis C virus genotype 1 infection (QUEST-2): a randomised, double-blind, placebo-controlled phase 3 trial.

BACKGROUND: Pegylated interferon (peginterferon) alfa 2a or 2b plus ribavirin regimens were the standard of care in patients with hepatitis C virus (HCV) infection, but the sustained virological response can be suboptimum in patients with HCV genotype 1 infection. The efficacy, safety, and tolerability of the combination of simeprevir, a one-pill, once-daily, oral HCV NS3/4A protease inhibitor versus placebo, plus peginterferon alfa 2a or 2b plus ribavirin was assessed in treatment-naive patients with HCV genotype 1 infection. METHODS: In the QUEST-2, phase 3 study, done at 76 sites in 14 countries (Europe, and North and South Americas), patients with confirmed chronic HCV genotype 1 infection and no history of HCV treatment were randomly assigned with a computer-generated allocation sequence in a ratio of 2:1 and stratified by HCV genotype 1 subtype and host IL28B genotype to receive simeprevir (150 mg once daily, orally), peginterferon alfa 2a (180 µg once weekly, subcutaneous injection) or 2b (according to bodyweight; 50 µg, 80 µg, 100 µg, 120 µg, or 150 µg once weekly, subcutaneous injection), plus ribavirin (1000-1200 mg/day or 800-1400 mg/day, orally; simeprevir group) or placebo (once daily, orally), peginterferon alfa 2a or 2b, plus ribavirin (placebo group) for 12 weeks, followed by just peginterferon alfa 2a or 2b plus ribavirin. Total treatment duration was 24 weeks or 48 weeks (simeprevir group) based on criteria for response-guided therapy (ie, HCV RNA <25 IU/mL undetectable or detectable at week 4 and undetectable week 12) or 48 weeks (placebo). Patients, study personnel, and the sponsor were masked to treatment assignment. The primary efficacy endpoint was sustained virological response at 12 weeks after the planned end of treatment (SVR12). Analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT01290679. Results from the primary (SVR12, week 60) analysis are presented. FINDINGS: 209 (81%) of 257 patients in the simeprevir group and 67 (50%) of 134 in the placebo group had SVR12 (adjusted difference 32·2%, 95% CI 23·3-41·2; p<0·0001). The incidences of adverse events were similar in the simeprevir and placebo groups at 12 weeks (246 [96%] vs 130 [97%]) and for the entire treatment (249 [97%] vs 132 [99%]), irrespective of the peginterferon alfa used. The most common adverse events were headache, fatigue, pyrexia, and influenza-like illness at 12 weeks (95 [37%) vs 45 [34%], 89 [35%] vs 52 [39%], 78 [30%] vs 48 [36%], and 66 [26%] vs 34 [25%], respectively) and for the entire treatment (100 [39%] vs 49 [37%], 94 [37%] vs 56 [42%], 79 [31%] vs 53 [40%], and 66 [26%] vs 35 [26%], respectively). Rash and photosensitivity frequencies were higher in the simeprevir group than in the placebo group (61 [24%] vs 15 [11%] and ten [4%] vs one [<1%], respectively). There was no difference in the prevalence of anaemia between the simeprevir and placebo groups (35 [14%] vs 21 [16%], respectively, at 12 weeks, and 53 [21%] vs 37 [28%], respectively, during the entire treatment). INTERPRETATION: Addition of simeprevir to either peginterferon alfa 2a or peginterferon alfa 2b plus ribavirin improved SVR in treatment-naive patients with HCV genotype 1 infection, without worsening the known adverse events associated with peginterferon alfa plus ribavirin. FUNDING: Janssen Infectious Diseases-Diagnostics.

Serum angiopoietin-2 and soluble VEGF receptor 2 are surrogate markers for plasma leakage in patients with acute dengue virus infection.

BACKGROUND: Endothelial cell dysfunction is believed to play an important role in the pathogenesis of plasma leakage in patients with acute dengue virus (DENV) infection. Several factors, produced by activated endothelial cells, have been associated with plasma leakage or severe disease in patients with infectious diseases. OBJECTIVES: The aim of this study was to investigate which of these markers could serve as a surrogate marker for the occurrence of plasma leakage in patients with acute DENV infection. STUDY DESIGN: A case-control study was performed in patients with acute DENV infection in Santos, Brazil. Plasma leakage was detected with X-ray and/or ultrasound examination at admission. Serum levels of soluble endoglin, endothelin-1, angiopoietin-2, VEGF, soluble VEGFR-2, MMP-2, MMP-9, TIMP-1 and TIMP-2 were determined using commercially available ELISAs. RESULTS: Increased levels of angiopoietin-2, endothelin-1 and MMP-2 and decreased levels of soluble VEGFR-2 were significantly associated with the occurrence of plasma leakage. An unsupervised cluster analysis confirmed that angiopoietin-2 and soluble VEGFR-2 were strongly associated with clinical apparent vascular leakage. CONCLUSION: Angiopoietin-2 and soluble VEGFR-2 can serve as surrogate markers for the occurrence of plasma leakage in patients with acute DENV infection.

Inter- and intra-host viral diversity in a large seasonal DENV2 outbreak.

BACKGROUND: High genetic diversity at both inter- and intra-host level are hallmarks of RNA viruses due to the error-prone nature of their genome replication. Several groups have evaluated the extent of viral variability using different RNA virus deep sequencing methods. Although much of this effort has been dedicated to pathogens that cause chronic infections in humans, few studies investigated arthropod-borne, acute viral infections. METHODS AND PRINCIPAL FINDINGS: We deep sequenced the complete genome of ten DENV2 isolates from representative classical and severe cases sampled in a large outbreak in Brazil using two different approaches. Analysis of the consensus genomes confirmed the larger extent of the 2010 epidemic in comparison to a previous epidemic caused by the same viruses in another city two years before (genetic distance = 0.002 and 0.0008 respectively). Analysis of viral populations within the host revealed a high level of conservation. After excluding homopolymer regions of 454/Roche generated sequences, we found 10 to 44 variable sites per genome population at a frequency of >1%, resulting in very low intra-host genetic diversity. While up to 60% of all variable sites at intra-host level were non-synonymous changes, only 10% of inter-host variability resulted from non-synonymous mutations, indicative of purifying selection at the population level. CONCLUSIONS AND SIGNIFICANCE: Despite the error-prone nature of RNA-dependent RNA-polymerase, dengue viruses maintain low levels of intra-host variability.

Microbial translocation is associated with extensive immune activation in dengue virus infected patients with severe disease.

BACKGROUND: Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. METHODS: Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of São Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. RESULTS: Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. CONCLUSIONS: The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis.

Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil, in 2010.

In 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.

Pharmacodynamics of PEG-IFN-[alpha]-2a and HCV response as a function of IL28B polymorphism in HIV/HCV-coinfected patients.

We examined the association between IL28B single-nucleotide polymorphism rs12979860, hepatitis C virus (HCV) kinetic, and pegylated interferon alpha-2a pharmacodynamic parameters in HIV/HCV-coinfected patients from South America. Twenty-six subjects received pegylated interferon alpha-2a + ribavirin. Serum HCV-RNA and interferon concentrations were measured frequently during the first 12 weeks of therapy and analyzed using mathematical models. African Americans and whites had a similar distribution of IL28B genotypes (P = 0.5). The IL28B CC genotype was overrepresented (P = 0.015) in patients infected with HCV genotype-3 compared with genotype-1. In both genotype-1 and genotype-3, the first-phase viral decline and the average pegylated interferon-alpha-2a effectiveness during the first week of therapy were larger (trend P <= 0.12) in genotype-CC compared with genotypes-TC/TT. In genotype-1 patients, the second slower phase of viral decline (days 2-29) and infected cells loss rate, [delta], were larger (P = 0.02 and 0.11, respectively) in genotype-CC than in genotypes-TC/TT. These associations were not observed in genotype-3 patients.

Characterization of Dengue virus type 2: new insights on the 2010 Brazilian epidemic.

Dengue viruses (DENV) serotypes 1, 2, and 3 have been causing yearly outbreaks in Brazil. In this study, we report the re-introduction of DENV2 in the coast of São Paulo State. Partial envelope viral genes were sequenced from eighteen patients with dengue fever during the 2010 epidemic. Phylogenetic analysis showed this strain belongs to the American/Asian genotype and was closely related to the virus that circulated in Rio de Janeiro in 2007 and 2008. The phylogeny also showed no clustering by clinical presentation, suggesting that the disease severity could not be explained by distinct variants or genotypes. The time of the most recent common ancestor of American/Asian genotype and the São Paulo and Rio de Janeiro (SP/RJ) monophyletic cluster was estimated to be around 40 and 10 years, respectively. Since this virus was first identified in Brazil in 2007, we suggest that it was already circulating in the country before causing the first documented outbreak. This is the first description of the 2010 outbreak in the State of São Paulo, Brazil, and should contribute to efforts to control and monitor the spread of DENVs in endemic areas.

Pharmacodynamics of PEG-IFN-alpha-2a in HIV/HCV co-infected patients: implications for treatment outcomes.

BACKGROUND & AIMS: The pharmacokinetics and pharmacodynamics of pegylated-interferon-alpha-2a (PEG-IFN) have not been described in HCV/HIV co-infected patients. We sought to estimate the pharmacokinetics and pharmacodynamics of PEG-IFN and determine whether these parameters predict treatment outcome. METHODS: Twenty-six HCV/human immunodeficiency virus (HIV)-co-infected patients were treated with a 48-week regimen of PEG-IFN (180 microg/week) plus ribavirin (11 mg/kg/day). HCV RNA and PEG-IFN concentrations were obtained from samples collected until week 12. A modeling framework that includes pharmacokinetic and pharmacodynamic parameters was developed. RESULTS: Five patients discontinued treatment. Seven patients achieved a sustained virological response (SVR). PEG-IFN concentrations at day 8 were similar to steady-state levels (p=0.15) and overall pharmacokinetic parameters were similar in SVRs and non-SVRs. The maximum PEG-IFN effectiveness during the first PEG-IFN dose and the HCV-infected cell loss rate (delta), were significantly higher in SVRs compared to non-SVRs (median 95% vs. 86% [p=0.013], 0.27 vs. 0.11 day(-1) [p=0.006], respectively). Patients infected with HCV genotype 1 had a significantly lower average first-week PEG-IFN effectiveness (median 70% vs. 88% [p=0.043]), however, 4- to 12-week PEG-IFN effectiveness was not significantly different compared to those with genotype 3 (p=0.114). Genotype 1 had a significantly lower delta compared to genotype 3 (median 0.14 vs. 0.23 day(-1) [p=0.021]). The PEG-IFN concentration that decreased HCV production by 50% (EC(50)) was lower in genotype 3 compared to genotype 1 (median 1.3 vs. 3.4 [p=0.034]). CONCLUSIONS: Both the HCV-infected cell loss rate (delta) and the maximum effectiveness of the first dose of PEG-IFN-alpha-2a characterised HIV co-infected patients and were highly predictive of SVR. Further studies are needed to validate these viral kinetic parameters as early on-treatment prognosticators of response in patients with HCV and HIV.

Patterns of viral load in chronic hepatitis B patients in Brazil and their association with ALT levels and HBeAg status.

Serum hepatitis B virus (HBV) DNA level is a predictor of the development of cirrhosis and hepatocellular carcinoma in chronic hepatitis B patients. Nevertheless, the distribution of viral load levels in chronic HBV patients in Brazil has yet to be described. This cross-sectional study included 564 participants selected in nine Brazilian cities located in four of the five regions of the country using the database of a medical diagnostics company. Admission criteria included hepatitis B surface antigen seropositivity, availability of HBV viral load samples and age >or=18 years. Males comprised 64.5% of the study population. Mean age was 43.7 years. Most individuals (62.1%) were seronegative for the hepatitis B e antigen (HBeAg). Median serum ALT level was 34 U/L. In 58.5% of the patients HBV-DNA levels ranged from 300 to 99,999 copies/mL; however, in 21.6% levels were undetectable. Median HBV-DNA level was 2,351 copies/mL. Over 60% of the patients who tested negative for HBeAg and in whom ALT level was less than 1.5 times the upper limit of the normal range had HBV-DNA levels > 2,000 IU/mL, which has been considered a cut-off point for indicating a liver biopsy and/or treatment. In conclusion, HBV-DNA level identified a significant proportion of Brazilian individuals with chronic hepatitis B at risk of disease progression. Furthermore, this tool enables those individuals with high HBV-DNA levels who are susceptible to disease progression to be identified among patients with normal or slightly elevated ALT.