Comparison of Multiplex Nested PCR, In-house Real-Time PCR and Commercial Real-Time PCR Methods for the Detection of Plasmodium spp. from Blood Samples

Malaria is a life-threatening parasitic disease caused by the parasites belonging to Plasmodium genus. Microscopic examination of Giemsa stained blood smears is accepted as the gold standard diagnostic method. It is recommended to use more than one method in order to strengthen the laboratory diagnosis of malaria which is an important health problem in our country as in the whole world. In this study, it was aimed to compare the results of three different molecular methods and determine which molecular method could be used in the diagnostic algorithm to be applied. DNA was extracted from 280 whole blood sample stored in EDTA tubes using a commercial kit. Three different polymerase chain reaction (PCR) methods were used for the detection of Plasmodium spp. in DNA samples obtained and the results were compared. First, multiplex nested PCR was applied and then in-house real-time PCR (Rt-PCR) which was validated in our laboratory and a commercial Rt-PCR kit were applied. Multiplex nested PCR was accepted as the gold standard and 182 samples that were evaluated as Plasmodium spp. positive and 98 samples that were evaluated as negative were also studied by in-house and commercial Rt-PCR methods. In multiplex nested PCR's first step reaction 1670 base pairs (bp) band was observed in Plasmodium spp. positive samples and 117 bp band was observed in Plasmodium vivax positive samples in the second step reaction. Tm values of P.vivax positive samples were determined as 78-79 in the melting analysis of the in-house Rt-PCR. CT values of the positive samples in in-house Rt-PCR were between 20.03-31.71 and were between 17.26-34.94 in the commercial Rt-PCR. With the in-house Rt-PCR method 180 cases were determined as positive, while with the commercial Rt-PCR method 178 cases were determined as positive. Two samples with the in-house Rt-PCR and 4 samples with the commercial Rt-PCR were considered as false negative. When the sensitivity and specificity of the both methods were calculated, the sensitivity of the in-house Rt-PCR method was 0.98, the specificity was 0.97, the positive predictive value (PPV) was 98%, the negative predictive value (NPV) was 97%, the sensitivity of the commercial Rt-PCR was 0.97, the specificity was 0.95, the PPV was 97%, the NPV was 95%. A high level of agreement (kappa: 0.953) was determined between the in-house and the commercial Rt-PCR methods. In order for a test to be accepted as a confirmatory test, its specificity must be high. It was decided that sensitivity and specificity of the in-house Rt-PCR were suitable for using this method in the laboratory diagnosis of Plasmodium species.