Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

INTRODUCTION: In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. METHODS: Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. RESULTS: Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. CONCLUSIONS: Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

Antifungal susceptibility and distribution of Candida spp. isolates from the University Hospital in the municipality of Dourados, State of Mato Grosso do Sul, Brazil

Introduction Hospital infections caused by Candida spp. are a leading cause of morbidity and mortality in hospitalized patients, particularly those that are critically ill or immunocompromised. In this study, the distribution of Candida species in isolates from the University Hospital of the Federal University at Grande Dourados and their in vitro susceptibility to antifungal drugs were analyzed. Methods Yeasts were phenotypically identified using classical methodologies. Antifungal susceptibility tests to amphotericin B and fluconazole were performed using the broth microdilution technique. Results A total of 50 Candida isolates were obtained from hospitalized patients during the study period. We analyzed yeast isolates from urine (n=31; 62%), blood (n=12; 24%), and tracheal secretions (n=7; 14%). The following Candida species were identified: C. tropicalis (n=21; 42%), C. albicans (n=18; 36%), C. glabrata (n=10; 20%), and C. krusei (n=1; 2%). Antifungal susceptibility tests demonstrated that C. albicans was susceptible to both antifungal agents. However, 31.2% of the non-C. albicans Candida isolates displayed dose-dependent susceptibility to fluconazole, and 3.1% were resistant to amphotericin B. Conclusions In contrast to previous reports, our results indicated that C. tropicalis was the most commonly isolated yeast species among the hospital patients. The predominance of non-C. albicans Candida infections confirms the importance of species-level identification for implementing appropriate antifungal therapies.

Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings

ABSTRACT INTRODUCTION: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS: DIF is not very sensitive under clinical laboratory settings.

Blocking tumor exosome release using nanovectorization systems

Exosomes are small membrane vesicles secreted by most cell types, either normal or malignant and are found in most body fluids such as saliva, plasma and breast milk. In the past decade, the interest in these vesicles has been growing more and more since it was found that besides their beneficial functions such as the removal of cellular debris and unnecessary proteins during cell maturation process, they can also interact with other cells and transfer information between them, thus helping diseases like cancer to progress. The present work intended to use gold nanoparticles as vehicles for gene silencing in an attempt to reduce the tumor-derived exosome secretion, regulated by Rab27a protein, and also aimed to compare the exosome secretion between two breast cell lines, MCF7 and MDA. Changes in RAB27A gene expression were measured by Real-time Quantitative PCR and it was revealed a decreased in RAB27A gene expression, as expected. Exosomes were isolated and purified by two different methods, ultracentrifugation and the commercial kit ExoQuick™ Solution, and further characterized using Western Blot analysis. ExoQuick™ Solution was proven to be the most efficient method for exosome isolation and it was revealed that MDA cells secrete more exosomes. Furthermore, the isolated MCF7-derived exosomes were placed together with a normal bronchial/tracheal epithelial cell line (BTEC) for an additional assay, which aimed to observe the uptake of exosomes by other cells and the exosomes’ capability of promoting cell-cell communication. This observation was made based on alterations in the expression levels of c-Myc and miR-21 genes and the fact that they both have an increased expression in BTEC cells incubated with tumor-derived exosomes when compared to control cells (without incubation with the exosomes) lead us to the conclusion that the exosome uptake and exchange of information between the exosomes and the normal cells did occurred.

Looking for mechanisms regulating lung growth in CDH: rat and human studies

Tese de Doutoramento em Ciências da Saúde

Anatomy and histology of Embolyntha batesi MacLahlan, 1877 (Embiidina)

The Embioptera are rather generalized insects whose internal anatomy is simple and not subject to great modifications. For this reason these insects form an ideal group for elementary anatomical and histological studies (fig. 2). The digestive tract is a long, simple tube without convolutions or diverticulae from the buccal cavity to the rectum. The buccal structures are of the chewing type. The oesophagus and ingluvia are differentiated only by slight dilation of their walls. In nymphs and females the proventriculus is very distinct due to folds which flatten as the structure becomes packed with food. The enteron is the largest in such forms and in both sexes limited caudally by the Malpighian tubules. The proctodeus has six large rectal papillae. The nervous system is complete with only the fifth abdominal segment lacking a ganglion in the metathorax includes the ganglion of the first abdominal segment. The brain exhibits very clear structure in histological sections. The tracheal system includes two pairs of thoracic spiracles and eight abdominal pairs. Only th metathoracic spiracle has an air expiration function; all others serve for inspiration. Various structures in the spiracles protect the atrium. The circulatory system includes a long, simple dorsal vessel which extends forward from the ninth abdominal segment into the cranium. It opens anteriorly near the circumoesophageal connectives. The dorsal vessel has a pair of ostia and valves corresponding to each abdominal and thoracic segment. It lacks the diverticulae or folds commonly found in more highly evolved insects. The excretory system is represented by Malphighian tubules, pericardial cells, and fat-body. The number and disposition of Malpighian tubules is variable within the order. The pericardial cells are localized around the entire dorsal vessel up to the opening of the aorta in the head. The fat-bodies form compact layers in the dorsal and ventral regions of the body. In males they are more developed in the abdominal region. The mandibles, maxillae, and salivary glands are of a simple type with very few cytological modifications. Only the salivary glands which extend into the mesothoracic region show appreciable specialization. The reproductive system is bi-sixual and shows considerable sexual dimorphism. Males have five pair of testes with a metameric disposition, two distinct ducts, two epidymis, and the ejaculatory organs. The accessory glands vary in number and size and open in the anterior portion of the ejaculatory duct. The female reproductive organs are of the panoistic type. The system includes five pairs of ovarioles, two long paired oviducts a small, unpaired oviduct and the spermatheca which opens in the vagina. Reproduction usually involves a union of male and female gametes, and eggs are usually laid in clusters attached to a substrate.

Videolaryngoscopy improves intubation condition in morbidly obese patients

Résumé : Contexte clinique et objectifs: l'intubation oro-trachéale peut être plus difficile chez les patients obèses morbides (index de masse corporelle BMI > 35 kg/m2) que chez les patients non-obèses. Récemment, de nouveaux instruments permettant une intubation assistée au moyen d'une caméra ont été développés. Notre expérience pratique avec la vidéolaryngoscopie nous a conduit à l'hypothèse que celle-ci pourrait améliorer la vision laryngoscopique chez cette population spécifique et de ce fait faciliter l'intubation. Le but de cette étude était donc d'évaluer le bénéfice du vidéolaryngoscope sur le grade de laryngoscopie chez le patient obèse morbide. Résultats : le grade laryngoscopique fut abaissé de manière significative avec le vidéolaryngoscope comparé à la vision directe avec un laryngoscope standard. Lorsque le grade laryngoscopique était plus grand que 1 à la laryngoscopie directe, il fut dans la grande majorité des cas (93% des patients) abaissé avec le vidéolaryngoscope. Chez les 7 % restant, le grade laryngoscopique resta identique. Conclusions : chez le patient obèse morbide, l'utilisation du vidéolaryngoscope améliore de manière significative la visualisation du larynx et de ce fait facilite l'intubation. Une application systématique de ce procédé pourrait donc permettre de réduire l'incidence d'une intubation difficile ainsi que ses conséquences chez cette population de patients. Summary : Background and objective: Tracheal intubation may be more difficult in morbidly obese patients (body mass index >35 kgM-2) than in the non-obese. Recently, new video-assisted intubation devices have been developed. After some experience with videolaryngoscopy, we hypothesized that it could improve the laryngoscopic view in this specific population and therefore facilitate intubation. The aim of this study was to assess the benefit of a videolaryngoscope on the grade of laryngoscopy in morbid obesity. Methods: We studied 80 morbidly obese patients undergoing bariatric surgery. They were randomly assigned to one of two groups. One group was intubated with the help of the videolaryngoscope and in the control group the screen of the videolaryngoscope was hidden to the intubating anaesthesiologist. The primary end-point of the study was to assess in both groups the Cormack and Lehane direct and indirect grades of laryngoscopy. The duration of intubation, the number of attempts needed as well as the minimal SPO2 reached during the intubation process were measured. Results: Grade of laryngoscopy was significantly lower with the videolaryngoscope compared with the direct vision (P < 0.001). When the grade of laryngoscopy was higher than one with the direct laryngoscopy (n = 30), it was lower in 28 cases with the videolaryngoscope and remained the same only in two cases (P < 0.001). The minimal SPO2 reached during the intubation was higher with the videolaryngoscope but it did not reach statistical significance. Conclusions: In morbidly obese patients, the use of the videolaryngoscope significantly improves the visualization of the larynx and thereby facilitates intubation.

Automatic Mallampati Classification Using Active Appearance Models

Difficult tracheal intubation assessment is an important research topic in anesthesia as failed intubations are important causes of mortality in anesthetic practice. The modified Mallampati score is widely used, alone or in conjunction with other criteria, to predict the difficulty of intubation. This work presents an automatic method to assess the modified Mallampati score from an image of a patient with the mouth wide open. For this purpose we propose an active appearance models (AAM) based method and use linear support vector machines (SVM) to select a subset of relevant features obtained using the AAM. This feature selection step proves to be essential as it improves drastically the performance of classification, which is obtained using SVM with RBF kernel and majority voting. We test our method on images of 100 patients undergoing elective surgery and achieve 97.9% accuracy in the leave-one-out crossvalidation test and provide a key element to an automatic difficult intubation assessment system.

Parasite Genotypically Related to a Monoxenous Trypanosomatid of Dog's Flea Causing Opportunistic Infection in an HIV Positive Patient

An HIV positive patient presenting a clinical picture of visceral leishmaniasis co-infection was submitted to a bone marrow aspiration after admission to hospital. Amastigotes forms were seen in the bone marrow aspirate and the parasite grew in culture as promastigotes. Molecular analyses showed that the flagellates isolated did not belong to the genera Leishmania, Trypanosoma or Sauroleishmania. It was not possible to establish infection in laboratory animals. In vitro culture of mouse peritoneal macrophages revealed the invasion of the host cells by the flagellates and their killing 48 hr after infection. Opportunistic infection with an insect trypanosomatid was suspected. Further hybridization analyses against a pannel of different monoxenous and heteroxenous trypanosomatids showed kDNA cross-homology with Leptomonas pulexsimulantis a trypanosomatid found in the dog's flea

Sensitivity of a Vacuum Aspiratory Culture Technique for Diagnosis of Localized Cutaneous Leishmaniasis in an Endemic Area of Leishmania (Viannia) braziliensis Transmission

Sixty eight patients with localized cutaneous leishmaniasis from an area with Leishmania (Viannia) braziliensis transmission had cultures performed with a modified Marzochi´s vacuum aspiratory puncture technique to establish sensitivity and contamination rate with this new method. Overall sensitivity of three aspirates was 47.1%; (CI95% 39.4; 59.4) significantly greater than the sensitivity of a single one aspirate. Fungal contamination was observed in 6/204 (2.9%) inoculated culture tubes. We recommend that this useful technique should be adopted as routine for primary isolation of L. (V.) braziliensis from localized cutaneous ulcers.

Repair of non-circumferential cervical trachea defects by three different latissimus dorsi flaps. A comparative studyin an experimental setting.

BACKGROUND: Large intrathoracic airway defects may be closed using a pedicled latissimus dorsi (LD) flap, with rewarding results. This study addresses the question of whether this holds true for extrathoracic non-circumferential tracheal defects. METHODS: A cervical segment of the trachea of 4 x 1 cm was resected in 9 white male pigs. The defect was stented with a silicone stent for 3 months and closed either by an LD flap alone (group a, n = 3), an LD flap with an attached rib segment covered by pleura (group b, n = 3), or an LD flap reinforced by a perforated polylactide (MacroPore) plate (group c, n = 3). The trachea was assessed by rigid endoscopy at 3 and 4 months and histologically at 4 months postoperatively. RESULTS: The degree of stenosis at the level of the reconstruction at 4 months was 25, 50 and 75% in group a, 15, 50 and 60% in group b, and 20, 95 and 95% in group c, respectively. The percentage of the defect covered by columnar epithelium was 100% in all animals of group a, 60, 100 and 100% in group b, and 10, 0 and 0% in group c. Resorption of the rib was seen in all animals of group b and obstructive inflammatory polyps were found in 2 animals of group c. CONCLUSION: Pedicled LD flaps provided less satisfactory results for closure of large non-circumferential extrathoracic airway defects than observed after intrathoracic reconstruction. A pedicled rib segment added to the LD flap did not improve the results obtained from LD flap repair alone, and an embedded MacroPore prosthesis may result in severe airway stenosis due to plate migration and intense inflammatory reaction protruding into the tracheal lumen.

Standardization of in-house polymerase chain reaction for the identification of Mycobacterium tuberculosis at the reference tropical disease hospital in the State of Goiás, Brazil

This study compares smear, growth in Lowenstein-Jensen medium, and in-house polymerase chain reaction (PCR) techniques for the detection of Mycobacterium tuberculosis. A total of 72 specimens from 72 patients with clinical symptoms of tuberculosis, including 70 sputum and two bronchial aspirate samples, were tested in parallel by smear, culture, and in-house PCR techniques. From these, 48 (66.6%) were negative by the 3 methods, 2 (2.8%) were smear positive and negative by culture and in-house PCR, 11 (15.3%) were both smear and culture negative, and in-house PCR positive, 7 (9.7%) were positive by the 3 methods, 2 (2.8%) were positive by smear and culture, and negative by PCR, 2 (2.8%) were positive by culture and PCR, but smear negative. After the resolution of discrepancies in PCR results, the sensitivity and specificity for in-house PCR technique to M. tuberculosis relative to the culture, were 81.8% and 81.9%, respectively. These results confirm that this method, in-house PCR, may be a sensitive and specific technique for M. tuberculosis detection, occurring in both positive and negative smear and negative cultures.

Clinical characteristics and outcome of patients admitted to a medico- surgical ICU requiring non invasive ventilation (NIV) for hypercapnic respiratory failure

Introduction Because it decreases intubation rate and mortality, NIV has become first-line treatment in case of hypercapnic acute respiratory failure (HARF). Whether this approach is equally successful for all categories of HARF patients is however debated. We assessed if any clinical characteristics of HARF patients were associated with NIV intensity, success, and outcome, in order to identify prognostic factors. Methods Retrospective analysis of the clinical database (clinical information system and MDSi) of patients consecutively admitted to our medico-surgical ICU, presenting with HARF (defined as PaCO2 > 50 mmHg), and receiving NIV between May 2008 and December 2010. Demographic data, medical diagnoses (including documented chronic lung disease), reason for ICU hospitalization, recent surgical interventions, SAPS II and McCabe scores were extracted from the database. Total duration of NIV and the need for tracheal intubation during the 5 days following the first hypercapnia documentation, as well as ICU, hospital and one year mortality were recorded. Results are reported as median [IQR]. Comparisons were carried out with Chi2 or Kruskal-Wallis tests, p<0.05 (*). Results Two hundred and twenty patients were included. NIV successful patients received 16 [9-31] hours of NIV for up to 5 days. Fifty patients (22.7%) were intubated 11 [2-34] hours after HARF occurence, after having receiving 10 [5-21] hours of NIV. Intubation was correlated with increased ICU (18% vs. 6%, p<0.05) and hospital (42% vs. 31%, p>0.05) mortality. SAPS II score was related to increasing ICU (51 [29-74] vs. 23 [12-41]%, p<0.05), hospital (37% [20-59] vs 20% [12-37], p<0.05) and one year mortality (35% vs 20%, p<0.05). Surgical patients were less frequent among hospital fatalities (28.8% vs. 46.3%, p<0.05, RR 0.8 [0-6-0.9]). Nineteen patients (8.6%) died in the ICU, 73 (33.2%) during their hospital stay and 108 (49.1%) were dead one year after HARF. Conclusion The practice to start NIV in all suitable patients suffering from HARF is appropriate. NIV can safely and appropriately be used in patients suffering from HARF from an origin different from COPD exacerbation. Beside usual predictors of severity such as severity score (SAPS II) appear to be associated with increased mortality. Although ICU mortality was low in our patients, hospital and one year mortality were substantial. Surgical patients, although undergoing a similar ICU course, had a better hospital and one year outcome.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

A natural case of co-infection by Leishmania and Trypanosoma is reported in a dog (Canis familiaris) in south- western state of Mato Grosso do Sul, Brazil. Both amastigote and trypomastigote forms were observed after Giemsa staining of cytological preparations of the dog's bone marrow aspirate. No parasite was detected using medium culture inoculation of the sample. DNA obtained from the bone marrow aspirate sample and from the blood buffy coat was submitted to polymerase chain reaction (PCR) with a set of rDNA-based primers S4/S12. The nucleotide sequence of the PCR product was identical to that of Trypanosoma (Trypanozoon) evansi. The S4/S12 PCR was then used as template in a nested-PCR using a specific Leishmania set S17/S18 as primers, to explain the amastigote forms. The nucleotide sequence of the new PCR product was identical to that of Leishmania (Leishmania) chagasi. This case, as far as we know, is the first report of a dog co-infected with these parasites, suggesting that besides L. (L.) chagasi, the natural transmission of T. (T.) evansi occurs in the area under study.

Closure of large intrathoracic airway defects using extrathoracic muscle flaps.

BACKGROUND: Prospective assessment of pedicled extrathoracic muscle flaps for the closure of large intrathoracic airway defects after noncircumferential resection in situations where an end-to-end reconstruction seemed risky (defects of > 4-cm length, desmoplastic reactions after previous infection or radiochemotherapy). METHODS: From 1996 to 2001, 13 intrathoracic muscle transpositions (6 latissimus dorsi and 7 serratus anterior muscle flaps) were performed to close defects of the intrathoracic airways after noncircumferential resection for tumor (n = 5), large tracheoesophageal fistula (n = 2), delayed tracheal injury (n = 1) and bronchopleural fistula (n = 5). In 2 patients, the extent of the tracheal defect required reinforcement of the reconstruction by use of a rib segment embedded into the muscle flap followed by temporary tracheal stenting. Patient follow-up was by clinical examination bronchoscopy and biopsy, pulmonary function tests, and dynamic virtual bronchoscopy by computed tomographic (CT) scan during inspiration and expiration. RESULTS: The airway defects ranged from 2 x 1 cm to 8 x 4 cm and involved up to 50% of the airway circumference. They were all successfully closed using muscle flaps with no mortality and all patients were extubated within 24 hours. Bronchoscopy revealed epithelialization of the reconstructions without dehiscence, stenosis, or recurrence of fistulas. The flow-volume loop was preserved in all patients and dynamic virtual bronchoscopy revealed no significant difference in the endoluminal cross surface areas of the airway between inspiration and expiration above (45 +/- 21 mm(2)), at the site (76 +/- 23 mm(2)) and below the reconstruction (65 +/- 40 mm(2)). CONCLUSIONS: Intrathoracic airway defects of up to 50% of the circumference may be repaired using extrathoracic muscle flaps when an end-to-end reconstruction is not feasible.