Application of nested PCR for malaria parasite detection and classification

Abstract

Objective To compare the performance of nested PCR assay with traditional microscopic examination of Giemsa-stained blood films and to discuss the value of nested PCR application for the diagnosis of malaria. Methods Blood samples were collected from affiliated hospital of Shandong Institute of Parasitic Diseases and 17 cities′ CDC reported cases in Shandong province. Finger prick blood samples were taken from each participating individual and used to prepare Giemsa-stained thick and thin blood films for traditional microscopy. Anticoagulant or filter paper blood samples were used to extract DNA from malaria parasites.Primers of plasmodium genus-species were designed on the basis of conserved sequence on 18S small subunit ribosomal RNA(SSU rRNA). Nested PCR was applied to the amplification of target gene segments on 4 kinds of plasmodium infected human. The results of PCR were compared with microscopy and were processed by statistical analysis (chi-square test). The target fragments of PCR amplification were used for species identification and compared with the standard sequence registered in Genbank. Results Among the 259 samples, 249 had been detected positive for malaria parasites by nested PCR. The electrophoresis banding size of 187 samples were 205bp, identified as plasmodium falciparum. The electrophoresis banding size of 45 samples were 120bp, identified as plasmodium vivax.The electrophoresis banding size of 8 samples were 800bp, identified as plasmodium ovale. The electrophoresis banding size of 1 sample was 144bp, identified as plasmodium malariae. No species amplification bands were found in 10 samples, identified as negative. Plasmodium falciparum occupied the highest detection rate of positive samples (75.10%) and plasmodium vivax occupied the second highest detection rate of positive samples (18.07%), in addition, 8 mixed infection were detected (3.21%). PCR products of 5 plasmodium falciparum, 5 plasmodium vivax, 4 plasmodium ovale and 1 plasmodium malariae were sequencing and compared with standard sequence registered in Genbank by blast.The comparison results showed that fragments size and product sequence was in accordance with the reference. Microscopy results showed that 171 samples were plasmodium falciparum, 60 samples were plasmodium vivax, 4 samples were plasmodium ovale, 24 samples were negative. Species proportion of microscopy results were similar to nested PCR, plasmodium falciparum occupied the highest detection rate of positive samples (72.77%), plasmodium vivax occupied the second highest detection rate of positive samples (25.53%). However, there was no plasmodium malariae and mixed infection samples detected by microscopy. Nested PCR were compared with traditional microscopy and the results showed that consistent rate of detected samples between 2 methods was 84.56%.But nested PCR positive rate(96.14%) was significantly higher than traditional microscopy(90.73%), the difference between 2 groups had statistical significance (χ2=12.07, P<0.05). Fourteen samples were detected positive by nested PCR but negative by microscopic examination.Ten samples were detected negative by the both methods.It was worth noting that no samples detected positive by microscopic examination but negative by nested PCR.Furthermore, all 4 kinds of plasmodium species and mixed infection were detected by nested PCR but only 3 kinds of plasmodium species without mixed infection were detected by traditional microscopic examination.The results showed that nested PCR was more objective than the traditional microscopy in malaria parasites′ classification. Conclusions The results indicate that nested PCR used for detection of malaria parasites with high sensitivity and specificity and capable of identifying malaria parasites at the species level when microscopy is equivocal. Nested PCR is also suitable for the mixed infection of malaria parasites and has a broad application prospect in the study of diagnosis and molecular epidemiology of malaria cases. Key words: Malaria; Microscopy; PCR, nested