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8 pág., West Nile virus (WNV) is a re-emergent mosquito-borne RNA virus that causes major outbreaks of encephalitis around the world. However, there is no therapeutic treatment to struggle against WNV, and the current treatment relies on alleviating symptoms. Therefore, due to the threat virus poses to animal and human health, there is an urgent need to come up with fast strategies to identify and assess effective antiviral compounds. A relevant target when developing drugs against RNA viruses is the viral RNA-dependent RNA polymerase (RdRp), responsible for the replication of the viral genome within a host cell. RdRps are key therapeutic targets based on their specificity for RNA and their essential role in the propagation of the infection. We have developed a fluorescence-based method to measure WNV RdRp activity in a fast and reliable real-time way. Interestingly, rilpivirine has shown in our assay inhibition of the WNV RdRp activity with an IC50 value of 3.3 μM and its antiviral activity was confirmed in cell cultures. Furthermore, this method has been extended to build up a high-throughput screening platform to identify WNV polymerase inhibitors. By screening a small chemical library, novel RdRp inhibitors 1-4 have been identified. When their antiviral activity was tested against WNV in cell culture, 4 exhibited an EC50 value of 2.5 μM and a selective index of 12.3. Thus, rilpivirine shows up as an interesting candidate for repurposing against flavivirus. Moreover, the here reported method allows the rapid identification of new WNV RdRp inhibitors., This work was supported by Spanish Ministry of Science and Innovation AEI/10.13039/501100011033 under grant PID2020-115432RB-I00 (to R.A) grant PID2019-105117RR-C21 (to MAM-A); PID2019-105117RR-C22 (to M-JP-P); and from AECSIC under grant PIE-201980E100 (to M-JP-P). This research work was also funded by the European Commission-NextGeneration EU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global).

L. feeleii is one of the most frequent Legionella species isolated from natural pools of the central region of Spain. This study aimed to evaluate its ecology and to identify this Legionella species as a respiratory pathogen. A PCR assay for detecting the L. feeleii mip gene was developed to identify it in clinical and environmental samples. Culture and PCR were performed in environmental samples from four drinking water treatment plants (DWTPs). Free L. feeleii was only detected in raw water samples (3.4%), while L. feeleii as an Acanthamoeba endosymbiont was found in 30.7% of raw water, 11.5% of decanter biofilm, and 32% of finished water samples. Therefore, Acanthamoeba spp. plays an essential role in the multiplication, persistence, and spread of Legionella species in the environment. The first case of Legionnaires' disease caused by L. feeleii in Spain is described in this study. The case was diagnosed in an older woman through PCR and sequencing from urine and sputum samples. A respiratory infection could be linked with health care procedures, and the patient presented several risk factors (age, insulin-dependent diabetes, and heart disease). The detection of non-L. pneumophila, such as L. feeleii, is a factor that must be considered when establishing or reviewing measures for the control and prevention of legionellosis. This work was supported by Instituto de Salud Carlos III (PI12/02725) (PI17/01670) (Co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”); and Fundación Universitaria San Pablo CEU (USPCEU-PC07/2013 and USP-PC07/2014). LV was supported by Universidad San Pablo CEU/Spain (FPI Grants). TG was supported by CAPES/Brazil (BEX 9132/13-9). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.

The present study analyzes at the national level, the presence of circulating Legionella in the artificial aquatic systems of different facilities of all of them state-owned centers throughout Spain for 12 months. 1754 water samples from various state-owned centers were collected from January to December 2014. Samples were collected from the cooling towers and evaporative condensers (CTC), and water distribution networks such as domestic hot water (DHW), cold water for human consumption (CW), sprinkler irrigation systems (SIS), fire sprinkler systems (FSS), and water from decorative fountains (DF). All these facilities are considered, according to current regulations, as potential amplifying systems for bacteria and possible sources of infection by the generation of droplets and aerosols. The isolation and counting of Legionella in water samples was carried out using microbiological culture following the international normative UNE-EN-ISO 11,731:2007 (ISO 11,731:1998) and UNE-EN ISO 8199:2008 (ISO 8199:2005).The quantification of Legionella colonization, the annual distribution, and the geographical distribution of the Legionella isolates recovered in the water were analyzed. Besides, molecular techniques were used for the characterization of the Legionella non-pneumophila isolates. Legionella was recovered from 15.79% of the analyzed water samples. High colonization was more frequently detected in water samples from CTC, DHW, CW, and DF. Regarding the geographic distribution, positive samples of Legionella were obtained in 14 of the 18 Spanish locations analyzed. Legionella non-pneumophila was the most prevalent and was isolated from water samples from 13 different geographical locations (72%). Legionella anisa and Legionella jordanis were the most frequently non-pneumophila species isolated. Legionella donaldsonii was isolated for the first time in the water distribution networks in Spain. Legionella pneumophila sg 2-14 was detected in 13 locations and Legionella pneumophila sg 1 in 11 locations. Therefore, our study concludes that the presence of Legionella pneumophila and Legionella non-pneumophila species in these systems can be a potential threat to public health and should be examined thoroughly with complementary techniques, such as molecular techniques as a screen for routine diagnosis.

Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia (Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi) in organ meats of European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits (0.8%; 95% CI: 0.0–1.7%) but not in hares (0.0%; 95% CI: 0.0–4.6%), whereas E. intestinalis DNA was found in one wild rabbit (0.3%; 95% CI: 0.0–0.8%) and three Iberian hares (3.8%; 95% CI: 0.0–8.0%). Neither E. hellem nor E. cuniculi infection were detected in the 462 (0.0%; 95% CI: 0.0–0.8%) lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors’ knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare., This work has benefited from the financial aid of research grants funded by the Spanish Ministry of Science and Innovation (PID2019-111080RB-C21) and by the University of Córdoba (UCO-FEDER-1264967). J. Caballero-Gómez is supported by an FPU grant from the Spanish Ministry of Science, Innovation, and Universities (FPU17/01319) and S. Jiménez-Ruiz holds a Ph.D. contract from the UCLM co-supported by the European Social Fund (2018/12504).

This work reports the application of photocatalytic disinfection to the inactivation of Acanthamoeba trophozoites, a free-living pathogenic amoeba. Two types of photocatalytic reactors configurations have been used: i) a slurry reactor using suspended titanium dioxide (TiO2); and, ii) a fixed-bed reactor using immobilized TiO2 onto glass Raschig rings. The effect of the chemical composition of water has been analysed, comparing the efficiency of the process in deionized water (DW) and synthetic wastewater treatment plant effluent (SWTPE). The inactivation of Acanthamoeba spp. has been compared to that of Escherichia coli bacteria, being also analysed the concomitant inactivation of both microorganisms. Our results show that 99% of inactivation of E. coli and Acanthamoeba spp. can be achieved using photocatalysis in both reactor configurations, but interestingly, the kinetics of inactivation of both microorganisms together differs from that found with them separately. Particularly, E. coli seems to be more resistant to the inactivation in the presence of Acanthamoeba spp. which has been justified by the screen effect caused by the bigger size of Acanthamoeba spp. This observation is more pronounced in DW as the composition of the SWTPE prevent the microorganisms from suffering osmotic and/or mechanical stress and protect cellular structures to the attack of reactive oxygen species (ROS). On the other hand, the difference between the inactivation rate of E. coli and Acanthamoeba, points out the importance of the different inactivation mechanisms, suggesting that the entry of small TiO2 particles into the cytoplasm of the Acanthamoeba cells provokes the attack of inner structures and as a consequence a faster inactivation. This mechanism is not possible when the catalyst is immobilized leading to a higher cell resistance to inactivation and consequently lower efficiency of the disinfection process.

Pneumocystis jirovecii and microsporidia species are recognized as opportunistic infectious pathogens in AIDS patients. Coinfection of both in one patient has been rarely reported. The aim of the present study was to investigate the coinfection of P. jirovecii and microsporidia in different tissues from AIDS deceased patients. Post mortem histological finding of P. jirovecii and microsporidia was demonstrated by means of the Grocott's methenamine silver and Brown Brenn staining, respectively. Molecular technique was used for identification and characterization of both fungi. Out of the 514 autopsied cases P. jirovecii and microsporidia species were identified in 53 (10.3%) and 62 (12.1%) cases respectively. A total of five cases (0.97%) coinfected with Pneumocystis and microsporidia were recovered from all analyzed autopsies. Coinfection of Pneumocystis and microsporidia is very challenging and raises interesting issues about host-parasite relationship. The early diagnosis of both pathogens must be crucial to establish correct and early treatments, improve the patient's evolution, reducing the risk of death., This work was supported by the ‘Red Iberoamericana sobre Pneumocystosis’ (212RT0450) in the framework of The Ibero-American Programme for Science, Technology and Development (CYTED).

open access article Eukaryotic parasites represent a serious human health threat requiring health professionals with parasitology skills to counteract this threat. However, recent surveys highlight an erosion of teaching of parasitology in medical and veterinary schools, despite reports of increasing instances of food and water borne parasitic infections. To address this we developed a web-based resource, DMU e-Parasitology®, to facilitate the teaching and learning of parasitology, comprising four sections: theoretical; virtual laboratory; virtual microscopy; virtual clinical case studies. Testing the package was performed using a questionnaire given to ninety-five Pharmacy students in 2017/18 to assess effectiveness of the package as a teaching and learning tool. 89.5% of students reported appropriate acquisition of knowledge of the pathology, prevention and treatment of some parasitic diseases. 82.1% also welcomed the clinical specialism of the package as it helped them to acquire basic diagnostic skills, through learning infective features/morphology of the parasites.

Free-living amoebae (FLA) are ubiquitous and many isolates have been shown to be infected with amoeba-resisting bacteria, as the example of Acanthamoeba and Legionella interaction. Due to the high environmental prevalence of Acanthamoeba. in the Castilian Plateau (Spain), the aims of this work were to investigate the occurrence of Acanthamoeba and other FLA in water from several sampling points from four Drinking Water Treatment Plants (DWTP) and to investigate the presence of Legionella spp. and other amoeba-resisting bacteria in biofilms in raw and finished water, taking into account that no legislation exists for this protozoa control. Acanthamoeba was detected at different sampling points, and sand filters seemed to contribute to amoebic enrichment. After ozonation, a temporary decrease in viable amoebae was observed. The genotypes detected were T3, T4, and T5, revealing the first report of genotype T5 in waters from this region. Moreover, Balamuthia mandrillaris, Vermamoeba vermiformis and Paravahlkampfia sp. were detected. Regarding Legionella, PCR detection in raw and finished water was higher than by agar culture, but even higher after Acanthamoeba co-culture. Also, Legionella's presence was higher in raw water than in finished water. The decrease of free Legionella observed from raw (27.5%, by PCR) to finished water (3.4% by PCR) contrasted with the increase of Legionella-infected FLA from raw (30.7%) to finished water (52%). At biofilm, free Legionella was not detected, and the percentage of infected FLA was low (3.8%). Legionella species identified in these samples were L. drozanskii, L. donaldsonii and L. feeleii. Additionally, Acanthamoeba co-culture led to the isolation of Pseudomonas aeruginosa, P. stutzeri, P. fluorecens, Achromobacter xylosoxidans and Stenotrophomonas maltophilia. The highly disseminated presence of Acanthamoeba and the detection of amoeba-resisting bacteria inside amoebae highlight the importance of developing methods for controlling FLA in order to limit human pathogenic amoeba-resisting bacteria survival to the water purification processes.

The genus Legionella comprises more than 60 species, and about half are associated with infection. Legionella pneumophila is the most commonly associated with these infections and by far the most studied, but L. non-pneumophila species, such as L. feeleii, L. anisa, etc., may also present clinical importance. Free-living amoebae are their preferred environmental host, where these bacteria not only survive but also succeed in multiplying, and this relationship can lead to an increase in bacterial virulence. The goal of this study was to evaluate the alterations of Legionella pathogenicity due to its interaction with Acanthamoeba. For this, the expression of protein effectors SdhA, LegK2, and SidK were evaluated in L. pneumophila and L. feeleii, before and after infecting Acanthamoeba. Additionally, the host response was evaluated by measuring the production of IL-6, IL-8, and IFN-γ in infected macrophages. Regarding the virulence factors, an increase in SdhA expression was observed after these bacteria infected Acanthamoeba, with a higher increase in the macrophage cultures infected with L. feeleii. Also, an increase in the expression of LegK2 was observed after infecting Acanthamoeba, but it was more intense in the cultures infected with L. pneumophila. With regard to SidK, it was increased in L. feeleii after infecting Acanthamoeba, however the same effect was not observed for L. pneumophila. In cytokine production, the effect on IL-6 and IL-8 was similar for both cytokines, increasing their concentration, but higher production was observed in the cultures infected with L. feeleii, even though it demonstrated slightly lower production with the inoculum obtained from Acanthamoeba. Concerning IFN-γ, induction was observed in both species but higher in the infection by L. pneumophila. Nevertheless, it is not known if this induction is enough to promote an efficient immune response against either L. pneumophila or L. feeleii. Altogether, these alterations seem to increase L. feeleii virulence after infecting Acanthamoeba. However, this increase does not seem to turn L. feeleii as virulent as L. pneumophila. More studies are necessary to understand the aspects influenced in these bacteria by their interaction with Acanthamoeba and, thus, identify targets to be used in future therapeutic approaches.

BACKGROUND: The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. METHODS: Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. RESULTS: Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. CONCLUSIONS: The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out. This project was supported by Grant PI12/ 02725 from Instituto de Salud Carlos III (FISS) and FEDER by grant USPCEU-PC07/2013 of the Fundación Universitaria San Pablo CEU and by grant CEU-Banco de Santander. TSG was supported by Research Program EADS Construcciones Aeronáuticas S.A. - Brasil I and by CAPES/Brazil (Grant BEX 9132/13-9). Sí

Molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi has improved our understanding of the transmission of both organisms in humans. In this study, to infer possible infection sources, Cryptosporidium spp. and E. bieneusi in fecal specimens from 90 HIV-infected patients attending antiretroviral clinics in Lagos, Nigeria were detected and genotyped by PCR and DNA sequencing. Cryptosporidium spp. and E. bieneusi were identified in four and five patients, respectively, including the occurrence of subtype IeA11T3G3 of Cryptosporidium hominis in two patients, subtype IIcA5G3k of Cryptosporidium parvum in one patient, and Type IV of E. bieneusi in four patients. Among the remaining positive patients, one had mixed infection of Cryptosporidium meleagridis and C. hominis and one had mixed E. bieneusi genotypes. These data highlight a possible difference in major transmission routes (anthroponotic vs. zoonotic) between Cryptosporidium spp. and E. bieneusi in HIV+ patients in the study area.

Emerging methods based on mass spectrometry (MS) can be used in the rapid identification of microorganisms. Thus far, these practical and rapidly evolving methods have mainly been applied to characterize prokaryotes. We applied matrix-assisted laser-desorption-ionization-time-of-flight mass spectrometry MALDI-TOF MS in the analysis of whole cells of 18 N. fowleri isolates belonging to three genotypes. Fourteen originated from the cerebrospinal fluid or brain tissue of primary amoebic meningoencephalitis patients and four originated from water samples of hot springs, rivers, lakes or municipal water supplies. Whole Naegleria trophozoites grown in axenic cultures were washed and mixed with MALDI matrix. Mass spectra were acquired with a 4700 TOF-TOF instrument. MALDI-TOF MS yielded consistent patterns for all isolates examined. Using a combination of novel data processing methods for visual peak comparison, statistical analysis and proteomics database searching we were able to detect several biomarkers that can differentiate all species and isolates studied, along with common biomarkers for all N. fowleri isolates. Naegleria fowleri could be easily separated from other species within the genus Naegleria. A number of peaks detected were tentatively identified. MALDI-TOF MS fingerprinting is a rapid, reproducible, high-throughput alternative method for identifying Naegleria isolates. This method has potential for studying eukaryotic agents.

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.

Resumen Las tecnicas de diagnostico molecular por PCR permiten distinguir entre las diferentes especies de Cryptosporidium morfologicamente identicas capaces de infectar a humanos. De las 23 especies actualmente reconocidas en el genero, al menos 9 son capaces de infectar a humanos. Por ello, y debido a que la intensidad de las manifestaciones clinicas, la patogenicidad, la excrecion de ooquistes y la incidencia varian entre ellas, la realizacion de estudios moleculares es crucial para una mejor comprension de la epidemiologia de la criptosporidiosis humana. En el presente trabajo se analizan muestras procedentes de 2 estudios independientes: uno formado por 23 muestras procedentes de Madrid y otro compuesto por 72 muestras procedentes de La Coruna, todas ellas positivas para Cryptosporidium spp. por metodos microscopicos y pertenecientes a casos aislados de criptosporidiosis. Para la identificacion a nivel de especie se utilizaron las regiones de diagnostico descritas para el ADNr 18S y las regiones de diagnostico del gen de la COWP. De las 95 muestras analizadas, se consiguio extraer y amplificar ADN en 77 casos, en los que las especies causantes de la infeccion fueron: C. parvum (40 casos: 2 Madrid y 38 La Coruna), C. hominis (30 casos: 10 Madrid y 20 La Coruna) y C. meleagridis (2 casos: uno Madrid y uno La Coruna). En otros 5 casos fue imposible detectar la especie responsable de la infeccion, aunque se confirmara su positividad por PCR (4 Madrid y uno La Coruna). Los genotipos aislados en estos pacientes se correlacionaron con los hallados en animales de las mismas regiones.

a b s t r a c t Background: Microsporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare. Objective: To report genital microsporidiosis in female AIDS patients. Methods: Tissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kouri were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified. Results: We report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium. Conclusions: Microsporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract

Legionnaires' disease is a severe form of pneumonia, with worldwide relevance, caused by Legionella spp. Approximately 90% of all cases of legionellosis are caused by Legionella pneumophila, but other species can also be responsible for this infection. These bacteria are transmitted by inhalation of aerosols or aspiration of contaminated water. In Spain, environmental studies have demonstrated the presence of Legionella non-pneumophila species in drinking water treatment plants and water distribution networks. Aware that this evidence indicates a risk factor and the lack of routine assays designed to detect simultaneously diverse Legionella species, we analyzed 210 urine samples from patients presenting clinical manifestations of pneumonia using a semi-nested PCR for partial amplification of the 16S rDNA gene of Legionella and a diagnostic method used in hospitals for Legionella antigen detection. In this study, we detected a total of 15 cases of legionellosis (7.1%) and the first case of Legionnaires' disease caused by L. anisa in Spain. While the conventional method used in hospitals could only detect four cases (1.9%) produced by L. pneumophila serogroup 1, using PCR, the following species were identified: Legionella spp. (10/15), L. pneumophila (4/15) and L. anisa (1/15). These results suggest the need to change hospital diagnostic strategies regarding the identification of Legionella species associated with this disease. Therefore, the detection of Legionella DNA by PCR in urine samples seems to be a suitable alternative method for a sensitive, accurate and rapid diagnosis of Legionella pneumonia, caused by L. pneumophila and also for L. non-pneumophila species.

Genus Cryptosporidium, has undergone major revisions in recent years. The identification of new species and their major reservoirs has contributed to the knowledge of the epidemiology of human infection. In Spain, although there are many publications, few studies have been conducted to identify the circulating species and genotypes. This fact has led us to review and update these new studies published in Spain, particularly those that use molecular methods in order to characterise the species and genotypes present in our country. © 2010 Elsevier Espa ˜

Nosema ceranae is currently one of the major pathogens of honeybees, related to the worldwide colony losses phenomenon. The genotyping of strains based on ribosomal DNA (rDNA) can be misleading if the repeated units are not identical. The analysis of cloned rDNA fragments containing the intergenic spacer (IGS) and part of the rDNA small-subunit (SSU) gene, from N. ceranae isolates from different European and Central Asia populations, revealed a high diversity of sequences. The variability involved single-nucleotide polymorphisms and insertion/deletions, resulting in 79 different haplotypes. Two sequences from the same isolate could be as different as any pair of sequences from different samples; in contrast, identical haplotypes were also found in very different geographical origins. Consequently, haplotypes cannot be organized in a consistent phylogenetic tree, clearly indicating that rDNA is not a reliable marker for the differentiation of N. ceranae strains. The results indicate that recombination between different sequences may produce new variants, which is quite surprising in microsporidia, usually considered to have an asexual mode of reproduction. The diversity of sequences and their geographical distribution indicate that haplotypes of different lineages may occasionally be present in a same cell and undergo homologue recombination, therefore suggesting a sexual haplo-diploid cycle.

Enterocytozoon bieneusi is a microsporidian parasite that infects many vertebrate animals, including humans. The rDNA internal transcribed spacer (ITS) shows a hypervariable sequence; however, so far no clear information has been inferred about strain evolution in this species. We reviewed all the sequences described and performed a phylogenetic study. Four groups of sequences strongly differentiated from each other were detected, although most of the isolates (94%) corresponded to group I. The highly diverse sequences of this group were analyzed using median-joining networks. The host species (humans, pets, swine, cattle, birds, and wild animals) and the continents of origin of the isolates were considered. Central haplotypes in the network were obtained from very diverse hosts and geographical origins. The results show that although E. bieneusi has a broad host specificity, transmission is not completely free: some strains were able to circulate within a given host species and were only occasionally transmitted to another host. Additionally, while not relevant for swine or cattle hosts, geography seems to be a relevant factor for human infection by E. bieneusi .

The efficacy of the anti-inflammatory drug Bobel-24 against experimental infection by Cryptosporidium parvum was evaluated in neonatal lambs. The animals were treated by oral administration of the drug at 50 or 500 mg/kg of body weight. The prophylactic/therapeutic treatment was started 4 h before inoculation of the lambs with oocysts and was continued for eight consecutive days. The therapeutic treatment was initiated at the onset of diarrhoea, after confirmation of infection, and was continued for six consecutive days. Infection was monitored by daily examination of faecal samples from the first day until 30 days post-inoculation. The criteria considered in evaluating development of the infection and the drug activity were: oocyst shedding, presence of diarrhoea and weight gain at 15 and 30 days post-inoculation. Bobel-24 was effective as a prophylactic/therapeutic treatment at the lowest dose (50 mg/kg of body weight); in the group treated with this dose of drug there was a longer prepatent period, a shorter patent period and a lower intensity of oocyst excretion than in the untreated control group, and the differences were all statistically significant (P

SUMMARYIn humanToxocara canisinfection, an association has been shown between high IgG avidity in the chronic phase and low IgG avidity in recently acquired toxocarosis. The evolution of the antibody response in terms of avidity has been carried out through aT. canisinfection in BALB/c mice. Infection withT. canisembryonated eggs (EE) was carried out with single doses (SD) of 6, 12, 50, 100, 200 or 1000 EE/mouse and with multiple doses (MD) of 200 and 1000 EE. Specific antibodies againstT. canis(IgM+G, IgG, IgG1 and IgM) were detected by ELISA and Western Blot (WB) techniques in the presence and absence of urea. With the ELISA method, an increase in the avidity index (AI) of around 50% was detected from days 40–80 p.i. to the end of the study, with all the doses studied. The WB method showed the presence of high avidity antibodies bound to 100 kDa and 75 kDaT. canisproteins in all the cases when the IgM+G and the IgG1 antibodies were investigated. Antibodies of variable avidity were observed in those sera that recognized the group of low molecular weight proteins, between 37 kDa and 25 kDa.

l-Arginine is an essential amino acid in Leishmania (Leishmania) amazonensis metabolism. A key enzyme for parasite l-arginine metabolism is arginase (ARG) that uses arginine to produce urea and ornithine, a precursor of polyamine pathway guaranteeing parasite replication in both insect and mammal hosts. There is an alternative pathway to produce ornithine via l-proline and glutamate, but this mechanism is not described in Leishmania. In the mammal host, two enzymes can use l-arginine as substrate, the host ARG and the induced nitric oxide synthase that produces nitric oxide. The competition between induced nitric oxide synthase and both parasite and host ARG can favor the success of the infection or its control. Here, we established the metabolomics profile of the polyamine pathway of wild type (WT) L. (L.) amazonensis, submitted or not to l-arginine starvation, and compared to the ARG-knockout mutant (arg- ). Our results indicated that arginine starvation induces a decrease in arginine, ornithine, and putrescine, but we could not detect the significative level changes of spermidine, spermine, or agmatine. However, the absence of ARG on the arg- induced an increase of arginine and citrulline levels, but decreased the levels of ornithine and putrescine. Similarly to the WT arginine-starved parasites, the arg- parasites presented lower levels of proline when compared to the WT ones. This could be indicative of an alternative pathway to surpass the enzyme or its substrate absence.

Los microsporidios son protozoos intracelulares obligados, implicados en procesos de diarrea persistente en pacientes con sida, aunque no son exclusivos de este grupo de pacientes. La prevalencia de microsporidios en diferentes países varía entre 8% y 52%. En nuestro medio no se conoce su frecuencia, por lo que este trabajo se propuso determinar la frecuencia de microsporidiosis intestinal en pacientes positivos para VIH, mediante la prueba del Gram cromotropo rápido (quick hot Gram) y la PCR; para esto se realizó un estudio prospectivo, descriptivo, con una población intencional de todos los pacientes positivos para VIH remitidos al Laboratorio del Grupo Interdisciplinario para el Estudio de las Parasitosis Intestinales por las diferentes instituciones de atención de pacientes positivos para VIH de Medellín en el periodo comprendido entre agosto de 2001 y septiembre de 2002. Se hizo una encuesta clínico-epidemiológica y se practicaron análisis coprológicos seriados que incluían examen directo, por concentración y tinciones especiales para coccidias y microsporidios intestinales; además, se solicitó recuento de linfocitos TCD4+ y carga viral. Se estudiaron 103 pacientes en edades comprendidas entre 2 y 74 años; el 70% (72/103) presentaba diarrea al ingreso al estudio; la mayoría (83,5%) fueron hombres. La frecuencia global de microsporidiosis intestinal fue de 3,9% (4/103); se encontraron tres pacientes positivos para Enterocytozoon bieneusi y uno con Encephalitozoon intestinalis; otras parasitosis intestinales representaron el 39,8%. La frecuencia de microsporidiosis en este estudio fue relativamente baja; además, como era de esperarse, la mayoría de los casos de microsporidios estuvieron asociados con diarrea prolongada y recuentos de LTCD4+ menores de 100 cél/?l y cargas virales superiores a 100.000 copias (3/4).

Microsporidia are ubiquitous parasites infecting all animal phyla and we present evidence that supports their zoonotic potential. Fecal samples taken from domestic (cats and dogs), farm (pigs, rabbits and ostriches) and wild animals (foxes) from different provinces of Spain were evaluated for microsporidia infection by light microscopy and PCR. After Microsporidia species identification, E. bieneusi genotypes were additionally studied by sequence analysis of the ITS region. Eighty-five samples out of 159 exhibited structures that were compatible with microsporidia spores by Webeŕs stain with 37 of them being confirmed by PCR. Microsporidia species identified included E. bieneusi, E. intestinalis and A. algerae. We report the first diagnosis of E. intestinalis and E. bieneusi in ostriches and A. algerae in pigs. We also provide new information on the molecular characterization of E. bieneusi isolates both in rabbits and ostriches. All of the E. bieneusi genotypes identified belonged to the zoonotic group of genotypes (Group I) including genotypes A (dogs), I (pigs), D (rabbits and foxes) and type IV (ostriches). Our results demonstrate that microsporidia are present in domestic, farm and wild animals in Spain, corroborating their potential role as a source of human infection and environmental contamination.

Objective: We recently demonstrated that the biggest reduction of T cells in septic patients was produced in the γδ T subset. This depletion was directly proportional to the severity of the septic process, and it was associated with mortality. We hypothesized that microsporidia can harness the deficit of γδ T cells in septic patients to proliferate and contribute to the worsening of the sepsis. Methods: In this retrospective study, we analyzed anti-Encephalitozoon cuniculi antibody levels in sera from 46 septic patients, and compared them with a similar control group of healthy subjects. As a secondary objective we aimed to relate anti-E. cuniculi antibody levels with αβ and γδ T cells in these patients. Results: Forty-eight percent of septic patients were positive for IgE anti-E. cuniculi vs 13.0% of healthy subjects (OR: 3.67, CI95% 1.64-8.20, P=0.001). The frequency of αβ and CD56+ γδ T cell subsets decreased in septic patients with positive anti-E. cuniculi IgE antibodies. This decrease was more potent in the CD3+CD56+ γδ T cell subset. The genitourinary focus (urinary tract infections and pyelonephritis) produced a significant higher percentage of anti-E. cuniculi positive cases (11/13 (84.6%), OR=11.0, CI 95% 2.1-58.5, P=0.003). Conclusion: There was a greater level expression of IgE anti-E. cuniculi in septic patients, reaching almost 50% of positive. The presence of IgE anti-E. cuniculi in septic patients was related to a decrease of αβ and γδ T cells in peripheral blood. This decrease was more potent in the CD3+CD56+ γδ T cell subset. Microsporidia may be heavily involved in the physio-pathological evolution of sepsis.

A new fluorimetric method for the diagnosis of microsporidia was compared to the indirect immunofluorescence (IIF) method. Plates were coated with Encephalitozoon cuniculi spores, sera were incubated and an anti-human IgG FITC-conjugate was added. Finally, the plates were read using a fluorimeter. The results obtained were compared using the IIF technique confirming the positive sera with Fluorescence Index (FI) values of 3.75 and 5.24 in the fluorimetric method. Sera with FI values of 2.03 and 2.35 had borderline results when the IIF technique was used. The present results confirm the usefulness of fluorimetric methods in the diagnosis of human microsporidia, both in cases of the absence of immunodeficiency as well as in epidemiological studies.

Intestinal microsporidiosis has been associated traditionally with severely immunocompromised patients with AIDS. We describe two new cases of intestinal microsporidiosis due to Enterocytozoon bieneusi in human immunodeficiency virus-negative adults. Both patients presented with chronic nonbloody diarrhea, and one had intestinal lymphangiectasia as well. Intestinal microsporidiosis was diagnosed by evaluation of stool samples, and the specific species was determined by use of polymerase chain reaction (PCR) in duodenal biopsy specimens. To our knowledge, this is the first report of confirmation of E. bieneusi in the intestinal epithelium of HIV-negative individuals by use of PCR in duodenal biopsy specimens. Cases of intestinal microsporidiosis in HIV-negative individuals reported in the English-language literature are reviewed. These two new cases along with those described previously corroborate the need to evaluate for microsporidia in HIV-negative individuals with unexplained diarrhea.

Background: The cause of Crohn's Disease (CD) remains unknown. Recently a decrease in the global lymphocyte population in the peripheral blood of CD patients has been reported. This decrease was more evident in gamma delta T lymphocytes, especially gamma delta CD8+T subsets. Furthermore, a decrease of IL-7 was also observed in these patients. We propose the hypothesis that microsporidia, an obligate intracellular opportunistic parasite recently related to fungi, in CD patients can take advantage of the lymphocytes and IL-7 deficits to proliferate and to contribute to the pathophysiology of this disease. Methods and Findings: In this case-control study, serum samples were collected from 36 CD patients and from 36 healthy individuals (controls), IgE and IgG anti-Encephalitozoon antibodies were determined by ELISA; and forty-four intestinal tissue samples were analyzed through real time Polymerase Chain Reaction (PCR), twenty CD patients, nine with others diseases and 15 healthy subjects. We observed that IgE anti-Encephalitozoon levels were significantly higher in patients with CD: 0.386(+/- 0.256) vs control group, 0.201(+/- 0.147), P

Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis , Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae . C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

Background Microsporidia are obligate intracellular parasites that infect a broad range of vertebrates and invertebrates. They have been increasingly recognized as human pathogens in AIDS patients, mainly associated with a life-threatening chronic diarrhea and systemic disease. However, to date the global epidemiology of human microsporidiosis is poorly understood, and recent data suggest that the incidence of these pathogens is much higher than previously reported and may represent a neglected etiological agent of more common diseases indeed in immunocompetent individuals. To contribute to the knowledge of microsporidia molecular epidemiology in HIV-positive patients in Nigeria, the authors tested stool samples proceeding from patients with and without diarrhea. Methodology/Principal Findings Stool samples from 193 HIV-positive patients with and without diarrhea (67 and 126 respectively) from Lagos (Nigeria) were investigated for the presence of microsporidia and Cryptosporidium using Weber’s Chromotrope-based stain, Kinyoun stain, IFAT and PCR. The Weber stain showed 45 fecal samples (23.3%) with characteristic microsporidia spores, and a significant association of microsporidia with diarrhea was observed (O.R. = 18.2; CI: 95%). A similar result was obtained using Kinyoun stain, showing 44 (31,8%) positive samples with structures morphologically compatible with Cryptosporidium sp, 14 (31.8%) of them with infection mixed with microsporidia. The characterization of microsporidia species by IFAT and PCR allowed identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis and E. cuniculi in 5, 2 and 1 samples respectively. The partial sequencing of the ITS region of the rRNA genes showed that the three isolates of E.bieneusi studied are included in Group I, one of which bears the genotype B. Conclusions/Significance To our knowledge, this is the first report of microsporidia characterization in fecal samples from HIV-positive patients from Lagos, Nigeria. These results focus attention on the need to include microsporidial diagnosis in the management of HIV/AIDS infection in Nigeria, at the very least when other more common pathogens have not been detected.

Microsporidiosis must be regarded as a late opportunistic infection when HIV is advanced. In this article we describe 2 cases of disseminated infection with Encephalitozoon intestinalis. The first case had a local intestinal infection for > 1 y before it disseminated and microsporidia were found intracellularly in sputum. In the second case, spores were initially found in conjunctival cells, sinus lavage, sputum and urine. This patient had clinical symptoms and radiological findings from the central nervous system. Signs of cerebral lymphoma developed after treatment of the opportunistic microsporidial infection.

Diarrhea is the main health problem caused by human-related microsporidia, and waterborne transmission is one of the main risk factors for intestinal diseases. Recent studies suggest the involvement of water in the epidemiology of human microsporidiosis. However, studies related to the presence of microsporidia in different types of waters from countries where human microsporidiosis has been described are still scarce. Thirty-eight water samples from 8 drinking water treatment plants (DWTPs), 8 wastewater treatment plants (WWTPs) and 6 recreational river areas (RRAs) from Galicia (NW Spain) have been analyzed. One hundred liters of water from DWTPs and 50 L of water from WWTPs and RRAs were filtered to recover parasites, using the IDEXX Filta-Max® system. Microsporidian spores were identified by Weber’s stain and positive samples were analyzed by PCR, using specific primers for Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem . Microsporidia spores were identified by staining protocols in eight samples (21.0%): 2 from DWTPs, 5 from WWTPs, and 1 from an RRA. In the RRA sample, the microsporidia were identified as E. intestinalis . To the best of our knowledge, this is the first report of human-pathogenic microsporidia in water samples from DWTPs, WWTPs and RRAs in Spain. These observations add further evidence to support that new and appropriate control and regulations for drinking, wastewater, and recreational waters should be established to avoid health risks from this pathogen.

Species of Cryptosporidium infect a broad variety of animals. Because morphological features of the secreted oocysts are not useful in identifying the parasite at the species level, molecular tools were used to accomplish this task, leading to discovery of new Cryptosporidium species. With the use of this approach, Cryptosporidium bovis has recently been described as a new species infecting bovines and several other hosts, but clearly distinct from C. parvum. In this report, we present a description of a Cryptosporidium sp. isolate from a newborn lamb from a farm in Spain. The isolate seemed to be very similar to C. bovis based on the analysis of the gene that codes for the 18S rRNA.

Ocular toxocarosis (OT) is a well-established disease. However, our understanding of the migratory route, time and circumstances that produce ocular invasion are not clear. To improve our knowledge of factors related to ocular invasion, BALB/c mice were inoculated with simple doses (SD) of 6, 12, 50, 100, 200 and 1000 embryonated eggs (EE) and multiple doses (MD) of 200 and 1000 EE. Brains and eyes were studied for the presence of larvae in animals sacrificed on days 3, 5, 7, 9, 11, 13, 15, 17, 19, 40, 80 and 120 in SD and on days 28, 30, 32, 34, 36, 38, 40, 42, 46, 87 and 127 in MD. The humoral immune responses were studied by ELISA using excretory-secretory antigen. Due to the considerable number of days tested, results showed are based on one set of experiments. However, each point studied represents the result obtained from a group of five mice. We have shown that the eye involvement with Toxocara canis larvae is a phenomenon mainly produced once larvae have reached the brain. There is a direct relationship between the parasitic load and the number of ocular larvae. Moreover, the arrival of larvae to the eye is an independent event, unrelated to the kind of administered dose (SD, MD), although the number of the brain larvae was higher in the cases of MD. High levels of specific antibodies were observed but they did not prevent the arrival of the larvae to the brain and the eye.