Density assessment and reporting for Phlebotomus perniciosus and other sand fly species in periurban residential estates in Spain.

Green periurban residential areas in Mediterranean countries have flourished in the last decades and become foci for leishmaniasis. To remedy the absence of information on vector ecology in these environments, we examined phlebotomine sand fly distribution in 29 sites in Murcia City over a 3-year period, including the plots of 20 detached houses and nine non-urbanized sites nearby. We collected 5,066 specimens from five species using "sticky" interception and light attraction traps. The relative frequency of the main Leishmania infantum vector Phlebotomus perniciosus in these traps was 32% and 63%, respectively. Sand fly density was widely variable spatially and temporally and greatest in non-urbanized sites, particularly in caves and abandoned buildings close to domestic animal holdings. Phlebotomus perniciosus density in house plots was positively correlated with those in non-urbanized sites, greatest in larger properties with extensive vegetation and non-permanently lived, but not associated to dog presence or a history of canine leishmaniasis. Within house plots, sand fly density was highest in traps closest to walls. Furthermore, the study provides a guideline for insect density assessment and reporting and is envisioned as a building block towards the development of a pan-European database for robust investigation of environmental determinants of sand fly distribution.

Chemical composition and bioactivity potential of the new Endosequence BC Sealer formulation HiFlow.

AIM: To evaluate in a laboratory setting the effects of Endosequence BC Sealer HiFlow (Brasseler USA, Savannah, GA, USA), a novel calcium silicate-based sealer developed for use in warm canal filling techniques, on human periodontal ligament stem cells (hPDLSCs). METHODOLOGY: Eluates of EndoSequence BC Sealer HiFlow (BCHiF) (Brasseler USA), EndoSequence BC Sealer (BCS) (Brasseler USA) and AH Plus (AHP) (Dentsply DeTrey GmbH, Konstanz, Germany) were placed in contact with hPDLSCs. The characterization of the chemical elements of the root canal sealers was assessed using scanning electron microscopy and energy-dispersive X-ray analysis (SEM-EDX). Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine the ion release of the sealers. MTT assay and wound healing techniques were used to determine cell viability and migration, respectively. Cell morphology and cell attachment were assessed using a direct contact technique of hPDLSCs onto the surface of the sealers and analysed by SEM. The bioactivity potential was carried out with the Alizarin Red and qPCR testing methods. The statistical differences were evaluated using one-way anova and Tukey's test (P < 0.05). RESULTS: ICP-MS and EDX revealed significantly more zirconium in BCHiF than BCS (P < 0.05), whereas BCS had slightly higher levels of Ca2+ than BCHiF (P < 0.05). The cell viability assay revealed no relevant differences between BCS and BCHiF when compared with the control group (P > 0.05). Both BCS and BCHiF had similar rates of cell migration to the control group at 24 and 48 h. Cell morphology and adhesion capacity were also similar for BCS and BCHiF groups, whilst the AHP group was associated with reduced adhesion capacity. The Alizarin Red assay revealed a significant difference between the BCS and the control group (P < 0.001), as well as for the BCHiF group (P < 0.001). Finally, BCS and BCHiF promoted overexpression of osteo/cementogenic genes. CONCLUSIONS: In general, EndoSequence BC Sealer HiFlow possesses suitable biological properties to be safely used as a root canal filling material and promote increased expression of oste/cementogenic genes by hPDLSCs.

Cytocompatibility, bioactivity potential, and ion release of three premixed calcium silicate-based sealers.

OBJECTIVE: Compositional modifications may alter the biological and physicochemical characteristics of calcium silicate-based sealers (CSBS) and, ultimately, their bioactivity. The main objective of this study was to evaluate the biological properties of three CSBS: EndoSequence BC Sealer, Ceraseal, and Endoseal mineral trioxide aggregate. MATERIALS AND METHODS: Human periodontal ligament stem cells (hPDLSCs) were exposed to several eluates of CSBS. The ion release profile and pH were determined, and metabolic activity and cell migration were assessed using the MTT and wound healing assays. hPDLSCs were cultured in direct contact with the surface of each material, and cell morphology and attachment were analyzed by scanning electron microscopy (SEM). Bioactivity potential was assessed by RT-qPCR and mineralization assays. Statistical differences between biomaterials were assessed using one- or two-way ANOVA (α < 0.05). RESULTS: All materials showed an alkaline pH, although Endoseal exhibited a significantly higher pH compared with the other CSBS (p < 0.05). Ceraseal released significantly more Ca2+ (p < 0.05) than EndoSequence BC Sealer and Endoseal. Interestingly, Endoseal induced a significant reduction in cell viability and cell migration compared with the control (p < 0.001). Moreover, SEM showed abundant cells adhering to EndoSequence BC Sealer and Ceraseal surfaces, whereas very few round cells were detected on the surface of Endoseal. Finally, Ceraseal and EndoSequence induced ALP, CAP, and CEMP-1 expression and a significantly higher mineralization capacity than Endoseal (***p < 0.001). CONCLUSIONS: The eluates from EndoSequence BC Sealer and Ceraseal displayed higher cell viability, cell attachment, cell migration rates, and ion release rates than Endoseal. Ceraseal and EndoSequence BC Sealer exhibited significantly more gene expression and mineralization capacity than Endoseal. CLINICAL RELEVANCE: The results obtained in the present work suggest that EndoSequence BC Sealer and Ceraseal possess biological properties that make them suitable materials for root canal treatment.

Evaluation of changes in ion release and biological properties of NeoMTA-Plus and Endocem-MTA exposed to an acidic environment.

AIM: To analyse in vitro changes in ion release and biological properties of Endocem-MTA (Maruchi, Wonju, Korea) and NeoMTA-Plus (Avalon Biomed Inc, Bradenton, FL, USA) exposed to acidic or neutral environment on human dental periodontal ligament stem cells (hPDLSCs). METHODOLOGY: Cell viability and wound healing assays were performed using eluates of each material. Cell death and changes in phenotype induced by the set endodontic sealer eluates were evaluated through flow cytometry. To evaluate cell attachment to the different materials, hPDLSCs were directly seeded onto the material surfaces and analysed by scanning electron microscopy. The chemical composition of the materials was determined by energy-dispersive X-ray (EDX), and ion release was evaluated by inductively coupled plasma-mass spectrometry. Statistical analysis was performed with analysis of variance and a Bonferroni or Tukey post-test (α < 0.05). RESULTS: The MTT assay revealed non-cytotoxic effects of NeoMTA-Plus and Endocem-MTA at pH 5.2 and 7.4. However, there were minor differences compared with the control, especially at pH 5.2, where both materials were associated with significantly greater cell viability (P < 0.05). In both environments, the materials stimulated hPDLSCs to migrate. hPDLSCs were attached to the bioactive cements, with multiple prolongations proliferated on the surface of the samples. Moreover, there were no changes to cell phenotype or apoptosis/necrosis rates, indicating that the acidic environment did not induce cell death. Prismatic crystalline structures were seen on the surface of the cements exposed to butyric acid and EDX analysis identified a marked peak of Ca2+ from NeoMTA-Plus and Endocem-MTA in acidic and physiological environments. CONCLUSIONS: An acidic environment favoured the release of Ca2+ ions from both bioactive cements, and the cytotoxicity of these bioactive cements was low in both environments studied.

GuttaFlow Bioseal promotes spontaneous differentiation of human periodontal ligament stem cells into cementoblast-like cells.

OBJECTIVES: To evaluate in vitro the cementogenic potential and the biological effects of GuttaFlow Bioseal, GuttaFlow 2, MTA Fillapex and AH Plus on human periodontal ligament stem cells (hPDLSCs). METHODS: Cell viability, cell migration and cell morphology assays were performed using eluates of each material. To evaluate cell attachment, hPDLSCs were directly seeded onto the material surfaces and analyzed by scanning electron microscopy (SEM). The effects of endodontic sealers on cementum protein 1 (CEMP1), cementum-derived attachment protein (CAP), bone sialoprotein (BSP), ameloblastin (AMBN), amelogenin (AMELX) and alkaline phosphatase (ALP) gene expression on hPDLSCs were investigated by qPCR and immunofluorescence (IF). Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α<0.05). RESULTS: More than 90% of viable cells were obtained using extracts of GuttaFlow Bioseal and GuttaFlow2 after 72h of culture. By contrast, AH Plus and MTA Fillapex induced significantly lower levels of cell viability. GuttaFlow2 and GuttaFlow Bioseal promoted wound closure in a concentration-dependent manner, comparable to that observed with control extracts (*p<0.05). However, with AH Plus and MTA Fillapex, cell migration was significantly lower than in the control (***p<0.0001). SEM analysis pointed to an organized stress fiber assembly and high degree of cell adhesion on GuttaFlow Bioseal disks but low rates on GuttaFlow2, MTA Fillapex and AH Plus. When hPDLSCs were cultured with GuttaFlow Bioseal-conditioned media, qPCR assays and IF showed a higher level of AMELX, AMBN, CEMP1 and CAP expression than the control (*p<0.05)), whereas no such expression was observed in the other sealers. SIGNIFICANCE: Our results showed that GuttaFlow sealers were more cytocompatible than AH Plus and MTA Fillapex, while GuttaFlow Bioseal favored cementoblast differentiation of hPDLSCs in the absence of any growth factors.

Success of benznidazole chemotherapy in chronic Trypanosoma cruzi-infected patients with a sustained negative PCR result.

Cure assessment in chronic Trypanosoma cruzi infection is controversial, mainly because of the lack of reliable tests to ensure parasite elimination. Here, we assess the impact of benznidazole therapy on the conventional serology and parasitaemia in chronic Chagas disease. A total of 455 patients with long-term Trypanosoma cruzi infection underwent specific chemotherapy with benznidazole. Their parasitological status was assessed by polymerase chain reaction (PCR) detection of T. cruzi DNA. Drops in the titres of antibody levels were serially measured by indirect immunofluorescence assay (IFI) and chemiluminescent microparticle immunoassay (CMIA). Patients were monitored during the treatment period and for a further 90, 150 and 240 days. Controls were repeated yearly during the 7-year follow-up. The PCR result was negative in all patients between 60-day (n = 22) and 90-day (n = 294) controls. Treatment failure was detected in 45 patients and was significantly more frequent in those who did not complete the therapy [12 out of 13 (92 %) vs. 33 out of 442 (7 %)] (p = 0.0001). A significant drop in serum titres was detected after the first follow-up year in patients with sustained negative PCR results: 2nd year (p = 0.029 by IFI; p = 0.002 by CMIA), 5th year (p = 0.036 by IFI; p = 0.039 by CMIA) and 6th year (p = 0.028 by IFI; p = 0.019 by CMIA). The results point to a beneficial effect of benznidazole and may be the cure of chronic patients who had a consistently negative PCR result throughout the follow-up period.

Spatial distribution of human asymptomatic Leishmania infantum infection in southeast Spain: a study of environmental, demographic and social risk factors.

Recent PCR studies indicate that asymptomatic L. infantum infection is common in people in southern Europe. Understanding its spatial distribution is a requisite to evaluate the public health implications and to design disease control schemes. We investigated infection in blood samples from 657 donors in southeast Spain using PCR and antibody ELISA. They came from 19 blood centers and were interviewed about their residence, occupation, dog ownership and Leishmaniosis awareness. The percentage of PCR and ELISA positives were 8% (49/618) and 2% (13/657). Donor's residences were spatially clustered around blood donning centers and PCR prevalence was 18% in rural municipalities with 20-1330 inhabitants, 12% in those with 1467-5088 inhabitants and 3% in larger communities, and was associated with dog ownership (p<0.05). Further analysis of data from rural donors indicated that PCR status was strongly related to the climate, altitude and soil type in the donor's residence area and not to other demographic or sociologic variables. Mixed logistic regression analysis predicted PCR prevalence to be greatest in the 200-300m altitude range with a mean spring-summer (time of highest vector activity) temperature of 18.4-19.0°C. A temperature and altitude risk map was generated that will provide the basis for elaborating evidence-based vector surveillance studies.

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain.

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n=53), 29% of foxes (n=48), 29% of stone martens (n=21) and in 25-50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.

Comparative value of microscopy, serology and real time pcr in the diagnosis of asymptomatic canine Leishmania infantum infection

Se investigó la sensibilidad (SE) del examen citológico mediante microscopia óptica (MO) de improntas de bazo y linfonodo, de la prueba de anticuerpos ELISA (inmuno-ensayo ligado a enzima) y de la PCR (reacción en cadena de la polimerasa) a tiempo real (tr), para diagnosticar la infección asintomática por Leishmania infantum en 110 perros aparentemente sanos, del sureste de España. El porcentaje de perros positivos a MO, ELISA y PCRtr fue 2% (2/110), 27% (26/97) y 67% (39/58), respectivamente, aunque el porcentaje de PCR-positivos osciló entre 35-41% para cada tejido individualmente y 9% en sangre. La SE estimada (intervalos de confianza del 95%) de la MO en relación a la PCRtr y al ELISA fue 5% (0-12) y 8% (0-18), respectivamente. Estos resultados confirman que la mayoría de perros aparentemente sanos de una población endémica de L. infantum están infectados, que aproximadamente solo la tercera parte de éstos desarrolla anticuerpos frente al parásito y solo unos pocos tienen suficiente carga parasitaria en tejido linfoide como para ser detectada mediante MO. Consecuentemente, el grado de concordancia de la PCRtr, el ELISA y la MO en el diagnóstico de leishmaniosis canina asintomática es escaso (AU)

The sensitivity (SE) of cytological examination of spleen and lymphnode smears by optical microscopy (OM), antibody-ELISA (enzyme-linked immunosorbent assays) and real-time (rt) PCR (polymerase chain reaction), for diagnosing asymptomatic canine Leishmania infantum infection was investigated in 110 apparently healthy dogs from southeast Spain. The percentage of OM, ELISA and rtPCR positive dogs were 2% (2/110), 27% (26/97) y 67% (39/58), respectively, although the percentage of rtPCR-positive dogs were 35-41% in individual tissues and 9% in blood. The estimated SE (95% confidence interval) of OM relative to the rtPCR and ELISA tests was 5% (0-12) and 8% (0-18), respectively. Results confirm that most apparently healthy dogs from L. infantum endemic areas are infected, that approximately only one third of these infected dogs develop antibodies and that very few have parasite loads that are high enough to allow detection by OM. As a result, the degree of agreement between rtPCR, ELISA and OM for L. infantum diagnosis in subclinnically infected dogs is low (AU)

Side effects of benznidazole treatment in a cohort of patients with Chagas disease in non-endemic country.

Chagas disease is a disease endemic in Latin America, caused by the parasite Trypanosoma cruzi. Benznidazole is the most commonly used drug for the etiological treatment of the disease although its effectiveness varies according to the phase of the same and toxic side effects are frequent. This prospective study describes the side effects of benznidazole treatment of a cohort of 373 chronic patients. Of these 40.2% presented adverse reactions. The most frequent side effect were dermatological reactions 32.4% (121 of 373) followed by digestive intolerance 9.1% (34 of 373). Surprisingly, three cases of migratory arthritis were observed. Patients treated with benznidazole must be followed up so that the long term incidence of side effects can be studied.

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain.

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.

[Cognitive profile of children with neurological disorders: II. Myelomeningocele]. / Perfil cognitivo de niños con trastornos neurológicos: II. Mielomeningocele.

The Myelomeningocele is a neurological dysfunction, as a consequence of some injury in the SNC. Since, this brain disease has an inevitable influence maturity and psychological development in children who suffer it, it is considered as essential as know the place and extension of the injury, finding out the knowledge competence of child who suffers Myelomeningocele. This would be the main objective of this research, specifically we study that the knowledge development of 25 children with Myelomeningocele (14 boys, 11 girls) aged 5-14 years. 18 suffers Myelomeningocele with Hydrocephalus and 7 are affected with Myelomeningocele without Hydrocephalus. This research wants to improve the knowledge of the cognitive development of these children, to achieve, from a psychological evaluation with psychometric instrument specialized (Wechsler Intelligence Scale for Children-Wisc) a tailed inventory about the development levels reached for these children. Therefore, intending to define the areas of potency and dysfunction in children that we know are affected with a SN pathology.