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Background  Malaria is caused by five  Plasmodium  species and transmitted by anopheline mosquitoes. It occurs in single and mixed infections. Mixed infection easily leads to misdiagnosis. Accurate detection of malaria species is vital. Therefore, the study was conducted to determine the level of mixed infection and misdiagnosis of malaria species in the study area using SYBR Green I-based real time PCR.    Methods  The study was conducted in seven health centres from North Gondar, north-west Ethiopia. The data of all febrile patients, who attended the outpatient department for malaria diagnosis, from October to December 2013, was recorded. Dried blood spots were prepared from 168 positive samples for molecular re-evaluation. Parasite DNA was extracted using a commercial kit and Plasmodium species were re-evaluated with SYBR Green I-based real time PCR to detect mixed infections and misdiagnosed mono-infections.    Results  Among 7343 patients who were diagnosed for malaria in six study sites within the second quarter of the Ethiopian fiscal year (2013) 1802 (24.54%) were positive for malaria parasite. Out of this, 1,216 (67.48%)  Plasmodium falciparum , 553 (30.68%)  Plasmodium vivax  and 33 (1.8%) mixed infections of both species were recorded. The result showed high prevalence of  P. falciparum  and  P. vivax , but very low prevalence of mixed infections. Among 168 samples collected on dried blood spot 7 (4.17%) were  P. vivax , 158 (94.05%) were  P. falciparum  and 3 (1.80%) were mixed infections of both species. After re-evaluation 10 (5.95%)  P. vivax,  112 (66.67%)  P. falciparum,  21 (12.50%)  P. falciparum  +  P. vivax  mixed infection, and 17 (10.12%)  Plasmodium ovale  positive rate was recorded .  The re-evaluation showed high level of mixed infection, and misdiagnosis of  P. ovale  and  P. vivax .    Conclusions  The result shows that  P. falciparum  prevalence is higher than  P. vivax  in the study area. The results, obtained from SYBR Green I-based real time PCR, indicated that the diagnosis efficiency of microscopy is very low for species-specific and mixed infection detection. Therefore, real time PCR-based species diagnosis should be applied for clinical diagnosis and quality control purposes in order to prevent the advent of drug resistant strains due to misdiagnosis and mistreatment.

malaria journal

Detection of mixed infection level of Plasmodium falciparum and Plasmodium vivax by SYBR Green I-based real-time PCR in North Gondar, north-west Ethiopia