Mullus barbatus L. and Mullus surmuletus L. from western Mediterranean waters (SE Spain) are infected by Hysterothylacium fabri, but not by zoonotic nematodes. Possible impact on fish hosts.

In order to know whether there is a risk of anisakiasis (or anisakidosis) by consumption of fish of the genus Mullus from the western Mediterranean Sea, which are appreciated for their quality, an epidemiological survey was carried out to evaluate the occurrence of zoonotic or potentially zoonotic nematodes in M. barbatus and M. surmuletus. Although the presence of the third larval stage (L3) of anisakids (Anisakis and Contracaecum) has been previously described in these fish, the results showed the absence of anisakids and the presence, never in muscle, of L3 and L4 of raphidascaridids of the genus Hysterothylacium, molecularly identified as H. fabri. Phylogenetic analysis groups them into the Mediterranean Sea clade, far from individuals isolated in the Pacific Ocean. Prevalence was slightly higher, but not significant, in M. barbatus versus M. surmuletus (72.3% vs 60.0%), but mean intensity (MI) and mean abundance (MA) parameters were approximately twice as high in M. barbatus as in M. surmuletus (MI 5.8 vs 2.8, p = .001; MA 4.2 vs 1.7, p < .001). The presence of the parasite seems to have different effects on these two sympatric species. In M. barbatus it seems to affect their growth, as it appreciably reduces the value of allometry coefficient in infected fish (2.78 vs. 2.18). On the other hand, in M. surmuletus the infection significantly (p < .04) affects the Fulton's condition factor, an indicator of the health status of the fish. It can be concluded that the ingestion of these fish by the people poses negligible risk of anisakiasis, but the consumer should continue to be urged to follow the rules of prevention against this illness.

Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3.

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer's disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1-3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood-brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.

Differential Cleaving of Specific Substrates for Cathepsin-Like Activity Shows Cysteine and Serine Protease Activities and a Differential Profile Between Anisakis simplex s.s. and Anisakis pegreffii, Sibling Species Major Etiologic Agents of Anisakiasis.

Humans can contract anisakiasis by eating fish or squid containing live larvae of the third stage (L3) of the parasitic nematodes of the genus Anisakis, majorly from Anisakis simplex s.s. and Anisakis pegreffii, sibling species of the A. simplex s.l. complex. Most cases diagnosed molecularly are due to A. simplex s.s., although A. pegreffii has also been identified in human cases. Cathepsins are mostly lysosomal multifunctional cysteine proteases and can participate in the pathogenicity of parasites. Cathepsin B and L activities were investigated in the two sibling species of Anisakis mentioned. L3 and L4 of both species were collected during their in vitro development, and cathepsin activity was determined in the range of pH 4.0-8.5, using specific fluorogenic substrates. The activity detected with the substrate Z-FR-AMC (N-α-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methyl-coumarin) was identified as cathepsin L (optimum pH = 5.0, range 4.0-6.0, p < 0.001). Activity was highest in L3 freshly collected from fish, especially in A. simplex s.s., and decreased during development, which could be related to virulence, invasion of host tissues, and/or intracellular digestion. Cathepsin B-like activity was not identified with either of the substrates used (Z-RR-AMC [N-α-benzyloxycarbonyl-L-arginyl-L-arginine-7-amido-4-methyl-coumarin] and Z-FR-AMC). With Z-RR-AMC, cleaving activity was detected almost exclusively in L4 of A. simplex s.s. (p < 0.05) with optimum pH = 8.0 (range 7.0-8.5). Assays with class-specific protease inhibitors showed that this activity was mainly due to serine proteases [up to 90% inhibition with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)], although metalloproteases (up to 40-45% inhibition with 1,10 phenanthroline) and slight cysteine protease activity (<15% inhibition with E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane]; putative cathepsin B-like) were also detected. These results show differential serine protease activity between sibling Anisakis species, regulated by larval development, at least in A. simplex s.s. The higher cathepsin L and serine protease activities detected in this species could be related to its greater pathogenicity, reported in experimental animals, compared to that of A. pegreffii.

A scanning electron microscopy study of Anisakis physeteris molecularly identified: from third stage larvae from fish to fourth stage larvae obtained in vitro.

The development of the fourth larval stage (L4) of Anisakis physeteris was studied using scanning electron microscopy (SEM), comparing it with third larval stage (L3) recently obtained from the host fish, blue whiting (Micromesistius poutassou), from the western Mediterranean Sea (east coast of Spain, zone FAO 37.1.1). After molting to L4, samples of the parasite were examined at different times in order to observe their development. Following collection of the L4, a small portion was taken from the middle of the larva for molecular identification, confirming in all cases that it was A. physeteris. The anterior and posterior sections of the larvae were prepared for morphological study by SEM. The development of a row of denticles on each of the three prominent lips, almost reaching the buccal commisures, was observed in the L4. Pores of unknown function were found in the upper external part of each lip. Clearly developed cephalic papillae, amphids, and deirids were also observed in L4, while, although present in L3, these were beneath the cuticle. Phasmids were detected in L4 but not in L3. The L4 tail finished in a conical lobe with a blunt point, absent in L3. In the oldest L4, some preanal papillae were observed beneath the cuticle in males, while, in females, the vulva could be seen by light microscopy, apparently still covered by the cuticle.

Early development and life cycle of Contracaecum multipapillatum s.l. from a brown pelican Pelecanus occidentalis in the Gulf of California, Mexico.

The initial developmental stages of Contracaecum multipapillatum (von Drasche, 1882) Lucker, 1941 sensu lato were studied using eggs obtained from the uteri of female nematodes (genetically identified) found in a brown pelican Pelecanus occidentalis from Bahía de La Paz (Gulf of California, Mexico). Optical microscopy revealed a smooth or slightly rough surface to the eggs. Egg dimensions were approximately 53 × 43 µm, although after the larvae had developed inside, egg size increased to 66 × 55 µm. Hatching and survival of the larvae were greater at 15°C than at 24°C, and increased salinity resulted in a slight increase in hatching but seemed to reduce survival at 24°C, but not at 15°C. The recently hatched larvae measured 261 × 16 µm within their sheath. When placed in culture medium, the larvae grew within their sheath, and a small percentage (~2%) exsheathed completely (314 × 19 µm). The larvae continued to grow and develop once they had exsheathed, attaining mean dimensions of 333 × 22 µm. Although they did not moult during culture, optical microscopy revealed a morphology typical of third-stage larvae. Finally, the genetic identity between the larval parasites collected from mullet Mugil curema and adult female parasites collected from the brown pelican suggests a life cycle of C. multipapillatum in which the mullet are involved as intermediate/paratenic hosts and the brown pelicans as final hosts in the geographical area of Bahía de La Paz.

A scanning electron microscopy study of early development in vitro of Contracaecum multipapillatum s.l. (Nematoda: Anisakidae) from a brown pelican (Pelecanus occidentalis) from the Gulf of California, Mexico.

Eggs obtained from the uteri of female nematodes, genetically identified as Contracaecum multipapillatum s.l., found in a brown pelican (Pelecanus occidentalis) from Bahía de La Paz, Gulf of California, Mexico, were used to study the early developmental stages of this anisakid by scanning electron microscopy (SEM). Egg dimensions were approximately 54 × 45 µm measured by SEM. Observation of the eggs revealed an outer surface of fibrous appearance. The newly hatched larvae were ensheathed and highly motile. Observation with SEM showed that the sheaths of the larvae were striated and revealed an excretory pore and a cleft near the anterior end of the sheath, presumably to facilitate the opening of the sheath for the emergence of the larva. The hatched larvae were placed in nutritive culture medium, where they grew within their sheath, some exsheathing completely 2 weeks later. The surface patterns of the sheath and the cuticle of the exsheathed larvae were clearly different. Although they did not moult during culture, SEM revealed a morphology typical of third-stage larvae of Contracaecum from fish, as previously observed by optical microscopy. Thus, we suggest that newly hatched larvae from eggs of C. multipapillatum are third larval stage but with sheath of the second larval stage, as occuring in other anisakids.

Anisakiasis and Anisakidae.

Parasitism as a lifestyle is much more common in nature than it seems [...].

Anisakis infection in anchovies (Engraulis encrasicolus) from Iberian waters, southwestern Europe: Post-mortem larval migration.

The Anisakis larvae presence in fish for human consumption is a health risk that needs to be monitored. The anchovy is a fish that is highly appreciated by consumers and that can harbour Anisakis. It is thus necessary to periodically evaluate the presence of anisakid larvae in them. So, anchovies from Iberian Peninsula coasts were analysed. Fish examination for macroscopic nematodes showed L3s of both Anisakis type I and Hysterothylacium aduncum. The Anisakis prevalence varies with the catching area and the fish size. The muscle prevalence was 7.45% (mean intensity 1.75; range 1-5). Molecular analysis showed 110 A. simplex s.s. (17 in muscle), 22 A. pegreffii (3) and 7 hybrid genotype individuals (1). Considering that most of the Iberian Peninsula coasts are a sympatry area between these two Anisakis species, it has been observed that A. simplex s.s./A. pegreffii ratio increases from south to north in a clockwise direction. Also, 19 larvae were detected on the fish surface from the Bay of Biscay, indicating the ability of these larvae to migrate after the fish death. The A. simplex s.s./A. pegreffii larvae proportion found on the anchovy surface is similar to the found in viscera and lower than in muscle, suggesting that most of the larvae migrating to the surface must have come from the visceral package. This confirms the importance of removing fish viscera immediately after capture, for those fish species where this is possible. As both species cause anisakiasis/anisakidosis, these data show a real risk to human health, especially in dishes highly prized in Mediterranean countries prepared with raw or semi-raw anchovies.

Anisakis Infection in the Spotted Flounder Citharus linguatula (Pleuronectiformes: Citharidae) Caught in the Gulf of Cadiz (Area FAO 27-ICES IXa) Appears to Negatively Affect Fish Growth.

Spotted flounder (Citharus linguatula L.) caught in the Gulf of Cadiz (area FAO 27 ICES IXa) were examined for Anisakis larvae and to assess the possible risk of anisakiasis in humans through consumption of this fish. Larvae of the genera Anisakis and Hysterothylacium were identified in the analysis of 128 purchased fish specimens. All Anisakis larvae corresponded to type I. Molecular analysis showed the presence of A. pegreffii, A. simplex s.s., and recombinant genotype between the two. The prevalence of Anisakis was 9.4% with a mean intensity of 1.42, while for Hysterothylacium the values were 12.5% and 1.06. The length and weight of the fish, but not Fulton's condition factor, varied significantly between infected and uninfected fish. The prevalence of Anisakis increased with fish length, with no fish parasitized with Anisakis measuring less than 15.5 cm (2-2.5 years old), which is probably related to the reported dietary change of these fish at around 2 years of age. Fish not parasitized with any of these nematodes showed positive allometric growth, while those parasitized only with Anisakis showed negative allometric growth. When comparing both groups including only fish ≥ 15.5 cm (the smallest size of Anisakis-infected fish), the difference is shown to be statistically significant (p = 0.01), suggesting that Anisakis infection of spotted flounder negatively affects fish growth even when parasite intensity is low, which may have important economic repercussions. Finally, the low prevalence and, above all, intensity of Anisakis in these fish, as well as the habit of consuming this fish fried in oil in our geographical area, means that the risk of acquiring anisakiasis through consumption of this fish is low.

Molecular Epidemiology of Anisakis spp. in Wedge Sole, Dicologlossa cuneata (Moreau, 1881), from Fishmarkets in Granada (Southern Spain), Caught in Two Adjacent NE and CE Atlantic Areas.

The presence of third stage larvae (L3) of Anisakis spp. in wedge sole, Dicologlossa cuneata (Moreau, 1881), purchased in fishmarkets in the city of Granada (Andalusia, southern Spain) was assessed. The wedge sole were caught in two FAO zones: area 27.IXa NE Atlantic (SW Spain coast) and area 34.1.11 CE Atlantic (NW Morocco coast). Only Anisakis larvae, type I, were detected in the largest fish (>20 cm) from the CE Atlantic. These were molecularly identified as A. simplex s.s. The prevalence (P) of Anisakis in this area was 12.5% and the mean intensity (MI) was 1. The presence of Hysterothylacium spp. larvae was also detected in the fish from both areas, with the prevalence being approximately double in the CE Atlantic area (12.5 vs. 5.7). A comparison between the Anisakis-infected and non-infected fish from this area showed that the former were significantly longer than the latter (p < 0.01). These results show that Anisakis parasitization of wedge sole sold in the markets of the city of Granada is of low prevalence and intensity (P = 4.5, MI = 1), especially in those from area 27.IXa (P = 0), indicating that the risk of human infection is low, particularly as this fish is traditionally prepared by deep-frying in oil in Andalusia (southern Spain).

Structure-Based Design of Potent Selective Nanomolar Type-II Inhibitors of Glycogen Synthase Kinase-3ß.

For the first time, the in silico design, screening, and in vitro validation of potent GSK-3ß type-II inhibitors are presented. In the absence of crystallographic evidence for a DFG-out GSK-3ß activation loop conformation, computational models were designed using an adapted DOLPHIN approach and a method consisting of Prime loop refinement, induced-fit docking, and molecular dynamics. Virtual screening of the Biogenics subset from the ZINC database led to an initial selection of 20 Phase I compounds revealing two low micromolar inhibitors in an isolated enzyme assay. Twenty more analogues (Phase II compounds) related to the hit [pyrimidin-2-yl]amino-furo[3,2-b]furyl-urea scaffold were selected for structure-activity relationship analysis. The Phase II studies led to five highly potent nanomolar inhibitors, with compound 23 (IC50 =0.087 µM) > 100 times more potent than the best Phase I inhibitor, and selectivity for GSK-3ß inhibition compared to homologous kinases was observed. Ex vivo experiments (SH-SY5Y cell lines) for tau hyperphosphorylation revealed promising neuroprotective effects at low micromolar concentrations. The type-II inhibitor design has been unraveled as a potential route toward more clinically effective GSK-3ß inhibitors.

Collagenolytic activity related to metalloproteases (and serine proteases) in the fish parasite Hysterothylacium aduncum (Nematoda: Anisakidae).

Proteases play a vital role in both the life cycle of parasites and the parasite-host relationship and are considered important virulence factors. In the present study, the presence of proteases with collagenolytic activity was investigated in the fish nematode Hysterothylacium aduncum during in vitro development. Collagenolytic activity was found in all studied developmental stages of the nematode (third [L3] and fourth [L4] larval stages and adults). In L3, the activity was maximum at pH 6.5 and, in the other stages, at 7.0. Pepsin is known to favour in vitro development of the worm, but, in this study, collagenolytic activity was shown to be significantly greater when no pepsin was added to the culture medium (at pH 6.5, p = 0.011). At pH 7.0, most activity was observed in the immature adult, after the final moult, suggesting that the collagenolytic activity may be involved in remodelling of the cuticle and in sexual maturity. On the other hand, at pH 6.5, activity may be related to tissue migration by L3 within the host. Using specific inhibitors, it was demonstrated that most of the collagenolytic activity detected in all the developmental stages was due to metalloproteases (40 to 100%), although serine proteases were also detected in L4 and adults (10 to 30%).

CO2-fixing enzymes and phosphoenolpyruvate metabolism in the fish parasite Hysterothylacium aduncum (Ascaridoidea, Anisakidae).

CO2 stimulates the development of many of the intestinal helminths that are able to fix CO2 by means of phosphoenolpyruvate carboxykinase (PEPCK), such as Hysterothylacium aduncum. We determined the activity of CO2-fixing enzymes such as PEPCK and phosphoenolpyruvate carboxylase (PEPC), although no significant activity was detected for pyruvate carboxylase or carboxylating-malic enzyme. The former act on phosphoenolpyruvate (PEP) to yield oxalacetate. In the helminths studied, PEP has a vital role in glucidic metabolism. Consequently, we determined the activity of other enzymes involved in the crossroad of PEP, such as pyruvate kinase (PK), lactate dehydrogenase and malate dehydrogenase. All enzymes detected showed significant variations in activity during the in vitro development of the parasite from the third larval stage to mature adult. Fixing of CO2 by PEPCK decreased during development (from 228 to 115 nmol min(-1) mg(-1) protein), while that by PEPC increased (from 19 to 46 nmol min(-1) mg(-1) protein). This enzyme, which is rare in animals, could play a part in detecting levels of free phosphate, releasing it from PEP when required for processes such as glycogenolysis, glycolysis and adenosine 5'-triphosphate (ATP) synthesis. PK, which showed increasing activity during development up to immature adult (from 56 to 82 nmol min(-1) mg(-1) protein), could act in combination with PEPC to obtain energy in the cytosol (in the form of ATP) and in the mitochondria (possible destination of the pyruvate formed), compensating for the decrease in activity of PEPCK.

Differential proteolytic activity in Anisakis simplex s.s. and Anisakis pegreffii, two sibling species from the complex Anisakis simplex s.l., major etiological agents of anisakiasis.

Proteolytic activity was studied in two sibling species of Anisakis (Nematoda: Anisakidae), A. simplex s.s. and A. pegreffii, throughout their in vitro development from third larval stage (L3) from the host fish (L3-0h) to fourth larval stage (L4) obtained in culture. Proteases have a significant role in the lifecycle of the parasite and in the pathogen-host relationship. Proteolytic activity peaks were detected at pH 6.0 and 8.5. Protease activity was detected in all the developmental stages of the two species studied at both pH values. These pH values were used for assaying with specific inhibitors which permitted the determination of metalloprotease activity, and, to a lesser extent, that of serine and cysteine protease. Aspartic protease activity was only detected at pH 6.0. At this pH, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p < 0.001), the majority of activity being due to metalloproteases and aspartic proteases, which could be related to nutrition, especially the latter, as occurs in invertebrates. At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p < 0.01). At this pH, the majority of activity was due to metalloproteases in all developmental phases of both species, although, in L3-0h, the activity of these proteases was significantly higher (p < 0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former. Serine proteases have frequently been implicated in the invasive capacity and pathogenicity of some parasites. This may be related to the significantly higher activity (p ≤ 0.05) of serine protease in all the larval stages of A. simplex studied at pH 6.0. Thus, there are interspecific differences in proteases that have been related to pathogenesis in nematodes. These differences could thus be contributing to the previously reported differences in pathogenicity between these two Anisakis species.

Características de recién nacidos hijos de madre con IgM positiva para el Virus Herpes Simplex

Introducción: El herpes connatal es una entidad infrecuente asociada a elevada morbimortalidad. La probabilidad de transmisión al recién nacido va de 5% al 85%. El diagnóstico se dificulta por falta de clínica, serología no confiable y por la no disponibilidad de PCR en los servicios públicos de países en vías de desarrollo. La IgM en gestantes podría ser utilizada como un marcador de sospecha para evaluar al neonato. Objetivo: Caracterizar a los recién nacidos, hijos de gestantes con IgM positiva para HVS 1-2 y la frecuencia de encefalitis en los infantes. Materiales y métodos : Estudio observacional, descriptivo, prospectivo, realizado de mayo de 2020 a octubre de 2021. Se incluyeron recién nacidos (RN) de madres con IgM positiva para Herpes Virus Simplex (HVS) a partir de la segunda mitad del embarazo. En el RN se realizó serología IgG e IgM, y además, PCR- RT para HVS 1-2 en sangre y/o LCR, excluyéndose los nacidos en otras maternidades y/o sin datos de serología materna. Resultados: 36 pacientes. Edad materna 28 años (DS + 4), 5% con antecedentes de HVS, 61% cesárea. 36% prematuros, 13% RCIU. Síntomas agudos en el RN 22%. De ellos, 19% plaquetopenia, 44% alteración de GOT. 63% PCR HVS en sangre y 44% en LCR. Se encontró hemorragia, hidrocefalia, leucomalacia en 27%. No se encontró diferencias en la expresión clínica por tipo de parto. Conclusiones: Los RN hijos de gestantes con IgM positiva para VHS desde la segunda mitad del embarazo o periparto, presentaron infección por VHS determinada por PCR en sangre o LCR, independiente de la vía del parto. El diagnóstico serológico en embarazadas permite la pesquisa, diagnóstico y tratamiento temprano del RN.

Introduction: neonatal herpes is a rare entity associated with high morbidity and mortality. The probability of transmission to the newborn ranges from 5% to 85%. The diagnosis is difficult due to the lack of clinical signs, unreliable serology and the non-availability of PCR in public services in developing countries. IgM in pregnant women could be used as a suspected marker to evaluate the neonate. Objective: To characterize newborn children of pregnant women with positive IgM for HSV 1-2 and the prevalence of encephalitis in infants. Materials and methods: Observational, descriptive, prospective study, carried out from May 2020 to October 2021. Newborns (NB) of mothers with positive IgM for Herpes Virus Simplex (HSV) from the second half of pregnancy were included. In newborns, IgG and IgM were performed, and in addition, PCR-RT for HSV 1-2 in blood and/or CSF, excluding those born in other hospitales and/or without maternal serology data. Results: We included 36 patients. Maternal age was 28 years (DS + 4), 5% with a history of HSV. 61% were delivered via cesarean section, 36% were premature, 13% had IUGR. 22% of the newborns had acute symptoms. 19% had thrombocytopenia, 44% had GOT alteration. 63% were PCR positive for HSV in serum and 44% were CSF-positive. Hemorrhage, hydrocephalus and leukomalacia were found in 27%. No differences were found in clinical expression by type of delivery. Conclusions: Newborns born to pregnant women with positive IgM for HSV from the second half of pregnancy or peripartum, presented HSV infection as determined by PCR in blood or CSF, regardless of the route of delivery. Serological diagnosis in pregnant women allows early screening, diagnosis and treatment of the NB.

Prediabetes in patients receiving tacrolimus in the first year after kidney transplantation: a prospective and multicenter study.

BACKGROUND: Tacrolimus-based immunosuppression, the most widely used regimen in kidney transplantation, increases the risk of new onset diabetes after transplantation (NODAT). However, the prevalence, evolution and risk factors of different prediabetic alterations: impaired fasting glucose, impaired glucose tolerance, and provisional diabetes, have not been established. METHODS: In this multicenter and prospective study we evaluated 154 nondiabetic kidney transplant recipients receiving tacrolimus, mycophenolate mofetil and low dose steroids. An oral glucose tolerance test was performed 3 and 12 months after transplantation and prediabetes was defined by American Diabetes Association criteria. RESULTS: Prediabetes was highly prevalent and showed little variation between 3 and 12 months (36% and 33%, respectively). Impaired glucose tolerance was the most frequent abnormality observed (23% and 25%, respectively) observed. In addition, 20% of recipients showed NODAT by 1 year. Multivariate analysis showed that age (odds ratio [OR]: 1.07, 95% confidence interval [CI]: 1.004-1.14), pretransplant body mass index (OR: 1.3, CI: 1.09-1.6) and triglyceride/high density lipoprotein-cholesterol ratio, a marker of insulin resistance, (OR: 1.4, CI: 1.05-1.9) were independent risk factors for prediabetes. CONCLUSION: One in two recipients with tacrolimus-based immunosuppresion showed prediabetes or NODAT by 1 year posttransplantation when properly investigated. Older age and high pretransplant body mass index and triglyceride/high density lipoprotein-cholesterol ratio were risk factors for prediabetes. These findings may help applying early interventions to prevent the disorder.

Molecular epidemiology of Anisakis spp. in blue whiting Micromesistius poutassou in eastern waters of Spain, western Mediterranean Sea.

The infection of blue whiting Micromesistius poutassou from the western Mediterranean Sea, off the eastern coast of Spain, with larvae of Anisakis spp. was studied. Between April 2016 and April 2017, 140 fish were analyzed. Total epidemiological data showed that the prevalence of Anisakis spp. was 29.3% and the mean intensity 1.8. Of the 74 larvae collected, 61% were type I and the remaining 39%, type II. Of the former, 91% were molecularly identified as Anisakis pegreffii (P = 19.3%; MI = 1.4), 2.2% as Anisakis simplex s.s. (P = 0.7%; MI = 1.0), while the rest (6.7%) showed a recombinant genotype between the two (P = 2.1%; MI = 1.0). All the type II larvae analyzed were molecularly identified as Anisakis physeteris (P = 10.0%; MI = 2.1). Three fish (2.1%) were found to have larvae in the muscle, while two were found with 1 larva of A. pegreffii and one with two larvae (1 A. simplex s.s. and 1 A. pegreffii). Statistical analysis showed that the prevalence of Anisakis spp. in blue whiting was higher in spring than in autumn (P < 0.001), probably due to the greater size (and age) of the fish and related to factors as diet shift, accumulation with age and higher food intake. Analysis of the data suggested that blue whiting were first infected with Anisakis type I (mean age 2.3 years) and later with Anisakis type II (mean age 2.7 years), probably due to the diet changing with age, with the incorporation of the paratenic/intermediate host species of these parasites. In any case, the public health authorities must continue to emphasize the need for suitable thermal treatment (freezing or cooking) of the fish prior to consumption.

Encefalitis por Virus Herpes Simplex con PCR persistentemente positivo en líquido cefalorraquídeo. A propósito de un caso neonatal

RESUMEN Las infecciones por herpes virus en la etapa neonatal pueden causar una alta morbimortalidad. La persistencia del virus, a pesar del tratamiento de primera línea, puede llevar a consecuencias devastadoras para el paciente. Presentamos el caso de un paciente neonato con persistencia de Virus Herpes Simplex en LCR, en el cual fue necesario iniciar foscarnet para contener la infección.

ABSTRACT Herpes virus infections in the neonatal stage can cause high morbidity and mortality. The persistence of the virus, despite first-line treatment, can lead to devastating consequences for the patient. We present the case of a neonatal patient with persistence of Herpes Simplex Virus in the CSF, in whic foscarnet treatment was required to contain the infection.

Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe.

The sardine (Sardina pilchardus) is a fish commonly consumed and appreciated in many countries, although they are more likely to be eaten fresh in western Mediterranean countries such as Spain, Portugal, France or Italy. A molecular epidemiological survey of sardines from 5 fishing areas of the Spanish Mediterranean (Málaga, southern Spain) and Atlantic coasts (southern: Cádiz and Isla Cristina; northern: A Coruña and Ondarroa) was carried out to determine the presence of Anisakis spp. larvae. The highest prevalence of these larvae was observed in fish from A Coruña (28.3%), followed by Ondarroa (5%) and Cádiz (2.5%). No Anisakis larvae were found in fish from Málaga and Isla Cristina. Three Anisakis genotypes were identified: Anisakis simplex sensu stricto, Anisakis pegreffii and a hybrid genotype between these two species. A. pegreffii was the most prevalent species in A Coruña (71% of larvae). Only three Anisakis larvae (9% collected larvae) were located in the musculature of sardines: two were identified as A. pegreffii while the other was a hybrid genotype. Sardine infection was associated with fishing area and fish length/weight (length and weight were strongly correlated; Pearson's correlation 0.82; p<0.001). Risk factor multivariate analysis showed that the risk of infection increases 1.6 times for every additional cm in the length of the sardines from the same fishing area. Comparison of fish of equal length showed that in sardines from A Coruña the risk of parasitization is 11.5 times higher than in those from other fishing areas. Although the risk of infection by Anisakis through consumption of sardines is generally low due to the low epidemiological parameter values (prevalence 10%, mean intensity 1.7 (range 1-5) and mean abundance 0.17), as larger fish are more heavily parasitized, there is an increased risk of infection by Anisakis through consumption of large sardines which are raw or have undergone insufficient treatment (undercooked, smoked, marinated, salted, pickled, freezing,…).

Factores pronósticos de mortalidad en niños con bacteriemias adquiridas en la comunidad (BAC) hospitalizados en un centro de referencia del Paraguay / Prognostic mortality factors in children with acquired bacteremia in the community (ABC) hospitalized at a tertiary medical center in Paraguay

Objetivo: Identificar etiologìa de BAC y factores pronósticos de mortalidad. Material y Método: Estudio retrospectivo de BAC en pacientes de 29 días a 16 años hospitalizados en un Centro de Referencia del Paraguay, periodo 2007 a 2015, se analizaron variables demográficas, clínicas y laboratoriales categorizando a los pacientes según el desenlace (Vivos o Muertos). Resultados: 187 casos de BAC , edad media 40 + 35 meses, M/F 1,4:1, edad < 1 año: 30,5% (57/187), 1 a 4 años 49,2% (92/187), 5 a 10 años 15,5% (29/187) y > 10 años 4,8 % (9/187). Diagnósticos de ingreso: Neumonía (78%), Infección de Piel y Partes Blandas (9%), Infección a Múltiples focos (7%), Meningitis (3,7%) Bacteriemia sin foco 2%. Las bacteriemias por Gram Positivos fueron más frecuentes que las bacteriemias por Gram Negativos: 91,4 vs 8,6% (p<0,05), aislamientos: S.pneumoniae, S.aureus, S.coagulasa negativo (2/2) y K. pneumoniae (46 %, 34%, 7,5% y 2%). Ingresaron a UCI 63 pacientes (33,7%), mortalidad 16,5 %. Variables asociadas a mortalidad: Edad <5 años (p<0,05), Desnutrición (p=0,02, RR= 2,4, IC95%: 1,1-5), Infección a múltiples focos(p=0,002, RR=3,2, IC 95%: 1,6-6,4), Choque p<0,001, RR19, IC95%:7,8-46), Glasgow <12 (p<0,001, RR=5,5, IC 95%:3,2-9,6), Sat O2 < 94% (p=0,007, RR 2, IC95%: 1,1-4) , Hb< 7 (p<0,01, RR: 11, IC95%: 6,8 a 19), GB > 15000, plaquetas < 100000 (p<0,001, RR=4,4, IC95%: 2,4- 7,8), Bacteriemia por Gram Positivos (p=0,001, RR=4,9, IC 95%: 1,5-15). Conclusión: En niños con BAC son más frecuentes las bacteriemias por Gram positivos. Se identificaron factores pronósticos de mortalidad.