A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene

Autor(es)

dos Santos, Emilly Henrique ^[1] ; Yamamoto, Lidia ^[1] ; Domingues, Wilson ^[1] ; di Santi, Silvia Maria ^{[2, 1, 3]} ; Kanunfre, Kelly Aparecida ^{[1, 4]} ; Okay, Thelma Suely ^{[5, 1, 6]}

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Inst Med Trop, Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Fac Med, Dept Molestias Infecciosas & Parasitarias, Sao Paulo, SP - Brazil

^[3] Superintendencia Controle Endemias SUCEN, Nucleo Estudos Malaria, Sao Paulo, SP - Brazil

^[4] Univ Sao Paulo, Fac Med, Dept Molestias Infecciosas & Parasitarias, Lab Invest Med, LIM 48, Sao Paulo, SP - Brazil

^[5] Univ Sao Paulo, Fac Med, Dept Pediat, Sao Paulo, SP - Brazil

^[6] Inst Med Trop, Lab Soroepidemiol & Imunobiol, Ave Dr Eneas Carvalho Aguiar 470, Predio 2, BR-05403000 Sao Paulo, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Parasitology International; v. 76, JUN 2020.
• Citações Web of Science: 0

Resumo

Plasmodium malariae mainly causes asymptomatic submicroscopic parasitemia in the endemic Amazon and non-endemic Atlantic Forest, where the number of cases and transmission of malaria through blood transfusion has increased. This study developed a P. malariae/P. brasilianum Real Time PCR (rtPCR) targeting the cytochrome b oxidase (cytb), a highly repetitive gene (20-150 copies/parasite) that should detect more cases than the 18S rRNA (4-8 copies/parasite) gene-based amplification systems. Cytb from human and non-human Plasmodium species (including P. brasilianum) aligned to the only 20 African P. malariae cytb sequences identified polymorphic regions within which we designed P. malariae species-specific primers. Non-human Plasmodium species, related parasites, anemia-causing microorganisms, normal human DNA and 47 blood bank donors samples that were truly negative to malaria accessed rtPCR specificity. Truly positive samples (n = 101) with species identification by semi-nested, nested or TaqMan PCR, and four samples from the Atlantic Forest that were suspected of malaria but three of them had negative genus TaqMan and 18S rRNA nested PCR. The cloned amplification product used in standard curves determined qPCR detection limit (0.5-1 parasite equivalent/mu L). The 10 positive P. malariae samples among truly positives yielded positive rtPCR results and more importantly, rtPCR detected the four samples suspected of malaria from the Atlantic Forest. The rtPCR specificity was 100%, reproducibility 11.1% and repeatability 6.7%. In conclusion, the proposed rtPCR is fast, apparently more sensitive than all 18S rRNA amplification systems for detecting extremely low parasitemia. The rtPCR is also specific to P. malariae/P. brasilianum species. This new molecular tool could be applied to the detection of P. malariae/brasilianum infections with submicroscopic parasitemias in the context of epidemiological studies and blood bank safety programs. (AU)

• Processo FAPESP: 18/20615-3 - Estudo piloto para o desenvolvimento de uma amplificação para a detecção e quantificação de Plasmodium malariae
• Beneficiário: Emilly Henrique dos Santos
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 17/26543-1 - Desenvolvimento de uma multiplex-nested-RT-PCR para a detecção simultânea de cinco RNA vírus associados a infecções congênitas.
• Beneficiário: Thelma Suely Okay
• Modalidade de apoio: Auxílio à Pesquisa - Regular