Molecular Genotyping of Giardia duodenalis Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid,Spain

Background

The flagellate protozoan Giardia duodenalis is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating G. duodenalis isolates in the country.

Methods

In this longitudinal molecular epidemiological study we report the diversity and frequency of the G. duodenalis assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013–January 2015. G. duodenalis cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. G. duodenalis assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (GDH) and β-giardin (BG) genes of the parasite. Sociodemographic and clinical features of patients infected with G. duodenalis were also analysed.

Principal findings

Of 188 confirmed positive samples from 178 giardiasis cases a total of 124 G. duodenalis isolates were successfully typed at the GDH and/or the BG loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in G. duodenalis assemblage B, but not assemblage A, isolates at both the GDH and BG loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of G. duodenalis found in individuals with symptomatic giardiasis in the population under study.

Conclusions

Human giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent G. duodenalis assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of G. duodenalis.     

Flow Cytometry for Determination of the Efficacy of Contact Lens Disinfecting Solutions against Acanthamoeba spp.

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10⁵ to 10⁶/ml) at 22°C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.     

Study of airborne fungal spores in Madrid, Spain

The concentration of fungal spores in the atmosphere of Madrid was recorded and analyzed for the year 2003. Airborne spores were sampled continuously with a Hirst-type spore trap located on the roof of a building of the School of Pharmacy, at about 8 m above ground level. Correlation between the mean daily spore concentrations and meteorological variables were explored by means of Spearman’s correlation analyses. Seventy spore types were identified, of which the most numerous were Cladosporium, Aspergillaceae (conidia), Coprinus, Agaricales (basidiospores), Ustilago (teliospores) and Pleospora (ascospores). These six types of spores represented more than 70% of the total. Cladosporium represented 41% of the total fungal spores, while Ustilago spores, the concentrations of which in May and June exceeded 47% of the monthly total spore count, constituted the second most important group. Spores reached their highest concentrations in the spring months, and in the autumn, mainly in October. A␣positive significant correlation was found between airborne spore counts and temperature and relative humidity. The results provide a picture of the spectrum of airborne fungal spores present in the atmosphere of Madrid and of the `peak' periods of their presence. Future studies will provide more detailed information on the seasonal dynamics of the spores most frequently found in the air as well as on the extent to which atmospheric conditions influence their release, dispersion and sedimentation processes.     

Interactions between Human Norovirus Surrogates and Acanthamoeba spp.

Human noroviruses (HuNoVs) are the most common cause of food-borne disease outbreaks, as well as virus-related waterborne disease outbreaks in the United States. Here, we hypothesize that common free-living amoebae (FLA)—ubiquitous in the environment, known to interact with pathogens, and frequently isolated from water and fresh produce—could potentially act as reservoirs of HuNoV and facilitate the environmental transmission of HuNoVs. To investigate FLA as reservoirs for HuNoV, the interactions between two Acanthamoeba species, A. castellanii and A. polyphaga, as well as two HuNoV surrogates, murine norovirus type 1 (MNV-1) and feline calicivirus (FCV), were evaluated. The results showed that after 1 h of amoeba-virus incubation at 25°C, 490 and 337 PFU of MNV-1/ml were recovered from A. castellanii and A. polyphaga, respectively, while only few or no FCVs were detected. In addition, prolonged interaction of MNV-1 with amoebae was investigated for a period of 8 days, and MNV-1 was demonstrated to remain stable at around 200 PFU/ml from day 2 to day 8 after virus inoculation in A. castellanii. Moreover, after a complete amoeba life cycle (i.e., encystment and excystment), infectious viruses could still be detected. To determine the location of virus associated with amoebae, immunofluorescence experiments were performed and showed MNV-1 transitioning from the amoeba surface to inside the amoeba over a 24-h period. These results are significant to the understanding of how HuNoVs may interact with other microorganisms in the environment in order to aid in its persistence and survival, as well as potential transmission in water and to vulnerable food products such as fresh produce.     

Isoenzyme patterns and phylogenetic relationships in Acanthamoeba spp. isolated from contact lens containers in Korea

In order to refer to the basic information regarding the identification of isolates obtained from a contact lens container in Korea, the isoelectric focusing gel electrophoresis was employed to compare the isoenzyme band patterns among Acanthamoeba spp. including eight isolates and the simple pairwise dissimilarity analysis was carried out. For an alkaline phosphate development, isolate 7 and Acanthamoeba polyphaga showed homologous band patterns, and isolates 1, 2, and 3 showed the same patterns. For lactate dehydrogenase, similar patterns were observed in isolates 2 and 3. Isolates 3 and 5 showed homologous band patterns for malate dehydrogenase and glucose phosphate isomerase. For hexokinase, isolates 4, 7, and A, hatchetti showed the same band patterns. In others, a considerable number of interstrain polymorphisms was observed in nine isoenzyme band patterns. In Acanthamoeba group II, genetic distances among isolates 1, 2, 3, 4, and 5 ranged from 0.104 to 0.200. In comparison to A. castellanii, A. hatchetti, and A. polyphaga, genetic distances of isolates 7 and 8 were 0.254 and 0.219, respectively. In Acanthamoeba group III, including A. culbertsoni, A. healyi, and A. royreba, isolate 6 had genetic distances which ranged from 0.314 to 0.336. Finally, when comparing to the six reference Acanthamoeba, it was possible to classify isolates 1, 2, 3, 4, and 5, as genetically close-related species and as independent species group. Furthermore, isolates 6, 7 and 8 were identified as independent species as well.     

Present Status of Corneal Contact Lenses in Medical Practice

Common indications for and contraindications to corneal contact lens wear are outlined. The processes of adaptation of the cornea to a correctly fitted contact lens are described. Biomicroscopic examination of the cornea revealed that 29 of 250 eyes in the authors'' series showed small abrasions at some time during the fitting period. The dangers of infection of a simple corneal abrasion by such pathogens as Pseudomonas aeruginosa are stressed. Prophylactic treatment of abrasions with a topical antibiotic preparation containing polymyxin is recommended. Serious criticism is levelled at the practice of estimating the fit of a contact lens by the fluorescein pattern only, and repeated keratometry readings combined with biomicroscopy are considered to be essential. It is proposed that the evaluation, fitting and after-care of the contact lens patient should be the responsibility of an ophthalmologist.     

Foliar Symptoms Triggered by Ozone Stress in Irrigated Holm Oaks from the City of Madrid,Spain

Background

Despite abatement programs of precursors implemented in many industrialized countries, ozone remains the principal air pollutant throughout the northern hemisphere with background concentrations increasing as a consequence of economic development in former or still emerging countries and present climate change. Some of the highest ozone concentrations are measured in regions with a Mediterranean climate but the effect on the natural vegetation is alleviated by low stomatal uptake and frequent leaf xeromorphy in response to summer drought episodes characteristic of this climate. However, there is a lack of understanding of the respective role of the foliage physiology and leaf xeromorphy on the mechanistic effects of ozone in Mediterranean species. Particularly, evidence about morphological and structural changes in evergreens in response to ozone stress is missing.

Results

Our study was started after observing ozone -like injury in foliage of holm oak during the assessment of air pollution mitigation by urban trees throughout the Madrid conurbation. Our objectives were to confirm the diagnosis, investigate the extent of symptoms and analyze the ecological factors contributing to ozone injury, particularly, the site water supply. Symptoms consisted of adaxial and intercostal stippling increasing with leaf age. Underlying stippling, cells in the upper mesophyll showed HR-like reactions typical of ozone stress. The surrounding cells showed further oxidative stress markers. These morphological and micromorphological markers of ozone stress were similar to those recorded in deciduous broadleaved species. However, stippling became obvious already at an AOT40 of 21 ppm•h and was primarily found at irrigated sites. Subsequent analyses showed that irrigated trees had their stomatal conductance increased and leaf life -span reduced whereas the leaf xeromorphy remained unchanged. These findings suggest a central role of water availability versus leaf xeromorphy for ozone symptom expression by cell injury in holm oak.     

Dermatophytes isolated in Hospital Universitario 12 de Octubre (Madrid, Spain).

Over a 10 year period (January 1988 - December 1997), 3,241 dermatophyte strains were isolated from 18,465 specimens from patients in whom dermatophytosis was suspected clinically. This represents a 17.5% rate of isolation. Trichophyton rubrum (38.44%), Microsporum canis (28.75%), Epidermophyton floccosum (14.5%) and Trichophyton mentagrophytes (13.5%) were the dominant species, and Trichophyton tonsurans (2.09%) has emerged, whilst in the previous decade it had virtually disappeared. Our study is basically based on an out-patient selected population, and tinea corporis (30.79%), followed by tinea cruris (16.69%) and tinea unguium (16.69%) were the most prevalent clinical forms.     

Wild food plants traditionally used in the province of Madrid, Central Spain

This paper reports an ethnobotanical investigation, performed between 1999 and 2002, to determine the wild food plants traditionally used in the province of Madrid (central Spain). One hundred and twenty-three vascular species belonging to 31 families were at one time used as vegetables, wild fruits, in the making of beverages, as seasonings, or as other preparations. Wild vegetables formed the largest group, withScolymus hispanicus, Silene vulgaris, andRumex pulcher the most frequently cooked vegetables, andRorippa nasturtium-aquaticum andMontia fontana the most commonly used in salads. Also common was the consumption of asparagus and several species of climbing plants such asBryonia dioica, Tamus communis, andHumulus lupulus. Ethnobotanical novelties includedSonchus crassifolius, the young sprouts of which were used in salads, and Inula salicina, used to make herbal tea. Some ecological and cultural aspects of the collection of wild plants for food are discussed, as well as the relationships between the food and medicinal uses of some species.     

Molecular surveillance of HIV-1 in Madrid, Spain: a phylogeographic analysis

The molecular epidemiology of HIV-1 is constantly changing, mainly as a result of human migratory flows and the high adaptive ability of the virus. In recent years, Spain has become one of Europe''s main destinations for immigrants and one of the western European countries with the highest rates of HIV-positive patients. Using a phylogeographic approach, we have analyzed the relationship between HIV-1 variants detected in immigrant and native populations of the urban area of Madrid. Our project was based on two coincidental facts. First, resistance tests were extended to naïve and newly diagnosed patients, and second, the Spanish government legislated the provision of legal status to many immigrants. This allowed us to obtain a large data set (n = 2,792) from 11 Madrid hospitals of viral pol sequences from the two populations, and with this unique material, we explored the impact of immigration in the epidemiological trends of HIV-1 variants circulating in the largest Spanish city. The prevalence of infections by non-B HIV-1 variants in the studied cohort was 9%, rising to 25% among native Spanish patients. Multiple transmission events involving different lineages and subsubtypes were observed in all the subtypes and recombinant forms studied. Our results also revealed strong social clustering among the most recent immigrant groups, such as Russians and Romanians, but not in those groups who have lived in Madrid for many years. Additionally, we document for the first time the presence of CRF47_BF and CRF38_BF in Europe, and a new BG recombinant form found in Spaniards and Africans is tentatively proposed. These results suggest that the HIV-1 epidemic will evolve toward a more complex epidemiological landscape.     

Acanthamoeba healyi n. sp. and the isoenzyme and immunoblot profiles of Acanthamoeba spp., groups 1 and 3.

Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti-Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni, Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba, it was considered as a strain of A. royreba. Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba.     

Acanthamoeba healyi N. Sp. and the Isoenzyme and Immunoblot Profiles of Acanthamoeba spp., Groups 1 and 3

ABSTRACT Two strains of Acanthamoeba isolated from human brain tissue and a strain of Acanthamoeba isolated from a fish were compared with 10 species of Acanthamoeba belonging to groups 1, 2 and 3 based on their isoenzyme profiles and antigenic characteristics. A total of 12 enzymes were studied. The isoenzymes and antigens were electrophoretically separated on polyacrylamide gradient gels, and the patterns obtained were compared after appropriate staining for particular enzymes and reactivities with homologous and heterologous rabbit anti- Acanthamoeba antisera. One of the human strains (CDC:1283:V013) was identified as A. healyi n. sp. because of its unique isoenzyme profiles for 11 of the 12 enzymes tested. The other human isolate was reidentified as A. culbertsoni because its isoenzyme profiles for 10 of 12 enzymes resembled those of A. culbertsoni , Lilly A-1 strain. Since the isoenzyme profiles and the antigenic patterns of the fish isolate as well were remarkably similar to those of A. royreba , it was considered as a strain of A. royreba . Polyacrylamide gradient gel electrophoresis appears to be a powerful technique for the study of isoenzymes and antigens of Acanthamoeba .     

Herpetomonas spp. isolated from tomato fruits (Lycopersicon esculentum) in southern Spain

A flagellate of the family Trypanosomatidae was isolated from fruits of Lycopersicon esculentum (tomato) in southeastern Spain. The isolate was successfully adapted to in vitro culture in monophasic media. The morphology showed the kinetoplast to be positioned towards the middle of the body, and the typical opistomastigote form characteristic of members of the genus Herpetomonas. Amplification of the mini-exon gene was negative, whilst for the 5S ribosomal rRNA gene the result was positive. The DNA sequence was obtained and its alignment with other trypasomatids, obtained using the BLAST algorithm, suggested it was closely related to Herpetomonas samuelpessoai.     

Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 × 10⁴ Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.     

Correlations between Morphological, Molecular Biological, and Physiological Characteristics in Clinical and Nonclinical Isolates of Acanthamoeba spp.

Eleven Acanthamoeba isolates, obtained from Acanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showed Acanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.     

Avian spatial segregation at edges and interiors of urban parks in Madrid, Spain

Few studies have investigated the effects of urban landscape composition on avian habitat selection at urban-park edges. I assessed how the number of species, density of guilds, and density of individual species varied between edge and interior habitats in six large wooded parks in Madrid (Spain), and analysed such patterns in relation to habitat structure, car traffic, and pedestrian traffic. Few differences in habitat structure were found; whereas car and pedestrian traffic were significantly higher at edges. Species foraging in trees and on the ground, and nesting in trees and in tree cavities had lower numbers and breeding densities at edges, probably as a result of the disturbance from traffic noise and pedestrians. Species highly habituated to human activities (House Sparrows Passer domesticus and Rock Doves Columba livia) displayed opposite patterns, with higher breeding densities at urban-park edges, probably due to their higher foraging opportunities (refuse, people leftovers, deliberate feeding) and nest site availability in adjacent buildings. Urbanisation sprawl may increase the prevalence of edge specialists and diminish the representation of species with specific habitat requirements.