A serological and epidemiological evaluation of risk factors for toxocariasis in children in central Poland

The level of specific anti-Toxocara IgG was examined in 343 children from central Poland with suspected Toxocara infection. Based on the presence of specific IgG antibodies and clinical signs and symptoms, toxocariasis was confirmed in 249 patients. The serological results of diagnosed cases were analysed in connection with epidemiological data (contamination of soil around the children's homes and presence of a dog or a cat). A higher prevalence of soil contamination with Toxocara eggs was found in suburban and rural areas (30.9% and 24.6%, respectively) whereas it was lower in urban areas (10.3%). In about 40% of the children whose households were not contaminated, a decrease in the antibody level was observed 2 years after treatment for toxocariasis, while there was no such decline in those living in contaminated places. The logistic regression model employed to determine the correlation between seropositivity in the children and selected epidemiological risk factors showed a statistically significant relationship in connection only with dog ownership (P = 0.0238). The present results have demonstrated the high risk of toxocariasis for children from rural and suburban areas in Poland. The sero-epidemiological investigations indicated that re-infection might be the reason for persistence of seropositivity after treatment.     

A Case of Ocular Toxocariasis Successfully Treated with Albendazole and Triamcinolon

We present a case of ocular toxocariasis treated successfully with oral albendazole in combination with steroids. A 26-year-old male visited the authors'' clinic with the chief complaint of flying flies in his right eye. The fundus photograph showed a whitish epiretinal scar, and the fluorescein angiography revealed a hypofluorescein lesion of the scar and late leakage at the margin. An elevated retinal surface and posterior acoustic shadowing of the scar were observed in the optical coherence tomography, and Toxocara IgG was positive. The patient was diagnosed with toxocariasis, and the condition was treated with albendazole (400 mg twice a day) for a month and oral triamcinolone (16 mg for 2 weeks, once a day, and then 8 mg for 1 week, once a day) from day 13 of the albendazole treatment. The lesions decreased after the treatment. Based on this study, oral albendazole combined with steroids can be a simple and effective regimen for treating ocular toxocariasis.     

A seroepidemiological survey for toxocariasis in apparently healthy residents in Gangwon-do,Korea

We investigated the sero-prevalence of toxocariasis among healthy Korean adults in 1999. A total of 314 sera from normal inhabitants in Whachon-gun, Gangwondo, Korea was examined for specific antibody levels against excretory-secretory products of second stage larvae of Toxocara (TES). The presence of cross-reactions with other helminthiases such as cysticercosis, paragonimiasis, sparganosis or clonorchiasis was also checked by specific IgG ELISA. Sera showing positive reaction against TES were also tested by IgG immunoblot and by IgE ELISA. Out of 314 subjects, 16 was found to be positive by TES IgG ELISA and immunoblot, among whom 12 were also positive by TES IgE ELISA. Among the 16 seropositive samples, two sera showed positive reaction against Paragonimus and sparganum antigen, respectively. These results inferred that cross-reactions were negligible between toxocariasis and other helminthiases. Toxocariasis seroprevalence among Korean rural adults was detected to be approximately 5%.     

Toxocariasis and ingestion of raw cow liver in patients with eosinophilia

Ingestion of raw animal liver has been suggested as a possible mode of infection of human toxocariasis. We evaluated the relationship between toxocariasis and the ingestion of raw meat in patients with eosinophilia of unknown etiology. The study population consisted of 120 patients presenting with peripheral blood eosinophilia (>500 cells/microliter or >10% of the white blood cell count). They were divided into 2 groups: 104 seropositive patients based on a Toxocara excretory-secretory IgG ELISA and 16 seronegative patients. While 25.0% of seronegative patients had a recent history of eating raw cow liver, 87.5% of seropositive patients had this history. Multivariate statistical analysis showed that a recent history of eating raw cow liver was related to an increased risk of toxocariasis. Collectively, it is proposed that raw cow liver is a significant infection source of toxocariasis in the patients with eosinophilia of unknown etiology.     

O型孕妇产前IgG血型抗体效价与新生儿溶血病的相关性

目的:探讨O型孕妇产前IgG血型抗体效价与新生儿溶血病(HDN)的相关性。方法:选择2013年1月至2015年12月期间,在我院分娩的夫妻ABO血型不合的O型Rh(D)阳性孕妇432例及新生儿为研究对象,观察孕妇血清IgG抗体效价情况,新生儿HDN发病情况,并分析二者相关性。结果:432例孕妇中血清IgG抗A(B)效价大于或等于1:64的有189例,占43.75%,随IgG抗A(B)效价升高,孕妇比例逐渐减少。随孕次增加,孕妇血清IgG抗A(B)效价逐渐增高,各组孕妇血清IgG抗A(B)效价比较有统计学差异(P0.05)。随年龄增加,孕妇血清IgG抗A(B)效价逐渐增高,各组孕妇血清IgG抗A(B)效价比较有统计学差异(P0.05)。随IgG抗A(B)效价升高,HDN发生率显著升高,各组比较有统计学差异(P0.05)。等级相关检验显示,孕妇血清IgG抗A(B)与HDN发生呈正相关(r=0.732,P0.05)。结论:O型孕妇产前IgG血型抗体是引起HDN的主要原因,其水平与HDN呈正相关,值得临床重视。     

Seropositivity of Toxoplasma gondii and Toxocara spp. in Children with Cryptogenic Epilepsy,Benha, Egypt

The present study aimed to investigate the possible association of Toxoplasma gondii and Toxocara spp. infections with cryptogenic epilepsy in children. The study was carried out between June 2014 and March 2015. Total 90 children (40 with cryptogenic epilepsy, 30 with non-cryptogenic epilepsy, and 20 healthy control children) were evaluated to determine the anti-Toxocara and anti-T. gondii IgG seropositivity using ELISA kits. Epileptic cases were selected from those attending the pediatrics outpatient clinic of Benha University Hospital, Pediatrics Neurology Unit, and from Benha Specialized Hospital of children. The results showed that the level of anti-T. gondii IgG seropositivity was significantly higher among children with cryptogenic epilepsy (20%) than among children with non-cryptogenic children (0%). In healthy controls (10%), there was no association between toxocariasis seropositivity and cryptogenic epilepsy (only 5.7%; 4 out of 70 cases) among cases and 10% (2 out of 20) among controls. Among toxocariasis IgG positive cases, 3 (7.5%) were cryptogenic, and only 1 (3.3%) was non-cryptogenic. These statistically significant results support the association between T. gondii infection and cryptogenic epilepsy while deny this association with toxocariasis.     

A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10(4) at 350 nm and 8.29 x 10(3) at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10(3) at 350 nm and 1.26 x 10(5) at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.     

Parasite-specific antibody profile in the aqueous humor of rabbits with ocular toxocariasis

Human ocular toxocariasis is diagnosed using ophthalmologic and immunologic examinations. Many researchers have suggested that intraocular parasite-specific antibody levels are indicative of ocular toxocariasis, but little is known about the time course of the changes in these levels. We therefore investigated the anti-Toxocara canis antibody profile in the aqueous humor in an animal model of ocular toxocariasis. We intravitreally injected T. canis larvae into the right eye of 4 rabbits; 2 rabbits were orally administered T. canis eggs. We collected serum, aqueous humor, and tear samples weekly and determined the serum and aqueous humor levels of anti-T. canis immunoglobulin (Ig)G, IgA, IgM, and IgE antibodies and the tear IgG antibody level by enzyme-linked immunosorbent assay (ELISA). The severity of vitreous opacity and the aqueous humor IgG levels (measured using optical density [OD]) changed concordantly in the larvae-injected eyes; the OD exceeded 0.1 from 2–4 weeks after infection and remained elevated during active intraocular inflammation. However, the aqueous humor IgG levels were also elevated in 6 out of 8 eyes without intraocular larvae in both groups, and were low in 1 eye with live intravitreal larvae. In contrast, the serum IgG and IgM levels and the tear IgG levels increased in all rabbits, regardless of the presence of intraocular inflammation. Vitreous opacity occurred in all intravitreally infected eyes, but significant histopathological evidence of retinal damage was not detected. Thus, besides the presence of intraocular larvae, some other factors in the host may be required for the development of retinal lesions.     

Isotype specific immune responses in murine experimental toxocariasis

In this work, a murine experimental model of toxocariasis has been developed in BALB/c, C57BL/10 and C3H murine strains orally inoculated with 4,000 Toxocara canis embryonated eggs, in order to investigate the isotype-specific immune responses against excretory-secretory antigens from larvae. T. canis specific IgG+M, IgM, IgG, IgA, IgG1, IgG2a and IgG3 were tested by ELISA. The dynamics of the specific immunoglobulins (IgG+IgM) production showed a contrasting profile regarding the murine strain. Conversely to the results obtained with the IgM isotype, the IgG antibody class showed similar patterns to those obtained with IgG+IgM antibodies, only in the case of the BALB/c strain, being different and much higher than the obtained with IgG+IgM antibodies, when the C3H murine strain was used. The antibodies IgG+IgM tested in BALB/c and C57BL/10 were both of the IgM and IgG isotypes. Conversely, in the C3H strain only IgG specific antibody levels were detected. The IgG1 subclass responses showed a similar profile in the three murine strains studied, with high values in BALB/c, as in the case of the IgG responses.     

Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

A dot enzyme-linked immunosorbent assay (dot-ELISA) was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.     

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis. Antibody aggregation control is also important for therapeutic antibody development. To date, there have been few reports of IgG3 aggregation during protein expression and the low pH conditions needed for purification and virus inactivation. This study explored the potential of IgG3 antibody for therapeutics using anti‐CD20 IgG3 as a model to investigate aggregate formation. Initially, anti‐CD20 IgG3 antibody showed substantial aggregate formation during expression and low pH treatment. To circumvent this phenomenon, we systematically exchanged IgG3 constant domains with those of IgG1, a stable IgG. IgG3 antibody with the IgG1 CH3 domain exhibited reduced aggregate formation during expression. Differential scanning calorimetric analysis of individual amino acid substitutions revealed that two amino acid mutations in the CH3 domain, N392K and M397V, reduced aggregation and increased CH3 transition temperature. The engineered human IgG3 antibody was further improved by additional mutations of R435H to obtain IgG3KVH to achieve protein A binding and showed similar antigen binding as wild‐type IgG3. IgG3KVH also exhibited high binding activity for FcγRIIIa and C1q. In summary, we have successfully established an engineered human IgG3 antibody with reduced aggregation during bioprocessing, which will contribute to the better design of therapeutic antibodies with high effector function and Fab arm flexibility.     

Long-term systemic and mucosal antibody responses measured in BALB/c mice following intranasal challenge with viable enterotoxigenic Escherichia coli

The immunogenicity induced in BALB/c mice following intranasal challenge with a viable nonlethal dose (1.2 x 10(8) CFU) of enterotoxigenic Escherichia coli (ETEC) strain E23477A (O139:H28:CS1:CS3:LT+:ST+) was studied over a 140-day period. Serum IgG and IgM antibodies against coli surface antigen 3 (CS3), O139 lipopolysaccharide and heat-labile enterotoxin were measured by day 14 and remained at elevated levels out to day 140. The serum IgG response to the somatic antigens (CS3 and O139 lipopolysaccharide) was significantly greater (P < 0.05) than the IgG response to heat-labile enterotoxin, and the serum IgG response to CS3 was significantly greater (P < 0.05) than the IgG response to O139 lipopolysaccharide. The predominant serum IgG subclasses to CS3 were IgG1 and IgG2a, and they were significantly greater (P < 0.05) than IgG2b and IgG3. The predominant serum IgG subclass response to O139 lipopolysaccharide was initially IgG3 until day 56, after which IgG1 was predominant. The serum subclass response to CS3 indicated a mixed T helper 1/2 (Th1/Th2) profile, whereas the response to O139 lipopolysaccharide was primarily that of a Th2-type, at least over time. Fecal IgG and IgA responses to CS3 and O139 lipopolysaccharide were detected by day 14 and were measured out to day 140, with the CS3 fecal antibody responses being significantly greater (P < 0.05) than the O139 lipopolysaccharide and heat-labile enterotoxin fecal antibody responses. The aim of this study is the development of the intranasal mouse model that can aid in better understanding the immunopathology of ETEC infection and in screening of vaccine candidates prior to volunteer trials.     

Concentration dependence of IgG-protein A affinity studied by wireless-electrodeless QCM

The binding affinity between human immunoglobulin G (IgG) and protein A was studied by the homebuilt wireless-electrodeless quartz crystal microbalance (QCM). Protein A was immobilized on the electrodeless AT-cut quartz plate of 0.05 mm thick and its fundamental resonance frequency near 34 MHz was measured by a noncontacting manner using a line antenna. The vibrational analysis was performed to ensure higher sensitivity of the electrodeless QCM. A flow-cell system was fabricated to continuously measure the resonance frequency during the injection sequence of the IgG solutions with concentrations of 1-20,000 ng/mL. The exponential frequency changes were recorded to determine the affinity based on the Langmuir kinetics. The equilibrium constant K(A) significantly varied between 6 x 10(6) and 6 x 10(10) M(-1), depending on the IgG concentration, which is attributed to various formations of IgG-protein A complexes.     

Immunodiagnosis of ocular toxocariasis using Western-blot for the detection of specific anti-Toxocara IgG and CAP for the measurement of specific anti-Toxocara IgE

A prospective multicentric study was carried out to assess both the performance of Western-blot (WB) detecting specific anti-Toxocara IgG and that of CAP measuring specific IgE titre for the immunodiagnosis of ocular toxocariasis. For 14 outpatients presenting ophthalmic symptoms (choroiditis, chorioretinitis, papillar oedema, hyalitis, retinal detachment and/or uveitis), samples of serum and aqueous fluid (AF) were sent to the Department of Parasitology, University Hospitals, Toulouse, France. All patients but two tested positive with WB on the serum; 13 WB tests were performed on the AF, 12 of which were positive. The two patients who had a negative WB serum result tested positive for the AF. Specific IgE detection was considered as a complementary test of WB. Two patients showed a greater specific IgE titre in the AF than in the serum, and one had a positive result in the AF, but not in the serum. These six patients were considered as clear cases of ocular toxocariasis. Western-blot coupled with specific anti-Toxocara IgE detection appeared therefore to be an accurate procedure for the immunodiagnosis of ocular toxocariasis, provided the testing was simultaneously performed on the serum and AF.     

卵巢癌抗独特型单链抗体的人源化——抗独特型微抗体的构建与表达

为了降低人抗鼠抗体反应 ,获得满意的免疫原性 ,将模拟人卵巢癌抗原的抗独特型单链抗体人源化 .采用重叠PCR和基因工程的技术 ,将 6B11ScFv基因的轻链和重链颠倒 ,成为 6B11VL VH.再与人IgG1铰链区和CH3区的基因进行融合 (VL VH CH3) ,构建抗独特型微抗体的原核表达载体 .转化E .coliBL2 1(DE3)后用IPTG诱导表达 .经SDS PAGE分离显示 ,在 5 0kD左右处有一诱导蛋白带 .不连续非变性凝胶电泳显示 ,表达产物分子量为 10 0kD左右 .采用ELISA、竞争抑制实验、West ern印迹对其进行活性测定 .结果表明 ,人源化的抗独特型微抗体具有特异性双价结合卵巢癌单克隆抗体COC16 6 9和识别人免疫球蛋白IgG1的活性 ,成功地将鼠源的scFv的人源化     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Detection and quantification of proteins using self-excited PZT-glass millimeter-sized cantilever

A composite self-excited PZT-glass cantilever (4mm in length and 2mm wide) was fabricated and used to measure the binding and unbinding of model proteins. A key feature of the cantilever is that its resonant frequency is dependent on its mass. The fabricated cantilever has mass change sensitivity in liquid of 7.2 x 10(-11)g/Hz. Resonant frequency change was measured as protein reacted or bound with the sensing glass cantilever surface. Protein concentrations, 0.1 and 1.0mg/mL, which resulted in nanogram mass change were successfully detected. The mass change sensitivity gave a total mass change of 54+/-0.45 ng for the binding of anti-rabbit IgG (biotin conjugated) to rabbit IgG immobilized cantilever and the subsequent binding of captavidin. The unbinding of anti-rabbit IgG and captavidin gave a total mass change of 54+/-1.70 ng. Fluorescence based assays showed the combined mass of both proteins in the released samples was 54+/-2.24 ng. The binding kinetics of the model proteins is modeled as first order. The initial binding rate constant of anti-rabbit IgG to rabbit IgG was 1.36+/-0.02(min(mg/mL))(-1). The initial binding rate constant of captavidin to biotinylated anti-rabbit IgG was (2.57 x 10(-1))+/-0.003(min(mg/mL))(-1). The significance of the results we report here is that millimeter-sized PZT-actuated glass cantilevers have the sensitivity to measure in real-time protein-protein binding, and the binding rate constant.     

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.     

A Cross-sectional Serological Study of Cysticercosis,Schistosomiasis, Toxocariasis and Echinococcosis in HIV-1 Infected People in Beira,Mozambique

Background

Helminthic infections are highly endemic in Mozambique, due to limited access to healthcare and resources for disease prevention. Data on the subclinical prevalence of these diseases are scarce due to the fact that an immunological and imaging diagnosis is not often available in endemic areas. We conducted a cross-sectional study on HIV1⁺ patients from Beira city in order to determine the seroprevalence of cysticercosis, schistosomiasis, toxocariasis and echinoccocosis and its possible interaction with HIV infection.

Methodology/Principal Findings

Patients (601) were voluntarily recruited at the Ponta Gea Health Center and their demographic and clinical data were recorded (including CD4⁺ cell count and antiretroviral regimen). Mean age was 39.7 years, 378 (62.9%) were women and 223 (37.1%) were men. Four hundred seventy-five (475) patients (79%) were already on highly active antiretroviral therapy (HAART), and 90 started therapy after being enrolled in the study. For serological testing we used a Multiplex Western Blot IgG from LDBIO Diagnostics. The overall seroprevalence was 10.2% for cysticercosis, 23% for schistosomiasis, 7.3% for toxocariasis and 17.3% for echinococcosis.

Conclusions/Significance

Neither age nor the CD4⁺ count were significantly associated with the seroprevalence of the helminths studied. However, patients with CD4⁺ between 200–500/µl had a higher seroprevalence to all helminths than those with less than 200/µl cells/and those with more than 500 cells/µl. Female gender was significantly associated with cysticercosis and schistosomiasis, and being in HAART with toxocariasis. Headache was significantly associated with cysticercosis and toxocariasis. There was no association between epilepsy and seropositivity to any of the parasites. The study concluded that a clear understanding of the prevalence and manifestations of these coinfections, how best to diagnose subclinical cases, and how to manage diseases with concomitant antiretroviral therapy is needed.