Effect of Bead-Beating on DNA Extraction from Trichuris trichiura Positive Stool Samples: A Meta-Analysis

Polymerase chain reactions are helpful diagnostic methods to accurately quantitate the intensities of infections of various soil-transmitted helminths, especially in the low-intensity infection samples. Method of DNA extraction hugely impacts the outcomes of the diagnostic method. Trichuris trichiura is one of the three major helminths prevalent world-wide and accurate estimation of their loads in stool is affected by the method of DNA extraction. We meta-analyze two studies by dividing them into all together 6 sub-studies. The objectives of the meta-analysis required the two studies to be divided into sub-studies as the different methods of DNA extractions could not be combined. We found that the inclusion of the beadbeating step during DNA extraction significantly increases the sensitivity of the test.


Introduction:
Soil-transmitted helminths (STH) claim high disease burden especially in the poor parts of the world (1) . The diseases, while being treatable by common deworming medications and preventable with common hygienic standards (2) , are a major contributor of worldwide morbidity. Roundworm (Ascaris lumbricoides, ascariasis), whipworm (Trichuris trichiura, trichuriasis) and hookworm (Ancylostoma duodenale and Necator americanus, ancylostomiasis and necatoriasis) are the three major worms among others that claim the highest disease morbidities. Round worm and whipworm are transmitted by faeco-oral route (3,4)  While the extraction of DNA from stool samples containing roundworms and hookworms pose no reported difficulties, the extraction of DNA from Trichuris eggs have been reported to require additional handling (10) . More specifically, studies have repeatedly pointed out the requirement of bead-beating step during DNA extraction for proper extraction of the Trichuris DNAs. A meta-analysis of comparative studies was carried to search for articles which assessed the relevance of bead-beating during DNA extraction. While many studies incorporate bead-beating into their DNA extraction methods, very few have done comparative studies to show the efficacy of inclusion of this step.

Material and Methods:
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 April 2020 doi:10.20944/preprints202004.0419.v1 Search strategy: Articles were searched in Pubmed by using "AND" Boolean operator to combine two search fields, "qPCR OR quantitative polymerase chain reaction OR quantitative PCR OR quantitative real-time PCR OR polymerase chain reaction OR PCR" and "Trichuris OR Trichuriasis OR Whip worm OR Whipworm OR Trichuris Trichiura".
Additional search was carried in Google search engine with search term "Trichuris bead beating DNA extraction".
Additional search were carried to study the biochemical structure of helminth eggs.
Statistical Analyses: Random effects model odds ratio was estimated for the meta-analysis. Meta-analysis was carried in MedCalc software which uses Mantel and Haenszel (1959) method for fixed effect model and DerSimonian and Laird (1986) method for random effects modelling (11) .

Results:
Search zirconium beads using NucliSENS easyMag DNA extraction system (Biomerieux, USA). They concluded (no data reported) that zirconium beads gave best results for DNA isolation. The study found that vortexing without any beads gave comparable results for Trichuris trichiura analytical sensitivity compared to bead beating with zirconium beads beaten for 30 secs at 7,000 oscillations. They reported that the clinical sample showed lower (better) limit of detection compared to the artificially spiked sample hinting the presence of microscopically invisible extra-cellular DNA in clinical stool samples.
Study by Kaisar (2017) (13) Fig. 2). None of the individual sub-studies showed a significant effect of bead-beating while the random effects model showed a significant effect. This could be due to small sample sizes in the individual studies. None of the small sub-studies showed a negative effect of bead-beating. Thus, it can be safely concluded that the significant effect in the meta-analysis is due to synergistic effect all the sub-studies whose  Biochemical studies of helminth eggs: The eggs of nematodes have been known to be to resist various environmental stresses and chemical. The egg of Ascaris alone has shown to remain viable against various acids, alkali and digestive enzymes (14-16) . This extreme resilience is hypothesized to be due to the eggshell, which has to be strong enough to protect the embryo inside (17) . Nematodes eggshells, including those of Hookworm, Trichuris and Ascaris, in general, are made up of 3 layers: outer vitelline layer, middle chitinous layer and inner lipid layer. The middle chitinous layer is a composite layer composed of chitin microfibrils surrounded by protein matrix, which is said to make the egg resistant to mechanical damage (18-20) . The inner lipid layer is impermeable to most chemical (21,22) and maybe a reason for the difficulty in DNA extraction using chemical process. In many nematodes, including Ascaris, it consist of ascarosides esters which could be a reason for the impermeability (23,24) , and is said to be responsible for resistance against chemical actions (25) .

Discussion:
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 April 2020 doi:10.20944/preprints202004.0419.v1 Increasing number of STH studies are using bead-beating steps in the DNA extraction method. While the possible need of this step have been raised earlier (7,26) , very few studies have actually reported the significance of this addition. Though Trichuris is considered to be the only STH that require the bead-beating, Ayana et al have found that this could also be required for hookworms (9) . They also reported that the blood and tissue kit gave better result than the stool kit. It may be because kits are usually designed for the isolation of bacterial DNA in the stools (27) . The sizes of helminth eggs for hookworm, Trichuris and Ascaris are comparable and the biochemical structures of the egg shells are similar too. While the hookworm eggs are known to be fragile and can rupture within hours of stool collection at room temperature, the Trichuris and Ascaris eggs remain in soil for longer durations. Present meta analysis shows that inclusion of bead beating makes significant impact on the DNA extraction of Trichuris DNA and recommends the use of this technique.
Limitations of this study include only two studies being analyzed due to paucity of systematic comparison studies. We have divided the two studies into total of 6 sub-studies. It was not possible to keep the sub-studies combined as the methods of stool preserved, kits used and the cohort of patients studied, if combined, could affect the outcome concluded. Another limitation could be non-inclusion of the cycle threshold data for the PCR which could differentiate the slight changes in the yield of Trichuris DNA extracted using various interventions during DNA extraction. But most of the studies also do not report the mass of the DNA template used in the PCR and only volumes of template DNA are mentioned.

Conclusion:
More systematic studies assessing the effect of bead-beating on DNA extraction of Trichuris is recommended.
As reported by the two studies above, preliminary optimization of the best bead types and/or sizes should also be carried so the impact is clearly concluded.