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Prospective population surveillance has been conducted for candidemia in Brazil (A. L. Colombo, M. Nucci, B. J. Park, et al., J. Clin. Microbiol. 44:2816-2823, 2006). In the present study, a total of 63 isolates from 61 patients, representing 11 medical centers from nine geographic regions, were characterized by multilocus sequence typing (MLST). A total of 48 unique profiles or diploid sequence types (DSTs) were observed, with nine new sequence types (STs) and 32 new DSTs. There were no apparent correlations between center/region and DST patterns. Subtypes were compared to those in a known characterized reference set, including a large database of strains obtained worldwide. Significantly, only one C. albicans group 2 isolate was found in our collection, although isolates from this particular group are commonly found worldwide. These data, combined with information from other previously reported studies, establish a statistically significant diminishment of group 2 strains in Central and South America, including Mexico and portions of the Southwestern United States.

Caenorhabditis elegans is a valuable tool as an infection model toward the study of Candida species. In this work, we endeavored to develop a C. elegans-Candida parapsilosis infection model by using the fungi as a food source. Three species of the C. parapsilosis complex (C. parapsilosis (sensu stricto), Candida orthopsilosis and Candida metapsilosis) caused infection resulting in C. elegans killing. All three strains that comprised the complex significantly diminished the nematode lifespan, indicating the virulence of the pathogens against the host. The infection process included invasion of the intestine and vulva which resulted in organ protrusion and hyphae formation. Importantly, hyphae formation at the vulva opening was not previously reported in C. elegans-Candida infections. Fungal infected worms in the liquid assay were susceptible to fluconazole and caspofungin and could be found to mount an immune response mediated through increased expression of cnc-4, cnc-7, and fipr-22/23. Overall, the C. elegans-C. parapsilosis infection model can be used to model C. parapsilosis host-pathogen interactions.

Background Cryptococcosis is the second most common cause of invasive fungal infections in renal transplant recipients in many countries, and data on graft outcome after treatment for this infection is lacking in less-resourced health care settings. Methods Data from 47 renal transplant recipients were retrospectively collected at a single institution during a period of 13 years. Graft dysfunction, graft loss, and mortality rates were evaluated. Predictors of mortality and graft loss were estimated. Results A total of 38 (97.4%) patients treated with amphotericin B deoxycholate (AMBd) showed graft dysfunction after antifungal initiation and 8 (18.2%) had kidney graft loss. Graft loss within 30 days after cryptococcosis onset was significantly associated with disseminated infection, greater baseline creatinine levels, and graft dysfunction concomitant to AMBd therapy and an additional nephrotoxic condition. The 30-day mortality rate was 19.2% and it was significantly associated with disseminated and pulmonary infections, somnolence at admission, high CSF opening pressure, positive CSF India ink, creatinine levels greater than 2.0 mg/dL at admission, graft dysfunction in patients treated with AMBd and an additional nephrotoxic condition and graft loss within 30 days. Conclusion Graft dysfunction was common in renal transplant recipients with cryptococcosis treated with AMBd. The rate of graft loss rate was high, most frequently in patients with concomitant nephrotoxic conditions. Therefore, the clinical focus should be on the use of less nephrotoxic lipid formulations of amphotericin B in this specific population requiring a polyene induction regimen for treatment of severe cryptococcosis in all health care systems caring for transplantation recipients.

The accuracy of antifungal susceptibility testing is important for reliable resistance surveillance and for the clinical management of patients with serious infections due to Candida spp. Our primary objective was to compare the results of fluconazole and voriconazole disk diffusion testing of 3227 Candida spp. performed by 47 centers participating in the ARTEMIS program with disk diffusion and MIC results obtained by the central reference laboratory. The overall categoric agreement between participant disk diffusion test results and reference MIC results was 87% for fluconazole and 95.2% for voriconazole. Likewise good agreement was observed between participant disk diffusion test results and reference laboratory disk diffusion test results, with an agreement of 90.5%, 1% very major error (VME), and 3.4% major error (ME) for fluconazole and 94.2%, 1.1% VME, and 2.5% ME for voriconazole. The disk diffusion test was reliable for detecting those isolates of Candida spp. that were characterized as resistant to fluconazole and voriconazole by MIC testing. External quality assurance data obtained by surveillance programs such as the ARTEMIS Global Antifungal Surveillance Program ensure the generation of useful surveillance data and result in the continued improvement of antifungal susceptibility testing protocols.

The reevaluation of the genus Trichosporon has led to the replacement of the old taxon Trichosporon beigelii by six new species. Sequencing of the ribosomal DNA (rDNA) intergenic spacer 1 (IGS1) is currently mandatory for accurate Trichosporon identification, but it is not usually performed in routine laboratories. Here we describe Trichosporon species distribution and prevalence of Trichosporon asahii genotypes based on rDNA IGS1 sequencing as well as antifungal susceptibility profiles of 22 isolates recovered from blood cultures. The clinical isolates were identified as follows: 15 T. asahii isolates, five Trichosporon asteroides isolates, one Trichosporon coremiiforme isolate, and one Trichosporon dermatis isolate. We found a great diversity of different species causing trichosporonemia, including a high frequency of isolation of T. asteroides from blood cultures that is lower than that of T. asahii only. Regarding T. asahii genotyping, we found that the majority of our isolates belonged to genotype 1 (86.7%). We report the first T. asahii isolate belonging to genotype 4 in South America. Almost 50% of all T. asahii isolates exhibited amphotericin B MICs of ≥2 μg/ml. Caspofungin MICs obtained for all the Trichosporon sp. isolates tested were consistently high (MICs ≥ 2 μg/ml). Most isolates (87%) had high MICs for 5-flucytosine, but all of them were susceptible to triazoles, markedly to voriconazole (all MICs ≤ 0.06 μg/ml).

Realizou-se um estudo observacional, prospectivo, de base laboratorial, para investigar a incidência de candidemia, distribuição de espécies e condições clínicas entre setembro 2003 e março 2004, em um hospital privado terciário em Recife, Nordeste do Brasil. Um caso de candidemia foi definido como isolamento de Candida spp de hemocultura. A taxa de incidência foi calculada por 1.000 admissões. Um total de 5.532 pacientes foram admitidos no hospital durante o período de estudo, e 1.745 culturas de sangue foram processadas. Foram observados 21 episódios de candidemia em 18 pacientes. A taxa de incidência de candidemia foi de 3,9 episódios por 1.000 admissões. Espécies não-albicans representaram mais de 50% dos casos, predominando Candida parapsilosis (33%) e Candida tropicalis (24%). Onze (61%) pacientes morreram. A incidência de candidemia foi mais alta que aquela observada em estudo multicêntrico brasileiro. Candidemia foi predominantemente causada por espécies não-albicans. We conducted a prospective, observational, laboratory-based study on candidemia to investigate the incidence of candidemia, species distribution and clinical conditions between September 2003 and March 2004 in a private tertiary hospital in Recife, northeastern Brazil. Cases of candidemia were defined as occurrences of isolation of Candida spp from blood cultures. The incidence rate was calculated per 1,000 admissions. A total of 5,532 patients were admitted to the hospital during the study period, and 1,745 blood cultures were processed. Twenty-one episodes of candidemia were observed in 18 patients. The incidence rate of candidemia was 3.9 episodes per 1,000 admissions. Non-albicans species accounted for more than 50% of the cases, and Candida parapsilosis (33%) and Candida tropicalis (24%) predominated. Eleven (61%) patients died. The incidence of candidemia was higher than that observed in a Brazilian multicenter study. Candidemia was caused predominantly by non-albicans species. Universidade Federal de Pernambuco Núcleo de Ensino, Pesquisa e Assistência em Infectologia Cerpe Diagnósticos Departamento de Microbiologia Universidade Federal de São Paulo (UNIFESP) Universidade Federal de São Paulo (UNIFESP) Hospital Universitário Clementino Fraga Filho Laboratório de Micologia Universidade Federal de Pernambuco Departamento de Estatística UNIFESP, Hospital Universitário Clementino Fraga Filho Laboratório de Micologia SciELO

The paradoxical growth (PG) of Candida sp. biofilms in the presence of high caspofungin (CAS) concentrations was previously unknown. We sought to characterize the PG at supra-MICs of CAS among clinical Candida sp. isolates grown as biofilms in 96-well polystyrene microtiter plates. The MICs of CAS were determined for 30 clinical Candida sp. isolates (4 Candida albicans , 6 C. tropicalis , 7 C. parapsilosis , 8 C. orthopsilosis , and 5 C. metapsilosis isolates) when they were grown as planktonic cells and biofilms and were defined as the lowest drug concentrations that resulted in a prominent decrease in growth and a 50% reduction in metabolic activity, respectively. PG was defined as a resurgence of growth (>50% of that in the drug-free growth control well) at drug concentrations above the MIC. With the exception of C. tropicalis , all isolates displayed PG more frequently when they were grown as biofilms than when they grown as planktonic cells. PG was undetectable among C. metapsilosis isolates in planktonic cell MIC tests but was present in 100% of the isolates in biofilm MIC tests. The drug concentration and the number of drug dilutions supporting PG were higher for biofilms than for planktonic cells. Microscopic changes in cell morphology were observed among both planktonic and biofilm cells with PG. Specifically, the accumulation of enlarged, globose cells was associated with PG, and we hypothesize that CAS-induced changes in the cell wall composition may be the explanation.

Fluconazole in vitro susceptibility test results for 205,329 yeasts were collected from 134 study sites in 40 countries from June 1997 through December 2005. Data were collected for 147,776 yeast isolates tested with voriconazole from 2001 through 2005. All investigators tested clinical yeast isolates by the CLSI M44-A disk diffusion method. Test plates were automatically read and results recorded with a BIOMIC image analysis system. Species, drug, zone diameter, susceptibility category, and quality control results were collected quarterly. Duplicate (same patient, same species, and same susceptible-resistant biotype profile during any 7-day period) and uncontrolled test results were not analyzed. Overall, 90.1% of all Candida isolates tested were susceptible (S) to fluconazole; however, 10 of the 22 species identified exhibited decreased susceptibility (C. glabrata and C. krusei . Among 137,487 isolates of Candida spp. tested against voriconazole, 94.8% were S and 3.1% were R. Less than 30% of fluconazole-resistant isolates of C. albicans , C. glabrata , C. tropicalis , and C. rugosa remained S to voriconazole. The non- Candida yeasts (8,821 isolates) were generally less susceptible to fluconazole than Candida spp. but, aside from Rhodotorula spp., remained susceptible to voriconazole. This survey demonstrates the broad spectrum of these azoles against the most common opportunistic yeast pathogens but identifies several less common yeast species with decreased susceptibility to antifungal agents. These organisms may pose a future threat to optimal antifungal therapy and emphasize the importance of prompt and accurate species identification.

Candida tropicalis is the 2nd most frequent agent of candidemia in Brazil (20-24%). We attempted to characterize the epidemiology, microbiology, and outcome of candidemia due to C. tropicalis by comparing patients with candidemia due to C. tropicalis with those with candidemia due to Candida albicans. Among the 924 episodes of candidemia, 188 (20%) were caused by C. tropicalis. These cases were compared with 384 candidemias due to C. albicans. C. tropicalis was the 2nd most frequent species in adults (21.6%) and elderly patients (23.2%), and 3rd in neonates (11.9%) and children (18.5%). Cancer was the most frequent underlying disease, and in adults and elderly patients, diabetes was the 2nd most frequent underlying disease. The only difference between C. tropicalis and C. albicans candidemia was a higher proportion of neutropenic patients in C. tropicalis candidemia. C. tropicalis is a leading cause of candidemia in Brazil, and its epidemiology is similar to that of C. albicans.

Fungal infections due to Candida species represent an important cause of nosocomial bloodstream infections. We report a large pseudo-outbreak of Candida guilliermondii fungemia that occurred in a university hospital in Brazil. C. guilliermondii was identified in 64 (43%) of the 149 blood samples drawn between June 2003 and July 2004. The samples were from patients in different wards of the hospital but concentrated in pediatric units. None of the patients had clinical signs of fungemia, and observational analysis revealed errors in the collection of blood samples. During the investigation of the pseudo-outbreak, C. guilliermondii was isolated from environmental surfaces and from the skin and nails of members of the nursing team. Through a subtyping analysis it was found that some of the nonpatient isolates were highly related to the patient isolates, and all the patient isolates were highly related. This is consistent with the hypothesis that the pseudo-outbreak was from a limited number of common sources. The adoption of intervention measures was effective in resolving the outbreak, supporting the hypothesis that the outbreak was due to poor techniques of drawing blood samples for culture.

The significant increase in the use of antifungal agents, both for the treatment of candidiasis and invasive aspergillosis and as azole fungicides in agricultural crop protection has resulted in the emergence of resistant clinical isolates, particularly to triazoles and echinocandins. Notably, among isolates that were primarily sensitive to fluconazole such as Candida parapsilosis and Candida tropicalis have witnessed an emerging resistance development. Also for echinocandins, the occurrence of Candida isolates with lower susceptibility to these drugs has been reported, which is possibly due to its broad clinical use. Triazole resistance among Aspergillus fumigatus and other Aspergillus species is commonly found in European and Asian countries. Specific mutations are associated with azole resistance in A. fumigatus and these mutations are now reported globally from six continents. Therefore, we highlight the need to conduct antifungal resistance surveillance studies using clinical isolates of Candida and Aspergillus in different geographical regions and monitoring of the infection rates in distinct population groups for early detection of resistance to these drugs and implementation of efficient policies for infection control and treatment.

Intraabdominal candidiasis (IAC) is a condition associated with a high morbidity and mortality in the Intensive Care Unit (ICU) [1]. Actual guidelines are centered on candidemia [2] and there are uncertainties in the diagnostic criteria for IAC, thus contributing to the scarcity of information.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens., This study was supported by an National Health and Medical Research Council of Australia (NH&MRC) grant [#APP1031952] to W Meyer, S Chen, V Robert, and D Ellis; CNPq [350338/2000-0] and FAPERJ [E-26/103.157/2011] grants to RM Zancope-Oliveira; CNPq [308011/2010-4] and FAPESP [2007/08575-1] Fundacao de Amparo Pesquisa do Estado de So Paulo (FAPESP) grants to AL Colombo; PEst-OE/BIA/UI4050/2014 from Fundacao para a Ciencia e Tecnologia (FCT) to C Pais; the Belgian Science Policy Office (Belspo) to BCCM/IHEM; the MEXBOL program of CONACyT-Mexico, [ref. number: 1228961 to ML Taylor and [122481] to C Toriello; the Institut Pasteur and Institut de Veil le Sanitaire to F Dromer and D Garcia-Hermoso; and the grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and the Fundacao de Amparo a Pesquisa do Estado de Goias (FAPEG) to CM de Almeida Soares and JA Parente Rocha. I Arthur would like to thank G Cherian, A Higgins and the staff of the Molecular Diagnostics Laboratory, Division of Microbiology and Infectious Diseases, Path West, QEII Medial Centre. Dromer would like to thank for the technical help of the sequencing facility and specifically that of I, Diancourt, A-S Delannoy-Vieillard, J-M Thiberge (Genotyping of Pathogens and Public Health, Institut Pasteur). RM Zancope-Oliveira would like to thank the Genomic/DNA Sequencing Platform at Fundacao Oswaldo Cruz-PDTIS/FIOCRUZ [RPT01A], Brazil for the sequencing. B Robbertse and CL Schoch acknowledge support from the Intramural Research Program of the NIH, National Library of Medicine. T Sorrell's work is funded by the NH&MRC of Australia; she is a Sydney Medical School Foundation Fellow., info:eu-repo/semantics/publishedVersion

Purpose: Clinical data on patients with intra-abdominal candidiasis (IAC) is still scarce. Methods: We collected data from 13 hospitals in Italy, Spain, Brazil, and Greece over a 3-year period (2011–2013) including patients from ICU, medical, and surgical wards. Results: A total of 481 patients were included in the study. Of these, 27 % were hospitalized in ICU. Mean age was 63 years and 57 % of patients were male. IAC mainly consisted of secondary peritonitis (41 %) and abdominal abscesses (30 %); 68 (14 %) cases were also candidemic and 331 (69 %) hadconcomitant bacterial infections. The most commonly isolated Candida species were C. albicans (n = 308 isolates, 64 %) and C. glabrata (n = 76, 16 %). Antifungal treatment included echinocandins (64 %), azoles (32 %), and amphotericin B (4 %). Septic shock was documented in 40.5 % of patients. Overall 30-day hospital mortality was 27 % with 38.9 % mortality in ICU. Multivariate logistic regression showed that age (OR 1.05, 95 % CI 1.03–1.07, P\0.001), increments in 1-point APACHE II scores (OR 1.05, 95 % CI 1.01–1.08, P = 0.028), secondary peritonitis (OR 1.72, 95 % CI 1.02–2.89, P = 0.019), septic shock (OR 3.29, 95 % CI 1.88–5.86, P\0.001), and absence of adequate abdominal source control (OR 3.35, 95 % CI 2.01–5.63, P\0.001) wereassociated with mortality. In patients with septic shock, absence of source control correlated with mortality rates above 60 % irrespective of administration of an adequate antifungal therapy. Conclusions: Low percentages of concomitant candidemia and high mortality rates are documented in IAC. In patients presenting with septic shock, source control is fundamental.

Candidemia studies have documented geographic differences in rates and epidemiology, underscoring the need for surveillance to monitor trends. We conducted prospective candidemia surveillance in Brazil to assess the incidence, species distribution, frequency of antifungal resistance, and risk factors for fluconazole-resistant Candida species. Prospective laboratory-based surveillance was conducted from March 2003 to December 2004 in 11 medical centers located in 9 major Brazilian cities. A case of candidemia was defined as the isolation of Candida spp. from a blood culture. Incidence rates were calculated per 1,000 admissions and 1,000 patient-days. Antifungal susceptibility tests were performed by using the broth microdilution assay, according to the Clinical and Laboratory Standards Institute guidelines. We detected 712 cases, for an overall incidence of 2.49 cases per 1,000 admissions and 0.37 cases per 1,000 patient-days. The 30-day crude mortality was 54%. C. albicans was the most common species (40.9%), followed by C. tropicalis (20.9%) and C. parapsilosis (20.5%). Overall, decreased susceptibility to fluconazole occurred in 33 (5%) of incident isolates, 6 (1%) of which were resistant. There was a linear correlation between fluconazole and voriconazole MICs ( r = 0.54 and P < 0.001 [Spearman's rho]). This is the largest multicenter candidemia study conducted in Latin America and shows the substantial morbidity and mortality of candidemia in Brazil. Antifungal resistance was rare, but correlation between fluconazole and voriconazole MICs suggests cross-resistance may occur.

Neuroparacoccidioidomycosis (neuroPCM) is the central nervous system infection by the fungus Paracoccidioides brasiliensis . Its diagnosis is a difficult task that depends on neuroimaging techniques such as computed tomography and magnetic resonance imaging. However, the detection of circulating P. brasiliensis antigens in body fluids by inhibition enzyme-linked immunosorbent assay (inh-ELISA) has provided encouraging results. In this study, 14 cerebrospinal fluid (CSF) and 11 serum samples of patients with neuroPCM were analyzed by inh-ELISA for detection of circulating glycoprotein antigens of 43 kDa (gp43) and 70 kDa (gp70). Circulating gp43 and gp70 antigens were detected in all CSF samples from patients with neuroPCM at mean concentrations of 19.3 and 6.8 μg/ml, respectively. In addition, both gp43 and gp70 antigens were detected in 10 of 11 serum samples analyzed at mean concentrations of 4.6 and 4.0 μg/ml, respectively. By immunodiffusion test, CSF samples were determined to be negative in 13 of 14 samples. The detection of anti-gp43 and anti-gp70 antibodies by conventional ELISA showed positive results for all CSF samples, with titers ranging from 1:50 to 1:51,200. Therefore, the high sensitivity of the inh-ELISA technique in detecting gp43 and gp70 antigens in the CSF of neuroPCM patients strongly indicates that this assay can be considered as a powerful diagnostic tool. In addition, the finding of anti-gp43 and anti-gp70 antibodies in CSF samples by conventional ELISA also seems to be a promising diagnostic method for this special modality of PCM.

Objective:To report an outbreak ofPichia anomalafungemia that occurred in a Brazilian pediatric intensive care unit (ICU) from October 2002 to January 2004.Design:Unmatched case-control study.Methods:We randomly selected four control-patients for each case-patient from a list of all patients admitted to the ICU for at least 48 hours during the outbreak. A second control group was composed of all consecutive patients with nosocomial candidemia in the ICU during the outbreak. An environmental study was performed, and genetic relatedness among the clinical isolates was characterized by randomly amplified polymorphic DNA assay.Results:During the study period, 1,046 children were admitted to the pediatric ICU, 17 of whom developedP. anomalafungemia (attack rate, 1.6%). The median age was 1.1 years, and the main underlying conditions were congenital malformations (35.3%) and neoplastic diseases (11.8%). The overall mortality rate was 41.2%. Two patients received no antifungal treatment; all of the others were treated with amphotericin B. On multivariate analysis, only the presence of a central venous catheter was significantly associated withP. anomalafungemia. The yeast was not found on healthcare workers' hands or in the environment. Molecular studies showed that the outbreak was caused by a single strain. The distribution of risk factors was similar between patients withP. anomalafungemia and control-patients with candidemia.Conclusions:This study highlights the importance ofP. anomalaas an emerging nosocomial fungal pathogen. Patients withP. anomalafungemia seem to have risk factors in common with those who have candidemia.

The continuous use of triazoles can result in the development of drug resistance. Azole-resistant clinical isolates, spontaneous and induced mutants of Aspergillus fumigatus have been documented. The azoles block the ergosterol biosynthesis pathway by inhibiting the enzyme 14-alpha-demethylase, product of the CYP51. Fungal azole resistance involves both amino acid changes in the target site that alter drug-target interactions and those that decrease net azole accumulation. The reduced intracellular accumulation has also been correlated with overexpression of multidrug resistance (MDR) efflux transporter genes of the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) classes. About 20 genes are involved in the A. fumigatus ergosterol biosynthesis pathway. There are several duplicated genes in this pathway. Interestingly, erg3 and erg11 showed two copies in A. fumigatus. In general, Aspergillus spp. have proportionally more MFS transporter encoding genes than Saccharomyces cerevisiae, S. pombe, and Neurospora crassa. The drug H+ (12 and 14 spanners) sub-families are also proportionally greater than in the other species. Although the numbers of ABC transporter encoding genes are comparable, again the Aspergillus spp. have more ABC transporters related to multidrug permease than the other fungal species.

In a retrospective study performed over 6 years in Brazil, Fusarium solani was found to be the most common species causing mycotic keratitis. the genetic diversity of 44 isolates from 39 patients was assessed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) fingerprinting. ERIC-PCR was more discriminatory than PCR-RFLP for differentiating the strains. By combining of the results of both techniques, we identified 40 genotypes. Molecular typing revealed a high genomic heterogeneity of the strains of F. solani studied. Univ Rovira & Virgili, Fac Med & Ciencies Salut, Dept Ciencies Med Bas, Unitat Microbiol, Tarragona 43201, Spain Universidade Federal de São Paulo, DIPA, São Paulo, Brazil Universidade Federal de São Paulo, Dept Oftalmol, São Paulo, Brazil Universidade Federal de São Paulo, DIPA, São Paulo, Brazil Universidade Federal de São Paulo, Dept Oftalmol, São Paulo, Brazil Web of Science

In this study, we describe resistance mechanisms in fluconazole-resistant isolates of C. albicans isolated from AIDS patients from nine Brazilian hospitals. These mechanisms include the presence of point mutations in the ERG11 gene and overexpression of ERG11, and several genes encoding efflux pumps, as measured by quantitative real-time reverse transcriptase polymerase chain reaction. Several fluconazole-resistant strains had multiple mechanisms of resistance. Four mutations previously described, Y132F, K143R, E266D, and V437I, were identified among the strains, whereas some isolates contained more than one mutation. Fourteen novel mutations were identified. Interestingly, all Brazilian fluconazole-resistant isolates showed homozygosity at mating-type loci (MTL) associated with fluconazole resistance. This is the first comprehensive assessment at molecular level of mechanisms of fluconazole resistance in C. albicans isolates from South America.

Objective: Amphotericin B failure is frequently seen in patients with candidaemia caused by Candida rugosa. We evaluated amphotericin 13, fluconazole, posaconazole and voriconazole as alternative treatments against infection in mice with two isolates of C. rugosa.Methods: Neutropenic mice were inoculated intravenously with C. rugosa. Amphotericin 13, fluconazole, posaconazole and voriconazole were administered for 7 days after infection. Efficacy of the antifungal treatment was assessed by survival and tissue burden of C. rugosa.Results: All of the four drugs significantly prolonged survival over controls. With both isolates, kidney counts were reduced significantly below controls for amphotericin B, fluconazole and posaconazole. However, voriconazole was less effective than the other antifungals.Conclusion: Despite poor clinical response to amphotericin B, in vivo data indicate that amphotericin B increases organ clearance and survival over untreated controls. However, although voriconazole improved survival over controls, increased tissue clearance was not seen. This discrepancy may be caused by rapid clearance of voriconazole in mice. These studies suggest fluconazole, posaconazole or voriconazole may be useful alternatives to amphotericin B in therapy of C. rugosa infection. Univ Texas, Hlth Sci Ctr, Dept Med, Div Infect Dis, San Antonio, TX 78229 USA Univ Texas, Hlth Sci Ctr, Dept Pathol, San Antonio, TX 78229 USA Univ Autonoma Nuevo Leon, Fac Med, Dept Microbiol, Monterrey, Mexico Universidade Federal de São Paulo, Div Infect Dis, São Paulo, Brazil Universidade Federal de São Paulo, Div Infect Dis, São Paulo, Brazil Web of Science

Paracoccidioidomycosis (PCM) is a systemic fungal disease that is particularly important among individuals living and working in rural areas of endemicity in Latin America. Detection of anti- Paracoccidioides brasiliensis antibodies is of limited value due to false-negative results. Detection of P. brasiliensis -gp43 circulating antigen is a practical approach for a specific diagnosis of the disease. In a previous study we described an inhibition enzyme-linked immunosorbent assay able to detect the 43-kDa P. brasiliensis antigen in sera of 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM. To investigate its potential application for the follow-up of PCM patients during treatment, antigen levels were monitored at regular intervals for up 8 to 12 months in serum samples from 23 patients. The results showed that treatment with itraconazole resulted in decreasing levels of circulating gp43 that were correlated with the reduction of anti-gp43 antibodies. It was also observed that by the end of 12 months of treatment gp43 levels were

PURPOSE: To report the antifungal susceptibility profile of yeast isolates obtained from cases of keratitis. METHODS: Susceptibility testing of 15 yeast strains isolated from corneal infections to amphotericin B, fluconazole, itraconazole and ketoconazole was performed using the NCCLS broth microdilution assay. RESULTS: Most episodes of eye infections were caused by Candida albicans. The antifungal drugs tested showed the following minimal inhibitory concentration values against yeast isolates: 0.125-0.5 µg/ml for amphotericin B; 0.125->64.0 µg/ml for fluconazole; 0.015-1.0 µg/ml for itraconazole and 0.015-0.125 µg/ml for ketoconazole. Despite the fact that all Candida isolates were judged to be susceptible to azoles, one isolate showed a minimal inhibitory concentration value significantly higher than a 90% minimal inhibitory concentration of all tested isolates. Rhodotorula rubra was resistant to fluconazole and itraconazole. CONCLUSIONS: Despite the fact that most yeast isolates from corneal infections are usually susceptible to amphotericin B and azoles, they exhibit a wide range of minimal inhibitory concentration values for antifungal drugs. The identification of strains at species level and their susceptibility pattern to antifungal drugs should be considered before determining the concentration to be used in topical antifungal formulations in order to optimize therapeutic response in eye infections.OBJETIVO: Relatar resultados e avaliar a aplicabilidade do teste de suscetibilidade a antifúngicos de leveduras isoladas de infecções corneais oculares. MÉTODOS: Realizou-se teste de suscetibilidade pelo método de microdiluição em caldo, padronizado pelo NCCLS-EUA, em 15 amostras de leveduras de infecções corneanas a anfotericina B, fluconazol, itraconazol e ketoconazol. RESULTADOS: A maioria dos episódios de infecção corneal foi causada por Candida albicans. As drogas antifúngicas testadas exibiram valores de concentração inibitória mínima (CIM) para as leveduras isoladas: 0,125-0,5 µg/ml para anfotericina B; 0,125->64,0 µg/ml para fluconazol; 0,015-1,0 µg/ml para itraconazol e 0,015-0,125 µg/ml para ketoconazol. Apesar de todas as amostras terem sido suscetíveis aos azólicos, uma exibiu valor de concentração inibitória mínima significantemente maior do que a concentração inibitória mínima 90% de todas as amostras testadas. Rhodotorula rubra foi resistente a fluconazol e itraconazol. CONCLUSÃO: Apesar da maioria das leveduras isoladas de infecções corneais serem usualmente suscetíveis a anfotericina B e azólicos, observa-se ampla variação de valores de concentração inibitória mínima obtidas com as diferentes drogas antifúngicas testadas. A identificação das cepas e a determinação do padrão de suscetibilidade devem ser consideradas antes da determinação da concentração das drogas a ser empregada em formulações antifúngicas tópicas com o objetivo de otimizar a resposta terapêutica às infecções oculares.

We investigated the presence of Candida dubliniensis among isolates previously identified as Candida albicans and maintained in a yeast stock collection from 1994 to 2000. All isolates were serotyped and further evaluated for antifungal susceptibility profile. After doing a screening test for C. dubliniensis isolates based on the capability of colonies to grow at 42°C, its final identification was obtained by randomly amplified polymorphic DNA (RAPD) analysis using three different primers. A total of 46 out of 548 screened isolates did not exhibit growth at 42°C and were further genotyped by RAPD. Eleven isolates were identified as C. dubliniensis with RAPD analysis. Regarding serotypes, 81.5% of C. albicans and all C. dubliniensis isolates belonged to serotype A. Of note, 9 out of 11 C. dubliniensis isolates were obtained from patients with acquired immunodeficiency syndrome (Aids) and all of them were susceptible to azoles and amphotericin B. We found 17 (3%) C. albicans isolates that were dose-dependent susceptibility or resistant to azoles. In conclusion, we found a low rate of C. dubliniensis isolates among stock cultures of yeasts previously identified as C. albicans. Most of these isolates were recovered from oral samples of Aids patients and exhibited high susceptibility to amphotericin B and azoles. C. albicans serotype A susceptible to all antifungal drugs is the major phenotype found in our stock culture.

Opportunistic infections, which affect acquired immunodeficiency syndrome (Aids) patients, are frequently disseminated and may cause bloodstream infections (BSI). The aim of this study was to evaluate the main causes of BSI in Aids patients with advanced stage of the disease, with special emphasis on the identification of fungemia. During a 21 months period, all patients with Aids (CD4 < 200) and febrile syndrome admitted to 3 university hospitals were systematically evaluated. For each patient presenting fever, a pair of blood cultures was collected and processed by using a commercial lysis-centrifugation system. One hundred and eleven patients (75 males) with a mean age of 36 years (median 33 years) and mean CD4 count of 64 cells/ml were included. Among the 111 patients evaluated we documented 54 episodes of BSI, including 46 patients with truly systemic infections and 8 episodes considered as contaminants. BSI were caused by gram-positive bacteria (43%), fungi (20%), gram-negative bacteria (15%), mycobacteria (15%), and mixed flora (7%). The crude mortality rate of our patients was 39%, being 50% for patients with BSI and 31% for the others. In conclusion, BSI are a common related to systemic infections on Aids patients with advanced stage of disease and is associated with a high rate of mortality. Universidade Federal de São Paulo (UNIFESP) Hospital São Paulo Hospital e Maternidade Santa Marcelina Hospital Heliópolis UNIFESP, Hospital São Paulo SciELO

From March 1999 to March 2000, we conducted a prospective multicenter study of candidemia involving five tertiary care hospitals from four countries in Latin America. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to the National Committee for Clinical Laboratory Standards microbroth assay method. During a 12 month-period we were able to collect a total of 103 bloodstream isolates of Candida spp. C. albicans was the most frequently isolated species accounting for 42% of all isolates. Non-albicans Candida species strains accounted for 58% of all episodes of candidemia and were mostly represented by C. tropicalis (24.2%) and C. parapsilosis (21.3%). It is noteworthy that we were able to identify two cases of C. lusitaniae from different institutions. In our casuistic, non-albicans Candida species isolates related to candidemic episodes were susceptible to fluconazole. Continuously surveillance programs are needed in order to identify possible changes in the species distribution and antifungal susceptibility patterns of yeasts that may occurs after increasing the use of azoles in Latin American hospitals.

We evaluated the antifungal susceptibility profile of 200 recent bloodstream isolates of Candida spp. sequentially obtained from patients admitted to five tertiary care hospitals in Brazil. Isolates were identified by classical methods and the antifungal susceptibility profile was determined by the NCCLS microbroth assay method. Candida albicans was the most frequent species (41.5%), followed by C. tropicalis (24%) and C. parapsilosis (20.5%). The frequency of C. glabrata and C. krusei was low (nine and two isolates, respectively). Only three strains were resistant to fluconazole (two C. krusei and one C. glabrata) and only one was resistant to itraconazole (the same C. glabrata strain that was resistant to fluconazole). Two strains were considered susceptible dose-dependent (SDD) to fluconazole and 13 isolates (6.5%) were SDD to itraconazole. Overall, the MIC50 value of non-C. albicans isolates for fluconazole was two dilutions higher than that of C. albicans isolates, and for itraconazole was one diluti...

Fungal infection is one of the most important causes of morbidity and mortality in bone marrow transplant (BMT) recipients. The growing incidence of these infections is related to several factors including prolonged granulocytopenia, use of broad-spectrum antibiotics, conditioning regimens, and use of immunosuppression to avoid graft-versus-host disease (GvHD). In the present series, we report five cases of invasive mold infections documented among 64 BMT recipients undergoing fluconazole antifungal prophylaxis: 1) A strain of Scedosporium prolificans was isolated from a skin lesion that developed on day +72 after BMT in a chronic myeloid leukemic patient. 2) Invasive pulmonary aspergillosis (Aspergillus fumigatus) was diagnosed on day +29 in a patient with a long period of hospitalization before being transplanted for severe aplastic anemia. 3) A tumoral lung lesion due to Rhizopus arrhizus (zygomycosis) was observed in a transplanted patient who presented severe chronic GvHD. 4) A tumoral lesion due to Aspergillus spp involving the 7th, 8th and 9th right ribs and local soft tissue was diagnosed in a BMT patient on day +110. 5) A patient with a history of Ph1-positive acute lymphocytic leukemia exhibited a cerebral lesion on day +477 after receiving a BMT during an episode of severe chronic GvHD. At that time, blood and spinal fluid cultures yielded Fusarium sp. Opportunistic infections due to fungi other than Candida spp are becoming a major problem among BMT patients receiving systemic antifungal prophylaxis with fluconazole.

Supplemental digital content is available in the text., Background Invasive fungal infections remain a leading cause of morbidity and mortality among liver transplant recipients (LTRs). In this patient population, invasive Candida infections (ICIs) account for the large majority of cases. To date, only small studies and case-series analysing clinical presentation and risk factors for mortality in LTRs with ICIs are available. Methods We performed a retrospective multicenter multinational study in 10 centers in Europe and Brazil. All consecutive LTRs developing ICIs during the period January 2011 to December 2013 were included in the study. Results A total of 42 LTRs were included. Median age was 52.5 years, and 78.6% of patients were men. Viral hepatitis was the most common cause for liver transplantation (42.9%). Candidemia represented the majority of cases (24, 57.1%), followed by intra-abdominal candidiasis (18, 42.9%). Overall 30-day mortality was 23.8%, with higher mortality in patients with candidemia compared with intra-abdominal candidiasis (37.5% vs 5.6%, P = 0.02). Multivariate analysis showed candidemia to be a risk factor associated with mortality among LTRs presenting ICIs (odds ratio, 11.86; 95% confidence interval, 1.5-280; P = 0.01). Candida albicans represented the most common isolate (59.5%). High rates of antifungal resistances were found, with 16.7% and 4.8% of isolates displaying resistance to azoles and caspofungin, respectively. Conclusions Our study confirms the occurrence of high mortality rates in LTRs developing ICIs. Mortality rates varied according to the type of infection, with candidemia representing a risk factor for mortality. The high rates of antifungal resistance should be considered in the choice of the empiric antifungal regimen.

Clinical and Laboratory Standards Institute (CLSI) conditions for testing the susceptibilities of pathogenicSporothrixspecies to antifungal agents are based on a collaborative study that evaluated five clinically relevant isolates ofSporothrixschenckii sensu latoand some antifungal agents. With the advent of molecular identification, there are two basic needs: to confirm the suitability of these testing conditions for all agents andSporothrixspecies and to establish species-specific epidemiologic cutoff values (ECVs) or breakpoints (BPs) for the species. We collected available CLSI MICs/minimal effective concentrations (MECs) of amphotericin B, five triazoles, terbinafine, flucytosine, and caspofungin for 301Sporothrix schenckii sensu stricto, 486S. brasiliensis, 75S. globosa, and 13S. mexicanamolecularly identified isolates. Data were obtained in 17 independent laboratories (Australia, Europe, India, South Africa, and South and North America) using conidial inoculum suspensions and 48 to 72 h of incubation at 35°C. Sufficient and suitable data (modal MICs within 2-fold concentrations) allowed the proposal of the following ECVs forS. schenckiiandS. brasiliensis, respectively: amphotericin B, 4 and 4 μg/ml; itraconazole, 2 and 2 μg/ml; posaconazole, 2 and 2 μg/ml; and voriconazole, 64 and 32 μg/ml. Ketoconazole and terbinafine ECVs forS. brasiliensiswere 2 and 0.12 μg/ml, respectively. Insufficient or unsuitable data precluded the calculation of ketoconazole and terbinafine (or any other antifungal agent) ECVs forS. schenckii, as well as ECVs forS. globosaandS. mexicana. These ECVs could aid the clinician in identifying potentially resistant isolates (non-wild type) less likely to respond to therapy.

Background Risk factors for invasive fusariosis (IF) have not been characterized. We attempted to identify risk factors for IF in a prospective cohort of hematologic patients treated in 8 centers in Brazil. Methods Patients with (cases) and without (controls) proven or probable IF diagnosed in a cohort of patients with acute myeloid leukemia (AML) or myelodysplasia (MDS), and in allogeneic hematopoietic cell transplant (HCT) recipients (early, until day 40; late, after day 40 posttransplant) were compared by univariate Cox regression analysis. Results Among 237 induction remission courses of AML/MDS and 663 HCTs (345 allogeneic and 318 autologous), 25 cases of IF were diagnosed. In the AML/MDS cohort, active smoking (hazard ratio [HR], 9.11 [95% confidence interval {CI}, 2.04-40.71]) was associated with IF. Variables associated with IF in the early phase of allogeneic HCT were receipt of antithymocyte globulin (HR, 22.77 [95% CI, 4.85-101.34]), hyperglycemia (HR, 5.17 [95% CI, 1.40-19.11]), center 7 (HR, 5.15 [95% CI, 1.66-15.97]), and AML (HR, 4.38 [95% CI, 1.39-13.81]), and in the late phase were nonmyeloablative conditioning regimen (HR, 35.08 [95% CI, 3.90-315.27]), grade III/IV graft-vs-host disease (HR, 16.50 [95% CI, 2.67-102.28]), and previous invasive mold disease (HR, 10.65 [95% CI, 1.19-95.39]). Conclusions Attempts to reduce the risk of IF may include smoking cessation, aggressive control of hyperglycemia, and the use of a mold-active agent as prophylaxis in patients receiving nonmyeloablative HCT or ATG in the conditioning regimen. Future research should further explore smoking and other prehospital variables as risks for IF.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundacao de Amparo a Pesquisa do Estado de Mato Grosso (FAPEMAT) Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. the genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole. Universidade Federal de São Paulo, Lab Especial Micol, Disciplina Infectol, São Paulo, Brazil Univ Fed Alfenas, Inst Ciencias Biomed, Dept Microbiol & Immunol, Alfenas, MG, Brazil Univ Fed Mato Grosso, Fac Med, Microbiol Lab, Cuiaba, MT, Brazil Universidade Federal de São Paulo, Lab Especial Micol, Disciplina Infectol, São Paulo, Brazil FAPESP: 2007/08575-1 FAPESP: 2010/17179-5 CAPES: PNPD 02640-09-0 Web of Science

Aspergillus spp. are among the most common causes of opportunistic invasive fungal infections in tertiary care hospitals. Little is known about the prevalence and in vitro susceptibility of Aspergillus species in Latin America, because there are few medical centers able to perform accurate identification at the species level. The purpose of this study was to analyze the distribution of cryptic and rare Aspergillus species among clinical samples from 133 patients with suspected aspergillosis admitted in 12 medical centers in Brazil and to analyze the in vitro activity of different antifungal drugs. The identification of Aspergillus species was performed based on a polyphasic approach, as well as sequencing analysis of the internal transcribed spacer (ITS) region, calmodulin, and β-tubulin genes and phylogenetic analysis when necessary. The in vitro susceptibility tests with voriconazole, posaconazole, and itraconazole were performed according to the CLSI M38-A2 document (2008). We demonstrated a high prevalence of cryptic species causing human infection. Only three isolates, representing the species Aspergillus thermomutatus , A. ochraceus , and A. calidoustus , showed less in vitro susceptibility to at least one of the triazoles tested. Accurate identifications of Aspergillus at the species level and with in vitro susceptibility tests are important because some species may present unique resistance patterns against specific antifungal drugs.

BACKGROUND: Sporotrichosis is a chronic subcutaneous mycosis of humans and animals, which is typically acquired by traumatic inoculation of plant material contaminated with Sporothrix propagules, or via animals, mainly felines. Sporothrix infections notably occur in outbreaks, with large epidemics currently taking place in southeastern Brazil and northeastern China. Pathogenic species include Sporothrix brasiliensis, Sporothrix schenckii s. str., Sporothrix globosa, and Sporothrix luriei, which exhibit differing geographical distribution, virulence, and resistance to antifungals. The phylogenetically remote species Sporothrix mexicana also shows a mild pathogenic potential. METHODS: We assessed a genetically diverse panel of 68 strains. Susceptibility profiles of medically important Sporothrix species were evaluated by measuring the MICs and MFCs for amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), posaconazole (PCZ), flucytosine (5FC), and caspofungin (CAS). Haplotype networks were constructed to reveal interspecific divergences within clinical Sporothrix species to evaluate genetically deviant isolates. RESULTS: ITC and PCZ were moderately effective against S. brasiliensis (MIC90 = 2 and 2 μg/mL, respectively) and S. schenckii (MIC90 = 4 and 2 μg/mL, respectively). PCZ also showed low MICs against the rare species S. mexicana. 5FC, CAS, and FLC showed no antifungal activity against any Sporothrix species. The minimum fungicidal concentration ranged from 2 to >16 μg/mL for AMB against S. brasiliensis and S. schenckii, while the MFC90 was >16 μg/mL for ITC, VRC, and PCZ. CONCLUSION: Sporothrix species in general showed high degrees of resistance against antifungals. Evaluating a genetically diverse panel of strains revealed evidence of multidrug resistant phenotypes, underlining the need for molecular identification of etiologic agents to predict therapeutic outcome.

During the past decade, studies on the composition of human microbiota and its relation to the host became one of the most explored subjects of the medical literature. The development of high-throughput molecular technologies allowed a deeper characterization of human microbiota and a better understanding of its relationship with health and disease. Changes in human habits including wide use of antimicrobials can result in dysregulation of host–microbiome homeostasis, with multiple consequences. The purpose of this review is to highlight the most important evidence in the literature of host–microbiome interactions and illustrate how these intriguing relations may lead to new treatment and prevention strategies. Keywords: Gastrintestinal microbiome, Human

We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS 2 gene was found in all five isolates. This mutation yielded a 1,3-β- d -glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Cross-reactivity of Fusarium species with serum galactomannan antigen (GMI) test has been observed. We sought to evaluate if GMI could help to early diagnose invasive fusariosis and to monitor treatment response. We reviewed the records of all patients with invasive fusariosis between 2008 and 2012 in three Brazilian hospitals. We selected patients who had at least 1 GMI test within 2 days before or after the date of the first clinical manifestation of fusariosis, and analyzed the temporal relationship between the first positive GMI test and the date of the diagnosis of invasive fusariosis, and the kinetics of GMI in relation to patients' response to treatment. We also selected 18 controls to determine the sensitivity and specificity of the test. Among 18 patients, 15 (83%) had at least one positive GMI (median 4, range 1-15). The sensitivity and specificity of was 83% and 67%, respectively. GMI was positive before the diagnosis of invasive fusariosis in 11 of the 15 cases (73%), at a median of 10 days (range 3-39), and after the diagnosis in 4 cases. GMI became negative in 8 of the 15 patients; 3 of these 8 patients (37.5%) were alive 90 days after the diagnosis of fusariosis compared with 2 of 7 (29%) who did not normalize GMI (p = 1.0). GMI is frequently positive in invasive fusariosis, and becomes positive before diagnosis in most patients. These findings may have important implications for the choice of antifungal therapy in settings with high prevalence of invasive fusariosis.

Background: Active surveillance is necessary for improving the management and outcomes of patients with candidemia. The aim of this study was to describe the epidemiologic and clinical features of candidemia in pediatric patients in Latin America. Method: Prospective, multicenter, surveillance study of candidemia in a pediatric population from 23 hospitals in 8 Latin America countries between November 2008 and October 2010. Results: Three hundred and two cases of candidemia were reported with a median incidence of 0.81/1000 admissions. Eighty nine (29%) were neonates. The main risk factors were prematurity, intensive care unit (ICU) admission, parenteral nutrition, respiratory disease and mechanical ventilation in neonates and malignancy, neutropenia, neurological disease and previous use of corticosteroids in children. The main species isolated in neonates and children were Candida albicans (43.8% and 35.7%), Candida parapsilosis (27.0% and 26.3%) and Candida tropicalis (14.6% and 14.6%), respectively. The most frequent antifungal therapy used in neonates and children was deoxycholate-amphotericin-B (43.8% and 29.1%) and fluconazole (28.1% and 53.1%). Seventeen neonates (19.1%) and 20 children (9.4%) did not receive antifungal therapy. The 30-day survival rate was 60% in neonates and 72% in children (P = 0.02). Survival was significantly higher in treated than in nontreated neonates (72% vs. 24%; P < 0.001). A multivariate analysis showed that independent predictors for 30-day mortality in children were renal disease (odds ratio: 4.38, 95% confidence interval: 1.92–10.1, P < 0.001) and receipt of corticosteroids (odds ratio: 2.08, 95% confidence interval: 1.04–4.17, P = 0.04). Conclusions: To our knowledge, this is the first prospective, multicenter surveillance study of candidemia in children in Latin America. This epidemiologic information may provide us with Methods: to improve preventive, diagnostic and therapeutic strategies in our continent.

Vaginal specimens for culture were obtained from two hundred and five immunocompetent, non-hospitalized patients selected among all women attending the Gynecology and Obstetric Ambulatory Clinic of the University of Espirito Santo, Brazil, during a 2-year period (From 1998 to 1999). Patients were checked for signs and symptoms of vulvovaginitis and previous use of topical and systemic antifungal drugs. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to NCCLS microbroth assay. The prevalence of vaginal yeast isolates from asymptomatic women was 25% (30/121) and 60% (50/84) among patients with symptoms of vulvovaginitis. Candida albicans was the most frequently isolated species in both groups (46% and 90%, respectively), followed by C. glabrata (13% and 6%, respectively). All isolates were susceptible to amphotericin B. Only ten isolates had dose dependent susceptibility (DDS) or resistance to azoles; and seven of these were non-albicans species. Based on our results we suggest that species identification and antifungal susceptibility testing need not be routinely performed in immunocompetent women, and may be reasonable only for the minority of patients with complicated vulvovaginal candidiasis that fail to respond to therapy.

This study was a non-comparative multicenter clinical trial to evaluate the efficacy and tolerability of itraconazole oral solution 200 mg/day (100 mg twice a day in the fasting state) for the treatment of oropharyngeal candidiasis in AIDS patients. We included 50 patients who were treated and followed for up to 3 weeks after ending therapy in the analysis. Mycological cures at the end of therapy occurred in 20/50 patients (40%), but colonization by Candida sp. was recorded in 42/50 (84%) by the end of follow-up. A high rate of clinical response was observed in 46/50 (92%), and the response was sustained for up to 21 days after stopping therapy in 24/46 patients (52%). Clinical relapses were documented among 22 patients, but all causative fungal organisms associated with a relapse were susceptible to itraconazole. There were many patients with persistence or recurrence of Candida, but without mucositis. Relapse of Candida mucositis was significantly related to low levels of CD(4) lymphocytes exhibited by symptomatic patients. The drug was well tolerated by all but 1 patient. We conclude that itraconazole oral solution (100 mg bid for 7-14 days) is a well tolerated and effective treatment for suppressing the symptoms of oropharyngeal candidiasis in AIDS patients. Patients with severe immunosuppression may relapse and require frequent cycles of treatment or longterm suppressive therapy.

The genetically heterogeneous taxon Candida parapsilosis was recently reclassified into three species: Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. The prevalences of these species among 141 bloodstream isolates tested in Brazil were 88% for C. parapsilosis, 9% for C. orthopsilosis, and 3% for C. metapsilosis. Except for three C. orthopsilosis isolates that were considered resistant to 5-flucytosine, all isolates representing the different species of this complex were susceptible to polyenes, triazoles and caspofungin.

Paracoccidioidomicose é considerada a micose sistêmica endêmica mais prevalente na América Latina. Apesar da maior parte da casuística de paracoccidioidomicose ocorrer entre trabalhadores rurais, há poucos casos documentados de ocorrência dessa micose entre índios brasileiros. São apresentados 2 casos de paracoccidioidomicose em índios Suruí, família linguística Tupi-Mondé, procedentes de Cacoal, Rondônia. Ambos apresentaram sorologia positiva à imunodifusão apenas com antígenos da fase miceliana do P. brasiliensis. Os autores apresentam revisão de literatura sobre a ocorrência dessa micose entre índios brasileiros e discutem a necessidade de futuras investigações buscando caracterizar as diferenças regionais de cepas de P. brasiliensis e seu impacto no diagnóstico sorológico dessa micose. Paracoccidioidomycosis has been considered the most frequent endemic systemic mycosis in Latin America. Although most cases of paracoccidioidomycosis involve rural workers, this systemic fungal disease has been scarcely reported among Amerindian populations from Brazil. We report two cases of paracoccidioidomycosis in Tupi-Mondé Amerindians from Cacoal, state of Rondônia, Brazil. Both cases exhibited positive serological results by a specific immunodiffusion test only when the assay was performed with antigens obtained from the mycelial form of P. brasiliensis. The authors present a literature review of paracoccidioidomycosis in Brazilian Amerindians and discuss the need for further investigations about the impact of the antigenic diversity of P. brasiliensis from different geographic areas on the serological diagnosis of PCM. Universidade Federal de São Paulo (UNIFESP) UNIFESP SciELO

We performed a prospective double-blind study to evaluate the correlation between inhibition zones obtained by a disk-diffusion test, using Neo-sensitabs of fluconazole (Rosco Diagnostica), and the MICs generated by the NCCLS macrobroth dilution assay. Eighty clinical isolates, representing 5 of the clinically relevant species of Candida, were tested simultaneously by both methods. A clear inverse correlation was found between the results obtained by both tests (r = -0.69). In addition, there was high degree of agreement between methods in the identification of susceptible isolates. However, the resistance definition by disk-diffusion test had a positive predictive value of only 17%. Our data support the hypothesis that Rosco Fluconazole Neo-sensitabs have potential as a screening test for the identification of Candida isolates susceptible to fluconazole. Resistant isolates should be further investigated by standardized broth procedures.

Fast and reliable identification of different species of the genus Candida is important to define adequate therapeutic decisions, because the different species have highly variable susceptibilities to antifungal drugs; azoles and amphothericin B. Accurate statistical records on case history and epidemiological studies also depend on effective identification. To address this problem we established a RAPD method that enabled direct identification of five very common species of Candida. Initially, reference band patterns were established for C. albicans, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. One of the primers, M2, showed remarkably conserved intra-specific patterns of approximately 10 bands each, ranging in size from 2.0 to 0.1 kb. These patterns were significantly different and species-specific. Few bands were conserved between different species of Candida, which was assumed to be consistent with their phylogenetic relatedness. In addition, band patterns were constant and reproducible and DNA isolated from single colonies yielded sufficient DNA for identification. The reference band patterns were then used, in blind experiments, to identify species of Candida in 50 randomly chosen samples, including clinical isolates and ATCC strains. RAPD results were 100% consistent with results obtained by conventional diagnostic methods and were achieved in one day instead of several days taken by conventional methods. Because ideal identification methods should be consistent with phylogeny and taxonomy we tested whether RAPD could be used to calculate genetic distances. Comparison of RAPD phylogenetic trees with 18S rRNA trees showed significant differences in tree topologies which indicated that RAPD data could not accurately measure the relative distances between different species. Also, computer simulations of RAPD random patterns were used to test whether the observed degree of RAPD band pattern similarities could occur at random. These simulations suggested that the level of inter-specific band pattern similarities observed in our data could be obtained at random, while intra-specific pattern similarities could not. RAPD would be helpful to discriminate between isolates but not to quantitate the differences. We suggest that the inaccurate estimate of genetic distances from RAPD is a general limitation of the technique and not a specific problem of our identification method. Because of the repetitive character of the target sequences, genetic distances calculated from RAPD could be affected by paralogy, namely, recombination and duplication events not parallel with speciation events.

The objective of this study was to characterize the epidemiology of candidemia in cancer patients in the city of Rio de Janeiro, Brazil. An 18-month survey of fungemia in patients with cancer was undertaken in three Hospitals in Rio de Janeiro. Forty-three episodes of candidemia were identified in 43 patients, 43 of which were episodes of candidemia; in ten cases the strains were not available for further identification of species and were excluded from this analysis. The overall distribution of fungi causing fungemia was: Candida albicans (5), Candida tropicalis (16), Candida parapsilosis (6), Candida guilliermondii (4), Candida lusitaniae (1) and Candida stellatoidea (1). Antifungal prophylaxis had been administered before the episode of fungemia in only six patients (18.2%): oral itraconazole in three patients and oral nistatin, low dose intravenous amphotericin B and oral fluconazole in one patient each. There was no difference in the presence of risk factors, clinical characteristics or in the outcome between albicans and non-albicans species, nor between Candida tropicalis and other non-albicans species. There was a clear predominance of non-albicans species, regardless of the underlying disease, antifungal prophylaxis or the presence of neutropenia.

The aim of our study was to evaluate the accuracy of the chromogenic media Albicans ID2O (bioMerieux, France) for the identification of Candida albicans among 330 yeast strains. All C. albicans (100) and C. dubliniensis (20) strains exhibited blue color when cultured on Albicans ID2O. However, the blue color was also exhibited by cultures of C. rugosa (30/30) and C. tropicalis (3/50) isolates.

The description of the association between the use of amphotericin-B (amB) and the development of systemic arterial hypertension was only anecdotal so far. We describe the case of a 19-year-old female patient who had acute lymphoblastic leukemia and developed prolonged neutropenia after reinduction chemotherapy. Candida parapsilosis was isolated from blood cultures, and amB was started. Sustained severe arterial hypertension developed shortly after amB administration and continued for several hours after the infusion. Aldosterone, blood urea nitrogen, and creatinine levels were normal. After clinical improvement, amB was replaced by fluconazole, and blood pressure normalized. Severe hypertension may be an adverse event associated with AmB treatment that requires intensive treatment.

In the last decade the number of systemic yeast infections has increased significantly. Although Candida albicans is the most frequently isolated yeast from clinical specimens, the emergence of non-albicans species has clearly been a recent concern. As a consequence, there is a greater need for rapid and accurate methods for yeast identification. The aim of this study was to evaluate the performance of the AUXACOLOR system (Sanofi Diagnostics Pasteur) for the identification of clinically relevant yeasts, as compared with the conventional method. Yeast isolates (n = 97) belonging to 12 species were identified by the commercial system and the classic method. Correct identifications were obtained by using AUXACOLOR system in 79.4% of the isolates tested. Misidentification occurred in 5.2% of the strains and 15.5% were not identified due to a failure in the manufacturer's data base. In order to improve its accuracy, there is a need for expanding the database or revamping the tests included in the system.

A 59-year-old female farm worker developed a dark-centered cutaneous lesion in her right leg, associated with progressive swelling for the past 35 years. During the past 4 years, she also had dry cough and dyspnea, which became more intense for the past 6 months. She complained of asthenia and weight loss of 10 kg in the previous year. The patient had diabetes mellitus and hypertension. On physical examination, she had crackling rale at the base of the lungs, edema of the distal right lower limb, with several sinuses and ulcerated lesions of ≈1 cm with purulent discharge (Figure 1). Figure 1. Right lower foot with several ulcerated lesions with purulent discharge. Radiographic studies showed lytic foci of destruction in the medullary cavity with bone expansion, reactive sclerosis, and thickening of the cortex in right tibia and in bones of the right foot. Metatarsal bones also had cortical scalloping (Figure 2A and 2B). T1-weighted magnetic resonance image of the right foot with fat suppression after intravenous gadolinium demonstrate numerous round lesions containing small low-signal foci, also known as dot-in-circle sign, representing granulation tissue surrounded …