Alterations of plasma circulating microRNAs in BALB/c mice with Toxocara canis visceral and cerebral larva migrans.

BACKGROUND: Human toxocariasis is a neglected parasitic disease characterised by the syndromes visceral, cerebral, and ocular larva migrans. This disease is caused by the migrating larvae of Toxocara roundworms from dogs and cats, affecting 1.4 billion people globally. Via extracellular vesicles (EVs), microRNAs have been demonstrated to play roles in host-parasite interactions and proposed as circulating biomarkers for the diagnosis and follow-up of parasitic diseases. METHODS: Small RNA-seq was conducted to identify miRNAs in the infective larvae of T. canis and plasma EV-containing preparations of infected BALB/c mice. Differential expression analysis and target prediction were performed to indicate miRNAs involved in host-parasite interactions and miRNAs associated with visceral and/or cerebral larva migrans in the infected mice. Quantitative real-time polymerase chain reaction (PCR) was used to amplify circulating miRNAs from the infected mice. RESULTS: This study reports host and parasite miRNAs in the plasma of BALB/c mice with visceral and cerebral larva migrans and demonstrates the alterations of these miRNAs during the migration of larvae from the livers through the lungs and to the brains of infected mice. After filtering unspecific changes in an irrelevant control, T. canis-derived miRNAs and T. canis infection-induced differential miRNAs are predicted to modulate genes consistently involved in mitogen-activated protein kinase (MAPK) signalling and pathways regulating axon guidance and pluripotency of stem in the infected mice with visceral and cerebral larva migrans. For these plasma circulating miRNAs predicted to be involved in host-parasite crosstalk, two murine miRNAs (miR-26b-5p and miR-122-5p) are experimentally verified to be responsive to larva migrans and represent circulating biomarker candidates for visceral and cerebral toxocariasis in BALB/c mice. CONCLUSIONS: Our findings provide novel insights into the crosstalk of T. canis and the mammalian host via plasma circulating miRNAs, and prime agents and indicators for visceral and cerebral larva migrans. A deep understanding of these aspects will underpin the diagnosis and control of toxocariasis in humans and animals.

Chronic Migraineurs Form Carboxyhemefibrinogen and Iron-Bound Fibrinogen.

Chronic migraine (CM) is a disabling painful condition that is associated with dementia and thrombotic disease. It has been proposed that carbon monoxide (CO) and iron may play a role in CM, and CO and iron are products of the heme oxygenase system which is widespread within the brain. Further, CO and iron enhance plasmatic coagulation in part via a fibrinogen-dependent mechanism. Thus, our goal was to determine whether patients with CM had experienced carboxyhemefibrinogen formation, iron bound fibrinogen formation and plasmatic hypercoagulability. Nonsmokers with CM were recruited after informed, written consent. Blood was collected, anticoagulated with sodium citrate, and then centrifuged with plasma stored at -80ºC. Carboxyhemefibrinogen formation, iron bound fibrinogen formation and coagulation kinetics were determined via thrombelastographic methods. Patient results were compared with laboratory values generated from normal control plasmas. Incidence (95% confidence intervals) of the various parameters was determined using the Clopper-Pearson method. Twenty-six CM patients (24 female) were recruited; they were 46±12 years old. With regard to fibrinogen modification, 88.5% (69.8%-97.6%) of CM patients had formation of carboxyhemefibrinogen, iron bound fibrinogen, or both. With regard to coagulation, 42.3% (23.4%-63.1%) of patients had abnormally decreased time to clot initiation, 80.8% (60.6%-93.4%) had abnormally large velocity of clot formation, and 46.2% (26.6%-66.7%) had abnormally strong clot strength. Patients with CM have a large incidence of carboxyhemefibrinogen and iron bound fibrinogen formation and hypercoagulability. Confirmatory and potential therapeutic clinical trials targeting CO and iron modified hypercoagulation as a source of pain and vascular disease in CM patients are indicated.

Visceral larva migrans syndrome: analysis of serum cytokine levels in children with hepatic lesions confirmed in radiological findings.

Human toxocariasis is a helminth zoonosis resulting from accidental infection of humans by the roundworms Toxocara canis (T. canis) and cati (T. cati). The infection occurs in five forms: systemic (VLM), ocular, neurological, covert and asymptomatic. The aim of this study was to characterize the radiological and immunological findings in hepatic inflammation during the course of systemic infection by Toxocara sp. in children. Fifteen children, 2 to 17 years of age, with serological diagnosis of T. canis infection underwent abdominal ultrasonography and computer tomography (CT). Eosinophil counts, immunoglobulin E titres, interleukins IL-1α, IL-4, Il-6, IL-10 and interferon gamma were measured for all patients. Abdominal ultrasound revealed multiple hypoechoic areas in the livers of all patients. On the CT images, the hepatic lesions were seen as multiple, ill-defined, oval low-attenuating nodules that measured 6 to 9 millimetres in diameter. The nodules were usually best seen in the portal venous phase and were not seen on arterial-phase images. Significant intergroup differences were observed in the concentrations of IL-1α, IL-4, IL-6 and IL-10. The level of IFN-γ was not significantly elevated in patient sera relative to controls. The analysis shows that the production of anti-inflammatory cytokines is insufficient for granuloma formation in children presenting liver lesions in the course of VLM.

[Technological direction in the diagnosis and therapy of parasittic diseases: state-of-the-art and promises].

The paper presents information on the technological methods aimed at improving the diagnosis and prevention of parasitic diseases.

Synthesis, antigenicity against human sera and structure-activity relationships of carbohydrate moieties from Toxocara larvae and their analogues.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galß1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meß1-3GalNAcα1-OR (A), Fucα1-2Gal4Meß1-3GalNAcα1-OR (B), Fuc2Meα1-2Galß1-3GalNAcα1-OR (C), Fucα1-2Galß1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meß1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1-->3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.

[Toxocariasis in children from a subtropical region]. / Toxocariosis en niños de una region subtropical.

Toxocariasis is present worldwide but people living in areas with sanitary deficiencies are considered at the highest risk of infection, particularly children. The aim of this work was to know clinical and immunological aspects of infantile toxocariasis in a subtropical region in Argentina. For this purpose, 182 children of both sexes, 0-16 years old and with eosinophilia higher than 10%, living in Resistencia City (Northeast Argentina) were studied. Clinical examination, personal and epidemiological data recording, parasitological fecal examination and dosage of Toxocara canis IgG and IgM levels by EIE were performed; all positive sera were confirmed by Western Blot. Out of 182 children, 122 were positive for T. canis-IgG (67.0%); 28.8% lack of potable drinking water at home, 58.8% lack of sewerage facilities, 91.1% referred a close contact with dogs or cats, 30.0% had a history of geophagia and 86.7% lived along streets without pavement. The clinical forms of the infection were: 77.8% asymptomatic, 6.7% ocular larva migrans and 15.5% visceral larva migrans. In 22 children the serological follow up until 18 months after treatment showed 10 children without change in IgG levels, in 11 it decreased but remained high, and in one it increased. There were 19 children with high levels of T. canis-IgM; in 8 it decreased along treatment, in one it remained stable and 10 of them became negative. There was one child considered as a re-infection case. Results highlight the importance that health authorities should assign to this infection in regions with sanitary deficiencies, where toxocariasis is usually not recognized as a relevant public health problem.

Toxocariosis en niños de una region subtropical / Toxocariasis in children from a subtropical region

La toxocariosis está presente en todo el mundo, pero se considera en mayor riesgo a los habitantes de zonas com deficiencias sanitarias y particularmente a los niños. El objetivo de este trabajo fue conocer aspectos inmunológicos y clínicos de la infección infantil en un área subtropical de Argentina, para lo cual se estudiaron 182 niños de ambos sexos de la ciudad de Resistencia (Noreste de Argentina), de 0 a 16 años, con eosinofilia mayor al 10%. Se realizaron exámenes clínicos, encuestas epidemiológicas, exámenes copropa-rasitológicos y dosajes de IgG e IgM anti Toxocara canis por EIE; los sueros positivos fueron confirmados por Western Blot. De los 182 niños estudiados, 122 resultaron seropositivos (67%), 28.8% no contaban con agua potable en su domicilio, 58.8% no tenían cloacas, 91.1% habían tenido contacto con perros y/o gatos, 30.0% tenían antecedentes de geofagia y 86.7% vivían sobre calles sin pavimento. La infección se presentó en forma asintomática en el 77.8% de los casos, como larva migrans ocular en el 6.7% y como larva migrans visceral en el 15.5 % de los casos. En 22 niños el seguimiento serológico post-tratamiento hasta los 18 meses mostró que la IgG se mantuvo estable en 10 casos, en 11 disminuyó pero manteniendo valores elevados y em uno aumentó. Hubo 19 casos con IgM positiva; 8 disminuyeron sus títulos, uno se mantuvo estable y 10 se negativizaron. Hubo un caso de reinfección. Estos resultados reafirman la importancia que las autoridades sanitárias deben asignar a esta infección, particularmente en las regiones carenciadas, en las que habitualmente no se reconoce a la toxocariosis como un problema relevante de salud pública.

Toxocariosis en niños de una region subtropical / Toxocariasis in children from a subtropical region

La toxocariosis está presente en todo el mundo, pero se considera en mayor riesgo a los habitantes de zonas com deficiencias sanitarias y particularmente a los niños. El objetivo de este trabajo fue conocer aspectos inmunológicos y clínicos de la infección infantil en un área subtropical de Argentina, para lo cual se estudiaron 182 niños de ambos sexos de la ciudad de Resistencia (Noreste de Argentina), de 0 a 16 años, con eosinofilia mayor al 10%. Se realizaron exámenes clínicos, encuestas epidemiológicas, exámenes copropa-rasitológicos y dosajes de IgG e IgM anti Toxocara canis por EIE; los sueros positivos fueron confirmados por Western Blot. De los 182 niños estudiados, 122 resultaron seropositivos (67%), 28.8% no contaban con agua potable en su domicilio, 58.8% no tenían cloacas, 91.1% habían tenido contacto con perros y/o gatos, 30.0% tenían antecedentes de geofagia y 86.7% vivían sobre calles sin pavimento. La infección se presentó en forma asintomática en el 77.8% de los casos, como larva migrans ocular en el 6.7% y como larva migrans visceral en el 15.5 % de los casos. En 22 niños el seguimiento serológico post-tratamiento hasta los 18 meses mostró que la IgG se mantuvo estable en 10 casos, en 11 disminuyó pero manteniendo valores elevados y em uno aumentó. Hubo 19 casos con IgM positiva; 8 disminuyeron sus títulos, uno se mantuvo estable y 10 se negativizaron. Hubo un caso de reinfección. Estos resultados reafirman la importancia que las autoridades sanitárias deben asignar a esta infección, particularmente en las regiones carenciadas, en las que habitualmente no se reconoce a la toxocariosis como un problema relevante de salud pública. (AU)

[Ocular toxocariasis. Diagnostic and therapeutic options]. / Okuläre Toxocariasis. Diagnostische und therapeutische Optionen.

BACKGROUND: The diagnosis of ocular toxocariasis can be difficult and the aim of this study was to evaluate the benefit of ELISA testing of vitreous body fluid. PATIENTS AND METHODS: We present five consecutively treated patients with ocular symptoms of toxocariasis, three of these patients presenting with epiretinal membranes and subretinal granulomas were vitrectomized. Vitreous and body fluid and serum were tested for toxocara antibodies by ELISA. Moreover vitreous body fluid of 10 patients with epiretinal membranes of other origins were examined by the same ELISA. RESULTS: In all three operated patients toxocara antibodies were detected in the vitreous fluid but ELISA testing of serum samples was negative in two of the three patients. Visual acuity increased or remained stable in the operated patients. The ELISA test was negative in all vitreous fluid samples of the control group. CONCLUSIONS: ELISA testing of vitreous body fluid can prove the presence of toxocara infection when no systemic signs of infection are present and no antibodies are detectable in the serum. Well-timed vitrectomy is a suitable therapy for vitreo-retinal complications in ocular toxocariasis to improve prognosis and to confirm the diagnosis.

Application of western blotting procedure for the immunodiagnosis of visceral larva migrans in mice by using excretory/secretory antigens.

A Western blotting procedure with excretory/secretory antigens from Toxocara canis larvae was developed for immunodiagnosis of visceral larva migrans in mice. In this study, eighty Swiss albino mice were allotted into two groups of 40 each as control and experimental groups, and T. canis ova containing infective larvae were given to mice in the latter group to form visceral larva migrans. Blood samples were taken from 5 infected and 5 control mice on days 25, 30, 35, 40, 45, 50, 55, and 60 after infection. After bleeding, the mice were necropsied. Slides were prepared from their brain tissues and examined for visceral larva migrans. Following this procedure, their guts were also examined for intestinal parasites. Protein bands of excretory/secretory antigens of 2nd stage larvae of Toxocara canis were determined by using SDS-PAGE. Sera from the mice were tested by Western blotting and results were compared to the protein bands obtained by SDS-PAGE to determine specific bands. Specific protein bands for visceral larva migrans were determined as 24, 28, and 48 kDa according to our test results.

Visceral larva migrans: migratory pattern of Toxocara canis in pigs.

The migratory pattern of Toxocara canis was investigated following infection of pigs with 60000 infective eggs. Groups of six pigs were slaughtered at 7, 14 and 28 days after infection (p.i.), and the number of larvae in selected organs and muscles was determined by digestion. A group of uninfected pigs was used as negative controls for blood parameters and weight gain. Toxocara canis migrated well in the pig, although the relative numbers of larvae recovered decreased significantly during the experiment. On day 7 p.i., high numbers of larvae were recovered from the lymph nodes around the small intestine and to some extent also from the lymph nodes around the large intestine, and from the lungs and the liver. On day 14, the majority of larvae were recovered from the lungs and the lymph nodes around the small intestine, and by day 28 p.i. most larvae were found in the lungs. Larvae were recovered from the brain on days 14 and 21, with a maximum on day 14 p.i. No larvae were found in the eyes. Severe pathological changes were observed in the liver and lungs, especially on day 14 p.i.; also, development of granulomas was observed in the kidneys. Finally, a strong specific antibody response towards T. canis L2/L3 ES products was observed from day 14 p.i. until termination of the experiment, and the maximum eosinophil response was observed 14 days p.i. The pig is a useful non-primate model for human visceral larva migrans, since T. canis migrate well and induce a strong immunological response in the pig. However, the importance of the pig as a paratenic host is probably minor, because of the relatively early death of most of the larvae.

Larval toxocariasis and its clinical manifestation in childhood in the Slovak Republic.

Results are presented of 90 children aged 1-15 years hospitalized with toxocariasis. Blood count analysis and laboratory examination were done by routine clinical laboratory methods. Anti-Toxocara antibodies were detected in the serum of patients using an ELISA method. Demographic analysis of the children's families exposed to the risk of disease allowed estimation of age-specific rates for clinical toxocariasis. The probability of toxocaral infection and the intensity of its clinical manifestations in children are determined by the epidemiology of this zoonosis and by the risk factors in the family. The presence of high titres of specific IgG antibodies in all age categories correlates with the clinical manifestations of toxocariasis. The highest admission rate is in the age categories of 3-5 years (43.3%) and 6-10 years (36.7%). Laboratory findings show that the most conspicuous changes occur in the age category 1-5 years. The high percentage of seropositive dog-keeping and puppy-breeding families and the possibility of infection with repeated doses of larvae stimulate eosinophilia, which prevails in children under the age of five years. We present the percentage of patients whose parameters showed deviations from the reference values for a particular age category. Analyses of laboratory indices and of clinical manifestations will contribute to the accuracy of diagnosis and effectiveness of treatment of this disease.

Visceral larva migrans and tropical pyomyositis: a case report

Os autores relatam o caso de um jovem com piomiosite tropical grave, eosinofilia e hiperimunoglobulinemia E. O paciente relatou historia de contato com um cao e o teste de ELISA para Toxocara canis revelou-se positivo. O Staphylococcus aureus foi a unica bacteria isolada da secrecao purulenta obtida dos abscessos musculares. Sugere-se que a piomiosite tropical possa ser iniciada pela presenca das larvas desse ou de outros parasitos com tropismo para os musculos. As alteracoes imunologicas e estruturais nos musculos acometidos pelas larvas e a presenca de bacteriemia podem favorecer a instalacao da bacteria e o desenvolvimento da piomiosite

Cross-reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique.

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenically related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis and A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed.

[2 cases of toxocariasis (visceral larva migrans)]. / Dos casos de toxocarosis (larva migrans visceral).

BACKGROUND: Different epidemiological studies have demonstrated that specific anti-Toxocara antibodies are detected in the serum of a high percentage of the Spanish population. But very few clinical cases of visceral larva migrans are being confirmed. METHODS AND RESULTS: Two cases of visceral toxocarosis, in two sisters, are described. In the first, the prevailing clinic was swelling of joints and upper respiratory tract symptoms; and asthma and cutaneous allergic manifestations in the second patient. Both cases presented with an elevated blood eosinophil count, high levels of total IgE and high titlers of anti-Toxocara antibodies. All symptoms disappeared after treatment with diethylcarbamazine and they remain asymptomatic several months after. CONCLUSIONS: In pediatric population, toxocarosis should be ruled out in every patient with respiratory symptoms, allergic cutaneous manifestations and elevated blood eosinophil count. The anti-Toxocara antibodies assay is of great value in establishing the diagnosis of this parasitic disease.

Clinical and laboratorial features of visceral toxocariasis in infancy.

Forty children with a diagnosis of Visceral Toxocariasis were evaluated prospectively from February 1982 to June 1989. Diagnosis was established by clinical, laboratory and serological (ELISA - ES Toxocara canis antigen) evaluations. A great clinical polymorphism was found in our patients, ranging from unspecific or absent manifestations to an exuberant symptomatology. The laboratory findings were: leukocytosis, eosinophilia and elevation of serum gammaglobulin and isohemagglutinin levels. No significant relationship between clinical findings and laboratory parameters was found. Serology (ELISA) was a method of great diagnostic support but did not show a correlation with clinical and laboratory findings in this study. There was a significant relationship between pulmonary manifestations and the presence of signs and/or symptoms, when the patients were sent to us. Our findings, especially the high incidence of pulmonary manifestations, suggest that Visceral Toxocariasis has to be included in the differential diagnostic of children with pulmonary manifestations, characteristic epidemiological data and associated eosinophilia.

"Covert toxocariasis" in a child treated with low-dose diethylcarbamazine.

A girl aged 2.5 years with "covert toxocariasis" was treated with low-dose diethylcarbamazine because of supposed noticeable disseminated Toxocara canis infection without ocular or visceral manifestations. There was marked blood and bone marrow eosinophilia, significant increased Toxocara canis antibody (ELISA) and immunoglobulins E, G and M, leucocytosis and an increased sedimentation rate. She had no geophagia, but often sucked small stones, probably contaminated with faeces from puppies. Symptoms were fever, inactivity, weakness, tiredness and loss of appetite. She was followed clinically and with blood samples throughout a period of three years and four months.

Toxocara antibodies in patients with visceral or ocular disorder in Slovenia.

The incidence of Toxocara antibodies was determined in 319 sera from patients suspected of having Toxocara infection. Serological responses were compared with clinical manifestations and age of the patients. A seropositive reaction was found in 24% of the patients. Highly positive results (OD 0.510-1.000 and OD > 1.000) by ELISA were obtained significantly more frequently in younger patients than in older ones (p < 0.005). The percentage of seropositive patients with visceral disorder was found to decrease with age, whereas the proportion of seropositive patients with ocular disorder increased with age. Serologically confirmed Toxocara infection in Slovenia alerted us to the fact that this health issue should be given due attention in the future.

Seroprevalence of Toxocara canis antibodies among Orang Asli (aborigines) in Peninsular Malaysia.

An enzyme-linked immunosorbent assay using excretory-secretory antigens of the second stage larvae maintained in vitro was used to determine the seroprevalence of Toxocara antibodies in Orang Asli (aborigines) of Peninsular Malaysia. The mean + 3 SD optical density of 30 healthy subjects was used as the cut-off point. Overall prevalence was found to be 31.9%. No significant relationship was found between positive rates with sex and age groups, though children between 0 to 9 years recorded the highest positive rates. Eosinophil counts were found to be closely related to the proportion of positivity to toxocaral infection and mean optical densities. There was some degree of cross-reaction with Trichuris trichuria positive sera.