Neutralization of SARS-CoV-2 (lineage B.1.1) by hyperimmune llama (Lama glama) serum in vero cell culture. / Neutralización del virus SARS-CoV-2 (linaje B.1.1) por suero hiperinmune de llama (Lama glama) en cultivo de células Vero.

OBJECTIVE.: To evaluate the serological antibody response of a llama (Lama glama) to SARS-CoV-2 (B.1.1 lineage) immunization and the neutralizing capacity of hyperimmune llama serum against SARS-CoV-2 virus (B.1.1 lineage) in Vero cells. MATERIALS AND METHODS.: A llama was immunized with inactivated SARS-CoV-2 (B.1.1 lineage). Serum samples were analyzed to evaluate the level of antibodies by ELISA, as well as reactivity to SARS-CoV-2 antigens by Western Blot. In addition, viral neutralization in cell cultures was assessed by the Plate Reduction Neutralization Test (PRNT). RESULTS: . Seroreactivity increased in the immunized llama from week 4 onwards. Antibody titers were the highest after the seventh immunization booster. Western blot results confirmed the positive ELISA findings, and immune serum antibodies recognized several viral proteins. The neutralization assay (PRNT) showed visible viral neutralization, which was in accordance with the ELISA and Western Blot results. CONCLUSIONS.: The findings suggest that hyperimmune llama serum could constitute a source of therapeutic antibodies against SARS-CoV-2 infections (lineage B.1.1), and should be studied in further research.

OBJETIVO.: Evaluar la respuesta serológica de anticuerpos de una llama (Lama glama) a la inmunización del virus SARS-CoV-2 (linaje B.1.1) y la capacidad neutralizante del suero de llama hiperinmune frente al virus SARS-CoV-2 (linaje B.1.1) en células Vero. MATERIALES Y MÉTODOS.: Se inmunizó una llama con el virus SARS-CoV-2 inactivado (Linaje B.1.1) y se analizaron muestras de suero para evaluar el nivel de anticuerpos mediante ELISA, así como la reactividad a antígenos de SARS-CoV-2 mediante Western Blot. Además, se evaluó la neutralización viral en cultivos celulares por la Prueba de Neutralización por Reducción de Placas (PRNT, por sus siglas en inglés). RESULTADOS.: Se observó un aumento en la serorreactividad en la llama inmunizada desde la semana 4 en adelante. Los títulos de anticuerpos fueron más elevados en el séptimo refuerzo de inmunización. Los resultados de Western Blot confirmaron los hallazgos positivos del ELISA, y los anticuerpos del suero inmune reconocieron varias proteínas virales. El ensayo de neutralización (PRNT) mostró una neutralización viral visible, concordante con los resultados de ELISA y Western Blot. CONCLUSIONES.: Los hallazgos sugieren que el suero hiperinmune de llama podría constituir una fuente de anticuerpos terapéuticos contra las infecciones por el virus SARS-CoV-2 (linaje B.1.1) y que deberá ser evaluado en estudios posteriores.

Evaluation of dengue NS1 antigen rapid tests and ELISA kits using clinical samples.

BACKGROUND: Early diagnosis of dengue virus (DENV) infection can improve clinical outcomes by ensuring close follow-up, initiating appropriate supportive therapies and raising awareness to the potential of hemorrhage or shock. Non-structural glycoprotein-1 (NS1) has proven to be a useful biomarker for early diagnosis of dengue. A number of rapid diagnostic tests (RDTs) and enzyme-linked immunosorbent assays (ELISAs) targeting NS1 antigen (Ag) are now commercially available. Here we evaluated these tests using a well-characterized panel of clinical samples to determine their effectiveness for early diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: Retrospective samples from South America were used to evaluate the following tests: (i) "Dengue NS1 Ag STRIP" and (ii) "Platelia Dengue NS1 Ag ELISA" (Bio-Rad, France), (iii) "Dengue NS1 Detect Rapid Test (1st Generation)" and (iv) "DENV Detect NS1 ELISA" (InBios International, United States), (v) "Panbio Dengue Early Rapid (1st generation)" (vi) "Panbio Dengue Early ELISA (2nd generation)" and (vii) "SD Bioline Dengue NS1 Ag Rapid Test" (Alere, United States). Overall, the sensitivity of the RDTs ranged from 71.9%-79.1% while the sensitivity of the ELISAs varied between 85.6-95.9%, using virus isolation as the reference method. Most tests had lower sensitivity for DENV-4 relative to the other three serotypes, were less sensitive in detecting secondary infections, and appeared to be most sensitive on Day 3-4 post symptom onset. The specificity of all evaluated tests ranged from 95%-100%. CONCLUSIONS: ELISAs had greater overall sensitivity than RDTs. In conjunction with other parameters, the performance data can help determine which dengue diagnostics should be used during the first few days of illness, when the patients are most likely to present to a clinic seeking care.

First international external quality assessment study on molecular and serological methods for yellow fever diagnosis.

OBJECTIVE: We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. STUDY DESIGN: For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. RESULTS: Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. CONCLUSION: This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.