Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR)

Abstract

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in Côte d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire (n = 141), Angola (n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world (n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene (ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

“…All the control and naturally infected (Touré et al 1999), again suggesting that the microscopic identification of Loa loa was incorrect. There remains the possibility of cross-contamination, but several measures to avoid it were taken (Rubio et al 2002). Another possibility is that there was a mixed infection in the sample and that the PCR methods were unable to detect both parasites simultaneously, perhaps because the presence of a second filaria inhibits the amplification reaction in the filaria and Loa loa nested PCRs, while the microscopist was able to identify the more prevalent pathogen in the sample.…”

Plasmodium malariae and Plasmodium ovale – the ‘bashful’ malaria parasites

“…These assays enable detection of the four Plasmodium parasite species that infect humans at densities ~100 times lower than the limit of LM detection [47] and have evolved from simple species-specific amplification strategies to multiplex approaches that incorporate semiquantitative assessments [31,[49][50][51][52][53][54][55][56]. We have greatly extended the potential use of PCR diagnosis for a wide range of epidemiological studies through our recent efforts to develop a ligase detection reaction fluorescent microsphere assay (LDR-FMA) [52,57] to evaluate all four malaria parasites of humans in a single-well multiplex format.…”

Vivax malaria presenting with cerebral malaria and convulsions

“…DNA extracted from blood using DNA extraction kit (Qiagen) was used for detection of malaria parasite as well as to rule out mixed infection by nested PCR assay as described by Rubio et al (2002). The results confirmed P. vivax monoinfection (Fig.…”