Rafaella Fortini Queiroz Grenfell

Antigenic extracts of Leishmania braziliensis and Leishmania amazonensis associated with saponin partially protects BALB/c mice against Leishmania chagasi infection by suppressing IL-10 and IL-4 production

This study evaluated two vaccine candidates for their effectiveness in protecting BALB/c mice against Leishmania chagasi infection. These immunogenic preparations were composed of Leishmania amazonensis or Leishmania braziliensis antigenic extracts in association with saponin adjuvant. Mice were given three subcutaneous doses of one of these vaccine candidates weekly for three weeks and four weeks later challenged with promastigotes of L. chagasi by intravenous injection. We observed that both vaccine candidates induced a significant reduction in the parasite load of the liver, while the L. amazonensis antigenic extract also stimulated a reduction in spleen parasite load. This protection was associated with a suppression of both interleukin (IL)-10 and IL-4 cytokines by spleen cells in response to L. chagasi antigen. No change was detected in the production of IFN-γ. Our data show that these immunogenic preparations reduce the type 2 immune response leading to the control of parasite replication.

Schistosoma mansoni in a low-prevalence area in Brazil: the importance of additional methods for the diagnosis of hard-to-detect individual carriers by low-cost immunological assays

Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.

New Approaches with Different Types of Circulating Cathodic Antigen for the Diagnosis of Patients with Low Schistosoma mansoni Load

Background Schistosomiasis mansoni is a debilitating and sometimes fatal disease. Accurate diagnosis plays a key role in patient management and infection control. However, currently available parasitological methods are laborious and lack sensitivity. The selection of target antigen candidates has turned out to be a promising tool for the development of more sensitive diagnostic methods. In our previous investigations, the use of crude antigens led to false-positive results. Recently, focus has been given to highly purified Schistosoma mansoni antigens, especially to circulating antigens. Method Thus, our main goal was to test different types of circulating cathodic antigen glycoprotein (CCA), as “crude antigen,” the protein chain of recombinant CCA and two individual peptides. These schistosome proteins/peptides were tested in a new diagnostic method employing immunomagnetic separation based on the improvement of antigen–antibody binding. Principal Findings Use of recombinant CCA as a diagnostic antigen allowed us to develop a diagnostic assay with high sensitivity and specificity with no false-negative results. Interestingly, the “crude antigen” worked as a good marker for control of cure after praziquantel treatment. Conclusions/Significance Our new diagnostic method was superior to enzyme-linked immunosorbent assay in diagnosing low endemicity patients.

Vaccine Self-Assembling Immune Matrix Is a New Delivery Platform That Enhances Immune Responses to Recombinant HBsAg in Mice

ABSTRACT Vaccination remains the most effective public health tool to prevent infectious diseases. Many vaccines are marginally effective and need enhancement for immunocompromised, elderly, and very young populations. To enhance immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create VacSIM (vaccine self-assembling immune matrix), a new delivery method. VacSIM solution can easily be mixed with antigens, organisms, and adjuvants for injection. Postinjection, the peptides self-assemble into hydrated nanofiber gel matrices, forming a depot with antigens and adjuvants in the aqueous phase. We believe the depot provides slow release of immunogens, leading to increased activation of antigen-presenting cells that then drive enhanced immunogenicity. Using recombinant hepatitis B virus surface antigen (rHBsAg) as a model immunogen, we compared VacSIM delivery to delivery in alum or complete Freunds adjuvant (CFA). Delivery of the rHBsAg antigen to mice via VacSIM without adjuvant elicited higher specific IgG responses than when rHBsAg was delivered in alum or CFA. Evaluating IgG subtypes showed a mixed Th1/Th2 type response following immunization with VacSIM, which was driven further toward Th1 with addition of CpG as the adjuvant. Increased specific IgG endpoint titers were observed in both C57BL/6 and BALB/c mice, representative of Th1 and Th2 environments, respectively. Restimulation of splenocytes suggests that VacSIM does not cause an immediate proinflammatory response in the host. Overall, these results suggest that VacSIM, as a new delivery method, has the potential to enhance immunogenicity and efficacy of numerous vaccines.

Biomarkers for schistosomiasis: Towards an integrative view of the search for an effective diagnosis

Human schistosomiasis, caused mainly by Schistosoma mansoni, S. japonicum, and S. hematobium, remains a prevalent and serious parasitic disease worldwide. Although it is a debilitating disease, a lack of sensitive methods for the precise diagnosis of active infection cases is important to prevent morbidity. The optimization of new diagnostic approaches may be accomplished by the selection of specific markers. In that manner, markers can be satisfactorily used for detection of different phases of infection, as acute and chronic phases, pre-patent and post-patent phases and after chemotherapy, improving the efficiency of methods. For that purpose, proteomics and glycomics analyses have been performed in schistosomes, in particular S. mansoni, using powerful high-throughput methodologies. These investigations have not only chartered protein, o-glycan and n-glycan profiles across developmental stages within mammalian host, but are also leading to the characterization of features of the surface tegument, the eggshell and excretory-secretory proteomes of schistosomes.

Immunodiagnostic Methods: What Is Their Role in Areas of Low Endemicity?

Worldwide Schistosomiasis mansoni continues to be a serious public health problem. Over the past decades, control programmes have made remarkable progress in reducing S. mansoni infections to a relatively low level in Brazil and African countries. Endemic regions are currently circumscribed in certain core areas where reinfection and repeated chemotherapy are frequent and, consequently, are related to residents with low parasite load. At present, diagnosis is predominately a key step for final disease control although low endemicity area residents are hardly detected by most of the available assays. In this paper, we review the current status and efforts made aiming at the improvement of diagnostic tools for S. mansoni in low endemicity infections. The establishment of diagnostic assays—simple, affordable, sensitive, and specific for field diagnosis of S. mansoni—is essential and should be given high priority.

Antischistosomal activity of a calcium channel antagonist on schistosomula and adult Schistosoma mansoni worms

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+channels.

Acute schistosomiasis diagnosis: a new tool for the diagnosis of schistosomiasis in a group of travelers recently infected in a new focus of Schistosoma mansoni

INTRODUCTION The diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group. METHODS We describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings. RESULTS ELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg. CONCLUSIONS Our data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.

Improvement of POC-CCA Interpretation by Using Lyophilization of Urine from Patients with Schistosoma mansoni Low Worm Burden: Towards an Elimination of Doubts about the Concept of Trace

Background Accurate diagnostic techniques for schistosomiasis are essential for prevalence determination and identification of positive patients. A point-of-care test for detecting schistosome circulating cathodic antigen (POC-CCA) has been evaluated for its accuracy in different endemic regions. This reagent strip/dipstick based assay has showed high sensitivity for individuals with high or moderate worm burden, but the interpretation of light infections is less clear, especially for trace readings. Methodology/Principal Findings We introduced a urine lyophilization step to the POC-CCA assay to improve its sensitivity and clarify the interpretation of traces. We evaluated POC-CCA sensitivity and specificity within individuals with low parasite burdens in a Brazilian endemic area where a high number of traces were detected. Patients that were positive for other helminths were also evaluated for cross reactions. In all cases, a combined parasitological diagnosis using Kato-Katz (24 slides) and Saline Gradient (1 g of feces) were used as reference. At baseline, diagnosis by POC-CCA (1–2 cassettes) showed 6% sensitivity, inaccurately predicting a low prevalence of Schistosoma mansoni infections (2 POC-CCA positives/32 egg positives). After urine lyophilization, the sensitivity was increased significantly (p < 0.05). Prevalence rates changed from 2% to 32% (27 POC-CCA positives/32 egg positives), equivalent to parasitological techniques. Most of the trace readings changed to positive after lyophilization while some negatives turned into traces. Cross reaction analysis confirmed the specificity of POC-CCA. Conclusions/Significance Trace readings cannot be primarily defined as positive or negative cases. It is critical to verify case-by-case by concentrating urine 10 fold by lyophilization for the diagnosis. Following lyophilization, persistent trace readings should be read as negatives. No trained technician is needed and cost is restricted to the cost of a lyophilizer and the electricity to run it.

Newly Established Monoclonal Antibody Diagnostic Assays for Schistosoma mansoni Direct Detection in Areas of Low Endemicity

Background Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity. Methodology/Principal Findings We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure. Conclusions/Significance The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.