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1.  Expansion and Evolution of a Virulent, Extensively Drug-Resistant (Polymyxin B-Resistant), QnrS1-, CTX-M-2-, and KPC-2-Producing Klebsiella pneumoniae ST11 International High-Risk Clone 
Journal of Clinical Microbiology  2014;52(7):2530-2535.
In this study, we report the early expansion, evolution, and characterization of a multiresistant Klebsiella pneumoniae clone that was isolated with increasing frequency from inpatients in a tertiary-care university hospital in Brazil. Seven carbapenem- and quinolone-resistant and polymyxin B-susceptible or -resistant K. pneumoniae isolates isolated between December 2012 and February 2013 were investigated. Beta-lactamase- and plasmid-mediated quinolone resistance (PMQR)-encoding genes and the genetic environment were investigated using PCR, sequencing, and restriction fragment length polymorphism (RFLP). Clonal relatedness was established using XbaI–pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and phylogenetic group characterization. Plasmid analyses included PCR-based replicon typing (PBRT) and hybridization of the S1-PFGE product, plasmid MLST, and conjugation experiments. Virulence potential was assessed by PCR by searching for 10 virulence factor-encoding genes (ureA, fimH, kfuBC, uge, wabG, magA, mrkD, allS, rmpA, and cf29a) and by phenotypic tests to analyze the hypermucoviscous phenotype. The genetic context of a multidrug-resistant and extensively drug-resistant K. pneumoniae ST11-KpI clone harboring IncFIIk-Tn4401a-blaKPC-2, qnrS1, and blaCTX-M-2 was found. Moreover, three isolates displayed high resistance to polymyxin B (MICs = 32, 32, and 128 mg/liter) as well as mucous and hypermucoviscous phenotypes. These bacteria also harbored ureA, fimH, uge, wabG, and mrkD, which code for virulence factors associated with binding, biofilm formation, and the ability to colonize and escape from phagocytosis. Our study describes the association of important coresistance and virulence factors in the K. pneumoniae ST11 international high-risk clone, which makes this pathogen successful at infections and points to the quick expansion and evolution of this multiresistant and virulent clone, leading to a pandrug-resistant phenotype and persistent bacteria in a Brazilian hospital.
doi:10.1128/JCM.00088-14
PMCID: PMC4097695  PMID: 24808234
2.  Serological Diagnosis of Paracoccidioidomycosis: High Rate of Inter-laboratorial Variability among Medical Mycology Reference Centers 
Background
Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded.
Methodology/Principal findings
We compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the “reference” titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the “reference” titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center's criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient's treatment. Surprisingly, all centers exhibited a high rate of “major” discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better.
Conclusion
There are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.
Author Summary
Paracoccidioidomycosis (PCM) is a neglected systemic fungal infection prevalent mostly in South America. Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and reagents are used. We compared here the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the “reference” titers. Each center then applied its serological routine test to the 150 sera from the other five centers blindly as regards to the “reference” titers. Surprisingly, all centers exhibited a high rate of discordances (mean of 31 discordant scores in 150 sera tested). When the scores given by one center to their own sera were compared with the scores given to their sera by the other centers, a high rate of major discordances was found (a mean of 14.8 sera in 30 presented a discordance with at least one other center). We concluded that there are inconsistencies among the laboratories that can potentially result in conflicting information regarding the patient's treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.
doi:10.1371/journal.pntd.0003174
PMCID: PMC4161321  PMID: 25211336
3.  Characterization of PbPga1, an Antigenic GPI-Protein in the Pathogenic Fungus Paracoccidioides brasiliensis 
PLoS ONE  2012;7(9):e44792.
Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), one of the most prevalent mycosis in Latin America. P. brasiliensis cell wall components interact with host cells and influence the pathogenesis of PCM. Cell wall components, such as glycosylphosphatidylinositol (GPI)-proteins play a critical role in cell adhesion and host tissue invasion. Although the importance of GPI-proteins in the pathogenesis of other medically important fungi is recognized, little is known about their function in P. brasiliensis cells and PCM pathogenesis. We cloned the PbPga1 gene that codifies for a predicted GPI-anchored glycoprotein from the dimorphic pathogenic fungus P. brasiliensis. PbPga1 is conserved in Eurotiomycetes fungi and encodes for a protein with potential glycosylation sites in a serine/threonine-rich region, a signal peptide and a putative glycosylphosphatidylinositol attachment signal sequence. Specific chicken anti-rPbPga1 antibody localized PbPga1 on the yeast cell surface at the septum between the mother cell and the bud with stronger staining of the bud. The exposure of murine peritoneal macrophages to rPbPga1 induces TNF-α release and nitric oxide (NO) production by macrophages. Furthermore, the presence of O-glycosylation sites was demonstrated by β-elimination under ammonium hydroxide treatment of rPbPga1. Finally, sera from PCM patients recognized rPbPga1 by Western blotting indicating the presence of specific antibodies against rPbPga1. In conclusion, our findings suggest that the PbPga1gene codifies for a cell surface glycoprotein, probably attached to a GPI-anchor, which may play a role in P. brasiliensis cell wall morphogenesis and infection. The induction of inflammatory mediators released by rPbPga1 and the reactivity of PCM patient sera toward rPbPga1 imply that the protein favors the innate mechanisms of defense and induces humoral immunity during P. brasiliensis infection.
doi:10.1371/journal.pone.0044792
PMCID: PMC3443090  PMID: 23024763
4.  Paracoccidioidomycosis Epidemiological Features of a 1,000-Cases Series from a Hyperendemic Area on the Southeast of Brazil 
Paracoccidioidomycosis has been known for over 100 years, and until now, there were only few estimates of the disease's incidence. We aim to analyze 1,000 cases treated between 1960 and 1999 at Ribeirão Preto city, São Paulo, Brazil, where the disease's incidence range detected was 1.6 to 3.7 cases per 100,000 habitants per year (mean = 2.7 cases/year). We observed a male to female ratio of 6:1 and an age distribution from 3 to 85 years. The acute/subacute form of the disease accounted for 25.4% of cases. Most of the patients (93.5%) had lived or worked in rural areas before the disease development. Smoking and alcoholism were reported by 64.7% and 37.2% of patients, respectively. Comorbidities identified included tuberculosis (8.3%), Chagas' disease (8.6%), and human immunodeficiency virus/acquired immunodeficiency syndrome (4.2%). The present study revealed an area in Brazil where paracoccidioidomycosis is hyperendemic (has the highest reported incidence of this disease); this endemic area is probably caused by geological and climatic conditions as well as intensive agriculture.
doi:10.4269/ajtmh.2011.11-0084
PMCID: PMC3163882  PMID: 21896820
5.  HIV Risk Behavior among Youth in the Dominican Republic: The Role of Alcohol and Other Drugs 
Existing literature related to HIV in the Dominican Republic has tended to neglect the unique role of tourism areas as distinct ecologies facilitative of sexual risk behavior, particularly HIV vulnerability and transmission. Furthermore, limited attention has focused on Dominican adolescents living in close proximity to tourism areas who have become increasingly exposed to alcohol due to the expanding tourism industry in the Dominican Republic. While most previous analyses of the effects of alcohol on adolescent sexual risk behavior have focused on the transient effects of alcohol on judgment and decision making, the effects of chronic alcohol use on sexual behavior has been a neglected area of research. Our study explores the relationship between chronic alcohol use, the parent–adolescent relationship, affective factors such as self-esteem, and intentions to engage in sex. We examine the above factors within the context of tourism areas which represent a unique ecology of alcohol availability and consumption and HIV risk. We discuss implications for developing applied family-based programs to target Dominican adolescent alcohol use and sexual risk behavior in tourism areas of high alcohol exposure.
doi:10.1177/1545109711419264
PMCID: PMC3319024  PMID: 21911848
adolescent risk behavior; alcohol; Dominican Republic; HIV; tourism
6.  Dissemination of blaKPC-2 by the Spread of Klebsiella pneumoniae Clonal Complex 258 Clones (ST258, ST11, ST437) and Plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae Species in Brazil ▿ 
This article reports the spread of blaKPC-2 in the Sao Paulo and Rio de Janeiro states, facilitated by globally spread K. pneumoniae clonal complex 258 (CC258) clones (ST258, ST11, and ST437) and a diversity of plasmids (IncFII, IncN, and IncL/M, two untypeable plasmids carrying Tn4401a or Tn4401b) successfully disseminated among species of the Enterobacteriaceae (Enterobacter cloacae, Serratia marcescens, and Citrobacter freundii). It also constitutes the first description of sequence type 258 (ST258) in Brazil, which was associated with a nosocomial hospital outbreak in Ribeirao Preto city.
doi:10.1128/AAC.01783-10
PMCID: PMC3122403  PMID: 21576442
7.  Development of an experimental model of infected bone void in the ulna of rabbits 
Acta Ortopedica Brasileira  2012;20(3):136-138.
Objective
Develop a model that allowed the study of bone regeneration in infection conditions.
Method
A 15 mm defect was surgically created in the rabbit ulna and inoculated with 5x108 colony-forming units (CFU) of S. aureus. Surgical debridement was performed two weeks after and systemic gentamicin was administered for four weeks. Animals were followed up to 12 weeks to evaluate infection control and bone regeneration.
Result
Bone regeneration was inferior to 25% of the defect in radiological and histological analysis.
Conclusion
Infected bone defect of 15 mm in the rabbit ulna was unable to achieve full regeneration without further treatment. Level of Evidence V, Experimental Study.
doi:10.1590/S1413-78522012000300002
PMCID: PMC3718429  PMID: 24453593
Bone diseases; Infectious; Bone regeneration; Rabbits
8.  Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis 
PLoS ONE  2011;6(6):e21423.
The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.
doi:10.1371/journal.pone.0021423
PMCID: PMC3120880  PMID: 21731741
9.  Detection and Selection of Microsatellites in the Genome of Paracoccidioides brasiliensis as Molecular Markers for Clinical and Epidemiological Studies 
Journal of Clinical Microbiology  2004;42(11):5007-5014.
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). Here, we describe the microsatellite patterns observed in a collection of P. brasiliensis random sequence tags. We identified 1,117 microsatellite patterns in about 3.8 Mb of unique sequences (0.47% of the total DNA used in the analysis). The majority of these microsatellites (87.5%) are found in noncoding sequences. We used two polymorphic microsatellites located on noncoding and coding sequences, as well as two microsatellites located on introns, as molecular markers to discriminate P. brasiliensis isolates, to look for relationships between the genetic background of the strains and the types of human disease they cause. We did not observe any correlation between the clinical form of human PCM and four simple sequence repeat patterns analyzed.
doi:10.1128/JCM.42.11.5007-5014.2004
PMCID: PMC525212  PMID: 15528688

Results 1-9 (9)