João Brandão

Validation of host-specific Bacteriodales 16S rRNA genes as markers to determine the origin of faecal pollution in Atlantic Rim countries of the European Union

The recent implementation of the Revised Bathing Water Directive in the European Union has highlighted the need for development of effective methods to differentiate between sources of faecal contamination. It had previously been shown that amplification of 16S rRNA genes of host-specific Bacteriodales species using the HF183F and CF128F primers could be used as markers for human and bovine faecal contamination in the United States. This paper determined the sensitivity and specificity of these markers in four Atlantic Rim countries (France, Ireland, Portugal and the United Kingdom) to evaluate their usefulness in determining the origin of faecal contamination. It was shown that the HF183F marker displayed high sensitivity (80-100%) and specificity (91-100%), and is reliable as an indication of human faecal contamination. The CF128F marker displayed 100% sensitivity in all four countries. However, strong regional variations in specificity (41-96%) were observed, highlighting the need for local validation before this marker is employed in source tracking of faecal contamination.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

Microbes in Beach Sands: Integrating Environment, Ecology and Public Health.

Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.

Mortality in Portugal associated with the heat wave of August 2003: early estimation of effect, using a rapid method

During the first two weeks of August 2003, Portugal was affected by a severe heat wave. Following the identification in Portugal of the influence of heat waves on mortality in 1981 and 1991 (estimated excess of about 1900 and 1000 deaths respectively), the Observatório Nacional de Saúde (ONSA) - Instituto Nacional de Saúde Dr. Ricardo Jorge, together with the Vigilância Previsão e Informação - Instituto de Meteorologia, created a surveillance system called ÍCARO, which has been in operation since 1999. ÍCARO identifies heat waves with potential influence on mortality [1]. Before the end of the 2003 heat wave, ONSA had produced a preliminary estimate of its effect on mortality. The results based on daily number of deaths from 1 June to 12 August 2003 were presented within 4 working days. Data was gathered from 31 National Civil registrars, covering the district capitals of all 18 districts of mainland Portugal, and representing approximately 40% of the mainlands mortality. The number of deaths registered in the period 30 July to 12 August was compared with the ones registered during 3 comparison periods: (in July): 1-14 July, 1-28 July, and 15-28 July). 15-28 July, the period best resembling the heat wave in time and characteristics, produced an estimation of 37.7% higher mortality rate then the value expected under normal temperature conditions. From this value, an estimate of 1316 death excess was obtained for mainland Portugal. The main purpose of this article is to present the method used to identify and assess the occurrence of an effect (excess mortality) during the heat wave of summer 2003.

Beach sand and the potential for infectious disease transmission: observations and recommendations

Recent studies suggest that sand can serve as a vehicle for exposure of humans to pathogens at beach sites, resulting in increased health risks. Sampling for microorganisms in sand should therefore be considered for inclusion in regulatory programmes aimed at protecting recreational beach users from infectious disease. Here, we review the literature on pathogen levels in beach sand, and their potential for affecting human health. In an effort to provide specific recommendations for sand sampling programmes, we outline published guidelines for beach monitoring programmes, which are currently focused exclusively on measuring microbial levels in water. We also provide background on spatial distribution and temporal characteristics of microbes in sand, as these factors influence sampling programmes. First steps toward establishing a sand sampling programme include identifying appropriate beach sites and use of initial sanitary assessments to refine site selection. A tiered approach is recommended for monitoring. This approach would include the analysis of samples from many sites for faecal indicator organisms and other conventional analytes, while testing for specific pathogens and unconventional indicators is reserved for high-risk sites. Given the diversity of microbes found in sand, studies are urgently needed to identify the most significant aetiological agent of disease and to relate microbial measurements in sand to human health risk.

Pathogenic fungi: an unacknowledged risk at coastal resorts? New insights on microbiological sand quality in Portugal

Whilst the potential impact on beach users from microorganisms in water has received considerable attention, there has been relatively little investigation into microbial contaminants in sand. Thirty three beaches across Portugal were analyzed during a five year period (2006-2010) to determine the presence of yeasts, pathogenic fungi, dermatophytes, total coliforms, Escherichia coli and intestinal enterococci in sand. Our results showed that 60.4% of the samples were positive for fungi and that 25.2% were positive for the bacterial parameters. The most frequent fungal species found were Candida sp. and Aspergillus sp., whereas intestinal enterococci were the most frequently isolated bacteria. Positive associations were detected among analyzed parameters and country-regions but none among those parameters and sampling period. Regarding threshold values, we propose 15 cfu/g for yeasts, 17 cfu/g for potential pathogenic fungi, 8 cfu/g for dermatophytes. Twenty-five cfu/g for E. coli, and 10 [corrected] cfu/g for intestinal enterococci.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi, and Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This may allow preventive and corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.

Epidemiology of candidemia in oncology patients: a 6-year survey in a Portuguese central hospital.

This study presents data on the incidence of candidemia in a Portuguese oncology hospital during a 6-year period. The species distribution and their antifungal susceptibility, as well as the clinical outcomes associated with candidemia were evaluated. A total of 119 episodes were reported, with the majority occurring among patients older than 56 years. The most common underlying medical conditions were solid tumors (64.5%) and hematological disease (28.2%). The most frequent species found was Candida albicans (48.7%), followed by C. parapsilosis (20.2%), C. tropicalis (8.4%), C. krusei (6.7%) and C. glabrata (5.0%), but Saccharomyces cerevisiae and Rhodotorula mucilaginosa were also isolated. Candida albicans was more frequently associated with solid tumors of the gastrointestinal and genitourinary tracts and breast (P=0.005), while non-C. albicans Candida species were most frequently recovered from hematological patients (P=0.007). The mortality rate associated with candidemia was 31.9% (P=0.016). All C....

Microbiological and mycological beach sand quality in a volcanic environment: Madeira archipelago, Portugal

Madeira forms a mid-Atlantic volcanic archipelago, whose economy is largely dependent on tourism. There, one can encounter different types of sand beach: natural basaltic, natural calcareous and artificial calcareous. Microbiological and mycological quality of the sand was analyzed in two different years. Bacterial indicators were detected in higher number in 2010 (36.7% of the samples) than in 2011 (9.1%). Mycological indicators were detected in a similar percentage of samples in 2010 (68.3%) and 2011 (75%), even though the total number of colonies detected in 2010 was much higher (827 in 41 samples) than in 2011 (427 in 66 samples). Enterococci and potentially pathogenic and allergenic fungi (particularly Penicillium sp.) were the most common indicators detected in both years. Candida sp. yeast was also commonly detected in the samples. The analysis of the 3rd quartile and maximum numbers of all indicators in samples showed that artificial beaches tend to be more contaminated than the natural ones. However, a significant difference between the variables was lacking. More monitoring data (number of bathers, sea birds, radiation intensity variation, and a greater number of samples) should be collected in order to confirm if these differences are significant. In general, the sand quality in the archipelagos beaches was good. As the sand may be a vector of diseases, an international common set of indicators and values and a compatible methodologies for assessing sand contamination, should be defined, in order to provide the bathers with an indication of beach sand quality, rather than only the water.

Workshop Report: Developing Pollution Source Tracking for Recreational and Shellfish Waters

Traditional methods, such as the detection of total and fecal coliforms, and more recently enterococci, that are used to detect fecal pollution levels do not identify the source of the pollution. Despite a significant volume of research in this field, at present there is no common methodology to identify sources of fecal contamination affecting bathing and shellfish waters in Europe.