Strategies for detection of Plasmodium species gametocytes

Abstract

Carriage and density of gametocytes, the transmission stages of malaria parasites, are determined for predicting the infectiousness of humans to mosquitoes. This measure is used for evaluating interventions that aim at reducing malaria transmission. Gametocytes need to be detected by amplification of stage-specific transcripts, which requires RNA-preserving blood sampling. For simultaneous, highly sensitive quantification of both, blood stages and gametocytes, we have compared and optimized different strategies for field and laboratory procedures in a cross sectional survey in 315 5-9 yr old children from Papua New Guinea. qRT-PCR was performed for gametocyte markers pfs25 and pvs25, Plasmodium species prevalence was determined by targeting both, 18S rRNA genes and transcripts. RNA-based parasite detection resulted in a P. falciparum positivity of 24.1%; of these 40.8% carried gametocytes. P. vivax positivity was 38.4%, with 38.0% of these carrying gametocytes. Sensitivity of DNA-based parasite detection was substantially lower with 14.1% for P. falciparum and 19.6% for P. vivax. Using the lower DNA-based prevalence of asexual stages as a denominator increased the percentage of gametocyte-positive infections to 59.1% for P. falciparum and 52.4% for P. vivax. For studies requiring highly sensitive and simultaneous quantification of sexual and asexual parasite stages, 18S rRNA transcript-based detection saves efforts and costs. RNA-based positivity is considerably higher than other methods. On the other hand, DNA-based parasite quantification is robust and permits comparison with other globally generated molecular prevalence data. Molecular monitoring of low density asexual and sexual parasitaemia will support the evaluation of effects of up-scaled antimalarial intervention programs and can also inform about small scale spatial variability in transmission intensity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow diagram of molecular analyses performed for detection of asexual and sexual parasite stages of P. vivax and P. falciparum in field samples from PNG.

Red and blue frames indicate assays done on DNA and on RNA, respectively. Orange and green boxes are P. falciparum and P. vivax-specific assays, respectively. P. malariae and P. ovale assays are not included in the diagram.

Figure 2. DNA- versus RNA-based quantification of Plasmodium parasites by qPCR and qRT-PCR of 18S rRNA genes or transcripts in comparison to light microscopy (LM).

P. falciparum (upper panel) and P. vivax (lower panel). Boxed values indicate the correlation coefficient (r2) and the conversion functions extracted from these data. All correlation coefficients (r2) were significant (p-value < 0.001).

Figure 3. Comparison of two blood sampling strategies for measuring gametocyte prevalence rates.

(A) P. falciparum, (B) P. vivax. Gametocyte positivity (left panel) and transcript copy numbers (right panel) are shown for RNAprotect solution versus filter paper soaked in TRIzol. Only samples were compared for which both measurements were available.

Figure 4. Gametocyte trend line generated with 3D7 P. falciparum in vitro culture for converting pfs25 transcript copy numbers into gametocyte counts.

Dashed lines indicate 95% confidence interval of intercept.

Figure 5. Effect of extended storage time on pfs25 transcripts.

20 samples were chosen to represent a wide range of transcript copy numbers at start of the 2 yr storage period. The initial copy numbers (black bars) are shown next to copy number detected 2 yrs later in the same RNA sample (white bars).