Dirofilariasis palpebral causada por Dirofilaria repens: un caso importado / Human dirofilariasis in the eyelid caused by Dirofilaria repens: An imported case

Caso clínico: Mujer de 33 años, ucraniana, acude a urgencias asegurando tener alojado en el párpado un gusano que se mueve y le muerde. La exploración reveló un cuerpo extraño subcutáneo, filiforme, que reptaba bajo la piel. Fue extirpado quirúrgicamente extrayéndose un parásito vivo identificado por PCR como Dirofilaria repens. Discusión: Los humanos son huéspedes accidentales en el ciclo de las dirofilarias. En España apenas se han publicado 8 casos de afectación subcutánea. Ante el calentamiento global D. repens se ha convertido en un agente infeccioso emergente en países de Centroeuropa como el originario de la paciente. Exponemos nuestras conclusiones (AU)

Clinical case: The case concerns a 33-year-old Ukrainian woman, who came to the Ophthalmology Emergency Service, stating that she had a «worm» under the skin of her upper left eyelid that wriggled at night and bit her. A complete examination revealed a subcutaneous foreign body, which showed a filarial appearance and seemed to crawl under the skin. It was surgically removed to discover a live parasite, PCR-identified as Dirofilaria repens. Discussion: Humans are accidental hosts in the cycle of Dirofilaria. There are only 8 reported cases of subcutaneous infection in Spain. Due to global warming, D. repens has become an emerging infectious agent in Central Europe countries such as Ukraine. The findings in this case are discussed (AU)

Present status of laboratory diagnosis of human taeniosis/cysticercosis in Europe.

Human cysticercosis (CC) is a parasitic zoonosis caused by the larval stage (cyst) of the Taenia solium. Cysts can establish in the human central nervous system (neurocysticercosis, NCC) and other organs and tissues; they also develop in pigs, the natural intermediate host. Human taeniosis may be caused by T. solium, Taenia saginata and Taenia asiatica tapeworms; these infections are usually asymptomatic, but show a significant relevance as they perpetuate the parasites' life cycle, and, in the case of T. solium, they are the origin of (N)CC. In European Union (EU) member states and associated countries, the occurrence of autochthonous T. solium cases is debated, and imported cases have significantly increased lately; the status of T. asiatica has been never reported, whereas T. saginata is prevalent and causes an economic impact due to condemned carcasses. Based on their effects on the EU society, the specific diagnosis of these pathologies is relevant for their prevention and control. The aims of this study were to know the diagnostic tests used in European laboratories for human taeniosis/cysticercosis by means of a questionnaire, to determine potential gaps in their detection, and to obtain preliminary data on the number of diagnosed taeniosis/CC cases.

Human dirofilariasis in the eyelid caused by Dirofilaria repens: An imported case. / Dirofilariasis palpebral causada por Dirofilaria repens: un caso importado.

CLINICAL CASE: The case concerns a 33-year-old Ukrainian woman, who came to the Ophthalmology Emergency Service, stating that she had a «worm¼ under the skin of her upper left eyelid that wriggled at night and bit her. A complete examination revealed a subcutaneous foreign body, which showed a filarial appearance and seemed to crawl under the skin. It was surgically removed to discover a live parasite, PCR-identified as Dirofilaria repens. DISCUSSION: Humans are accidental hosts in the cycle of Dirofilaria. There are only 8 reported cases of subcutaneous infection in Spain. Due to global warming, D. repens has become an emerging infectious agent in Central Europe countries such as Ukraine. The findings in this case are discussed.

Molecular diagnosis of diphyllobothriasis in Spain, most presumably acquired via imported fish, or sojourn abroad.

Human diphyllobothriasis is sporadically detected in Spain. Diphyllobothrium latum and Diplogonoporus balaenopterae have been identified. In the study, four cases of presumably imported diphyllobothriasis in Spanish patients were appraised. Molecular diagnosis allowed us to identify 'exotic' fish tapeworms such as Diplogonoporus balaenopterae in one patient and Diphyllobothrium pacificum in the others.

Analysis of an expressed sequence tag library from Dicrocoelium dentriticum.

Dicrocoeliosis caused by Dicrocoelium dendriticum is an important liver disease, which affects ruminants all around the world. Despite the significant economic losses caused by this trematode, molecular knowledge is very scarce. In fact, there is no information in the expressed sequence tag (EST) database about the parasite. Furthermore, the immunological diagnosis of dicrocoeliosis remains unsatisfactory, and there aren't available recombinant proteins that could be tested in the diagnosis. For this reason a cDNA library was constructed with mRNA extracted from D. dendriticum adults for first time. A random preliminary screening of 230 phage plaques from the library resulted in the identification of 173 new EST. The deduced proteins expressed by these genes have been described as possible vaccine targets in other trematodes, and/or as relevant diagnosis antigens. Then, our goal was to identify D. dentriticum diagnosis genes to be used as recombinant antigens in the specific immunological diagnosis of the trematodoses. A D. dendriticum cDNA encoding an 8-kDa recombinant protein has been cloned, expressed in Escherichia coli and evaluated in dicrocoeliosis diagnosis using both Western Blot and enzyme-linked immunosorbent assay (ELISA). The recombinant expression molecule has demonstrated its value as a diagnosis antigen of dicrocoeliosis, able to discriminate between positive and controls on day 30 post infection. This is the first research conducted for identification and characterization of D. dendriticum ESTs, which can serve as a starting point for future research on immunodiagnosis and immunoprofilaxis of dicrocoeliosis.

Histological and molecular biology diagnosis of neurocysticercosis in a patient without history of travel to endemic areas: case report.

BACKGROUND: in endemic areas, neurocysticercosis appears mainly as a single, large, spherical and non-enhancing intracranial cyst. CASE PRESENTATION: an atypical case of neurocysticercosis (NCC) in a French Caucasian, without history of travel to endemic areas, was confirmed by histology and molecular speciation. Imaging was atypical, showing several hook-bearing scolices visible in the cyst, while the serology employed was non-contributary. CONCLUSIONS: NCC should be considered when multiple taeniid scolices are observed within the same cystic lesion.

Ani s 1 and Ani s 7 recombinant allergens are able to differentiate distinct Anisakis simplex-associated allergic clinical disorders.

Diagnosis in gastro-allergic anisakiasis (GAA) is straightforward, when clinical history is combined with further allergological evaluation of specific IgE by means of skin prick test and serum specific IgE. In Anisakis simplex sensitisation associated chronic urticaria (CU+), clinical evaluation of possible previous parasitism is difficult, and positive serum specific IgE could be due to cross-reactivity or other unknown factors. In this study, we evaluated the association between IgE seropositivity to the recombinant allergens Ani s 1 and Ani s 7 and several A. simplex-associated allergic disorders. Twenty-eight patients with GAA and 40 patients with CU+ were studied and their IgE responses were compared with a control group composed of patients with chronic urticaria not sensitized to A. simplex (CU-) according to the skin prick test, as well as a group of 15 healthy subjects not referring urticaria or currently A. simplex associated symptoms. 82.1% of GAA patients and 42.5% of CU+ patients were positive for Ani s 1 (P < 0.001), while the Ani s 7 allergen was recognized by 92.9 and 92.5% of sera from patients with GAA and CU+, respectively. The combined positivity obtained for both allergens reached 100% in GAA, and 95% in CU+. IgE determinations to Ani s 1 and Ani s 7 allergens are useful to diagnose the Anisakis infections and to differentiate among several A. simplex-associated allergic disorders. The IgE responses to Ani s 1 are mainly associated with GAA, while this molecule cannot be considered a major allergen in CU+ patients.

Molecular and immunological characterization of Fasciola antigens recognized by the MM3 monoclonal antibody.

Fascioliasis is a re-emerging parasitosis produced by liver flukes of the genus Fasciola. In this study we used protein fingerprinting (PMF) and MS/MS analysis to investigate the Fasciola secretory antigens that are recognized by mAb MM3. The results showed that mAb MM3 binds to several Fasciola cathepsins L1 and L2, but also co-purifies a Kunitz-type protein previously described in F. hepatica, which appears to bind to Fasciola cathepsins L. After identifying the target antigens for mAb MM3, we cloned and expressed a cathepsin L1 isoform in E. coli (gb|FR848428), which after refolding exhibited the MM3-recognized epitope and displayed cysteine protease activity. Using native, folded-recombinant and denatured-recombinant Fasciola cathepsins L as targets, we demonstrated that during F. hepatica infections in sheep, antibody responses to linear and conformational epitopes present on cathepsins L are promoted. However, the antibody response to linear epitopes was only detected in significant amounts in animals suffering from repeated infections. A different antibody response to linear and conformational epitopes also appears to occur in rabbits immunized with native or recombinant unfolded cathepsins, as sera from animals immunized with the latter did not react with native cathepsins and vice versa. In addition, the ELISA inhibitions showed that the MM3 epitope is not recognized by rabbits, which explains the usefulness of these species for producing capture antibodies for use in MM3-ELISA assays.

The Anisakis simplex Ani s 7 major allergen as an indicator of true Anisakis infections.

Ani s 7 is currently the most important excretory/secretory (ES) Anisakis simplex allergen, as it is the only one recognized by 100% of infected patients. The allergenicity of this molecule is due mainly to the presence of a novel CX(17-25)CX(9-22)CX(8)CX(6) tandem repeat motif not seen in any previously reported protein. In this study we used this allergen as a model to investigate how ES allergens are recognized during Anisakis infections, and the usefulness of a recombinant fragment of Ani s 7 allergen (t-Ani s 7) as a marker of true Anisakis infections. The possible antigenic relationship between native Ani s 7 (nAni s 7) from Anisakis and Pseudoterranova decipens antigens was also investigated. Our results demonstrate that nAni s 7 is secreted and recognized by the immune system of rats only when the larvae are alive (i.e. during the acute phase of infection), and that this molecule is not present in, or is antigenically different from, Pseudoterranova allergens. The t-Ani s 7 polypeptide is a useful target for differentiating immunoglobulin E antibodies induced by true Anisakis infections from those induced by other antigens that may cross-react with Anisakis allergens, including P. decipiens. The results also support the hypothesis that the Ani s 7 major allergen does not participate in maintaining the antigenic stimulus during chronic infections.

A novel phylogeny for the genus Echinococcus, based on nuclear data, challenges relationships based on mitochondrial evidence.

The taxonomic status of Echinococcus, an important zoonotic cestode genus, has remained controversial, despite numerous attempts to revise it. Although mitochondrial DNA (mtDNA) has been the source of markers of choice for reconstructing the phylogeny of the genus, results derived from mtDNA have led to significant inconsistencies with earlier species classifications based on phenotypic analysis. Here, we used nuclear DNA markers to test the phylogenic relationships of members of the genus Echinococcus. The analysis of sequence data for 5 nuclear genes revealed a significantly different phylogeny for Echinococcus from that proposed on the basis of mitochondrial DNA sequence data, but was in agreement with earlier species classifications. The most notable results from the nuclear phylogeny were (1) E. multilocularis was placed as basal taxon, (2) all genotypes of Echinococcus granulosus grouped as a monophyletic entity, and (3) genotypes G8 and G10 clustered together. We conclude that the analysis of nuclear DNA data provides a more reliable means of inferring phylogenetic relationships within Echinococcus than mtDNA and suggest that mtDNA should not be used as the sole source of markers in future studies where the goal is to reconstruct a phylogeny that does not only reflect a maternal lineage, but aims to describe the evolutionary history at species level or higher.

Identification of Spanish Trichinella isolates by ISSR-PCR: intra-specific variability of Trichinella britovi.

In the present work, we investigated genetic variability of the Spanish Trichinella isolates by ISSR-PCR (inter-simple sequence repeat polymerase chain reaction), a technique that is being successfully used to study diversity among related populations. We recovered a total of 43 isolates from different host and geographic localization and identified them by molecular techniques (RAPD and multiplex-PCR) and by Western blot with monoclonal antibodies US5 and US9. Nineteen (44.2%) out of 43 were identified as Trichinella spiralis and 24 (55.8%) as Trichinella britovi. When these samples were analysed by the ISSR technique, all the T. spiralis isolates presented a pattern similar to the T. spiralis ISS116. By contrast, the ISSR-PCR analysis of the isolates identified as T. britovi, showed two different banding profiles compatible with the European T. britovi isolate pattern (ISS2), and the autochthonous Spanish T. britovi isolate (ISS11). Three of these 43 isolates were involved in human outbreaks; the three were identified as T. britovi and showed a pattern similar to the European isolate ISS2. As conclusion, we highlight that an intra-species variability within the Spanish T. britovi isolates analysed was observed, with a predominant group similar to T. britovi ISS2, while T. spiralis group isolates were more homogeneous. No correlations were found between the different ISSR-PCR T. britovi types and the host/geographical origin of the isolates.

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification.

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.

Massive presence of Echinococcus granulosus (Cestoda, Taeniidae) cysts in a wild boar (Sus scrofa) from Spain.

We report a heavy infestation of a free-living wild boar sow from Spain with Echinococcus granulosus cysts and state its molecular characterization. We found >65 hydatid cysts in the thoracic and abdominal cavities of the sow. Parasites were routinely processed for their identification and histopathology and DNA molecular characterization of the E. granulosus cysts were carried out. The polymerase chain reaction results confirmed the E. granulosus identity of the cysts and restriction fragment length polymorphism and sequencing revealed its G1 genotype. Our results suggest that wild boar could be involved in the epidemiology of E. granulosus, particularly considering that large amounts of carcass remains are available to dogs and wolves during the hunting season. The recent population increase of the wild boar in Spain and the DNA confirmation that the wild boar isolate shared identical sequences to the sheep strain emphasize the importance of the reported finding in public health.

Neurocysticercosis: detection of Taenia solium DNA in human cerebrospinal fluid using a semi-nested PCR based on HDP2.

Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. This disease is usually diagnosed by radiology but the results are not always clear-cut and so immunological assays are often also used. A semi-nested PCR, based on the non-coding HDP2 sequence of T. saginata, has now been developed for detecting DNA from T. solium cysticerci and confirming NC. This PCR, which amplifies a 171-bp T. solium product, allowed the specific detection of just 174 attograms of T. solium DNA. The efficacy of the PCR was tested using cerebrospinal fluid (CSF) from neurological patients, including 46 confirmed Mexican cases of NC and 32 patients from non-endemic Spain. Eighteen of the confirmed cases [including 10 (71%) of the 14 with vesicular extraparenchymal cysticerci and four (17%) of the 24 with damaged cysticerci] and two (33%) of the six patients with 'uncertain' diagnosis (in whom a diagnosis of NC could not be established by radiological and immunological studies) were found PCR-positive. The 36 patients known to have neurological problems other than NC were found PCR-negative. The HDP2 PCR offers a new tool in the diagnosis of NC and in exploring the pathogenesis of this serious disease.

Parasite contamination of soil in households of a Mexican rural community endemic for neurocysticercosis.

High neurocysticercosis (NC) prevalence was recently determined by a computed tomography (CT) scan study in the community of Tepetzitzintla, State of Puebla, Mexico. The aim of the present work was to evaluate the magnitude of fecal and parasite contamination by Taenia spp. in the soil of households of this community during the four seasons of the year. The toilet, backyard, kitchen, washboard, water containers and corrals of 14 to 26 households were sampled during each season. High Taenia spp. egg intensity was found in 24.2% of the sampled areas. The highest percentage was detected in Spring and the lowest in Summer. Significantly higher levels of Taenia spp. eggs were present in kitchen soil samples. A significant correlation was found between the presence of Taenia spp. eggs in household soil during the Summer, and NC diagnoses of the inhabitants by CT scan. Coproparasitological examinations and anti-cysticercal antibodies were determined in a cohort of inhabitants of the sampled households. Antibody levels and coproparasitological results were not associated with NC. Overall, these results illustrate the high degree of fecal contamination of potential risk to human health in rural communities and could be of use for control programmes.

Novel sequences and epitopes of diagnostic value derived from the Anisakis simplex Ani s 7 major allergen.

BACKGROUND: Anisakis simplex allergens may cause severe allergic reactions in infected patients. Human anisakiasis can be specifically diagnosed by detection of immunoglobulin E (IgE) antibodies against O-deglycosylated nAni s 7 allergen captured by monoclonal antibody (mAb) UA3 (UA3-ELISA), although the nature of this important allergen is unknown. The aim of this study was to clone and characterize the Ani s 7 major allergen, and to obtain a recombinant fragment suitable for serodiagnosis. METHODS: An Anisakis cDNA library was screened with mAb UA3 and a cDNA clone (rAni s 7) encoding a 1096-amino acid fragment of Ani s 7 (GenBank: EF158010) was identified. Bioinformatic tools and immunological and biochemical techniques were used to characterize the allergen obtained. RESULTS: The rAni s 7 fragment comprised 19 repeats of a novel CX(17-25)CX(9-22)CX(8)CX(6) tandem repeat motif not seen in any previously reported protein sequence. An internal (435)Met-(713)Arg fragment of the rAni s 7 (t-Ani s 7) was expressed in Escherichia coli and evaluated for serodiagnostic utility. Indirect enzyme-linked immunosorbent assay (ELISA) with t-Ani s 7 identified as positive the same 60 sera as UA3-ELISA. The sequence MCQCVQKYGTEFCKKRLA from rAni s 7 was identified as the epitope recognized by mAb UA3, and is the target for over 60% of human IgE antibodies that react with O-deglycosylated nAni s 7. CONCLUSIONS: In addition to their clear value for serodiagnosis of human anisakiasis, the nature of the novel sequences and epitopes identified in the Ani s 7 allergen are of interest for a better understanding of the mechanisms operating in Anisakis-induced allergy.

[Consensus document on bath-PUVA therapy. The Spanish Photobiology Group of the Spanish Academy of Dermatology and Venereology]. / Documento de consenso sobre la modalidad terapéutica del baño-PUVA. Grupo Español de Fotobiología de la Academia Española de Dermatología and Venereología.

Bath PUVA is a variant of phototherapy as efficacious as oral PUVA therapy that avoids many of the adverse effects associated to this treatment. Nevertheless, the special features and the specialized equipment required for its employment have limited its application in the dermatologic clinics of our country. Following the trend initiated after the publication of the consensus document on oral PUVA therapy and narrow band (NB) UVB therapy, the Spanish Photobiology Group from the Spanish Academy of Dermatology and Venereology has developed a therapeutic guideline for bath PUVA therapy based on the literature review and the experience of its members. The document aims to be a practical reference guide for those dermatological centres that include phototherapy among their services. It reviews the concept and indications of this type of treatment and proposes recommendations concerning therapeutic procedures, drug associations of interest and prophylaxis and management of adverse effects.

Molecular identification of Echinococcus granulosus genotypes (G1 and G7) isolated from pigs in Mexico.

With the aim of genotyping Echinococcus granulosus cysts found in Mexican livestock, we collected hydatid cysts from the livers and lungs of pigs in slaughterhouses in the state of Morelos, Central Region of Mexico. DNA was extracted from the parasites and examined by polymerase chain reaction (PCR) of rDNA internal transcribed spacer 1 (ITS1-PCR), Eg9-PCR, Eg16-PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP). In addition, fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase 1 (ND1) were sequenced. Two different genotypes of E. granulosus were unequivocally identified, the common sheep genotype, G1, and the common pig genotype, G7. The G1 genotype of E. granulosus has not been previously demonstrated in Mexico. Because of its recognized infectivity in humans, G1 genotype is a direct threat to human health and its presence in Mexico is consequently of immediate public health importance and epidemiological relevance.

Documento de consenso sobre la modalidad terapéutica del baño-PUVA / Consensus document on bath-PUVA therapy

El baño-PUVA se reconoce como una variante de fototerapia tan eficaz como la terapia PUVA oral, pero que permite evitar buena parte de los efectos adversos asociados a este tratamiento. Sin embargo, las particularidades de su empleo y la necesidad de utillaje especializado han limitado su uso en los centros dermatológicos de nuestro país. En continuidad con la línea iniciada tras la publicación del documento de consenso de la terapia PUVA oral y la terapia UVB de banda estrecha (BE), el Grupo Español de Fotobiología (GEF) de la Academia Española de Dermatología y Venereología (AEDV), ha llevado a cabo la elaboración, a través de la revisión de la literatura al respecto y la experiencia propia de sus miembros, de una guía terapéutica del baño-PUVA. El documento pretende ser una guía práctica de referencia para aquellos centros dermatológicos que incluyan la fototerapia entre sus prestaciones. En él se revisan el concepto y las indicaciones de esta forma de tratamiento y se proponen recomendaciones referidas a los procedimientos terapéuticos, las asociaciones farmacológicas de interés, la prevención y el manejo de los efectos secundarios

Bath PUVA is a variant of phototherapy as efficacious as oral PUVA therapy that avoids many of the adverse effects associated to this treatment. Nevertheless, the special features and the specialized equipment required for its employment have limited its application in the dermatologic clinics of our country. Following the trend initiated after the publication of the consensus document on oral PUVA therapy and narrow band (NB) UVB therapy, the Spanish Photobiology Group from the Spanish Academy of Dermatology and Venereology has developed a therapeutic guideline for bath PUVA therapy based on the literature review and the experience of its members. The document aims to be a practical reference guide for those dermatological centres that include phototherapy among their services. It reviews the concept and indications of this type of treatment and proposes recommendations concerning therapeutic procedures, drug associations of interest and prophylaxis and management of adverse effects