Malaria and tuberculosis co-infection:role for hemozoin immunosuppression?

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2014

Human factors required for mycobacterium tuberculosis macrophage infection

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

Novel particulate antibiotic-loaded platforms as sustained drug delivery systems for bone infection treatment

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2015

Back-trajectories Show Export of Airborne Fungal Spores (Ganoderma sp.) From Forests to Agricultural and Urban Areas in England

We propose here the hypothesis that all of United Kingdom (UK) is likely to be affected by Ganoderma sp. spores, an important plant pathogen. We suggest that the main sources of this pathogen, which acts as a bioaerosol, are the widely scattered woodlands in the country, although remote sources must not be neglected. The hypothesis is based on related studies on bioaerosols and supported by new observations from a non-forest site and model calculations to support our hypothesis. Hourly concentrations of Ganoderma sp. spores were measured from 2006 to 2010 using a 7-day volumetric spore trap at the city of Worcester. The concentrations peak during the night and early in the morning. This suggests that the main spore sources are located a few hours away with respect to air masses transport and reach urban areas thanks to air masses transport. The back-trajectory analysis was applied to determine the location of Ganoderma sp. spore sources. The analysis of back-trajectories demonstrated that 78% of the air masses reached Worcester from a 180° arc direction from the East to West. Three episodes were selected for detailed investigation and they revealed that during the episodes air masses always passed main UK woodlands before the arrival in Worcester, independently of their origin, but the long distance transport under certain conditions might be possible. Our studies suggest that the sources of UK Ganoderma sp. spores are mainly to be found in UK. Hence our studies suggest that research and mitigation strategies in UK should give their main attention to national sources, without neglecting the contribution from long distance transport.

Potential Impact of Climate Change on Fungal Distributions: Analysis of 2 Years of Contrasting Weather in the UK

The impact of climate change on fungal growth and spore production is less well documented than for allergenic pollen grains, although similar implications for respiratory tract diseases in humans occur. Fungal spores are commonly described as either “dry” or “wet” according to the type of weather associated with their occurrence in the air. This study examined the distribution of selected fungal spores (Alternaria spp., Cladosporium spp., Didymella spp., Epicoccum spp., Leptosphaeria spp. and rusts) occurring in the West Midlands of UK during 2 years of contrasting weather. Spore specimens were collected using a 7-day volumetric air sampler and then analysed with the aid of light microscopy. Distributions of spores were then studied using normality tests and Mann–Whitney U test, while relationships with meteorological parameters were investigated using Spearman’s rank test and angular-linear correlation for wind direction analysis. Our results showed that so-called wet spores were more sensitive to the weather changes showing statistically significant differences between the 2 years of study, in contrast to “dry” spores. We predict that in following years we will observe accelerated levels in allergenic fungal spore production as well as changes in species diversity. This study could be a starting point to revise the grouping system of fungal spores as either “dry” or “wet” types and their response to climate change

Investigating and Predicting Atmospheric Concentrations of Allergenic Fungal Spores (Alternaria, Cladosporium, Didymella and Ganoderma)

Air quality is an increasing concern of the European Union, local authorities, scientists and most of all inhabitants that become more aware of the quality of the surrounding environment. Bioaerosols may be consisted of various elements, and the most important are pollen grains, fungal spores, bacteria, viruses. More than 100 genera of fungal spores have been identified as potential allergens that cause immunological response in susceptible individuals. Alternaria and Cladosporium have been recognised as the most important fungal species responsible for respiratory tract diseases, such as asthma, eczema, rhinitis and chronic sinusitis. While a lot of attention has been given to these fungal species, a limited number of studies can be found on Didymella and Ganoderma, although their allergenic properties were proved clinically. Monitoring of allergenic fungal spore concentration in the air is therefore very important, and in particular at densely populated areas like Worcester, UK. In this thesis a five year spore data set was presented, which was collected using a 7-day volumetric spore trap, analysed with the aid of light microscopy, statistical tests and geographic information system techniques. Although Kruskal-Wallis test detected statistically significant differences between annual concentrations of all examined fungal spore types, specific patterns in their distribution were also found. Alternaria spores were present in the air between mid-May/mid-June until September-October with peak occurring in August. Cladosporium sporulated between mid-May and October, with maximum concentration recorded in July. Didymella spores were seen from June/July up to September, while peaks were found in August. Ganoderma produced spores for 6 months (May-October), and maximum concentration could be found in September. With respect to diurnal fluctuations, Alternaria peaked between 22:00h and 23:00h, Cladosporium 13:00-15:00h, Didymella 04:00-05:00h and 22:00h-23:00h and Ganoderma from 03:00h to 06:00h. Spatial analysis showed that sources of all fungal species were located in England, and there was no evidence for a long distance transport from the continent. The maximum concentration of spores was found several hours delayed in comparison to the approximate time of the spore release from the crops. This was in agreement with diurnal profiles of the spore concentration recorded in Worcester, UK. Spores of Alternaria, Didymella and Ganoderma revealed a regional origin, in contrast to Cladosporium, which sources were situated locally. Hence, the weather conditions registered locally did not exhibit strong statistically significant correlations with fungal spore concentrations. This has had also an impact on the performance of the forecasting models. The best model was obtained for Cladosporium with 66% of the accuracy.

Comparisons of Fungal Spore Distributions Using Air Sampling at Worcester, England (2006-2010)

This study determined annual and monthly fluctuations in concentration of 20 fungal genera. The selection of taxa was made based upon their high frequency in the air as well as their well-known allergenic properties. Air samples were collected using a spore trap of Hirst design at an urban site where the trap continuously worked throughout a 5-year survey. Weather data were acquired from a meteorological station co-located with the air sampler. Influence of several meteorological parameters was then examined to reveal species–environment interactions and the potential location of fungal spore sources within the urban area. The maximum monthly sum of mean daily spore concentration varied between genera, and the earliest peaks were recorded for Pleospora sp. in April and Ustilago sp. in June. However, the majority of investigated spore types occurred in the greatest concentrations between August and September. Out of the 20 studied taxa, the most dominant genus was Cladosporium sp., which exceeded an allergenic threshold of 3000 s m-3 40 times during very rainy years and twice as much during dry years. A Spearman’s rank test showed that statistically significant (p B 0.05) relationships between spore concentration and weather parameters were mainly rs B 0.50. Potential sources of spores at Worcester were likely to be localised outside the city area.

Telaprevir or boceprevir based therapy for chronic hepatitis C infection: Development of resistance-associated variants in treatment failure

The use of triple-therapy, pegylated-interferon, ribavirin and either of the first generation hepatitis C virus (HCV) protease inhibitors telaprevir or boceprevir, is the new standard of care for treating genotype 1 chronic HCV. Clinical trials have shown response rates of around 70–80%, but there is limited data from the use of this combination outside this setting. Through an expanded access programme, we treated 59 patients, treatment naïve and experienced, with triple therapy. Baseline factors predicting treatment response or failure during triple therapy phase were identified in 58 patients. Thirty seven (63.8%) of 58 patients had undetectable HCV RNA 12 weeks after the end of treatment. Genotype 1a (p = 0.053), null-response to previous treatment (p = 0.034), the rate of viral load decline after 12 weeks of previous interferon-based treatment (p = 0.033) were all associated with triple-therapy failure. The most common cause of on-treatment failure for telaprevir-based regimens was the development of resistance-associated variants (RAVs) at amino acids 36 and/or 155 of HCV protease (p = 0.027) whereas in boceprevir-based regimens mutations at amino acid 54 were significant (p = 0.015). SVR12 rates approaching 64% were achieved using triple therapy outside the clinical trial setting, in a patient cohort that included cirrhotics.

Fungal contamination in two Portuguese wastewater treatment plants

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Characterizing the fungal and bacterial microflora and concentrations in fitness centres

Fitness centres are special places where conditions for microbiological proliferation should be considered. Moisture due to human perspiration and water condensation as a result of human physical activities are prevalent in this type of buildings. Exposure to microbial contaminants is clinically associated with respiratory disorders and people who work out in polluted environments would be susceptible to contaminants. This work studied the indoor air contamination in three gymnasiums in Lisbon. The sampling was performed at two periods: at the opening (morning) and closing (night) of the three gymnasiums. The airborne bacterial and fungal populations were sampled by impaction directly onto Tryptic Soy Agar (for bacteria) and Malt Extract Agar (for fungi) plates, using a Merck MAS-100 air sampler. Higher bacterial concentrations were found at night as compared to the morning but the same behaviour was not found for fungal concentrations. Gram-negative catalase positive cocci were the dominant bacteria in indoor air samples of the studied gymnasiums. In this study, 21 genera/species of fungal colonies were identified. Chrysosporium sp., Chrysonilia sp., Neoscytalidium hialinum, Sepedonium sp. and Penicillium sp. were the most prevalent species identified in the morning, while Cladosporium sp., Penicillium sp., Chrysosporium sp., Acremonium sp. and Chrysonilia sp. were more prevalent at night. A well-designed sanitation and maintenance program for gymnasiums is needed to ensure healthier space for indoor physical activity.

Candidemia in Acute Leukemia Patients

Fungal infections are an important cause of morbidity and mortality in patients with acute leukemia (AL). Candidemia, once rare, is now a common nosocomial infection because of the intensity of chemotherapy, prolonged neutropenia, administration of broad-spectrum antibiotics and use of central venous catheters (CVC). We retrospectively identified patients treated for AL from 6/86 to 6/95 who also had candidemia. We describe 28 patients (incidence 6.3%) with a median age of 39 years, 24 of whom were on remission induction and 4 on postremission chemotherapy. All patients had CVC and empiric antimicrobial therapy, 4 had been given prophylactic antifungal drugs, and 2 had parenteral nutrition. Neutropenia was profound (median leukocyte nadir 200/microliters, median duration 19 days). Candida was isolated in blood cultures 10 days (median) after the start of neutropenia. The clinical presentation included fever (100%), respiratory symptoms (71.4%), skin lesions (39.2%) and septic shock (17.8%). Amphotericin B was given to 17 patients and liposomal amphotericin to 5 patients. Infection resolved in 18 patients (64.2%). 10 of whom were in complete remission. Mortality from candidemia was 17.8% (5/28). In conclusion, fungal infections are responsible for death in a significant number of patients. In our series treatment success was related to its rapid onset and to the recovery of neutropenia.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore, our results confirm the potential usefulness of PCR serial screening and the clinical applicability in everyday routine. PCR screening offers a noninvasive repeatable aid to the diagnosis of IFI.

The Intensive Care Global Study on Severe Acute Respiratory Infection (IC-GLOSSARI): a Multicenter, Multinational, 14-Day Inception Cohort Study

PURPOSE: In this prospective, multicenter, 14-day inception cohort study, we investigated the epidemiology, patterns of infections, and outcome in patients admitted to the intensive care unit (ICU) as a result of severe acute respiratory infections (SARIs). METHODS: All patients admitted to one of 206 participating ICUs during two study weeks, one in November 2013 and the other in January 2014, were screened. SARI was defined as possible, probable, or microbiologically confirmed respiratory tract infection with recent onset dyspnea and/or fever. The primary outcome parameter was in-hospital mortality within 60 days of admission to the ICU. RESULTS: Among the 5550 patients admitted during the study periods, 663 (11.9 %) had SARI. On admission to the ICU, Gram-positive and Gram-negative bacteria were found in 29.6 and 26.2 % of SARI patients but rarely atypical bacteria (1.0 %); viruses were present in 7.7 % of patients. Organ failure occurred in 74.7 % of patients in the ICU, mostly respiratory (53.8 %), cardiovascular (44.5 %), and renal (44.6 %). ICU and in-hospital mortality rates in patients with SARI were 20.2 and 27.2 %, respectively. In multivariable analysis, older age, greater severity scores at ICU admission, and hematologic malignancy or liver disease were independently associated with an increased risk of in-hospital death, whereas influenza vaccination prior to ICU admission and adequate antibiotic administration on ICU admission were associated with a lower risk. CONCLUSIONS: Admission to the ICU for SARI is common and associated with high morbidity and mortality rates. We identified several risk factors for in-hospital death that may be useful for risk stratification in these patients.

the liver endoplasmic reticulum in malaria liver infection

Malaria, a disease caused by Plasmodium, represents a major health problem with a still disconcertingly high mortality rate (655 000 malaria deaths were estimated by the World Health Organization in 2012), mainly in Africa [1]. After a bite by an infected Anopheles mosquito occurs, Plasmodium sporozoites reach their target organ, the liver, within minutes. After traversing several hepatocytes, the parasite invades a final one and establishes a parasitophorous vacuole, where it replicates exponentially generating thousands of infective merozoites, the red blood cell infectious forms that are released in the blood stream. The liver stage is the first obligatory phase of malaria infection and, although no symptoms are associated with it, it is absolutely crucial to the establishment of a successful infection.(...)

Host genetic factors in mouse malaria liver stage infection

Fundação para a Ciência e a Tecnologia