Rational Primer and Probe Construction in PCR-Based Assays for the Efficient Diagnosis of Drifting Variants of SARS-CoV-2

The genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been evolving via genomic drifts resulting in “emerging/drifting variants” circulating worldwide. The construction of polymerase chain reaction (PCR) assays for the reliable, efficient, and specific diagnosis of the drifting variants of SARS-CoV-2 is specifically governed by the selection and construction of primers and probes. The efficiency of molecular diagnosis is impacted by the identity/homology of the genome sequence of SARS-CoV-2 with other coronaviruses, drifting variants or variants of concern (VOCs) circulating in communities, inherent capacity of mutation(s) of various target genes of SARS-CoV-2, and concentration of genes of interest in host cells. The precise amplicon selection and construction of primers and probes for PCR-based assays can efficiently discriminate specific SARS-CoV-2 drifting variants. The construction of single nucleotide polymorphism (SNP)-specific primers and probes for PCR assays is pivotal to specifically distinguish SARS-CoV-2 variants present in the communities and contributes to better diagnosis and prevention of the ongoing COVID-19 pandemic. In this study, we have utilized in silico-based bioinformatic tools where the alignment for genes, the positions and types of SNPs/mutations of VOCs, and the relative number of SNPs per nucleotide in different genomic regions were investigated. Optimal and specific genome region (amplicon) selection with comparatively lower mutability in the SARS-CoV-2 genome should be prioritized to design/construct PCR assays for reliable and consistent diagnosis in various regions of the world for a longer duration of time. Further, the rational selection of target genes that is at an optimal detectable concentration in biological samples can bolster PCR assays of high analytical sensitivity. Hence, the construction of primers and probes with the rational selection of targeting specific E gene, genomic regions with highly conserved sequences, multiple target genes with relatively lower mutability and detectable level of concentration, SNP-specific binding regions of spike (S gene) protein, and shorter amplicon size (100–150 bp) are vital for the PCR assays to achieve optimal efficiency in the point-of-care laboratory diagnosis of circulating drifting variants of SARS-CoV-2 with optimal accuracy.


Introduction
Cutting-edge molecular assays, specifically quantitative polymerase chain reaction (qPCR), have proved to be a crucial technology to give high sensitivity and specificity for the diagnosis since the early days of coronavirus disease 2019 (COVID-19) emergence [1]. Along with real-time reverse transcription polymerase chain reaction (rRT-PCR), other tests have been designed for the diagnosis of SARS-CoV-2 including serological lateral flow assay test for antigens and/ or antibodies, enzyme-linked immunosorbent assay (ELISA), and reverse transcription loop-mediated isothermal amplification (RT-LAMP) [2][3][4]. In the initial days, clinical diagnosis was even relied upon, in addition to, less specific computed tomography (CT) scans [5]. e laboratory diagnosis of SARS-CoV-2 is a time-bound process as it is highly dependent upon the viral load of the patient, which can fluctuate along the duration of the disease [2].
Among these entire tests, RT-PCR can provide high sensitivity and specificity for the longest duration of disease state, i.e., from the disease incubation phase till recovery phase [2]. e rRT-PCR is considered as a gold standard for SARS-CoV-2 diagnosis and has continuously been used widely in public health/diagnostic laboratories from resource-constrained/limited to well-resourced settings in the fight against the ongoing COVID-19 pandemic. Wholegenome sequencing (WGS), an invaluable tool for genomic surveillance, has helped to identify novel infections [6] and variants and develop diagnostic kits for rapid detection and outbreak containment [7]. However, it is still not applicable for the point of care for routine laboratory diagnosis in poor/ limited resourced settings due to costly next gene sequencing (NGS) machine, reagents, and consumables, requiring wellequipped and highly sophisticated laboratory and welltrained/skilled human resources. Approximately 4.3 billion tests have been performed with 287 million confirmed positive cases worldwide for 7.8 billion world population by the end of December 2021 [8].
Coronaviruses, or Nidovirales order in general, have the largest RNA genomes of all RNA viruses known, and due to high-fidelity RNA replication and transcription machineries, the number of new mutations occurring per replication is relatively low (S G) [9] in comparison with other RNA virus such as orthomyxovirus. However, a large population of infected individuals and the selective pressure of evading the immune system and having a better target cell attachment factors have given rise to new variants of concerns (VOCs), such as Alpha, Beta, Gamma, Delta, and Omicron variants.
ese variants have accumulated a considerable number of mutations, especially in their structural genes such as S and N, and as a result, some variants such as Omicron variant may not be detectable by currently available diagnostic tests due to S gene target failure or S gene dropout [10]. Due to resource constraints, commercial diagnostic kits that detect only one to two genes have also come to be used [11], and this may increase the chance of false-negative results in case qPCR fails to diagnose one or two genes.
Studies have reported variable sensitivities for the rRT-PCR assays used to diagnose SARS-CoV-2 [12,13]. Although test sensitivity could be lowered by errors in methodology, instrument, and diagnostic kits, a decrease in PCR efficacy due to mutation in the primer or probe binding sites is very hard to account for [12] unless sequencing of the PCRtargeted genomic region is carried. Primer and probe design plays particularly important roles to ensure target detection and quantification, replicate accuracy, sensitivity, and amplification efficiency of all targets, and reduce variant-specific cross-reactivity. Each primer and probe set needs to be evaluated on an individual basis (reference genes and genes of interest) to determine designs and conditions that are ideal for the target amplification via standard curve methodology with dilution series to achieve close to 100% efficiency. e combination of proper functional validation primers and probes in a reaction is also desired as different primer pairs and/or probes of combination in a reaction should not interact with each other. e prime objectives of the PCR assays are to provide a reliable diagnosis for a longer duration of time during the evolution/mutations of the virus with optimal accuracy. Currently available commercial molecular rRT-PCR kits have lower efficiencies and/or inefficient to detect/differentiate the drifting variants of SARS-CoV-2 [10]. Primers and probe design/construction are arguably the most crucial factor in a multiplex assay where more than one target is analyzed in the same real-time PCR. erefore, the construction of primers and probes based on the latest genetic information/data of emerging SARS-CoV-2 variants/drifting variants is desired to enhance real-time PCR efficiency and the optimal accuracy necessary to discriminate variants for COVID-19 diagnosis.
is bioinformatic study provides the rational primer and probe construction techniques/deep understanding of PCR-based assays for the reliable detection of SARS-CoV-2 variants ensuring optimal accuracy and replicability.

Methods
In silico-based bioinformatic assessments were performed comparing SARS-CoV-2 Wuhan-Hu-1 (reference genome) to VOCs (Alpha, Beta, Gamma, Delta, and Omicron), bat SARS-like coronavirus, SARS-CoV, and bat coronavirus. Whole-genome sequence (WGS) or specific gene sequence data of SARS-CoV-2 were collected from NCBI Nucleotide GenBank and http://www.GISAID.org. MUltiple Sequence Comparison by Log-Expectation (MUSCLE) [14] was used for multiple sequence alignment (MSA) among genes of interest or gene fragments by MEGA11 software version 0.1. MUSCLE claims to achieve relatively higher average accuracy and better speed than ClustalW2 or T-Coffee, on the chosen options [14]. Positions and types of SNPs or mutations for variants of SARS-CoV-2 are described as specified in the GISAID and NCBI GenBank databases.
We have used supplementary data provided by Mercatelli et al. [15] for the analysis of mutations in various target genes to aid in the selection of specific primers and probes for better diagnosis of SARS-CoV-2 variants. Sample (SARS-CoV-2 genomes) extracted from http://www. GISAID.org exhibited their distribution and size (N � 48635) according to regions that are presented in Table 1. e sampling distribution has been skewed towards more developed regions of the world. Two-third of samples were contributed from Europe, one-third of samples jointly were contributed from North America, Asia, and Oceania, and the rest (2% sample) were contributed from Africa and South America. We also included 0.02% sample (11 genomes) with no origin of sample submission (GISAID.org) for our analysis, which does not necessarily impact our results/findings with this comparably minimal quantity of sample size (Table 1).
A preprint of the first draft of this study has previously been uploaded in bioRxiv [16] (https://www.biorxiv.org/ content/10.1101/2021.04.04.438420v1.full.pdf ), and an updated version is presented in this article. For the specific analysis of the collected genetic data, WPS Spreadsheet version 11 and GraphPad Prism 5 were utilized. We have calculated the relative number of SNPs per nucleotide in different genomic regions. Even though these data only include variant data as of June 2020, this analysis will help to understand the general trend of genes to mutate and give an idea about their relative mutability. We have determined unique mutation events in the genome of SARS-CoV-2, by grouping entries into "refpos," "refvar," and "qvar" categories in WPS Spreadsheet and by removing the entries with duplicate SNP variants. However, we have retained the SNP entries where the same nucleotide may have undergone different kinds of mutations.
To accurately quantify mutations in the 3′ UTR region, entries were removed for sequences corresponding to 3′ UTR at or before nucleotide 29674, i.e., the last nucleotide for ORF10, and only sequence from 3′ to ORF10 was included as true 3′ UTR. We also removed intergenic SNPs, which were 3 in total. We combined entries for NSP12a and NSP12b into NSP12, which corresponds to the RdRp gene. We calculated the number of mutations or SNPs, prevalent or unique, per nucleotide (Nt) per 10,000 genomes for each of the SARS-CoV-2 genes by dividing the number of SNPs in a given gene by product of nucleotide size of that given gene and number of genomes analyzed and then multiplying it by ten thousand. Prevalent mutation here includes all the mutations contained in each and every viral genome. e numbers were rounded off to two decimal places. Figures were drawn in GraphPad Prism v5 and WPS Excel. Gene coordinates of SARS-CoV-2 Wuhan-Hu-1 genome (NCBI GenBank Accession ID : NC_045512.2) were used as reference. As mutations of all kinds, sense or nonsense, impact PCR, mutations discussed here do not represent the evolutionary implications.

Selection of Target Genes for PCR Assays.
Among the genes of choice used by WHO-collaborating laboratories, the Corman group (Charite, Germany) [1] used E gene and RdRp genes for the diagnosis of COVID-19, while other laboratories used N and/or ORF1 genes [17]. Commercially, the S gene has also been used [11]. "ORF1ab" has been frequently stated to be used for COVID-19 detection in the commercial PCR kits, but it is not clear whether it is the RdRp gene (NSP12). e first set of primers designed by the Corman group [1] selected RdRp and E gene as genes of choice. ough primers for N gene were also designed, the gene was abandoned due to low sensitivity during optimization. During the initial days of the pandemic, the exact strain of the virus was not confirmed and the Corman group recommended a set of primer for E gene, which could amplify the newly sequenced virus and similar SARS-like viruses and was considered a screening gene. is could be due to the relatively low synonymous and non-synonymous mutational differences in the SARS-CoV-2 virus compared with orthologous sequences from other bat and pangolin coronaviruses and SARS-CoV-2 [18], but this does not mean that the E (envelope) gene is the same in all of the related viruses.
We utilized MUSCLE, a high-throughput application for multiple sequence alignment, to achieve the highest score in sequence alignment and reductions in computational complexity [14].
We conducted MSA for the entire E gene (228-231 bp) (   We distinguished a total of 7 nucleotide position mismatches within 113 nucleotide-long E gene PCR amplicon fragments (6.2%) among the 8 GenBank sequences analyzed. We also observed that the major mismatch (4 of total 7 mismatches � 57%) was contributed by bat coronavirus BM48-31/BGR/2008 (NC_014470.1) in 113 bp long E gene PCR amplicon fragment among the 8 sequences tested. Of 7 mismatches in E gene PCR amplicon fragment, 4 (57%) mismatches were identified in the nucleotide seq. region of forward primer, probe, and reverse primer position. In forward primer, probe, and reverse primer nucleotide seq. region, 3 (75%) nucleotide position mismatches were attributed to bat coronavirus BM48- 31  ese analyses signify that both E and N gene primers and probes designed by the Corman group could reliably identify the SARS-CoV-2 strain at that time and the dominant variants that would come at least until early 2022. Most of the heterogeneities in MSA were attributed to bat SARS-like coronavirus, SARS-CoV, and bat coronaviruses. Mutations in the 5' ends of primers and 3′ ends of probes may not significantly hamper PCR efficiency. As E gene was the most conserved among the coronaviruses, the selection of this gene as screening gene was obvious. e use of degenerate nucleotides in the primers and probes or selection and optimization of primer and probe binding sites from more conserved areas could have more correctly screened the novel SARS virus at that time.

Advances in
China-CDC targeted ORF1ab and N genes and Institut Pasteur, Paris, France, targeted two regions of RdRp gene. Genes targeted by US CDC were two regions of N gene and by the National Institute of Infectious Diseases, Japan, were ORF1ab and S genes for conventional PCR and N gene for RT-PCR. Hong Kong University, China, targeted NSP14 in ORF1b and N genes, and the National Institute of Health, ailand, used N gene [19].
As of now, no significant problems in the PCR-based diagnosis of COVID-19 have occurred. Concerns and issues related to false-positive tests due to late-Ct value produced by the Corman primers (Eurosurveillance Editorial Team 20210) [20,21] have been reported. e selection of multiple gene targets can solve this problem and efficiently and precisely detect SARS-CoV-2.

Drifting Variants/VOCs of SARS-CoV-2.
Various SAR-S-CoV-2 variants have been evolving and circulating worldwide since their emergence. Different VOCs have been reported till date. WHO has defined VOCs as "having either increased transmissibility or detrimental epidemiology, or increased virulence or clinical presentation, or decreased effectiveness of public health and social measures or available diagnostics, vaccines and therapeutics" [6]. One of the first variants (not recorded as "variant of concern") was a D614G mutation, which was first reported in March 2020. e variant was called the "G clade" and was found to spread faster [22]. It represented 10% of global sequenced infection before March 2020 to around 80% by mid-May 2020. Regarding G clade, in vitro study found increased infectivity [22]. In fact, all of the VOCs today are subtypes of the G clade. Since then, the pandemic has been dominated by the Delta variant and recently by the Omicron variant. Figure 2 shows the increasing prevalence of G clade in 2020 and the domination of the G clade VOCs in 2021. e defining mutations of all of the VOCs are present in spike protein, while mutations in other genes may also affect the biological characteristics of the variants. e major prevalence/distribution of SARS-CoV-2 G clades was observed in 2020, while the domination of the G clade VOC distribution was seen in 2021 ( Figure 3). ree VOCs, Alpha, Delta, and Omicron variants, have dominated the SARS-CoV-2 pandemic. e Alpha variant (B.1.1.7) was first detected in the United Kingdom in November 2020 and remained a dominant variant till the emergence of Delta variant. It has 17 mutations, 8 in spike protein among which mutation N501Y enhanced its binding affinity and increased this variant transmissibility by more than 40% of the original strain [25]. e Delta variant (B.1.617.2 lineage), which was first reported from India in December 2021, remained the most prevalent variant till the emergence of the Omicron variant. Delta variant is considered to be 40-60% more transmissible than the Alpha variant [24]. According to the US FDA, N gene and S gene dropouts are not typically seen in Delta variant [10]. As of March 2022, the Omicron variant (BA.1), which was first reported from Botswana and South Africa in November 2021, has become the most dominant variant.
e Omicron variant has an N gene with ninenucleotide deletion from 28370 till 28362 bp, and an S gene with a specific deletion (delE31/R32/S33) can cause genetargeted failure for N and S gene and reduced test sensitivity [10]. Although the PCR assay/test that has multiple targets may not have a reduction in sensitivity, the defining mutations of all of the VOCs are present in spike protein while mutations in other genes may also affect the biological characteristics of the variants. e loss in sensitivity in PCR diagnosis has occurred for commercial kits targeting the genomic sites where mutations have been emerging. Commercial kits will not have decrease in PCR sensitivity if they are specifically targeting genomic regions where mutations are not detected. Table 2 demonstrates mutations present in spike proteins of the VOCs. e Sanger sequencing of the given nucleotide regions of the spike proteins may help to identify the strains/ variants at a local level. To identify SNPs or mutations in 6 Advances in Virology VOCs, cutting-edge molecular PCR assays can be utilized. Optimization to differentiate these variants can prevent the cost of sequencing, but SNP-specific PCR may be cumbersome if there are many variants. Also, this SNP-specific PCR may be easily hampered by any new insignificant SNP near the target SNPs. e molecular construction of SNP-specific primers and probe targeting genes of interest is essential to efficiently differentiate the drifting variants of SARS-CoV-2, which can identify VOCs.

Rational Selection of PCR Primer and Probe Binding Sites.
Reference [15] has analyzed over 48 thousand SARS-CoV-2 genomes till June 26, 2020, deposited them in the GISAID database till June 26, 2020, and found over 350 thousand mutations in the viral genomes [15]. e effect of mutations on qPCR sensitivities has been exemplified in a previous influenza pandemic [25]. e impact of mutations on PCR sensitivity carried at the community-or country-level depends upon two factors: first, the relative propensities of the target gene areas to undergo mutation and, second, the prevalence of such mutated clades/strains in the population [26]. e first scenario is described as prevalent mutations (Table 3A, Figure 4(a)) and the second by unique mutations (Table 3B, Figure 4(b)) in this article. We attempted to identify which genes had lesser mutability and whether it could be used as reliable target sites for PCR.
Based on the number of unique SNPs per nucleotide, we found that NSP10 was the most conserved region (Figure 4), and 3' UTR and 5′ UTR were the least conserved regions with a high tendency to undergo mutation compared with other regions. Similar results were seen when the prevalence of total/ prevalent SNPs was compared among different genomic regions with NSP10 being the most conserved and 5′ UTR being the least (Figures 4(a) and 4(b)). In general, nonstructural proteins were more conserved compared with structural ones (Table 3). During the multiplication process of the virus, the whole plus-stranded genomic RNAs are synthesized from minus-strand templates by a single type of replication machinery [9], and thus, the nonstructural genes in open reading frame 1ab and the nonstructural genes in the 3' end of the coronavirus genome should have similar mutation rates. While there could be different factors involved in this phenomenon, one of them could be evolutionary pressure for structural proteins to evolve [27]. Structural proteins are exposed to antibodies in the respiratory mucosa or blood during infection and transmission from one cell to another. e nonstructural proteins help in the intracellular physiology, particularly related to replication and transcription, and thus are unexposed to antibody. us, the structural proteins need to evolve to evade antibody-based suppression [27] of infection to new cells. Similarly, based on the results (prevalent and unique mutations) obtained, E protein was the most stable followed by M, N, NSP12ab/RdRp, and S. A similar study carried out in the United States found leader sequence, NSP2, NSP3, RdRp, helicase (NSP13), spike, ORF3a, ORF8, and nucleocapsid proteins to have accumulated mutations during a 4-month period (January to April) in 2020 and few other (NSP7, NSP9, NSP10, NSP11, Envelope, ORF6, and ORF7b proteins) did not accumulate mutation [28]. e study looked into non-synonymous mutations, whereas our study included both synonymous and non-synonymous mutations as both kinds of mutations impact PCR. As found in their study, NSP9 and NSP10 are also among the least mutating genes in our study. Similarly, 5′ UTR leader, ORF3, N, and ORF8 are highly mutating in both of these studies.
Usually, synonymous (or silent) mutations are not responsible for changes in amino acid sequences. Only nonsynonymous changes alter the amino acid sequence in proteins, but PCR deals with nucleotide sequences in DNA or cDNA.
us, both synonymous and non-synonymous   Advances in Virology Advances in Virology changes impact the binding of primers and/or probes. Hence, we used both synonymous and non-synonymous sequences for our analyses.

Rational Primer and Probe Concentration Targeting Genes of Interest in Host Cells.
Transcriptomic analysis of SARS-CoV-2 has repeatedly shown a higher prevalence of reads from the 3' sub-genomic RNAs in the infected host cells [29]. While this could be due to a higher concentration of the sub-genomic RNAs located in the 3′ end, the methodological bias due to sequencing from the 3' end of the genome might have impacted the viral sequence reads [29]. e higher reads in the 3′ region have been supported by translational studies [30]. Reference [31] has iterated that the cellular concentration of plus-stranded RNAs, which are synthesized using minus-strand as a template, is 50-to 100-fold higher than the minus-stranded RNA for coronaviruses (Stanley G) [31]. e transcription mechanism of the coronavirus causes the 3′ end of its genome to have higher reads. e so-called nested sub-genomic structures are formed during the coronavirus transcription process where the genes in the one-third 3' end of the viral genome are translated alone but not transcribed alone. is means, whenever a gene, 3′ to the ORF genes, is transcribed for its translation, all other genes in 3' direction to that particular gene are redundantly transcribed too. Considering that the concentration of each of the plusstranded translatable sub-genomic RNA units is 50-100 times more than their corresponding minus-strands, this results in a higher number of sequence reads for genes as we go towards the 3′ end of the genome causing the highest number of reads for N gene RNA followed by M, E, and S [29]. is could be the reason why the Ct values for structural genes N and E have better readings (lower Ct values) than that of the RdRp genes [32,33]. us, the 3' end sub-genomic RNAs for the structural proteins, which are present at higher concentration in the clinical samples, can be better regions for primer and probe design in terms of better analytical sensitivity. Genetic positions within specific genes may have variable propensities to mutate depending on the exposure of different motifs to the antibody environment or abilities to cope with changes in amino acid combinations. e mutation patterns, prevalent ( Figure 5(a)) and unique ( Figure 5(b)) along 5′ to 3′ direction for target genes used in qPCR diagnosis (NSP12ab/RdRp, S, E, M, and N), are shown in Figure 5. Visual screening of the mutation status along the nucleotide length of the genes can aid the selection of precise regions of each gene for primer and probe design. One should be careful to interpret that the shown mutation pattern in Figure 5 represents more than 45000 viral genomes, and thus, any one clinical sample is highly unlikely to contain all the mutations.

Conclusion
For routine point-of-care SARS-CoV-2 confirmatory laboratory diagnosis, rRT-PCR/qPCR is considered as a gold standard technique and is still widely used in the battle against the ongoing pandemic threat posed by the emergence of COVID-19 drifting variants/VOCs. e selection of better PCR target amplicon regions depends upon various factors. It may depend upon whether the investigators want to detect SARS-CoV-2 along with other related viruses. Investigators may also wish to determine and/or discriminate exact variants of interest present in the communities.
Evolving new mutation or drifting variants in SARS-CoV-2 genomes may render PCR assay to have variable sensitivities and specificities. We recommend low mutating structural genes to be used to better discriminate drifting variants of SARS-CoV-2 diagnosis. Recently, the S gene target failure in the UK variant of concern (Alpha strain) strains tested by Applied Biosystems TaqPath RT-PCR COVID-19 Kit was found to be due to 6-nucleotide deletion mutation in the spike gene region targeted by the kit [34]. If a diagnostic kit targets only S gene at the 6-nucleotide deletion region, the diagnostic result could be interpreted as negative for the clinical  samples tested. us, commercial kits with multiple genetic targets are advisable for the precise diagnosis of COVID-19. While the construction of primers and probes and the product sizes of individual genes in multiplex real-time PCR, in general, are maintained to be of equal or near-equal and shorter lengths (100-150 bp) and of equivalent GC contents among the target amplicons in addition to other factors [35], primers with higher GC contents (40 to 60%) help to prevent mismatch stabilization and ensure stable binding of primers and template. Designing primers to amplify a segment ranging from 60 to 150 bp enhances PCR efficiency. is allows the selection of regions with lower SNP loads within target genes, to design reliable primers and probes. e structural proteins 3′ end sub-genomic RNAs exhibiting relatively higher concentration in clinical samples could be the suitable region for designing primers and probe set for better analytical sensitivity. e findings of this study revealed that the gene of interest "E gene," which is the most conserved sequence and highly expressible structural gene of SARS-CoV-2 genomes, needs to be prioritized for the design of primers and probes for PCR-based assays for efficient diagnosis. In addition, SNP-specific binding regions of spike (S gene) protein should be considered for the construction of primers and probes with shorter PCR amplicon size, which enhances the efficiency and precision for VOC differentiation in SARS-CoV-2 diagnosis.
is study recommends the rational primer and probe design targeting the conserved sequence region of E gene, SNP-specific binding regions of spike (S gene) protein, multiple genetic targets with relatively lower mutability and detectable concentration level (ORF7a, ORF7b, etc.), target amplicons with equivalent GC contents and lower SNP/ mutation loads, and shorter amplicon size (100-150 bp) to be necessary for the PCR assays to achieve optimal efficiency, sensitivity, and reproducibility in the diagnosis of SARS-CoV-2 variants. However, each primer and probe set designed needs to be evaluated, optimized, and validated prior to being used in routine laboratory diagnosis.

Data Availability
Raw data available in the supplementary files in this study were taken from [15] (permission for using data has been sought and granted from the corresponding author), which were originally extracted/downloaded from http://www. GISAID.org. Excel files are available in the supplementary section. GraphPad files used during the analysis in this study are available on request.

Conflicts of Interest
e authors declare they have no conflicts of interest.

Authors' Contributions
SD and DRSJBR conceived the study. SD, DRSJBR, and NP analyzed and wrote the manuscript. SD, DRSJBR, and NP agreed on the final manuscript.