Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

• Published in: Memórias do Instituto Oswaldo Cruz Vol. 109; no. 1; pp. 21 - 28
• Main Authors: Costa, Daniela Camargos, Madureira, Ana Paula, Amaral, Lara Cotta, Sanchez, Bruno Antônio Marinho, Gomes, Luciano Teixeira, Fontes, Cor Jésus Fernandes, Limongi, Jean Ezequiel, Brito, Cristiana Ferreira Alves de, Carvalho, Luzia Helena
• Format: Journal Article
• Language: English
• Published: Brazil Instituto Oswaldo Cruz, Ministério da Saúde 01.02.2014; Fundação Oswaldo Cruz (FIOCRUZ)
• Subjects: Adolescent; Adult; Aged; Aged, 80 and over; Carrier State - diagnosis; Carrier State - parasitology; Chi-Square Distribution; Coinfection - diagnosis; DNA, Protozoan - analysis; Female; Genes, rRNA - genetics; Humans; malaria; Malaria - diagnosis; Malaria - parasitology; Male; Microscopy; Middle Aged; Parasitemia - diagnosis; Parasitemia - parasitology; PCR; Plasmodium - classification; Plasmodium - genetics; Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - methods; reproducibility; Reproducibility of Results; Sensitivity and Specificity; submicroscopic infection; Young Adult
• ISSN: 0074-0276; 1678-8060; 1678-8060; 0074-0276
• DOI: 10.1590/0074-0276140102

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.