An algorithm based on molecular protocols to improve the detection of Plasmodium in autochthonous malarial areas in the Atlantic Forest biome

Autor(es)

Farinas, Maria de Lourdes Rego Neves ; Aschar, Mariana ; Costa-Nascimento, Maria de Jesus ; Di Santi, Silvia Maria

• Tipo de documento: Artigo Científico
• Fonte: Revista do Instituto de Medicina Tropical de São Paulo; v. 64, p. 10-pg., 2022-01-01.

Resumo

Malaria is the most important vector-borne disease in the world and a challenge for control programs. In Brazil, 99% of cases occur in the Amazon region. In the extra-Amazonian region, a non-endemic area, epidemiological surveillance focuses on imported malaria and on autochthonous outbreaks, including cases with mild symptoms and low parasitemia acquired in the Atlantic Forest biome. In this scenario, cases are likely to be underreported, since submicroscopic parasitemias are not detected by thick blood smear, considered the reference test. Molecular tests are more sensitive, detecting asymptomatic individuals and mixed infections. The aim of this study was to propose a more efficient alternative to detect asymptomatic individuals living in areas of low malaria endemicity, as they are reservoirs of Plasmodium that maintain transmission locally. In total, 955 blood samples from residents of 16 municipalities with autochthonous malaria outbreaks in the Sao Paulo State were analyzed; 371 samples were collected in EDTA tubes and 584 in filter paper. All samples were initially screened by a genus-specific qPCR targeting ssrRNA genes (limit of detection of 1 parasite/ mu L). Then, positive samples were subjected to a nested PCR targeting ssrRNA and dihydrofolate reductase-thymidylate synthase genes (limit of detection of 10 parasites/mu L) to determine Plasmodium species. The results showed a statistically significant difference (K = 0.049; p < 0.0001) between microscopy positivity (6.9%) and qPCR (22.9%) for EDTA-blood samples. Conversely, for samples collected in filter paper, no statistical difference was observed, with 2.6% positivity by thick blood smear and 3.1% for qPCR (K = 0.036; p = 0.7). Samples positive by qPCR were assayed by a species-specific nested PCR that was in turn positive in 26% of samples (16 P. vivax and 4 P. malariae). The results showed that molecular protocols applied to blood samples from residents in areas with autochthonous transmission of malaria were useful to detect asymptomatic patients who act as a source of transmission. The results showed that the genus-specific qPCR was useful for screening positives, with the subsequent identification of species by nested PCR. Additional improvements, such as standardization of blood plotting on filter paper and a more sensitive protocol for species determination, are essential. The qPCR-based algorithm for screening positives followed by nested PCR will contribute to more efficient control of malaria transmission, offering faster and more sensitive tools to detect asymptomatic Plasmodium reservoirs. (AU)

• Processo FAPESP: 12/18014-5 - Plataforma para diagnóstico de malária aplicada em amostras de doadores de sangue de áreas endêmicas e não endêmicas brasileiras processadas em pool: determinação da frequência de positividade utilizando marcadores moleculares e sorológicos
• Beneficiário: Silvia Maria Fátima Di Santi
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 18/07890-5 - Detecção molecular de Plasmodium em áreas de malária autóctone localizadas em bioma de Mata Atlântica do Estado de São Paulo - o papel das infecções assintomáticas na manutenção da transmissão
• Beneficiário: Maria de Lourdes Rego Neves Farinas
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 11/07380-8 - Marcadores moleculares para análise do polimorfismo genético relacionado à resposta de Plasmodium falciparum aos antimaláricos
• Beneficiário: Silvia Maria Fátima Di Santi
• Modalidade de apoio: Auxílio à Pesquisa - Regular