Immunoinformatics and Pepscan strategies on the path of a peptide-based serological diagnosis of COVID19.

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

Schistosoma mansoni Adult Worm Protective and Diagnostic Proteins in n-Butanol Extracts Revealed by Proteomic Analysis.

The S. mansoni adult worm n-butanol extract (Sm-AWBE) has been previously shown to contain specific S. mansoni antigens that have been used for immunodiagnosis of schistosomiasis in solid phase alkaline phosphatase immunoassay (APIA) and western blot (WB) analyses. Sm-AWBE was also used in immunoprotection studies against a fatal live-cercariae challenge in experimental mouse vaccination (~43% protection). The Sm-AWBE fraction was prepared by mixing adult worm membranous suspensions with aqueous-saturated n-butanol, centrifuging and recovering n-butanol-resistant proteins in the aqueous phase. Here we report a preliminary identification of Sm-AWBE protein components as revealed from a qualitative proteomic study after processing Sm-AWBE by 1D-gel electrophoresis, in-gel and in-solution tryptic digestions, and mass spectrometry analyses. We identified 33 proteins in Sm-AWBE, all previously known S. mansoni proteins and antigens; among them, immunomodulatory proteins and proteins mostly involved in host-parasite interactions. About 81.8% of the identified Sm-AWBE proteins are antigenic. STRING analysis showed a set of Sm-AWBE proteins configuring a small network of interactive proteins and a group of proteins without interactions. Functional groups of proteins included muscle contraction, antioxidant, GPI-anchored phosphoesterases, regulatory 14-3-3, various enzymes and stress proteins. The results widen the possibilities to design novel antigen combinations for better diagnostic and immunoprotective strategies for schistosomiasis control.

In silico modeling and structural analysis of asparaginyl endopeptidase of schistosoma mansoni (Sm32): Immunological and drug target implications.

Asparaginyl endopeptidase (AE) of Schistosoma mansoni (Sm32), also known as legumain, is a cysteine protease indirectly involved in the digestion of hemoglobin of Schistosoma sp. in the gastrodermis, being a vaccine candidate against this trematode and a potential drug target. This study presents a model for the three-dimensional structure of Sm32 determined by means of homology modeling and a molecular dynamics simulation with explicit solvent refinement. The structure proved to be consistent with other AEs of known crystal structures described in their proenzyme form, revealing a catalytic domain that has a caspase-like overall structure and a C-terminal prodomain that adopts a death-domain-like architecture. We identified amino acid mutations in the ßIV strand, differences in the active site and in the surface electrostatic potentials between Sm32 and its homologous proteins of mouse and human. Additionally, amino acid changes in the activation peptide (AP) of the S. mansoni protein were determined. Our results strongly suggest that Sm32 can be exploited as a potential target for drug design and for the development of biomarkers used in diagnosis and in novel vaccines for the control of parasitic infection, opening the perspective of medicinal chemistry developments.

Use of Synthetic Peptides and Multiple Antigen Blot Assay in the Immunodiagnosis of Hepatitis C Virus Infection.

Acute hepatitis C virus (HCV) infection is usually asymptomatic, therefore, early diagnosis is rare. It may remain undiagnosed in individuals who progress to chronic infection, often until serious liver damage has developed. To incorporate the diagnosis of this viral disease in a multiple-diagnostic assay, we first analyzed by immunoinformatics the HCV subtype 1a polyprotein (specifically Core, E2, NS3, NS5A proteins) to select antigenic peptides to be tested initially by the Pepscan technique. Next, we performed the immunodiagnosis of HCV infection, using the Multiple Antigen Blot Assay (MABA). In 22 patients' sera included in this study, a 20-mer linear peptide belonging to the N-terminus of the worldwide conserved Core protein showed 100% sensitivity and specificity; other sequences showed different levels of antibody recognition. The use of MABA in combination with synthetic peptides as a source of multiple, specific, and nonexpensive antigens for other infectious diseases could represent a rapid, integrated, and inexpensive diagnostic methodology.

Orally-transmitted Chagas disease: Epidemiological, clinical, serological and molecular outcomes of a school microepidemic in Chichiriviche de la Costa, Venezuela.

Oral transmission of Trypanosoma cruzi is a frequent cause of acute Chagas disease (ChD). In the present cross-sectional study, we report the epidemiological, clinical, serological and molecular outcomes of the second largest outbreak of oral ChD described in the literature. It occurred in March 2009 in Chichiriviche de la Costa, a rural seashore community at the central littoral in Venezuela. The vehicle was an artisanal guava juice prepared at the local school and Panstrongylus geniculatus was the vector involved. TcI genotype was isolated from patients and vector; some showed a mixture of haplotypes. Using molecular markers, parasitic loads were high. Eighty-nine cases were diagnosed, the majority (87.5%) in school children 6-15 years of age. Frequency of symptomatic patients was high (89.9%) with long-standing fever in 87.5%; 82.3% had pericardial effusion detected by echocardiogram and 41% had EKG abnormalities. Three children, a pregnant woman and her stillborn child died (5.6% mortality). The community was addressed by simultaneous determination of specific IgG and IgM, confirmed with indirect hemagglutination and lytic antibodies. Determination of IgG and IgA in saliva had low sensitivity. No individual parasitological or serological technique diagnosed 100% of cases. Culture and PCR detected T. cruzi in 95.5% of examined individuals. Based on the increasing incidence of oral acute cases of ChD, it appears that food is becoming one of the most important modes of transmission in the Amazon, Caribbean and Andes regions of America.

Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease.

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease

Orally transmitted Chagas disease (ChD), which is a well-known entity in the Brazilian Amazon Region, was first documented in Venezuela in December 2007, when 103 people attending an urban public school in Caracas became infected by ingesting juice that was contaminated with Trypanosoma cruzi. The infection occurred 45-50 days prior to the initiation of the sampling performed in the current study. Parasitological methods were used to diagnose the first nine symptomatic patients; T. cruzi was found in all of them. However, because this outbreak was managed as a sudden emergency during Christmas time, we needed to rapidly evaluate 1,000 people at risk, so we decided to use conventional serology to detect specific IgM and IgG antibodies via ELISA as well as indirect haemagglutination, which produced positive test results for 9.1%, 11.9% and 9.9% of the individuals tested, respectively. In other more restricted patient groups, polymerase chain reaction (PCR) provided more sensitive results (80.4%) than blood cultures (16.2%) and animal inoculations (11.6%). Although the classical diagnosis of acute ChD is mainly based on parasitological findings, highly sensitive and specific serological techniques can provide rapid results during large and severe outbreaks, as described herein. The use of these serological techniques allows prompt treatment of all individuals suspected of being infected, resulting in reduced rates of morbidity and mortality.

A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate.

Understanding the mode of Schistosoma mansoni larval invasion and the mechanism of immune evasion utilized by larvae and adult worms is essential for a rational development of vaccines or drugs to prevent or cure the disease. This parasite has a very complex molecular organization in all parasite stages, and identifying the major parasite proteins would give clues to schistosome metabolism and to the interaction of the parasite with the host immune system. Our goal was the evaluation of the protein parasite repertoire using a proteomic approach, and the characterization of protein extracts from two different parasite stages of a Venezuelan isolate, such as cercariae and adult worms, previously performed by other authors in some other strains. A comparison among authors was made. Besides, we aimed to identify different isoforms of one of the vaccine candidates, the gluthation-S-transferase protein (Sm28GST), by 2D SDS-PAGE and mass spectrometry, and to achieve its immunologic detection using sera from rabbits immunized with synthetic peptides derived from the Sm28GST protein. These techniques allowed the identification of some of the target molecules of the protective immune response that are being evaluated as potential members of a multi-component and multi-stage anti-S. mansoni vaccine and to clarify if the selected peptides induce antibodies that are able to recognize different isoforms of the Sm28GST.

A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate / Aproximación al análisis proteómico e inmunológico de extractos proteicos de cercaria y verme adulto de Schistosoma mansoni y detección de uno de los candidatos a vacuna, Sm28GST, de un aislado venezolano

Understanding the mode of Schistosoma mansoni larval invasion and the mechanism of immune evasion utilized by larvae and adult worms is essential for a rational development of vaccines or drugs to prevent or cure the disease. This parasite has a very complex molecular organization in all parasite stages, and identifying the major parasite proteins would give clues to schistosome metabolism and to the interaction of the parasite with the host immune system. Our goal was the evaluation of the protein parasite repertoire using a proteomic approach, and the characterization of protein extracts from two different parasite stages of a Venezuelan isolate, such as cercariae and adult worms, previously performed by other authors in some other strains. A comparison among authors was made. Besides, we aimed to identify different isoforms of one of the vaccine candidates, the gluthation-S-transferase protein (Sm28GST), by 2D SDS-PAGE and mass spectrometry, and to achieve its immunologic detection using sera from rabbits immunized with synthetic peptides derived from the Sm28GST protein. These techniques allowed the identification of some of the target molecules of the protective immune response that are being evaluated as potential members of a multi-component and multi-stage anti-S. mansoni vaccine and to clarify if the selected peptides induce antibodies that are able to recognize different isoforms of the Sm28GST.

Es esencial comprender la forma como las larvas de Schistosoma mansoni invaden y los mecanismos de evasión inmune utilizados por larvas y adultos, para el desarrollo racional de vacunas o drogas para prevenir o curar la esquistosomiasis. Este parásito tiene una organización molecular muy compleja en todos sus estadíos, por lo que la identificación de las proteínas más importantes es clave para investigar el metabolismo del esquistosoma y la interacción del parásito con el sistema inmune del hospedero. El objetivo de este trabajo fue evaluar el repertorio proteico del parásito utilizando una aproximación proteómica y la caracterización de extractos proteicos de dos estadios parasitarios diferentes de un aislado venezolano, como la cercaria y el verme adulto, previamente realizado por otros autores en otras aislados. Se realizó una comparación entre autores. Además, se identificaron diferentes isoformas de uno de los candidatos a vacuna, la glutation S transferasa (Sm28GST) por 2D SDS-PAGE y espectrometría de masas y se logró su detección inmunológica, usando sueros de conejos inmunizados con péptidos sintéticos derivados de la proteína Sm28GST. Estas técnicas permitieron identificar algunas de las moléculas blanco de la respuesta inmune protectora que están siendo evaluados como miembros potenciales de una vacuna multi-estadio y multi-componente y aclarar si los péptidos seleccionados indujeron anticuerpos capaces de reconocer diferentes isoformas de la Sm28GST.

The multiple antigen blot assay: a simple, versatile and multipurpose immunoenzymatic technique.

This technique is based on the sensitization of different antigens in a single nitrocellulose strip, which react when exposed to an immune serum and thereafter with the appropriate peroxidase conjugate and the corresponding substrate. Signals in those reactive spots are recorded as black squares in a negative photographic film, using a chemiluminiscent substrate or as blue spots when a precipitable colorimetric substrate is used. This technique allows the simultaneous demonstration of antigenicity of different antigens (peptides, recombinant molecules, and crude preparations), with a high sensitivity and specificity. Its major value is based on its versatility, since it is possible to rapidly evaluate and to compare various antigenic preparations and to use it for diagnosis of different infectious, allergic and autoimmune diseases, at a low cost.

Low transmission areas of schistosomiasis in Venezuela: consequences on the diagnosis, treatment, and control

Schistosomiasis low transmission areas as Venezuela, can be defined as those where the vector exists, the prevalence of active cases is under 25 percent, individuals with mild intensity of infection predominate and are mostly asymptomatic. These areas are the consequence of effective control programs, however, "silent" epidemiological places are difficult to trace, avoiding the opportune diagnosis and treatment of infected persons. Clinic and abdominal ultrasound have not shown to discriminate infected from uninfected persons in areas where besides Schistosoma mansoni, intestinal parasites are the rule. Under these conditions, serology remains as a very valuable diagnostic tool, since it gives a closer approximation to the true prevalence. In this sense, circumoval precipitin test, ELISA-SEA with sodium metaperiodate, and alkaline phosphatase immunoassay joined to coprology allow the identification of the "schistosomiasis cases". In relation to public health, schistosomiasis has been underestimated by the sanitary authorities and the investment on its control is being transferred to other diseases of major social and political relevance neglecting sanitary efforts and allowing growth of snail population. Some strategies of diagnosis and control should be done before schistosomiasis reemergence occurs in low transmission areas.

Low transmission areas of schistosomiasis in Venezuela: consequences on the diagnosis, treatment, and control.

Schistosomiasis low transmission areas as Venezuela, can be defined as those where the vector exists, the prevalence of active cases is under 25%, individuals with mild intensity of infection predominate and are mostly asymptomatic. These areas are the consequence of effective control programs, however, "silent" epidemiological places are difficult to trace, avoiding the opportune diagnosis and treatment of infected persons. Clinic and abdominal ultrasound have not shown to discriminate infected from uninfected persons in areas where besides Schistosoma mansoni, intestinal parasites are the rule. Under these conditions, serology remains as a very valuable diagnostic tool, since it gives a closer approximation to the true prevalence. In this sense, circumoval precipitin test, ELISA-SEA with sodium metaperiodate, and alkaline phosphatase immunoassay joined to coprology allow the identification of the "schistosomiasis cases". In relation to public health, schistosomiasis has been underestimated by the sanitary authorities and the investment on its control is being transferred to other diseases of major social and political relevance neglecting sanitary efforts and allowing growth of snail population. Some strategies of diagnosis and control should be done before schistosomiasis reemergence occurs in low transmission areas.

Prevalencia de las parasitosis intestinales y esquistosomosis en comunidades de área centro norte de Venezuela / Prevalence of the intestinal parasite and schistosomiasis in communinities of the center-north area of Venezuela

Con el fin de evaluar las prevalencias de parásitos intestinales y los casos de esquistosomosis, se estudiaron seis comunidades ertenecientes a tres Estados, Aragua, Carabobo y Vargas, del centronorte de Venezuela. Se evaluaron 1603 personas por varios exámenes coprológicos para el diagnóstico de helmintos y protozoarios y 2175 personas para descartar esquistosomosis, en 1532 de los cuales se practicaron exámenes coprológicos y serológicos simultáneamente. Los helmmintos más frecuentes en las comunidades estudiadas fueron: Trichuris trichiura (29,63 por ciento) y Ascaris lumbricoides (12,91 por ciento). Entre los protosuarios destacaron, Entamoeba coli (12,41 por ciento), Blastocystis hominis (12,10 por ciento), Endolimax nana (9,85) y Giardia duodenalis (9,35 por ciento). Se diagnosticaron 251 casos de esquistosomosis, resultando una prevalencia de 11,5 por ciento. El rango de eliminación de huevos de Schistosoma mansoni fue de 24 a 1928 huevos/gramo de heces, siendo el 71 por ciento de las personas con leve intensidad de infección. En dos localidades del Estado Carabobo, Belén y Barrio José Leonardo Chirinos, se encontraron casos con eliminación de huevos en las heces en personas menores de 30 años. Los estudios de prevalencias de parasitosis intestinales y los trabajos de campo basados en coprología y serología para esquistosomosis, deben ser la base del Programa de Control para la vigilancia epidemiológica de estas entidades

Péptidos sintéticos para el diagnóstico y vigilancia epidemiológica en esquistosomosis mansoni / Synthetics peptides for the diagnose and vigilance epidemiologic in schitosomiasis mansoni

El diagnóstico de laboratorio de la esquistosomosis es uno de los problemas por resolver, especialmente en áreas de bajas cargas parasitarias, como Venezuela, además de ser también una de las limitantes la evaluación de las nuevas vacunas contra esta parasitosis. Por esta razón, nos planteamos desarrollar métodos de inmunodiagnóstico basados en la detección de anticuerpos y de antígenos circulantes utilizando la estrategia de la síntesis química, para elaborar péptidos derivados de la captesina B (Sm31) y la asparraginil endopeptidasa (Sm32). Tres de los péptidos de la Sm31 fueron reconocidos al menos por el 49 por ciento de los pacientes y de ellos el IMT-180, lo fue por el 86 por ciento, exhibiendo además una alta especificidad (100 por cierto). Dos de los péptidos de la Sm31 y 3 de la Sm32 indujeron en conejos, el reconocimiento de las respectivas moléculas, inclusive en cortes histológicos. Resultados preliminares de inmunoensayos de captura anti-Sm32 han revelado una baja sensibilidad

Desprendimiento de calor en los sistemas rotatorios Profile(R), Kavo 29 CH(R) y la técnica manual mecánica crown-down: estudio in vitro / Heat release in the Profile(R) and Kavo 29 CH(R) rotary systems and in the crown-down manual mechanical technique: an in vitro study

Propósito: el propósito del presente estudio in vitro fue medir el aumento de la temperatura en la superficie externa radicular de premolares extraídos, a 1 y 6 mm del ápice, ocurrido durante la preparación biomecánica del conducto radicular, utilizando termocuplas, con dos sistemas rotatorios (Profile(R), Kavo 29 CH(R)) y la técnica mecánica manual crown-down. Métodos: 78 especímenes se decoronaron a 16 mm y se montaron en una olla Hanau(R), simulando el medio bucal, a una temperatura constante de 37§C. Cada instrumento fue utilizado 30 segundos dentro del conducto y los cambios de temperatura se midieron empleando dos termocuplas tipo K adosadas a la cara mesial de los dientes a 1 y 6 mm del ápice. Resultados: el mayor desprendimiento de calor fue obtenido por la técnica manual mecánica crown-down, seguido por el sistema Kavo 29CH(R). El menor desprendimiento de calor se obtuvo empleando el sistema Profile(R). Para los tres sistemas, el mayor desprendimiento de calor se obtuvo con los dos primeros instrumentos y el incremento no fue mayor a 5§C. Conclusiones: los resultados de esta investigación sugieren que el aumento de temperatura en la superficie externa radicular no tiene efectos sobre los tejidos adyacentes al diente