Necrotizing scleritis due to Hormographiella aspergillata.

PURPOSE: To report the management and outcome of a case of necrotizing scleritis due to Hormographiella aspergillata. METHODS: Case report. RESULTS: A 79-year-old woman developed scleral inflammation following accidental trauma with a gorse plant in her left eye. An abscess formed at the site of the injury, which was surgically drained. Filamentous fungi were identified from the abscess contents, and oral voriconazole and topical voriconazole and natamycin drops were prescribed. Phenotypic analysis confirmed the presence of Hormographiella aspergillata, with low minimum inhibitory concentrations (MIC) for voriconazole and amphotericin B. Two weeks later the patient presented with an area of necrotizing scleritis which required surgical debridement and scleral grafting. Three months later, the scleral inflammation had resolved leaving an area of scleromalacia. CONCLUSIONS: Hormographiella aspergillata is a common environmental fungus that has recently emerged as a human pathogen and a rare cause of scleritis. To the best of our knowledge, this is the first report of scleritis in which a pure culture of H. aspergillata was obtained. Successful management poses a challenge as there are limited reports on antifungal susceptibility and a combination of medical and surgical treatment is often required.

Antifungal Combination Eye Drops for Fungal Keratitis Treatment.

Fungal keratitis (FK) is a corneal mycotic infection that can lead to vision loss. Furthermore, the severity of FK is aggravated by the emergence of resistant fungal species. There is currently only one FDA-approved formulation for FK treatment forcing hospital pharmacy departments to reformulate intravenous drug preparations with unknown ocular bioavailability and toxicity. In the present study, natamycin/voriconazole formulations were developed and characterized to improve natamycin solubility, permanence, and safety. The solubility of natamycin was studied in the presence of two cyclodextrins: HPßCD and HPγCD. The HPßCD was chosen based on the solubility results. Natamycin/cyclodextrin (HPßCD) inclusion complexes characterization and a competition study between natamycin and voriconazole were conducted by NMR (Nuclear Magnetic Resonance). Based on these results, several eye drops with different polymer compositions were developed and subsequently characterized. Permeability studies suggested that the formulations improved the passage of natamycin through the cornea compared to the commercial formulation Natacyn®. The ocular safety of the formulations was determined by BCOP and HET-CAM. The antifungal activity assay demonstrated the ability of our formulations to inhibit the in vitro growth of different fungal species. All these results concluded that the formulations developed in the present study could significantly improve the treatment of FK.

Prospective comparative multi-centre study on imported Plasmodium ovale wallikeri and Plasmodium ovale curtisi infections.

BACKGROUND: Few previous retrospective studies suggest that Plasmodium ovale wallikeri seems to have a longer latency period and produces deeper thrombocytopaenia than Plasmodium ovale curtisi. Prospective studies were warranted to better assess interspecies differences. METHODS: Patients with imported P. ovale spp. infection diagnosed by thick or thin film, rapid diagnostic test (RDT) or polymerase chain reaction (PCR) were recruited between March 2014 and May 2017. All were confirmed by DNA isolation and classified as P. o. curtisi or P. o. wallikeri using partial sequencing of the ssrRNA gene. Epidemiological, analytical and clinical differences were analysed by statistical methods. RESULTS: A total of 79 samples (35 P. o. curtisi and 44 P. o. wallikeri) were correctly genotyped. Males predominate in wallikeri group (72.7%), whereas were 48.6% in curtisi group. Conversely, 74.3% of curtisi group were from patients of African ethnicity, whilst 52.3% of Caucasians were infected by P. o. wallikeri. After performing a multivariate analysis, more thrombocytopaenic patients (p = 0.022), a lower number of platelets (p = 0.015), a higher INR value (p = 0.041), and shorter latency in Caucasians (p = 0.034) were significantly seen in P. o. wallikeri. RDT sensitivity was 26.1% in P. o. curtisi and 42.4% in P. o. wallikeri. Nearly 20% of both species were diagnosed only by PCR. Total bilirubin over 3 mg/dL was found in three wallikeri cases. Two patients with curtisi infection had haemoglobin under 7 g/dL, one of them also with icterus. A wallikeri patient suffered from haemophagocytosis. Chemoprophylaxis failed in 14.8% and 35% of curtisi and wallikeri patients, respectively. All treated patients with various anti-malarials which included artesunate recovered. Diabetes mellitus was described in 5 patients (6.32%), 4 patients of wallikeri group and 1 curtisi. CONCLUSIONS: Imported P. o. wallikeri infection may be more frequent in males and Caucasians. Malaria caused by P. o. wallikeri produces more thrombocytopaenia, a higher INR and shorter latency in Caucasians and suggests a more pathogenic species. Severe cases can be seen in both species. Chemoprophylaxis seems less effective in P. ovale spp. infection than in P. falciparum, but any anti-malarial drug is effective as initial treatment. Diabetes mellitus could be a risk factor for P. ovale spp. infection.

Epidemic Dynamics of Vibrio parahaemolyticus Illness in a Hotspot of Disease Emergence, Galicia, Spain.

Galicia in northwestern Spain has been considered a hotspot for Vibrio parahaemolyticus infections. Infections abruptly emerged in 1998 and, over the next 15 years, were associated with large outbreaks caused by strains belonging to a single clone. We report a recent transition in the epidemiologic pattern in which cases throughout the region have been linked to different and unrelated strains. Global genome-wide phylogenetic analysis revealed that most of the pathogenic strains isolated from infections were associated with globally diverse isolates, indicating frequent episodic introductions from disparate and remote sources. Moreover, we identified that the 2 major switches in the epidemic dynamics of V. parahaemolyticus in the regions, the emergence of cases and an epidemiologic shift in 2015-2016, were associated with the rise of sea surface temperature in coastal areas of Galicia. This association may represent a fundamental contributing factor in the emergence of illness linked to these introduced pathogenic strains.

Ophthalmic Econazole Hydrogels for the Treatment of Fungal Keratitis.

Econazole is a feasible alternative treatment in the management of fungal keratitis. Nevertheless, its low water solubility is considered the main limitation to the incorporation into ophthalmic formulations. In this work, econazole nitrate is solubilized by using cyclodextrins to achieve an optimum therapeutic concentration. Phase solubility diagrams suggest α-cyclodextrin as the most effective cyclodextrin and later the inclusion complex formed with this one was characterized in solution by 1D, 2D-NMR, and molecular modeling. Econazole-α-cyclodextrin inclusion complex was included in 2 types of ocular hydrogels: a natural polysaccharides ion-sensitive hydrogel and a hyaluronic acid hydrogel. Both of them show no ocular irritation in the hen's egg test on chorioallantoic membrane assay and a controlled econazole release over time. Permeability studies suggest that hydrogels do not modify the econazole nitrate permeability through bovine cornea in comparison with an econazole-α-cyclodextrin inclusion complex solution. Finally, ocular biopermanence studies performed using positron emission tomography show these hydrogels present a high retention time on the eye. Results suggest the developed formulations have a high potential as vehicles for the econazole topical ocular administration as fungal keratitis treatment.

High primary antibiotic resistance of Helicobacter Pylori strains isolated from dyspeptic patients: A prevalence cross-sectional study in Spain.

BACKGROUND: The rate of H. pylori resistance to different antibiotics is increasing and determines the selection of eradication therapy. The aim of this study was to determine the resistance patterns of H. pylori strains in our area. METHODS: Biopsies from gastric corpus for microbiological culture and antibiotic resistance were obtained in patients undergoing upper gastrointestinal endoscopy for dyspepsia. Selective Agar Pylori for isolation of the bacteria and Agar Mueller-Hinton supplemented with blood to test the sensitivity to antibiotics were used. Presence of H. pylori was confirmed using direct observation with phase-contrast microscopy and/or smears stained with acridine orange. In vitro bacterial susceptibility to amoxicillin, clarithromycin, rifampicin, tetracycline, metronidazole, and levofloxacin was tested using diffusion MIC test strips. Minimum inhibitory concentration values were determined based on the 6th version of the EUCAST (European Committee on Antimicrobial Susceptibility Testing) Clinical Breakpoint (2016). RESULTS: Two hundred and seventeen patients were included (58.1% female, median age 64 years, range 25-92). H. pylori was identified in 108 patients (49.8%); culture and antibiogram were completed in 77 of them (71.3% of H. pylori-positive patients). The resistance rates were as follows: levofloxacin 38.7%, rifampicin 33.3%, metronidazole 27% and clarithromycin 22.4%. No case of amoxicillin or tetracycline resistance was identified. Dual clarithromycin-metronidazole resistance was observed in 10% of strains, whereas multiple drug-resistant was observed in 14.2%. CONCLUSIONS: Resistance rate of H. pylori to antibiotics is high in the northwest of Spain. The high resistance to levofloxacin and clarithromycin advises against their wide empirical use of these antibiotics in eradication regimens.

Caracterización microbiológica de las gastroenteritis agudas virales atendidas en un servicio de pediatría en un área de alta cobertura vacunal frente a rotavirus / Microbiological profile of acute viral gastroenteritis attended in a paediatric department of an area with high vaccine cover against rotavirus

INTRODUCCIÓN: La vacunación frente a rotavirus ha condicionado un descenso significativo de la enfermedad. El presente trabajo pretende evaluar las características clínicas y epidemiológicas de la gastroenteritis aguda (GEA) por virus en un área de alta cobertura vacunal frente a rotavirus. Método Evaluación prospectiva microbiológica mediante coprocultivo y reacción en cadena de la polimerasa en tiempo real (RT-PCR) para virus gastroentéricos, y genotipificado de las cepas de rotavirus de los casos de GEA en menores de 5 años que acudieron a urgencias o fueron hospitalizados en nuestro centro de noviembre a marzo de 2009-2010 y 2010-2011.ResultadosSe incluyeron 51 pacientes con una edad media (desviación estándar) de 19,1 (13,9) meses. El coprocultivo fue negativo en 23 muestras (45% de los casos), identificándose mediante RT-PCR un microorganismo causal en 16 de estas muestras (el 70%). El rotavirus fue el microorganismo más detectado (53%) y el genotipo G1[P8] el más abundante. En el 14% de los casos (7 pacientes) se identificó coinfección, siendo el rotavirus y el astrovirus los agentes más frecuentemente involucrados. Conclusiones El rotavirus, principalmente el G1[P8], se ha identificado como la causa más frecuente de GEA en nuestro estudio. La utilización de RT-PCR mejora significativamente la sensibilidad diagnóstica en el contexto de la GEA, y pone de relieve un porcentaje elevado de coinfecciones virales

INTRODUCTION: Vaccination against rotavirus has led to a significant decline of the disease. The aim of the present work is to evaluate the clinical and epidemiological features of the viral acute gastroenteritis(AGE) in an area with high immunization coverage against rotavirus. METHOD: A prospective microbiological evaluation was made of stool culture and Real Time Polymerase Chain Reaction (RT-PCR) to gastroenteric virus and genotyping of rotavirus strains in < 5 year-old with AGE episodes attended by or admitted to our hospital from November-March of 2009-2010 and 2010-2011.RESULTS: A total of 51 patients were included, with a mean age (standard deviation) of 19.1 (13.9) months. Stool culture was negative in 23 samples (45% of the samples analyzed), and it was identified a responsible microorganism in 70% by the RT-PCR (16 samples). Rotavirus was the most common isolated microorganism (53%), and G1[P8] the most frequent genotype. A co-infection was detected in 14% of samples(7 patients), and rotavirus and astrovirus were the most frequent etiological agents involved CONCLUSIONS: Rotavirus, basically G1[P8], is the most common AGE responsible agent identified in our study. The use of RT-PCR enhances the AGE diagnostic sensitivity, and uncovers an important number of viral co-infections

[Microbiological profile of acute viral gastroenteritis attended in a paediatric department of an area with high vaccine cover against rotavirus]. / Caracterización microbiológica de las gastroenteritis agudas virales atendidas en un servicio de pediatría en un área de alta cobertura vacunal frente a rotavirus.

INTRODUCTION: Vaccination against rotavirus has led to a significant decline of the disease. The aim of the present work is to evaluate the clinical and epidemiological features of the viral acute gastroenteritis (AGE) in an area with high immunization coverage against rotavirus. METHOD: A prospective microbiological evaluation was made of stool culture and Real Time Polymerase Chain Reaction (RT-PCR) to gastroenteric virus and genotyping of rotavirus strains in < 5 year-old with AGE episodes attended by or admitted to our hospital from November-March of 2009-2010 and 2010-2011. RESULTS: A total of 51 patients were included, with a mean age (standard deviation) of 19.1 (13.9) months. Stool culture was negative in 23 samples (45% of the samples analyzed), and it was identified a responsible microorganism in 70% by the RT-PCR (16 samples). Rotavirus was the most common isolated microorganism (53%), and G1[P8] the most frequent genotype. A co-infection was detected in 14% of samples (7 patients), and rotavirus and astrovirus were the most frequent etiological agents involved. CONCLUSIONS: Rotavirus, basically G1[P8], is the most common AGE responsible agent identified in our study. The use of RT-PCR enhances the AGE diagnostic sensitivity, and uncovers an important number of viral co-infections.

Caracterización molecular de aislados humanos de Cryptosporidium spp. procedentes de 2 diferentes localizaciones de España / Molecular characterization of Cryptosporidium spp. isolated in humans in two different locations in Spain

Las técnicas de diagnóstico molecular por PCR permiten distinguir entre las diferentes especies de Cryptosporidium morfológicamente idénticas capaces de infectar a humanos. De las 23 especies actualmente reconocidas en el género, al menos 9 son capaces de infectar a humanos. Por ello, y debido a que la intensidad de las manifestaciones clínicas, la patogenicidad, la excreción de ooquistes y la incidencia varían entre ellas, la realización de estudios moleculares es crucial para una mejor comprensión de la epidemiología 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 Coruña, todas ellas positivas para Cryptosporidium spp. por métodos microscópicos y pertenecientes a casos aislados de criptosporidiosis. Para la identificación a nivel de especie se utilizaron las regiones de diagnóstico descritas para el ADNr 18S y las regiones de diagnóstico del gen de la COWP. De las 95 muestras analizadas, se consiguió extraer y amplificar ADN en 77 casos, en los que las especies causantes de la infección fueron: C. parvum (40 casos: 2 Madrid y 38 La Coruña), C. hominis (30 casos: 10 Madrid y 20 La Coruña) y C. meleagridis (2 casos: uno Madrid y uno La Coruña). En otros 5 casos fue imposible detectar la especie responsable de la infección, aunque se confirmara su positividad por PCR (4 Madrid y uno La Coruña). Los genotipos aislados en estos pacientes se correlacionaron con los hallados en animales de las mismas regiones (AU)

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions

[Molecular characterization of Cryptosporidium spp. isolated in humans in two different locations in Spain]. / Caracterización molecular de aislados humanos de Cryptosporidium spp. procedentes de 2 diferentes localizaciones de España.

Molecular PCR based diagnostic techniques have enabled us to distinguish between the different, morphologically identical, Cryptosporidium species that can infect humans. Of the 23 recognized species in the genus, at least 9 are able to infect humans. As the intensity of the clinical manifestations, pathogenicity, excretion of oocysts, and incidence, are different between this species, molecular studies are crucial for a better understanding of the epidemiology of human cryptosporidiosis. Samples form two independent studies are analyzed in this publication. One included 23 samples from Madrid, and the other, 72 samples from La Coruña. All of them positive for Cryptosporidium spp. by microscopic methods and belonging to isolated cases of human cryptosporidiosis. For the identification of the species responsible for the infection, the 18S rDNA diagnostic region and the COWP gene diagnostic regions were used. Out of the 95 samples tested, in 77 cases we were able to extract and amplify DNA. In those cases the species responsible for the infection were: C. parvum (40 cases, 2 Madrid and 38 La Coruña), C. hominis (30 cases, 10 Madrid and 20 La Coruña) and C. meleagridis (2 cases, 1 Madrid and 1 La Coruña). In 5 samples it was impossible to detect the species responsible for the infection, but their positivity was confirmed by PCR (4 Madrid and 1 La Coruña). The genotypes of the isolates from patients correlated well with animals from the same regions.

Uso combinado de amplificación aleatoria de polimorfis model ADN (RAPD) y reacción en cadena de la polimerasa (touchdown PCR) en el estudio epidemiológico de Aspergillus fumigatus / Combined used of RAPD and touchdown PCR for epidemiological studies of Aspergillus fumigatus

INTRODUCCIÓN. Aspergillus fumigatus es un hongo filamentoso que se comporta como patógeno oportunista y constituye una de las complicaciones infecciosas más importantes en los pacientes inmunocomprometidos. Los brotes nosocomiales de aspergilosis son cada vez más frecuentes, pero su identificación y caracterización epidemiológica es lenta y laboriosa. OBJETIVO. Describir un método rápido, sensible y específico para la identificación de A. fumigatus y su caracterización genotípica dentro de las posibilidades diagnósticas habituales en un laboratorio clínico de microbiología. MÉTODOS. Se utilizaron cepas de A. fumigatus procedentes de pacientes con aspergilosis invasivas (n = 4), medio ambiente hospitalario (n = 5) y colecciones de referencia (n = 1), así como otras especies fúngicas filogenéticamente próximas aisladas de pacientes (n = 1), del medio hospitalario (n = 6) o de colecciones de referencia (n = 1). La identificación de A. fumigatus se realizó tanto por métodos fenotípicos clásicos como mediante touchdown PCR (reacción en cadena de la polimerasa). La caracterización genotípica se llevó a cabo por RAPD (polimorfismo derivado de la amplificación aleatoria de ADN), comparando distintos protocolos de amplificación y tipos de primer (OPZ-19 y R-108) en relación con su poder resolutivo y reproducibilidad. RESULTADOS. Los resultados de la identificación fenotípica y molecular coincidieron plenamente. La caracterización molecular por RAPD presentó los mejores resultados, en cuanto a reproducibilidad y resolución se refiere, con el primer R-108 y tiempo prolongado de transición entre hibridación y elongación. CONCLUSIÓN. El análisis por RAPD es un método seguro y rápido para la caracterización genotípica de A. fumigatus cuyos patrones de bandas son fáciles de interpretar y reproducir cuando se prolonga drásticamente el tiempo de transición entre la hibridación y la extensión. El uso combinado de touchdown PCR y análisis por RAPD constituye un método sensible y exacto para la resolución de brotes nosocomiales por A. fumigatus (AU)

Combined used of RAPD and touchdown PCR for epidemiological studies of Aspergillus fumigatus.

INTRODUCTION: Aspergillus fumigatus is a filamentous fungus that acts as an opportunistic pathogen and has emerged as a major problem in immunosuppressed patients. Nosocomial outbreaks of aspergillosis are becoming more frequent, but their identification and epidemiological characterization is slow and difficult. OBJECTIVE: Description of a fast, sensitive, specific method to identify and fingerprint A. fumigatus using methodology available in clinical laboratories. METHODS: We studied several strains of A. fumigatus isolated from patients with invasive aspergillosis (n = 4), the hospital environment (n = 5) and reference cultures (n = 1), as well as other close phylogenetic fungal species from patients (n = 1), hospital environment (n = 6) and reference cultures (n = 1). A. fumigatus was identified by both touchdown PCR and conventional phenotyping methods. Genotyping was performed with random amplification of polymorphic DNA (RAPD) analysis, comparing the results from two primers (OPZ-19 and R-108) and different amplification protocols with regard to band resolution and reproducibility. RESULTS: Touchdown PCR and phenotype results were identical. Best RAPD results were obtained with the R-108 primer and considerably longer ramp times between annealing and extension. CONCLUSION: RAPD analysis is a fast, reliable tool for DNA fingerprinting. Patterns may be easier to repeat and interpret when longer ramp times are used. Touchdown PCR combined with RAPD analysis is a sensitive, accurate method for managing clinical outbreaks of Aspergillus fumigatus.