Abstract
Human skin microorganisms have been associated with various skin diseases. However, most studies have focused on bacterial communities, and little is known about normally resident skin viruses such as the Polyomaviridae and their association with cutaneous disorders.
We investigated the infection levels of Merkel cell polyomavirus (MCPyV), human polyomavirus 6 (HPyV6), and human polyomavirus 7 (HPyV7), using triplet skin swabs collected from lesional and nonlesional skins of 86 Japanese patients with inflammatory skin diseases and mycosis fungoides, and from 149 healthy control individuals.
This age-matched case-control study provides the first analyses of the loads of polyomaviruses in association with various skin diseases. The viral loads were significantly higher for HPyV6/HPyV7 and lower for MCPyV in patients with psoriasis. The viral load variation was observed not only at lesion sites, but also at clinically unaffected skin sites in most of the patients. The viral strains tested were all of the Asian/Japanese genotype.
Our findings suggest a covariation in the infection levels of cutaneous polyomaviruses in certain inflammatory skin conditions. Worldwide prospective longitudinal studies are warranted to understand the influence of such alterations on the pathogenesis of inflammatory skin disorders.
Cutaneous human polyomaviruses (HPyVs), such as Merkel cell polyomavirus (MCPyV), human polyomavirus 6 (HPyV6), and human polyomavirus 7 (HPyV7), infect children asymptomatically and persist in the skin with low viral loads [1–4]. These viruses have been shown to compose a ubiquitous part of the skin’s virome [5–8]. Studies have demonstrated that the genotypes of MCPyV, HPyV6, and HPyV7 vary between strains prevalent in the healthy skins of white (defined here as people of European descent) and Asian/Japanese populations [1, 2, 9, 10], assuming that the ecology of commensal HPyVs on the skin might differ between the 2 population types.
MCPyV can cause cutaneous Merkel cell carcinoma (MCC) [11], possibly in the skin harboring high MCPyV loads [12]. The incidence of this malignancy is higher in white persons than in Asian populations [13, 14]. HPyV7 has been associated with chronic pruritic eruption characterized by a unique parakeratosis pattern [15]. A similar clinical and histological presentation has also been described in association with HPyV6 infection [16]. Moreover, HPyV6 has been linked with a chronic inflammatory disorder with subcutaneous nodules known as Kimura disease [17], which is endemic in East Asia [18, 19]. These findings suggest that HPyV6 and HPyV7 appear to be associated directly or indirectly with the pathogenesis of inflammatory skin conditions and that the incidence of HPyV-associated skin diseases might vary globally. In addition, HPyV6 and HPyV7 have been detected in several types of epithelial neoplasms, suggesting that these viruses might also have oncogenic potential [20–22].
In recent years, the numbers of skin microbiome studies in association with skin diseases have been rising. Specifically, atopic dermatitis (AD) has been associated with imbalances of the skin microbiome characterized by increased colonization with Staphylococcus aureus [8, 23, 24]. AD is a chronic inflammatory skin disorder with a much higher prevalence in adult Asian patients (7%–10%) than in adult patients from Europe and the United States (European and American patients: EA patients) [25–27]. Asian patients with AD have a unique immune phenotypic characteristic with a blended phenotype between EA patients with AD and patients with psoriasis [28]. It is conceivable that members of the skin microbiome might affect immunity by activation of immune cells in the skin and that microbial conditions might be altered in certain inflammatory skin diseases. However, to date, most skin microbiome studies have focused on bacterial and fungal communities, and skin viral communities and their relationship with skin diseases remain poorly understood.
Given this background, here we investigated for the first time the prevalence and loads of MCPyV, HPyV6, and HPyV7 in swab specimens obtained from both normal-appearing skin and flared lesions of Japanese patients with chronic inflammatory skin diseases, including AD, psoriasis, and palmoplantar pustulosis. Studies have suggested a possible association of S. aureus with the etiology of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma [29, 30]. Parapsoriasis is considered to be an early stage of MF [31]. Therefore, we also investigated whether these skin lesions would have different populations of HPyVs and S. aureus compared with normal skin of healthy individuals.
METHODS
Study Participants and Specimen Collection
This study cohort included 86 Japanese patients having active skin diseases, including 21 patients with AD (median age, 35 [range, 16–57] years), 24 with psoriasis (median age, 62 [range, 22–88] years), 20 with palmoplantar pustulosis (median age, 56 [range, 32–69] years), 11 with parapsoriasis (median age, 67 [range, 43–77] years), and 10 with MF (median age, 59 [range, 46–78] years). There were 43 male and 43 female patients. The subjects had not received antibiotics or ultraviolet phototherapy at the time of sampling. Skin swab specimens were collected from an area of about 50 cm2 of the worst-affected rash lesion from the arm or the trunk by rubbing back and forth 5–10 times with sterile cotton swabs. Specimens were also collected in a similar manner from 2 normal-appearing skin sites at a distance from the lesion, usually on the contralateral side of the upper extremity or the trunk and on the forehead of the same patient’s body. In addition, as age-matched controls, 149 healthy Japanese volunteers (age range, 16–88 years; 70 men and 79 women) agreed to participate in this study, and specimens were obtained from 2 healthy skin regions of the arm or the trunk and forehead to match the skin sites of patients. They had no history of skin diseases and did not receive any special skin care at the time of sampling. All swabs were placed in a separate vial containing sterile phosphate-buffered saline, and DNA was extracted within 2 hours of sample collection.
This study was approved by the Ethics Committee of Kochi Medical School, Kochi University, Japan. Written informed consent was obtained from all participants. When individuals <18 years of age or elderly persons with impaired decision-making capacity were enrolled, their parents or families provided written informed consent on their behalf.
Quantification of Human Polyomavirus Loads
DNA extraction was performed using the standard phenol-chloroform method. Twenty-nanogram aliquots of extracted DNA were analyzed for the quantification of MCPyV, HPyV6, and HPyV7, using TaqMan-based quantitative real-time polymerase chain reaction (qPCR), as described elsewhere [1, 2, 32]. Primers and probes were prepared to amplify the viral small T antigen (ST) gene for detection of MCPyV and the large T antigen (LT) gene for detection of HPyV6 and HPyV7 [1, 2]. The sequences of primers and probes are listed in Supplementary Table 1. Results are expressed as viral DNA copies/ng DNA. Because samples with cycle threshold values ≤40–41 were considered to be positive for targeted viral nucleic acids, we considered swab specimens with ≥0.1 copies/ng DNA and ≥0.3 × 10–2 copies/ng DNA to be positive for MCPyV and HPyV6/HPyV7, respectively, according to published criteria [1, 2, 33]. We confirmed the detection specificity of the assay, finding no cross-reactions among HPyV6, HPyV7, and MCPyV [2].
Viral DNA Sequencing and Phylogenetic Analysis
The viral sequences of the LT gene of MCPyV (nucleotide positions 2148–2547 based on the GenBank MCPyV sequence MCC350; accession number EU375803 [11]), HPyV6 (nucleotide positions 3944–4926; accession number HM011560 [4]), and HPyV7 (nucleotide positions 4406–4969; accession number HM011564 [4]) were amplified by PCR, using a primer set for the MCPyV sequence and a combination of the 2 primer sets for the HPyV6 and HPyV7 sequences (Supplementary Table 1). The purified PCR products were sequenced directly, as described previously [32]. Phylogenetic trees were constructed using the maximum-likelihood method in MEGA 5.2 software [34]. Representative sequences of MCPyV, HPyV6, and HPyV7 obtained in this study have been deposited in the GenBank database under accession numbers LC416383–LC416384, LC416385–LC416386, and LC416387–LC416388, respectively.
Quantification of S. aureus Loads
The S. aureus 209P strain was used as the control for optimization. The S. aureus–specific nuc gene was selected as a detection target [35]. The qPCR assay was performed in the same conditions as described above. The sequences of primers and probes are listed in Supplementary Table 1. A standard PCR run was conducted using the same primers and the product was cloned into the pMD20-T vector (TaKaRa Bio, Shiga, Japan). We prepared 7-fold serial dilutions using 108copies of the cloned plasmid DNA to generate a standard curve and, from this, we calculated the copy number in each sample.
Immunohistochemistry
Immunohistochemistry was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections using the mouse monoclonal antibody 2t10 (1:100 dilution) based on an indirect biotin-avidin system using a biotinylated universal secondary antibody and diaminobenzidine substrate with hematoxylin counterstaining. The 2t10 antibody was kindly provided by Christopher B. Buck (National Cancer Institute, Bethesda, Maryland). This antibody recognizes HPyV7 ST antigen and cross-reacts weakly with HPyV6 [15]. The specificity of staining with this primary antibody was controlled by testing the isotype-matched control antibody in parallel.
Statistical Analyses
Any correlations between viral detection rates were analyzed using Fisher exact test. The differences in viral loads were compared using the Mann–Whitney nonparametric U test or Wilcoxon signed-rank test. All the statistical analyses were performed using R software, version 3.2.0, with its graphical user interface EZR [36]. A P value <.05 was considered significant.
RESULTS
Prevalence and Viral Loads of Human Polyomaviruses in Patients With Cutaneous Diseases
We evaluated the prevalence and the copy numbers of 3 cutaneous HPyVs in swab specimens obtained from patients with chronic skin disorders according to disease group (AD, psoriasis, palmoplantar pustulosis, parapsoriasis, and MF) compared with those from the control skins of healthy individuals. The age-matched case-control study was performed with control subjects of similar age (±3 years) using a control-to-case ratio of 3:1 [37]. We first analyzed swab specimens of dry skin regions obtained from the arm or the trunk of patients and control subjects to match the skin sites. All specimens were tested at least twice independently. As a result, the viral copy numbers were reproducible, demonstrating the reliability of our qPCR system for determination of viral loads.
Among the AD and psoriasis groups, the HPyV6 prevalence was significantly higher in both the lesional and nonlesional skin of patients than that in the control group (P < .01), whereas the MCPyV detection rates were significantly lower (lesional skin of patients with AD vs control, P = .002; nonlesional skin of patients with AD vs control, P = .023; lesional skin of patients with psoriasis vs control, P < .001; nonlesional skin of patients with psoriasis vs control, P = .001; Figure 1). The HPyV7 prevalence was also significantly higher in the skin of patients with psoriasis compared with the control group (lesional skin of patients with psoriasis vs control, P = .014; nonlesional skin of patients with psoriasis vs control, P < .001). We found lower detection rates of MCPyV in the skin samples of patients with palmoplantar pustulosis, parapsoriasis, and MF.
DNA detection rates of Merkel cell polyomavirus, human polyomavirus 6, and human polyomavirus 7 in triplet skin swabs from lesional skin and clinically unaffected nonlesional skin samples of patients and from the skin of age-matched control healthy individuals. These specimens were collected from dry skin environments from the arm or the trunk. Percentages of detection are presented above the graph. Comparisons of the detection rates between the groups were performed using Fisher exact test, and significant differences are shown as *P < .05 and **P < .01. Abbreviations: HI, healthy individuals; HPyV6, human polyomavirus 6; HPyV7, human polyomavirus 7; LS, lesional skin; MCPyV, Merkel cell polyomavirus; NLS, nonlesional skin.
Figure 2 shows box plots of viral DNA loads in triplet swab specimens from the lesional and nonlesional skins of patients and from the normal skin of control subjects. There was a trend toward higher loads of HPyV6 and HPyV7 in both the lesional and nonlesional skins of AD patients than those in the control subjects, but a significant difference was found only in HPyV6 copy numbers compared between the 2 groups (P < .01). Conversely, the copy numbers of MCPyV were significantly lower in the lesional AD skin samples (P < .01). Patients with psoriasis harbored significantly higher loads of HPyV6 and HPyV7 and lower loads of MCPyV in both the lesional and nonlesional skin regions compared with the control group (P < .01). A similar detection pattern was found in the palmoplantar pustulosis group. Patients with parapsoriasis carried significantly more abundant HPyV6 DNA in the lesional skin vs the control skin (P = .023). Lower MCPyV loads were found in the lesional and nonlesional skin specimens collected from patients with parapsoriasis and MF (P < .01). Overall, the detection rate and loads of the HPyV tested here did not reach statistical difference between pairs of specimens from lesional and nonlesional skin of the same patients according to the disease group.
Box plots showing viral DNA loads of Merkel cell polyomavirus, human polyomavirus 6, and human polyomavirus 7 in triplet skin swabs from lesional skin (LS) and clinically unaffected nonlesional skin (NLS) samples of patients and from the skin of age-matched control healthy individuals (HI). These specimens were collected from dry skin environments of the arm or the trunk. Horizontal lines within the boxes indicate median values, and the bars extend the median by 1.5 times the interquartile range. Comparisons of the viral loads between the HI and LS groups and between the HI and NLS groups were performed using Mann–Whitney nonparametric U test, and the Wilcoxon signed-rank test was used for comparison between the LS and NLS groups in the same patients. Significant differences are shown as *P < .05 and **P < .01. Abbreviations: HI, healthy individuals; HPyV6, human polyomavirus 6; HPyV7, human polyomavirus 7; LS, lesional skin; MCPyV, Merkel cell polyomavirus; NLS, nonlesional skin.
We next analyzed viral loads in specimens of forehead skin obtained from healthy control individuals and from the nonlesional forehead skin of the patients and compared them with those in lesional skin specimens of the patients. Results were similar to those described above (Figure 3). Thus, our qPCR results were reproducible when control samples and patients’ nonlesional samples were taken from sebaceous skin regions.
Box plots showing viral DNA loads of Merkel cell polyomavirus, human polyomavirus 6, and human polyomavirus 7 in triplet skin swabs from lesional skin (LS) and clinically unaffected nonlesional skin (NLS) samples of patients and from the skin of age-matched control healthy individuals (HI). The NLS and HI specimens were collected from the sebaceous skin environment of the forehead. Horizontal lines within the boxes indicate median values, and the bars extend the median by 1.5 times the interquartile range. Comparisons of the viral loads between the HI and LS groups and between the HI and NLS groups were performed using the Mann–Whitney nonparametric U test, and the Wilcoxon signed-rank test was used for comparison between the LS and NLS groups in the same patients. Significant differences are shown as *P < .05 and **P < .01. Abbreviations: HI, healthy individuals; HPyV6, human polyomavirus 6; HPyV7, human polyomavirus 7; LS, lesional skin; MCPyV, Merkel cell polyomavirus; NLS, nonlesional skin.
Based on the DNA results, we looked for FFPE lesional skin biopsy samples from patients whose skin swabs were positive for HPyV DNA at relatively high levels. Eventually, 4 biopsy samples from HPyV7 DNA–positive patients with psoriasis were available for immunohistochemistry. Of these, 2 cases showed a nuclear positivity for HPyV7 ST antigen within keratinocytes in the epidermis (Figure 4), which was a similar staining pattern observed in a case of HPyV7-associated rash reported previously [38].
Immunohistochemistry on lesional tissue sections from 2 patients with psoriasis. A, Hematoxylin and eosin staining. B, Immunohistochemical analysis with the 2t10 antibody, showing positivity for the small T antigen of human polyomavirus 7 in the nuclei of keratinocytes throughout the epidermis. C, Immunohistochemical analysis with an isotype-matched negative control antibody, showing no immunoreactivity. Insets show higher-magnification views.
Identification of Geographically Related Viral Genotypes
Because the LT sequences have been used widely for determining the genotypes of MCPyV, HPyV6, and HPyV7 [1, 2, 9, 39, 40], we constructed phylogenetic trees based on the LT sequences successfully amplified from our skin swabs, along with GenBank-retrieved reference sequences available in July 2018, whose countries of origin had been disclosed. Our sequences included 17 MCPyV strains, 11 HPyV6 strains, and 8 HPyV7 strains, which could be recovered from specimens of 3 patients with AD, 5 with psoriasis, 7 with palmoplantar pustulosis, 5 with parapsoriasis, and 6 with MF. As expected, the phylogenetic analyses formed 2 main clades, and all of our HPyV strains belonged to an Asian/Japanese clade (Supplementary Figure 1). This indicated that the HPyV strains evaluated in this study were indeed of the Asian/Japanese genotype.
Loads of S. aureus in Patients With Skin Diseases
We also evaluated the loads of S. aureus on the skins in our patients. The loads of S. aureus were significantly higher in lesional skin specimens of those with AD and MF than in control subjects (Figure 5). By contrast, patients with psoriasis and parapsoriasis had significantly lower levels of S. aureus infection in lesional skin specimens. There was a significant difference in the loads of S. aureus between the lesional and nonlesional skin regions of patients with AD, palmoplantar pustulosis, and MF, whereas such differences were not found in patients with psoriasis or parapsoriasis.
Box plots showing Staphylococcus aureus loads in triplet skin swabs from lesional skin (LS) and clinically unaffected nonlesional skin (NLS) samples of patients and from the skin of age-matched control healthy individuals (HI). These specimens were collected from dry skin environments of the arm or the trunk. Horizontal lines within the boxes indicate median values, and the bars extend the median by 1.5 times the interquartile range. Comparisons of the viral loads between the HI and LS groups and between the HI and NLS groups were performed using the Mann–Whitney nonparametric U test, and the Wilcoxon signed-rank test was used for comparison between the LS and NLS groups in the same patients. Significant differences are shown as *P < .05 and **P < .01. Abbreviations: HI, healthy individuals; LS, lesional skin; NLS, nonlesional skin.
DISCUSSION
The present study had several strengths. We assessed for the first time covariation of viral loads of 3 skin-tropic HPyVs on the skin in the context of inflammatory conditions, including the most common inflammatory skin diseases, AD and psoriasis. Our study appears to be timely, as HPyVs, particularly HPyV7, have been linked recently to a pruritus eruption affecting immunosuppressed patients [15, 16], and this new clinical entity, HPyV-associated rash and pruritus (PVARP), is receiving increasing attention [38, 41]. Viral loads have been noted to be higher in PVARP lesions than in normal skin control samples of healthy individuals [15, 16]. Therefore, we postulated that the HPyV loads might vary on the skin of patients with long-lasting inflammation. In this study, increased prevalences and loads of HPyV6 and HPyV6/HPyV7 were found in the fresh skin swab specimens taken from our patients with AD and psoriasis, respectively, compared with age-matched healthy skin controls. Notably, some patients with psoriasis harbored more abundant HPyV7 DNA along with detectable viral antigen levels. By contrast, significantly lower viral loads of MCPyV were seen in the skin specimens of patients with AD and psoriasis. Thus, the infection levels tended to be higher for HPyV6/HPyV7 and lower for MCPyV in patients with AD and psoriasis. In a smaller prevalence study by Fava et al [42], neither HPyV6 nor HPyV7 could be detected in 8 patients with psoriasis. A possible explanation for this discrepancy is in the different selection of materials and/or patients: they used optimal cutting temperature-embedded biopsied tissues with a likely lower sensitivity regarding viral DNA detection than fresh swabs [40], and their data were all obtained from subjects living in Europe.
This study has provided the first data on the Asian/Japanese variants of HPyVs in a series of patients with various cutaneous diseases. We and other groups have demonstrated the existence of 2 major ethnologically related variant genotypes of MCPyV, HPyV6, and HPyV7 prevalent in the skin, compared between white and Asian/Japanese individuals [1, 2, 9, 10]. Interestingly, there are HPyV-associated skin diseases that have striking differences in these incidences between the 2 ethnic/geographical groups: MCPyV-positive MCC developed preferentially in the white population [13, 14], and Kimura disease, which was proved to be associated with HPyV6 infection, is endemic mostly in East Asia and has rarely been reported in white individuals [18, 19]. Of note, we have demonstrated previously that the HPyV6 strain HPyV6-LN1 (GenBank accession number KU596573 [17]) that was recovered from the lesional tissue of a patient with Kimura disease had genetic alterations common to the Asian/Japanese strains [2]. In addition, the HPyV7 strain UTSW7.1 (KX771235) that was found in a patient with PVARP had several genomic alterations in the LT regions [16], and this strain belongs to the Asian/Japanese genotype group according to phylogenetic analysis based on the LT sequence [2]. Although our findings are limited by the small sample size, the data presented here should stimulate worldwide studies on whether specific HPyV6/HPyV7 genotypes are linked to particular cutaneous inflammatory diseases. In fact, the prevalence of AD is higher in Asia, and different cytokine expression patterns have been noted in Asian patients with AD compared with EA patients with AD [25–28], although it remains to be addressed to what extent such AD-linked phenotypic differences are actually microbiome related. Moreover, we found that our patients with MF and parapsoriasis had significantly lower detection and quantification rates of MCPyV compared with skin from healthy control individuals. In contrast, Du-Thanh et al [43] showed that the MCPyV detection rate was higher in the lesional skin of European patients with MF (14/33 [42%]) than in samples from healthy individuals (3/22 [14%]). Such differential MCPyV status also deserves to be further investigated by worldwide studies, especially in regard to geographically related MCPyV variant genotypes.
In addition to cutaneous HPyVs, we have also provided data on S. aureus loads. Our results are consistent with findings that increased S. aureus loads were noted in lesional skins of patients with AD and MF [24, 29, 30], and decreased S. aureus loads in patients with psoriasis [44, 45]. However, some studies have shown contrary results, that patients with psoriasis seem to be colonized with S. aureus more often than healthy individuals [46]. Thus, further studies are required to establish an association between the skin microbiome and psoriasis. Although our observations here were made in a small patient cohort, this is the first report on the presence of cutaneous HPyVs in palmoplantar pustulosis and parapsoriasis. The first data on skin bacterial communities in patients with parapsoriasis were reported by Salava et al [47], and they did not find any differences between the lesional and nonlesional skins of 13 patients. In the present study, we have added new data showing that the lesional skin of patients with parapsoriasis had a decreased S. aureus load compared with healthy individuals.
Finally, we also present here the first comparative data on the HPyV infection levels in lesional vs nonlesional skins in the same patients. Importantly, the effect of viral load variation was observed not only at the lesion sites but also at clinically unaffected skin sites in our series, especially in patients with AD for HPyV6, and psoriasis for MCPyV, HPyV6, and HPyV7. These observations suggest that AD and psoriasis are conditions that affect HPyV infections at the systemic level, leading to shifts of the clinically unaffected viral status toward that of the lesions, and not specifically limited to the lesion sites. Indeed, patients with psoriasis have been found to have common variations in bacterial communities in both lesional and nonlesional skins [48]. Our study has also shown no significant difference in the infection levels of S. aureus between the site samples in patients with psoriasis. An alternative explanation is that the lesion sites serve as reservoirs for transfer of the microbes to the unaffected skin sites by touching or scratching, resulting in the lack of differences between these sites. However, this is less likely as our data regarding S. aureus showed clear differences in loads between lesional and nonlesional skins. In addition, we did not find a statistically significant difference in MCPyV load between lesional and nonlesional skin specimens of patients with parapsoriasis and MF, consistent with previous data for patients with MF [43]. These results could be reproduced when nonlesional specimens were collected from the sebaceous forehead skin of the patients. It is plausible that the dynamics of cutaneous HPyV status appear to be specific to the disease rather than to the anatomical site.
One limitation of the current study was that our findings did not distinguish whether the variation in HPyV loads was one of the primary events leading to the cutaneous diseases, or secondary events resulting from the abnormal epithelial environment. Nguyen et al [16] reported a case of PVARP in which HPyV infection remained detectable on the skin even after resolution of the disease. This suggests that the disorder develops in regions of skin harboring primarily high viral loads. In addition, a criticism of using DNA-based technology to estimate viral loads is that it does not distinguish between dead and viable viruses, although the methods used in our study have been used previously for quantification of cutaneous HPyVs [1, 2, 9]. In the future, complementary DNA–based analysis might help to minimize the detection of dead microorganisms [49].
In summary, we have shown variations in viral loads of MCPyV, HPyV6, and HPyV7 according to skin disease type in a panel of inflammatory and malignant conditions in an Asian population. Despite the limitations inherent to such observational studies, this work constitutes a first step in the characterization of changes in cutaneous HPyVs for their potential roles in the pathogenesis of skin diseases, and our findings are expected to promote prospective longitudinal studies with a larger cohort of patients throughout their clinical course. In addition, identifying viral genotypes that tend to persist and cause skin diseases is worthy of study.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgments. We thank Christopher B. Buck for the gift of the anti-HPyV antibody.
Financial support. This work was supported by the Japan Society for the Promotion of Science (grant number JP16K19612); the Uehara Memorial Foundation; a GSK Japan research grant 2017; and the Takeda Science Foundation.
Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
