Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity.

Optimización en la recuperación de inmunoglobulinas, como molécula entera y fragmento F(ab´)2, en la producción de antivenenos / Optimization in the immunoglobulins recovery, as whole molecule and F(ab´)2 fragment, in the antivenim production

En la actualidad se utilizan, principalmente, dos métodos de purificación de anticuerpos a partir de plasmas equinos hiperinmunes para la producción de antivenenos a nivel industrial, obteniéndose preparaciones enriquecidas en moléculas de inmunoglobulinas G ó fragmentos F(ab´)2. Con ambos métodos, luego de la precipitación, se observa una importante pérdida de capacidad neutralizante en comparación con la capacidad neutralizante de los plasmas de partida. En este trabajo, se realizó el fraccionamiento de plasmas equinos hiperinmunes utilizando ácido caprílico con y sin digestión enzimática con pepsina. El objetivo del trabajo fue dar a conocer la proporción de recuperación de la capacidad neutralizante luego del fraccionamiento; resultando ésta menor cuando el plasma se trató enzimáticamente. Adicionalmente, se propuso establecer cuál sería la etapa responsable de la diferencia en la recuperación de anticuerpos entre una metodología y otra. Cuando se purificaron las inmunoglobulinas enteras, se recuperó aproximadamente un 53% de la capacidad neutralizante mientras que cuando la muestra se purificó luego de ser tratada enzimáticamente, se obtuvo alrededor del 30% de esa actividad. Una relación de similar magnitud se verifica en la recuperación de la masa de proteínas solubles luego de remover los contaminantes, entre una metodología y otra. La insolubilización del fragmento Fc generado durante la digestión sería el responsable de esa pérdida adicional de proteína y capacidad neutralizante.

Today two methods are mainly used for the purification of antibodies from hyperimmune equine plasma at industrial level obtaining enriched preparations of immunoglobulin G (IgG) molecules or F(ab´)2 fragments. In both methods, after the precipitation, an important loss in the neutralizing capability was observed compared to the one of the original plasma. In this work, we performed the fractionation of hyperimmune equine plasma using caprylic acid, with and without enzymatic digestion with pepsin. The aim was to explain the percentage of recovery of the neutralizing capability after the fractionation; which resulted minor when the plasma was enzymatically treated. Additionally, we intended to establish which stage, in the purification process, was the responsible for the difference in the antibody recovery between one methodology and the other. When entire immunoglobulins were purified, approximately 53% of the neutralizing capacity was recovered, but when the sample was purified after the enzymatic treatment, around the 30% of the activity was obtained. A ratio of similar magnitude is verified on the recovery of the soluble protein mass after the removal of contaminants, between the two methods. The insolubilization of the fragment Fc generated during digestion would be responsible for the additional loss of protein and neutralizing capacity.

O papel de imunoglobulinas na nefropatia da leptospirose em suínos / The role of immunoglobulins in nephropathy of leptospirosis in pigs

A leptospirose é uma antropozoonose endêmica em todo o mundo, que afeta o homem e várias espécies de animais domésticos e silvestres. No início da infecção há produção de IgM para o controle da infecção e após alguns dias, IgG são produzidas e provocam lise das leptospiras circulantes. Objetivou-se neste estudo identificar depósitos de antígeno de leptospiras e imunoglobulinas no tecido renal, para avaliar o papel de imunoglobulinas na patogênese da nefropatia da leptospirose em suínos. Foram colhidas 139 amostras de sangue e rim de suínos das cidades de Teresina/PI e Timon/MA, que foram avaliadas pela SAM, imunoistoquímica e PCR. Nefrite intersticial, fibrose, vasculite, tumefação do tufo glomerular e hipercelularidade difusa foram as principais alterações histopatológicas encontradas. A imunoistoquímica detectou antígeno de leptospira em 60 suínos. Depósitos de IgG, IgM e IgA foram observados no endotélio de capilares glomerulares, dos capilares intertubulares e na cápsula de Bowman, com marcação focal, difusa, global e segmentar. A deposição de IgM e IgA foi significantemente maior nos suínos infectados. Estranhamente depósitos de IgG foi significantemente maior nos suínos não infectados, onde não havia presença de antígeno de leptospiras e nem lesão túbulo-intersticial. Concluímos que antígeno de leptospiras no rim de suínos está relacionado a depósitos de IgM e IgA mas não a depósitos de IgG.

Leptospirosis is an endemic worldwide anthropozoonosis, affecting humans and several species of domestic and wild animals. At the beginning of infection is the production of IgM to control the infection and after a few days, IgG is produced and cause lysis of circulating leptospires. The objective of this study was to identify deposits of immunoglobulins and antigens of leptospires in kidney tissue, to assess the role of immunoglobulins in the pathogenesis of leptospirosis nephropathy in pigs. We collected 139 blood samples and kidney of pigs from the cities of Timon/MA and Teresina/PI, to be evaluated by SAM, immunohistochemistry and PCR. Interstitial nephritis, fibrosis, vasculitis; swollen glomeruli hypercellularity and diffuse in a pig were main pathological changes found. Immunohistochemistry leptospira antigen detected in 60 pigs. Deposits of IgG, IgM and IgA were observed in the endothelium of glomerular capillaries, the capillaries intertubulares and the Bowman's capsule, marked focal, diffuse, global and segmental. The deposition of IgM and IgA was significantly higher in infected pigs, strangely deposits of IgG was significantly higher in non-infected pigs, where there was no presence of antigen leptospires nor tubulointerstitial injury. We conclude that Leptospira antigen in porcine kidney relates to deposits of IgM and IgA but not IgG deposits.

Detecção de anticorpos IgG anti-Trypanosoma vivax em bovinos através do teste de Imunofluorescência indireta / Detection of IgG antibodies against Trypanosoma vivax in cattle by indirect immunofluorescence test

Trypanosoma vivax infecta uma grande variedade de animais ungulados selvagens e domésticos, podendo causar grande impacto na produção de ruminantes. Este trabalho teve como objetivo avaliar a detecção de anticorpos IgG anti-Trypanosoma vivax em bovinos provenientes do estado de Pernambuco, Brasil. Para tanto, foram analisadas 2,053 amostras de soro sanguíneo de bovinos provenientes de rebanhos de municípios do estado de Pernambuco, os quais foram analisados através da Reação de Imunofluorescência Indireta. Das amostras testadas 13,93% (286/2.053) foram reagentes para anticorpos IgG anti-Trypanosoma vivax. As freqüências, por mesorregião, variaram de 11,90% a 15,99%. Assim, os dados obtidos permitiram a caracterização do estado de Pernambuco como uma área de instabilidade enzoótica e sugere que o estado Pernambuco é área endêmica para Trypanosoma vivax e este parasito está distribuído por todo o estado.

Trypanosoma vivax infects a wide range of wild and domestic ungulates, causing important losses for the livestock industry. The aim of the present study was to assess the detection of IgG antibodies against T. vivax in cattle from the state of Pernambuco, Brazil. Therefore, we analyzed 2.053 blood serum samples from cattle herds of municipalities in Pernambuco, what was made by Immunofluorescence Assay. The overall seroprevalence of IgG antibodies against T. vivax in cattle was 13.93% (286/2053). The frequencies, by region, varied from 11.90% to 15.99%. Thus, the data obtained allowed to characterize the state of Pernambuco as an area of enzootic instability for T. vivax. The frequency herein reported (i.e., 13.93%) indicates that Pernambuco is an endemic area for T. vivax, this parasite being spread throughout the state.

Detection of human parvovirus B19 in cancer patients using ELISA and real-time PCR.

Purpose: Parvovirus B19 (B19) is associated with a wide range of diseases in humans, whose severity depends on the immunological and haematological status of the host. Objective: To determine the incidence of B19 DNA and specific IgM and IgG frequency among patients suffering from different haematological malignancies and to determine the viral load using real-time PCR. Materials and Methods: A total of 70 patients were included in the study, in addition to a control group consisting of 20 apparently healthy volunteers. B19 DNA quantitative analysis was performed using real-time PCR while screening for IgM and IgG anti-B19 antibodies was performed using ELISA. Results: B19 DNA was detected in 26 patients (36.14%) and 3 controls (15%) using real-time PCR. Anti-parvovirus B19 IgM antibodies were detected in 9 patients (12.6%) and 2 controls (10%). Anti-parvovirus B19 IgG antibodies were detected in 32 patients (45.71%) and 5 controls (25%). The difference between the patient and control groups was found to be statistically non-significant in all of the three tests (P < 0.05). The difference in B19 incidence among patients receiving multiple transfusions and non-transfused patients was also found to be statistically non-significant (P < 0.05). Conclusion: We found a high incidence of B19 infection among patients diagnosed with different types of haematological malignancies. We recommend that all cases of haematological disorders should be examined for specific antibodies and tested for the presence of B19 DNA in serum by PCR technique.

ELISA indireto para detecção de IgG antivírus da doença de Newcastle em soro de codorna / Indirect ELISA for the detection of IgG specific to Newcastle disease virus in quail serum

An indirect ELISA for the detection of japanese quail IgG specific to Newcastle disease virus (NDV) was developed. The secondary anti-quail IgG was produced in Balb/c mice, by inoculating Freund's complete adjuvant emulsified japanese quail-IgG extract. The purification of IgG was achieved using the caprilic acid method. The ELISA was compared to the haemagglutination-inhibition (HI) test for antibodies to NDV. ELISA cut-off point was established through TG-ROC analysis. Total correlation was observed between the ELISA and the HI, being the ELISA efficient in the identification of positive and negative sera, with high sensitivity and specificity (100 percent). These results validate the use of the indirect ELISA as an alternative for the detection of NDV-specific IgG in japanese quail sera, with the advantage of high sensitivity and automation

Activated protein C resistance and lupus anticoagulant activity induced by plasma and purified monospecific human IgG anti-β2-glycoprotein-I antibodies / Resistencia a la proteína C activada y actividad de anticoagulante lúpico inducidas por plasma y por anticuerpos purificados humanos del IgG anti β2-glicoproteína I

ABSTRACT Introduction. We investigated the activated protein C resistance (APCR) phenotype and the lupus anticoagulant (LA), activity induced by anti-β2-glycoprotein-I (anti-β2GP-I) antibodies. Patients and methods. We studied plasma and sera samples from 29 patients with persistently positive anti-β2GP-I: 22 with thrombosis (12 with primary APS, 10 with APS secondary to SLE) and seven without thrombosis (all with SLE); 25 healthy subjects were studied as controls. We detected anticardiolipin antibodies (ACA); IgG (and its subclasses) and IgM anti-β2GP-I, on irradiated and non-irradiated plates by ELISA. APCR was assessed by the activated partial thromboplastin time (APTT)-based assay and by the modified test. The FV Leiden mutation was studied by PCR. LA determination included screening and confirmatory dRVVT. Serum anti-β2GP-I were affinity purified on sepharose columns and their isotype, subclass, and reactivity against various antigens were studied by ELISA. Results. We found that titers of IgG anti-β2GP-I on irradiated plates were higher than on non-irradiated plates (p = 0.002), IgG2 was the predominant subclass. Fifteen patients (13 with thrombosis) had LA and 15 (also 13 with thrombosis) induced the APCR phenotype. Eleven (all with thrombosis) had both. Two patients were heterozygous for the Leiden mutation. Two purified antibodies, monospecific for β2GP-I, induced an in vitro APCR phenotype and LA activity. Conclusions. Our results seem to indicate that the inhibition of the APC anticoagulant function by IgG2 anti-β2GP-I with LA activity may be one of the responsible mechanisms of thrombophilia in patients with APS.

Introducción. Investigamos la resistencia a la proteína C activada (RPCA) y la actividad de anticoagulante lápico (AL), inducidas por anticuerpos anti-β2-glicoproteína-I (anti-β2GP-I). Pacientes y métodos. Estudiamos los plasmas y sueros persistentemente positivos para anti-β2GP-I de 29 pacientes: 22 tuvieron trombosis (12 con síndrome de antifosfolípidos (SAF) primario y 10 con SAF secundario a lupus erítematoso generalizado (LEG)) y siete sin trombosis (todos con LEG). Como controles estudiamos 25 sueros de personas clínicamente sanas. Detectamos anticuerpos anticardiolipina, anti-β2GP-I IgG (y sus subclases) e IgM por ELISA en placas irradiadas y no irradiadas. Evaluamos la RPCA por medio del tiempo parcial de tromboplastina activada y por la prueba modificada. Estudiamos la mutación FV de Leiden por PCR y el anticoagulante lápico con el método de dRVVT screening y confirmatorio. Después de purificar los anti-β2GP-I séricos con una columna de antígeno unido a sefarosa, analizamos por ELISA sus isotipos, subclases y reactividad contra β2GP-I y algunos fosfolípidos. Resultados. Los títulos de anti-β2GP-I IgG fueron más altos en placas irradiadas que en no irradiadas (p = 0.002), predominó la subclase IgG2. Quince plasmas (13 de pacientes con trombosis) tuvieron AL y 15 (13 también de pacientes con trombosis) indujeron el fenotipo de RPCA. Once plasmas (todos de pacientes con trombosis) indujeron ambas actividades. Dos pacientes fueron heterocigotos para la mutación de Leiden. Dos anticuerpos purificados monoespecíficos para β2GP-I indujeron el fenotipo de la RPCA y la actividad de AL in vitro. Conclusiones. Nuestros resultados sugieren que la RPCA, inducida por los anti-β2GP-I que concomitantemente tienen actividad de AL, puede tener implicaciones patogénicas en la trombofílía del SAF.

Inmunoglobulinas en pacientes con actinomicetoma por Nocardia brasiliensis / Immunoglobulins in patients with Nocardia brasiliensis actinomycetoma

Considerando que algunos autores han reportado un aumento en la cantidad de algunas inmunoglobulinas en los pacientes con actinomicetoma, en este trabajo nos propusimos determinar diferencias en la producción de IgG1, IgG2, IgG3, IgG4 e IgM en 25 pacientes con actinomicetoma por Nocardia brasiliensis y 25 personas sanas provenientes de una zona endémica de micetoma. La determinación de inmunoglobulinas se realizó por medio de la técnica de ELISA. Para sensibilizar las placas se emplearon 6 antígenos de N. brasiliensis: un antígeno crudo denominado NB y cinco derivados del mismo (NB2, NB4, NB6, NB8 y NB10) separados por punto isoeléctrico. Los niveles de las cuatro subclases de IgG fueron mayores en los sueros de los pacientes que en el suero de los controles, con una diferencia máxima en IgG3 e IgG4; para esta última subclase, los seis antígenos fueron altamente reactivos. La concentración de IgM fue igual en ambos grupos. Es probable que como ocurre en otras infecciones, en la fisiopatogenia del actinomicetoma influya no sólo el aumento o deficiencia de una clase de inmunoglobulina, sino la relación que existe entre las diferentes subclases.

Considering that some authors have reported an increasing of some immunoglobulins in actinomycetoma patients, in this study we propose to determine differential production of IgG1, IgG2, IgG3, IgG4 and IgGM in 25 patients with actinomycetoma and 25 healthy individuals from a mycetoma endemic area. Immunoglobulins were determined by ELISA technique. To sensibilize the plates, six Nocardia brasiliensis antigens were used: a crude antigen denominated NB and five derivatives (NB2, NB4, NB6, NB8 and NB10) obtained by their isoelectric point. Results showed that all IgG subclasses were higher in the patients’ sera than in control sera, with a maximal difference to IgG3 and IgG4. To the latter subclass, six antigens were highly reactives. IgM levels were similar in both groups. As it occurs in other infections, in the actinomycetoma pathogenesis probably participate the increase or deficiency of a determined immunoglobulin class, as well as the relationship between different subclasses.

Seroprevalencia de anticuerpos anti-caga de helicobacter pylori y su relacion con factores epidemiologicos wn Santa Fe / Seroprevalence of helicobacter pylori anti-caga antibodies and its relationship with epidemiologic factors in Santa Fe

La detección precoz de anticuerpos anti-CagA en adultos jóvenes tendría gran impacto clínico en la prevención del cáncer gástrico. El objetivo del presente trabajo fue determinar en nuestra región la seroprevalencia de los anticuerpos IgG y anti-CagA de Helicobacter pylori, con una técnica no invasiva y fácil de realizar; valorando su relación con diferentes factores de riesgos epidemiológicos. Se incorporaron 435 voluntarios de diferentes centros de salud con edad promedio de 40 años. Mediante una encuesta personalizada se registraron variables demográficas, condiciones socieconómicas, entre otros datos de interés. En el suero de los individuos se determinó la presencia de anticuerpos IgG específicos y anti-CagA del Helicobacter pylori mediante una técnica de enzimoinmunoensayo. La prevalencia de los anticuerpos IgG fue de 52.2%, siendo positivo en 152 (53.7%) mujeres y 75 (49%) varones. Los anticuerpos IgG estaban presentes en el 65.0% de los individuos con síntomas y en el 44.1% de los asintomáticos. La prevalencia de los anticuerpos anti-CagA en la muestra estudiada fue 33.1%, presentándose en el 63.4% de los sujetos seropositivos (IgG), siendo positivo en 96 (33.9%) mujeres y 48 (31.6%) varones. La prevalencia fue 45.4% y 25.7% en los sintomáticos y asintomáticos respectivamente. Se demostró que los anticuerpos IgG están asociados a la edad, zona de residencia, nivel educacional y número de dormitorios por vivienda; mientras que los anticuerpos anti-CagA dependen de la zona de residencia y de la presencia de síntomas. La influencia de la variable predictiva síntomas sobre la presencia de los anticuerpos anti-CagA revelan la importancia selectiva en la corroboración de parámetros clínicos de las enfermedades gastroduodenales asociadas a Helicobacter pylori.

Anticuerpos anti-albúmina bovina (BSA) en niños diabéticos tipo 1 recién diagnosticados y su asociación con lactancia materna y exposición a leche de vaca / Bovine serum albumin (BSA) antibodies in children with recently diagnosed type 1 diabetes with breast feeding and milk exposition

BACKGROUND: Environmental and genetic factors (viruses, toxins and diet) are involved in the aetiology of type 1 diabetes. Among the dietary factors, the role of breast feeding and exposure to cow's milk proteins deserve special attention. AIM: To determine the anti-BSA-IgG levels in type 1 diabetic children and to analyse the possible association with breast feeding duration, exposure to cow's milk and beta pancreatic auto-antibodies. PATIENTS AND METHODS: Blood samples were collected from 161 diabetic children and 144 controls to measure anti-BSA-IgG level, GAD65, IA-2 and ICA autoantibodies. All children answered a questionnaire about dietary habits during infancy. RESULTS: anti-BSA-IgG was positive (using a cut off point of 25.6 ng/ml) in 98 per cent of diabetic children and 0 per cent of the control population. The length of breast feeding or early exposure to cow's milk did not influence the concentration of anti-BSA-IgG. Positive BSA titers did not increase the beta pancreatic reactivity (ICA+, GAD+, IA2+). CONCLUSIONS: Our data confirm the high frequency of anti-BSA-IgG among diabetic children. However, a specific role in the immunological process of type 1 diabetes cannot be attributed to this protein.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2 percent and 91.1 percent, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis

Detection of immunoglobulin G in the lung and liver of hamsters with visceral leishmaniasis

Several organs are affected in visceral leishmaniasis, not only those rich in mononuclear phagocytes. Hypergammaglobulinemia occurs during visceral leishmaniasis; anti-Leishmania antibodies are not primarily important for protection but might be involved in the pathogenesis of tissue lesions. The glomerulonephritis occurring in visceral leishmaniasis has been attributed to immune complex deposition but in other organs the mechanism has not been studied. In the current study we demonstrated the presence of IgG in the lung and liver of hamsters with visceral leishmaniasis. Hamsters were injected intraperitoneally with 2 x 10(7) amastigotes of Leishmania (Leishmania) chagasi and the presence of IgG in the liver and lung was evaluated at 7, 15, 30, 45, 80 and 102 days postinfection (PI) by immunohistochemistry. The parasite burden in the spleen and liver increased progressively during infection. We observed a deposit of IgG from day 7 PI that increased progressively until it reached highest intensity around 30 and 45 days PI, declining at later times. The IgG deposits outlined the sinusoids. In the lung a deposit of IgG was observed in the capillary walls that was moderate at day 7 PI, but the intensity increased remarkably at day 30 PI and declined at later times of infection. No significant C3 deposits were observed in the lung or in the liver. We conclude that IgG may participate in the pathogenesis of the inflammatory process of the lung and liver occurring in experimental visceral leishmaniasis and we discuss an alternative mechanism other than immune complex deposition

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2 percent) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2 percent of the patients with CMV infection were symptomatic

Evaluation of an enzyme immunoassay for hepatitis C virus antibody detection using a recombinant protein derived from the core region of hepatitis C virus genome

This study was undertaken to evaluate an enzyme immunoassay (EIA) for hepatitis C virus antibody detection (anti-HCV), using just one antigen. Anti-HCV EIA was designed to detect anti-HCV IgG using on the solid-phase a recombinant C22 antigen localized at the N-terminal end of the core region of HCV genome, produced by BioMérieux. The serum samples diluted in phosphate buffer saline were added to wells coated with the C22, and incubated. After washings, the wells were loaded with conjugated anti-IgG, and read in a microtiter plate reader (492 nm). Serum samples of 145 patients were divided in two groups: a control group of 39 patients with non-C hepatitis (10 acute hepatitis A, 10 acute hepatitis B, 9 chronic hepatitis B, and 10 autoimmune hepatitis) and a study group consisting of 106 patients with chronic HCV hepatitis. In the study group all patients had anti-HCV detected by a commercially available EIA (Abbott(r)), specific for HCV structural and nonstructural polypeptides, alanine aminotransferase elevation or positive serum HCV-RNA detected by nested-PCR. They also had a liver biopsy compatible with chronic hepatitis. The test was positive in 101 of the 106 (95 percent) sera from patients in the study group and negative in 38 of the 39 (97 percent) sera from those in the control group, showing an accuracy of 96 percent. According to these results, our EIA could be used to detect anti-HCV in the serum of patients infected with hepatitis C virus.

Toxocara seroprevalence in children from a subtropical city in Argentina

Most studies from Argentina have focused on toxocariasis as an environmental problem of big cities, and there are no available data about children infection from small or middle-sized cities. In order to assess the prevalence of anti-Toxocara antibodies in infantile population, 206 children from Resistencia, of both sexes, aged 1-14 years old were studied by Elisa testing with E/S T. canis L2 antigens. Hematological parameters and immunoglobulin levels were determined; five days' stool samples were studied and epidemiological data were obtained by means of a questionnaire to parents. Results showed that 73 per cent of the children had one or more dogs living at home, 57 per cent reported geophagia and 37.9 per cent were positive for Toxocara serology, but there was no significant difference in prevalence neither for boys and girls, nor concerning age. An increased risk of infection was observed in age groups 5-6 and 7-8 for boys, and in age groups 3-4 and 5-6 for girls.

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

High quality human immunoglobulin G purified from Cohn fractions by liquid chromatography

In order to obtain intravenous immunoglobulin G (iv IgG) of high quality from F-I+II+III or F-II+III pastes prepared by the Cohn method, we developed a chromatography process using ion exchange gels, Q-Sepharose FF and CM-Sepharose FF, and Sephacryl S-300 gel filtration. Viral inactivation was performed by incubating the preparation with pepsin at pH 4.0 at 35oC for 18 h. The characteristics of 28 batches produced by us were: yield 4.3 + or - 0.2 g/l plasma, i.e., a recovery of 39.1 + or - 1.8 percent; IgG subclasses distribution: IgG1 = 58.4 percent, IgG2 = 34.8 percent, IgG3 = 4.5 percent and IgG4 = 2.3 percent; IgG size distribution was 98.4 percent monomers, 1.2 percent dimers and 0.4 percent polymers and protein aggregates; anticomplement activity was less than 0.5 CH50/mg IgG, and prekallikrein activator activity (PKA) was less than 5 IU/ml. These characteristics satisfied the requirements of the European Pharmacopoea edition, and the regulations of the Brazilian Health Ministry (M.S. Portaria No. 2, 30/10/1998)

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100 per cent of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100 per cent, specificity of 92 per cent, a positive predictive value of 43 per cent and a negative predictive value of 100 per cent for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.