Genotyping of malaria parasites has become an integral part of many malariological field studies. Since more than a decade genotyping has been considered imperative for clinical trials of antimalarials performed in endemic countries, The hallmark of PCR correction of clinical trial outcomes is discrimination of new infections versus recrudescences. Quantification of clone detectability at any time point of blood sampling contributes relevant information on the reliability of PCR corrections. Furthermore, the number of newly acquired clones per time interval might be a suitable outcome measurement of antimalarial interventions; the parameter “clone detectability” might also correct this estimate.
Imperfect detectability for P. falciparum has been common knowledge, but to date this effect has been quantified only in few molecular epidemiological studies. The detectablity of P. vivax clones has been largely ignored due to the reported absence of sequestration. However, the generally lower parasite densities in P. vivax compared to P. falciparum has potential to contribute to compromised detectablity in a major way. The precise estimation of the detection probabilities of both species, undertaken in the same field study and under perfectly matching experimental conditions, allows assessing the combined effects of parasite sequestration, synchronicity and low parasitaemia and differences among Plasmodium species. It is clear that the individual factors, contributing jointly to detectability, cannot be determined by our genotyping approach.
Our analysis of samples collected 24 hours apart revealed limited day-to-day fluctuations in the detection of P. falciparum and P. vivax infection. This indicates that short-term sampling has only a small impact on prevalence estimates – regardless of whether infections are detected by light microscopy or PCR. For both species the observed prevalence by PCR increased, when 2 days were combined, by less than 10%. A more pronounced difference in prevalence based on one versus two days of sampling was only observed for microscopic detection of P. falciparum, where prevalence increased by 24%. It is unclear in how far the effect of parasite synchronization and sequestration add to this discrepancy between PCR and microscopy in P. falciparum detectability. These finding suggests that for Plasmodium species conducting repeated sampling within 24 h does not substantially increase the observed prevalence.
Detection of individual clones was very high in P. falciparum
0.79). Accordingly, combining genotypes from both days resulted in a small increase in observed mean MOI rising from 1.52 based on one day to 1.68 for both days. Children <5 years have not yet developed a strong immunity to P. falciparum 
and therefore carry high parasite densities (mean parasite density: 2558 parasites/µl), which leads to a better chance to detect by PCR most of the parasite clones present. Recently in a similar study in Ghana blood samples from individuals up to 20 years were repeatedly collected in intervals of 1, 4, 5 and 7 days 
. Detectability was calculated using the same approach as in our study. Clone detectability was around 0.6 in sample pairs collected 24 hours apart 
In contrast to P. falciparum, sequestration of late stage parasites has not been reported from P. vivax. Despite this biological difference, the detectability of individual parasite clones was lower in P. vivax than in P. falciparum. A larger number of P. vivax clones was only detected on either day for both P. vivax markers analyzed. In P. vivax parasite densities are generally much lower than in P. falciparum, in our study mean P. vivax density was 498 parasites/µl compared to the 5-fold higher P. falciparum density. Our results suggest that the overall low parasitaemia combined with synchronized replication, as generally seen in P. vivax, has a larger impact on detectability than sequestration plus sporadic low parasitaema, as observed in P. falciparum.
The number of concurrent P. falciparum
or P. vivax
infections had a pronounced effect on detectability: Increasing MOI lead to decreasing detectability. It has been suggested that in multiple-clone infections, clones representing a minority of the total parasite population in a host might escape detection by PCR 
. In experimental mixtures of DNA from two different P. falciparum
clones up to ratios of 1
100, both genotypes were detected by PCR-CE 
, as well as in mixtures of DNA from two different P. falciparum
clones in a ratio of 1
. However, our observation that detectability decreased with increasing MOI, suggests impaired amplification of minority clones. Hence, the higher mean MOI in P. vivax
3.37) compared to P. falciparum
1.68) could be seen as a further reason for the slightly lower detectability in P. vivax
Detectability of the two P. vivax
0.73) and MS16 (
0.61) differed. Overall, more samples were positive for P. vivax
and more clones were detected with the MS16 PCR. This suggests a higher detection threshold of the msp1
F3 PCR in combination with a lower influence of fluctuations in parasite density on detectability. In addition, a difference in the genetic diversity of these markers could add to the discrepant values: two independent clones are expected to share more often the same msp1
Our results highlight that a single bleed does not reflect the full complexity of concurrently infecting P. vivax clones. The true MOI of P. vivax is underestimated to a greater extent than the MOI of P. falciparum. The effect of repeated sampling on prevalence, however, was in the same range in P. falciparum and P. vivax: for both species 6 to 9% of all infections were missed on any single day. The known biology suggests that low parasite densities should have different causes in P. falciparum and P. vivax. P. falciparum parasites sequester periodically but a clone is absent from the peripheral circulation only if its erythrocytic cycle is tightly synchronized, which according to our data seems not to be the rule. P. vivax generally occurs at lower densities, and the timing of a parasite clone within the erythrocytic cycle seems to be well synchronized. These are major differences between both species, but with respect to detectability of parasite clones in the blood stream, both species differ less than previously thought.
In our study participants parasite densities for P. vivax
dropped with increasing age, while for P. falciparum
densities remained at the same level 
. If parasite densities would directly impact detectability, a similar decrease in detectability with increasing age would thus be expected for P. vivax
. However, we did not observe a clear effect of parasite density or age on detectability. Despite the fact that we did not detect an age trend in detectability in our study participants aged 1 to 4.5 years, this relationship might be different in older individuals. It remains open whether the often observed decrease of prevalence over the entire age range in moderate to high levels of transmission might be due at least partially to lower detectability associated with decreasing parasite densities.
Our P. falciparum
results were generated by using a similar experimental and analytical approach as in a previous study, conducted in a highly endemic area in Ghana, and results can thus be compared. When all age groups were included in the Ghana study a strong age-dependency of detectability was noted, with a detectability of over 60% in younger individuals and only 10% in adults 
. Overall, only 35% of all clones present in the host were detected in a single blood sample 
. In individuals of the same age group as our participants, detectability ranged from 0.51 to 0.55 
. This lower detectability in Ghana could be affected by higher malaria endemicity and higher mean MOI. A lower transmission and therefore slower acquisition of immunity in PNG might lead to lower age dependency in detectability. It remains open whether in older children the often-observed decrease of prevalence by age in moderate to high levels of transmission might be due at least partially to lower detectability associated with decreasing parasite densities. Our current results reflect the situation in children harbouring high parasite densities. The situation in adults might be different.
In conclusion, when both 24 h bleeds were combined, we observed an increase in precision of estimates of epidemiological parameters in our study for both P. falciparum and P. vivax. While the increase in observed prevalence was limited, the effect on detection of individual alleles was more pronounced. Especially in highly endemic countries where most patients carry multiple clone infections repeated sampling substantially increases the precision of observed epidemiological parameters such as MOI. There was surprisingly little difference between the two parasites; just as studies of P. falciparum should recognize that they only detect a proportion of infections, the same is true for P. vivax.