Marcelo Andreetta Corral

MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.

PARASITOLOGICAL AND MOLECULAR DIAGNOSIS IN EXPERIMENTAL Strongyloides venezuelensis INFECTION

Strongyloides venezuelensis is a parasitic nematode of rats which is frequently used as a model to study human and animal strongyloidiasis. The aim of this study was to evaluate the correlation between parasitological and molecular diagnosis in Strongyloides venezuelensis infection. PCR assays were used to detect S. venezuelensis DNA in fecal samples obtained from experimentally infected Rattus norvegicus. The results showed a higher sensitivity of the PCR assay in detecting the infection compared to parasitological methods.

IS THE AGAR PLATE CULTURE A GOOD TOOL FOR THE DIAGNOSIS OF Strongyloides stercoralis IN CANDIDATES FOR TRANSPLANTATION

Identification and treatment of chronic strongyloidiasis is the most important factor for avoiding the severe form, particularly in transplant candidates, in whom immunosuppressive therapy (including the use of corticosteroids) is a risk factor for the occurrence of severe disease. Definitive diagnosis of strongyloidiasis is usually made through detection of the larvae in stool samples, which have low sensitivity. Agar Plate culture is considered more efficient than other conventional methods in the parasitological diagnosis of S. stercoralis with high sensitivity. However, it has not been used to screen candidates for transplantation.

DIAGNOSIS OF Strongyloides stercoralis INFECTION IN IMMUNOCOMPROMISED PATIENTS BY SEROLOGICAL AND MOLECULAR METHODS

SUMMARY Strongyloidiasis is a potentially serious infection in immunocompromised patients. Thus, the availability of sensitive and specific diagnostic methods is desirable, especially in the context of immunosuppressed patients in whom the diagnosis and treatment of strongyloidiasis is of utmost importance. In this study, serological and molecular tools were used to diagnose Strongyloides stercoralis infections in immunosuppressed patients. Serum and stool samples were obtained from 52 patients. Stool samples were first analyzed by Lutz, Rugai, and Agar plate culture methods, and then by a quantitative real time polymerase chain reaction (qPCR). Serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) using a soluble (AS) or a membrane fractions antigen (AM) obtained from alkaline solutions of the filariform larvae of Strongyloides venezuelensis. Of the 52 immunosuppressed patients, three (5.8%) were positive for S. stercoralis by parasitological methods, compared to two patients (3.8%) and one patient (1.9%) who were detected by ELISA using the AS and the AM antigens, respectively. S. stercoralis DNA was amplified in seven (13.5%) stool samples by qPCR. These results suggest the utility of qPCR as an alternative diagnostic tool for the diagnosis of S. stercoralis infection in immunocompromised patients, considering the possible severity of this helminthiasis in this group of patients.

IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS: USE OF SIX DIFFERENT ANTIGENIC FRACTIONS FROM Strongyloides venezuelensis PARASITIC FEMALES.

SUMMARY The aim of this study was to evaluate six different antigenic fractions from Strongyloides venezuelensis parasitic females for the immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from S. venezuelensis parasitic females were prepared in phosphate-buffered saline (SSF and SMF, respectively), Tris-HCl (TSF and TMF, respectively), and an alkaline buffer (ASF and AMF, respectively). Serum samples obtained from patients with strongyloidiasis or, other parasitic diseases, and healthy individuals were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions SSF, TSF, and ASF showed 85.0%, 75.0%, and 80.0% sensitivity and 93.1%, 93.1%, and 87.5% specificity, respectively. Membrane fractions SMF, TMF, and AMF showed 80.0%, 75.0%, and 85.0% sensitivity, and 95.8%, 90.3%, and 91.7% specificity, respectively. In conclusion, the present results suggest that the fractions obtained from parasitic females, especially the SSF and SMF, could be used as alternative antigen sources in the serodiagnosis of human strongyloidiasis.

Immunofluorescence assay for diagnosis of strongyloidiasis in immunocompromised patients.

Abstract Background: Strongyloides stercoralis is a parasite that causes human strongyloidiasis. The disease ranges from asymptomatic to severe forms, which are often fatal in immunocompromised individuals. Laboratory diagnosis is challenging owing to limitations in the use of conventional parasitological techniques. The present study aimed to evaluate the indirect immunofluorescence assay (IFA) using infective larvae of S. venezuelensis as an antigen for the immunodiagnosis of human strongyloidiasis in immunocompromised patients. Methods: Serum and stool samples from 200 immunocompromised patients (HIV-positive, HTLV-1-positive, and renal, liver, and/or bone marrow transplantation candidates) were used. Stool samples were examined using three parasitological methods: Lutz, Rugai, and culture agar plate. IFA was performed using sections of infective larvae of S. venezuelensis as antigens, and showed 95.4% sensitivity and 95.8% and specificity. Results: Among the 200 patients, 17 (8.5%) were positive for S. stercoralis by at least one parasitological method, and 43 (21.5%) were positive by IFA. Conclusions: IFA can be used as a screening method for the detection of S. stercoralis in immunocompromised patients.

Could mucin 16 and colony-stimulating factor 2-receptor beta possible graft versus host disease biomarkers? Medical hypotheses

Graft versus host disease (GVHD) occurs after bone marrow transplantation and is one of the most important causes of death worldwide. Reviews demonstrated GVHD patients with involvement of various tissues and organs, such as salivary glands. The diagnosis of acute GVHD has been the biopsies and the histopathologic evaluation of tissue from an involved organ. These procedures are invasive. Saliva proteins as possible biomarker for GVHD could facilitate the management and diagnosis accuracy. For support the proposed hypotheses, in this pilot study we collected whole saliva samples from patients with undergoing allogeneic hematopoietic cell transplantation (HCT) and from healthy subjects. Samples were collected prospectively between pre-transplant, thirty days, one hundred and, two hundred days after transplant. The proteomic profile was analyzed using SDS-PAGE and LCMS-ESI-IT-TOF mass spectrometry. The relevant personal data, past medical history were also recorded. The most relevant proteins found exclusively in GVHD patients were: CSF2RB, Protocadherin (Pcdh) Fat 2 precursor, protein capicua homolog isoform CIC-S, MUC16 and RGPD8_HUMAN RANBP2. This study aims to conduct an initial evaluation of the possible presence of such biomarkers in saliva from GVHD patients, and suggested a potential application of proteomics analysis as a alternative method to diagnose GVHD.

Molecular diagnosis of Strongyloides stercoralis among transplant candidates

Strongyloidiasis can occur without any symptoms or as a potentially fatal hyperinfection or disseminated infection, principally in immunosuppressed patients. Our study aimed to evaluate the application of conventional polymerase chain reaction (cPCR) and real‐time PCR (qPCR). Polymerase chain reaction (PCR) and real‐time PCR (qPCR) targeting the 18S rRNA gene for detection of Strongyloides stercoralis infection among transplant candidates were applied in stool samples obtained from 150 transplant candidates, preliminarily analyzed by parasitological methods. S. stercoralis larvae were visualized in 15/150 (10.0%) transplant candidates by parasitological methods. DNA from S. stercoralis was amplified in 26/150 (17.3%) and 49/150 (32.7%) stool samples of transplant candidates, using cPCR and qPCR, respectively. The results suggest that molecular methods, especially qPCR, should be used as an additional tool for diagnostic of S. stercoralis infection among transplant candidates.

Potential immunological markers for diagnosis of human strongyloidiasis using heterologous antigens

Strongyloides venezuelensis is a parasitic nematode of rodents that is frequently used to obtain heterologous antigens for immunological diagnosis of human strongyloidiasis. The aim of this study was to identify antigens from filariform larvae of S. venezuelensis for immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from filariform larvae of S. venezuelensis were obtained in phosphate saline (SS and SM) and in Tris-HCl buffer (TS and TM), and were analysed by Western blotting. Different antigenic components were recognized by IgG antibodies from the sera of strongyloidiasis patients. Highest recognition was observed for a 30-40 kDa mass range present in all antigenic fractions. The band encompassing this mass range was then excised and subjected to mass spectrometry for protein identification. Immunoreactive proteins identified in the soluble fractions corresponded to metabolic enzymes, whereas cytoskeletal proteins and galectins were more abundant in the membrane fractions. These results represent the first approach towards identification of S. venezuelensis antigens for use in immunodiagnostic assays for human strongyloidiasis.

Diagnosis of the strongyloid nematode Strongyloides venezuelensis in experimentally infected rats.

Strongyloides venezuelensis is an intestinal nematode of rats, frequently used as a model for studying human and animal strongyloidiasis. In the present study, we evaluated parasitological, serological and molecular methods for the diagnosis of experimental S. venezuelensis in rats, Rattus norvegicus. Blood and faecal samples were collected and analysed up to 60 days post infection (pi) with adult worm recovery occurring from 5 to 45 days pi. Using an enzyme-linked immunosorbent assay (ELISA), serum levels of IgG antibodies increased up to 28 days pi, thereafter decreasing by day 60 pi. Polymerase chain reaction (PCR) assays detected S. venezuelensis DNA in faecal samples of rats from 5 to 21 days pi. The present study therefore represents the first step towards improving the diagnosis of experimental strongyloidiasis.