Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

Gama, Bianca Ervatti; Silva-Pires, Felipe do Espirito Santo; Lopes, Mauro N'Kruman R; Cardoso, Maria Angélica; Britto, Constança; Torres, Kátia Luz; Lima, Leila de Mendonça; Souza, José Maria de; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

doi:10.1016/j.exppara.2007.02.011

Cited by 63 publications

(70 citation statements)

References 22 publications

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“…Mais recentemente, a técnica de PCR em tempo real mostrou poder contribuir não apenas para diminuir as chances de contaminação das amostras, mas também para diminuir os custos e o tempo para a obtenção dos resultados 9 . A alta co-positividade da PCR com a gota espessa na detecção de infecção malárica reforça sua utilidade em estudos epidemiológicos de prevalência, estudos de eficácia terapêutica e vacinal, detecção de portadores assintomáticos de plasmódio e na avaliação de novos métodos diagnósticos para malária 10 15 21 .…”

Section: Discussionunclassified

Diagnóstico molecular da malária em uma unidade de atenção terciária na Amazônia Brasileira

Costa

Vieira

Ferreira

et al. 2008

Rev. Soc. Bras. Med. Trop.

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O exame de rotina para o diagnóstico da malária continua sendo a gota espessa, apesar da comprovada diminuição da sensibilidade e especificidade em situações de densidade parasitária baixa e infecções mistas. A reação em cadeia da polimerase vem sendo cada vez mais utilizada para a detecção molecular e identificação das espécies de plasmódio, por apresentar maior sensibilidade e especificidade. Foi realizada a nested-PCR em amostras de sangue total de 344 pacientes com síndrome febril aguda que se apresentaram para o diagnóstico de malária, em uma unidade terciária de saúde, em Manaus (Amazonas). Nenhum caso de malária por Plasmodium malariae foi diagnosticado à gota espessa ou PCR. Observou-se co-positividade de 96,7%, co-negatividade de 62,2% e coeficiente kappa de 0,44 entre PCR e gota espessa para Plasmodium falciparum. Para Plasmodium vivax, co-positividade de 100%, co-negatividade de 78,1% e coeficiente kappa de 0,56. Na detecção da malária mista, co-positividade de 100%, co-negatividade de 84,9% e coeficiente kappa de 0,26. A reação em cadeia da polimerase detectou alto número de infecções mistas nas amostras analisadas, mas seu uso rotineiro no diagnóstico da malária merece ainda ampla discussão.

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Section: Discussionunclassified

Diagnóstico molecular da malária em uma unidade de atenção terciária na Amazônia Brasileira

Costa

Vieira

Ferreira

et al. 2008

Rev. Soc. Bras. Med. Trop.

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“…Because the thick blood film technique requires long periods of observation by highly trained professionals (Kain et al 1998), there is a need for molecular methodologies based on the amplification of DNA (Snounou et al 1993, Kimura et al 1997, Rubio et al 1999. Gama et al (2007) modified a realtime PCR protocol (Lee et al 2002) into a conventional genus-specific PCR that could detect 0.5 parasites/mm³. Because this modified technique was found to have the same threshold of detection as the real-time PCR, this protocol was adopted for our study.…”

Section: Discussionmentioning

confidence: 99%

“…Real-time PCR -The assay used for genus-specific amplification described by Gama et al (2007) was modified by reducing the reagent volume and increasing the number of cycles to obtain a protocol with a lower cost and a greater sensitivity. The genus-specific M60 and M61 primers and the M62 probe targeted the gene encoding the small subunit 18S rRNA of Plasmodium.…”

Section: Methodsmentioning

confidence: 99%

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Lima

Levi

Geraldi³

et al. 2011

Mem. Inst. Oswaldo Cruz

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“…Although the qPCR method requires specific material and is more expensive than mi-Prevalence of P. falciparum and P. vivax in Pará State croscopy and conventional PCR (Berry et al, 2005), it presents advantages of rapidity, lower contamination, and better standardization and can be used for routine testing, particularly for the study of populations in endemic areas in which patients may be asymptomatic (Mens et al, 2007;Boonma et al, 2007;Gama et al, 2007;Veron et al, 2009) and in patients who have negative results on routine methods but are strongly suspected of having malaria (Bourgeois et al, 2010). Thus, qPCR assay based on the detection of mitochondrial cytochrome c oxidase genes may be the method of choice for specific situations, including detection in low-parasitized individuals.…”

Section: Discussionmentioning

confidence: 99%

Prevalence of Plasmodium falciparum and P. vivax in an area of transmission located in Pará State, Brazil, determined by amplification of mtDNA using a real-time PCR assay

Souza

Carvalho²,

Amaral³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. The need for a more sensitive and time-efficient assay for malaria has led to the development of molecular assays involving real-time PCR (qPCR), a procedure that has the potential to detect low levels of parasitemia, identify mixed infections, and allow for precise differentiation of species via melting curve analysis or TaqMan fluorescence-labeled probes. Since the first study published in 2001 at least 17 assays have been developed, most of them using SSUrRNA as the target gene. We used qPCR to detect Plasmodium falciparum and P. vivax by amplification of mtDNA; this technique was evaluated on whole-blood samples from people living in areas of malaria transmission in the Brazilian Amazon region located in the area of inclusion of highway BR-163 (Cuiabá-Santarém) in Pará State: São Luiz do Tapajós, a municipal district of Itaituba (N = 74); Três Boeiras, a municipal district of Trairão (N = 134), and São Raimundo, a municipal district of Aveiro (N = 62). The results from the real-time PCR-based method were compared to conventional microscopy and to an established mtDNA-PCR assay. The qPCR (mtDNA) method was 16-19 times more efficient than the conventional PCR (mtDNA) and microscopy for detecting plasmodial infections.

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Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia

Cited by 63 publications

References 22 publications

Diagnóstico molecular da malária em uma unidade de atenção terciária na Amazônia Brasileira

Diagnóstico molecular da malária em uma unidade de atenção terciária na Amazônia Brasileira

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Prevalence of Plasmodium falciparum and P. vivax in an area of transmission located in Pará State, Brazil, determined by amplification of mtDNA using a real-time PCR assay

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