How much remains undetected? Probability of molecular detection of human Plasmodia in the field

Abstract

Background: In malaria endemic areas, most people are simultaneously infected with different parasite clones. Detection of individual clones is hampered when their densities fluctuate around the detection limit and, in case of P. falciparum, by sequestration during part of their life cycle. This has important implications for measures of levels of infection or for the outcome of clinical trials. This study aimed at measuring the detectability of individual P. falciparum and P. vivax parasite clones in consecutive samples of the same patient and at investigating the impact of sampling strategies on basic epidemiological measures such as multiplicity of infection (MOI).

Methods: Samples were obtained in a repeated cross-sectional field survey in 1 to 4.5 years old children from Papua New Guinea, who were followed up in 2-monthly intervals over 16 months. At each follow-up visit, two consecutive blood samples were collected from each child at intervals of 24 hours. Samples were genotyped for the polymorphic markers msp2 for P. falciparum and msp1F3 and MS16 for P. vivax. Observed prevalence and mean MOI estimated from single samples per host were compared to combined data from sampling twice within 24 h.

Findings and conclusion: Estimated detectability was high in our data set (0.79 [95% CI 0.76-0.82] for P. falciparum and, depending on the marker, 0.61 [0.58-0.63] or 0.73 [0.71-0.75] for P. vivax). When genotyping data from sequential samples, collected 24 hours apart, were combined, the increase in measured prevalence was moderate, 6 to 9% of all infections were missed on a single day. The effect on observed MOI was more pronounced, 18 to 31% of all individual clones were not detected in a single bleed. Repeated sampling revealed little difference between detectability of P. falciparum and P. vivax.

Figure 1. Dynamics of parasite clones over 24 hours.

Schematic overview of possible outcomes of 24 h bleeds. A sample is positive as soon as a parasite is detected on either day. Different colors of PCR results indicate different clones detected. The combined multiplicity of infection includes all clones detected in two corresponding bleeds.

Figure 2. Examples of results obtained on two consecutive days by PCR-capillary electrophoresis.

Capillary electrophoresis chromatograms obtained with the P. vivax marker msp1F3 from two patients on two consecutive days. X-axis: size of PCR product in base pairs. Y-axis: relative fluorescent units. In patient 1 one clone was detected on day 1, and two additional clones were detected on day 2, combined MOI = 3. Patient 2 was negative on day 1, but one clone was found on day 2, combined MOI = 1.

Figure 3. Detectablity of Plasmodium infections and parasite clones in different age groups.

Values for microscopy and “P. vivax PCR any marker” refer to detection of parasites without distinction of clones. Values for the molecular markers P. falciparum msp2, P. vivax msp1F3 and P. vivax MS16 refer to the detection of parasite clones. Larger 95% CI of P. falciparum detectability are mainly caused by smaller sample size.

Figure 4. Detectablity of parasite clones vs. multiplicity of infection.

Figure 5. Detectablity of parasite clones in patients harbouring different parasite densities.