Dried blood spots: a robust tool for malaria surveillance in countries targeting elimination : Journal of Vector Borne Diseases

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Dried blood spots

a robust tool for malaria surveillance in countries targeting elimination

Nain, Minu1; Sinha, Abhinav1; Sharma, Amit1,2,

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Journal of Vector Borne Diseases 60(1):p 11-17, Jan–Mar 2023.
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With the advancements in analytical and molecular techniques, Dried Blood Spots (DBS) are re-emerging as attractive and cost-effective alternatives for global health surveillance. The use of DBS has been well-characterized in the neonatal screening of metabolic diseases, therapeutic screening as well as in epidemiological studies for biomonitoring. Malaria is one such infectious disease where DBS use can expedite molecular surveillance for assessing drug resistance and for refining drug usage policies. In India, malaria cases have reduced significantly over the past decade but to achieve malaria elimination by 2030, country-wide DBS-based screening should be conducted to identify the presence of molecular markers of artemisinin resistance and to study parasite reservoirs in asymptomatic populations. DBS has wide applications in genomics, proteomics, and metabolomic studies concerning both host and pathogen factors. Hence, it is a comprehensive tool for malaria surveillance that can capture both host and parasite information. In this review, we elucidate the current and prospective role of DBS in malaria surveillance and its applications in studies ranging from genetic epidemiology, parasite and vector surveillance, drug development and polymorphisms to ultimately how they can pave the roadmap for countries aiming malaria elimination


The history of DBS goes long back to 1913 when Ivar Christian Bang isolated glucose from it for the first time[1]. Later, Gutherie developed a method for phenylketonuria (PKU) diagnosis from DBS samples which became a routine practice in neonatal screening for congenital and inherent metabolic disorders[2]. Since then the DBS technique has become a standard sample collection method for epidemiological surveys, monitoring drug resistance, disease prevalence, and seromarkers. The stability of DNA in DBS depends on several factors including storage duration, temperature, humidity, and kind of filter paper used. The quality of filter paper determines the variables like the chromatographic effect of filter paper, elution efficiency, homogeneity, and analyte recovery[3]. The limiting factor in field epidemiological studies is the lack of infrastructure for the collection, storage, and transport of venous blood samples. The advantages of DBS in such cases are (i) minimal invasiveness and low blood volume requirement, (ii) low biohazard risk, (iii) no centrifugation required, (iv) ease of shipment, regular mail, and room temperature can suffice for genetic markers, and (v) storage at 4°C for a short duration and at -20°C/-80°C for a longer term without analyte deterioration[4,5]. Apart from sample stability and easy handling, usual DNA extraction methods from DBS can detect parasite DNA of as low as 0.5 to 2 parasites /μl which is comparable to PCR results from reference blood/serum samples i.e. 0.5 p/μΝ[6] This makes DBS a popular and preferred tool for surveillance and epidemiological studies. The potential and emerging applications of DBS include therapeutic drug monitoring, tracking environmental contamination, pharmacokinetic, genomics, proteomics, metabolomics, and lipidomics study[7]. Nucleic acid extraction from DBS is currently used in the diagnosis, surveillance, and monitoring of several infectious diseases including HBV, HCV, HIV, neonatal screening, and malaria[8].

Malaria is a vector-borne tropical disease caused by Plasmodium parasites. Among the five Plasmodium species known to cause disease in humans, P. falciparum and P. vivax are the most crucial ones posing maximum threats. Despite a century-long effort, malaria is still a leading global health concern with 627000 deaths reported worldwide in 2020 as per the World Malaria Report[9]. Sub-saharan Africa region leads with the maximum malaria cases and related mortality in the world followed by Southeast Asia[9]. WHO has set a pioneering goal of malaria elimination in 35 countries and at least a 90% reduction in malaria cases by 2030[10,11]. India has also launched its malaria elimination program in accordance with the global technical strategy of WHO and aims elimination by 2030[10]. Among several other necessary actions, malaria elimination majorly needs active surveillance of low density/asymptomatic infections, malaria epidemiology (parasite and vector) studies, and tracking drug/insecticide resistance. Around 16,500 international (as per Google patents) and 24 Indian patents (Indian patents advanced search system) have been granted to date for multiple methods of DBS use in several stages of malaria management including diagnostics, surveillance, serosurveys, drug development, and monitoring. This shows the potential of the DBS sampling method in malaria management and elimination, given its advantage over venous blood samples. So, here we have discussed the role of DBS in disease surveillance and management concerning epidemiological studies, parasite and vector surveillance, drug development and polymorphism studies, the crucial arms for malaria elimination [Figure 1].

Figure 1:
DBS workflow with downstream applications in malaria management, control and elimination.

DBS use in malaria epidemiology

Efficient diagnosis of malaria infection and individual case management (including treatment and transmission prevention) is the primary tool to achieve elimination in the coming future. More so, it is crucial to identify, monitor, and tackle low density/asymptomatic infections which might act as silent parasite reservoirs in the population[12,13]. To guide future treatment strategies, field-based molecular surveys need to be conducted to generate accurate and efficient data on geographical locations with drug resistance and clusters of high disease prevalence. This information is critical for interrupting local transmission in malaria hot spots through suitable therapeutic interventions. The surveillance should be done with validated and reliable genetic tests and biological assays. Moreover, it should reach even the most remote places with minimal resources and minimum expertise. The genomic deoxyribonucleic acid (gDNA) from DBS is suitable for PCR and can be used for genotyping and genome sequencing once the whole genome is amplified from extracted gDNA[14]. Indeed, the whole plasmodium genome has been sequenced using selective whole genome amplification from clinical DBS samples[15]. This technique has hence been profusely used in low-cost screening for drug and insecticide resistance in malaria epidemiological surveys. Several ultrasensitive technologies have been developed in recent years for enhancing malaria surveillance. The traditional microscopy and rapid diagnostic test (RDTs) fail to detect these very low parasite levels in low transmission settings and need more sensitive DNA-based strategies such as photo-induced electron transfer polymerase chain reaction (PET-PCR), COX-III single direct PCR, DBS-based ultrasensitive PCR that can detect parasite at ~1parasite/DBS[16], multiplex Real-time PCR assay[17] and Loop-mediated isothermal amplification (LAMP) with a detection limit of <5000 parasites/ml[18,19] to detect these low-density infections and track their hotspots in a population[20,21]. More recently, a low-cost, robust method for high throughput screening of plasmodium from DBS in both clinical and non-clinical settings has been developed[22]. The technique utilizes mid-infrared spectroscopy in combination with machine learning for direct screening of malaria from DBS[22]. Bead-based enzyme-linked immunosorbent assay and quantitative suspension array technology are also developed recently to screen pLDH (Plasmodium falciparum lactate dehydrogenase) and pfHRP2 (Plasmodium falciparum histidine-rich proteins 2) from DBS, which can be used simultaneously or independently with pLDH and pfHRP2-based RDTs for malaria diagnostics[23,24]. DBS also serves as the stable sample source for measuring gametocytaemia which can determine the transmission capacity of asymptomatic/low-density infections[25,26]. Apart from traditional antimalarial/insecticide resistance surveys, DBS is being currently used to detect pfHRP2 deletion in the population which is presenting a serious threat to pfHRP2-based RDT diagnostics[27,28]. Kayvan et al., has developed an advanced and sensitive DNA isolation technique from DBS for drug resistance studies in low transmission settings as well as in non-symptomatic infections within high transmission settings with low parasite densities[16,29]. In malaria clinical trials and surveillance studies, thousands of samples are collected from geographically challenging areas where DBS can provide operational and economic advantages. DBS has emerged as an effective tool for serological surveys to monitor malaria transmission intensity, pattern, and past exposure biomarkers of both parasites and vectors of different species, especially in malaria-endemic regions[30,31,32,33,34][35]. Parasite biomarkers for malaria prevalence vary between high (MSP3) and low transmission settings (Pfs230)[36]. Screening of antibodies from DBS elutes can be done by multiple downstream assays including ELISA, multiplex bead assay, protein microarrays, and multiplex immunoassays as in the case of VAR2CSA antibody detection in pregnant women[30,31,32,37]. The crucial host genetic polymorphism of genes such as G6PD (Glucose-6-phosphate dehydrogenase), PCSK9, and P450 which play important roles in disease protection and drug metabolism respectively can be studied from DBS and hence can act as a comprehensive tool to study the role of host genetics in malaria outcome and drug effectiveness[38,39]. These studies can guide the future course of malaria prophylaxis and drug dosage in the target populations.

DBS use in malaria drug development and monitoring

Besides the widespread use of DBS in drug resistance surveys and for anti-malarial drug quantification, it can also be used to identify the presence of confirmed or potential molecular markers for malaria diagnostics and malaria severity including LDH, HRP2, hemozoin, aldolase, glutamate dehydrogenase (pGDH), CRP (C-reactive protein), primaquine (PQ) and multiple serological markers[40,41]. Due to ease of handling, transport, and storage, DBS as a sample source, is preferably used for pharmacokinetic-pharmacodynamics (PK-PD) studies of antimalarial drugs during routine therapeutic drug efficacy trials as well as qualitative metabolite profiling of drugs[42,43]. Besides the traditional method of drug elution using chemical methods, a more advanced method for drug concentration semi-quantitation from untreated DBS using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is being currently used[42,44,45,46]. LC-MS/MS is successfully used for PK-PD analysis of several antimalarial drugs and their metabolites including lumefantrine, chloroquine, and ivermectin[42,47,48]. Apart from the DBS use for biomarker screening and PK-PD analysis, it has found wide use in toxicology to screen toxins, substances of abuse, and trace elements[49].

Role of DBS in malaria-associated hemoglobinopathies

Besides the direct role of DBS in malaria surveillance, it has emerged as a dynamic tool for molecular and epidemiological studies to determine multiple aspects of co-morbid conditions like sickle cell anemia or trait, thalassemia, anemia, and G6PD deficiency[50,56]. These conditions play a crucial role in malaria susceptibility, severity, and drug response, and hence it is crucial to monitor the prevalence of these conditions in a population as a part of routine malaria surveillance[57,58,59,60,61,62]. These co-morbid hemoglobinopathies in their homozygous state present a wide range of phenotypic characteristics and affect malaria pathogenesis, drug response (PQ-induced cytotoxicity in G6PD deficient individuals), and disease outcome[52,53,55,63,64]. The sickle cell trait protects against malaria[65]. DBS is being widely used for routine neonatal screening of sickle cell anemia/trait in sub-Saharan Africa where it is highly prevalent and its early diagnosis might result in better care and survival rate[57,59,61]. G6PD is highly polymorphic and its genetic variants with reduced enzyme activity are prevalent in malaria-endemic areas as these variants have been shown to protect against P. falciparum infection and cerebral malaria[55,56,62,66,67]. Also, primaquine-induced cytotoxicity is well established in G6PD deficient individuals[68]. Therefore, it is crucial to screen and survey G6PD deficient variants that can help in predicting and avoiding drug-induced hemolysis in a population upon PQ therapy. Also, DBS has been shown as a successful sampling method for thalassemia diagnosis in low transmission settings by HPLC, gap PCR, and mass spectrometry[69,70,71]. A recent large-scale Indian study has demonstrated the successful use of DBS for screening multiple hemoglobinopathies using HPLC[58]. So, DBS use for molecular genotyping of these co-morbid blood disorders in the population can help not only to study the correlation or co-prevalence with malaria[50] but also better malaria control and management.

Applications of DBS in host metabolomics

The use of DBS for proteomics is also continuously evolving[72,73]. The highly specific multiple reaction monitoring-electrospray tandem mass spectrometry, MRM-MS has been coupled with DBS and has replaced the traditional screening methods like High-Performance Liquid Chromatography (HPLC) or Isoelectric Focusing (IEF)[74]. This uses a single-reaction multiple-analyte approach where hundreds of host and parasite markers from numerous diseases can be screened in a single DBS sample and generate high throughput data[75]. This technique can be used to screen multiple metabolic diseases in a single DBS by quantifying multiple therapeutic and endogenous proteins at once and hence, it has found widespread use in newborn screening for treatable congenital disorders and several metabolic disorders such as homocystinurea[76], Menkes disease[77] and medium-chain acyl-CoA dehydrogenase deficiency (MCADD)[78]. Not only the host proteins but pathogen markers such as viral antigens can also be isolated from DBS as in the case of Hepatitis C via Enzyme Immunoassays (EIA)[79]. So, specific host and pathogen protein biomarkers of a disease can be identified and extracted from DBS to aid the development of novel and improved therapeutics and diagnostics. DBS has been adopted as an alternative for analyzing RNA-based biomarkers such as circulating miRNA[80,81] and for characterization and isolation of disease specific miRNA in hypoxic-ischemic encephalopathy in newborns[80] and high altitude sickness[81].

Lately, the use of antioxidants on DBS collection filter paper has made it possible to isolate and characterize certain vitamins such as vitamins A, D, E, B, K and other micronutrients like carotenoids from DBS with up to 70 % stability[82,83,84,85,86]. Nutritional profiling in a population can also be used to establish links between levels of these micronutrients and several infectious and non-infectious diseases.


Though DBS provides a vast source of opportunities with its ease of sample collection, storage, and transport, it has its share of limitations as well. Some of the factors which can affect sample quality and recovery from DBS include matrix, hematocrit effect, filter paper quality, and requirement of sensitive detection techniques due to less amount of sample in DBS. DNA extracted is less accurate for P. vivax PCR and hence diagnosis when compared to whole blood samples[87]. However, the difference between, PCR performance of DNA from P. falciparum and mixed infection remains the same for the DNA extracted from DBS and whole blood[87]. Hematocrit effect is still a major shortcoming of DBS use as standard sampling tool for quantitative bioanalytical studies[88,89]. Several strategies have been coming up to cope with this hematocrit based spot area bias, recovery bias and matrix effect including different base/substrate for spotting, hematocrit prediction, in situ generated dried plasma spots and the most recent method of hct prediction using noncontact diffuse reflectance spectroscopy[90,91,92,93,94]. Still there is no universally, accepted method to minimize or completely remove this effect. So, a proper assessment of analyte stability must be established by the researcher before designing any study with DBS as sample source. Specific DBS guidelines must be followed at pre-analytical stage i.e. collection, storage and transport to assure analyte stability and quality.


Currently, when the world is aiming malaria elimination in near future, DBS can prove as the key sample source for stringent and regular epidemiological surveys[95] as traditional venous blood sample method is not feasible for such large-scale population-based studies due to limited economic and human resources. Individual case management, artemisinin combination therapy (ACT) and vector control are currently the main pillars of malaria control and management across the world[11]. Still, drug and insecticide resistance along with low density infections pose major challenges to these efforts of malaria control and elimination, especially in low transmission settings like South East Asia where low density/asymptomatic/subpatent infections act as parasite reservoirs and hinders malaria elimination plans. So, it is necessary to have active and robust surveillance and epidemiological surveys to achieve and sustain malaria elimination. Hence, DBS, as a sampling method, can be employed in countries targeting malaria elimination as an effective surveillance tool that will allow immediate access to these underlying molecular factors associated with malaria.

Ethical statement: Not applicable

Conflict of interest:



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      Malaria; Dried Blood Spots; disease surveillance; molecular markers; genomics; proteomics; metabolomics

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