title: Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?
abstract: The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.
authors.affiliation: Centro de Pesquisas René Rachou, Fiocruz, Belo HorizonteMG, Brasil, Centro de Pesquisas René Rachou - Fiocruz , Belo Horizonte , MG , Brasil.
authors.forename: Daniela Camargos
authors.initials: DC
authors.lastname: Costa
authors.affiliation: Departamento de Bioengenharia, Universidade Federal de São João Del Rei, São João Del ReyMG, Brasil, Departamento de Bioengenharia , Universidade Federal de São João Del Rei , São João Del Rey , MG , Brasil.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Madureira
authors.affiliation: Centro de Pesquisas René Rachou, Fiocruz, Belo HorizonteMG, Brasil, Centro de Pesquisas René Rachou - Fiocruz , Belo Horizonte , MG , Brasil.
authors.forename: Lara Cotta
authors.initials: LC
authors.lastname: Amaral
authors.affiliation: Universidade Federal de Mato Grosso, CuiabáMT, Brasil, Universidade Federal de Mato Grosso , Cuiabá , MT , Brasil.
authors.forename: Bruno Antônio Marinho
authors.initials: BA
authors.lastname: Sanchez
authors.affiliation: Universidade Federal de Mato Grosso, CuiabáMT, Brasil, Universidade Federal de Mato Grosso , Cuiabá , MT , Brasil.
authors.forename: Luciano Teixeira
authors.initials: LT
authors.lastname: Gomes
authors.affiliation: Universidade Federal de Mato Grosso, CuiabáMT, Brasil, Universidade Federal de Mato Grosso , Cuiabá , MT , Brasil.
authors.forename: Cor Jésus Fernandes
authors.initials: CJ
authors.lastname: Fontes
authors.affiliation: Centro de Controle de Zoonoses de Uberlândia, UberlândiaMG, Brasil, Centro de Controle de Zoonoses de Uberlândia , Uberlândia , MG , Brasil.
authors.forename: Jean Ezequiel
authors.initials: JE
authors.lastname: Limongi
authors.affiliation: Centro de Pesquisas René Rachou, Fiocruz, Belo HorizonteMG, Brasil, Centro de Pesquisas René Rachou - Fiocruz , Belo Horizonte , MG , Brasil.
authors.forename: Cristiana Ferreira Alves de
authors.initials: CF
authors.lastname: Brito
authors.affiliation: Centro de Pesquisas René Rachou, Fiocruz, Belo HorizonteMG, Brasil, Centro de Pesquisas René Rachou - Fiocruz , Belo Horizonte , MG , Brasil.
authors.forename: Luzia Helena
authors.initials: LH
authors.lastname: Carvalho
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002353:Carrier State / Q000175:diagnosis / Q000469:parasitology*; D016009:Chi-Square Distribution; D060085:Coinfection / Q000175:diagnosis; D016054:DNA, Protozoan / Q000032:analysis*; D005260:Female; D020459:Genes, rRNA / Q000235:genetics; D006801:Humans; D008288:Malaria / Q000175:diagnosis / Q000469:parasitology*; D008297:Male; D008853:Microscopy; D008875:Middle Aged; D018512:Parasitemia / Q000175:diagnosis / Q000469:parasitology; D010961:Plasmodium / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D016054:DNA, Protozoan