ABSTRACT
OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.
Subject(s)
Humans , SARS-CoV-2 , COVID-19 , Brazil , Sensitivity and Specificity , Clinical Laboratory Techniques , COVID-19 Testing , Antibodies, Viral , Antigens, ViralABSTRACT
The increased incidence of visceral leishmaniasis (VL) in Brazil is due to a lack of effective disease control measures. In addition to that, no effective treatment exists for canine VL in response to synthetic drugs. Thus, the objective of this study was to evaluate the effect of the essential oils of Coriandrum sativum and Lippia sidoides, and oleoresin from Copaifera reticulata, on Leishmania chagasi promastigotes and amastigotes. We also examined the toxicity of these treatments on the murine monocyte cell line RAW 264.7. To determine the IC50 a MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on promastigotes, and an in situ ELISA assay was conducted on amastigotes. Here, we demonstrate that oleoresin from C. reticulata was effective against both promastigotes (IC50 of 7.88 µg.mL-1) and amastigotes (IC50 of 0.52 µg.mL-1), and neither of the two treatments differed significantly (p > 0.05) from pentamidine (IC50 of 2.149 µg.mL-1) and amphotericin B (IC50 of 9.754 µg.mL-1). Of the three plant oils tested, only oleoresin showed no toxicity toward monocyte, with 78.45% viability after treatment. Inhibition of promastigote and amastigote growth and the lack of cytotoxicity by C. reticulata demonstrate that oleoresin may be a viable option for analyzing the in vivo therapeutic effects of leishmanicidal plants.
O aumento na incidência da Leishmaníase Visceral (LV) no Brasil deve-se à ineficácia das medidas de controle da doença. Além disso, não há tratamento efetivo para LV canina com drogas sintéticas. Assim, o objetivo deste trabalho foi avaliar o efeito dos óleos essenciais de Coriandrum sativum e de Lippia sidoides e do óleo-resina de Copaiferareticulata sobre promastigotas e amastigotas de Leishmania chagasi e analisar o grau de toxicidade sobre células monocíticas murinas RAW 264.7. Para determinar a CI50 sobre promastigotas foi usado teste MTT (brometo de 3-[4,5-dimetil-tiazol-2-il]-2,5-difeniltetrazólio) e sobre amastigotas foi realizado imunoensaio in situ pela técnica de ELISA. Os resultados obtidos comprovaram que o óleo-resina de C. reticulata foi o mais eficaz contra as formas promastigotas (CI50 de 7,88 µg.mL-1) e amastigotas (CI50 de 0,52 µg.mL-1) e em nenhum dos dois testes diferiu do controle pentamidina que obteve CI50 de 2,149 µg.mL-1, no teste sobre promastigotas, e anfotericina B que obteve CI50 de 9,754 µg.mL-1, nos testes com amastigotas (p > 0.05). Quanto à citotoxicidade apenas o óleo-resina não apresentou toxicidade com 78,45% de monócitos viáveis. Os resultados obtidos sobre promastigotas e amastigotas e a ausência de citotoxicidade do óleo-resina de C. reticulata evidenciam que este óleo-resina pode ser viável para a análise de seus efeitos terapêuticos em testes in vivo.