João N. de Almeida Júnior

Invasive Trichosporon Infection: a Systematic Review on a Re-emerging Fungal Pathogen

Objectives: This review aimed to better depict the clinical features and address the issue of therapeutic management of Trichosporon deep-seated infections. Methods: We comprehensively reviewed the cases of invasive Trichosporon infection reported in the literature from 1994 (date of taxonomic modification) to 2015. Data from antifungal susceptibility testing (AST) studies were also analyzed. Results: Two hundred and three cases were retained and split into four groups: homeopathy (n = 79), other immunodeficiency conditions (n = 41), miscellaneous (n = 58) and newborns (n = 25). Trichosporon asahii was the main causative species (46.7%) and may exhibit cross-resistance to different antifungal classes. The unfavorable outcome rate was at 44.3%. By multivariate analysis, breakthrough infection (OR 2.45) was associated with unfavorable outcome, whilst the use of an azole-based therapy improved the prognosis (OR 0.16). Voriconazole-based treatment was associated with favorable outcome in hematological patients (73.6 vs. 41.8%; p = 0.016). Compiled data from AST demonstrated that (i) T. asahii exhibits the highest MICs to amphotericin B and (ii) voriconazole has the best in vitro efficacy against clinical isolates of Trichosporon spp. Conclusions: Trichosporon infection is not only restricted to hematological patients. Analysis of compiled data from AST and clinical outcome support the use of voriconazole as first line therapy.

Identification of Candida haemulonii Complex Species: Use of ClinProToolsTM to Overcome Limitations of the Bruker BiotyperTM, VITEK MSTM IVD, and VITEK MSTM RUO Databases

Candida haemulonii is now considered a complex of two species and one variety: C. haemulonii sensu stricto, Candida duobushaemulonii and the variety C. haemulonii var. vulnera. Identification (ID) of these species is relevant for epidemiological purposes and for therapeutic management, but the different phenotypic commercial systems are unable to provide correct species ID for these emergent pathogens. Hence, we evaluated the MALDI-TOF MS performance for the ID of C. haemulonii species, analyzing isolates/strains of C. haemulonii complex species, Candida pseudohaemulonii and Candida auris by two commercial platforms, their databases and softwares. To differentiate C. haemulonii sensu sctricto from the variety vulnera, we used the ClinProToolsTM models and a single-peak analysis with the software FlexAnalysisTM. The BiotyperTM database gave 100% correct species ID for C. haemulonii sensu stricto, C. pseudohaemulonii and C. auris, with 69% of correct species ID for C. duobushaemulonii. Vitek MSTM IVD database gave 100% correct species ID for C. haemulonii sensu stricto, misidentifying all C. duobushaemulonii and C. pseudohaemulonii as C. haemulonii, being unable to identify C. auris. The Vitek MSTM RUO database needed to be upgraded with in-house SuperSpectra to discriminate C. haemulonii sensu stricto, C. duobushaemulonii, C. pseudohaemulonii, and C. auris strains/isolates. The generic algorithm model from ClinProToolsTM software showed recognition capability of 100% and cross validation of 98.02% for the discrimination of C. haemulonii sensu stricto from the variety vulnera. Single-peak analysis showed that the peaks 5670, 6878, or 13750 m/z can distinguish C. haemulonii sensu stricto from the variety vulnera.

INCIDENCE OF DIARRHEA BY Clostridium difficile IN HEMATOLOGIC PATIENTS AND HEMATOPOIETIC STEM CELL TRANSPLANTATION PATIENTS: RISK FACTORS FOR SEVERE FORMS AND DEATH

We describe the rate of incidence of Clostridium difficile-associated diarrhea (CDAD) in hematologic and patients undergone stem cell transplant (HSCT) at HC-FMUSP, from January 2007 to June 2011, using two denominators 1,000 patient and 1,000 days of neutropenia and the risk factors associated with the severe form of the disease and death. The ELISA method (Ridascreen-Biopharm, Germany) for the detections of toxins A/B was used to identify C. difficile. A multivariate analysis was performed to evaluate potential factors associated with severe CDAD and death within 14 days after the diagnosis of CDAD, using multiple logistic regression. Sixty-six episodes were identified in 64 patients among 439 patients with diarrhea during the study period. CDA rate of incidence varied from 0.78 to 5.45 per 1,000 days of neutropenia and from 0.65 to 5.45 per 1,000 patient-days. The most common underlying disease was acute myeloid leukemia 30/64 (44%), 32/64 (46%) patients were neutropenic, 31/64 (45%) undergone allogeneic HSCT, 61/64 (88%) had previously used antibiotics and 9/64 (13%) have severe CDAD. Most of the patients (89%) received treatment with oral metronidazole and 19/64 (26%) died. The independent risk factors associated with death were the severe form of CDAD, and use of linezolid.

Streptococcus pyogenes strains in Sao Paulo, Brazil: molecular characterization as a basis for StreptInCor coverage capacity analysis

BackgroundSeveral human diseases are caused by Streptococcus pyogenes, ranging from common infections to autoimmunity. Characterization of the most prevalent strains worldwide is a useful tool for evaluating the coverage capacity of vaccines under development. In this study, a collection of S. pyogenes strains from Sao Paulo, Brazil, was analyzed to describe the diversity of strains and assess the vaccine coverage capacity of StreptInCor.MethodsMolecular epidemiology of S. pyogenes strains was performed by emm-genotyping the 229 isolates from different clinical sites, and PCR was used for superantigen profile analysis. The emm-pattern and tissue tropism for these M types were also predicted and compared based on the emm-cluster classification.ResultsThe strains were fit into 12 different emm-clusters, revealing a diverse phylogenetic origin and, consequently, different mechanisms of infection and escape of the host immune system. Forty-eight emm-types were distinguished in 229 samples, and the 10 most frequently observed types accounted for 69 % of all isolates, indicating a diverse profile of circulating strains comparable to other countries under development. A similar proportion of E and A-C emm-patterns were observed, whereas pattern D was less frequent, indicating that the strains of this collection primarily had a tissue tropism for the throat. In silico analysis of the coverage capacity of StreptInCor, an M protein-conserved regionally based vaccine candidate developed by our group, had a range of 94.5 % to 59.7 %, with a mean of 71.0 % identity between the vaccine antigen and the predicted amino acid sequence of the emm-types included here.ConclusionsThis is the first report of S. pyogenes strain characterization in Sao Paulo, one of the largest cities in the world; thus, the strain panel described here is a representative sample for vaccine coverage capacity analysis. Our results enabled evaluation of StreptInCor candidate vaccine coverage capacity against diverse M-types, indicating that the vaccine candidate likely would induce protection against the diverse strains worldwide.

Trichosporon inkin as an Emergent Pathogen in Patients With Severe Pemphigus

IMPORTANCE To our knowledge, these are the first reports of bloodstream infections by Trichosporon inkin in patients with pemphigus. OBSERVATIONS Trichosporon inkin, a novel organism causing bloodstream infection, was detected in 2 patients with pemphigus. An elderly man with pemphigus foliaceus died despite treatment with liposomal amphotericin B, 3 mg/kg/d, and a young girl with pemphigus vulgaris responded to treatment with voriconazole, 8 mg/kg/d, for 24 days. One of the T inkin isolates had a minimal inhibitory concentration of 2 mg/L against amphotericin B, suggesting resistance to the drug. CONCLUSIONS AND RELEVANCE Delayed suspicion of invasive infection by T inkin may result in a poor outcome in patients with severe forms of pemphigus. This opportunistic infection is highly refractory to conventional potent antifungal treatment.

Rhizopus arrhizus and Fusarium solani Concomitant Infection in an Immunocompromised Host.

Neutropenic patients are at risk of the development of hyalohyphomycosis and mucormycosis. Correct identification is essential for the initiation of the specific treatment, but concomitant mold infections are rarely reported. We report one unprecedented case of concomitant mucormycosis and fusariosis in a neutropenic patient with acute myeloid leukemia. The patient developed rhino-orbital infection by Rhizopus arrhizus and disseminated infection by Fusarium solani. The first culture from a sinus biopsy grew Rhizopus, which was consistent with the histopathology report of mucormycosis. A second sinus biopsy collected later during the patient’s clinical deterioration was reported as hyalohyphomycosis, and the culture yielded F.solani. Due to the discordant reports, the second biopsy was reviewed and two hyphae types suggestive of both hyalohyphomycetes and mucormycetes were found. The dual mold infection was confirmed by PCR assays from paraffinized tissue sections. Increased awareness of the existence of dual mold infections in at-risk patients is necessary. PCR methods in tissue sections may increase the diagnosis of dual mold infections. In case of sequential biopsies showing discrepant results, mixed infections have to be suspected.

Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

ABSTRACT We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed.

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.

Bactec™ blood culture bottles allied to MALDI-TOF mass spectrometry: rapid etiologic diagnosis of bacterial endophthalmitis

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been used for direct identification of pathogens from blood-inoculated blood culture bottles (BCBs). We showed that MALDI-TOF MS is an useful technique for rapid identification of the causative agents of endophthalmitis from vitreous humor-inoculated BCBs with a simple protocol.

Diferença de tempo de positividade: método útil no diagnóstico de infecção de corrente sanguínea relacionada com cateter?

INTRODUCTION: Not only do catheter related bloodstream infections (CRBSIs) have considerable impact on morbidity and mortality in hospitalized patients, but they also raise hospital costs. The use of automated equipment in blood culture processing has allowed an alternative diagnosis of CRBSI by analyzing the differential time to positivity (DTP) of paired blood cultures (collected simultaneously) of peripheral blood and catheter blood. A rapid and accurate diagnosis of these infections may optimize clinical and therapeutic management, which prevents early catheter removal. OBJECTIVES: To assess DTP as an auxiliary tool for the diagnosis of CRBSI as well as to determine the main isolated microorganisms. METHODS: We evaluated blood cultures that had previously been collected in the complex Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (HC/FMUSP) from May to August 2008. According to the laboratory criteria, only DTP higher than 120 minutes was regarded as possible CRBSI. RESULTS: During the investigation period 11,017 aerobic blood cultures were processed, from which only 5% were paired samples. One hundred forty-eight (28%) samples were positive, from which 9% showed growth in peripheral blood, 41% only in catheter blood and 50% in both blood samples with 88% homology of identified microorganisms. DTP higher than 120 minutes occurred in 50% of the cases. The most common isolated microorganisms were: Staphylococcus aureus (22%), Candida spp. (18%), Klebsiella spp (7%). and Enterobacter spp (7%). CONCLUSION: The determination of the DTP as an auxiliary tool for the diagnosis of CRBSI is feasible and easily performed in clinical laboratories with automation, although the process of paired sample collection must be rigidly standardized.