West Nile and Usutu virus infections in wild birds admitted to rehabilitation centres in Extremadura, western Spain, 2017-2019.

West Nile virus (WNV) is an emerging flavivirus transmitted generally by mosquitoes of Culex genus. It is maintained in an enzootic life cycle where birds act as reservoir hosts. Humans and horses are also susceptible to infection, and occasionally, they suffer from neurological complications. However, they do not transmit the virus to other vectors, behaving as dead-end hosts. Sporadic WNV outbreaks observed in horses and wild birds from Extremadura (western Spain) during 2016 and 2017 seasons prompted to carry out this survey in wild birds, focused on specimens coming from two wildlife rehabilitation centres. Between October 2017 and December 2019, samples from 391 wild birds, belonging to 56 different species were collected and analysed in search of evidence of WNV infection. The analysis of serum samples for WNV-specific antibodies by ELISA, whose specificity was subsequently confirmed by virus-neutralisation test (VNT) showed positive results in 18.23 % birds belonging to 18 different species. Pelecaniformes (33.33 %), Accipitriformes (25.77 %) and Strigiformes (22.92 %) orders had the higher seroprevalences. Remarkably, WNV-specific antibodies were found in a black stork for the first time in Europe. Analysis by real time RT-PCR in symptomatic birds confirmed the presence of WNV lineage 1 RNA in griffon vulture and little owls. Specificity analysis of ELISA positive and doubtful sera was performed by differential VNT titration against WNV and two other cross-reacting avian flaviviruses found in Spain: Usutu virus (USUV) and Bagaza virus (BAGV). Only four samples showed USUV-specific antibodies (1.04 %) corresponding to three species: Eurasian eagle-owl, griffon vulture and great bustard (first detection in Europe) whereas no samples were found reactive to BAGV. Differential VNT yielded undetermined flavivirus result in 16 samples (4.17 %). This is the first study carried out on wild birds from Extremadura (western Spain). It highlights the widespread circulation of WNV in the region and its co-circulation with USUV.

[Recovering lost vaccine coverage due to COVID-19 pandemic]. / Recuperando las coberturas vacunales perdidas en la pandemia de COVID-19.

The 2019 coronavirus disease pandemic can have an alarming impact on vaccination coverage. WHO, UNICEF and Gavi warn that at least 80 million children under the age of 1 are at risk of contracting diseases such as diphtheria, measles and polio due to the interruption of routine immunization and the temporary suspension of 93 campaigns of large-scale vaccination.In Spain, a new healthcare scenario, which prioritizes telematics over in person, fear of contagion by going to health centers, and recommendations for physical distance and restricted mobility, reduce attendance at primary care centers. Despite recommendations established by the health authorities, vaccination coverage has decreased in all Autonomous Communities between 5% and 60%, depending on the age and type of vaccine. School vaccinations have been suspended and only vaccination of pregnant women against tetanus, diphtheria and pertussis has been maintained. The decrease has been more evident for non gratuity vaccines: the first dose of meningococcal vaccine B has decreased by 68.4% in the Valencian Community, and Andalusia has observed a 39% decrease in the total doses of this vaccine and of 18% for that of rotavirus.The recovering of vaccinations should be planned, organized and carried out in the shortest possible time.This article discusses some aspects of the recovery of vaccination coverage for different groups: children, adolescents and adults, and patients at risk and in special situations.

Occurrence, prevalence, and explanatory environmental variables of Spirocerca vulpis infestation in the foxes of western Spain.

The main aim of this study was to not only establish the prevalence of the recently described Spirocerca vulpis parasite in the wild-life cycle of carnivores in western Spain but to also elaborate a model to explain the risk of infestation based on 16 topo-climatic and habitat variables. During the period from June 2016 to November 2017, 1644 carcasses of red foxes (Vulpes vulpes) and another 105 wild mammals, legally hunted or killed in car accidents, were analyzed. Parasitic nodules of Spirocerca were found in 6% of the foxes, and the molecular analyses established a homology of our samples with the species S. vulpis. There were no differences in the occurrence of the infestation between sexes, but there were differences in terms of age, such that infestation was proportionally more frequent among young individuals. In terms of temporality, a higher percentage of positive cases was observed during the late-autumn and winter months, especially between December and February. This study provides new data on the factors that predispose S. vulpis infection in the red fox. Model results indicate that a spatial pattern exists in the occurrence and prevalence of this species in the studied area (higher probabilities to the west), and that this pattern seems to mainly be associated with topo-climatic variables.

Corrigendum: PAM50 Subtypes in Baseline and Residual Tumors Following Neoadjuvant Trastuzumab-Based Chemotherapy in HER2-Positive Breast Cancer: A Consecutive-Series From a Single Institution.

[This corrects the article DOI: 10.3389/fonc.2019.00707.].

PAM50 Subtypes in Baseline and Residual Tumors Following Neoadjuvant Trastuzumab-Based Chemotherapy in HER2-Positive Breast Cancer: A Consecutive-Series From a Single Institution.

Introduction: HER2-enriched subtype has been associated with higher response to neoadjuvant anti-HER2-based therapy across various clinical trials. However, limited data exist in real-world practice and regarding residual disease. Here, we evaluate the association of HER2-enriched with pathological response (pCR) and gene expression changes in pre- and post-treatment paired samples in HER2-positive breast cancer patients treated outside of a clinical trial. Methods: We evaluated clinical-pathological data from a consecutive series of 150 patients with stage II-IIIC HER2-positive breast cancer treated from August 2004 to December 2012 with trastuzumab-based neoadjuvant chemotherapy. Expression of 105 breast cancer-related genes, including the PAM50 genes, was determined in available pre-and post-treatment formalin-fixed paraffin-embedded tumor samples using the nCounter platform. Intrinsic molecular subtypes were determined using the research-based PAM50 predictor. Association of genomic variables with total pCR was performed. Results: The pCR rate was 53.3%, with higher pCR among hormonal receptor (HR)-negative tumors (70 vs. 39%; P < 0.001). A total of 89 baseline and 28 residual tumors were profiled, including pre- and post-treatment paired samples from 26 patients not achieving a pCR. HER2-enriched was the predominant baseline subtype not only in the overall and HR-negative cohorts (64 and 75%, respectively), but also in the HR-positive cohort (55%). HER2-enriched was associated with higher pCR rates compared to non-HER2-enriched subtypes (65 vs. 31%; OR = 4.07, 95% CI 1.65-10.61, P < 0.002) and this association was independent of HR status. In pre- and post-treatment paired samples from patients not achieving a pCR, a lower proportion of HER2-enriched and twice the number of luminal tumors were observed at baseline, and luminal A was the most frequent subtype in residual tumors. Interestingly, most (81.8%) HER2-enriched tumors changed to non-HER2-enriched, whereas most luminal A samples maintained the same subtype in residual tumors. Conclusions: Outside of a clinical trial, PAM50 HER2-enriched subtype predicts pCR beyond HR status following trastuzumab-based chemotherapy in HER2-positive disease. The clinical value of intrinsic molecular subtype in residual disease warrants further investigation.

Rare but evolutionarily consequential outcrossing in a highly inbred zoonotic parasite.

Recurrent self-mating can result in nearly clonal propagation of biological lineages, but even occasional outcrossing can serve to redistribute variation in future generations, providing cohesion among regional populations. The zoonotic parasite Trichinella spiralis has been suspected to undergo frequent inbreeding, resulting in genetically uniform larval cohorts which differ markedly from one another. Here, we explored the extent of inbreeding for this parasite by determining how genetic variation (at variable microsatellite markers) is distributed among 1379 larvae derived from 41 wild boars in Extremadura, Spain. In particular, we sought to determine how much of the genetic variation in this region's parasites occurs among the larvae of any given wild boar, and whether each derives from one, or more, parental lineages. We found strong evidence for inbreeding, resulting in genetically distinct parasite subpopulations among the parasites derived from many pairs of wild boar. Fully two-thirds of these parasite cohorts appear to derive from inbred parents; in 10% of the wild boars, parasites were so inbred as to become absolutely fixed in all of the assayed genetic loci. In spite of this, more than one pair of parents appear to have given rise to the infections in one-third of the sampled wild boars, resulting in mixed infections. These mixed infections should slow losses of heterozygosity and multi-locus polymorphism in any given parasite lineage. Such outcrossing should limit distinctions that would otherwise accumulate among transmission chains, thereby enforcing cohesion through the region's population in spite of its marked departure from panmixia. Conditions of transmission may differ in other regions, where such epidemiological features may engender different evolutionary outcomes.

Detection of Leishmania DNA and blood meal sources in phlebotomine sand flies (Diptera: Psychodidae) in western of Spain: Update on distribution and risk factors associated.

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012-2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.

Detection of Zoonotic Protozoa Toxoplasma gondii and Sarcocystis suihominis in Wild Boars from Spain.

Food safety regulations require the control of the presence of protozoa in meats destined for human consumption. Wild boar (Sus scrofa) meat may constitute a source of zoonoses. A 23.8% (688/2881) seroprevalence of anti-Toxoplasma gondii antibodies and 72.2% (662/910) Sarcocystis sarcocysts prevalence were detected among wild boars hunted in Southwestern areas of Spain. Identity of Sarcocystis spp. was performed by RFLP-PCR and sequencing, detecting S. miescheriana (7/8) and the zoonotic S. suihominis (1/8). Risk assessment studies of these coccidian in meats destined to human consumption are needed.

[Varicella vaccine effectiveness in schoolchildren in outbreaks in a semi-urban area]. / Efectividad de la vacuna de la varicela en el contexto de brotes escolares en una zona semiurbana.

OBJECTIVE: To estimate the effectiveness of the varicella vaccine in school outbreaks during the academic year 2009-2010. MATERIAL AND METHOD: Retrospective cohort study in public day-care centers and/or schools in an area in the region of Murcia. Spain. The participants were all children of 1 to 12 years who were in the same classroom where there was an outbreak of varicella. The main measurements were the sociodemographic, clinical and vaccination data, as well as variables related to varicella disease obtained through a questionnaire self-completed by parents, and from the computerized regional immunization registry (VACUSAN). RESULTS: A total of 51 varicella outbreaks were detected, with a median of 3 cases per outbreak at both educational levels. The overall vaccination coverage was 10.7% (95% CI 8.63 to 13.18), always being higher in Spanish schoolchildren versus foreign (OR: 21.21, 95% CI: 2.92 to 153.92, P<.001). Discrepancies were found between the vaccine questionnaire data and vaccination program (kappa=0.50, 95% CI: 0.43 to 0.58, P<.001). According to VACUSAN, the overall attack rate was 59.7 (95% CI: 55.82 to 63.43) in unvaccinated and 6.5 (95% CI: 2.54 to 15.45) in vaccinated children. An overall effectiveness of 89.1% (95% CI: 74.55 to 95.35) and 100% was obtained for 1 and 2 doses of vaccine, respectively. CONCLUSIONS: There is a high effectiveness of varicella vaccine, emphasizing that the administration of two doses of vaccine produces an adequate and optimal protection against varicella disease. A discrepancy was found between the information provided by parents and official records. Finally, there was a lower vaccination coverage in the immigrant community.

Computerised vaccination register for the Murcia region, Spain, 1991 to 2011.

We describe the Murcia regional vaccination register in Spain, which was set up in 1991, detailing its main features, advantages and limitations. We also report on some recent special actions carried out that led to an improvement in vaccination coverage against measles, rubella and mumps (MMR): by using the vaccination register, we were able to identify and vaccinate persons aged under 20 years in a measles outbreak in 2010 in the town of Jumilla who were not adequately vaccinated for their age with MMR vaccine. From spring 2012, use of our register will enable us to identify susceptible individuals in our region under 40 years of age who have received fewer than two doses of MMR vaccine and call them for the appropriate vaccination. We also set out our experience in the use of barcodes to identify individuals and collect vaccine data: our data show that the barcodes help to improve data quality and completeness. Finally, we identify certain challenges in search of greater standardisation for systems and encoding that is necessary to enable an easy exchange of data between different registers.

Toxicological evaluation of three contaminants of emerging concern by use of the Allium cepa test.

Di(2-ethylhexyl)phthalate, triclosan and propylparaben are contaminants of emerging concern that have been subjected to extensive toxicological studies, but for which limited information is currently available concerning adverse effects on terrestrial plant systems. The Allium cepa test, which is considered one of the most efficient approaches to assess toxic effects of environmental chemicals, was selected to evaluate the potential risks of these ubiquitous pollutants. Our data demonstrate that all three compounds studied may in some way be considered toxic, but different effects were noted depending on the chemical and the end point analysed. Results derived from the analysis of macroscopic parameters used in testing for general toxicity, revealed that while di(2-ethylhexyl)phthalate had no apparent effects, the other two chemicals inhibited A. cepa root growth in a dose-dependent manner. On the other hand, although all three compounds caused alterations in the mitotic index of root-tip cells, propylparaben was the only one that did not show evidence of genotoxicity in assays for chromosome aberrations and micronuclei. The results of the present study clearly indicate that sensitive plant bioassays are useful and complementary tools to determine environmental impact of contaminants of emerging concern.

Histopathology of trichinellosis in wild boar.

Histopathological study of Trichinella constitutes an important knowledge base to understand the pathogenesis of this disease. This study analyses cell response and macroscopic lesions in wild boar for the two species of Trichinella present in Spain: Trichinella spiralis and T. britovi. We carried out both trichinelloscopy and artificial digestion to calculate the parasitic load and relate this to the macroscopic lesions. The results obtained prove a lesser adaptation of T. britovi in wild boar. From a histological point of view, the organic region that was most affected was the skeletal muscle, where inflammatory infiltrates were observed around the larvae, and they were most abundant in calcified cysts.

An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines.

The ever growing anthropogenic pressure to the environment has lead in 2007 to the revision of the existing legislation and the approval of the new European law regarding the production and importation of chemicals, known as REACH. This new legal framework supports the development of alternative methods to animal experimentation encouraging the improvement and/or design of new methodological strategies for the toxicological evaluation of chemical compounds. Even though cytotoxicity studies are a reductionist approach to acute toxicity in vivo, they offer the best agreement between obtaining relevant information about the mechanism of toxic action and the use of alternative methods. Following this trend, this work presents an integrated cellular strategy in order to know the toxicity and mechanism of action of chemical compounds, using simple and reproducible in vitro systems. The experimental procedures are performed in two steps. The first one involves the systematic analysis of the main cellular targets using proliferation, viability and morphological probes. The second step relies upon the results obtained in the first step, including specific assays that focus on the mechanism of toxic action and the cellular response. The benefits of this strategy are exemplified with two real cases: pentachlorophenol and rotenone.

In vitro assessment of the cytotoxic and mutagenic potential of perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is a perfluorinated compound ubiquitously detected in the environment, including wildlife and humans. Despite the available information, research on the cytotoxicity of PFOA in non-tumoral mammalian cells is relatively limited. In this work, two in vitro toxicity systems were employed to provide further insight into the cytotoxic and mutagenic potential of PFOA. The cytotoxicity of the chemical towards Vero cells was assessed using biochemical and morphological parameters, while mutagenicity was evaluated according to Ames test. High doses of PFOA cause oxidative stress in Vero cells, that was closely linked to cell cycle arrest at the G1 phase and induction of apoptosis. Our results corroborate previous findings in human tumoral cells and suggest that the mode of action of this perfluorinated compound is not a peculiarity among mammalian cell types. On the other hand, the compound was not mutagenic in the Ames test, using four strains of Salmonella typhimurium in the presence or absence of rat S9 metabolic activation system.

Carbamazepine induces mitotic arrest in mammalian Vero cells.

We reported recently that the anticonvulsant drug carbamazepine, at supratherapeutic concentrations, exerts antiproliferative effects in mammalian Vero cells, but the underlying mechanism has not been elucidated. This motivates us to examine rigorously whether growth arrest was associated with structural changes in cellular organization during mitosis. In the present work, we found that exposure of the cells to carbamazepine led to an increase in mitotic index, mainly due to the sustained block at the metaphase/anaphase boundary, with the consequent inhibition of cell proliferation. Indirect immunofluorescence, using antibodies directed against spindle apparatus proteins, revealed that mitotic arrest was associated with formation of monopolar spindles, caused by impairment of centrosome separation. The final consequence of the spindle defects induced by carbamazepine, depended on the duration of cell cycle arrest. Following the time course of accumulation of metaphase and apoptotic cells during carbamazepine treatments, we observed a causative relationship between mitotic arrest and induction of cell death. Conversely, cells released from the block of metaphase by removal of the drug, continued to progress through mitosis and resume normal proliferation. Our results show that carbamazepine shares a common antiproliferative mechanism with spindle-targeted drugs and contribute to a better understanding of the cytostatic activity previously described in Vero cells. Additional studies are in progress to extend these initial findings that define a novel mode of action of carbamazepine in cultured mammalian cells.

Cytotoxicity of butylated hydroxyanisole in Vero cells.

Butylated hydroxyanisole (BHA) is perhaps the most extensively used synthetic antioxidant in the food and cosmetic industry, although considerable controversy exists in the literature regarding the safety of this compound. Most in vitro studies describing the effects of BHA have been performed in cancer cells, but it is unclear whether normal cells are equally susceptible to BHA exposure. The present study investigate the toxic potential of BHA in mammalian cells, using biochemical and morphological parameters, which reveal interference with structures essential for cell survival, proliferation and/or function. Cell growth inhibition was assessed by using colorimetric assays, whereas cellular alterations after BHA exposure, were evaluated using conventional light and fluorescence microscopy. Low doses of BHA exerted a significant cytotoxic effect, associated with loss of mitochondrial function. As the concentration of BHA was increased, morphological alterations in critical subcellular targets such as lysosomes, mitochondria and actin cytoskeleton, were observed. In parallel, BHA induced an irreversible loss of cell proliferative capacity, preceding apoptosis induction. Thus, the dose-dependent activity of BHA on Vero cells appears to be cytotoxic as well as cytostatic. Our observations, although simplified with respect to the in vivo situations, allowed the assessment of the specific damage at the cellular level, and provide some clue about the effects of BHA in non-tumoral mammalian cells.

Cytotoxic effects in mammalian Vero cells exposed to pentachlorophenol.

The effects of pentachlorophenol have been studied on diverse biological systems both in vivo and in vitro, however the cellular basis of the pronounced cytotoxicity of this organochlorine compound is poorly understood. In this work, morphological and biochemical analyses were carried out to identify the primary targets of pentachlorophenol toxicity in mammalian cells. Our results show that pentachlorophenol is a very potent cytotoxic drug that displays an unusual and interesting mode of action in Vero cells. Although this compound is a powerful uncoupler of oxidative phosphorylation, we present the novel finding that lysosome destabilization is an early cytotoxic response that precedes the mitochondrial dysfunction. In addition, soon after exposure to moderate doses of pentachlorophenol, a significant number of cells initiate an apoptotic death process identified by the condensed and fragmented state of their nuclei. These results demonstrate that there are multiple potential targets of PCP-induced toxicity in mammalian cells, and the need to develop further experimental studies for the risk assessment of this environmental pollutant.

CandidaDB: a genome database for Candida albicans pathogenomics.

CandidaDB is a database dedicated to the genome of the most prevalent systemic fungal pathogen of humans, Candida albicans. CandidaDB is based on an annotation of the Stanford Genome Technology Center C.albicans genome sequence data by the European Galar Fungail Consortium. CandidaDB Release 2.0 (June 2004) contains information pertaining to Assembly 19 of the genome of C.albicans strain SC5314. The current release contains 6244 annotated entries corresponding to 130 tRNA genes and 5917 protein-coding genes. For these, it provides tentative functional assignments along with numerous pre-run analyses that can assist the researcher in the evaluation of gene function for the purpose of specific or large-scale analysis. CandidaDB is based on GenoList, a generic relational data schema and a World Wide Web interface that has been adapted to the handling of eukaryotic genomes. The interface allows users to browse easily through genome data and retrieve information. CandidaDB also provides more elaborate tools, such as pattern searching, that are tightly connected to the overall browsing system. As the C.albicans genome is diploid and still incompletely assembled, CandidaDB provides tools to browse the genome by individual supercontigs and to examine information about allelic sequences obtained from complementary contigs. CandidaDB is accessible at http://genolist.pasteur.fr/CandidaDB.

Ultrastructural changes induced in HeLa cells after phototoxic treatment with harmine.

In the present work, we have continued our studies on harmine phototoxicity in human tumour cells. The toxicity of harmine in the dark was analysed by a quantitative neutral red uptake assay, and subcellular sensitive targets following harmine photosensitization were de fi ned by electron microscopic analysis of HeLa cells. The results obtained indicated that this compound shows a clear dose-dependent cytotoxic effect in the dark. The combined treatment with suitable doses of harmine and UV radiation was very effective at an early stage, although maximal cell killing appeared 48 h after photodynamic activation. Ultrastructural examination of HeLa cells immediately after the photodynamic treatment revealed lysosomal destabilization and profound cytoplasmic vacuolization that evolved to cytolysis, which is typical of necrotic cell death. It is concluded that harmine could be a valuable photosensitizer whose biological applications merit further evaluation.