Sensitive detection and accurate quantification of low-density P. falciparum infections from DBS has become increasingly important in the context of describing residual malaria transmission in close-to-elimination settings. In areas of highly heterogeneous transmission it is of interest to identify risk factors of residual infections and to understand the infectious reservoir in the population. To provide optimal protocols for large-scale molecular-epidemiological studies in a pre-elimination setting, a simple but sensitive method combining pooling, extraction and amplification was developed. This strategy consisted of direct pre-PCR of pooled 3-mm punches from DBS, followed by varATS qPCR.
Despite using a single DBS punch in a pool of 5 samples, the tested method was able to reliably identify 1 parasite/µl blood in a dilution series where DBS were reconstituted with WHO parasite density standard mixed with uninfected blood. The ultra-sensitive qPCR assay applied in this study targets multiple copies of P. falciparum var genes. A higher number of templates per parasite contributes to robust results in low-density infections and pools of these . Nonetheless, the uneven distribution of the parasites in blood spotted on DBS poses an additional limiting factor. Hence, the sensitivity of our method might be slightly overestimated, as the WHO density standard was added as a DNA solution directly into the PCR mastermix, thus more even distribution of templates compared to spotting parasites. This discrepancy cannot be bypassed, because the use of an international reference standard is indispensable for inter-laboratory comparisons. The level of sensitivity of our method complies with the malERA consultative group-recommended detection limit for malaria pre-elimination settings of 1 parasite/µl blood . Another previously published pooling approach used chelex extraction followed by cytochrome b nested PCR and reported a 100% sensitivity to detect a single positive sample with a density of 100 parasites/µl blood in a pool of 5 samples, and a 80% sensitivity to detect an infection of 10 parasites/µl in pooled analysis . Compared to this previous study, the limit of detection was substantially more sensitive with 95% sensitivity to detect 1.12 parasites/µl blood on a single positive DBS punch screened in pools of five punches, i.e. together with 4 negative DBS. As both studies use multi-target qPCRs, this difference seems to be due to the chelex extraction used in this previous study. The chelex protocol has the advantage of being low-cost, but its disadvantage consists in a dilution of DNA in a larger volume than the original volume of blood, thus, sensitivity is compromised. It should be emphasized that for standardization sake, we measured both, LOD and CV, using the WHO standard DNA mixed with negative blood on DBS. The difference between introducing DNA or parasites into a reaction is that DNA in solution is more evenly distributed than when all target copies were contained in parasites. Owing to multi-copy genomic targets of the varATS qPCR assay, the values for LOD and CV could prove slightly less sensitive, if whole parasites were analysed.
The ultra-sensitive diagnostic assay used in this study targets the P. falciparum var genes. These species-specific middle-repetitive sequences are dispersed throughout the genome . Other Plasmodium species also occur in the study area , but these were not investigated so far. qPCR assays of comparable sensitivity were not yet available. For P. vivax, qPCR  and LAMP  assays were developed that detect mitochondrial DNA, of which numerous concatenated copies exist per parasite . Mining of the P. vivax genome for species-specific, repetitive sequences identified a non-coding subtelomeric repeat, Pvr47 , yet, the Pvr47 copy number per genome (n=14) was not higher than that of mitochondrial DNA. To our knowledge, no middle-repetitive sequences of higher copy-number than mtDNA have been identified and validated so far for qPCR diagnosis of the other human Plasmodium species.
Other methods than qPCR could be employed for reactive case detection. Loop-mediated isothermal amplification (LAMP) was used in Zanzibar in earlier studies . However, LAMP was less sensitive than qPCR for detection of asymptomatic low-density infections . Other disadvantages of LAMP are high prize for commercial LAMP kits, false positive results often arising from home-made master mixes, and the lack of parasite quantification (own unpublished observations, ). In a previous study in febrile children from Tanzania, the performance of the us-qPCR assay used here was compared with that of conventional RDT or highly sensitive RDT . This earlier study showed that us-qPCR was substantially more sensitive in detecting low-density infection in children suffering from non-malarial fevers.
The advantage of direct on DBS pre-PCR is that the time-consuming extraction and purification of DNA is omitted, such as an overnight incubation in saponin as required in the chelex extraction. Punching disks from dried blood spots (DBS) is the most time-consuming step in the processing of samples. All DNA extraction methods equally require this initial step. Substantial time is saved by performing a pre-PCR amplification instead of chelex extraction, which reduces the processing time for eighty samples from two to one day. Pooling of punches from several DBS permits analysis of even more blood samples within one day. Reduction of processing time by pre-PCR justifies higher costs incurring from the requirement for a Phusion Blood Direct PCR Kit. The price per sample for the direct-on-punch pre-PCR method is around 2.4US$ compared to 0.03US$ per sample for chelex extraction. Introduction of pre-PCR permitted to introduce the highest possible concentration of parasite DNA into the amplification reaction. All alternative methods tested would have introduced less template into the qPCR mix, as pre-PCR overcomes loss of DNA during the extraction process. The major disadvantage of performing pre-PCR directly on filter paper was its high potential for contamination. Ten cycles of pre-amplification directly on DBS harbors dangers, mainly because it requires transfer of amplified product from pre-PCR into a second reaction tube or plate for qPCR. Such an open tube system requires utmost care because amplicons can potentially be transferred via aerosols or spills to neighboring wells leading to false positive results. The risk of contamination increases with increasing parasite density on the DBS. This risk requires an upscale in safety measures and controls as well as an adequate laboratory set up. In this study, the risk for contamination was minimized by dedicated rooms for master mix preparation, punching of samples, handling of post amplification product, and final qPCR reaction setup. Importantly, surfaces were decontaminated by exposure to UV light and bleach prior to and after completion of pipetting. Several negative controls were included in pre-PCR as well as qPCR to monitor any contamination. Despite installing preventive measures to avoid contamination, occasionally a no template control turned out positive. This could derive from aerosols or pipetting error. In case of contamination, all samples analysed in that experiment were repeated starting from new DBS punches. One way to further minimize cross-contamination, also employed in this study, was to analyse pools with high parasite concentrations (including all cases of symptomatic malaria) separately from low-density samples.
A relevant consideration in a molecular-epidemiological study is that pooling of samples from several individuals trades off test-sensitivity against the potential for high-throughput processing. This is because pooling of DBS reduces sensitivity of parasite detection because of a smaller blood volume processed. Due to space limits in reaction tubes, only five punches of 3mm diameter corresponding to 3µl blood each could be processed using our method. When samples were analysed individually, all five punches derived from one DBS, whereas for analysis of sample pools, only one punch per DBS could be processed.
During evaluation of the difference in LOD between the analysis of one sample (5 punches per DBS) versus a pool of 5 samples (one punch each), 8.6-fold loss in sensitivity was observed for pooling compared to individual screening. The thus reduced sensitivity by processing pools is in the sensitivity range of current molecular diagnostic assays used for screening of community samples. Using the varATS us-qPCR compensated partly for the loss of sensitivity through pooling.
For processing a large set of samples from low-transmission settings as in this study, performing an initial screen on sample pools was necessary. When evaluating the potential to miss samples through pooling, 7.5% (18/240) low-positive samples would be gained by individual analysis, 10/240 (4.2%) would be positive above the cut-off. These numbers highlight the limitations in large studies and reporting the potential for false negatives is relevant. However, in the context of investigating the extent of the asymptomatic parasitaemia in the community, there is no need to identify the full depth of the subclinical reservoir, as such very low-density infections are unlikely transmitted .
This equally applies to definition of a density cut-off. A cut-off for qPCR- positivity of 0.13 parasites/µl blood was introduced to compensate for the variance caused by stochastic distribution of scarce parasites. Using the cut-off of 0.13 parasites/µl leads to omission of all low positive samples that would not be detected with certainty of less than 95%. Although samples below this cut-off were detected in some independent replicates, a very robust data set was created with records of positive samples that would be reproduced if repeatedly analysed.
Earlier Mass Screening and Treatment (MSAT) campaigns relying on RDT-based or LM-based diagnosis have not produced convincing results: Studies in Burkina Faso and Zanzibar found no sustained effect on incidence of anti-malarial treatment of asymptomatic P. falciparum carriers after screening and treatment campaigns [38,39]. A population-wide malaria testing and treatment with RDTs and artemether-lumefantrine in southern Zambia, an area with heterogeneous transmission, showed an overall modest impact on decreasing the malaria infection burden . A recent study in Indonesia reported similar results; after two or three rounds of MSAT using microscopy, little or no impact on malaria incidence was found . Such little effect on incidence and prevalence is likely due to the large proportion of missed low-density infections, which will sustain transmission despite treatment of RDT-positive infections. A recent study in Zambia, performed in a close-to-elimination setting, showed that almost half of all PCR-diagnosed infections remained undetected by RDT, and about a quarter of these RDT-negative infections carried gametocytes and, therefore, may be infectious to mosquitoes .
The results obtained in Zanzibar are in line with previous observations of additional detection of P. falciparum infections by PCR [16,42,43]. The absolute numbers of infections detected by performing us-qPCR were small, with 45 infections detected additionally to RDT-positive individuals in a total of 4590 blood samples screened. RDT detected only 29 of the 78 qPCR-positive individuals and had a diagnostic sensitivity of 37%. Thus, to inform targeted response interventions, such as focal testing and treatment, RDT alone might not be sufficiently sensitive. However, it remains to be shown by further epidemiological analyses of these data from the Zanzibar household surveys, whether both diagnostics reveal the same epidemiological patterns and risk factors for infection in the various household types. Performing molecular diagnostics in the framework of elimination research represents a relevant expansion into a not yet well characterized, potential reservoir of infection.
It has to be emphasized that detection of very low-density infections is not trivial, and their detection is not necessary in many malariological studies . However, low-density infections are relevant in studies like this one, aiming at a better understanding of transmission patterns. Even though low-density infections are unlikely to be transmitted at the time point of sampling, they might be transmitted later in the course of the infection. Thus, recording low-density infections with parasite densities from 1-10 p/µl generates more accurate and meaningful prevalence data compared to RDT-based data [2,45]. Applying molecular tools in elimination research is useful for better understanding transmission patterns and underlying transmission risk in residual transmission scenarios and for the design and evaluation of targeted interventions.
In contrast to research studies, only cheap and simple-to-use methods, such as LM or RDT, are generally available for routine surveillance. Although the developed approach simplifies malaria diagnosis from DBS and supports high throughput screening, molecular diagnostic for programmatic use and routine implementation does not seem realistic currently, mainly due to a lack of funding, capacity and appropriate laboratory set-up. On the other hand, molecular parasite detection is very useful as a research tool for gaining knowledge on foci of residual malaria or the silent reservoir of transmission, as well as for informing mathematical modelling.