Genomic diversity of the human pathogen Paracoccidioides across the South American continent.

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis widely reported in the Gran Chaco ecosystem. The disease is caused by different species from the genus Paracoccidioides, which are all endemic to South and Central America. Here, we sequenced and analyzed 31 isolates of Paracoccidioides across South America, with particular focus on isolates from Argentina and Paraguay. The de novo sequenced isolates were compared with publicly available genomes. Phylogenetics and population genomics revealed that PCM in Argentina and Paraguay is caused by three distinct Paracoccidioides genotypes, P. brasiliensis (S1a and S1b) and P. restrepiensis (PS3). P. brasiliensis S1a isolates from Argentina are frequently associated with chronic forms of the disease. Our results suggest the existence of extensive molecular polymorphism among Paracoccidioides species, and provide a framework to begin to dissect the connection between genotypic differences in the pathogen and the clinical outcomes of the disease.

Biochemical characterization of a Kunitz inhibitor from Inga edulis seeds with antifungal activity against Candida spp.

We describe the characterization of IETI, the first trypsin inhibitor purified from Inga edulis, a tree widely distributed in Brazil. Two-step chromatography was used to purify IETI, a protein composed of a single peptide chain of 19,685.10 Da. Amino-terminal sequencing revealed that IETI shows homology with the Kunitz family, as substantiated by its physical-chemical features, such as its thermal (up to 70 °C) and wide-range pH stability (from 2 to 10), and the value of its dissociation constant (6.2 nM). IETI contains a single reactive site for trypsin, maintained by a disulfide bridge; in the presence of DTT, its inhibitory activity was reduced in a time- and concentration-dependent manner. IETI presented activity against Candida ssp., including C. buinensis and C. tropicalis. IETI inhibitory activity triggered yeast membrane permeability, affecting cell viability, thus providing support for the use of IETI in further studies for the control of fungal infections.

Non-clonal occurrence of pmrB mutations associated with polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae in Brazil.

BACKGROUND: Polymyxins are currently used as a "last-line" treatment for multidrug-resistant Gram-negative infections. OBJECTIVES: To identify the major mechanisms of resistance to polymyxin and compare the genetic similarity between multi-drug resistant Klebsiella pneumoniae strains recovered from inpatients of public hospitals in the Mid-West of Brazil. METHODS: 97 carbapenems non-susceptible K. pneumoniae were studied. ß-lactamases (bla OXA-48, bla KPC, bla NDM, bla CTX-M, bla SHV, bla TEM, bla IMP, bla VIM) and mcr-1 to mcr-5 genes were investigated by polymerase chain reaction (PCR). Mutations in chromosomal genes (pmrA, pmrB, phoP, phoQ, and mgrB) were screened by PCR and DNA sequencing. Clonal relatedness was established by using pulsed-field gel electrophoresis and multilocus sequence typing. FINDINGS: K. pneumoniae isolates harbored bla KPC (93.3%), bla SHV (86.6%), bla TEM (80.0%), bla CTX-M (60%) genes. Of 15 K. pneumoniae resistant to polymyxin B the authors identified deleterious mutations in pmrB gene, mainly in T157P. None K. pneumoniae presented mcr gene variants. Genetic polymorphism analyses revealed 12 different pulsotypes. MAIN CONCLUSIONS: Deleterious mutations in pmrB gene is the main chromosomal target for induction of polymyxin resistance in carbapenem-resistant K. pneumoniae in public hospitals in the Mid-West of Brazil.

The Simultaneous Occurrence of Histoplasmosis and Cryptococcal Fungemia: A Case Report and Review of the Literature.

Simultaneous infections with Cryptococcus neoformans and Histoplasma capsulatum are rare and typically occur in immunocompromised individuals, particularly AIDS patients. Because both of those fungi can spur the development of latent infections, it is generally unknown which organism was first present. The diagnosis of one fungus can hide the diagnosis of the other, leading to underdiagnoses and poor prognosis. We report a case of C. neoformans and H. capsulatum co-infection. We also performed a systematic review of the literature. Our patient, a 69-year-old HIV-negative man, presented with a 9-month history of a productive cough, dyspnea and wheezing. His history showed chronic obstructive pulmonary disease and type II diabetes mellitus (DM). Pulmonary lesions of unidentified etiology were noted. Cryptococcal involvement of the central nervous system was confirmed, and C. neoformans and H. capsulatum were also isolated from blood cultures. In the literature, we found ten cases of simultaneous histoplasmosis and cryptococcosis. Including our patient, all of the cases were located in North America and Latin America, except for two, one Indian and one African diagnosed in France. AIDS was the main predisposing factor, followed by DM and age >60 years. Several diagnostic methods were employed. A blood culture and bronchoalveolar lavage (BAL) culture resulted in the growth of C. neoformans in most of the cases. H. capsulatum was more often detected in BAL and bone marrow cultures. Although co-infection by these pathogens is severe, it can be cured if it is diagnosed and treated early. We emphasized the importance of clinical suspicion and differential diagnosis of systemic mycoses.

First molecular typing of cryptococcemia-causing cryptococcus in central-west Brazil.

Molecular epidemiology studies on cryptococcemia are limited. This study aimed to describe the clinical features of patients with bloodstream infections by Cryptococcus sp. in a public tertiary hospital in Mato Grosso do Sul, as well as identify the fungus' molecular type and determine its antifungal susceptibility. Molecular typing was performed using URA5 restriction fragment length polymorphism PCR, and antifungal susceptibility was determined by microdilution method standardized by the Clinical and Laboratory Standards Institute. Over 14 years, 48 patients were diagnosed with cryptococcemia. The majority (72.9 %) was male with a median age of 40 years; 81.3 % of the patients had HIV/AIDS and 72.9 % died. Cryptococcus neoformans was the most commonly isolated species (97.9 %). Molecular analysis identified the genotypes C. neoformans VNI (93.7 %), C. neoformans VNII (4.2 %), and Cryptococcus gattii VGII (2.1 %). In vitro, these fungi were not resistant to fluconazole, itraconazole, voriconazole, and amphotericin B. This is the first description of the molecular types of cryptococcemia agents in central-west Brazil. Its high lethality, especially in HIV-negative patients, suggests that early diagnosis and prompt antifungal therapy are crucial for a good clinical outcome.

Antimicrobial activity of some medicinal plants from the cerrado of the centralwestern region of Brazil.

Ethanol extracts from six selected species from the Cerrado of the Central-Western region of Brazil, which are used in traditional medicine for the treatment of infectious diseases and other medical conditions, namely Erythroxylum suberosum St. Hil. (Erythroxylaceae), Hyptis crenata Pohl. ex Benth. (Lamiaceae), Roupala brasiliensis Klotz. (Proteaceae), Simarouba versicolor St. Hil. (Simaroubaceae), Guazuma ulmifolia Lam. (Sterculiaceae) and Protium heptaphyllum (Aubl.) March. (Burseraceae), as well as fractions resulting from partition of these crude extracts, were screened in vitro for their antifungal and antibacterial properties. The antimicrobial activities were assessed by the broth microdilution assay against six control fungal strains, Candida albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and Cryptococcus neoformans, and five control Gram-positive and negative bacterial strains, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Toxicity of the extracts and fractions against Artemia salina was also evaluated in this work. All plants investigated showed antimicrobial properties against at least one microorganism and two species were also significantly toxic to brine shrimp larvae. The results tend to support the traditional use of these plants for the treatment of respiratory and gastrointestinal disorders and/or skin diseases, opening the possibility of finding new antimicrobial agents from these natural sources. Among the species investigated, Hyptis crenata, Erythroxylum suberosum and Roupala brasiliensis were considered the most promising candidates for developing of future bioactivity-guided phytochemical investigations.

Molecular Epidemiology of blaKPC-Encoding Klebsiella pneumoniae Isolated from Public Hospitals in Midwest of Brazil.

This study was conducted to determine the molecular epidemiology of blaKPC-encoding Klebsiella pneumoniae recovered from three public hospitals in Brazil. Molecular investigation of blaOXA-48, blaKPC, blaNDM, blaCTX-M, blaSHV, blaTEM, blaIMP, and blaVIM resistance genes was performed in 99 K. pneumoniae isolates from inpatients of intensive care units. Antimicrobial susceptibility was determined with a Vitek-2 System, except for polymyxin B, which was evaluated by the microbroth dilution test. Clonal relatedness was established by pulsed-field gel electrophoresis and multilocus sequence typing. Screening resistance genes showed that K. pneumoniae isolates carried the blaKPC (88.9%), blaSHV (73.5%), blaTEM (72.2%), and blaCTX-M (43.9%) genes. The most frequent sequence types (STs) were ST273, ST11, ST 1298, ST13, ST2687, and ST37. We report new STs in K. pneumoniae that have not been detected previously in Brazil. K. pneumoniae belonging to the same clone is present in different hospitals in the same region, showing the spread of multidrug-resistant K. pneumoniae.

Combination efficacy of voriconazole and amphotericin B in the experimental disease in immunodeficient mice caused by fluconazole-resistant Cryptococcus neoformans.

The therapeutic efficacy of amphotericin B and voriconazole alone and in combination with one another were evaluated in immunodeficient mice (BALB/c-SCID) infected with a fluconazole-resistant strain of Cryptococcus neoformans var. grubii. The animals were infected intravenously with 3 × 10(5) cells and intraperitoneally treated with amphotericin B (1.5 mg/kg/day) in combination with voriconazole (40 mg/kg/days). Treatment began 1 day after inoculation and continued for 7 and 15 days post-inoculation. The treatments were evaluated by survival curves and yeast quantification (CFUs) in brain and lung tissues. Treatments for 15 days significantly promoted the survival of the animals compared to the control groups. Our results indicated that amphotericin B was effective in assuring longest-term survival of infected animals, but these animals still harbored the highest CFU of C. neoformans in lungs and brain at the end of the experiment. Voriconazole was not as effective alone, but in combination with amphotericin B, it prolonged survival for the second-longest time period and provided the lowest colonization of target organs by the fungus. None of the treatments were effective in complete eradication of the fungus in mice lungs and brain at the end of the experiment.

A Rare Case of Aspergillus Mediastinitis After Coronary Artery Bypass Surgery: A Case Report and Literature Review.

BACKGROUND Mediastinitis is a serious complication after cardiac surgery; it is a deep sternal wound infection following sternotomy, with clinical evidence and/or microbiological involvement and sternal osteomyelitis. The most common pathogens are Staphylococcus spp (S. aureus), followed by gram-negative organisms. Establishing an etiological diagnosis of fungal mediastinitis is often a challenging issue, given the nonspecific clinical presentation. CASE REPORT A 74-year-old man was diagnosed with a three-vessel coronary artery disease in a university hospital. The patient had as clinical background hypertension, a body mass index (BMI) of 29.78 kg/m², and no diabetes mellitus. After an uneventful coronary artery bypass surgery, he presented clinical and radiological mediastinitis manifestations on the 9th postoperative day. He was treated with a range of antibiotics, with no clinical improvement until the 33rd postoperative day. Then, mediastinal fluid and biopsied tissue were collected and he was started on voriconazole due to growing Aspergillus spp. On the 93rd postoperative day, he had clinical improvement and, after several exams, was released from the hospital. We present the first report of Aspergillus fumigatus mediastinitis after cardiac surgery in Brazil, successfully treated with voriconazole. CONCLUSIONS Aspergillus infection should be considered in the differential diagnosis of mediastinitis after coronary surgery, especially in a clinical case of unexplained sepsis, negative blood culture, and no clinical improvement despite antibiotic therapy. This case report highlights that the mediastinal fluid and biopsy tissue culture can be useful for the diagnosis of fungal mediastinitis.

Antifungal activity of Annona coriacea Mart. ethanol extracts against the aetiological agents of cryptococcosis.

Cryptococcosis is an opportunistic disease with a worldwide distribution. This disease is caused by fungi of the genus Cryptococcus, and its treatment is limited to several antifungals. In this study, the antifungal, cytotoxic and mutagenic properties of ethanol extracts from the bark and leaves of Annona coriacea were evaluated against the standard Cryptococcus species and clinical yeast specimens. Both extracts of A. coriacea showed inhibitory activity of 1.5 mg/mL for all of the yeasts tested. The number of viable cells at the lowest tested concentration was 0.187 mg/mL. The extracts that were tested showed inhibitory activity and reduced the fungal growth of the Cryptococcus gattii species and Cryptococcus neoformans species complexes, suggesting that this plant may be an effective alternative treatment for cryptococcosis.

Treatment compliance of patients with paracoccidioidomycosis in Central-West Brazil.

OBJECTIVE: To evaluate the treatment compliance of patients with paracoccidioidomycosis. METHODS: We studied 188 patients with paracoccidioidomycosis admitted to a tertiary referral hospital in the Central-West Region of Brazil from 2000 to 2010, to assess their compliance to treatment. In order to be considered compliant, patients needed to present two established criteria: (1) receive medicines from the pharmacy, and (2) achieve a self-reported utilization of at least 80% of the dispensed antifungal compounds prescribed since their previous appointment. RESULTS: Most patients were male (95.7%), had the chronic form of the disease (94.2%), and were treated with cotrimoxazole (86.2%). Only 44.6% of patients were treatment compliant. The highest loss to follow-up was observed in the first 4 months of treatment (p < 0.02). Treatment compliance was higher for patients with than for those without pulmonary involvement (OR: 2.986; 95%CI 1.351-6.599), and higher for patients with than without tuberculosis as co-morbidity (OR: 2.763; 95%CI 1.004-7.604). CONCLUSIONS: Compliance to paracoccidioidomycosis treatment was low, and the period with the highest loss to follow-up corresponds to the first four months. Pulmonary paracoccidioidal involvement or tuberculosis comorbidity predicts a higher compliance to paracoccidioidomycosis therapy.

Post-neurosurgical meningitis caused by KPC-producing Klebsiella pneumoniae: report of two cases.

Nosocomial bacterial infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) is associated with high mortality in neurosurgical patients. There are few reports in the literature on meningitis caused by CRKP. We report two cases of CRKP meningitis after neurosurgery. The K. pneumoniae identification and antimicrobial susceptibility testing were performed using the Vitek Compact System. Minimum inhibitory concentrations of polymyxin B were determined using the broth microdilution method. Molecular typing of K. pneumoniae isolates was investigated using multilocus sequence typing. Antimicrobial susceptibility testing showed that the K. pneumoniae isolates were multidrug resistant and co-produced extended-spectrum ß-lactamases and KPC enzymes. The patients were treated with intrathecal polymyxin. Genetic polymorphism analyses revealed two different K. pneumoniae clones (ST1298 and ST2687), which were observed for the first time in CRKP infections. We recommend intravenous administration of intrathecal polymyxin for treating meningitis caused by multidrug-resistant K. pneumoniae .

Clinical and epidemiological features of 123 cases of cryptococcosis in Mato Grosso do Sul, Brazil.

To identify the clinical and epidemiological profile of cryptococcosis diagnosed at the University Hospital of the Federal University of Mato Grosso do Sul, Brazil, medical records of 123 patients admitted from January 1995 to December 2005 were analyzed. One hundred and four cases (84.5%) had HIV infection, six (4.9%) had other predisposing conditions and 13 (10.6%) were immunocompetent. Male patients predominated (68.3%) and their age ranged from 19 to 69 years (mean: 35.9). Most patients (73.2%) were born and lived lifelong in the state of Mato Grosso do Sul. Involvement of the central nervous system occurred in 103 patients (83.7%) and headache and vomiting were the most frequent symptoms. In 77 cases it was possible to identify the Cryptococcus species: 69 (89.6%) C. neoformans and eight (10.4%) C. gattii. Amphotericin B was the drug of choice for treatment (106/123), followed by fluconazole in 60% of cases. The overall lethality rate was 49.6%, being 51% among the HIV infected patients and 41.2% among the non-HIV infected (p > 0.05). Although cryptococcosis exhibited in our region a similar behavior to that described in the literature, the detection of an important rate of immunocompetent individuals and five C. gattii cryptococcosis in HIV-infected patients is noteworthy.

Non-viral microbial keratitis in adults: clinical and laboratory aspects.

This study compares patients with and without non-viral microbial keratitis in relation to sociodemographic variables, clinical aspects, and involved causative agent. Clinical aspects, etiology and therapeutic procedures were assessed in patients with and without keratitis that were diagnosed in an Eye Care Center in Campo Grande, MS, Brazil. Patients were divided into two groups: (a) cases: 64 patients with non-viral microbial keratitis diagnosed at biomicroscopy; and (b) controls: 47 patients with other eye disorders that were not keratitis. Labor activity related to agriculture, cattle raising, and contact lens use were all linked to keratitis occurrence (p<0.005). In patients with keratitis, the most common symptoms were pain and photophobia, and the most frequently used medicines were fourth-generation fluoroquinolones (34.4%), amphotericin B (31.3%), and natamycin (28.1%). Microbial keratitis evolved to corneal perforation in 15.6% of cases; transplant was indicated in 10.9% of cases. Regarding the etiology of this condition, 23 (42.2%) keratitis cases were caused by bacteria (Pseudomonas aeruginosa, 12.5%), 17 (39.1%) by fungi (Fusarium spp., 14.1% and Aspergillus spp., 4.7%), and 4 (6.3%) by Acanthamoeba. Patients with keratitis present with a poorer prognosis. Rapid identification of the etiologic agent is indispensable and depends on appropriate ophthalmological collection and microbiological techniques.

Clinical and laboratorial features of oral candidiasis in HIV-positive patients.

INTRODUCTION: We describe the clinical and laboratorial features of oral candidiasis in 66 HIV-positive patients. METHODS: Polymerase chain reaction-based techniques were performed for differentiation of Candida spp. isolated from patients at a public teaching hospital in Midwest Brazil. RESULTS: Oral lesions, mainly pseudomembranous, were significantly related to higher levels of immunosuppression. Of 45 Candida isolates, 66.7% were C. albicans. Most of the isolates were susceptible to the antifungal drugs tested. CONCLUSIONS: Oral lesions were associated with higher immunosuppression levels. Lower susceptibility to antifungals by non-albicans isolates supports the importance of surveillance studies using susceptibility tests to aid in the treatment.

Cryptococcal meningitis epidemiology: 17 years of experience in a State of the Brazilian Pantanal.

INTRODUCTION: This study aimed to describe cryptococcal meningitis (CM) cases and the associated demographic, clinical, and microbiological data obtained from cities in the State of Mato Grosso do Sul in the Midwestern region of Brazil. METHODS: The data from 129 patients with laboratory-confirmed CM admitted from 1997 to 2014 were retrospectively reviewed. The molecular types of Cryptococcus neoformans and Cryptococcus gattii isolated from cerebrospinal fluid were analyzed to determine their geographic distribution. RESULTS: The patients had a mean age of 37 years and consisted mostly of men (76.7%). Most of the Cryptococcus isolates were obtained from patients infected with human immunodeficiency virus (HIV) and included 105 (87.5%) and 5 (55.6%) isolates of C. neoformans and C. gattii complexes, respectively. A restriction fragment length polymorphism (RFLP) analysis of URA5 revealed that most of the isolates were C. neoformans molecular type VNI (89.1%), whereas the molecular types VGII (7%) and VNII (3.9%) were observed less frequently. Notably, 65% of the cases with a time from symptom onset to laboratory diagnosis of more than 60 days resulted in fatalities, and sequelae were observed among the patients who survived. CONCLUSIONS: The present study documents the occurrence of neurocryptococcosis, which is mainly caused by C. neoformans VNI, in Mato Grosso do Sul, Brazil, with probable autochthonous cases in the Brazilian Pantanal, the world's largest tropical wetland, and a biome where cryptococcosis has not yet been explored.

Neuroparacoccidioidomycosis: analysis of 13 cases observed in an endemic area in Brazil.

The epidemiological, clinical and laboratory features of 13 cases of neuroparacoccidioidomycosis (NPCM) were analysed. All patients were men, with a mean age of 41.6 years. The lungs were involved in 11 cases (84.6%) and only two cases had mycosis limited to the central nervous system. Co-morbidity was observed in four patients (malignant neoplasm in three and diabetes mellitus in one). The most frequent neurological manifestations were paresis (eight cases), headache (five cases) and gait disturbance (four cases). Neuroimaging diagnosis showed a predominance of multiple round lesions with ring enhancement following contrast medium injection. Lesions were seen in the brain hemispheres (nine cases), thalamus (nine cases), cerebellum (four cases), brainstem (four cases) and spinal cord (four cases). Most cases responded well to therapy. Lesions with enhancement following contrast medium injection persisted in four patients for a period of 6 months to 8 years. These findings emphasize the importance of considering NPCM in the differential diagnosis of brain and spinal cord lesions in endemic areas of paracoccidioidomycosis.

Study of 30 cases of histoplasmosis observed in the Mato Grosso do Sul State, Brazil.

Thirty cases of histoplasmosis observed at the University Hospital of the Federal University of Mato Grosso do Sul (HU-UFMS) from January 1998 to December 2005 are reported. Most (83.3%) of the patients were men, average 33.4 years old, 63.3% of them were born and living in Mato Grosso do Sul and 83.3% presented AIDS as an underlying disease. In almost all cases (96.7%) the disease occurred in its disseminated form and the most frequent clinical manifestations were: fever (83.3%), weight loss (70.0%), cough (63.3%), hepatomegaly and splenomegaly (40.0%), and lymph node enlargement (36.7%). The laboratory diagnosis was obtained in 29 patients by isolation of Histoplasma capsulatum from various clinical specimens cultivated in Sabouraud dextrose and brain heart infusion agar and in 16 patients the fungus was observed by direct microscopy of Giemsa-stained smears. The observed mortality was 40%. This is the first report in the literature of the occurrence of histoplasmosis in Mato Grosso do Sul State.