Implementation of the ANA HEp-2 consensus guidelines in Brazilian clinical laboratories / Implantação das diretrizes dos consensos de FAN HEp-2 nos laboratórios clínicos brasileiros

ABSTRACT Introduction: The detection of autoantibodies in HEp-2 cells represents a relevant tool for the diagnosis of autoimmune diseases, especially rheumatic autoimmune diseases. As a result of the methodological advances, the technique gradually increased the sensitivity, as well as the need for standardization. Objective: To evaluate the implementation of the Brazilian Consensus recommendations for autoantibody determination in HEp-2 cells. Methods: A structured form in a virtual platform was filled in by experts in clinical laboratories that carry out the methodology across the country. The questionnaire addressed the adoption of the Brazilian Consensus guidelines, detailing the technical aspects, quality control, the strategy for reading slides and the release of reports. Results: The study included 53 laboratories responsible for more than 300,000 antinuclear antibody (ANA) tests/month; more than half (58.5%) reported fully adopting the recommendations of the Brazilian Consensus. The majority (83.1%) used the 1:80 for screening dilution, and 75.5% of laboratories, perform education and quality control programs. Only 39.6% reported using more than one kit brand to perform the test, and 32.1% did not report observing all phases of the cell cycle during slide reading. The study also detected some heterogeneity among participants in the identification of patterns. Conclusion: The results confirm the adoption of the Brazilian Consensus recommendations by most of participating laboratories, although with variable extent. There is need for improvement in some aspects, especially those related to the quality control.

RESUMO Introdução: A pesquisa de autoanticorpos em células HEp-2 representa uma relevante ferramenta no auxílio diagnóstico de doenças autoimunes, especialmente as reumáticas. Em virtude dos avanços metodológicos, a técnica aumentou gradativamente a sensibilidade, bem como a necessidade de padronização. Objetivo: Avaliar a implantação das recomendações dos consensos brasileiros de pesquisa de autoanticorpos em células HEp-2. Métodos: Preenchimento de formulário em plataforma virtual direcionada aos laboratórios clínicos que realizam a metodologia. Os participantes responderam a um questionário sobre a adoção das diretrizes dos consensos brasileiros, detalhando os aspectos técnicos, o controle de qualidade, a leitura de lâminas e a emissão de laudos. Resultados: Participaram do estudo 53 laboratórios responsáveis por mais de 300 mil testes de fator antinuclear (FAN)/mês; mais da metade (58,5%) informou adotar integralmente as recomendações dos consensos. A maioria (83,1%) utiliza a diluição 1:80 para triagem, e 75,5% dos laboratórios, programas de educação e controle de qualidade. Apenas 39,6% utilizam mais de uma marca de kit para a realização do teste, e 32,1% não relataram observar todas as fases do ciclo celular na leitura da lâmina. O estudo detectou ainda discreta heterogeneidade entre participantes na identificação de padrões. Conclusão: Os resultados evidenciam a adoção das recomendações dos consensos de forma absoluta pela maioria dos laboratórios participantes, bem como a necessidade de aperfeiçoamento em alguns aspectos relevantes para a qualidade do ensaio.

Leishmania (Viannia) braziliensis amastigotes from patients with mucosal leishmaniasis have increased ability to disseminate and are controlled by nitric oxide at the early stage of murine infection.

Mucosal leishmaniasis (ML) caused by Leishmania (Vianna) braziliensis usually appears after the healing of the primary lesion when amastigotes disseminate from the infection site to the mucosal area. Here, we investigated murine infection with amastigotes obtained from patients with ML or localized cutaneous leishmaniasis (LCL). Amastigotes were used to infect wild type, IFN-γ KO and inducible nitric oxide synthase (iNOS) KO mice. Amastigotes from patients with LCL induced lesions that appeared earlier in IFN-γ KO than parasites from ML. The lesion after infection with ML appeared early in iNOS KO than in IFN-γ KO mice and in iNOS KO mice parasites from ML and LCL cause similar lesions at the initial phase of infection, while parasites from ML induced greater lesions than the ones from LCL at the late phase. A greater number of parasites were observed in spleen of IFN-γ KO and iNOS KO mice infected with amastigotes from patients with ML than those with LCL. Parasites from ML infect a lower percentage of macrophages and are killed independent on IFN-γ and dependent on NO. The data suggest that amastigotes responsible for mucosal lesion in humans develop slowly on the initial phase of infection due to high susceptibility to NO and they have an increased ability to disseminate.

Electroacupuncture at the ST36 acupoint increases interleukin-4 responsiveness in macrophages, generation of alternatively activated macrophages and susceptibility to Leishmania major infection.

BACKGROUND: Electroacupuncture (EA) has been used to treat inflammatory diseases. Alternatively activated macrophages (AAMo) stimulated by cytokines such as interleukin (IL)-4, IL-10 and IL-13 are anti-inflammatory and mildly microbicidal. This study aimed to evaluate whether EA at the Zusanli acupoint (ST36) would change the profile of healthy murine macrophages, particularly the generation of AAMo and susceptibility to Leishmania major infection. METHODS: BALB/c mice were treated with EA (15/30 Hz) at the ST36 acupoint for 20 min/d for 5 d. After the final EA session, the mice were euthanized and their peritoneal cells were harvested and counted for determination of arginase activity, nitric oxide (NO) production and microbicidal activity after culture in the presence or absence of IL-4, interferon-γ (IFNγ) or lipopolysaccharide (LPS) or both IFNγ and LPS. Twelve mice were infected with L. major promastigotes into the footpads after the final EA session and the infection course was monitored. RESULTS: Peritoneal cells freshly obtained from EA-treated mice had similar arginase and microbicidal activities to cells from sham-treated mice. After culture with IL-4, cells from EA-treated mice exhibited significant increases in the arginase activity (sham: 58 ± 11.3 vs. EA: 80.7 ± 4.6%, P = 0.025) and number of parasites/infected cell (sham: 2.5 ± 0.4 vs. EA: 4.3 ± 0.8 cells, P = 0.007). The NO production was lower in cells from EA-treated mice cultured in the presence of a combination of IFNγ and LPS (sham: 31.6 ± 6.5 vs. EA: 22.3 ± 2.1 µM, P = 0.025). The lesion size in mice infected with L. major promastigotes was larger in EA-treated mice (sham: 3.26 ± 0.29 vs. EA: 2.23 ± 0.4 mm, P = 0.039). CONCLUSION: EA at the ST36 acupoint increases IL-4 responsiveness in macrophages, Generation of AAMo and susceptibility to L. major infection.