Immune complexes as a tool for strongyloidiasis immunodiagnosis in kidney and liver transplant candidate.

Strongyloidiasis is a chronic and asymptomatic infection in immunocompetent patients. Immunocompromised patients, such as organ transplant candidates, can develop severe forms of this disease, and the best way to prevent progression to these forms is early diagnosis. Serological techniques using specific IgG and immune complexes (IC) detection can help in the diagnosis of these patients. This study aimed to detect specific anti-Strongyloides IC and IgG antibodies in kidney transplant (KT) and liver transplant (LT) candidates. A total of 100 blood samples was collected from transplant candidates (50 blood samples each from KT and LT candidates). Serum was obtained and analysed using enzyme-linked immunosorbent assay for IC and IgG detections. The IC levels showed frequencies of 18% and 2% in the KT and LT groups, respectively, whereas anti-Strongyloides IgG was detected in 34% and 12% of KT and LT candidates, respectively. The correlation between IC and IgG detection is poor in KT candidates, while in LT candidates, there is a significant positive correlation. The detection of IC can be an additional tool for the diagnosis of strongyloidiasis, especially when associated with the detection of specific IgG anti-Strongyloides antibodies.

Strongyloides-specific IgA, IgG and IgG immune complex profile in patients with pulmonary tuberculosis.

AIMS: To describe an anti-Strongyloides IgA, IgG and IgG immune complex antibody response profile in patients with pulmonary tuberculosis. METHODS AND RESULTS: Saliva and serum samples were collected from 100 individuals: group I, 50 apparently healthy individuals; and group II, 50 pulmonary tuberculosis patients. The IgA, IgG and IgG immune complex detection were carried out via an ELISA immunoenzymatic test. Optical density medians in saliva samples of IgA antibody (median of 7.21) and IgG-IC (median of 4.95) were significantly higher in tuberculosis group compared to control individuals (median IgA of 3.93 and IgG-IC of 2.38). CONCLUSION: This study presents antibody data to the field of pulmonary tuberculosis and strongyloidiasis coinfection, including saliva samples, and especially IgG immune complex detection.

Mimotope-based antigens as potential vaccine candidates in experimental murine cysticercosis.

Human cysticercosis is a public health problem caused by Taenia solium metacestodes; thus, eradication of T. solium transmission by vaccination is an urgent requirement. The Cc48 mimotope from T. solium cysticerci was tested expressed in phage particles (mCc48) and chemically synthesized (sCc48) as a vaccine candidate in experimental murine cysticercosis. For this, BALB/c mice were immunized with mCc48 (G1; n = 40), sCc48 (G2; n = 40) and phosphate-buffered saline (PBS) (G3; n = 40, positive control) and challenged with Taenia crassiceps metacestodes. Another PBS group without parasite challenge was used as a negative control (G4; n = 40). Mice were sacrificed 15, 30, 45 and 60 days post-infection for cysticerci and serum collection. Immunization efficacy was determined by cysticerci counting. Serum samples were tested by ELISA to verify antibody (IgM, IgG, IgA and IgE) and cytokine (IFNγ and IL-4) levels. The sCc48 achieved the highest rates of protection and efficacy (90 and 98%, respectively). The group immunized with mCc48 presented the highest reactivity for IgM, IgG and IgE. All groups presented IL-4, but IFNγ was quite variable among groups. The protection induced by sCc48 synthetic peptide supports further studies of this mimotope as a potential vaccine candidate against cysticercosis.

Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection.

Definitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

Neurocysticercosis serodiagnosis: mimotope-based synthetic peptide as potential biomarker.

Herein, we evaluate a mimotope-based synthetic peptidenamed NC41 to diagnose neurocysticercosis (NC), a neglected parasitic disease and a major cause of epilepsy worldwide. NC41 synthetic peptide was evaluated to diagnose NC, and total saline extract from Taenia solium metacestodes (SE) was used as control. Serum samples from patients with NC (n = 40), other parasitic diseases (n = 43), and healthy individuals (n = 40) were tested. Diagnostic parameters such as sensitivity (Se), specificity (Sp), likelihood ratio (LR), and area under curve (AUC) were calculated using receiver operating characteristic (ROC) curves. The sequence from T. solium phosphoenolpyruvate carboxykinase (PEPCK) was used for epitope prediction, resulting in one high-scoring patch centered at residue L247. NC41 synthetic peptide reached high diagnostic performance (Se 97.5% and Sp 97.5%, LR+ 39 and AUC 0.997). Data from diagnostic parameters and in silico analyses proved the usefulness of NC41 synthetic peptide as a diagnostic marker for human NC.

Inducible nitric oxide synthase controls experimental Strongyloides infection.

Infection with Strongyloides sp. induces a host immune response, predominantly the Th2 type, that is able to eliminate the parasite. However, little is known about the role of the nitric oxide (NO) mediator, induced by the enzyme nitric oxide synthase (NOS), in strongyloidiasis. Therefore, in this study, we investigated the immune response of mice genetically deficient in the enzyme inducible nitric oxide synthase (iNOS-/- ), infected with Strongyloides venezuelensis. C57BL/6 wild-type (WT) and iNOS-/- mice were individually inoculated by subcutaneous injection of 3000 S. venezuelensis L3 larvae. In the absence of iNOS, mice were more susceptible to the infection than WT animals, in which the parasite was completely eliminated. The overall production of cytokines and specific IgG, IgG1 or IgE antibodies against the parasite was significantly lowered in infected iNOS-/- mice. The expression of iNOS was observed in the intestine of WT hosts but mainly in the wall of the parasite, despite the presence of iNOS in mice. Altogether, we concluded that iNOS expression may play an important role in the control of S. venezuelensis infection.

Immunoblotting using Strongyloides venezuelensis larvae, parthenogenetic females or eggs extracts for the diagnosis of experimentally infected immunosuppressed rats.

The nematode Strongyloides stercoralis is responsible for strongyloidiasis in humans. Diagnosis of infection occurs through detection of larvae in feces, but low elimination of larvae often hampers the detection of disease, particularly in cases of patient immunosuppression. Immunodiagnostic tests have been developed; however obtaining S. stercoralis larvae for the production of homologous antigen extract is technically difficult. Thus, the use different developmental forms of Strongyloides venezuelensis has become an alternative method for the production of antigen extracts. The aim of this study was to evaluate immunoblotting using alkaline extracts from S. venezuelensis L3 larvae, parthenogenetic females or eggs to test detection of experimental strongyloidiasis associated with immunosuppression. Immunocompetent and immunosuppressed male rats were experimentally infected, and serum sample from all animals were obtained at 0, 5, 8 13, and 21 days post infection (d.p.i.). Immunoblotting was evaluated for use in detection of anti-S. venezuelensis IgG in both experimental rat groups. The larval extract immunoblotting profile had the most immunoreactive fractions in the immunosuppressed group beginning at 5 d.p.i., while the immunocompetent group reactivity began on 8 d.p.i. Immunoreactive protein fractions of 17 kDa present in larval alkaline extract presented as possible markers of infection in immunosuppressed rats. It is concluded that all extracts using immunoblotting have diagnostic potential in experimental strongyloidiasis, particularly larval extract in immunosuppressed individuals.

Increased susceptibility to Strongyloides venezuelensis infection is related to the parasite load and absence of major histocompatibility complex (MHC) class II molecules.

In human and murine models strongyloidiasis induce a Th2 type response. In the current study we investigated the role of different loads of Strongyloides venezuelensis in the immune response raised against the parasite and the participation of the major histocompatibility complex (MHC) class II molecule in the disease outcome in face of the different parasite burden. The C57BL/6 wild type (WT) and MHC II(-/-) mice were individually inoculated by subcutaneous injection with 500 or 3000 S. venezuelensis L3. The MHC II(-/-) mice infected with 3000L3 were more susceptible to S. venezuelensis infection when compared with WT groups, in which the parasite was completely eliminated. The production of Th2 cytokines and specific IgG1 or IgE antibodies against parasite were significantly lowered in MHC II(-/-) infected mice with different larvae inoculums. The infection of MHC II(-/-) mice with S. venezuelensis induced slight inflammatory alterations in the small intestine, and these lesions were lower when compared with WT mice, irrespective of the parasite load utilized to infect animals. Finally, we concluded that MHC class II molecules are essential in the immune response against S. venezuelensis mainly when infection occurs with high parasite inoculum.

Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography.

The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

Multiple Stool Sampling and Specific Parasitological Technique are Crucial to Diagnose Strongyloidiasis in Alcoholic Patients.

PURPOSE: Chronic alcoholism is a well-known risk factor for strongyloidiasis, in these patients the disease is potentially more severe, probably due to the breakdown of local protective barriers and immunosuppression caused by alcohol, which can lead to autoinfection and dissemination. The aim of this study was to evaluate multiple stool sampling and a specific parasitological assay agar plate culture (APC) for the diagnosis of Strongyloides stercoralis in alcoholics. METHODS: APC was compared to sedimentation technique (HPJ; Hoffman, Pons and Janer), as parasitological methods to detect S. stercoralis infection in alcoholic individuals. Three stool samples from 60 alcoholic and 60 non-alcoholic individuals were analyzed. RESULTS: S. stercoralis larvae were observed in 11 (18.3%) alcoholic individuals and 1 (1.7%) nonalcoholic individual (P = 0.0042). In view of the combined results, sensitivity for the APC method was 63.6% (CI 31.6-87.6%) with the first sample reaching 100% (CI 67.8-100%) after analyzing three fecal samples. The HPJ sensitivity was 36.4% (CI 12.4-68.4) in the first sample, reaching 72.7% (CI 39.3-92.7) after three samples analyzed. CONCLUSION: The present results suggest that in alcoholic patients, it is important to repeat stool sampling with specific techniques, especially using the APC method, to avoid misdiagnosis in cases that could evolve to disseminated strongyloidiasis.

Route of dexamethasone administration influences parasite burden in Strongyloides hyperinfection model.

Rodents infected with Strongyloides venezuelensis are experimental models applied to strongyloidiasis research. This study evaluated oral and subcutaneous dexamethasone (DEX) treatments to establish immunosuppression in an experimental model of Strongyloides hyperinfection. Rattus norvegicus Wistar were divided: G I (-): untreated and uninfected animals, G II (+): untreated and infected, G III (o -) orally treated and uninfected, G IV (o +) orally treated and infected, G V (sc -) subcutaneously treated and uninfected, G VI (sc +) subcutaneously treated and infected. For oral administration, DEX was diluted in sterile water (5 µg/ml) and made available to the animals on intervals in experimental days - 5-0, 8-13 and 21-26. For subcutaneous administration, animals received daily injections of DEX disodium phosphate (2 mg/kg). Infection was established by the subcutaneous inoculation of 3000 S. venezuelensis filarioid larvae. Groups were evaluated by egg per gram of feces and parasite females counts and IgG, IgG1 and IgG2a detection. GIV (o +) had egg peaks count on days 13 and 26 and maintained egg elimination until the last experimental day. Parasitic females recovery at day 30 was significantly higher in G IV (o +) when compared to G VI (sc +). Levels of IgG, IgG1 and IgG2a of all groups, except the positive control GII (+), were below the detection threshold. Pharmacological immunosuppression induced by oral administration of DEX produced high parasitic burden, and is a noninvasive method, useful to establish immunosuppression in strongyloidiasis hyperinfection model in rats.

Seropositivity to Cysticercosis in School-Age Children Living in a Low-Income Municipality in the Midwest Region of Brazil.

Background: Human cysticercosis (CC) is a global public health problem, especially in Latin America, including Brazil. We aimed to analyze the seroprevalence of CC among school-age children and adolescents. Methods: We analyzed the presence of specific IgG antibodies against Taenia solium metacestodes in 500 serum samples from elementary school children and adolescents in Jataí City, state of Goiás, Brazil. IgG antibodies against the antigenic extract of the parasite were detected and analyzed by ELISA, and specific peptides were identified by confirmatory Western Blotting test. Results: Of the 500 study participants, 205 (41%) were male, and 295 (59%) were female. Participants aged between 4 and 18 years (mean age 8.4 years). The percentage of serum samples reactive by ELISA was 37.2%. These samples were analyzed by Western Blotting, which confirmed that the seropositivity rate was 6.2% (95% CI 2.4-14.7) in 31 samples reactive for CC-specific bands, determined in serum samples from 18 male (5-11 years old) and 13 female (4-12 years old) students. Conclusion: The CC seroprevalence demonstrated in schoolchildren suggests that this parasitosis is endemic in the study area. Further investigations are necessary to clarify the local epidemiology of this parasitosis.

IgG4 and IgE anti-Strongyloides stercoralis as additional parameters in characterizing patients with diabetes from a hyperendemic area.

We report the detection of IgG, IgG1, IgG4 and IgE anti-Strongyloides stercoralis as complementary tool for screening in patients with diabetes in hyperendemic areas for strongyloidiasis. A panel of 119 serum samples were analyzed: 76 from patients with DM2 and 43 patients with other endocrine diseases and a positive correlation for total IgG levels with IgG4 (rs = 0.559; P = 0.024; n = 16) and IgG and IgE (rs = 0.585; P < 0.0001; n = 76) was found in the diabetes group.

Genetic polymorphism in Taenia solium metacestodes from different Brazilian geographic areas.

The aim of the present study is to investigate genetic polymorphisms in Taenia solium metacestodes from different Brazilian geographical areas and to relate them to antibody recognition in serum samples of neurocysticercosis (NC) patients. Metacestodes were obtained from the Distrito Federal (DF), Bahia, Minas Gerais (MG) and São Paulo (SP) regions of Brazil. Samples of human sera from 49 individuals with NC, 68 individuals with other helminthiasis and 40 healthy volunteers were analysed (157 individuals in total). Antigens were prepared and used in enzyme-linked immunosorbent assay and western blotting assays to detect specific immunoglobulin G antibodies. Genetic distances between metacestode populations were analysed using random amplified polymorphic DNA (RAPD) analysis. Our results show that there was a higher frequency of reactivity in the DF region in the sera from NC patients (p < 0.05), while discrimination between active and inactive NC was seen only in extracts from the MG and SP regions (p < 0.05). Using RAPD, the sample from the DF region presented a greater increase compared to the other regions. A relationship between genetic polymorphisms among T. solium metacestodes from different areas in Brazil and the differences in antibody detection in patients with NC were established.

Infectivity of Strongyloides venezuelensis is influenced by variations in temperature and time of culture.

The present research investigated the influence of temperature and time of larvae culture on the infectivity of Strongyloides venezuelensis. Mice were infected s.c. with 1500 larvae of S. venezuelensis maintained at 28 °C for three days of culture (dc), 28 °C for seven dc or 18 °C for seven dc. On days 1, 3, 5, 7, 14 and 21 post-infection the animals were sacrificed and cell numbers in the blood, peritoneal cavity fluid (PCF), broncoalveolar fluid (BALF), cytokines, immunoglobulins, number of parasites and eggs/g of feces were quantified. Results demonstrated an increase in eosinophils and mononuclear cells in the blood, PCF and BALF of infected mice. Larvae at 28 °C/3dc induced earlier eosinophils in the PCF and BALF as opposed to larvae at 28 °C/7dc and 18 °C/7dc. Larvae at 28 °C/7dc induced higher synthesis of IL-4, IL-5 and IL-10 on days 5 and 7 post-infection. Larvae at 28 °C/3dc in culture induced higher synthesis of IL-12 than larvae of seven dc, but time in culture induced better synthesis of IFN-γ after larval migration had ceased and only adult worms were present. Larvae at 28 °C/3dc in culture induced higher synthesis of IgG and IgG1 and expelled less female parasites than larvae cultivated for seven days. In conclusion, it was observed that the infectivity of S. venezuelensis is influenced by variations in temperature and time of culture.

Diagnosis of human strongyloidiasis: Application in clinical practice.

This review considers the advantages and disadvantages of parasitological techniques, methods of detecting antibodies and antigens, as well as molecular biology techniques in the diagnosis of human strongyloidiasis. In addition, it elucidates the potential of different techniques for rapid and effective detection of clinical cases, thus enabling early treatment and preventing fatal consequences of this helminthiasis.

Innovative approaches to improve serodiagnosis of Strongyloides stercoralis infection.

Strongyloidiasis is a helminthiasis of neglected condition that has no gold standard parasitological diagnosis due to the intermittent release of larvae in feces. This study aimed to use an scFv (single chain variable fragment) obtained by Phage Display, previously validated to detect immune complexes in serum samples from individuals infected with Strongyloides stercoralis by enzyme-linked immunosorbent assay (ELISA). Now the ability of scFv to detect the immune complexes was verified by immunofluorescence, flow cytometry using magnetic beads and surface plasmon resonance (SPR). As ELISA, the SPR, immunofluorescence and flow cytometry demonstrated the ability of scFv to detect immune complexes in sera from individuals with strongyloidiasis and discriminate them from sera of individuals with other parasitic diseases and healthy individuals. Besides de conventional ELISA, the novel approaches can also be promptly applied as auxiliary diagnostic tools to the existing parasitological method for accurate diagnosis of human strongyloidiasis.

Corrigendum to "scFv against HSP60 of Strongyloides sp. and Its Application in the Evaluation of Parasite Frequency in the Elderly".

[This corrects the article DOI: 10.1155/2020/4086929.].

Immunoreactivity of proteins within 30-40 kDa range during the acute and the recovery phases in rats experimentally infected with Strongyloides venezuelensis.

In experimental infection with Strongyloides venezuelensis, the acute and recovery phases can be distinguished, unlike human infections caused by Strongyloides stercoralis. The objective of this study was to evaluate the production of anti-Strongyloides IgG antibodies and the recognition of immunogenic protein bands during the acute and the recovery phases in rats experimentally infected with S. venezuelensis. Rats were infected subcutaneously with 400 or 4,000 S. venezuelensis infective larvae. The acute phase was characterized by elimination of a large number of eggs in the faeces on days 6-14 post infection; the recovery phase was characterized by the resolution of the infection between days 30 and 35 post infection. Differences in IgG levels were observed in the acute and the recovery phases. Different antigenic fractions were recognized in both phases of infection. It is concluded that proteins within the 30-40 kDa range are immunoreactive markers for both the acute and the recovery phases in rats experimentally infected with S. venezuelensis, particularly using membrane antigen.

Parasitological and immunological aspects of oral and subcutaneous prednisolone treatment in rats experimentally infected with Strongyloides venezuelensis.

Strongyloides venezuelensis is a model to study human strongyloidiasis, which infects wild rodents and shares common antigenic epitopes with Strongyloides stercoralis. This study aimed to evaluate parasitological and immunological parameters of prednisolone immunosuppression protocols in rats (Rattus novergicus) infected with S. venezuelensis. Rats were divided into six groups (n = 36): untreated and uninfected (-) or infected (+); oral treatment and uninfected (o-) or infected (o+); subcutaneous treatment and uninfected (sc-) or infected (sc+). For oral immunosuppression, 5 mg/mL of water diluted prednisolone were given five days before infection, and in the days 8 and 21 (for 5 days). For subcutaneous immunosuppression, 10 mg/kg of prednisolone were given daily. The infection was established by the subcutaneous injection of approximately 3,000 S. venezuelensis filarioid larvae per animal. All animals from the (+) and (o+) groups survived, while four rats from the (sc+) died prior to necropsy date. Parasitological analysis showed higher egg elimination in (o+) in comparison to (+) and (sc+) on 7, 13 and 26 days post infection (d.p.i.).The recovery of parasitic females at day 30 was significantly higher in (o+), compared to (+). The (+) and (o+) groups showed a clear increase in anti-S. venezuelensis IgG, IgG1 and IgG2 from 13th d.p.i. Oral immunosuppression led to a higher number of adult females and increased egg output while maintaining IgG and subclasses antibody levels comparable to the positive control.