Table 1.
Characteristics of real-time PCR methods.
Table 2.
Sensitivity comparison of real-time PCR methods applying five different dilutions of P.falciparum reference material.
a.
Fig 1.
Ct value comparisons of DNA samples extracted from 200 μl EDTA whole blood versus filter paper 50 μl blood (N = 37).
Positive P.falciparum material from 37 patients had been collected and extracted by two different methods resulting in DNA from whole blood purified by spin-column, and DNA from filter paper purified by Chelex-100 [5, 18]. The two Ct trends showed a constant difference of 4–9 cycles, and for 31 out of 37 samples the difference was 7–9 cycles.
Fig 2.
Boxplots showing how research microscopy and RDT results correlate to positive Ct values among Tanzanian field samples.
(A) A boxplot showing how research microscopy results negative/positive correlate to positive cytb SYBR real-time PCR results (N = 54). (B) A boxplot showing how rapid diagnostic test (RDT) results negative/positive correlates to positive cytb SYBR real-time PCR results (N = 47).
Table 3.
Sensitivity assessment of real-time PCR methods applying positive P. falciparum Tanzanian field DNA samples (N = 111).
a.
Fig 3.
Sensitivity analysis and method comparison of PCR methods applying positive P.falciparum field DNA samples with extreme low parasitaemia (N = 42).
Extreme low parasitaemia samples that counted negative or Ct value above 30 by cytb SYBR real-time PCR were further analyzed. This was done to investigate if the stored DNA had been degraded over time, as well as compare the performance of the four PCR methods. The varATS TaqMan real-time PCR had a higher detection rate for low parasitaemia. However, for all of the methods eight of the previously positive P. falciparum samples were now negative. A positive sample was defined by at least two out of three parallels detected, and only one of the eight negatives had no positive detections of parallels by any of the methods. For several of the samples it was a randomly/non-consistently detection trend, depending on the actual amount of amplification target in the template. Detailed data are given in S1 Dataset.
Table 4.
Univariate cross tabulation analysis of factors associated with low parasitaemia (≤1000 copies/rxn).