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One of the fundamental aspects of a parasitic infection diagnosis is the use of adequate antigens to develop specific and sensitive immunoassays. This fact is especially complicated in nematode infection cases because of the high cross-reactivity among different parasites in this group. We performed an evaluation of Anisakis simplex antigens purified by affinity chromatography. We used sera from 38 patients diagnosed with Anisakis sensitization and sera from 35 patients with clinical suspicion of visceral larva migrans (VLM). These sera were assayed by the ELISA method against the crude extracts (CEs) and the purified antigens. When the sera from patients diagnosed with Anisakis sensitization were tested against the A. simplex CE, the IgG was the most abundant immunoglobulin. When the A. simplex larval antigens were purified using a column of IgG anti-A. simplex (PAK) or a column of IgG anti-Ascaris suum (PAS) were tested, we observed a higher diminution in the IgG levels, which coincides with the augmentation of the mean values against the "eluted of Ascaris" (EAS antigen). When the IgE was detected, only 18.4% of the sera reacted with the PAS antigen. We have observed that in the purification process of A. simplex antigen by affinity chromatography, the majority of the proteins that produced cross-reactivity against A. suum and Toxocara canis were eliminated.

The infection efficiency of different strains of Encephalitozoon hellem of human origin was tested in Vero E6 cell cultures, scoring the number of infection foci (NIF) after 9, 14, 20, and 24 days of inoculation. The results revealed a strong interaction of the strain type with time: different strains showed different proliferative dynamics. Number of infection foci was lower on the first sampling day for CDC:V257, EHVS-96, and PV6-96, with a subsequent increase at a higher rate for the first strain and lower for the latter. In contrast, PV7-96 showed the highest NIF at the first sampling, followed by a slight decrease. Since these strains were selected by their genotype for the polar tube protein (PTP)—1A, 1B, 1C, and 2C, respectively, it is tempting to suggest a major role of this protein in the differences detected, although the influence of other genes that hypothetically may also differ among the strains employed cannot be discarded. The different in vitro infection efficiencies raise the possibility that some strains of E. hellem will also produce more aggressive features in infected patients.

Article available at http://www.jimmunol.org/cgi/content/abstract/176/1/451, African swine fever virus (ASFV) is able to inhibit TNF--induced gene expression through the synthesis of A238L protein. This was shown by the use of deletion mutants lacking the A238L gene from the Vero cell-adapted Ba71V ASFV strain and from the virulent isolate E70. To further analyze the molecular mechanism by which the viral gene controls TNF-, we have used Jurkat cells stably transfected with the viral gene to identify the TNF- regulatory elements involved in the induction of the gene after stimulation with PMA and calcium ionophore. We have thus identified the cAMP-responsive element and 3 sites on the TNF- promoter as the responsible of the gene activation, and demonstrate that A238L inhibits TNF- expression through these DNA binding sites. This inhibition was partially reverted by overexpression of the transcriptional factors NF-AT, NF-B, and c-Jun. Furthermore, we present evidence that A238L inhibits the activation of TNF- by modulating NF-B, NF-AT, and c-Jun trans activation through a mechanism that involves CREB binding protein/p300 function, because overexpression of these transcriptional coactivators recovers TNF- promoter activity. In addition, we show that A238L is a nuclear protein that binds to the cyclic AMP-responsive element/3 complex, thus displacing the CREB binding protein/p300 coactivators. Taken together, these results establish a novel mechanism in the control of TNF- gene expression by a viral protein that could represent an efficient strategy used by ASFV to evade the innate immune response, This work was supported by grants from Ministerio de Educación y Ciencia (BFU2004-00298/BMC), the Wellcome Trust (075813/C/04/z), and by an institutional grant from the Fundación Ramón Areces. C.H. was a fellow from Fundación Ramón Areces.

Microsporidia are intracellular obligate parasites, today mainly associated with diarrhea in AIDS patients. Microsporidia prevalence ranges from 8% to 52% in different countries, as evaluated by several diagnostic methods, such as the stain test and PCR. In Medellín, Colombia, its frequency is unknown, and hence, a study was undertaken to determine the frequency of intestinal microsporidiosis in HIV patients, by means of the quick-hot Gram chromotrope test and the PCR. A prospective and descriptive study of an intentional population of all HIV-positive patients was sent to the Grupo Interdisciplinario para el Estudio de las Parasitosis Intestinales laboratory by institutions treating the HIV-positive patients of Medellín between August 2001 and September 2002. The clinical-epidemiological survey included a serial stool test with direct concentration and special stains for coccidiae and intestinal microsporidia. In addition, counts of lymphocytes TCD4+ and viral load were requested. One hundred and three patients with ages ranging from 2-74 years were evaluated. Seventy percent presented with diarrhea--mostly in men (83.5%). The overall frequency of intestinal microsporidiosis was 3.9% and that of other intestinal parasitic infections was 39.8%. Three of the four patients positive for microsporida were infected with Enterocytozoon bieneusi and one with Encephalitozoon intestinalis. The microsporidiosis frequency was relatively low with 3 of the 4 cases associated with protracted diarrhea, counts of LTCD4+ below 100 cel/microl and viral loads up to 100,000 copies.

LUIS NAVARRO-i-MARTINEZ, FERNANDO J. BORNAY-LLINARES, CRISTINA RUEDA, CARMEN del AGUILA, ALEXANDRE J. da SILVA, ANA OLEAGA, VICENTE RAMAJO, SOLEDAD FENOY and NORMAN J. PIENIAZEK Universidad Miguel Hernandez de Elche, Alicante, Spain, and Universidad San Pablo-CEU, Madrid, Spain, and Instituto de Recursos Naturales y Agrobiologia-CSIC, Salamanca, Spain, and Centers for Disease Control and Prevention, Atlanta, GA, USA

The aim of this study was to verify whether cross-reactivity appeared between Toxocara canis and Anisakis simplex in an experimental rodent model. No cross-reactions were detected using sera from mice infected with T. canis eggs. When responses obtained against T. canis ES antigen using sera from BALB/c and C57BL/10 mice infected with T. canis eggs were compared with those obtained by testing sera from mice infected with one A. simplex L3, an increase in cross-reactions was observed using the C57BL/10 strain.

Seven isolates of Encephalitozoon hellem from human immunodeficiency virus-positive patients were genotyped through a series of markers: the internal transcribed spacer (ITS) of ribosomal DNA, the polar tube protein (PTP) gene, and two intergenic spacers (IGS-TH and IGS-HZ) whose polymorphism is newly reported. The genome markers were all analyzed at three levels: PCR amplification followed by polyacrylamide gel electrophoresis, single-strand conformation analysis (SSCA), and DNA sequencing. The polymorphisms detected involve insertions/deletions and point mutations. SSCA can distinguish any pair of sequences, even those differing by a single base pair. The different isolates studied fit into the previously described ITS genotype 1A, except one which seems to be a 2A derivative variant (2D). When PTP and the new markers IGS-TH and IGS-HZ were analyzed, most of the isolates displayed different genotypes, demonstrating that E. hellem has a strong intraspecies variability. A set of markers such as those used here may be very useful in genotyping of clinical samples and in the assessment of epidemiological relationships among E. hellem strains.

En: Acta Tropica. ISSN 0001-706x. vol. 89, 2003, págs 85-89. The aim of this work was to study cross-reactivity in the diagnosis of two related ascaridosis. Nineteen patients diagnosed with recidivous acute urticaria (RAU) caused by Anisakis simplex and 26 patients diagnosed with visceral larva migrans (VLM) caused by Toxocara canis were studied employing commercial diagnostic kits and “in house” assay kits. Cross-reactivity observed was greater when using “in house” assay kits, suggesting that T. canis excretory–secretory antigens were not only recognized by antibodies from patients with RAU but with greater intensity compared to the A. simplex excretory–secretory antigens.

We report what is, to our knowledge, the first study in which microsporidial infection was detected in elderly human immunodeficiency virus (HIV)--negative patients. Of the 60 elderly patients studied, 47 had diarrhea. Intestinal microsporidiosis due to Enterocytozoon bieneusi was diagnosed in 8 patients (17.02%) by use of Weber's chromotrope-based stain and polymerase chain reaction with species-specific primers. The mean age of these 8 patients was 75 years; 7 had chronic diarrhea and 1 had nonchronic diarrhea. Six of the patients with chronic diarrhea had no other pathogens isolated. In our opinion, elderly patients, because of their special immunological characteristics, should be considered a group at risk for the acquisition of intestinal microsporidiosis.

In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as Encephalitozoon cuniculi , type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.

Background: Microsporidia are protozoa which mainly affect severely immunodepressed AIDS patients in developed countries as well as those in developing ones. Traveler's diarrhea affects approximately 40% of people traveling from industrialized countries to developing ones, and no pathogens are identified in many of those patients on their returning, suggesting that known enteropathogens escape detection or entirely new ones could be responsible. Very few reports of travel-related microsporidiosis have been described. Methods: Between January, 1996 and January, 1998, a total of 40 European travelers from the tropics with a clinical picture of protacted diarrhea (three or more loose stools per day lasting for more than 3 weeks) were evaluated. Weber's trichrome modified by Kokoskin stain for microsporidial spores were performed in stool samples of every patient. Microsporidial DNA extraction and PCR amplification were attempted in every stool sample where microsporidial spores were observed. Results: Four cases of imported Enterocytozoon bieneusi were detected: one HIV-infected short term traveler, a pregnant long term traveler, and two immunocompetent short term travelers. Diarrhea was self-limited, and the spores cleared from the stools in all HIV-non infected travelers, but showed a chronic course in the HIV-infected one. Conclusions: Available data is too limited to affirm that residence or travel in tropical countries increases the risk for microsporidial infection, but the cases presented here suggest that E. bieneusi could be a cause of self-limited diarrhea in immunocompetent travelers returning from the tropics or could chronically affect immunocompromised HIV-infected travelers.

This study describes the production, characterization and use of an anti-idiotype serum raised against the monoclonal antibody TC-1 which recognizes a T. canis excretory/secretory antigen (ES Ag) epitope. Anti-idiotypic (anti-Id or Ab2) antibodies were produced in rabbits using TC-1 F)ab')2 fragments; these anti-Id inhibited ES Ag binding to biotinylated TC01, and also inhibited a larval microprecipitation assay using TC-1. Assays show that the Ab2β or “internal image” of a T. canis ES Ag epitope was obtained. The antibodies have been used as an idiotypic copy of ES Ag in a diagnostic ELISA for murine toxocariosis. Affinity-purified anti-Id antibodies were used to raise a homologous anti-anti-Id (Ab3) response in rabbits. Antibody formation was followed in the sera of BALB c mice inoculated with embryonated eggs of T. canis during a 12-month infestation. A 3-week latency period was observed before specific anti-TC-1 epitope antibodies were detected. High levels were reached at 7 weeks post-inoculation with a maximum at the ninth month, and were then maintained until the end of the experiment. The results show the possible utility of anti-Id antibodies as an ES Ag molecular replica.

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 μm/0.4-0.6 μm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking. © 2012 Optical Society of America., The authors acknowledge funding from a CSIC Proyecto Intramural Frontera PIF80F0171/2/3; Ministerio de Ciencia e Innovación, Spain, FIS2008-02065 (S. M.), FIS2011-25637 (S. M.), RYC2006-2920 (J. R. I.), FIS2009-07966 (J. R. I.), CTQ2010-16457 (A. U. A.), PROFIT CIT-300100-2007-50 (J. M. L.); Instituto de Salud Carlos III ISCIII- (RICET)-FEDER RD 06/0021/0006 (L. R.), and FIS 2009-01928 (L. R.); European Union FP7 HEALTH-2007-223414 (L. R.); EURHORCS-ESF Grant EURYI-05-102-ES (S. M.).

The antibody titer was followed in a group of patients, clinically diagnosed with toxocariasis, during a 5 year period. We observed that larvae can survive for at least 5 years in humans. Antigenic stimulation was enough to keep high levels of immunoglobulins over this period. Antibody levels decreased slowly and this pattern is similar to that shown by animal models.

Parasitism by the larval phase ofToxocara canisis a chronic process in which the larvae survive in the tissues, resulting in the constant stimulation of the immune system. As a result, the detection of specific antibodies may not reflect the active state of the parasite. We have studied the dynamics of the production of specific immune complexes by ELISA with the monoclonal antibody TC-1 in rabbits inoculated with single and multiple doses ofT. caniseggs. We also compared this with the production of specific antibodies and their possible modification after treatment with mebendazole. The specific antibodies against excretory-secretory antigen were detected with peaks at 10 and 12 weeks depending on the dose and remained positive during the entire experiment (62 weeks). Treatment caused an increase in the level of detectable antibodies dropping to similar levels to the controls. Specific immune complexes were detected only in multiple doses, and were then positive during the entire experiment. From the beginning of treatment the values of immune complexes fell quickly, remaining at undetectable levels during the rest of the experiment. For this reason the detection of specific immune complexes is a valid technique for monitoring the efficiency of treatment.

A sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

A prospective study was carried out to determine the prevalence rates of microsporidiosis and other enteroparasites in HIV-positive children in the Madrid area. HIV-positive pediatric patients from three hospitals were enrolled in the study. A total of 293 samples (158 stool and 127 urine) were collected from 83 children whose mean age was 6.3 years and had a mean CD4 count of 504.7/mm3 (range 1-2,220/mm3), 48 of whom suffered diarrhea at the time of the study. Microsporidia identification was investigated in stool and urine samples using Weber's chromotrope-based stain, IIF and PCR species-specific tests. Enteric parasites were identified in 32.5% of the children. Cryptosporidium sp. was the most common parasite encountered (14.4%), followed by Blastocytis sp. (9.6%) and Giardia duodenalis (8.4%). Microsporidia was only found in the stools of one child (1.2% of total and 2% of those with diarrhea) and Enterocytozoon bieneusi was demonstrated by PCR. The patient was 10 years old, presented non-chronic diarrhea and his CD4 count was 298/mm3. These data differ from those previously reported by us in HIV-positive adults (13.9%) in the same area, although this group showed more severely depressed CD4 lymphocyte counts than children. New epidemiological studies should be carried out to elucidate whether additional risk factors exist between these groups.