Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1-kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (+/- 931.7)- and 295,400 (+/- 75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (+/- 493,700)- and 308,000 (+/- 139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/mu g protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirao Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm(3) [standard deviation (SD) = 99] and a mean viral load of 5.09 log(10) copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno([clinical20%]) algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naive HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

Differential MiRNA expression in childhood acute lymphoblastic leukemia and association with clinical and biological features

The present study aimed to analyze the expression profile of the microRNAs previously described as associated with childhood ALL, miR-92a, miR-100, miR-125a-5p, miR-128a, miR-181b, miR-196b and let-7e, and their association with biological/prognostic features in 128 consecutive samples of childhood acute lymphoblastic leukemia (ALL) by quantitative real-time PCR. A significant association was observed between higher expression levels of miR-196b and T-ALL, miR-100 and patients with low white blood cell count at diagnosis and t(12;21) positive ALL. These findings suggest a potential activity of these microRNAs in pediatric ALL biology. (C) 2011 Elsevier Ltd. All rights reserved.

Vancomycin-resistant enterococcus outbreak in a pediatric intensive care unit: report of successful interventions for control and prevention

The objective of this study is to retrospectively report the results of interventions for controlling a vancomycin-resistant enterococcus (VRE) outbreak in a tertiary-care pediatric intensive care unit (PICU) of a University Hospital. After identification of the outbreak, interventions were made at the following levels: patient care, microbiological surveillance, and medical and nursing staff training. Data were collected from computer-based databases and from the electronic prescription system. Vancomycin use progressively increased after March 2008, peaking in August 2009. Five cases of VRE infection were identified, with 3 deaths. After the interventions, we noted a significant reduction in vancomycin prescription and use (75% reduction), and the last case of VRE infection was identified 4 months later. The survivors remained colonized until hospital discharge. After interventions there was a transient increase in PICU length-of-stay and mortality. Since then, the use of vancomycin has remained relatively constant and strict, no other cases of VRE infection or colonization have been identified and length-of-stay and mortality returned to baseline. In conclusion, we showed that a bundle intervention aiming at a strict control of vancomycin use and full compliance with the Hospital Infection Control Practices Advisory Committee guidelines, along with contact precautions and hand-hygiene promotion, can be effective in reducing vancomycin use and the emergence and spread of vancomycin-resistant bacteria in a tertiary-care PICU.

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of Sao Paulo, Brazil

Background: The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives: This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of Sao Paulo, Brazil. Study design: RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results: From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naive patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion: The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of Sao Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

In vitro cytotoxicity of Selol-loaded magnetic nanocapsules against neoplastic cell lines under AC magnetic field activation

The goals of this study are to evaluate in vitro compatibility of magnetic nanomaterials and their therapeutic potential against cancer cells. Highly stable ionic magnetic fluid sample (maghemite, gamma-Fe2O3) and Selol were incorporated into polymeric nanocapsules by nanoprecipitation method. The cytotoxic effect of Selol-loaded magnetic nanocapsules was assessed on murine melanoma (B16-F10) and oral squamous cell carcinoma (OSCC) cell lines following AC magnetic field application. The influence of different nanocapsules on cell viability was investigated by colorimetric MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. In the absence of AC magnetic field Selol-loaded magnetic nanocapsules, containing 100 mu g/mL Selol plus 5 x 10(12) particle/mL, showed antitumoral activity of about 50% on B16-F10 melanoma cells while OSCC carcinoma cells demonstrated drug resistance at all concentrations of Selol and magnetic fluid (range of 100-500 mu g/mL Selol and 5 x 10(12) -2.5 x 10(13) particle/mL). On the other hand, under AC applied fields (1 MHz and 40 Oe amplitude) B16-F10 cell viability was reduced down to 40.5% (+/- 3.33) at the highest concentration of nanoencapsulated Selol. The major effect, however, was observed on OSCC cells since the cell viability drops down to about 33.3% (+/- 0.38) under application of AC magnetic field. These findings clearly indicate that the Selol-loaded magnetic nanocapsules present different toxic effects on neoplastic cell lines. Further, the cytotoxic effect was maximized under AC magnetic field application on OSCC, which emphasizes the effectiveness of the magnetohyperthermia approach. (C) 2012 American Institute of Physics. [doi: 10.1063/1.3680541]

Streptococcus pneumoniae: susceptibility to penicillin and moxifloxacin

Objective: To determine the minimum inhibitory concentrations (MICs) of parenteral penicillin and moxifloxacin against Streptococcus pneumoniae strains isolated at a hospital center. Methods: In-vitro, prospective study involving 100 S. pneumoniae isolates collected from patients who had been treated, between October of 2008 and July of 2010, at the Hospital das Clinicas complex of the University of Sao Paulo School of Medicine, located in the city of Sao Paulo, Brazil. The isolates were obtained from respiratory tract cultures or blood samples unrelated to meningeal infections, and they were tested for penicillin and moxifloxacin susceptibility by E-test. The MIC category interpretations were based on updated standards. Results: All isolates were fully susceptible to parenteral penicillin (MIC <= 2 mu g/mL), and, consequently, they were also susceptible to amoxicillin, ampicillin, third/fourth generation cephalosporins, and ertapenem. Of the S. pneumoniae strains, 99% were also susceptible to moxifloxacin, and only one strain showed an MIC = 1.5 mu g/mL (intermediate). Conclusions: Our results showed high susceptibility rates to parenteral penicillin and moxifloxacin among S. pneumoniae isolates unrelated to meningitis, which differs from international reports. Reports on penicillin resistance should be based on updated breakpoints for non-meningitis isolates in order to guide the selection of an antimicrobial therapy and to improve the prediction of the clinical outcomes.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naive patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Missed opportunities for prevention of mother-to-child transmission of HIV-1 in the NISDI Perinatal and LILAC cohorts

Objective: To evaluate cases of mother-to-child transmission of HIV-1 at multiple sites in Latin America and the Caribbean in terms of missed opportunities for prevention. Methods: Pregnant women infected with HIV-1 were eligible for inclusion if they were enrolled in either the NISDI Perinatal or LILAC protocols by October 20, 2009, and had delivered a live infant with known HIV-1 infection status after March 1, 2006. Results: Of 711 eligible mothers, 10 delivered infants infected with HIV-1. The transmission rate was 1.4% (95% CI, 0.7-2.6). Timing of transmission was in utero or intrapartum (n = 5), intrapartum (n = 2), intrapartum or early postnatal (n = 1), and unknown (n = 2). Possible missed opportunities for prevention included poor control of maternal viral load during pregnancy; late initiation of antiretrovirals during pregnancy; lack of cesarean delivery before labor and before rupture of membranes; late diagnosis of HIV-1 infection; lack of intrapartum antiretrovirals; and incomplete avoidance of breastfeeding. Conclusion: Early knowledge of HIV-1 infection status (ideally before or in early pregnancy) would aid timely initiation of antiretroviral treatment and strategies designed to prevent mother-to-child transmission. Use of antiretrovirals must be appropriately monitored in terms of adherence and drug resistance. If feasible, breastfeeding should be completely avoided. Presented in part at the XIX International AIDS Conference (Washington, DC; July 22-27, 2012); abstract WEPE163. (c) 2012 Published by Elsevier Ireland Ltd. on behalf of International Federation of Gynecology and Obstetrics.

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of São Paulo, Brazil

Abstract Background The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of São Paulo, Brazil. Study design RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naïve patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of São Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

HIV-1 tropism and CD4 T lymphocyte recovery in a prospective cohort of patients initiating HAART in Ribeirão Preto, Brazil

While human immunodeficiency virus (HIV)-1 chemokine co-receptors 5 tropism and the GWGR motif in the envelope third variable region (V3 loop) have been associated with a slower disease progression, their influence on antiretroviral response remains unclear. The impact of baseline V3 characteristics on treatment response was evaluated in a randomised, double blind, prospective cohort study with patients initiating highly active antiretroviral therapy with lopinavir or efavirenz plus azithothymidine/3TC (1:1) over 48 weeks. Similar virological and immunological responses were observed for both treatment regimens. The 43 individuals had a mean baseline CD4 T cell count of 119 cells/mm³ [standard deviation (SD) = 99] and a mean viral load of 5.09 log10 copies/mL (SD = 0.49). The GWGR motif was not associated with a CD4 T cell response, but predicted R5 tropism by the geno2pheno[clinical20%] algorithm correlated with higher CD4 T cell levels at all monitoring points (p < 0.05). Moreover, higher false-positive rates (FPR) values from this analysis revealed a strong correlation with CD4 T cell recovery (p < 0.0001). Transmitted drug resistance mutations, documented in 3/41 (7.3%) cases, were unrelated to the assigned antiretroviral regimen and had no impact on patient outcomes. In conclusion, naÏve HIV-1 R5 infected patients exhibited higher CD4 T cell counts at baseline; this difference was sustained throughout therapy. The geno2pheno[clinical] option FPR positively correlated with CD4 T cell gain and may be useful in predicting CD4 T cell recovery.

A study of multidrug-resistant tuberculosis in risk groups in the city of Santos, São Paulo, Brazil

Monitoring the extent of and trends in multidrug-resistant tuberculosis (MDR-TB) is a priority of the Brazilian National Tuberculosis Control Programme. The current study aimed to estimate the incidence of MDR-TB, describe the profile of TB drug resistance in risk groups and examine whether screening for MDR-TB adhered to the recommended guidelines. A descriptive study that examined diagnosed cases of pulmonary TB was conducted in the city of Santos, Brazil, between 2000-2004. Of the 2,176 pulmonary TB cases studied, 671 (30.8%) met the criteria for drug sensitivity testing and, of these cases, 31.7% (213/671) were tested. Among the tested cases, 9.4% were resistant to one anti-TB drug and 15% were MDR. MDR was observed in 11.6% of 86 new TB cases and 17.3% of 127 previously treated cases. The average annual incidence of MDR-TB was 1.9 per 100,000 inhabitants-years. The extent of known MDR-TB in the city of Santos is high, though likely to be underestimated. Our study therefore indicates an inadequate adherence to the guidelines for MDR-TB screening and suggests the necessity of alternative strategies of MDR-TB surveillance.