Roadmap to DILI research in Europe. A proposal from COST action ProEuroDILINet.

In the current article the aims for a constructive way forward in Drug-Induced Liver Injury (DILI) are to highlight the most important priorities in research and clinical science, therefore supporting a more informed, focused, and better funded future for European DILI research. This Roadmap aims to identify key challenges, define a shared vision across all stakeholders for the opportunities to overcome these challenges and propose a high-quality research program to achieve progress on the prediction, prevention, diagnosis and management of this condition and impact on healthcare practice in the field of DILI. This will involve 1. Creation of a database encompassing optimised case report form for prospectively identified DILI cases with well-characterised controls with competing diagnoses, biological samples, and imaging data; 2. Establishing of preclinical models to improve the assessment and prediction of hepatotoxicity in humans to guide future drug safety testing; 3. Emphasis on implementation science and 4. Enhanced collaboration between drug-developers, clinicians and regulatory scientists. This proposed operational framework will advance DILI research and may bring together basic, applied, translational and clinical research in DILI.

Assessing cognitive decline in the aging brain: lessons from rodent and human studies.

As life expectancy continues to increase worldwide, age-related dysfunction will largely impact our societies in the future. Aging is well established to promote the deterioration of cognitive function and is the primary risk factor for the development of prevalent neurological disorders. Even in the absence of dementia, age-related cognitive decline impacts specific types of memories and brain structures in humans and animal models. Despite this, preclinical and clinical studies that investigate age-related changes in brain physiology often use largely different methods, which hinders the translational potential of findings. This review seeks to integrate what is known about age-related changes in the brain with analogue cognitive tests used in humans and rodent studies, ranging from "pen and paper" tests to virtual-reality-based paradigms. Finally, we draw parallels between the behavior paradigms used in research compared to the enrollment into clinical trials that aim to study age-related cognitive decline.

'Cloudbuster': a Python-based open source application for three-dimensional reconstruction and quantification of stacked biological imaging samples.

Three-dimensional (3D) spheroid cultures are generating increasing interest in cancer research, e.g. for the evaluation of pharmacological effects of novel small molecule inhibitors. This is mainly due to the fact that such 3D structures reflect physiological characteristics of tumours and the cellular microenvironments they reside in more faithfully than two-dimensional (2D) cell cultures; in addition, they allow the reduction of animal experiments while providing significantly relevant human-based models. Quantification of such organoid structures as well as the mainly slice-based acquisition and thus forced 2D representation of 3D spheroids provide a challenge for the interpretation of the associated generated data. Here, we provide a novel open-source workflow to reconstruct a 3D entity from slice-recorded microscopical images with or without treatment with anti-migratory small molecule inhibitors. This reconstruction produces distinct point clouds as basis for subsequent comparison of basic readout parameters using average computer processor, memory and graphics resources within an acceptable time frame. We were able to validate the usefulness of this workflow using 3D data generated by various imaging techniques, including z-stacks from confocal microscopy and histochemically labelled spheroid sectioning, and demonstrate the possibility to accurately characterize inhibitor effects in great detail.

Serologic evidence for Schmallenberg virus circulation at high altitude, Central Portugal, 2015-2016.

A few years after the initial 2011 large scale Schmallenberg virus (SBV) epidemic that affected Europe, a subsequent decrease in new SBV infections was observed presumably associated with natural substitution of previously exposed animals. In the present work, a 2-year prospective serosurvey was performed to evaluate SBV circulation in a population of sheep living at high altitude in the central region of Portugal and with restricted movement. Blood from a representative sample of 168 autochthonous sheep was collected in 2015 and again in 2016, and tested for the presence of anti-SBV IgG by ELISA. Of the 2015 sample collection, seven animals tested positive for anti-SBV IgG, corresponding to a seroprevalence of 4.2% while of the 2016 sample collection, 10 presented SBV antibodies, showing a seroprevalence of 6.0% (p = 0.619). Results show that SBV is endemic in sheep of central Portugal, even in herds at high altitude locations. When comparing anti-SBV seroprevalences of 2015/2016 found in this study, to one detected in 2014 in the same region, a steep decrease could be observed (p < 0.001). This is in accordance with what has been documented in Western European countries, where a decrease in the number of SBV-infected sheep has been found, a fact which may pose a new threat for SBV re-emergence.

A biocompatible decellularized pulp scaffold for regenerative endodontics.

AIM: To investigate the feasibility of decellularizing the entire dental pulp using a mild treatment protocol to develop a decellularized biological extracellular matrix scaffold for use in regenerative endodontic procedures. METHODOLOGY: Decellularized human dental pulps were assessed using histological and immunohistochemical methods, scanning electron microscope and DNA quantification assay. Cytotoxicity assays to determine decellularized scaffold biocompatibility were also performed. Decellularized scaffolds were seeded with human dental pulp stem cells and cell viability assessed using Live/Dead® stain. Quantitative data were analysed statistically using Student's t-test and one-way analysis of variance to compare mean values between groups depending on group numbers. RESULTS: Assessment of decellularized tissues revealed an acellular matrix with preservation of native tissue histoarchitecture and composition. Decellularized tissues showed no evidence of cytotoxicity, with cell growth in direct contact with the scaffold and no reduction in cellular activity following extract incubation. Furthermore, the scaffold was able to support human dental pulp stem cell viability and attachment following recellularization. CONCLUSIONS: Promising results were observed in developing a decellularized biological scaffold derived from the dental pulp with the perseveration of extracellular structural components which are required for tissue-specific regeneration.

Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

Assessment of Corneal Epithelial Thickness in Asymmetric Keratoconic Eyes and Normal Eyes Using Fourier Domain Optical Coherence Tomography.

Purpose. To compare the characteristics of asymmetric keratoconic eyes and normal eyes by Fourier domain optical coherence tomography (OCT) corneal mapping. Methods. Retrospective corneal and epithelial thickness OCT data for 74 patients were compared in three groups of eyes: keratoconic (n = 22) and normal fellow eyes (n = 22) in patients with asymmetric keratoconus and normal eyes (n = 104) in healthy subjects. Areas under the curve (AUC) of receiver operator characteristic (ROC) curves for each variable were compared across groups to indicate their discrimination capacity. Results. Three variables were found to differ significantly between fellow eyes and normal eyes (all p < 0.05): minimum corneal thickness, thinnest corneal point, and central corneal thickness. These variables combined showed a high discrimination power to differentiate fellow eyes from normal eyes indicated by an AUC of 0.840 (95% CI: 0.762-0.918). Conclusions. Our findings indicate that topographically normal fellow eyes in patients with very asymmetric keratoconus differ from the eyes of healthy individuals in terms of their corneal epithelial and pachymetry maps. This type of information could be useful for an early diagnosis of keratoconus in topographically normal eyes.

Multicentre study highlighting clinical relevance of new high-throughput methodologies in molecular epidemiology of Pneumocystis jirovecii pneumonia.

Pneumocystis jirovecii causes severe interstitial pneumonia (PcP) in immunosuppressed patients. This multicentre study assessed the distribution frequencies of epidemiologically relevant genetic markers of P. jirovecii in different geographic populations from Portugal, the USA, Spain, Cuba and Mozambique, and the relationship between the molecular data and the geographical and clinical information, based on a multifactorial approach. The high-throughput typing strategy for P. jirovecii characterization consisted of DNA pooling using quantitative real-time PCR followed by multiplex-PCR/single base extension. The frequencies of relevant P. jirovecii single nucleotide polymorphisms (mt85, SOD110, SOD215, DHFR312, DHPS165 and DHPS171) encoded at four loci were estimated in ten DNA pooled samples representing a total of 182 individual samples. Putative multilocus genotypes of P. jirovecii were shown to be clustered due to geographic differences but were also dependent on clinical characteristics of the populations studied. The haplotype DHFR312T/SOD110C/SOD215T was associated with severe AIDS-related PcP and high P. jirovecii burdens. The frequencies of this genetic variant of P. jirovecii were significantly higher in patients with AIDS-related PcP from Portugal and the USA than in the colonized patients from Portugal, and Spain, and children infected with P. jirovecii from Cuba or Mozambique, highlighting the importance of this haplotype, apparently associated with the severity of the disease and specific clinical groups. Patients from the USA and Mozambique showed higher rates of DHPS mutants, which may suggest the circulation of P. jirovecii organisms potentially related with trimethoprim-sulfamethoxazole resistance in those geographical regions. This report assessed the worldwide distribution of P. jirovecii haplotypes and their epidemiological impact in distinct geographic and clinical populations.

Clinical and epidemiological profile of women with breast cancer managed in a public referral hospital in northeastern Brazil.

AIM: To evaluate the clinical and epidemiological profile of women with breast cancer, managed in the Breast Service at Hospital Get6lio Vargas (HGV). MATERIALS AND METHODS: A descriptive, observational cross-sectional study, involving 174 breast cancer patients, managed at the Breast Service at HGV in Teresina, in the northeast of Brazil, from May 2011 to June 2014. Epidemiological variables included age group, age at menarche, age at first childbirth, and menopause. The tumor characteristics studied were histological type, TNM classification, and staging. The results were organized in distribution tables. RESULTS: Breast carcinoma was more common in patients aged 60 and over, in multiparous women, and in women who were menopausal before age 55. The most common histological type was ductal invasive carcinoma T2N1MO (Stage IIB) in 83 (48%) women. CONCLUSION: The current study shows that the majority of carcinomas were Stage II or advanced tumors, with clinically positive axillary lymph node status and weak correlation with reproductive risk factors.

Tumor necrosis factor-α and interleukin-6 gene polymorphism association with susceptibility to celiac disease in Italian patients.

The aim of this research was to study polymorphisms in the genes encoding cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in patients with celiac disease (CD) antigens DQ2 (DQ2-positive) or DQ8 (DQ8-positive). We compared the results with healthy controls to determine whether any of the polymorphisms have a role in susceptibility to CD. A case-control of 192 patients with CD (96 DQ2-positive and 96 DQ8-positive) and 96 healthy controls from northeast Italy were included in the study. Analysis of single nucleotide polymorphisms (SNPs) was carried out using the polymerase chain reaction-restriction fragment length polymorphism method. Significant differences for the TNF-α(-308 G>A) polymorphism were observed when we compared the flowing groups: DQ2-positive with controls [odds ratio (OR) = 0.45, P = 0.0002]; DQ8-positive with controls (OR = 3.55, P < 0.0001); and DQ2-positive with DQ8-positive (OR = 0.12, P < 0.0001). We did not observe a statistically significant association between IL-6 (-174 G>C) polymorphism and CD (P > 0.05). Our results suggest that TNF-α(-308 G>A) polymorphism may play a role in susceptibility to CD in Italian patients.

Interaction between resource identity and bacterial community composition regulates bacterial respiration in aquatic ecosystems.

Resource identity and composition structure bacterial community, which in turn determines the magnitude of bacterial processes and ecological services. However, the complex interaction between resource identity and bacterial community composition (BCC) has been poorly understood so far. Using aquatic microcosms, we tested whether and how resource identity interacts with BCC in regulating bacterial respiration and bacterial functional diversity. Different aquatic macrophyte leachates were used as different carbon resources while BCC was manipulated through successional changes of bacterial populations in batch cultures. We observed that the same BCC treatment respired differently on each carbon resource; these resources also supported different amounts of bacterial functional diversity. There was no clear linear pattern of bacterial respiration in relation to time succession of bacterial communities in all leachates, i.e. differences on bacterial respiration between different BCC were rather idiosyncratic. Resource identity regulated the magnitude of respiration of each BCC, e.g. Ultricularia foliosa leachate sustained the greatest bacterial functional diversity and lowest rates of bacterial respiration in all BCC. We conclude that both resource identity and the BCC interact affecting the pattern and the magnitude of bacterial respiration in aquatic ecosystems.

Interaction between resource identity and bacterial community composition regulates bacterial respiration in aquatic ecosystems / A interação entre a identidade do recurso e a composição da comunidade bacteriana regula a respiração bacteriana em ecossistemas aquáticos

Abstract Resource identity and composition structure bacterial community, which in turn determines the magnitude of bacterial processes and ecological services. However, the complex interaction between resource identity and bacterial community composition (BCC) has been poorly understood so far. Using aquatic microcosms, we tested whether and how resource identity interacts with BCC in regulating bacterial respiration and bacterial functional diversity. Different aquatic macrophyte leachates were used as different carbon resources while BCC was manipulated through successional changes of bacterial populations in batch cultures. We observed that the same BCC treatment respired differently on each carbon resource; these resources also supported different amounts of bacterial functional diversity. There was no clear linear pattern of bacterial respiration in relation to time succession of bacterial communities in all leachates, i.e. differences on bacterial respiration between different BCC were rather idiosyncratic. Resource identity regulated the magnitude of respiration of each BCC, e.g. Ultricularia foliosa leachate sustained the greatest bacterial functional diversity and lowest rates of bacterial respiration in all BCC. We conclude that both resource identity and the BCC interact affecting the pattern and the magnitude of bacterial respiration in aquatic ecosystems.

Resumo A identidade e a composição do recurso estruturam a comunidade bacteriana, que, por sua vez, determina a magnitude dos processos bacterianos e seus serviços ecológicos. Porém, a complexa interação entre a identidade do recursos e a composição da comunidade bacteriana (CCB) tem sido pouco avaliada até o momento. Utilizando microcosmos aquáticos, nós testamos quando e como a identidade do recurso interage com a CCB na regulação da respiração bacteriana e da diversidade funcional bacteriana. Diferentes lixiviados de macrófitas aquáticas foram utilizados como diferentes fontes de carbono, enquanto que a CCB foi manipulada através de mudanças sucessionais das populações bacterianas em culturas de recrescimento. Nós observamos que tratamentos com a mesma CCB respiraram diferentemente em cada fonte de carbono; diferentes fontes também suportaram diferentes valores de diversidade funcional bacteriana. Não houve padrão linear claro de mudança na respiração bacteriana em relação ao tempo de sucessão das comunidades bacterianas nos lixiviados, i.e. diferenças na respiração bacteriana entre diferentes CCB foram idiossincráticas. A identidade do recurso regulou a magnitude da respiração, em cada CCB, e.g. o lixiviado de Ultricularia foliosa sustentou os maiores valores de diversidade funcional bacteriana e as menores taxas de respiração bacteriana em todas as CCB. Nós concluímos que a identidade do recurso e a CCB interagem afetando o padrão e a magnitude da respiração bacteriana em ecossistemas aquáticos.

Interaction between resource identity and bacterial community composition regulates bacterial respiration in aquatic ecosystems

Abstract Resource identity and composition structure bacterial community, which in turn determines the magnitude of bacterial processes and ecological services. However, the complex interaction between resource identity and bacterial community composition (BCC) has been poorly understood so far. Using aquatic microcosms, we tested whether and how resource identity interacts with BCC in regulating bacterial respiration and bacterial functional diversity. Different aquatic macrophyte leachates were used as different carbon resources while BCC was manipulated through successional changes of bacterial populations in batch cultures. We observed that the same BCC treatment respired differently on each carbon resource; these resources also supported different amounts of bacterial functional diversity. There was no clear linear pattern of bacterial respiration in relation to time succession of bacterial communities in all leachates, i.e. differences on bacterial respiration between different BCC were rather idiosyncratic. Resource identity regulated the magnitude of respiration of each BCC, e.g. Ultricularia foliosa leachate sustained the greatest bacterial functional diversity and lowest rates of bacterial respiration in all BCC. We conclude that both resource identity and the BCC interact affecting the pattern and the magnitude of bacterial respiration in aquatic ecosystems.

Resumo A identidade e a composição do recurso estruturam a comunidade bacteriana, que, por sua vez, determina a magnitude dos processos bacterianos e seus serviços ecológicos. Porém, a complexa interação entre a identidade do recursos e a composição da comunidade bacteriana (CCB) tem sido pouco avaliada até o momento. Utilizando microcosmos aquáticos, nós testamos quando e como a identidade do recurso interage com a CCB na regulação da respiração bacteriana e da diversidade funcional bacteriana. Diferentes lixiviados de macrófitas aquáticas foram utilizados como diferentes fontes de carbono, enquanto que a CCB foi manipulada através de mudanças sucessionais das populações bacterianas em culturas de recrescimento. Nós observamos que tratamentos com a mesma CCB respiraram diferentemente em cada fonte de carbono; diferentes fontes também suportaram diferentes valores de diversidade funcional bacteriana. Não houve padrão linear claro de mudança na respiração bacteriana em relação ao tempo de sucessão das comunidades bacterianas nos lixiviados, i.e. diferenças na respiração bacteriana entre diferentes CCB foram idiossincráticas. A identidade do recurso regulou a magnitude da respiração, em cada CCB, e.g. o lixiviado de Ultricularia foliosa sustentou os maiores valores de diversidade funcional bacteriana e as menores taxas de respiração bacteriana em todas as CCB. Nós concluímos que a identidade do recurso e a CCB interagem afetando o padrão e a magnitude da respiração bacteriana em ecossistemas aquáticos.

Diagnosis of Pneumocystis pneumonia: evaluation of four serologic biomarkers.

The diagnosis of Pneumocystis pneumonia (PCP) relies on microscopic visualization of Pneumocystis jirovecii organisms or DNA detection in pulmonary specimens. This study aimed to assess the usefulness of (1-3)-ß-d-glucan (BG), Krebs von den Lungen-6 antigen (KL-6), lactate dehydrogenase (LDH) and S-adenosyl methionine (SAM) as serologic biomarkers in the diagnosis of PCP. Serum levels of BG, KL-6, LDH and SAM were investigated in 145 Portuguese patients, 50 patients from the Netherlands, 25 Spanish patients and 40 Portuguese blood donors. Data on clinical presentation, chest imaging and gasometry tests were available. PCP cases were confirmed by microscopy and PCR techniques. A cost-effectiveness analysis was performed. BG was found to be the most reliable serologic biomarker for PCP diagnosis, followed by KL-6, LDH and SAM. The BG/KL-6 combination test was the most accurate serologic approach for PCP diagnosis, with 94.3% sensitivity and 89.6% specificity. Although less sensitive/specific than the reference standard classic methods based on bronchoalveolar lavage followed by microscopic or molecular detection of P. jirovecii organisms, the BG/KL-6 test may provide a less onerous procedure for PCP diagnosis, as it uses a minimally invasive and inexpensive specimen (blood), which may be also a major benefit for the patient's care. The BG/KL-6 combination test should be interpreted within the clinical context, and it may be used as a preliminary screening test in patients with primary suspicion of PCP, or as an alternative diagnostic procedure in patients with respiratory failure or in children, avoiding the associated risk of complications by the use of bronchoscopy.

Providing preoperative information for children undergoing surgery: a randomized study testing different types of educational material to reduce children's preoperative worries.

This study developed three types of educational preoperative materials and examined their efficacy in preparing children for surgery by analysing children's preoperative worries and parental anxiety. The sample was recruited from three hospitals in Lisbon and consisted of 125 children, aged 8-12 years, scheduled to undergo outpatient surgery. The participants were randomly assigned to one of the seven independent conditions that were combined into the following three main groups: an experimental group, which received educational materials with information about surgery and hospitalization (a board game, a video or a booklet); a comparison group, which received entertaining material with the same format type; and a control group, which did not receive any material. Children's preoperative worries and parental anxiety were evaluated after the experimental manipulation. Children who received educational materials were significantly less worried about surgery and hospital procedures than children in the comparison and the control groups, although no statistically differences were found between the type of materials within the experimental group, and no significant effect occurred on parental state anxiety. These results do however support the hypothesis that providing preoperative materials with educational information reduce children's preoperative worries.

(1-3)-beta-D-glucan in association with lactate dehydrogenase as biomarkers of Pneumocystis pneumonia (PcP) in HIV-infected patients.

Pneumocystis pneumonia (PcP) is a major HIV-related illness caused by Pneumocystis jirovecii. Definitive diagnosis of PcP requires microscopic detection of P. jirovecii in pulmonary specimens. The objective of this study was to evaluate the usefulness of two serum markers in the diagnosis of PcP. Serum levels of (1-3)-beta-d-glucan (BG) and lactate dehydrogenase (LDH) were investigated in 100 HIV-positive adult patients and 50 healthy blood donors. PcP cases were confirmed using indirect immunofluorescence with monoclonal anti-Pneumocystis antibodies and nested-PCR to amplify the large subunit mitochondrial rRNA gene of P. jirovecii in pulmonary specimens. BG and LDH levels in serum were measured using quantitative microplate-based assays. BG and LDH positive sera were statistically associated with PcP cases (P ≤ 0.001). Sensitivity, specificity, positive/negative predictive values (PPV/NPV), and positive/negative likelihood ratios (PLR/NLR) were 91.3 %, 61.3 %, 85.1 %, 79.2 %, 2.359, and 0.142, respectively, for the BG kit assay, and 91.3 %, 35.5 %, 75.9 %, 64.7 %, 1.415 and 0.245, respectively, for the LDH test. Serologic markers levels combined with the clinical diagnostic criteria for PcP were evaluated for their usefulness in diagnosis of PcP. The most promising cutoff levels for diagnosis of PcP were determined to be 400 pg/ml of BG and 350 U/l of LDH, which combined with clinical data presented 92.8 % sensitivity, 83.9 % specificity, 92.8 % PPV, 83.9 % NPV, 5.764 PLR and 0.086 NLR (P < 0.001). This study confirmed that BG is a reliable indicator for detecting P. jirovecii infection. The combination between BG/LDH levels and clinical data is a promising alternative approach for PcP diagnosis.

[Circumferential viscocanalostomy with suture tensioning in Schlemm canal (canaloplasty)-one year experience]. / Viscocanalostomía circunferencial con sutura de tensión en el canal de Schlemm (canaloplastia): un año de experiencia.

PURPOSE: To study the efficacy and the safety of ultrasound biomicroscopy assisted canaloplasty in the treatment of open-angle glaucoma (OAG) METHODS: A prospective study of uncontrolled open-angle glaucoma patients, or patients with cataracts associated with controlled or uncontrolled OAG under maximal medical therapy who had been subjected to canaloplasty alone or combined with cataract surgery, respectively. Complications, Goldmann intraocular pressure (IOP) and mean number of drugs (ND) were evaluated at 1 day, 1 week, 1 month, and every 3 months. RESULTS: Thirty five surgeries were performed (11 canaloplasties, 24 phaco-canaloplasties). The mean IOP (mmHg) dropped from 24.5±5.1, in canaloplasty, and from 19.8±6.4, in phacocanaloplasty, preoperatively, to medium-low levels at all follow-up periods (13.5±1.0 and 11.0±4.2, at 1 year in canaloplasty and phaco-canaloplasty, respectively). The number of grugs used dropped from 3.3±0.5 before surgery to less than of 1 in all follow-up periods (0.5±0.8 at 1 year). The complications were 2 microruptures of the trabeculodescemetic window, 5 entries in the collector channels, 5 choroidal space/anterior chamber passages, 10 hyphemas, 3 hypotonies, one peripheral Descemet detachment, one intracorneal hematoma, two peripheral anterior synechia, one internal iris prolapse, and two suture extrusions to the anterior chamber. CONCLUSIONS: Ultrasound biomicroscopy assisted Canaloplasty, alone or combined, provided a sustained IOP reduction to medium-low levels, led to a decrease in the number of drugs and had a good safety profile, making this a good alternative to trabeculectomy.

Unusual coexistence of opportunistic lung infections in a human immunodeficiency virus positive patient suffering from persistent Pneumocystis jirovecii pneumonia: a case report.

It is well established that HIV patients are at high risk of opportunistic infections (OI), like the ones caused by Pneumocystis jirovecii, a worldwide pathogen implicated in interstitial pneumonia (PcP). We present a case of a newly diagnosed HIV-1 patient with multiple OI, including a persistent form of PcP, an invasive aspergillosis (IA), cytomegalovirus and Mycobacterium xenopi lung infection. We describe the combination of laboratorial screening, surgery and antimicrobial therapy that were crucial for patient recovery.

Effect of sediment composition on methane concentration and production in the transition zone of a mangrove (Sepetiba Bay, Rio de Janeiro, Brazil).

The aim of this research was to evaluate the effect of sediment composition on methane (CH4) dynamics in sediments of different areas in the transition zone between a mangrove and the sea. This research was conducted in a mangrove at Coroa Grande, on the southern coast of Rio de Janeiro. Samples were collected at three stations: (1) region colonised by Rhizophora mangle L. on the edge of the mangrove, (2) region colonised by seagrasses and (3) infra-littoral region without vegetation. Samples were collected from the surface layer of the sediment to determine the concentrations of nutrients (C, N and P) and CH4 concentration and production. We observed that concentrations of CH4 and carbon (C) were significantly higher (p < 0.05) in station 1 than station 3. The molar ratios (C:N, C:P and N:P) suggest that the origin of the substrate is mainly autochthonous. Methanogenesis was initially low, possibly due to competition between methanogens and sulfate reducers, and increased significantly (p < 0.05) on the twenty-sixth day in the sediment of station 1, probably due to higher organic matter (OM) availability in this region. Results indicate that methanogenic activity observed herein is not regulated by the amount or quality of OM, but by other factors. The concentration of CH4 in the sea-land ecotone at Mangrove Coroa Grande is a function of available OM suggesting a possible inhibition of methanotrophy by intense oxygen consumption in the soil surface covered by detritus of Rhizophora mangle vegetation.

Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies.

Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia.