Dear Editor,1. The Cas effector can be guided by a CRISPR RNA (crRNA) to target the invading nucleic acids by base-pairing and then cleaves the target to provide the host with immunity3. To cope with the immunity pressure imposed by the host CRISPR system, phages have evolved different kinds of anti-CRISPR (Acr) systems to inactivate the Cas nucleases and close the host immunity system in turn4. The Acr proteins can directly interact with the Cas proteins to prevent the crRNA loading or target binding; alternatively, they possess enzymatic activities to cleave the crRNAs or post-translationally modify the Cas proteins4. Among them, there exists a recently reported GNAT-family acetyltransferase, namely AcrVA5, which is encoded by the MGEs and specifically inhibits the type V-A Cas12a effector through acetyl modification of a key lysine site. The AcrVA5-inactivated Cas12a loses the ability to interact with the protospacer adjacent motif (PAM) site and fails in protecting the hosts by cleaving the invading MGEs6. Through inactivating the Cas12a system, MGEs containing the acrVA5 gene can effectively shut down the bacterial immunity system and have the potential to widely spread among the Cas12a-harboring hosts. However, to our great surprise, the acrVA5 gene exists only in three Moraxella bovoculi strains which have lost their deacetylases /CRISPR associated (Cas) system protects bacteria and archaea from mobile genetic elements (MGEs) such as bacteriophagesLachnospiraceae bacterium (Lb) Cas12a lost both cis- and trans-cleavage activities towards double-stranded target DNA (dsDNA)   and Acidaminococcus sp. (AsCas12a) and demonstrated that AcrVA5 was able to inactivate both orthologs  and transformed it into E. coli strains with or without a high level of CobB expression (Supplementary Fig. cobB gene in E. coli10 (Supplementary Fig. cobB-deleted strains were considered as the cobB-negative host, while the cobB overexpression strain was the cobB-positive host. To prepare a reporter plasmid without the targeting sequence of the Cas12a/crRNA complex, we mutated the Cas12a-targeting DNA sequence in the apr gene without changing the amino acid sequence as well as the apramycin resistance (Supplementary Fig. apr gene. Because the nonsense mutation changed both the PAM site and the guide sequence, Cas12a failed to cleave the target sequence both in vitro (Supplementary Fig. apr effectively escaped from the host defense system and showed high transformation efficiencies in an acrVA5-independent manner (Fig. We further explored the physiological role of CobB in protecting the Cas12a-containing host from the invasion of MGEs that harbor the ner Fig. , which wapr gene was then transformed into E. coli strains expressing Cas12a to mimic the invasion of MGEs, and as expected, in the absence of acrVA5 gene, no transformants could be obtained in all tested strains independent on the expressional level of CobB, representing the invasion of the foreign plasmid could be completely blocked by the hosts (Fig. apr and acrVA5, mimicking MGEs harboring the acrVA5 cassette, the high transformation efficiency of the reporter plasmid was obtained in the cobB-negative hosts, which was consistent with the above findings that AcrVA5 effectively inactivated Cas12a. As the overexpression of CobB in the cobB-positive host could deacetylate and reactivate the AcrVA5-acetylated Cas12a, the transformation of the reporter plasmid was largely blocked. As expected, the efficiency of the reporter plasmid was still much lower than the control, although overexpression of CobB and AcrVA5 may severely reduce the E. coli transformation efficiencies (Fig. The reporter plasmid containing the wild type sts Fig. . Howeveries Fig. . Based o6, the CobB-mediated deacetylation reactivation of Cas12a through modification can be taken as both a prevention strategy to the anti-CRISPR elements and a safeguard to the CRISPR defense system, protecting hosts from the invading of the acrVA5-containing MGEs (Fig. 11.As the AcrVA5-mediated acetyl modification of Cas12a has been considered as an effective anti-Cas12a strategy by MGEsGEs Fig. . MoreoveE. coli cellular extracts and purified AcrVA5 and found a large number of proteins could be acetylated by AcrVA5 (Supplementary Fig. E. coli, the whole cellular acetylation level was greatly increased, and the acetylated proteins could then be efficiently deacetylated by CobB at the presence of NAD+ (Supplementary Fig. 12, and the present study may possibly provide a clue for the unclear mechanisms.Besides Cas12a, AcrVA5 may function as a broad-spectrum acetyltransferase and influences cellular metabolic processes. We next performed the in vitro western blot analysis through using Taken together, one could infer that the wars between invading MGEs and the microbial hosts will never end, and besides acetyl- and deacetyl-modification, there probably exist other kinds of competitive mechanisms subject to future investigation. While just as the Chinese idiom goes, one can believe a vice rises one foot, but virtues rise ten.Supplementary Information


Little is known about the prevalence of driving among teenagers who have not yet obtained a license. The primary objective of the present study was to estimate the prevalence of unlicensed driving among young drivers using the quasi-induced exposure (QIE) approach and to determine whether unlicensed driving was more common among minority and lower-income teenagers. Additionally, we examined whether unlicensed driving among adolescents increased following the implementation of a graduated driver licensing (GDL) system and whether GDL differentially affected minority and low-income adolescents.Using North Carolina crash and driver license data, we identified 90,267 two-vehicle crashes from 1991 through 2016 where only one driver was considered contributory and the non-contributory driver was a White or Black 16 or 17 years old. In the QIE approach, these non-contributory young drivers are assumed to be representative of all adolescents driving in the state during this time period. The prevalence of unlicensed driving among adolescents by age and year was estimated by identifying the proportion of non-contributory drivers who had never been licensed by the time of their involvement in these two-vehicle crashes. We further conducted logistic regression analyses to examine the likelihood of a non-contributory young driver being unlicensed as a function of race, neighborhood income level, and licensing era (prior to or after GDL was implemented).During the 26 years for which data were available, the mean annual prevalence of unlicensed driving was 1.2% for 16-year-olds and 1.7% among 17-year-olds. Young Black drivers and individuals living in lower-income neighborhoods were somewhat more likely to drive before obtaining a license, but the rates of unlicensed driving among these groups were also quite low. Unlicensed driving increased slightly for 17-year-olds following the implementation of GDL, but returned to previous levels after a few years.Unlicensed driving among adolescents in North Carolina is substantially less common than suggested by previous self-report studies and analyses of fatal crash data. The move to graduated driver licensing (GDL) for young beginning drivers in the USA began a quarter century ago. Since then, there has been a dramatic increase in research on young drivers, along with a marked advance in the sophistication of that research. Nonetheless, little is known about the prevalence of driving among teenagers who have not yet obtained a license. This is somewhat puzzling given the concerns that have long been expressed about the possible unintended effects of GDL on unlicensed driving.Self-report has been the most common approach to studying unlicensed driving (or unlicensed drivers) among adolescents. In a nationally representative school-based survey, 4% of 9th–11th grade students in the U.A reported driving at least one hour a week without a license  approach allows researchers to estimate the relative risk of a certain behavior or outcome while accounting for exposure  What is the prevalence of unlicensed driving among adolescents in North Carolina (NC)? (2) Is unlicensed driving more common among adolescents in minority and low-income families? (3) Did unlicensed driving among adolescents increase following the implementation of a GDL system, and did GDL differentially affect minority and low-income adolescents? Unlicensed driving can include driving with an expired, revoked, or suspended license, but in the case of adolescents, unlicensed driving is primarily a matter of driving before getting a license. Accordingly, we conceptualized unlicensed driving here as driving done by not-yet-licensed drivers, which has the added advantage of avoiding the conceptual complexity and resulting analytic complications of including the quite different phenomenon of losing a license through suspension or revocation due to misbehavior.We examined data from motor vehicle crashes that involved two vehicles, in which the police reported that the actions of one of the drivers contributed to the occurrence of the crash (hereafter referred to as the “contributory driver”), while no action of the other driver was noted as contributing (“non-contributory driver”), and in which the non-contributory driver was 16 or 17 years old (16 is the minimum age to drive without adult supervision in NC). We assumed the non-contributory drivers played no role in the occurrence of these crashes other than by their mere presence, and thus that the distributions of various characteristics among non-contributory drivers in crashes approximate the corresponding distribution among all drivers on the road. Using this approach, we estimated the proportion of the total driving exposure of drivers aged 16–17 who were not yet licensed. The data analyzed were selected from all police-reported crashes that occurred in NC during 1991–2016.Crash data are reported by investigating officers to the NC Department of Transportation (NCDOT), which maintains the NC Crash Data File. Crash report data include extensive detail about the characteristics of each crash, each vehicle involved in the crash, and each crash-involved person including drivers, all passengers, and non-occupants . We examined crash data from 1991 through 2016 for the present study.Driver licensing information is collected by the NCDOT whenever a license is issued, suspended, renewed, or any other license action is taken. For this study, we obtained licensing history data from 1980 through 2019, with information on the date and type of each license issued and all subsequent license actions. NCDOT grants permission to use both crash and license data for research purposes, so long as unique identifiers are not included in publications, re-disclosed, or used to contact individuals .We geocoded drivers’ addresses as recorded in crash reports to the Census tract level and linked them with tract-level demographic information available from the US Census Bureau. The standard Census hierarchy includes : regions, divisions, states, counties, tracts, block groups, and blocks. The shapefiles used for geocoding changed between 1990 and 2010 in NC. Consequently, the same address might belong to a different block or block group with decennial updates. Addresses are much less likely to shift between larger area groupings like tracts. Therefore, we geocoded at the Census tract level because it generated a more stable solution. Although tracts comprise larger areas and are less precise than block groups and blocks, they provide a better indicator of neighborhood characteristics than county or zip code.The NC driver license file maintains a complete history of an individual’s license actions, including all applications as well as licenses issued. This enabled us to determine whether crash-involved, non-contributory drivers had ever been licensed in NC by the time of their crash. Non-contributory crash-involved 16- and 17-year-old drivers for whom there was no record on any license issuance were coded as a not-yet-licensed driver.Driver race/ethnicity is included in NC crash and licensing data as a single variable with the following categories: White, Black, Indian (Native American), Hispanic, Asian, and Other. Drivers self-identify into only one of these categories, which precludes separate analyses of race and ethnicity. Prior to 2000, Hispanics and Asians were categorized as “Other,” and there were not enough crash-involved Native American drivers to analyze separately. Accordingly, analyses included only self-identified White and Black drivers. We examined whether drivers’ race may have influenced officers’ judgment of their contribution to the crash and found no differences between White and Black drivers by comparing the proportion in each group for which no driver action was indicated as contributing to the crash. These were virtually identical for White and Black 16–17-year-olds (32.1% vs. 31.8%).We measured the income level of drivers’ home neighborhoods using the median household income of the Census tract in which they resided. We considered Census tracts whose median income was in the lowest quartile for the state in a particular year to be low-income neighborhoods. Yearly income levels were adjusted to reflect 1999 US dollars.To get a better sense of the validity of neighborhood median household income as an indicator of socioeconomic status, we scored drivers in the lowest income quartile in 2015 using the Area Deprivation Index .  We used this information instead of traffic citations to identify drivers who were involved in a crash merely because of their presence at the road location. Although citations are sometimes used for this purpose, driver actions are only one of several factors that influence whether officers issue citations , neighborhood income level (lowest 25% vs. upper 75%), and licensing era (pre- vs. post-enactment of GDL). We examined the effect of GDL by comparing 5-year periods before  and after  it was implemented, which reduced the analytic sample to 41,878 cases. Further, we examined whether the introduction of GDL was associated with a differential change in unlicensed driving for drivers of different races and/or income levels. Separate logistic regression models were estimated for 16-year-olds and 17-year-olds. In keeping with sound epidemiological practice Savitz , as wellDuring the 26 years for which data were available, the mean annual prevalence of unlicensed driving was 1.2% for 16-year-olds and 1.7% among 17-year-olds. As indicated in Fig. Table Unlicensed driving was more common—though still relatively rare—among Black adolescents and those residing in the lowest-income neighborhoods. The combined rate of unlicensed driving among 16- and 17-year-old Black adolescents was 3.3% and 2.9% for adolescents in the lowest-income neighborhoods (combined age data not shown in table). Racial differences were slightly larger among 16-year-olds, whereas income level differences were somewhat larger among 17-year-olds. Controlling for race reduced income level effects slightly, and vice versa, as is indicated by differences between the crude and adjusted odds ratios in Table Table The goal of this study was to identify the prevalence of unlicensed driving among 16- and 17-year-olds in the NC driving population and to examine the associations of race, neighborhood income level, and GDL with unlicensed driving. Using a QIE approach, we found that for the 20-year period from 1991 through 2010, the prevalence of driving without having gotten a license was consistently between one and two percent among 16- and 17-year-olds. More recently (2011–2016), it declined, fluctuating around one percent yearly. Our detailed analyses focused on the 10-year period surrounding the date when GDL took effect . Unlicensed driving among 16-year-olds did not change after GDL was enacted and increased only slightly for 17-year-olds. Overall, our findings indicate that unlicensed driving is not nearly as common among adolescents as suggested by previous studies. It is possible, of course, that unlicensed driving in NC differs markedly from other states, but we know of no reason to think this would be the case.By avoiding several shortcomings that routinely characterize self-report surveys, such as respondent self-selection, response set biases and other measurement difficulties, and sampling complications or inadequacies Babbie , and by One of the key motivations for this study was to examine whether unlicensed driving was more common among adolescents in minority and low-income families. Previous research has indicated that being Black and living in poor neighborhoods is associated with driving without having gotten a license. Our results replicated those findings; however, the difference in unlicensed driving we found in NC between Black and White drivers is about half what Elliot et al.  reportedDozens of studies indicate that well-designed GDL programs are highly effective in reducing adolescent driver crashes Williams . NonetheAlthough the QIE approach offers many advantages over previous studies, this study does have a few limitations. First, to the extent that not-yet-licensed adolescent drivers were more likely than those with a license to leave the scene of a crash, our findings would underestimate the prevalence of unlicensed driving. Another question is whether not-yet-licensed drivers are more likely than licensed drivers to contribute to crashes. Although drivers with revoked or suspended licenses are more likely to crash Brar , virtualUsing a unique analytic approach, this study found that unlicensed driving among adolescents is substantially less common than suggested by self-report studies and analyses of the license status of crash-involved drivers. Young Black drivers and individuals living in lower-income neighborhoods are somewhat more likely than young White drivers and less disadvantaged adolescents to drive before obtaining a license, but the rates of unlicensed driving among these groups are also quite low. Unlicensed driving increased slightly for 17-year-olds following implementation of GDL but returned to previous levels within a few years.


The primary aims of our study were to determine if hospital readmissions within one year following primary total joint arthroplasty (TJA) and their relative timing influence patients’ ability to achieve the two-year Patient-Reported Outcomes Measurement Information System (PROMIS) physical, PROMIS mental, and PROMIS Physical-Function-Short-Form-10a (SF-10a) minimal clinically important difference (MCID). This is a retrospective study conducted using data from a multi-institutional, arthroplasty registry. Only patients with paired patient-reported outcome measure (PROM) assessments (preoperatively and two years postoperatively) were included. Five separate readmission cohorts were formed: (1) any-cause readmission within one year, (2) any-cause readmission within 90 days, (3) non-index-surgery-related readmission within 90 days, (4) index-surgery-related readmission within one year, and (5) index-surgery-related readmission within 90 days. A propensity score match was used to match each of the patients to one of the 972 patients (1:1 basis) in the non-readmission group. The association between failure to achieve each of the three two-year MCIDs and Readmission status was analyzed using logistic regression. We found that all readmissions within one year and index-surgery-related readmissions within one year resulted in an increased risk of failure to achieve the two-year MCID across all three collected PROMs. Index surgery-related readmissions within 90 days  sustained significantly different rates of two-year PROMIS physical MCID achievement compared to matched controls. Postoperative complications requiring readmission, particularly those related to the joint arthroplasty and those within 90 days of index surgery, significantly impact the ability to achieve the two-year MCID of PROMs. The increasing demand and cost for arthroplasty care have necessitated a need to monitor outcomes -3. MetriThere is a growing understanding of how adverse surgical events, such as periprosthetic infections, revisions, and hospital readmissions may affect patient satisfaction, PROM scores, and the ability to achieve MCID. While some of these events are discrete and easy to categorize, hospital readmissions are quite diverse and require a deeper analysis due to their frequency and lack of uniformity. For example, readmission for a periprosthetic joint infection or a myocardial infarction constitutes two vastly different pathophysiologic events and therefore can impact postoperative outcomes in varying ways -18. FurtThe primary aim of this study was to investigate whether hospital readmission within one year following primary TJA and its timing relative to the index surgery influenced patients’ ability to achieve the two-year PROMIS physical, PROMIS mental, and SF-10a MCID.Level of Evidence III. This retrospective study was conducted with Institutional Review Board approval using data from a regional, multi-institutional, arthroplasty registry. This registry is populated from the electronic medical records of patients treated in a healthcare network comprised of seven hospitals. All cases of primary total hip arthroplasty (THA) and total knee arthroplasty (TKA) performed since 2016 within this hospital network are captured in the registry as well as all postoperative readmissions with the same network, with an approximate 10% lost-to-follow-up rate within one year of surgery . The PROA registry query for all primary THA and primary TKA patients with complete preoperative and two-year postoperative PROM score sets resulted in 1,302 patients. Variables collected for each patient included: demographics , Charlson Comorbidity Index (CCI), preoperative diagnoses , discharge disposition, length of stay, and identity of the institution where the THA or TKA was performed. PROM scores for these patient populations were used to derive the three distinct MCIDs at the two-year postoperative timepoint using the distribution-based method, which defines the MCID as one-half of the standard deviation of the preoperative to postoperative change in each PROM for the entire population -22. Of tA rigorous chart review was performed to classify the primary reason for the initial hospital readmission. Patients were then categorized into those having any readmission within one year of the index TJA and those having no readmissions. Patients undergoing subsequent elective procedures within 90 days of index TJA (n=34) were excluded from further analysis because of the inherent difficulty in attributing the complication readmission as related to the first elective procedure or the second elective procedure. Patients with multiple readmissions were categorized by only the readmission related to index TJA. If none of the readmissions pertained to index TJA, only the first readmission was categorized. With respect to timing and indication, readmission events were classified according to timing as early (within 90 days of index surgery) or any (0-365 days) and the reason for the readmission . The index-surgery-related readmissions were further classified according to the chief complaint identified by the consulted orthopaedic surgeon in the discharge summary. For non-index-surgery-related readmissions, the chief complaint as identified in the patient’s discharge summary was recorded and further classified by organ system. A complete list of readmission events is included in Appendix A. Readmission events are further stratified by readmission group in Appendix B.Five separate readmission cohorts were formed: (1) any-cause readmission within one year (n=296), (2) any-cause readmission within 90 days (102), (3) non-index surgery-related readmission within 90 days (n=58), (4) index surgery related readmission within one year (n=90), and (5) index-surgery related readmission within 90 days (n=44).Statistical analysisA propensity score match was used to match each patient in the five subgroups to one of the 972 patients (1:1 basis) in the non-readmission group. A separate propensity score match was performed for each PROM. Patients were matched on age, BMI, history of myocardial infarction, cancer, or cerebrovascular accident, CCI, congestive heart failure, type II diabetes, discharge disposition, hospital, length of stay, sex, and joint. Chi-squared test of independence and student’s t-test were used to compare categorical and continuous demographic variables respectively, between all patients (unmatched) as well as the matched cohorts. For continuous variables used in the propensity score matching, matching was performed for values within 0.5 standard deviations of the mean of the variable. For each patient in the non-readmission and readmission cohorts, the change from preoperative to two-year postoperative PROM assessments was calculated and used to determine whether that patient achieved the two-year MCID. The three primary outcomes of interest were failure to achieve PROMIS physical, PROMIS mental, and SF-10a MCIDs at two years. Readmission status was interpreted as a binary variable in regression analysis. For each matched readmission cohort, every candidate predictor variable was included in multivariable logistic regression so as to not omit potential interactions. For only the variables that were significantly associated (p<0.05) with the outcome of interest after regression analysis, odds ratios (OR) and corresponding 95% confidence intervals (CI) were reported. All statistical analyses were performed using R  and RStudio .Achieving MCID and readmissions (matched cohorts)For patients with any readmission  within one year n=296), 133 (44.9%), 194 (65.5%), and 157 (53.0%) patients failed to achieve the two-year PROMIS physical, PROMIS mental, and SF-10a MCIDs, respectively , PROMIS mental , and SF-10a  MCID compared to matched non-readmission cohort  MCID achievement compared to matched controls Table . AppendiStratification of readmissionsAnalysis of the all-cause within one-year readmission cohort (n=296) revealed index surgery-related complications were the leading cause of readmissions ; gastrointestinal , cardiac , and orthopaedic complications not related to the index surgery . A complete list of complications stratified by readmission group is included in Appendix E. Infection-related complications  were the leading cause of index surgery-related admissions; lysis of adhesions following TKA  was the second reason for index surgery-related admissions.As PROMs continue to enhance patient care by incorporating a patient’s subjective appraisal of a surgical procedure, metrics such as the MCID may help in interpreting the clinical significance of these outcomes. In investigating the association between readmissions and achievement of MCID, the authors found that patients with any hospital readmission within one year of TJA were less likely to achieve two-year MCID across PROMIS physical, PROMIS mental, and SF-10a compared to patients without a readmission event. It is important to note that these lower patient-reported outcomes were driven primarily by index surgery-related readmissions as opposed to readmissions for non-index surgery-related reasons. Furthermore, after stratifying by time, index surgery-related readmissions within 90 days of TJA were more strongly associated with failure to achieve PROMIS physical MCID as compared to matched controls. These findings can provide guidance for physicians when managing patient expectations and when providing postoperative care following TJA. To the authors’ knowledge, the current study is the first to distinguish between readmissions related to the arthroplasty procedure and those unrelated as it pertains to impact on three commonly used PROMs. The data supports the notion that readmissions following TJA are heterogeneous and can impact the ultimate outcome differently based on the indication for readmission and time relative to arthroplasty. It also provides reassurance to surgeons when asked about the impact of non-index surgery-related readmissions on ultimate recovery after TJA.Previous studies have focused on pre-operative factors that can predict satisfaction following joint replacement . Recent Beyond the actual measures, it is also critical to consider the time point at which these measurements are taken. Previous evidence has suggested that TJA patients continue to experience gains for up to one year after TJA . HoweverConceptually, it is logical to infer that any interruption in a patients’ rehabilitation caused by a complication necessitating readmission during the acute postoperative period may impede them from achieving critical milestones, thus compromising the ultimate function of the joint. Similarly, the majority of readmissions unrelated to the index procedure may not be predictive of the patients’ ability to achieve MCID, with the rarer exceptions of more debilitating reasons for readmission such as unrelated lower extremity fractures, stroke with motor deficits, and intensive care unit admissions. Conversely, those readmissions occurring after 90 days are less impactful as the majority of the functional gains and recovery may have already been achieved and the patient has had an opportunity to experience the benefits of their replaced joint thus buoying their subjective assessment of the surgery.This study, however, is not without limitations and its results should be interpreted within the context of its strengths and weaknesses. Given its retrospective nature, the current study is limited in identifying additional potentially confounding variables that were not collected in the registry. Additionally, readmissions to facilities outside of the institutional network were unable to be included and could impact the analysis as a source of measurement bias. It is worth noting that the authors’ institution does include a multitude of community and tertiary referral hospitals in the area, so the effects of uncaptured readmissions were theoretically minimized. Furthermore, loss-to-follow-up is a limitation inherent to any PROM analysis and this study is no exception. However, the arthroplasty registry utilized reports an approximate 10% lost-to-follow-up rate within one postoperatively, thus helping to mitigate such biases . AnotherIn this study, we derived our own MCID values using a distribution-based method as opposed to using an anchor-based method given the retrospective nature of our study and lack of anchor questionnaire available . The disAdditionally, the current study includes patients from a variety of surgeons at multiple hospitals lending credence to the applicability of the results. Another possible limitation involves the omission of one-year postoperative PROM scores when assessing readmissions that occurred in the first postoperative year. Only the two-year postoperative period was assessed as it would account for time-dependent completion of the postoperative assessment and allow for an adequate period of recovery prior to assessment completion. We also chose to classify patients with multiple readmissions according to the readmission related to index TJA, which may be a source of bias as patients with multiple readmissions are expected to have worse outcomes. The number of patients included, and the two-year follow-up scores should be considered strengths of this study.In conclusion, postoperative complications requiring readmission, particularly those related to joint arthroplasty, significantly impact patient-reported outcomes and especially the ability to achieve the two-year MCID. Moreover, complications within 90 days are more impactful than complications that occur later on during the first postoperative year. This information is important as measures to mitigate index surgery-related complications and readmissions will have a significant impact in improving the proportion of patients achieving MCID on PROMs.


Previous studies have shown the clinical effect of 2 Hz repetitive transcranial magnetic stimulation (rTMS) for depression; however, its underlying neural mechanisms are poorly understood. The aim of this study was to examine the effects of rTMS on the activity of the prefrontal cortex in patients with depression, using functional near-infrared spectroscopy (fNIRS).Forty patients with major depressive disorder (MDD) and 40 healthy controls were enrolled in this study. Patients underwent 4 weeks of 2 Hz TMS delivered to the right dorsolateral prefrontal cortex (DLPFC). fNIRS was used to measure the changes in the concentration of oxygenated hemoglobin ([oxy-Hb]) in the prefrontal cortex during a verbal fluency task (VFT) in depressed patients before and after rTMS treatment. The severity of depression was assessed using the Hamilton Rating Scale for Depression-24 item (HAMD-24).Prior to rTMS, depressed patients exhibited significantly smaller [oxy-Hb] values in the bilateral prefrontal cortex during the VFT compared with the healthy controls. After 4 weeks of 2 Hz right DLPFC rTMS treatment, increased [oxy-Hb] values in the bilateral frontopolar prefrontal cortex (FPPFC), ventrolateral prefrontal cortex (VLPFC) and left DLPFC during the VFT were observed in depressed patients. The increased [oxy-Hb] values from baseline to post-treatment in the right VLPFC in depressed patients were positively related to the reduction of HAMD score following rTMS.These findings suggest that the function of the prefrontal cortex in depressed patients was impaired and could be recovered by 2 Hz rTMS. The fNIRS-measured prefrontal activation during a cognitive task is a potential biomarker for monitoring depressed patients’ treatment response to rTMS. Major depressive disorder (MDD) is a common psychiatric disease, with over 300 million individuals worldwide suffering from the disease . UnfortuFor patients with MDD, repetitive transcranial magnetic stimulation (rTMS) has emerged as a promising treatment , 4. rTMSAlong with the rapid development of recent neuroimaging technologies, the brain activity in patients with MDD has gradually come to be visible. Previous functional neuroimaging studies have shown the dysfunction of the PFC in patients with MDD, which may be related to their clinical symptoms, including both depressive mood and cognitive impairment –13. TherThe verbal fluency task (VFT) is a frequently used cognitive task in the fNIRS studies, in which participants are asked to generate as many words as possible beginning with a certain semantic category or letter within a limited time. The VFT mainly reflects the executive function, which is associated with some basic neurocognitive activities, such as working memory, motivation and attention. Many studies have reported that neurocognitive impairments are associated with PFC dysfunctions in numerous psychiatric disorders , 16. AccIn past decades, two main rTMS strategies for depression treatment have been developed: high-frequency rTMS on the left DLPFC and low-frequency rTMS on the right DLPFC , 18, 19.In our study, fNIRS was used to examine the hemodynamic changes in the PFC in both patients with MDD and healthy counterparts during VFT, and then patients were treated with 4-week of 2 Hz right DLPFC rTMS treatment. We compared the change in the PFC before and after rTMS treatment. We hypothesized that  patientsPatients were recruited from the outpatient department of West China Hospital, Sichuan University from May 2021 to December 2021. We included 40 patients aged 20–59 years who were diagnosed with moderate MDD according to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorder (DSM-5) with a Hamilton Rating Scale for Depression-24 item (HAMD-24) total score between 20 and 35. All patients had to be right-hand dominant. In order to eliminate the influence of antidepressant medications, we included the patients who had not taken antidepressants at least 1 month before treatment start. Considering that it is common for patients with depression comorbid with anxiety and insomnia , the useForty healthy controls (HCs) were recruited from the local community and matched to the MDD patients in terms of age, gender, level of education. They were required to be right-hand dominant and in a good healthy condition with no any known history of neurologic and psychiatric diseases, or a family history of psychotic disorder.Before this study, all participants provided written informed consent. This study was approved by the West China Hospital Clinical Trials and Biomedical Ethics Committee of Sichuan University  on April 30, 2021 and registered in the Chinese Clinical Trials Registry (registration No. ChiCTR2100046806) on May 29, 2021.Major depressive disorder patients received rTMS treatment for 4 weeks. Depression severity was assessed by HAMD-24. Before and after 4 weeks of rTMS treatment, all patients were assessed with HAMD-24 and fNIRS by a trained research staff to ensure consistency. In addition, considering acute cognitive enhancing effects of rTMS, which were typically observed when the test was administered immediately following stimulation, usually within several minutes , we thusRepetitive transcranial magnetic stimulation was performed with a CCY-I Magnetic Stimulator  with an air-cooled, figure-of-eight 70 mm coil. At the first TMS session, the resting motor threshold (RMT) of the right abductor pollicis brevis muscle was determined as the lowest strength of transcranial magnetic stimulation needed to elicit at least 5 electromyographic responses in the form of motor evoked potential  ≥50 μV in 10 trials . The sitThe 24-item HAMD , 30 inclThe primary outcome measure for this study was the total score of HAMD-24. Clinical response was defined as a reduction in HAMD-24 scores of at least 50% from baseline.),” “shi ,” “shuo ,” and “jia ,” which indicate upper, time, speak and home, respectively, was audibly presented to the subjects. And subjects were instructed to generate as many words as possible which began with the same syllable. All the participants were given the same syllable cues and no changes were made to the order of presentation. We provided all participants with a practice session before the formal testing, in order to ensure the participants fully understand the tasks. During the task, an investigator monitored the performance of the participants, in order to ensure the participants were fully engaged in the assessment.The task procedure in the present study was a Chinese-language phonological VFT developed by Quan et al.  for ChinThe 37 multi-channel fNIRS instrument  measures the concentration changes of [oxy-Hb] and [deoxy-Hb] in cerebral cortex using two wavelengths (695 and 830 nm) of infrared light, based on the modified Beer Lambert law . The absAccording to a previous study of anatomical craniocerebral correction via the international 10–20 system , 37, we The toolbox HOMER2, a MATLAB-based graphical user interface program was used to analyze the NIRS data . First, t-test (age) or Mann-Whitney test (education), comparing the MDD and HCs groups. Symptom change, i.e., difference between the two total HAMD-24 scores (pre – post over the full treatment course), was compared by paired t-test. To analyze our NIRS data, Mann-Whitney U tests were used to compare [oxy-Hb] values during the VFT for each channel between the MDD and HCs groups at baseline. Then, to assess [oxy-Hb] responses to rTMS treatment in patients with depression, the differences in [oxy-Hb] values during the VFT for each channel were compared between pre- and post-treatment in the MDD group, using Wilcoxon test.Statistical analysis was performed using SPSS software version 26.0. Baseline demographic characteristics were assessed by means of a chi-squared test (sex), To examine the relationships between [oxy-Hb] values before treatment during the VFT and HAMD-24 total scores and to test whether the former was related to clinical outcome, correlation analysis was carried for MDD patients. For channels showing a significant difference in [oxy-Hb] value in MDD group between pre- and post-treatment, we also examined the association between the [oxy-Hb] changes from baseline to post-treatment and the degree of improvement in depressive symptoms.p < 0.05.We adopted a false discovery rate (FDR)  in order2 = 0.621, p = 0.600), age  or education  between the MDD patients and the healthy controls. For patients, the duration of illness was 5.80 ± 7.37 years. A 60% (24/40) patients were diagnosed with MDD with a first episode and 40% (16/40) had recurrent episodes. A 72.5% (29/40) patients were comorbid with anxiety and 75% (30/40) patients were comorbid with insomnia. A 67.5% (27/40) patients had never taken antidepressant medications. A 55% (22/40) patients were medicated with benzodiazepine drugs. The demographic and clinical characteristics of the participants are presented in There was no significant difference in gender (chi-square test: χt-test: p < 0.001). A 27.50% (11/40) of MDD patient were responded to treatment in our study.After 4-weeks of rTMS treatment, the HAMD-24 scores in the MDD patients significantly decreased, from 26.08 to 17.73 . After 4-weeks of rTMS treatment, the significant increase in [oxy-Hb] changes were observed in the MDD group compared with the pre-treatment levels in the 7 channels located over the bilateral FPPFC, left DLPFC and bilateral VLPFC . The prefrontal cortical activation during the VFT in different group are shown in At baseline, the [oxy-Hb] values in the MDD group during the VFT were significantly lower than that of HCs group in the 31 channels located over the bilateral FPPFC, DLPFC and VLPFC .In the MDD group, the increased [oxy-Hb] values from baseline to post-treatment was positively related to the total HAMD-24 score reductions in the right VLPFC in 37 (Pearson’s However, after FDR correction, there was no significant correlations between NIRS data and clinical data.To the best of our knowledge, this is the first study to examine the effects of DLPFC rTMS on modulating to cognitive task in MDD patients using fNIRS. We found that patients with MDD had significantly less activation in the PFC in response to the VFT compared to their healthy counterparts, whereas that the level of the activation in patients was not related to their depression severity. After 4-weeks of 2 Hz DLPFC rTMS treatment, the decreased depressive severity and the increased activation in response to the VFT were observed in MDD patients, and the increased activation in the right VLPFC from baseline to post-treatment was positively related to the improvement of depressive symptoms.The overall results of previous NIRS studies of the VFT revealed reduced PFC activation in MDD patients compared with HCs , 47, 48.We found that the [oxy-Hb] values in the bilateral FPPFC, left DLPFC and bilateral VLPFC during the VFT were gradually increased after 4-weeks of 2 Hz rTMS applied on right DLPFC than before rTMS. Our results suggest that 2 Hz rTMS could evoke an increased cerebral cortex activation during a cognitive task. Similarly, previous study using fMRI indicated that 2-week of rTMS applied on the DLPFC had focal and remote effects on several brain areas involved in working memory in healthy subjects during an n-back task . Also, CIn the present study, 2 Hz right DLPFC rTMS was effective as a monotherapy for MDD who were not undergoing any antidepressant medication. The finding was consistent with previous studies , 64, whiMoreover, our study demonstrated, for the first time, the correlation of the increase of NIRS activation in the prefrontal region with improvements in the depressive symptoms of patients during the rTMS treatment. In our 4-week rTMS treatment period, the longitudinal increases in the right VLPFC were shown to be positively correlated with improvements in the severity of depressive symptoms for MDD patients. This finding is consistent with the previous study conducted by Shinba et al.  which inThere are some limitations of our study. First, our fNIRS signals in typical source-detector channels were possibly contaminated with systemic interference occurring in the superficial layers of the head . AlthougOur study demonstrated that patients with MDD had significantly reduced brain activation in the PFC during VFT when compared with HCs, and these functional deficits can be improved after 4 weeks of 2 Hz right DLPFC rTMS treatment. Furthermore, there was correlation between improvements in the depression severity in MDD and increases of hemodynamic response to VFT in the right VLPFC during treatment. These results suggest that hemodynamic response to VFT in PFC, measured by fNIRS, is a potential biomarker for monitoring MDD patients’ treatment response to rTMS. How to improve the cognitive and brain function of MDD and how to predict the prognosis of the patients are important issues that need more exploration in the future.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the West China Hospital Clinical Trials and Biomedical Ethics Committee of Sichuan University. The patients/participants provided their written informed consent to participate in this study.ZZ conceived and designed the experiments. JH performed the experiments and data analysis. TZ checked the processed experimental data. JZ and JH wrote the manuscript with input from all authors. PW supervised the project. All authors discussed the results and contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although our pilot subjects had significant co-morbidities, extremely high viral loads and moderately severe symptoms during the Delta phase of the pandemic, the response to treatment was highly encouraging. Rapid reductions in viral loads were observed and negative PCR tests were documented within a median of 4 days. These laboratory findings occurred in parallel with significant clinical improvement, mostly within 12–24 h of commencing the treatment protocol. There were no significant adverse effects and none of the subjects who completed the protocol required in-patient hospitalization. The outcomes were similarly encouraging at one-year follow-up with virtual absence of “long COVID” symptoms or of COVID-19 re-infection. Our results indicate that the protocols may be a safe and promising approach to challenging COVID-19 disease. Moreover, due its broad spectrum of activity, this approach has the potential to address the prevailing and future COVID-19 variants and other infections transmitted via the upper respiratory tract. Extensive studies with a large cohort are warranted to validate our results.Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 virus was first recognized in late 2019 and remains a significant threat. We therefore assessed the use of local methylene blue photodynamic viral inactivation (MB-PDI) in the oral and nasal cavities, in combination with the systemic anti-viral, anti-inflammatory and antioxidant actions of orally ingested methylene blue (MB) and photobiomodulation (PBM) for COVID-19 disease. The proposed protocol leverages the separate and combined effects of MB and 660nm red light emitted diode (LED) to comprehensively address the pathophysiological sequelae of COVID-19. A total of eight pilot subjects with COVID-19 disease were treated in the Bahamas over the period June 2021–August 2021, using a remote care program that was developed for this purpose. Although not a pre-requisite for inclusion, none of the subjects had received any COVID-19 vaccination prior to commencing the study. Clinical outcome assessment tools included serial cycle threshold measurements as a surrogate estimate of viral load; serial online questionnaires to document symptom response and adverse effects; and a one-year follow-up survey to assess long-term outcomes. All subjects received MB-PDI to target the main sites of viral entry in the nose and mouth. This was the central component of the treatment protocol with the addition of orally ingested MB and/or PBM based on clinical requirements. The mucosal surfaces were irradiated with 660 nm LED in a continuous emission mode at energy density of 49 J/cm More than two years after declaration of a pandemic, COVID-19 remains a serious concern globally, with successive waves of increasingly more resistant variants [Photodynamic therapy offers great potential in this regard with established ability to address bacterial, fungal, protozoan and viral infections, with very low likelihood of drug resistance due to its rapid, multi-targeted cidal effects .A fundamental aspect of COVID-19 disease is the tissue and organ damage caused by a hyper-inflammatory state  often reMB is FDA approved as a lifesaving treatment for methemoglobinemia. There is growing evidence of many other health benefits, particularly as an enhancer of mitochondrial function, cognitive function and mental health ,7,8,9. M50 = 3 μM) [With respect to anti-viral activity, MB has been reported to inhibit the attachment of the SARS-CoV-2 spike protein to the angiotensin-converting enzyme-2 (ACE2) receptor even at very low, physiologically relevant concentrations (IC = 3 μM) . MB incr = 3 μM) ,18. MB i = 3 μM) . The ant = 3 μM) ,21,22. OThe formation of micro-clots is a critically important sequela in the pathogenesis of COVID-19 disease and is responsible for a number of severe outcomes and death. MB is a potential inhibitor of platelet aggregation especially in the context of hyper-inflammation and may therefore be of significant benefit in this regard .MB has proven to possess intrinsic anti-bacterial , anti-fuBased on the actions described above it is not surprising that improvement of fever, malaise and hypoxia was often observed within a few hours (h) of oral administration of MB in study subjects. Furthermore, MB is generally well-tolerated and regarded as a very safe drug. The two main precautions are a limited number of drug interactions and G6PD deficiency. The major concern relating to MB drug interactions is serotonin syndrome. This appears to be a significant factor associated with intravenous infusion , as oppoA recent evidence-based review conducted by Hanna et al., 2021 highlighted the molecular mechanisms of COVID-19 and potential therapeutic strategies of which phototherapy can be considered, as a potential treatment modality in COVID-19 management ,31.2+ sensitivity, which accounts for its anti-inflammatory effects [The literature has well-documented that photobiomodulation (PBM) therapy acts as an immunomodulator, inducing antioxidant and anti-inflammatory effects ,33,34. I effects ,34. Addi effects ,36,37, r effects ,39,40.The review conducted by Hanna et al. 2020  documentAdditionally, the actions of both MB and PBM on mitochondrial function are widely recognized. Several studies have shown the impact of dysfunctional mitochondria on the immune response. Recent studies revealed that human alveolar epithelial cells with dysfunctional mitochondria displayed increased production of pro-inflammatory cytokines  all of which were found to be increased in COVID-19 ,43.In contrast to systemic steroids which broadly suppress the immune response, PBM stimulates complexes of the mitochondrial respiratory chain to optimize and modulate the immune response, resulting in an improvement of its ability to eradicate infection via stimulation of the innate and adaptive immune cells ,45,46, wPBM is often applied to local target tissues to exploit its many beneficial effects. However, several reports indicate that a multi-organ systemic effect of PBM can be achieved by directly targeting the blood circulation. These efforts were pioneered by Russian scientists in 1981 and other scientists have now documented cardiovascular and other organ system benefits ,53,54,55Sublingual and nasal PBM provide a non-invasive alternative means of targeting the blood circulation and have been shown to provide comparable effects to intravenous blood irradiation ,69. AbsoNotwithstanding the multiple additive benefits of MB and PBM, the authors consider photodynamic inactivation (PDI) to be the central and critically important component of our treatment protocol. It is noteworthy that PDI has a very long history of use in various clinical applications and is regarded as a very safe method of treatment including on mucosal surfaces ,71,72.PDI involves activation of a photosensitizer compound by a compatible light energy in the presence of oxygen ,74. The MB-PDI results in oxidation of carbonyl moieties on proteins; oxidation of guanine residues; and single strand breakage and cross-linkage of RNA ,77,78. TA systematic review by Dalvi et al., 2021 of several in vitro, in vivo and clinical studies has confirmed the significant efficacy of MB-PDI, as an antimicrobial measure . MB-PDI Moreover, as the SARS-CoV-2 infection and many other respiratory viral infections appear to involve predominantly upper airway disease at the early stages of infection, targeting effective treatment to these mucosal surfaces appears logical. The application of local anti-microbial PDI to the oro-pharynx and nasal passages for SARS-CoV-2 disease, is supported by evidence that ACE-2 positive epithelial cell lining salivary gland ducts are early target cells of SARS-CoV-1 and SARS-CoV-2, and a likely source of the viral particles detected in saliva droplets, particularly during the early stages of infection ,87.8 RNA copies per throat swab on day four. Within one week of symptom onset, a significant number of viruses were bound to ACE2 receptors in the mucosa of the oral cavity and both throat and nasal cavities [A clinical case series conducted by Woelfel et al., 2020 showed that pharyngeal shedding of SARS-CoV-2 was very high during the first week of symptoms with a peak at 7.11 × 10cavities . Schikorcavities  proposedcavities . Therefocavities .Another potential benefit of local PDT is broad based viral antigen presentation and possible promotion of mucosal and systemic immunity. This phenomenon has been described in vivo in the context of anti-tumor therapy  and bactTo confirm the reduction in viral load with the use of serial quantitative polymerase chain reaction (PCR) tests (cycle threshold (CT) measurements).To evaluate symptom progression and recovery time over follow up time points.To appraise the feasibility and convenience of the home-based device and treatment of COVID-19 disease.To assess the long-term outcomes following treatment.This pilot study was modelled on two preceding successful clinical studies which employed PDI in the early stages of COVID-19 infection ,94. The A total of eight subjects with COVID-19 infection were tested and treated for COVID-19 disease over the period June 2021–August 2021.The study was conducted in accordance with the Declaration of Helsinki and based on the initial results of the exploratory cases described, approved was granted by the National COVID-19 Medical Ethics Committee in the Bahamas on 18 July 2021 to conduct a standardized case series study (Research Identification Number MROS/311971/PC).1.Subjects who were residents of Nassau, Bahamas, presenting with COVID-19 symptoms of 10 days or less with a positive test for COVID-19, using reverse transcription polymerase chain reaction (RT-PCR) test.2.Subjects were at least 18 years of age, of both genders, and of any ethnicity.3.Subjects with or without systemic diseases (ASA I and II).1.Concomitant therapy: patients who were taking any FDA/MHRA/EMA/WHO approved anti-viral drugs. .2.2 saturation < 90% or severe hypertension .Any enrolled subjects who needed urgent medical care, such as O3.Subjects who were unable to properly comply with the treatment instructions and complete the course of the treatment protocols.The measurements of the PCR-CT were obtained from a single lab for all tests carried out. All the samples were maintained between 2 and 8 degrees Celsius and delivered to the laboratory within 12 h or less. All samples were processed by the same professional laboratory using the Bioneer AccuPower SARS-CoV-2 Multiplex Real Time RT-PCR Kit manufactured by Bioneer Corporation, Daejeon, South Korea. The following two gene target groups were measured: the E gene and SARS-CoV-2 genes (RdRp and/or N gene). The E gene negative threshold value was 35 and SARS-CoV-2 gene negative threshold value was 34. Estimated changes in the viral load were calculated using the initial SARS-CoV-2 gene target CT and the negative SARS-CoV-2 gene target threshold (34). This calculation is based a log base 2 scale: The estimated viral load reduction (EVLR) was calculated with the following formula.We recognize that CT values are not a direct measurement of viral load present but can provide a useful measure of the relative viral loads over time in a given patient, particularly if a standardized swab technique is utilized and specimens are processed by the same laboratory professional using the same test equipment.Patient clinical data were obtained remotely by serial phone and serial online questionnaires. Online questionnaires confirmed COVID-19 vaccination history, past medical history, medications, supplements, allergies, symptom onset, graded symptom severity and the presence of any other symptomatic persons in the household. Follow up questionnaires were requested 12-hourly for the first 24 h and then daily until recovery time. Each follow-up questionnaire specifically elicited any adverse effects or difficulties experienced with the home treatments. The follow up questionnaire also graded symptom severity and provided a free text section for any additional symptoms or comments regarding clinical progress. Home visits were organized in response to any concerning signals indicated during the telephone review calls or from the online questionnaire feedback.All of the above data were collected and analyzed by an independent research assistant who was not involved in either the study design or in subjects’ treatments. All the subjects were given a number for identification. A single experienced clinician was involved in treatment and in overseeing the subjects’ progress.The treatment supplies were delivered to subjects’ homes including the treatment device, MB solution and oral MB capsules, if indicated.Our pilot study utilized a treatment device that is shown in 2. The duration of each treatment session to both the nasal and oro-pharynx was 5 min (300 s) resulting in a maximum calculated energy density of 49 J/cm2 emitted by the device. Effective energy density lessened with increasing distance from the device, particularly at the midline of the tongue and throat. However, the fact that subjects had to elevate the tongue (which heavily adsorbed the MB solution), in order to avoid discomfort during the PDI treatment session, indicates that an adequate light energy dose reached this area. Of note, the MB solution and the light energy alone did not have this effect.The pilot study device provided a non-thermal, non-coherent light source emitting non-ionizing radiation within the visible light spectrum. The device parameters are as follows: wavelength: λ 660 nm, total power output: 2928 mW, irradiance 163 mW/cm2 over 25 min using a laser probe with an aperture of approximately 1 cm2. In the present study the laser probe was held just outside the subject’s mouth which was held wide open for the duration of the treatment. As a result, the emitted energy was dispersed over a wider area. The average surface area of the adult mouth is reported to be approximately 215 cm2, but this is likely to be a significant overestimate in the context of light irradiation, as the tongue papillae account for a large proportion of the oral mucosa surface area [2 successfully employed in a case series study reported by Tardivo et al., 2005 [Schikora et al., 2020  reportedace area . In contace area  were higl., 2005  to treatFor the PDI phase of treatment, 2 for superficial PBM applications.For the administration of PBM therapy, the device was positioned to treat the ventral surface of the tongue without applying MB solution prior . The ran2 based on 60 s exposure.However, in administering PBM to the blood circulation, the fact that the blood components are constantly moving past the light source must be considered in calculating the energy density. Blood flow rates vary widely depending on vessel type, location, size and other influences. The sublingual veins are the major target of sublingual PBM. They are prominent veins extending dorsally from the tip to the root of tongue and drain directly into the large internal jugular vein. Based on sublingual vein blood flow rates , the bloWhen administering PBM, the pilot study device was positioned to treat the undersurface of the tongue without applying MB solution prior .Patients were provided with a web link that provided detailed instructions on how to carry out treatments including brief training videos. This was reinforced by instructions given by phone and photographic confirmation of proper device placement sent to the treating physician.The initial goal of our study was to confirm the results of the German COVID-19 MB-PDI clinical study conducted by Schikora et al., 2020  which reThe protocol was therefore progressively adapted and fine-tuned over the course of the eight documented exploratory cases, in order to achieve more efficient viral clearance and clinical recovery. This culminated in an establishment of our standardized three-phase Luminnova Upper Respiratory Protocol (LURP) by the end of this pilot series . Of noteMB capsules were initially administered orally in a dose range of 1.5 to 2 mg/kg 12-hourly. We subsequently transitioned to 8-hourly based on observed patient responses, with a maximum dosage of 200 mg. MB capsules were withheld if there were any potential drug interactions or medical contraindications. Patients were not screened for Glucose-6-phosphate dehydrogenase (G6PD) deficiency, as studies have shown that the dosing level that is commonly used can be safely administered to both adults and children with G6PD ,100.PBM was introduced as an additional measure to address the pronounced hyper-inflammatory and hypoxic state encountered in the majority of the subjects based on their presenting symptom profiles. The advantage of this non-pharmacologic measure is that there are no medical or pharmaceutical contra-indications and it was very easy to incorporate from a logistical and safety perspective. The LED-PBM phase of therapy was achieved by inserting the LED device directly under the tongue with the ends of the device lateral to the teeth and gums. It must be emphasized that PBM treatments were administered without prior application of topical MB solution. This step was carried out just prior to the PDI phase in order to minimize the presence of MB staining in the mouth which could lead to a photodynamic rather than a photobiomodulation response. We found that, apart from the superior surface of the tongue, MB was quickly cleared from the oral cavity by saliva flow, certainly within 4 h.Local ONPDI was administered to target SARS-CoV-2 virus in the epithelial lining of the salivary glands, oral cavity, nasal and throat passages. The LED device that emits red light (λ 660 nm) was utilized to activate 1% MB solution, which was topically administered on the mucosal surfaces within the oral and nasal cavities.Patients were instructed to gently apply the MB solution to the nasal mucosa with a cotton tipped swab and wait 10 min to allow absorption before activating the MB solution with the LED device. While waiting for absorption to occur at the nasal mucosa, the oral cavity was rinsed with 7.5 mL of MB solution for 1 min. PDI treatment was carried out immediately after rinsing with MB in order to avoid the solution being washed away or excessively diluted by saliva. The device was held in place  for 5 miOnce the oro-pharynx PDI was completed, subjects then irradiated the nasal passages for 5 min with the LED device also shown in A solution was provided to rapidly remove MB staining from the lips and teeth after the treatment, but most patients did not use it until the end of their treatment course as they were in isolation, and this was not considered a high priority considering that they were significantly unwell. This option was more extensively used when the treatment had been applied as a prophylactic measure by asymptomatic close contacts of COVID-19 patients.Of note, the local ONPDI phase III effect may also have been augmented by activation of orally administered MB capsules which could potentially achieve therapeutic concentrations suitable for a photodynamic response within the vascular network of the nasal and oral passages. This phase of treatment may have also conceivably resulted in a whole body systemic PDI component with activity against transient episodes of viraemia.Have you had any persistent symptoms after your initial COVID-19 infection which lasted for a period 1–3 months or greater?Have you had any recurrence of COVID-19 symptoms after your initial infection?Have you had a positive COVID-19 test after your initial infection?In view of the high prevalence of post-acute sequelae of SARS-CoV-2 disease (PASC) and repeat infections, subjects were followed-up after a period of one year. They were asked three questions listed below:A 61-year-old Afro-Caribbean female with a history of recurrent bronchitis, BMI 31.1 and no history of previous COVID-19 vaccination, presented three days after symptom onset on 22 June 2021. She commenced the following treatments one day prior to Luminnova COVID-19 protocol: Bisolvon, Paracetamol, Ivermectin, Zithromax and Aerius.Two ONPDI treatments were administered. Her symptoms improved initially, but after two days her symptoms escalated once again and oral-MB was therefore prescribed, as a supportive measure at 1 mg/kg twice daily. We noted an improvement after each dose with flares after about 6–8 h so the dosage frequency was increased to 8-hourly.Four days after commencing oral/nasal PDT treatments, we were able to obtain PCR CT values. To our dismay these were E gene 17.19 and SARS-CoV-2 gene (nucleocapsid/RdRp genes) 16.34. Based on this very high viral load and the fact that diarrhea was a prominent symptom, we suspected the Delta variant. At this point, we discovered that although she had been given written, pictorial and verbal instructions, she had not placed the device correctly in the mouth and therefore had not had effective photodynamic treatments. By the time we obtained CT results, she was at Day 8 of her illness. A second series of treatments were then carried out with proper device placement, which is explained in ONPDI treatments were administered daily for three consecutive days. There was a gradual rise in the CT levels consistent with a reduction in the viral load. It can be argued that the viral load could have been gradually reducing at this point even without treatment. However, when the treatments were paused for 2 days, the CT values decreased and then increased again upon resumption of the protocol, signaling that the viral load reduction resulted from the treatment. Furthermore, the progress in terms of symptoms also followed the same pattern of improvement, relapse and further improvement upon resumption of treatment.In order to gain a better understanding of the treatment response and the optimal timing for the follow-up swabs, we decided to perform paired swabs immediately before and after the treatments on two consecutive days. The paired CT value results on the 2 July 2021 confirmed an increase in the CT value immediately after the treatment. Interestingly, the CT value treatment that was carried out on the next day (3 July 2021) had increased to the negative range even prior to the treatment. The difference in these readings may be related to the presence of non-viable viral RNA, which had cleared by the following morning. A summary of the subject’s symptoms progression is shown in Based on the EVLR calculation, there was a 207,000-fold reduction in viral load between Day 0 when the second treatment series was commenced and Day 7 when the negative PCR test was obtained.Subject one reported that she had no post-acute sequelae of SAR-CoV-2 (“long COVID”) symptoms, recurrence of symptomatic COVID-19 re-infection or subsequent positive test for SARS-CoV-2 after recovering from the initial infection .the worst he had ever experienced in his entire life”.A 55-year-old Afro-Caribbean male with a history of hypertension and who is a cigarette smoker, BMI 34 with no history of previous COVID-19 vaccination, presented 5 days after symptom onset on 6 July 2021. The treatments that he received prior to commencing the oral/nasal PDT were as follows: Gravol, Aerius, Levofloxacin, Aeroflux and Vitamin C. The subject had travelled to a funeral in the USA where other close relatives had tested positive for COVID-19, including his sister (Subject #1). However, he had two negative COVID-19 test results. He was receiving treatment for a bacterial pneumonia, but his condition had become progressively worse, and he was very weak. He stated that the symptoms were “A repeat RT-PCR test was carried out which was positive for COVID-19 with a CT value of 22.12 E gene/20.17 SARS gene. Intravenous fluid was administered at the subject’s home along with a single ONPDI treatment late at night on 6 July. The patient made a remarkable recovery by the next morning and refused additional PCR tests, as he declared that he had fully recovered . At the Subject two reported that he had no post-acute sequelae of SAR-CoV-2 (“long COVID”) symptoms, recurrence of symptomatic COVID-19 re-infection or subsequent positive test for SARS-CoV-2 after recovering from the initial infection .A 40-year-old Afro-Caribbean male who had no significant past medical history with BMI of 31.7 and no history of previous COVID-19 vaccination, presented 5 days after symptom onset on 6 July 2021. The treatments prior to commencing the oral/nasal PDI included Hydroxychloroquine, Theraflu, zinc and vitamin D3.One session per day of ONPDI: on Days 0 to 2.Three sessions of ONPDI on Day 3.Oral-MB was administered from 6 July to 10 July 2021 and PBM therapy was administered from 7 July to 10 July 2021.The treatment regimen was as follows:The subject did not initially admit that he had been taking hydroxychloroquine. A relative had been prescribed this the year before with a great clinical benefit. Hence, he had self-treated, but the symptoms were significantly worsening, so he sought assistance with the Luminnova COVID-19 protocol.In response to complaints about discomfort from nasal swabbing the technique of obtaining samples was adjusted after the initial four paired samples  in this case. More importantly, based on the response observed after each treatment session, it was decided to administer three-treatment sessions in 1 day in order to achieve a more significant reduction in the viral load. PBM therapy was also added to the regimen in an attempt to bring about a greater symptom response in terms of ongoing diarrhea and chills which had persisted 24 h after commencing our treatment protocol .Once the accelerated treatment schedule and other changes were introduced, there was excellent clinical progress. There was a steep increase in the CT values  and a marked reduction in symptoms. In fact, the E gene target was negative immediately after the third treatment session carried out on Day 3 and both gene targets were negative by Day 4 with no additional ONPDI treatments. By Day 7, he resumed heavy manual tasks with no difficulties.Based on the EVLR calculation, there was a 32,995-fold reduction in viral load between Day 0 when treatment was commenced and Day 4 when the negative PCR test was obtained.Subject three reported that he had no post-acute sequelae of SAR-CoV-2 (long COVID) symptoms, recurrence of symptomatic COVID-19 re-infection or subsequent positive test for SARS-CoV-2 after recovering from the initial infection .A 38-year-old Afro-Caribbean female who had no significant past medical history, BMI 31.3 and no history of previous COVID-19 vaccination, presented 3 days after symptom onset on 11 July 2021. The treatments prior to the Luminnova COVID-19 protocol were as follows: hydroxychloroquine, Cetamol Cold and Flu, zinc and vitamin D3.Three sessions of topical oral/nasal PDT on Days 0 and 1.Two sessions of topical oral/nasal PDT on Day 3.Oral MB and sublingual PBM 8-hourly Day 0 to Day 2.The treatment regimen was as follows:The subject was in the same household as Subject three. Of note, she had taken hydroxychloroquine prior to starting the protocol and her treatment was commenced at an earlier stage of the infection than Subject three (3 days after symptom onset). She nevertheless had very low CT values and was very unwell just prior to the treatment, as shown in Based on the EVLR calculation, there was a 4,312,220-fold reduction in viral load between Day 0 when the treatment series was commenced and Day 4 when the negative PCR test was obtained.Subject four reported that she had no post-acute sequelae of SAR-CoV-2 (“long COVID”) symptoms, recurrence of symptomatic COVID-19 re-infection or subsequent positive test for SARS-CoV-2 after recovering from the initial infection .A 42-year-old Afro-Caribbean male with no significant past medical history, BMI of 32.5 and no history of previous COVID-19 vaccination, presented 5 days after symptom onset on 16 July 2021. The subject had not received any treatment prior to commencing the Luminnova COVID-19 protocol.Number of ONPDI treatments prescribed 8-hourly.Oral MB-prescribed 8-hourly, but this treatment schedule was not maintained.The Treatment Regimen was as follows:The subject was initially commenced with only ONPDI treatments, but oral-MB was also advised once the high viral load was confirmed. He was poorly compliant due to nausea. The authors believe that nausea was most likely secondary to the infection and the oral-MB capsules. Reduction in both viral load and symptoms were noted 24 h after commencing the treatment regimen including oral MB. At approximately 36 h, MB was temporarily discontinued. The third PCR at 72 h revealed that the viral load had increased once again and his symptoms had worsened. Upon re-commencing MB once again, there was a reduction in the viral load by Day 4  and gainSubject five reported marked PASC or “long COVID” symptoms for 2–3 months after his acute infection including weakness, difficulty breathing and postural dizziness  He did hA 31-year-old Caucasian female who had no significant past medical history was referred by a colleague for treatment with the Luminnova protocol, as she was experiencing progressively worsening symptoms, due to COVID-19 infection. She was pregnant at 8 months gestation at the time of presentation with BMI of 27.6 (pregnant). She had no history of previous COVID-19 vaccination and presented five days after symptom onset on 18 July 2021. The treatments prior to the Luminnova COVID-19 protocol were Acetaminophen and antenatal supplements. The subject’s obstetrician was consulted, and approval was given to proceed with ONPDI. Despite orally ingested MB having been safely used in rare instances in late pregnancy , this waThe Treatment Regimen was as follows:1.ONPDI:First course of eight treatments; Day 0 to Day 2.Second course of eight treatments; Day 4 to Day 6.2.Sublingual PBM; Day 4 to Day 7.3.No oral-MB was administered as the patient was pregnant.Improvement in her symptoms was observed on commencing ONPDI, although at a much slower rate than previous cases . The chaThe subject was assessed at the Emergency Room (ER) of her local hospital on Day 3 of the Luminnova protocol and chest imaging was carried out. The ER team concluded that her condition was stable and did not warrant admission. Therefore, she was discharged the following day. Repeat CT values from a PCR swab were obtained on Day 3, but were not available on Day 4; however, they indicated that her viral load had reduced significantly even before attending the ER.As the option of hospital support was unavailable, we continued ONPDI and added sublingual PBM therapy along with nutritional supplements at home on Day 4. After the brief period of rest and re-hydration in the ER, there was much better response to the treatments administered as shown in the symptom progression chart of Based on the EVLR calculation, there was a 450,000-fold reduction in viral load between Day 0 when the second treatment series was commenced and Day 7 when the negative PCR test was obtained.Subject six reported that she had no post-acute sequelae of SAR-CoV-2 (“long COVID”) symptoms, recurrence of symptomatic COVID-19 re-infection or subsequent positive test for SARS-CoV-2 after recovering from the initial infection .A 47-year-old Afro-Caribbean female with no significant past medical history, BMI 29.2 and no history of previous COVID-19 vaccination, presented 2 days after symptom onset on 21 July 2021. The subject has not received any treatment prior to the Luminnova COVID-19 protocol.Topical ONPDI-course of eight treatments, Day 0 to Day 2No oral-MB administered as patient presented early and had mild symptomsThe treatment regimen was as follows:The patient was referred by a close contact who had been treated with this protocol. As she presented early and only had a moderately high viral load, only ONPDI was administered. She experienced mild symptoms only and made a rapid recovery. RT-PCR was repeated on Day 4 and confirmed negative .Based on the EVLR calculation, there was a 128-fold reduction in viral load between Day 0 when the treatment was commenced and Day 4 when the negative PCR test was obtained.Subject seven reported that she had no post-acute sequelae of SAR-CoV-2 (“long COVID”) symptoms, recurrence of symptomatic COVID-19 re-infection or subsequent positive test for SARS-CoV-2 after recovering from the initial infection .Oral-MB capsules.Sublingual PBM.ONPDI.A thirty-eight-year-old Afro-Caribbean male with no significant past medical history. His body mass index (BMI) was 32.5 and no history of previous COVID-19 vaccination, presented 3 days after symptom onset on 21 July 2021. There was no treatment prior to commencing the Luminnova COVID-19 protocol. The treatment regimen was based on a three-phase protocol as follows:The initial treatment regimen was a total of eight treatments given every 8 h, Day 0 to Day 2, whereas the repeat treatment regimen was a total of eight treatments given every 8 h, Day 4 to Day 6.Subject eight presented with prominent symptoms and an extremely high viral load. The initial CT value was 6.05/9.41 . These rThis treatment protocol regimen proved to be very successful. Repeat PCR at Day 3 confirmed an 11-point increase in the CT values, indicating a 2000-fold decrease in the viral load within 72 . This waBased on the EVLR calculation, there was a 259-million-fold reduction in viral load between Day 0 when the treatment was commenced and Day 8 when the negative PCR test was obtained.Subject eight complained of mild persistent symptoms which he described as “mucous on his chest”, but no additional symptoms such as fatigue, cardiac symptoms, brain fog or other neurological symptoms. There was no recurrence of symptomatic COVID-19 infection or subsequent positive test for SARS-CoV-2 .Our pilot study of eight cases (eight subjects) was supported by objective data  and self-reported outcome questionnaires completed by each subject. This study provides an early indication that the combination of local ONPDI, sublingual PBM and orally administered MB may be an effective means of managing even challenging COVID-19 disease in the home environment. Seven subjects were of Afro-Caribbean ethnicity and one subject was Caucasian. To the best of the authors’ knowledge, this is the first COVID-19 clinical case series published in an Afro-Caribbean population. Subject ages were between 31–61 years. Most subjects were at a higher risk for poor COVID-19 outcomes. One subject was pregnant (8 months gestation) and the majority had BMI of 31 or greater. Apart from one subject who had moderately high viral load, viral loads at presentation ranged from very high to extreme in one subject. Despite these risk factors, expedited clinical recovery was observed in parallel with very rapid of SARS-CoV-2 viral clearance. The median negative PCR test and clinical recovery time of the seven subjects who completed the treatment was 4 days. This underlines the significance and the effectiveness of our treatment protocols even in the context of high BMI, very high viral loads and suspected Delta variant COVID-19 disease. We have outlined the key points of our results and their significance below.Our pilot study  offered There were progressive increases in CT values and a parallel reduction in symptoms with successive treatment sessions. Moreover, a slowing or reversal in the trend of these improvements was also observed when the treatments were paused temporarily, and continued improvement was then noted upon resumption of the protocol . We therefore concluded that responses observed were likely due to the treatments administered.Large reductions in viral loads were documented over very short periods . This waWe were unable to document serial CT changes for Subject two as he refused repeat PCR and insisted that he had fully recovered 24 h after a single ONPDI session. Notwithstanding the lack of laboratory confirmation, there was an excellent clinical recovery and good long-term outcomes as indicated by his questionnaire responses  and 1 yeThe CT values observed in the other six subjects suggests that these interventions may have great potential in high viral load COVID-19 disease. The most striking response was noted for subject eight who presented with extremely low CT values (6.05/9.41). A more aggressive treatment approach was taken with the aim of expediting clearance of the SARS-CoV-2 virus and protecting the lungs and other organs. The treatment schedule was therefore accelerated. The dramatic reductions in viral load (259-million-fold) and rapid recovery in this subject with a high BMI and suspected Delta variant COVID-19 was highly encouraging. We, therefore, adopted the protocol administered to this subject as a standardized approach going forward.Notable reduction in symptoms were observed within 12–24 h of commencing treatment in all the subjects apart from subjects; one and six. The slow response observed initially for Subject 1 was due to incorrect device placement. Once this issue was addressed there was good clinical response. Subject six was not administered oral MB as she was pregnant (8 months gestation). Although there was a marked reduction in viral load, symptomatic response was significantly slower in this subject in comparison to the other subjects. This may have been at least partially due to pregnancy. However, a rapid clinical response to MB in other subjects along with the regression in symptoms on discontinuing MB in Subject five, suggests that withholding MB may also have been a factor in Subject six. Despite the fact that MB had to be avoided, significant symptom reduction, including reduced cough and weakness were noted for Subject six following the introduction of sublingual PBM on Day 4. Overall, her response was encouraging considering that systemic pharmacological agents were avoided due to pregnancy. Summary of the results illustrated in We also observed that there was a correlation between CT levels and the severity of symptoms. Contrary to prevailing opinion, the CT values appeared to be a highly predictive tool in assessing clinical prognosis and progress in the pilot cases during the Delta phase of the pandemic. This was likely aided by the use of a single laboratory for PCR results. However, the cost and 12–24 h delays in getting PCR results at times presented a challenge in utilizing these tests to support clinical decision making.Adverse effect data were elicited via daily online follow up questionnaires and telephone conversations. Subjects were specifically asked to describe in as much detail as possible if they experienced any difficulties or adverse effects from the treatment with each follow up questionnaire completed. The responses obtained indicated that the treatment protocol was well tolerated. There were a few reports of minor issues, but no serious adverse events were reported.Subject five was unable to consistently comply with and did not complete the recommended protocol. He had complained of nausea on taking oral MB. This improved somewhat once his dosing schedule was adjusted to space the oral MB dosing without reducing the total daily dosage. However, this was still a challenge and subsequently led him to abandon the treatment protocol.Three subjects  had to attend the ER due to dehydration. In these three cases, in-patient hospital admission was not considered necessary as no significant lung or other organ impairment was detected. Dehydration was at least partially due to diarrhea, nausea and vomiting which were commonly associated with the Delta variant. However, oral MB was also considered a potential factor as it is almost exclusively excreted via the kidneys. Once a more concerted effort at oral hydration was emphasized, this problem was largely avoided, and oral MB was generally well tolerated.Subject one complained of mild stinging of the throat after the first ONPDI treatment, but this had resolved in less than 30 min. This appeared to be related to gargling with the MB solution. Hence, subject one and subsequent subjects were advised to actively rinse the oral cavity, but to minimize gargling. Subjects were also instructed to hold the tongue upwards and back in order to avoid burning the upper surface of the tongue which heavily absorbed MB solution. No other study subjects complained of burning of the throat, tongue or other sites.When employing a light-based therapy, it is important to consider the skin type of subjects treated as melanin is an active chromophore of specific wavelengths. Potential problems resulting from treatment of melanin rich sites include reduced absorption of the light energy and/or excessive heating of the target tissues. Fortunately, the mucosal surfaces of the nose and mouth do not contain melanin and therefore we did not anticipate that these issues would arise. Seven of the eight subjects were skin types VI and one subject was skin type II. The treatment protocols were demonstrably effective in all subjects who completed the protocol, indicating adequate light penetration. Secondly, the ONPDI and PBM were well tolerated overall, with no perception of heat at the treatment sites.Post-acute sequelae of SARS-CoV-2 (“long COVID”) and recurrent COVID-19 infection have proven to be frequent and highly problematic issues associated with SARS-CoV-2 viral infections. Six of the seven subjects who completed the protocols reported no long COVID symptoms, recurrence of symptomatic COVID-19 infection or subsequent positive SARS-CoV-2 test results .Subject eight reported mild persistent chest congestion up to one year post treatment. This was perhaps not surprising as his initial viral load was the highest documented of all the patients treated with the Luminnova protocols over the course of two years. There was also a pause in his treatment over two days due to attendance at a local hospital Emergency Room due to dehydration and discharged after a few hours. Extremely high viral load and the pause in treatment may have led to a higher risk of lung injury, albeit mild. He reported no recurrence of symptomatic COVID-19 infection or subsequent positive SARS-CoV-2 test results .Subject five was unable to complete the protocol and was the only subject that had marked and prolonged symptoms after recovering from acute SARS-CoV-2 infection. He was also the only subject that reported recurrence of symptomatic COVID-19 infection with positive SARS-CoV-2 test results .These findings may indicate the need to clear SARS-CoV-2 as quickly and as thoroughly as possible, in order to avoid long-COVID symptoms. Subject 5 was also the only subject to report repeat symptomatic infection with a positive SARS-CoV-2 test. The finding that all subjects who completed the protocol had no recurrence of SARS-CoV-2 symptoms or positive tests, may also point to a durable immunity conferred in the context of these subjects. However, further studies in a larger population would be required to confirm this.In late June 2021, there was a deluge of significantly ill-COVID-19 patients in the Bahamas and elsewhere. Hospitals and acute care clinics were overwhelmed, and cross-infection was a significant concern. As a result, COVID-19 patients were advised to quarantine at home and only seek hospital intervention, if they developed breathing difficulties. Naturally this led to significant anxiety, as many patients were very unwell and were aware of a number of reports of deaths in the community. There was a large unmet need for effective early treatment and home support for these patients.Our treatment protocol was designed to facilitate minimal contact between patients and the healthcare team and allow patients a viable option of continuing self-care at home, thereby reducing the burden on healthcare facilities. A single-person LED treatment device was customized for this purpose. This approach avoided the risk of laser eye injury and the need for on-site medical supervision. However, a significant amount of time was required to provide remote support and advice and there were communication challenges, which had to be addressed.Subject one underlined the challenge of ensuring that subjects understood precisely how to place the device for effective treatments. In this case the device had been placed incorrectly initially resulting in inadequate clinical progress. As a result, more reliable ways of communicating this information were developed and Subject one and subsequent subjects were able to reliably carry out the treatments independently in the home setting with minimal on-site medical intervention. We therefore concluded that the treatment protocol administered was feasible provided adequate patient education was provided.To our knowledge, this is the fourth publication to document a clinical response to ONPDI for COVID-19 infection. The first study was a controlled clinical study of 1200 subjects carried out in Germany in early 2020, employing MB-ONPDI . This stInterestingly, we had also noted a rapid response to one to two ONPDI treatment sessions during the early phase of the pandemic when the original Wuhan strain was still dominant in the Bahamas. Treatment was administered using the same LED device used in this pilot study. We had noted that patients remained well after treatment without any recurrence of symptoms, providing that the entire household was treated. However, as noted earlier, an expanded and accelerated protocol was needed to manage the most aggressive COVID-19 disease encountered during the Delta wave .A smaller controlled clinical study by Weber et al., 2020  employedAnother group who demonstrated that SARS-CoV-2 can be reduced to undetectable levels in the nose utilizing an established MB-PDI platform for pre-surgical infection prophylaxis . HoweverN-acetyl cysteine in severely ill COVID-19 subjects in ICU. They documented impressive responses with statistically significant improvements in laboratory results  and clinical status  [The benefits of oral-MB and PBM for COVID-19 disease have also been previously reported. Randomized controlled trials Phase I, II and III conducted by Alamdari et al., 2020, 2021 and 2021, respectively ,104,105,ubjects) ,101,102.Sigman et al., 2020  describeWilliams et al., 2022 successfully employed transdermal PBM in a non-randomized 50-person case study . They coOur pilot study employed sublingual PBM as opposed to transdermal PBM due to the absence of melanin and the proximity of the target cells in the blood to the light source, a much lower fluence is needed at this site. The use of a single-person LED device also facilitated independent treatment in the home-setting with much lower potential for cross-infection between the medical team and patients. Further studies assessing the magnitude of response to sublingual and/or intra-nasal PBM used independently of other treatment interventions for COVID-19 are warranted.A significant limitation of our pilot study was the inclusion of only a small number of subjects in which a single, standardized protocol was not used. Nevertheless, despite these important limitations, the magnitude of viral load reduction and rapidity of the clinical response in a population who were at risk was indeed promising. This provides an early indication that these protocols may be a viable means of addressing COVID-19 disease in the community.Inflammatory markers, basic metabolic and hematologic assessments and chest scans were not documented. Additional understanding of the immunological response and its impact on the organ systems could be obtained by including these investigations before and after use of our protocols.As MB has independent anti-viral actions, a control arm including application of the solution without photodynamic activation would be helpful to clarify whether the progressive changes in viral load were due at least in part to the solution alone. The controlled study by Schikora et al., 2020  providedAlthough the use of three separate treatment interventions  achieved great clinical response in our pilot subjects, a further limitation we noted is it did not provide us with a clear understanding on how much each intervention contributed to these positive outcomes. Hence, gaining a greater understanding of each intervention effect would be very beneficial in further protocol adjustments, but was not considered ethical in the context of a high-risk disease where the overwhelming priority was to expedite recovery.Lastly, statistical analysis was not feasible in view of the evolving protocols utilized. However, based on the findings of our pilot study, a second case series using a standardized protocol was subsequently documented also during the Delta phase and will be published separately. A similar protocol is now being successfully utilized to target a series of Omicron sub-variants. The durability of this response underlines the value of PDI, MB and PBM to target COVID-19 in a non-variant specific manner.Due to the broad spectrum of action against multiple microbial species, coupled with lack of documented evidence of microbial resistance over decades of use, PDI has the potential to target current and emerging COVID-19 variants. This is a critically important advantage of this treatment approach. Moreover, there is potential to address many other pathogens contracted via the nasal and oral mucosa, including influenza, the common cold, respiratory syncytial virus, adenovirus infections and possibly pox viruses, using these combined protocols ,109,110.The combination of oral-MB, PBM therapy and local ONPDI provided a comprehensive and effective treatment approach to at risk, high viral load associated COVID-19 disease in the pilot cases documented. Firstly, we demonstrated rapid clearance of the SARS-CoV-2 virus as evidenced by CT data. Secondly, the immune modulating effects of oral-MB and PBM therapy are both well recognized and their inclusion in our protocol appears to have offered critical benefits in managing COVID-19 with expedited recovery of the lungs and other affected organs. This approach may potentially benefit other severe infectious diseases, where morbidity and death result not only from the direct consequences of infection but where the magnitude and type of host response may be even more problematic.The deployment of a home-based protocol offered multiple benefits both for patients and the healthcare team, including reduced contact between patients and healthcare providers and far greater convenience for patients. Additionally, the simplicity of using a single drug/device protocol to successfully address multiple aspects of COVID-19 intervention, reduced the inherent risks of drug interactions and reduced the costs and other associated challenges of a poly-drug regimen. Despite the markedly high viral loads and morbidity at presentation, in-patient hospital admission was avoided in all pilot subjects who completed the protocol. Reducing the need for hospital admission represents a significant benefit to patients while reducing the burden on local health care facilities.Based on the above factors and the broad scope of PDI application, our protocol can potentially be expanded to target not only an unlimited number of future COVID-19 variants, but also many other infections contracted via the upper respiratory tract or oral mucosa in the future. While the data presented are indeed promising, further extensive studies with larger cohorts utilizing our standardized LURP are warranted to validate our results.An international patent was filed resulting from the work reported in this manuscript: International Patent Application No. PCT/EP2021/062061-SCHIKORA, Detlef; Luminnova Health.