Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ?aoxA, ?cycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.

Expanding the Clinical and Genetic Spectrum of Human CD40L Deficiency: The Occurrence of Paracoccidioidomycosis and Other Unusual Infections in Brazilian Patients

CD40 ligand (CD40L) deficiency or X-linked hyper-IgM syndrome (X-HIGM) is a well-described primary immunodeficiency in which Pneumocystis jiroveci pneumonia is a common clinical feature. We have identified an unusual high incidence of fungal infections and other not yet described infections in a cohort of 11 X-HIGM patients from nine unrelated Brazilian families. Among these, we describe the first case of paracoccidioidomycosis (PCM) in X-HIGM. The molecular genetic analysis of CD40L was performed by gene sequencing and evaluation of CD40L protein expression. Nine of these 11 patients (82%) had fungal infections. These included fungal species common to CD40L deficiency (P. jiroveci and Candida albicans) as well as Paracoccidioides brasiliensis. One patient presented with PCM at age 11 years and is now doing well at 18 years of age. Additionally, one patient presented with a simultaneous infection with Klebsiella and Acinetobacter, and one with condyloma caused by human papilloma virus. Molecular analysis revealed four previously described CD40L mutations, two novel missense mutations (c.433 T>G and c.476 G>C) resulting in the absence of CD40L protein expression by activated CD4(+) cells and one novel insertion (c.484_485insAA) within the TNFH domain leading to a frame shift and premature stop codon. These observations demonstrated that the susceptibility to fungal infections in X-HIGM extends beyond those typically associated with X-HIGM (P. jiroveci and C. albicans) and that these patients need to be monitored for those pathogens.

Epidemiological and clinical characteristics of nosocomial candidiasis in university hospitals in Cuiaba - Mato Grosso, Brazil

Background: Fungal infections are emerging as an important cause of human disease, especially among hospitalized patients with serious underlying disease and several risk factors. Aims: To evaluate epidemiological and clinical characteristics of patients with nosocomial candidiasis in university hospitals in Cuiaba - MT, Brazil. Methods: A descriptive study of 91 patients admitted to university hospitals in Cuiaba - MT, with clinical and laboratory diagnosis of nosocomial candidiasis, over a 20-month period. Results: A rate for nosocomial infections by Candida spp. of 5 per 1000 admissions, proportional mortality of 14.4% and lethality of 53.8% were determined. The patient age ranged from 29 days to 82 years-old, among which, 74.7% were adults and 25.3% children. The intensive care units contributed with the highest number of cases of infection by Candida spp. (69.2%). The most important underlying disease was gastrointestinal tract disease (11%). Prematurity and low birth weight were the most important risk factors among newborns. The use of antibiotics, invasive procedures, H-2 blockers, multiple blood transfusions and stay length of >= 21 days were the most frequent risk factors among adults. Candida albicans was the most common species in all cases. Conclusions: In this study, C. albicans was the most frequently detected species in candidiasis and risk factors increased the susceptibility of hospitalized patients to acquiring a nosocomial infection by Candida spp. (C) 2011 Revista Iberoamericana de Micologia. Published by Elsevier Espana, S.L. All rights reserved.

Candida spp. isolated from inpatients, the environment, and health practitioners in the Pediatric Unit at the Universitary Hospital of the Jundiai Medical College, State of Sao Paulo, Brazil

Introduction: This study aimed to isolate and identify Candida spp. from the environment, health practitioners, and patients with the presumptive diagnosis of candidiasis in the Pediatric Unit at the Universitary Hospital of the Jundiai Medical College, to verify the production of enzymes regarded as virulence factors, and to determine how susceptible the isolated samples from patients with candidiasis are to antifungal agents. Methods: Between March and November of 2008 a total of 283 samples were taken randomly from the environment and from the hands of health staff, and samples of all the suspected cases of Candida spp. hospital-acquired infection were collected and selected by the Infection Control Committee. The material was processed and the yeast genus Candida was isolated and identified by physiological, microscopic, and macroscopic attributes. Results: The incidence of Candida spp. in the environment and employees was 19.2%. The most frequent species were C. parapsilosis and C. tropicalis among the workers, C. guilliermondii and C. tropicalis in the air, C. lusitanae on the contact surfaces, and C. tropicalis and C. guilliermondii in the climate control equipment. The college hospital had 320 admissions, of which 13 (4%) presented Candida spp. infections; three of them died, two being victims of a C. tropicalis infection and the remaining one of C. albicans. All the Candida spp. in the isolates evidenced sensitivity to amphotericin B, nystatin, and fluconazole. Conclusions: The increase in the rate of hospital-acquired infections caused by Candida spp. indicates the need to take larger measures regarding recurrent control of the environment.

Disseminated Scedosporium prolificans infection and survival of a child with acute lymphoblastic leukemia

Scedosporium prolificans is a saprophytic fungus responsible for an increasing number of infections among immumocompromised hosts. Historically, disseminated infection with this organism has resulted in death. We report on a pediatric patient who developed overwhelming S. prolificans sepsis after induction chemotherapy for acute lymphoblastic leukemia. She is well 18 months after the diagnosis of fungal sepsis and continues to receive chemotherapy for leukemia, which remains in remission.

Ocular mycoses:infections of the eye caused by fungi

Fungi are ubiquitous organisms in nature and can be found in association with healthy eyes. The incidence of actual fungal infection of the eye, however, is relatively low compared with that attributable to viruses and bacteria. Nevertheless, fungal infection of the eye is increasing especially in immuno-compromised patients and a wide variety of fungal infections have now been described worldwide with species of Fusarium, Aspergillus, Candida, and dematiaceous fungi predominating. At present there are a limited number of compounds available to control ocular mycoses while resistance to anti-fungal agents has been growing in recent years, especially to azoles. Several mechanisms of resistance have been identified including modification of sterol synthesis pathways by the fungus, modification of enzymes to reduce the binding of azoles to fungal components and increased efficiency of removal of the azole within fungal cells. Although resistance to amphotericin-B has been reported, it continues to be the most important treatment for life-threatening conditions and more severe ophthalmic infections. Natamycin is often first choice for filamentous fungal keratitis and topical amphotericin-B for Candida keratitis. Continued monitoring of the behaviour of ocular fungi will be essential in future together with the development of new anti-fungal agents.

A multi-faceted diagnostic approach to lung infections in patients with cystic fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

EVALUATION OF THE ANTI-FUNGAL PROPERTIES OF EXTRACTS OF Daniella oliveri

The in vitro anti-fungal activity of leaf and stem bark of Daniella oliveri Rolfe was investigated against selected yeasts and moulds including dermatophytes. Water and methanol were used to extract the powdered leaf and stem bark using cold infusion. Antimicrobial activity was assessed by agar-well diffusion. Phytochemical analysis was carried out using standard procedures. The plant extracts were active against the test organisms at concentrations ranging from 3.125-100 mg/mL. The methanol extracts were more active than the aqueous extracts with the highest inhibition against the yeasts, Candida albicans and Candida krusei (MIC values of 3.125 mg/mL and 6.25 mg/mL respectively). Epidermophyton floccosum and Trichophyton interdigitale were the least inhibited of all the fungal strains. Phytochemical screening revealed the presence of tannins, anthraquinones, flavonoids, cardiac glycosides, alkaloids and saponins. The anti-fungal activity of Daniella oliveri as shown in this study indicates that the plant has the potential of utilisation in the development of chemotherapeutic agents for the treatment of relevant fungal infections.

Orbital apex syndrome due to aspergillosis: case report

We report the case of a 73-year-old female who presented facial numbness and pain in the first division of the trigeminal nerve, ptosis, diplopia and visual loss on the right side for the previous four months. The neurological, radiological and histological examination demonstrated a rare case of invasive fungal aspergillosis of the central nervous system, causing orbital apex syndrome, later transformed in temporal brain abscess. She died ten months later due to respiratory and renal failure in spite of specific antimycotic therapy.

Inpatient dermatological consultations in a university hospital

INTRODUCTION: Dermatology is primarily an outpatient specialty, but it also plays an important role in the care of inpatients. METHODS: We conducted a prospective study that recorded data from inpatient dermatology consultation request forms over a period of four months. The study evaluated 313 requests that led to 566 visits, 86 biopsies, 35 laboratory exams, 41 direct microscopic studies, 18 direct immunofluorescence analyses, 14 skin cultures and a few other exams. RESULTS: The most frequent requesting service was internal medicine (24%), followed by neurology (12%), cardiology (11%), infectious diseases and pediatrics (8% each) and psychiatry and general surgery (6% each). The most frequent diagnostic groups were infectious diseases (25%, divided into fungal infections (13%), bacterial infections (7%) and viral infections (5%)), eczemas (15%) and drug reactions (14%). To our knowledge, this is the first study to attempt to evaluate the impact of the consultations by asking multiple-choice questions that were analyzed by the authors. In 31% of the cases, the consultation was considered extremely relevant because it aided in managing the disease that led to admission or treated a potentially severe dermatological disease. In 58% of the cases, the consultation was considered important because it facilitated diagnosis and/or treatment of a dermatological disease that was unrelated to the reason for admission.

T Helper 1-Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis

Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1-stimulating adjuvant in P. brasiliensis infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

Cell-mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that specific antibodies show different degrees of protection against mycosis. In a previous study, antigens secreted by Sporothrix schenckii induced a specific humoral response in infected animals, mainly against the 70-kDa molecule, indicating a possible participation of antibodies to this antigen in infection control. in the present study, an IgG1 mAb was produced against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. Moreover, in a second schedule treatment, the mAb was injected after infection was established, and again we observed a significant reduction in CFU associated with an increase of IFN-gamma production. Also, the 70-kDa antigen is shown to be a putative adhesin present on the surface of this fungus. In conclusion, we report for the first time the protective effect of a specific antibody against S. schenckii.