Malaria is a serious global health problem causing over 600,000 deaths in 2021 [1]. Correct diagnosis is necessary for prevention, control, and suitable treatment of malaria, and commonly performed by microscopy, rapid diagnostic tests (RDTs), and molecular methods [2]. Success of a molecular method for malaria diagnosis depends critically on the quality of genomic DNA being used for examination, which in turn depends on accuracy of the employed isolation procedure.

Multitudes of different DNA extraction methods have been published [3] since the first DNA isolation in 1869 [4], and each method must overcome specific extraction problems such as DNA shearing, background contamination, low purity, and low yield. Of these, dried blood spot (DBS) is one of the most common methods used to collect and extract DNA in field studies of malaria molecular epidemiology, where transport and storage conditions are often suboptimal. This technique is minimally invasive (requiring only microliters of typically peripheral blood), inexpensive, and easy to multiplex and automate [5]. DBS samples are compatible with many bioanalytical methods, among them chromatography, mass spectrometry, DNA, and immunoassays. However, DNA extraction is particularly challenging in resource-limited regions, where scissors are used to cut filter papers, possibly introducing contamination between samples due to poor scissor cleaning.

Many DBS methods use 70% ethanol to clean the scissors, but it is unknown whether ethanol or other cleaning solutions may be a source of sample contamination. We drafted this letter to shed light on concerns regarding contamination of samples extracted by scissors which may impact interpretation of molecular epidemiology results.

Results of the nested PCR revealed that no bands were detected in samples washed with DNase. Schematic representation of agarose gel electrophoresis of nested PCR products from SW1, SE1 and SD1 show the presence of bands at 565 bp and 534 bp in msp1 (Figure 3) and msp-2 (Figure 4) respectively. In the nested PCR assay using msp-1 and msp-2, no band was detected in SW1, SW2, SE2, SD1, SD2, RE and RD tested. We detected bands in SE2 in msp-1 and SE1 and SE2 in msp-2, respectively. Only blank confetti catted by scissors washed with ethanol presented bands, detected in C1 and C2 on agarose gel electrophoresis of digestion PCR products, but no band was detected with C3 (Figure 5).

Ethanol is a non-additive precipitant fixative that shifts isoelectric points of proteins less than other fixatives [7]. It fixes proteins by dehydration and precipitation, the degree of which being dependent on the amount of water present and the solubility of materials in the mixture. The basic aims of fixation are to preserve the tissue nearest to its living state, and to prevent any change in shape and size of tissue at the time of processing. This could possibly explain how pieces of blood-stained confetti attached to scissors after being dipped in ethanol. In addition, we observed one band from residual of water (RW) in msp-2. Water is by far the most studied chemical compound [8] and called the universal solvent since it can dissolve more substances than any other liquid [9]. After dipping in water, particles of confetti on scissors detached themselves and remained at the bottom of the water. DNase is an endonuclease that digests single and double-stranded DNA; it hydrolyzes phosphodiester bonds producing mono- and oligodeoxyribonucleotides with 5’-phosphate and 3’OH groups. The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions: 1) in the presence of Mg2+, DNase cleaves each strand of dsDNA independently in a statistically random fashion [10]; 2) In the presence of Mn 2+, the enzyme cleaves both. DNA strands at approximately the same site, producing DNA fragments with blunt ends or with one or two nucleotide overhangs [10]. We did not detect any band with msp-1 and msp-2 in scissors washed with DNase.

Scissors cleaned with DNase prevented cross-contamination between samples during processing of dried blood spots. Ethanol cleaning of scissors, which is commonly used, failed to avert cross-contamination. Sample cross-contamination can reduce diagnosis accuracy; we recommend DNase wash as the standard cleaning solution to improve DNA extraction.