Comparison on simultaneous capillary and venous parasite density and genotyping results from children and adults with uncomplicated malaria: a prospective observational study in Uganda

The World Health Organization (WHO) estimated 219 million malaria infections and 435,000 deaths in 2017 with Africa accounting for 90% of these cases [1]. Uganda accounts for approximately 4% of total malaria cases worldwide with approximately 7.8 million cases in 2016 [1]. The gold standard for malaria diagnosis is examination of a Giemsa-stained blood film with light microscopy [2]. Blood films can be prepared using capillary or venous blood. In clinical settings, capillary finger-pricks are most commonly utilized for the detection and quantification of parasite density [2]. However, in research settings, either capillary or venous sampling may be employed over the course of the study, depending on the amount of blood needed at each visit. Notably, in these cases, venous and capillary smear results have been used interchangeably.

In treatment efficacy and other types of research studies, parasite DNA is typically extracted from blood samples, and genotyping of polymorphic markers using microsatellites is used to compare the parasite strains at the time of diagnosis to those present at the time of clinical or parasitological failure [3]. In addition, these same microsatellites are used to quantify the number of unique strains infecting an individual, which represents the complexity of infection (COI) [4]. Similar to parasite density measurements, parasite DNA fingerprinting results from either capillary or venous blood are used interchangeably.

Capillary and venous blood compartments have been studied for differences in measurements of hematocrit, leukocyte populations, glucose, G6PD-deficiency status, and other analytes [5‐15]. Results of these studies vary, for example from finding slightly higher leukocyte counts in capillary than venous samples to platelet counts being higher in venous than capillary blood [5, 6, 8‐10]. Notably, certain species of another intraerythrocytic parasite, Babesia, have been found to exhibit higher parasite density levels in capillary than in venous compartments [16, 17]. In the case of malaria, Plasmodium falciparum parasites are known to sequester in capillaries, a property which may impact the characterization of parasite density and/or strain dynamics in these matrices [18‐21]. Indeed, a handful of studies, limited to asymptomatic or suspected malaria cases, have compared the two compartments [22‐25]. While a majority have found no significant difference in the magnitude of asexual density in either compartment, a few studies have found a higher sensitivity for detection of asexual and gametocyte stages in capillary versus venous blood.

No data, to our knowledge, is available on the comparison of capillary and venous parasite density following treatment for symptomatic malaria, in pregnancy or in HIV-infected individuals, nor any data on comparing genotyping results from capillary and venous blood. We report the following findings with the goal of informing the interpretation of malaria parasite density and genotyping results from research studies.

Our initial hypothesis was that systematic differences in parasite density may be present between matrices given that Plasmodium falciparum parasites are known to sequester in capillaries [18‐21]. In addition, we hypothesized that pregnant women may demonstrate differences from other demographic groups, due to changes in hemodynamic parameters during pregnancy [30]. However, measurements in clinical malaria from capillary and venous compartments did not demonstrate a significant difference from one another throughout the study time points, aside from a slightly higher parasite density in capillary versus venous specimens by 21% on Day 1 (p = 0.056), a magnitude which is not of clinical significance. This lack of difference was seen in all demographic groups.

Our results are largely in alignment with previous studies, however, there are some notable differences (see summary of studies in Additional file 1: Table S2) [23‐25, 31]. Similar to our results, a lack of difference in asexual parasite density measurements was reported in two distinct Cameroonian studies, one including 137 asymptomatic gametocyte-positive Cameroonian children (age 4–15 years), and another involving 150 Cameroonians presenting with symptoms of malaria (ages 7 to 66 years) [22, 23]. However, two studies have reported differences, although the magnitude of differences was small. To our knowledge, the first study to compare parasite density in both compartments was conducted in Burkina Faso in 73 asymptomatic children, where densities were found to be statistically higher in capillary than in venous blood compartments (median parasitemia capillary and venous, 300 and 200 parasites/uL, respectively [24]. The most recent study to explore this was in Gabon, and similarly found a higher median asexual density in those presenting with symptoms of malaria [25]. However, parasite densities were low in this study, with minor differences between compartments (median parasitemia capillary and venous, 495 and 429 parasites/uL, respectively) that are of unclear clinical significance. One fairly consistent finding, however, is that the detection of parasites may be more sensitive in capillary versus venous blood in those presenting with low initial parasite density [22, 24, 25]. In the Cameroonian study, those presenting with symptoms of malaria had positive capillary smears in 29% of cases, but only 17% of venous smears (p = 0.011), while the Gabon study found that capillary blood had an 8% higher sensitivity than venous blood [22, 25]. While our study was not powered to detect this outcome, at the time of recurrent parasitemia, 4 samples were positive only in the capillary compartment, and one sample only in the venous compartment. Nonetheless, overall parasite density did not differ between compartments at the time of detection with recurrent parasitemia.

Another aspect which has been compared in these compartments is the ability to detect gametocytes. P. falciparum gametocyte densities, as determined by microscopy did not differ between capillary and venous blood in the Cameroonian study of asymptomatic children [23], but an exploratory study in Ethiopia found a trend towards a higher absolute count of Pfs25-mRNA copies in capillary blood, and the Gabon study found both a higher sensitivity for gametocyte detection and higher densities in capillary versus venous blood by microscopy, both involving symptomatic individuals [31]. Thus, additional studies are needed, but data may suggest that capillary specimens have higher gametocyte loads than venous samples at the time of presentation with symptomatic malaria, when overall parasite densities are higher. Such data may have implications on our understanding of malaria transmission.

Thus, in totality, results support either a lack of difference or slightly higher asexual parasite density in capillary as compared to venous blood. Furthermore, while there may be large differences in the magnitude of density between the compartments, these differences are not consistently in one direction, and do not appear to be of a magnitude that is clinically relevant. Additionally, the magnitude in variability in parasite density measurements may be in line with other sources of variability such as inter-observer agreement among microscopists [32‐34]. Indeed, previous studies of intra-observer agreement from microscopy have revealed up to a 17% median discrepancy in parasite counts when two microscopists examined the same slide [34]. Thus, in comparing the magnitude of variability in our results, with those seen in studies of readings between microscopists, we do not think the variability in parasite density measurements between the two compartments is of clinical significance. Additional areas warranting further exploration are the possibility of different sensitivities for detection of low density asexual parasite and gametocyte densities in capillary compared to venous blood samples.

To our knowledge, we also present the first data on the comparison of genotyping results from capillary and venous blood [3]. The use of MSP-2-based genotyping alone, we found venous samples had a statistically higher number of strains detected than capillary samples on the day of diagnosis (p = 0.02), but not at the time of recurrent malaria (p = 0.12), when parasite densities were comparatively lower than at initial presentation. The explanation for the higher number of strains detected in venous than in capillary blood by MSP-2, the marker demonstrating the highest level of polymorphism in our study (data not shown) is unclear and will require further study to see if results are reproducible in other settings.

Finally, we sought to assess whether differences in genotyping results from capillary and venous blood impact PCR-corrected outcome classifications. While the WHO recommends the use of multiple markers (typically MSP-1, MSP-2, and GLURP), a large number of studies continue to use MSP-2-based genotyping alone [3]. In our study, genotype-corrected outcomes displayed a high degree of variability depending on the pairing of compartments on the day of presentation and the day of failure, ranging from 19 to 30%. We currently use a step-wise approach of six markers (MSP-2, MSP-2, and 4 microsatellites) to assess for recrudescent malaria, and, not surprisingly, the use of these additional markers greatly reduced both the reported proportion of recrudescent infections and the variability in calls depending on the pairing of sample compartments (2–5.9%).

Our study has a few limitations. Notably, we identified individuals for enrollment based on the presence of a fever with a capillary blood smear that was positive for parasites. Thus, we would have missed individuals who were positive only by venous smear at diagnosis. In addition, we did not include any asymptomatic or sub-patent infections. Additionally, our study took place in a region with high transmission intensity and strain diversity. It is likely that strains may have been missed due to known limitations of PCR-based genotyping in multi-clonal infections. Lastly, the use of PCR-based genotyping from dried blood spots has inherent limitations in the sensitivity, due both to the technique, as well as the limitation of blood volume. It is likely that a larger blood volume and/or newer sequence-based techniques may reveal other patterns not seen at our level of discrimination.

Quantitative parasite density measurements do not statistically differ between capillary and venous compartments in children, adults, or pregnant women at the time of presentation with uncomplicated malaria, nor over the course of standard 42-day follow-up. However, more strains were detected by MSP-2 genotyping in venous samples than in capillary samples at the time of diagnosis. The variability in genotype-corrected treatment outcomes is high when using a single marker, but is dramatically reduced by the use of multiple polymorphic markers. These findings indicate that both capillary and venous compartments can be used for sampling with confidence in clinical research settings.