This non-comparative cohort study investigated long-term donor cell survival after allogenic simple/cultivated limbal epithelial transplantations  by genetic analysis. Transplanted corneal epithelial cells, which underwent impression cytology and/or corneal-button biopsy, were examined for personal identities of autosomal short-tandem repeats; the percentages of donor cells were calculated based on matching recipient or donor buccal-DNA references. Twelve patients were included; 4 underwent allo-CLET, 8 underwent allo-SLET. Eight patients (67%) had total limbal stem cell deficiency (LSCD). Genetic analysis was performed postoperatively . Donor cells were detected in 4 of 12 patients (25%), all of whom underwent allo-SLET; 1 patient had a donor genotype and 3 patients had a mixed donor/recipient genotype. The longest time of donor cell detection was 30 months. Seven patients (58%) used systemic immunosuppressives at the time of genetic analysis . Allogenic donor cells survived in both procedures for the long term postoperatively, which encourages the long-term use of systemic immunosuppressives. Donor cells may not be the only factor in graft survival, in that most successful cases had a recipient profile. Their presence for a specific time may promote niches for the patients’ own cells to repopulate, especially for partial LSCD. Limbal stem cell transplantation is the mainstay treatment to restore the corneal epithelium. Conventional surgeries2, when cultivated limbal epithelial transplantation (CLET), which requires the harvesting of only a small amount of donor limbal tissue, was described. However, this technique requires expensive laboratory cell culture facilities. Simple limbal epithelial transplantation (SLET), introduced in 20123, requires only diced limbal tissue expanded in vivo on the corneal surface, thus eliminating the cost of laboratory facilities. During this novel era of limbal stem cell transplantation, the two techniques were among the most commonly performed worldwide5.A significant advance in the field was introduced in 19976, this should be balanced between their various side effects10 and graft survival. In addition, the optimal site of origin of the corneal epithelial cells covering the entire corneal surface in the successful cases also is not definitive. It is interesting to explore if the cells originating from the donor gradually disappear and are ultimately replaced by the recipient cells.Bilateral LSCD is especially challenging because allogeneic limbal epithelial transplantation  is required, and postoperative immunosuppression is necessary to prevent allograft rejection. Because the appropriate duration of using systemic immunosuppressive medications is not definitive12, and no previous studies of SLET have been published. The aim of the current study was to investigate the persistence of donor epithelial cells over the long term in patients who underwent allo-LET transplantation in which we used a new method of specimen collection and genetic analysis.To our knowledge, only two studies have reported allogenic donor survival after CLETThis non-comparative cohort study followed the tenets of the Declaration of Helsinki and the institutional review board of Faculty of Medicine Siriraj Hospital, Mahidol University  approved the study. All patients provided informed consent before enrollment. The study was registered in Thai Clinical Trials Registry . At the time of registration, the protocol with a sample size of 20 and a minimum follow-up of 3 months postoperatively was initially planned. However, we later extended the follow-up time to gain information on DNA analysis in the long term. Eventually, 12 patients were recruited.15. Briefly, the criteria included the presence of total or predominant corneal epithelial cells in the central cornea by IVCM and/or the presence of total or predominant specific markers of corneal epithelial cell (CK12) by ICIF.The inclusion criteria included patients with bilateral LSCD who had undergone allo-CLET or allo-SLET with a follow-up of at least 1 year, age of at least 18 years, and a successful outcome defined by slit-lamp examination and laboratory investigation. Clinical success was determined by the absence of epithelial defects, absence of conjunctivalization in the central 5-mm cornea, and no/mild ocular surface inflammation. Moreover, laboratory investigations for phenotypic identification to prove the existence of corneal epithelial cells by in vivo confocal microscopy (IVCM) and/or impression cytology with immunofluorescence staining (ICIF) were included. The full protocols of both investigations were reported previouslyThe specimen collection techniques for genotyping analyses were from impression cytology (IC) and/or the superficial layer of the corneal button of penetrating keratoplasty (PK). The IC was the mainstay of the sampling in most patients. However, some patients with deep corneal stromal opacity required PK after allo-LET, and superficial dissection for the corneal epithelial layer of the corneal buttons at the time of PK was an alternative sampling technique.The genotyping reference profiles of recipients and donors were obtained using buccal swabs. With allo-SLET, all underwent living-related allo-SLET, so all donor-reference samples were received from living relatives. However, in allo-CLET, all donors were from cadavers and the donor-reference sample collection was impossible. To avoid false donor-positive results, a buccal swab also was obtained from all specimen collectors and laboratory technicians involved in all processes. The genotypic result was interpreted as donor, recipient, or mixed (donor and recipient) genotype, depending on the matching of the results between the patients and the references after DNA genotypic analysis.Corneal epithelial cell samples were collected by IC using Biopore membrane . After application of tetracaine hydrochloride 0.5% eye drops, the membrane was pressed lightly on the entire corneal surface for about 5 s. The procedure was performed using a sterile technique with a negative control, during which another membrane was left in the air and later underwent the same DNA genotypic analysis process.DNA extraction The IC specimen and the buccal swab from the recipient, living-related donor for reference profiles, and controls were extracted using a QIAamp DNA Micro Kit  and subsequently quantified by spectrophotometry.PCR amplification 16 loci, each containing a polymorphic short tandem repeat, of the DNA samples were amplified following the manufacturer’s instructions for the AmpFlSTR Identifiler Plus PCR Amplification Kit . The 16 loci were D451179, D21511, D75420, CSF1PO, D381368, TH01, IM38317, IM88038, H781338, D195433, uWA, TPOX, 010561, AMH, D58818, and HGA.DNA genotyping The PCR amplification product was separated by capillary electrophoresis using ABI 3500 Genetic Analyzers (Applied Biosystems) according to the instruction manual. GeneMapper ID-X Software (Applied Biosystems) was used for data analysis.Twelve eyes of 12 patients  who underwent allo-LET with successful outcomes were included. Eight (67%) and four (33%) eyes had been treated with living-relative-allo-SLET and allo-CLET, respectively. The demographic data and clinical characteristics of the patients are shown in Table The DNA genotypic results of all patients after successful allo-LET are shown in Table Among 4 eyes in the subgroup with partial LSCD and the partial limbal areas that were transplanted, all eyes were classified as having a recipient genotype. Interestingly, among the 8 eyes in the other subgroup with total LSCD with total limbal areas that were transplanted, 1 eye was classified as having a donor genotype, but 3 eyes appeared to have a mixed genotype, and 4 eyes appeared to have a recipient genotype. The eyes with the recipient genotype in the total LSCD subgroup surprisingly had successful outcomes validated by total or prominent corneal epithelia in vivo confocal microscopy or K12 by ICIF.Among the patients with residual donor cells (donor and mixed genotypes), 3 of the 4 patients were receiving systemic immunosuppression between 12 and 30 months.The representative clinical findings of the ocular surface at the time of genotypic analysis are shown in Fig. 18 and SLET14. However, few studies have reported the survival of donor cells over the long term.After the introduction of the advanced surgical techniques of allo-LET for bilateral LSCD in recent decades, clinically successful outcomes have consistently been reported worldwide for CLETThe current study included patients with total and partial LSCD. Of the patients with long-term survival of the donor cells, all had total LSCD and all underwent SLET. Actually, the donor genotype remained in the transplanted cornea in half of the patients with total LSCD including 38% (3 of 8 eyes) with the mixed genotype and 12% (1 of 8 eyes) with the donor genotype. Moreover, the donor cells exceeded 50% in all patients with the mixed genotype and 100% in the eyes with the donor genotype.The extent of LSCD and surgical techniques and other factors that follow might entirely affect the results.12 that studied the fate of the donor cells after successful allo-CLET or allo-SLET and showed that CLET failed to demonstrate long-term survival of the donor cells. Chen et al.12 did not detect any donor cells after long-term allo-CLET , and Daya et al.11 detected donor cells up to 7 months after allo-CLET . Only the absence or presence of the donor cells without mention of the specific amounts or percentages was reported in those studies.The longest time that the donor cells were detected was 30 months postoperatively after CLET or SLET. All patients with long-term survival of the donor cells underwent SLET. This result supported by the only two previous reports22. These results might not be applicable to the recent CLET and SLET techniques, which require only small amounts of transplanted donor tissue. Conversely, the traditional procedures used much larger sizes of donor tissue, thus providing more donor LSCs at the beginning. In addition, the amounts or percentages of the remaining donor cells were not reported in any previous studies, so we could not compare the current results.Compared to the traditional surgical procedures, the studies on KLAL and CLAL tended to report longer donor cell survival times that varied from 3 to 56 monthsThe differences between the SLET and CLET surgical techniques may have been a factor in donor cell detection. SLET is similar to KLAL regarding direct transplantation of the stem cells onto ocular surface, which may result in outcomes similar to KLAL. The multiple steps during the cultivation process also may have interfered with the survival of the original stem cells instead of the 1 step of biopsy harvest and transplantation during SLET.Using living-relative donor (ABO group matched) and performing cell expansion on the patient’s ocular surface during SLET, instead of using cadaveric donor and cell expansion in the laboratory during CLET, may result in different donor survival outcomes.24. A previous study found that adult stem cells had tissue-protective mechanisms against immunologic reactions and apoptosis25. Moreover, a murine study showed that a small population of LSCs regulated the immune reaction by inhibiting lymphocyte proliferation and altering cytokine production and highly expressed genes for various anti-apoptotic proteins26. This explanation might apply especially to patients with partial LSCD when the donor graft helps support the patient’s remaining stem cell to repopulate the corneal epithelium, which gradually grows and eventually replaces the decaying donor cells, and maintain a normal ocular surface27.Interestingly, despite that all current patients had a successful clinical outcome, the recipient genotype was found in most patients . A possible explanation is that the donor grafts themselves may not be the only LSC source in the repopulation of the corneal epithelial cells but may have other roles, especially in providing a niche for the patients’ own cells to repopulate. Those effects of the stem cell may be enhanced by the properties of the amniotic membrane, which also was used in all eyesThe previous statements might be confirmed by the finding that all current patients with partial LSCD had a recipient genotype after either CLET or SLET. Another explanation for this result of partial LSCD might be the different surgical techniques with different amounts of donor LSCs transplanted at the beginning. In contrast to the surgery in which 360 degrees of the fibrovascular tissue on the cornea was removed and transplanted, the surgery for partial LSCD was performed only on certain areas of the diseased corneas and limbus, so fewer donor cells were transplanted.29 have reported preliminary results that autologous conjunctival epithelial cell cultivated on amniotic membrane in a suitable environment can differentiate into the corneal epithelial phenotype and maintain a healthy ocular surface after transplantation in patients with total LSCD. Alternately, the circulating donor DNA of the graft cells may play a role in chimerism similar to solid organ transplantations, which was indicated in animal studies31 and in one study of patients after kidney transplantation in whom the donor DNA was detected in recipient whole blood at 2 years postoperatively32.For the patients with a recipient genotype who presented with total LSCD, the mechanism that maintains the long-term clinical successful outcome without evidence of viable stem cells remains to be elucidated, i.e., the reason for why the surface remained clear and the epithelial phenotype still demonstrated corneal epithelial cells by IVCM and CK12 marker by ICIF. Previous studies33. An examination of all layers of corneal epithelial cells would be ideal and specimen collection from a corneal button might be more reliable. This was achievable in only two patients who underwent a subsequent PK and the donor cells were seen in both corneal buttons with a mixed genotype.The possibility to identify the donor cells may be limited partly by the specimen collection techniques. To illustrate, even though IC is minimally invasive and repeatable and used in most cases, the technique collected only the superficial cell layers. The LSCs, however, were mostly in the basal layer of the corneal epithelium11, which was 7 months and Chen et al.12 in which the cells were not detected at any time. The difference might have resulted from the different techniques of SLET and CLET as discussed previously.The postoperative time may be the other factor that affects donor cell longevity. In this study, the donor cells were detected in all patients for 2 years, after which they were not detected in all cases except for 1 case after SLET in which the donor cells were detected at 30 months. The Moreover, while the donor cells were detected up to 30 months, the availability tended to fade, especially after 2 or 3 years. The patient outlier may be explained by the discussion regarding the contributory factor of partial LSCD. The finding was longer than in the previous reports of Daya et al..12 used only a short course of systemic steroid and stopped within 4 months, in the current study immunosuppressive medications (mycophenolate mofetil and/or cyclosporin) were administered for a mean of 22.5 months. Daya et al.11 also used long-term cyclosporine 2–3 mg/kg and the cells were maintained indefinitely.The use of postoperative systemic immunosuppressive agents may be another explanation. While Chen et al34 found in an animal study that immunosuppression prolonged graft survival but not donor cell survival. They hypothesized that although clinical graft rejection was avoidable with immunosuppression, subclinical rejection was ongoing and continued to slow eradication of the donor cell population. In clinical practice, a recent suggestion was continuation of immunosuppressive agents for longer periods after a minimum of 6 months following allogenic CLET and SLET36. Considering the long-term recognition of the donor genotype at 30 months after allo-SLET in this study, with 1 patient (patient 6) with acute allograft rejection at a similar timeline of 32 months, we suggest continuing the immunosuppressive agents for at least 2–3 years postoperatively.Logically, graft rejection destroys donor cells and, thus, inhibiting graft rejection using immunosuppressive agents might prolong donor cell survival. Most current patients with remaining donor cells in this study still were still receiving systemic immunosuppression. However, among patients who had ongoing systemic immunosuppression at the time of genetic analysis, the donor cells disappeared with the recipient genotype in 4 of 7 patients (57%), suggested that the donor cell population may be depleted despite ongoing immunosuppression. Mills et al.37. To minimize contamination, the protocol in the current study included using a sterile technique during IC and coupling every collection with a negative control, leaving another IC imprinting instrument in the air. This study found no false positive results in any negative controls.Finally, the accuracy in interpreting the results was a concern. A previous publication that studied a technique to detect donor cell survival after allo-LET reported a false positive result in the negative controlsA study limitation was the small number of patients. Even though the 12 eyes are sufficient to interpret the results, there are multiple confounding factors that may have affected the outcome measures. A larger number of cases with a more homogenous group of patients regarding the underlying cause and extent of LSCD and the surgical technique should be studied to analyze the survival and other related factors of donor survival such as HLA matching of donors and recipients and the specific and type/duration of immunosuppressive agents. The strengths of this study were the long-term period (up to 10 years) of investigation of the donor survival in successful allo-LET cases using recent techniques of specimen collection and genetic analysis and the ability to demonstrate the longest persistence of the donor cells of 30 months after allo-CLET and allo-SLET.In conclusion, although the donor cells were detectable during the early years postoperatively, they disappeared in most successful cases. The essential role of the LSC donor in promoting corneal epithelial repair should be studied further to explain those clinically successful cases. The long-term use of postoperative systemic immunosuppressive agents was encouraging.


To characterize personal exposures and measures of eye and respiratory tract irritation in controlled environmental chamber studies of 44 healthy adult volunteers simulating upper-bound use of peracetic acid (PAA)–based surface disinfectant for terminal cleaning of hospital patient rooms.Experimental, within-subject, double-blinded cross-over design.Objective and subjective exposure effects were assessed for PAA and its components: acetic acid (AA) and hydrogen peroxide (HP). Deionized water was included as a control. Breathing-zone concentrations of PAA, AA, and HP were assessed for 8 female multiday volunteers (5 consecutive days) and 36 single-day volunteers . Wetted cloths were used to wipe high-touch surfaces for 20 minutes per trial. Also, 15 objective measures of tissue injury or inflammation and 4 subjective odor or irritation scores were assessed.Disinfectant trials showed 95th percentile breathing zone concentrations of 101 ppb PAA, 500 ppb AA, and 667 ppb HP. None of the volunteers observed over 75 test days exhibited significant increases in IgE or objective measures of eye and respiratory tract inflammation. Subjective ratings for disinfectant and AA-only trials showed similar increases for odor intensity and nose irritation, with lower ratings for eye and throat irritation. Females were 2.5-fold more likely than males to assign moderate + irritation ratings.Simulated upper-bound hospital use of PAA-based disinfectant led to no significant increases in objective markers of tissue injury, inflammation, or allergic sensitization, and no frank signs of eye or respiratory tract irritation. Due to the potential for such infections to be transmitted widely with serious consequences, hospitals have established infection tracking and surveillance systems in place, along with robust prevention strategies to reduce the rate of hospital-acquired infections. Beginning in 2013, peracetic acid (PAA)–based disinfectants were introduced into a number of hospitals because of their superior ability to eliminate Clostridium difficile and other dangerous pathogens. Confirmation of this efficacy was recently demonstrated in a study showing that hospital-wide surface hygiene protocols with a PAA-based surface disinfectant containing active ingredients PAA (0.13%) and hydrogen peroxide  reduced hospital-acquired Clostridioides difficile infections by 50% on average in a study across 8 hospitals over 3 years. In 2022, Carling et al noted that such use of PAA-based disinfectant reduced a variety of other hospital-acquired infections: Staphylococcus aureus (MRSA), Enterococci (VRE), norovirus, Klebsiella pneumoniae, Candida auris, and other multidrug-resistant organisms (MDROs). Despite the demonstrated efficacy of a PAA-based disinfectant in the control of hospital-acquired infection transmission, concerns have been raised regarding the sensory and health impacts of exposure to PAA disinfectants on environmental service workers.Hospital-acquired infections, also known as healthcare-associated infections (HAIs), are nosocomial infections that are not present at the time of admission. Subjective responses of “difficulty breathing” in the 2018 study were most strongly associated with “sensitizer and irritant use” or “total stress.” In the 2019 study, participants frequently attributed their symptoms as work-related regardless of their frequency or magnitude of PAA-based disinfectant exposure. Work-shift average exposures were assessed in both studies, showing 95th-percentile PAA exposures up to 48 ppb, well below the 160-ppb level that is considered to be generally well tolerated. The final evaluations in both the 2018 and 2019 NIOSH reports did not find a robust statistical relationship between the self-reported health effects and work-shift concentrations of active ingredients PAA and HP. However, in 2017, Hawley et al concluded that their findings suggested that PAA and HP “contributed to eye and respiratory symptoms reported by hospital cleaning staff at low levels of measured exposure.”National Institute for Occupational Safety and Health (NIOSH) investigations of 2 US hospitals using PAA-based surface disinfectant evaluated self-reported health complaints relating to skin, eye, and respiratory tract irritation among staff. However, there is substantial variability in the acceptance of PAA-based cleaner. For example, staff members in a South Australian hospital using a chlorine-based disinfectant reported adverse respiratory reactions to the cleaner. Replacing the chlorine-based disinfectant with a hydrogen peroxide disinfectant eliminated respiratory complaints but resulted in an increase of slip incidents and the need for additional cleaning due to a build-up of product residue on surfaces. A 1-year trial of a buffered PAA-based disinfectant, however, resolved the residue problem and, despite the vinegar odor, resulted in no respiratory issues reported by staff, including those originally affected by the chlorine-based cleaner.Many disinfectant cleansers, particularly those used in hospital settings, have strong pungent odors that can cause worker complaints and discomfort, particularly upon initial use.Thus, in present study, we evaluated the irritant and inflammatory potential in the eyes and respiratory tract from a PAA-based disinfectant under conditions of simulated upper-bound use in a hospital setting. Although allergic hypersensitivity -mediated immediate or delayed-type hypersensitivity) to PAA has not been reported in the scientific literature to date, we also sought to evaluate whether 5 consecutive days of exposure to a PAA-based disinfectant increased serum IgE levels. Auxiliary studies characterizing disinfectant product dispenser calibration and environmental service-worker use patterns for hospital patient room cleaning and mass transfer characteristics of surface application and breathing zone PAA concentrations when applying a high-end disinfectant concentration will be reported separately.Two subgroups of volunteers participated: (1) single-day volunteers  to assess the range of individual exposures and irritant responses, and (2) multiday volunteers  whose exposures and irritant responses were assessed over 5 consecutive-use days. and they exhibit irritant receptor-triggered cough reflex at lower average airborne-irritant concentrations compared to males . None of the participants reported a history of any respiratory conditions, including asthma, and none reported any prior exposure to any PAA-based disinfectant.The study was approved by the Advarra Institutional Review Board and full consent was obtained from each of the healthy volunteers. Study participants were recruited by Monell Chemical Senses Center and were compensated for their participation. Predominantly female participants were included because they are significantly more prone to report odor-related irritation compared to male participants,1).In this study, we utilized an experimental, within-subject, double-blind, crossover design for evaluating eye and respiratory irritation responses in healthy human volunteers to a PAA-based disinfectant and its components in an environment chamber, which was set up to simulate a hospital room Fig. . The env3). Figure Participants were exposed during each of 4 conditions during a single day: (1) PAA-based disinfectant (positive odor or irritant), 2) acetic acid (AA) only (positive odor or irritant), (3) HP only (negative odor or irritant), and (4) water only (negative odor and negative irritant control) . The AA-only solution (0.38%) and the HP-only solution (0.63%) were utilized to generate similar airborne exposures as observed with the disinfectant mixture. Solutions were made fresh daily and were evenly distributed over a designated number of microfiber cloths (dimensions 38 cm × 40 cm) at a saturation level of 125 mL per cloth that could be used for surface cleaning without dripping, in accordance with the disinfectant manufacturer’s training materials for use (Supplementary Appendix 2).The PAA-based disinfectant use solution  contained 0.13% PAA, 0.16% AA, and 0.64% HP (Supplementary Appendix  surface area)  Fig. . Volunte3.The following objective measures were applied: eye imaging for measurement of eye-blink frequency and hyperemia ; nasal assessment of right and left sinus volume by nasal rhinometry; nasal nitric oxide; nasal mucus collection for measuring selected cytokines of respiratory tract inflammatory response ; and respiratory assessment of exhaled breath nitric oxide and spirometry . Multiday volunteers were also assessed for allergic sensitization by analysis of blood immunoglobulin E before the first exposure trial and after the last trial on day 5. Each trial was video-recorded, and participants were observed for any frank signs of eye or respiratory irritation . Further details on the irritation metric methods and data collected are reported in Supplementary Appendix  a log scale for sensation intensity, was used by each volunteer to assign ratings anchored by corresponding magnitude descriptors . The general labeled magnitude scale,ors Fig. .For the statistical analysis, summary statistics were calculated by solution for each exposure and irritation metric. For the exposure characterization, analysis of variance models (ANOVAs) were implemented using the Tukey multiple comparison test to identify significantly different group means. All analyses were performed in SAS version 9.4 software .For the subjective irritation scores, ANOVAs were used with conditions (A–D) and time (before or after and hour) as factors to understand the effects of OxyCide™ exposure. Any interactions revealed by analysis were explored using a Tukey HSD post hoc analysis, with Bonferroni corrections as appropriate. All analyses were performed in Statistica software .P = .0065). None of the volunteers exhibited increases in objective measures of eye and respiratory tract inflammation. We observed no increase in ocular redness or vascularity following any exposure condition. In addition, no significant elevations were detected for nasal or lung nitric oxide, and no decreases were observed for any of the spirometry end points . None of the 8 multiday volunteers exhibited increased serum IgE after exposure day 5 compared to their pre-study value (Supplementary Appendix 3).Mean ± standard deviation personal exposures for the disinfectant trials were 66 ± 23 ppb PAA, 287 ± 121 ppb HP, and 387 ± 148 ppb AA; the relevant component exposures were comparable in the AA-only and HP-only trials (Table 3).Subjective irritation scores for the disinfectant trials (PAA/AA/HP) and for the AA-only trials were similarly elevated for odor and nose, with lower scores for eye and throat Table . Female  was reported as significantly lower than nasal irritation. The similarity between the ratings of odor intensity and the nasal irritation ratings to disinfectant solution and AA-only trials suggests that the combined sensations of a strong and unusual odor with tingling sensations in the nose served to elevate the reporting of nasal irritation. Importantly, none of the volunteers had significant increases in objective markers of inflammation or allergic sensitization, and none exhibited any ocular or respiratory tract irritation. These findings demonstrate that no significant increase in objective markers of tissue injury or inflammation of the eye or respiratory tract were observed for healthy volunteers exposed during upper bound use of the PAA-based disinfectant or its components (HP only and AA only) despite the broad range of subjective irritation scores describing the intensity of unpleasant sensation for the 44 volunteers studied over 75 days of testing.This study of upper-bound personal exposures during simulated hospital use of PAA-based disinfectant demonstrated that, based on mean subjective irritation scores for the disinfectant solution trials (PAA/AA/HP) and the AA-only trials, the volunteers considered their personal exposures to be significantly more irritating on average compared to HP-only or deionized water trials. Notably, eye irritation, which is typically a more sensitive locus for airborne irritants, particularly among women, and show a lower threshold for irritant receptor-triggered cough reflex. This element was part of the upper-bound response analysis that was incorporated into the study design. Consistent with the published findings showing greater sensitivity to odors and inhaled irritants, female participants in the current study were significantly more likely (2.5-fold) than male participants to rate subjective odor and nasal irritation as “moderately sensed” or greater. The small subset of men (n = 4) showed a statistically significant 30% lower mean breathing zone PAA compared to the 40 women and had lower means for all other exposure metrics that were not significantly different at P < .05. These lower breathing-zone measurements may be attributable to greater male volunteer height , differences in wiping technique, and/or chance related to the far fewer observations for men relative to women studied.Our study design included predominantly female participants because compared to males they are significantly more prone to report odor-related irritation1) evaluated 49 EVS workers across 15 hospitals and demonstrated that the selected 20 minutes wiping period for PAA-based disinfectant exceeded the interquartile range (IQR) of 8.8–18 minutes in the field. An IQR of 3–7 cloths used per patient room or bathroom and an IQR of 1.7–4.1 minutes for cloth handling time were observed versus the current study protocol utilizing 4 cloths handled for 5 minutes each. Separate studies (Supplementary Appendix 1) evaluating the mass transfer characteristics of PAA-based disinfectant at a higher concentration  showed 24 ppb higher average breathing zone PAA concentrations  (Supplementary Appendix 1). Compared to longer periods of individual wetted cloth use , using cloths for <5 minutes led to significantly higher disinfectant film density  and application time . It also led to significantly increased cleaning efficiency , longer dwell time (>5 minutes), and significantly reduced PAA transfer to the user’s breathing zone (an average of 7.0% PAA transfer vs 14.9% at 1 cloth per 20 minutes). These findings suggest that applying a heavier film density of PAA-based disinfectant reduces the fraction immediately released to air, thereby increasing the PAA mass retained on surfaces to accomplish antimicrobial efficacy goals.The current study was designed to simulate upper-bound environmental service (EVS) worker use of PAA-based disinfectant for terminal cleaning of hospital patient room and bathroom spaces. Each trial included 20 minutes of continuous wiping high-touch surfaces with wetted cloths according to the product manufacturer’s training guidelines for cleaning. Auxiliary studies  compared to an expected floor space (patient room + bathroom) in hospitals of 24–30 m. The volunteers completed 3–5 full cycles of simulated cleaning in 20 minutes, corresponding to a total surface area cleaned at 25–34 m, comparable to the total floor space of typical patient room and bathroom spaces. Considered collectively, these study design elements reasonably represent upper bounds for use conditions and breathing zone exposures to PAA, HP, and AA for EVS workers during patient room terminal cleaning.The current study chamber had a fresh-air exchange rate of 5.4 to 5.5 per hour, which was below the minimum recommended 6 hours for patient rooms.In conclusion, the current study findings provide important dose–response evidence for eye and respiratory irritation/inflammatory responses among healthy human volunteers exposed during upper-bound use conditions of PAA-based hospital surface disinfectant or its components, HP and AA. The range of breathing-zone concentrations reported for these studies demonstrate no objective effects on eye or respiratory tract tissue injury or inflammation, and, among a subset of the volunteers, did not induce allergic sensitization following 5 consecutive days of exposure to the disinfectant or its components. With continued exposure, sensory adaptation is a common feature of human sensory systems—odor and irritation—has been demonstrated for numerous substances, including acetic acid. Confirmation of this shift in response is shown by the dramatic reduction in health complaints made to the manufacturer or its independent agent of the disinfectant from 2013 to 2020 (Supplementary Appendix 2). The data presented here demonstrate the range of subjective irritation responses of naïve individuals to a PAA-based disinfectant and AA-only under conditions of simulated environmental service worker use, but no significant increases in objective irritation measures of eye and respiratory irritation, inflammation, or sensitization.Although environmental service workers have far longer-term exposure to PAA-based disinfectants than the participants in these studies, the irritation effects of PAA are known to be concentration dependent rather than concentration-times-duration dependent.


As both the industry and academia become more focused on biomass-based smart materials, they are attracting a lot of attention. There has been a significant effort in the field of polyurethane (PU) synthesis to replace polyols used in synthesis with bio-derived polyols. Bio-derived polyols have limited application potential for bio-based PU due to their low functionality. Here, we reported castor oil (CO) based multifunctional polyols prepared by grafting thiols such as 1-mercaptoethanol or α–thioglycerol via a facile thiol-ene click reaction method . Subsequently, bio-based shape memory polyurethanes (SMPU) crosslinked with prepared polyols were synthesized using a 2-step prepolymer method. By confirming the functionality of the synthesized polyols, it was determined that COT has an OH value of 380 mg KOH/g, which is approximately three times that of CO. The successful synthesis of SMPUs was confirmed through chemical structural analysis. It was also proved that the phase separation between the soft and hard segments was limited due to the increase in crosslinking density. As compared to SMPU crosslinked with CO, the mechanical strength of SMPU crosslinked with COT was improved by 80%, while the elongation was decreased by about 26%. As a result of shape memory behavior analysis, it was confirmed that the outstanding SMPU can be synthesized using CO-based multifunctional polyols. SMPUs are typically synthesized by reacting diisocyanate with a mixture of macroglycol and a low-molecular-weight diol or diamine used as a chain extender. Typical polyol types include polycarbonate polyols, polyether polyols, adipate-based polyester polyols, and caprolactone-based polyester polyols9. Among these, caprolactone-based polyester polyols exhibit excellent heat resistance, water resistance, and processability and have a wide hardness range; therefore, they are widely used in PU development. The characteristics of SMPU strongly depend on the hydrogen bonding in a PU structure. There are hydrogen bonds within the hard segment and also between the hard and soft segments in PU. Hydrogen bonding within the hard segments occurs between two urethane units, which causes strong phase separation. Hydrogen bonds formed between the hard and soft segments are known to lead to phase mixing. Therefore, the analysis of hydrogen bonding in SMPUs is essential to characterize and design SMPUs10.Shape memory polyurethanes (SMPU) are an important class of polymeric smart materials which can be programmed into temporary shape and returned their original shape by various external stimulus such as heat, light, humidity, pH, microwave, ultrasound or electricityTg) of the PU11. Zhang et al.12 prepared green polyols by grafting fatty acids extracted from CO into various vegetable oils, such as olive, canola, grape seed, linseed, and CO, which were used to produce PUs, and investigated PU properties. It was found that the higher the hydroxyl value of the modified vegetable oil, the higher the crosslinking density and Tg of a PU.In recent years, research on PU using bio-based materials has been conducted to replace petrochemical-based chemicals. Oprea synthesized a PU elastomer using polyether-based polyols, hexamethylene diisocyanate (HDI), and a mixture of Castor oil (CO) and 1,4-butanediol as a chain extender and reported that the increased hard segment molar ratio and the presence of a dangling structure formed by introducing CO into the hard segment resulted in an increase in the glass transition temperature  which is an aliphatic unsaturated chain with a double bond at the C-9 position1H-NMR analyses. After confirming that the hydroxyl value of CO gradually increased from 160 mg KOH/g to 270 mg KOH/g and 380 mg KOH/g after mercaptoethanol or α-thioglycerol were introduced into CO, respectively. PUEs were designed using them as crosslinking agents. Semicrystalline polycaprolactone-diol (PCL-diol) was used for the soft segment, and the hard segment comprised HDI and the as-prepared CO-based multifunctional polyol as a crosslinking agent. For comparison, SMPU using CO as a crosslinking agent was also prepared. The chemical structural properties of the prepared PUs and the change of hydrogen bonding in PU according to the type of crosslinking agent were analyzed by FT-IR spectroscopy. The structural, thermal properties, crystallinity, and thermal stability of the SMPUs were analyzed by Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD) analysis, and thermogravimetric analysis (TGA). Finally, the mechanical and shape memory characteristics were analyzed by using a universal testing machine.In this study, multifunctional polyols were prepared by grafting mercaptoethanol or α-thioglycerol  into CO via a thiol-ene click reaction. The success of the grafting reactions for the as-prepared polyols was confirmed by FT-IR and N,N-dimethylformamide (DMF), magnesium sulfate anhydrous (MgSO4) and sodium chloride (NaCl) were supplied from Daejung Chemical (Korea). HDI was supplied from Wako chemical. Polycaprolactone-diol, CAPA 2200A (molecular weight 2000) was purchased from Perstorp chemical (Sweden). All reagents were used as received without further purification.CO was purchased from Yakuri Pure Chemical Co., Ltd. Mercaptoethanol, α-thioglycerol, 2,2-dimethoxy-2-phenylacetophenone (DMPA), and dibutyltin dilaurate (DBTDL) were purchased from Sigma-Aldrich (USA). Ethyl acetate, 24. Briefly, the quartz tubes sealed with rubber septa containing the mixture of CO, thiols, photo initiators (DMPA), and solvent (ethyl acetate) were rolled on a tube-roller placed in modified incubator in order to ensure thorough mixing during the reaction. The molar ratio of thiols was set to 4:1 for the C=C double bonds of CO, and DMPA was incorporated as much as 2 wt.% of the entire reactant. After 24 h of the reaction, the products were washed with deionized water and NaCl aqueous for at least five times. The polyols were dried using MgSO4 and the solvent was eliminated by rotary evaporation. The final products were dried under vacuum for 24 h prior to use. The polyols were coded as COM and COT treated with mercaptoethanol and 1-thioglycerol respectively. The successful of the COM and COT synthesis was confirmed according to the results of our previous studies24.The CO based multifunctional polyols with high functionalities were prepared by our previous method Fig. a22–24. B2 atmosphere with a few drops of DBTDL as a catalyst. In the second step, CO, COM, and COT dissolved in DMF were added dropwise and stirred at 80 °C for 3 h. No additional catalyst was added. The solutions were degassed in vacuo at room temperature and cast into a PTFE-coated mold. The solvent was allowed to evaporate at 80 °C in a convection oven for 24 h. The final ratios of NCO:OH of the products were 1.05:1. The detailed composition of the PUs obtained are listed in Table Before preparation of the polyurethane films, the CO, COM and COT were dried at 80 °C for 10 h in vacuo. The polyurethanes were synthesized in a two-step polymerization process as shown in Fig. 3 according to the ASTM 638 standard and the crosshead speed was set to 20 mm/min. The mechanical cyclic recovery test was carried out 5 times up to 100% strain. To quantify the mechanical hysteresis (HM) for the prepared SMPUs, according to Fig. HM were calculated by equation given below from the difference in area under the loading and unloading curves.ATR-FTIR (Nicolet 760 MAGNa-IR spectrometer) was used to determine the structure of the polyurethanes. XRD patterns were scanned in a 2θ range of 5‒70° at scan rate of 3°/min using X-ray diffractometer . TGA for the PUs were carried out from 30 to 800 °C at 20 °C/min under nitrogen atmosphere using PerkinElmer Pyris 1. Thermal behavior of the SMPUs were characterized by DSC . All SMPUs were heated from − 80 to 160 °C at 10 °C/min under nitrogen atmosphere and held at 160 °C for 3 min and quenched to − 80 °C, followed by heating again to 160 °C at the same heating rate. The mechanical properties of the SMPUs were measured using an AND MCT-1150 universal testing machine. Testing specimens were prepared in a dimension of 63 × 3 × 0.3 mmShape-memory test was carried out using Instron 5960 UTM equipped with environmental chamber.SMPUs were successfully synthesized by applying PCL-diol (2000 g/mol) as a soft segment and either CO or the as-prepared COM or COT as a crosslinker. As shown in Fig. −1. Compared to the PCL-diol spectrum, the spectra of the SMPU samples showed new peaks corresponding to N–H and C=O stretching bands in the urethane bonding at 3380 and 1728 cm−1, respectively, and a peak ascribed to N–H bending at 1533 cm−1. Moreover, peaks due to the symmetric sp2 and asymmetric sp3 stretching bands from the aliphatic chain were observed at 2937 and 2859 cm−1, respectively25. No significant change in the FT-IR spectra was observed after introducing the crosslinking agents with different OH values into the hard segment. According to several studies, however, we can obtain meaningful information about hydrogen bonding in PUs from the FT-IR spectra26. The most widely known approach is to determine the amount of hydrogen bonds by separating the carbonyl peaks. In the PCL-based PU, it is not straightforward to separate the hydrogen-bonded carbonyl peak (about 1700 cm−1) and the free carbonyl peak (about 1730 cm−1)27. In our study, the carbonyl peaks were also overlapped because of the carbonyl peaks of PCL. In such a case, however, the N–H stretching peak can provide meaningful information on hydrogen bonding. It has been found that the sharper the N–H stretching peak, the greater the number of hydrogen bonds; whereas, the broader it is, the fewer hydrogen bonds. The magnified N–H stretching peaks of the SMPU samples are shown in Fig. To analyze the chemical structures of the synthesized SMPUs, FT-IR analysis was performed; the results are shown in Fig. θ = 21.3°, whereas the XRD patterns of the SMPU samples showed a broad diffraction peak at 2θ = 15–30°, which represents the semi-crystalline properties of SMPU; this suggests that the crosslinked bonds were formed between the hard and soft segments after CO, COM, or COT was introduced as a crosslinking agent. This bond formation limited the mobility required for the soft segment to form a crystal structure. In particular, the full width at half maximum (FWHM) of the broad peak was in the order of CO < COM < COT, which confirms that the crosslinking density increased with increasing functionality of the polyols used, resulting in the formation of a more amorphous structure.XRD analysis was performed to study the crystallinity of the SMPU samples. The patterns are shown in Fig. Tm), transition temperature (Tg), degree of crystallinity (Xc), and others are listed in Table Tg, crystallization temperature (Tc), and melting temperature (Tm) were observed in all SMPU samples. It is known that Tg does not appear in PCL-diol, but Tg became detectable after PCL-diol was incorporated into the crosslinked PU structure. However, almost no change in Tg was observed upon varying the introduced crosslinking agent. It was found in other studies that Tg increased due to inter-domain mixing between the soft and hard segments when small molecule triols such as trimethylolpropane were used as a crosslinking agent. In the cases of CO, COM, and COT, this distinction could not be made because of their very bulky structures. The heat of fusion per gram of PCL-diol in the PU of SMPU-CO, SMPU-COM, and SMPU-COT decreased to 43.9, 34.4, and 32.2 J/g, respectively, as shown in Table The thermal properties of SMPU-CO, SMPU-COM, and SMPU-COT were analyzed through DSC and the 2nd heating scan are shown in Fig. 29. Table 31.To verify the thermal stability of the synthesized SMPU samples, TGA was performed under a nitrogen atmosphere. TG thermograms and their derivative thermogravimetry (DTG) curves are depicted in Fig. 32. Therefore, the number of crosslinking points increased after the crosslinking agents having high functionalities was introduced, which resulted in a considerable increase in tensile strength.Figure 25. Moreover, if the initial elongation were not exceeded in subsequent cycles, no further deformation of the hard segment occurred and, thus, high elastic recovery could be maintained.To investigate the elastic recovery characteristics of the synthesized SMPUs, five cyclic mechanical tests were performed; the results are shown in Fig. L0. The melting temperature and crystallization temperature of the soft segment of the prepared SMPUs were considered as the transition temperatures. After sufficient time was given, the sample was stretched to 200% and 400%. The stretched length of sample is nL0 (n = 2 and 4). Then, the specimen is cooled below its transition temperature, followed by removing tensile load. When the temperature was raised above the transition temperature again, shape recovery of the specimen occurred. A detailed description is shown in Fig. L2. Shape fixity (Rf) and shape recovery ratios (Rr) are calculated by using follow equations:In order to evaluate the shape memory characteristics, shape memory test using UTM was performed with the following procedures: The sample is first heated above the transition temperature. The original length of the sample is Typical shape-memory testing profile of SMPU-COT at strains of 200 and 400% are shown in Fig. In this study, two types of multifunctional polyols having increased hydroxyl values were prepared using a thiol-ene click reaction in CO. Three types of PU were prepared by a two-step synthesis based on the PCL-diol/HDI system by applying either CO or the COM or COT as a crosslinking agent. The successful synthesis of SMPU was confirmed by ATR-FTIR analysis; it was confirmed that hydrogen bonding in the molecule decreased as the functionality of the crosslinking agent increased. Based on XRD and DSC results, it was confirmed that the phase separation between the soft and hard segments was limited because of the increase in the crosslinking density, which decreased crystallinity. TGA results confirmed that SMPU-COT, which decomposed at the highest temperature, exhibited high thermal stability. Based on mechanical property measurements, the tensile strength of SMPU-COT was 23 MPa and the tensile elongation was 840%. Compared to SMPU-CO, the strength was improved by ~ 80%, and the elongation was decreased by about 26%. By introducing a crosslinking agent having a higher functionality into the hard segment, improvement of the elastic recovery property of SMPU was confirmed. As a result of the shape memory behavior analysis, it was confirmed that the higher the functionality of the crosslinking agent introduced into the SMPU led the better shape fixity and recovery characteristics of the SMPU.Supplementary Information.


CSF viral escape was associated with increased IL-1β, IFN-γ, IP-10, ICAM-1, and VCAM-1 in CSF but not plasma; IP-10 had the strongest association (p = 0.0008). CSF VL and WBC correlated with IFN-γ, IP-10, ICAM-1, and VCAM-1 (p < 0.05). Although markers of CNS injury showed no significant association with asymptomatic CSF viral escape, CSF YKL-40 correlated positively with CSF IL-1β (p = 0.003), IFN-γ (p = 0.0008), IP-10 (p < 0.0001), and NFL (p = 0.06) and negatively with neurocognitive T scores (p = 0.02). These findings identify CSF inflammation and glial activation markers that may serve as surrogate measures of HIV persistence in the CNS for future studies on therapeutics targeting the CNS reservoir.HIV establishes a viral reservoir in the CNS despite viral suppression in the blood on antiretroviral therapy (ART). In a minority of people with HIV (PWH), HIV RNA is detectable in CSF when HIV RNA in plasma is undetectable or HIV RNA levels are higher in CSF compared with plasma, an event termed CSF viral escape that can occur with or without neurological symptoms. Asymptomatic CSF viral escape occurs in 3–20% of PWH on ART, yet associated biomarkers are unclear. To identify biomarkers associated with asymptomatic CSF viral escape, we performed a matched group study of PWH on ART with vs. without CSF viral escape  and 50 HIV-negative controls. PWH were on 3 or more ART drugs for >1 year, and the group with no CSF viral escape was suppressed below 50 copies/mL in plasma and CSF. Biomarkers of inflammation , cell adhesion , CNS injury (NFL), and glial activation  were measured in paired plasma and CSF using the Meso Scale Discovery platform. PWH with vs. without CSF viral escape had more individuals (40%) with a plasma viral load (VL) > 50 copies/mL, higher CSF VL (median 156 vs. 40 copies/mL;  HIV infects the CNS and establishes a persistent viral reservoir in the brain despite viral suppression in the blood to below detectable levels on antiretroviral therapy (ART). HIV can be detected in cerebrospinal fluid (CSF) soon after acute infection, while early ART initiation has been shown to decrease the size of the brain viral reservoir and associated CNS injury ,2. Most HIV-associated neurocognitive disorders (HAND), consisting of asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and HIV-associated dementia (HAD), remain prevalent in people with HIV (PWH) despite viral suppression on ART ,8. AlthoIn most PWH on ART, HIV RNA is suppressed to below detectable levels in both plasma and CSF; when plasma HIV RNA is detectable, HIV RNA levels are typically more than one log10 copies/mL lower in CSF than in plasma ,26,27,28The identification of biomarkers associated with CSF viral escape is relevant for clinical management and developing strategies to achieve HIV cure. However, few reports have been published on biomarkers in CSF viral escape; limited data in these studies detected biomarkers of CNS immune activation and neuroinflammation, particularly neopterin, IP-10, and VCAM-1 ,37,38,39Plasma and CSF samples from 120 individuals  were collected between 2006 and 2016. HIV+ individuals were enrolled at four sites in the National NeuroAIDS Tissue Consortium (NNTC)  and six sites in the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) study . Recruitment procedures and eligibility criteria for NNTC and CHARTER have been described ,45. All Eligible HIV+ participants were adults on a stable combination ART regimen including three or more ART drugs for ≥1 year with one or more paired plasma and CSF samples with viral load (VL) measurements between 2006–2016 and sufficient sample volumes available for the proposed studies. Lumbar punctures were performed for research purposes. Plasma and CSF VL were measured using assays considered to have a lower limit of detection of 50 HIV RNA copies/mL ; three CSF samples had additional available measurements to detect HIV RNA below 50 copies/mL using a single copy assay . We exclHIV+ individuals were administered a comprehensive neuropsychological test battery designed to assess seven neurocognitive domains . Demographically corrected global neurocognitive T scores were generated from individual test T scores, as described ,46. T schttps://www.mesoscale.com/~/media/files/handout/assaylist.pdf, accessed on 26 August 2023.Plasma and CSF samples were centrifuged at 1200 rpm for 5 min at 4 °C, and the supernatants were aliquoted and stored at −80 °C. Plasma and CSF concentrations of inflammation markers , cell adhesion molecules , CNS injury marker (NFL), and glial activation markers (GFAP and YKL-40) were measured using the Meso Scale Discovery (MSD) platform  according to the manufacturer’s protocols and data analyzed using MSD discovery workbench 4.0 software, as described . DetectiCSF and plasma albumin levels were measured using the BCG albumin assay kit , as described . The CSFp-value < 0.05 was considered statistically significant (no Bonferroni correction was applied). Relationships between continuous variables were analyzed by Spearman’s rank correlation. Statistical analyses were performed using GraphPad Prism, version 9.0.Demographics and clinical characteristics were compared between groups of interest by the nonparametric Mann–Whitney U test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables. Differences in log10 transformed biomarker levels between paired plasma and CSF samples were analyzed by the paired Wilcoxon signed-rank test and between groups of interest by the Mann–Whitney U test. For these statistical analyses, a two-sided p = 0.27), and CSF/plasma albumin ratio , but they have a higher proportion of detectable plasma VL > 50 copies/mL , a higher CSF VL , a lower CD4 count , and an increasing trend for CSF white blood cell counts . The most commonly used ART drugs were emtricitabine/tenofovir (53%), efavirenz (29%), 3TC (27%), ritonavir-boosted atazanavir (22%), abacavir (20%), and darunavir (19%); the most commonly used integrase inhibitor was raltegravir (14%). There were no significant differences between groups by CSF VL > 50 copies/mL vs. <50 copies/mL in percentage with a HAND diagnosis (ANI or MND) , global neurocognitive T score (median 47 vs. 47), and BDI score (median 6 vs. 7).The demographic and clinical characteristics of the study cohort are summarized in p = 0.005, <0.0001, 0.003, 0.01, 0.0004, respectively) and CSF VL < 50 copies/mL , while plasma IL-1β, IL-8, MCP-1, and VCAM-1 were higher only in HIV+ individuals with CSF VL < 50 copies/mL . In contrast, plasma IFN-γ, IL-6, and IL-15 and CSF IL-6, IL-15, and MCP-1 levels were not significantly different between groups by HIV status. In HIV+ individuals with CSF VL > 50 copies/mL vs. <50 copies/mL, IFN-γ, IL-1β, IP-10, ICAM-1, and VCAM-1 levels were increased in CSF  but not plasma. Among these biomarkers, CSF IP-10 had the strongest association with CSF viral escape (p = 0.0008).Next, we compared plasma and CSF inflammation biomarker levels by HIV status and CSF VL > 50 copies/mL vs. <50 copies/mL . Comparep = 0.004), while IFN-γ, IL-1β, ICAM, and VCAM-1 levels were higher in plasma compared with CSF among both HIV+ individuals with CSF VL < 50 or >50 copies/mL. In contrast to the upregulation of IP-10 in CSF vs. plasma observed among HIV+ individuals with CSF VL > 50 copies/mL, IP-10 levels were similar in CSF compared with plasma among HIV+ individuals with CSF VL < 50 copies/mL.Next, we compared levels of IFN-γ, IL-1β, IP-10, ICAM-1, and VCAM-1 in paired plasma vs. CSF of HIV+ individuals on ART according to CSF VL < 50 copies/mL and >50 copies/mL A. IP-10 p = 0.06; r = 0.791 p = 0.001, respectively), IP-10 , ICAM-1 , and VCAM-1 ; IL-1β showed similar increasing trends   B.p = 0.03, 0.03, and <0.0001, respectively). In contrast, CSF GFAP and YKL-40 levels were increased in both HIV+ individuals with CSF VL < 50 copies/mL and >50 copies/mL vs. HIV- controls , while CSF NFL levels showed no significant differences between groups. Among these CNS injury and glial activation biomarkers, CSF YKL-40 showed the most significant upregulation in HIV+ individuals with CSF VL > 50 copies/mL when contrasting differences between groups according to CSF VL > 50 copies/mL and <50 copies/mL vs. HIV- controls.Given that some previous studies have detected increased CNS injury biomarkers in HIV+ individuals with CSF VL > 50 copies/mL, particularly those with associated neurological symptoms ,28,31,40p = 0.003), IFN-γ (p = 0.0008), and IP-10 (p < 0.0001). Additionally, CSF IFN-γ, IL-1β, and IP-10 were strongly correlated with each other . Both ICAM-1 and VCAM-1 were also correlated with IL-1β, IFN-γ, and IP-10 . These findings suggest there may be functional inter-relationships between CSF IL-1β, IFN-γ, IP-10, ICAM-1, VCAM-1, and glial activation marker YKL-40 in the CNS of HIV+ individuals on ART that are upregulated in the setting of asymptomatic CSF viral escape.The preceding analyses suggest that the glial activation marker CSF YKL-40 may be upregulated in HIV+ individuals with asymptomatic CSF viral escape. Therefore, we evaluated inter-relationships between YKL-40 and other biomarkers of interest by Spearman’s rank correlation analyses . CSF YKLp = 0.0008 and p < 0.0001, respectively). In contrast, there was no significant difference in plasma YKL-40 level between HIV+ individuals without HAND and HIV- controls. CSF YKL-40 levels were significantly increased only in HIV+ individuals with HAND compared with HIV- controls (p = 0.0007), while there was no significant difference between HIV+ individuals without HAND vs. HIV- controls or HIV+ individuals with vs. without HAND. We did not detect significant differences in plasma/CSF NFL, GFAP, or other biomarkers between HAND vs. no HAND. CSF YKL-40 levels correlated negatively with global neurocognitive T scores  and positively with CSF NFL levels   B,C, albeIn this study of PWH on ART with vs. without asymptomatic CSF viral escape, in which groups with CSF VL > 50 copies/mL or <50 copies/mL were matched for age, duration of HIV infection, nadir CD4 count, and type of ART regimen, we showed that asymptomatic CSF viral escape was associated with increased IL-1β, IFN-γ, IP-10, ICAM-1, and VCAM-1 in CSF but not plasma. CSF IP-10 had the strongest association with CSF viral escape, consistent with previous studies ,37. FurtOur finding that IFN-γ and IP-10 levels correlate with HIV RNA levels and WBC counts in the CSF of PWH on ART is consistent with previous studies ,14,16,49We found that CSF IL-1β, IFN-γ, IP-10, ICAM-1, and VCAM-1 levels were increased in PWH on ART with CSF viral escape and inter-correlated with each other, implying functional inter-relationships between these pro-inflammatory molecules. VCAM-1 and ICAM-1 are cell adhesion molecules that increase the attachment of circulating leukocytes to endothelial cells and thereby promote immune cell trafficking into the CNS. Within the CNS compartment, these adhesion molecules are expressed not only on endothelial cells that form the blood-brain barrier but also on epithelial cells in the choroid plexus that form the blood-CSF barrier. VCAM-1, ICAM-1, and other cell adhesion molecules are induced during CNS immune activation and neuroinflammation, which in turn increases immune cell trafficking across the blood-brain and blood-CSF barriers. Our finding that VCAM-1 is strongly upregulated in CSF viral escape is consistent with a previous study , and we Although CSF NFL, GFAP, and YKL-40 levels were not significantly different between PWH with or without asymptomatic CSF viral escape, CSF YKL-40 correlated positively with CSF IL-1β, IFN-γ, IP-10, and NFL and negatively with neurocognitive T scores. Previous studies have suggested that CSF YKL-40 is associated with increased plasma and CSF HIV/SIV RNA, HIV/SIV encephalitis, and markers of axonal injury ,48,51,52As expected, PWH with vs. without asymptomatic CSF viral escape had more individuals (40%) with plasma VL > 50 copies/mL, a higher CSF VL, a lower CD4 count, and a higher CSF WBC, consistent with previous studies ,27,29,36We acknowledge several limitations of the study, including a relatively small sample size and small group of PWH with asymptomatic CSF viral escape, which reduces statistical power and the ability to detect some associations. Additionally, this was a cross-sectional study, so we could not evaluate the prognostic associations of biomarkers with future CSF viral escape and neurocognitive impairment. We did not have data on neurological signs or symptoms for the HIV- control group, which could have given a clearer picture of the potential value of some biomarkers. The viral load assays used to measure CSF HIV RNA levels had a detection limit of 50 copies/mL, so we could not evaluate associations between measurable CSF HIV RNA below 50 copies/mL and biomarkers. We did not have data to address some factors that impact durable viral suppression and the development of CSF viral escape, particularly ART adherence, treatment interruptions, viral blips, HIV genotypes, and drug resistance mutations. Additionally, we did not evaluate any macrophage- or microglia-specific markers. The majority of PWH were receiving outdated protease-inhibitor-based ART regimens , and a minority were on an integrase inhibitor, which is now a mainstay of therapy , so some findings may reflect the use of older ART drugs. Lastly, the studies were correlative, so we could not establish causality. Longitudinal studies of larger cohorts on newer ART regimens that incorporate sensitive single-copy assays to detect HIV below 50 copies/mL may overcome these limitations and determine the prognostic significance of CSF biomarkers.In summary, we identified CSF biomarkers associated with asymptomatic CSF viral escape in PWH on ART in a matched-group cross-sectional study of HIV+ individuals with CSF viral escape and CSF HIV RNA > 50 copies/mL vs. <50 copies/mL. The studies identified a panel of CSF biomarkers reflecting inflammation and glial activation in the CNS that may serve as surrogate measures of viral persistence in the CNS and may be useful for future studies evaluating therapeutics targeting the CNS reservoir.


Cerebrospinal fluid (CSF) plays an important role in the homeostasis of the brain. We previously reported that CSF major glycoproteins are biosynthesized in the brain, i.e., lipocalin-type prostaglandin D2 synthase (L-PGDS) and transferrin isoforms carrying unique glycans. Although these glycoproteins are secreted from distinct cell types, their CSF levels have been found to be highly correlated with each other in cases of neurodegenerative disorders. The aim of this study was to examine these marker levels and their correlations in other neurological diseases, such as depression and schizophrenia, and disorders featuring abnormal CSF metabolism, including spontaneous intracranial hypotension (SIH) and idiopathic normal pressure hydrocephalus (iNPH). Brain-derived marker levels were found to be highly correlated with each other in the CSF of depression and schizophrenia patients. SIH is caused by CSF leakage, which is suspected to induce hypovolemia and a compensatory increase in CSF production. In SIH, the brain-derived markers were 2–3-fold higher than in other diseases, and, regardless of their diverse levels, they were found to be correlated with each other. Another abnormality of the CSF metabolism, iNPH, is possibly caused by the reduced absorption of CSF, which secondarily induces CSF accumulation in the ventricle; the excess CSF compresses the brain’s parenchyma to induce dementia. One potential treatment is a “shunt operation” to bypass excess CSF from the ventricles to the peritoneal cavity, leading to the attenuation of dementia. After the shunt operation, marker levels began to increase within a week and then further increased by 2–2.5-fold at three, six, and twelve months post-operation, at which point symptoms had gradually attenuated. Notably, the marker levels were found to be correlated with each other in the post-operative period. In conclusion, the brain-derived major glycoprotein markers were highly correlated in the CSF of patients with different neurological diseases, and their correlations were maintained even after surgical intervention. These results suggest that brain-derived proteins could be biomarkers of CSF production. Cerebrospinal fluid (CSF) plays a pivotal role in the homeostasis of the central nervous system by distributing growth factors, removing metabolic waste, controlling pH, and maintaining intracranial pressure (ICP) . CushingThe composition of major proteins in the CSF is similar to that of blood ,6. For iPsathyrella velutina lectin (rPVL) [Iron plays many important roles in the brain, and has involvement in myelination, neurotransmission and electron transfer in the respiratory chain . Howevern (rPVL) . Co-locan (rPVL) . Cortican (rPVL) .Another major glycoprotein in the CSF is lipocalin-type prostaglandin D2 synthase (L-PGDS). Urade et al., reported that L-PGDS is biosynthesized by the leptomeninges and the choroid plexus, which are CSF-producing tissues . The mol111indium-labelled radioisotope (RI) diethylenetriamine penta-acetic acid (111In-DTPA) is injected into the CSF and its decline is followed over time by RI cisternography. A considerable amount of radioactivity drains from the CSF into the blood and is then excreted via the urine. Some of the radioactivity is retained in the CSF, detected in the form of residual RI radioactivity. More than 30% of the radioactivity is maintained in the CSF of the control patients 24 h after RI injection; this is compared to SIH patients, where less than 10% remains, thus attesting to the CSF leakage [SIH is caused by the leakage of CSF, which induces CSF loss and intracranial hypotension ,17,18. T leakage . This leAnother abnormality showing altered CSF metabolism is iNPH, which is a senile hydrocephalus that induces dementia and gait disturbance, among other symptoms. ,23. The In the present study, we quantified the levels of brain-derived glycoproteins in various neurological diseases and found that their levels were well correlated with each other across different pathophysiological conditions.We previously demonstrated that several CSF glycoproteins, such as Man-Tf, GlcNAc-Tf, and L-PGDS, were derived from the cerebrum and were correlated with each other in neurodegenerative disorders, including AD, other tauopathies, and synucleinopathy ,11. In tSambucus sieboldiana agglutinin (SSA) for Sia-Tf, recombinant Burkholderia cenocepaciar (rBC2L-A) for Man-Tf, and rPVL for GlcNAc-Tf. Patients were classified into two groups based on the peak ages of onset: one group included depression, schizophrenia, SIH, and non-SIH patients with peak ages in the thirties and forties; another group included iNPH and non-iNPH patients with peak ages in the seventies. The two groups were analyzed statistically, avoiding the potential confounding effects of aging. In depression and schizophrenia patients, the levels of all markers were slightly higher than those of non-SIH patients, but the differences were not significant. SIH patients showed very high and variable levels of all markers compared to non-SIH patients. Man-Tf, GlcNAc-Tf, L-PGDS, Sia-Tf, and total protein levels were increased by 2.9-, 2.2-, 2.0-, 1.7-, and 1.6-fold, respectively, indicating that both brain- and blood-derived proteins were markedly increased in SIH. In iNPH, GlcNAc-Tf and L-PGDS levels were significantly higher than the levels in non-iNPH patients.Prior to the correlation analysis, we quantified levels of Man-Tf, GlcNAc-Tf, and L-PGDS as brain-derived markers . We alsoWe analyzed correlations between CSF markers in CSF samples from patients with depression. Scatter diagrams and Spearman’s rank correlation coefficients are indicated for each marker combination , Table 1Correlations between CSF markers were analyzed in schizophrenia patients. Scatter diagrams and Spearman’s rank correlation coefficients are indicated in SIH is caused by CSF leakage, which induces intracranial hypotension and the dilatation of veins. An increase in CSF production may occur to compensate for this leakage. All marker levels were elevated and varied widely in CSF samples from SIH patients . In spitCSF marker correlations were analyzed in CSF samples from non-SIH patients. Scatter diagrams and Spearman’s rank correlation coefficients are indicated for marker combinations , Table 4iNPH is caused by a reduced absorption of CSF, which induces the accumulation of CSF in the ventricles. MRI imaging reveals enlarged ventricles with excess CSF, which compress the brain parenchyma, leading to dementia, gait disturbance, and urinary incontinence. Marker correlations were analyzed in CSF of iNPH patients, with scatter diagrams and Spearman’s rank correlation coefficients shown below for each marker combination , Table 5Marker correlations were analyzed in CSF samples from non-iNPH patients , Table 6We next examined the diagnostic accuracy of CSF markers for SIH and iNPH, which showed significant differences in marker levels. The criteria for good markers are a sensitivity and specificity of more than 85%, together with an area under the ROC curve (AUC) greater than 0.9. SIH is mainly diagnosed on the basis of MRI imaging and the measurement of intracranial hypotension. As biochemical markers for SIH are barely established, CSF markers were examined for their diagnostic accuracy. Sensitivity values for Man-Tf, GlcNAc-Tf, L-PGDS, Sia-Tf, and the total protein levels were 97%, 77%, 77%, 46%, and 62%, respectively, while the specificity values were 95%, 75%, 75%, 95%, and 85%, respectively . Man-Tf Levels of GlcNAc-Tf and L-PGDS in CSF samples from iNPH patients were significantly lower than those of non-iNPH patients. CSF markers were examined for their diagnostic accuracy to differentiate iNPH from non-iNPH. The sensitivity values for Man-Tf, GlcNAc-Tf, L-PGDS, Sia-Tf, and the total protein levels were 62%,72%, 82%, 81%, and 46%, respectively, whereas their respective specificity values were 38%, 28%, 57%, 19%, and 54% . All marPatients with iNPH usually undergo a “shunt operation”, in which excess CSF is bypassed from the ventricles to the peritoneal cavity. Following the operation, prior symptoms such as dementia and gait disturbance usually attenuate gradually (shunt responder), suggesting that the shunt system functions well and that CSF metabolism has normalized. Responders were examined for their marker levels with the expectation that the operation would change pre-operative marker levels. CSF specimens were withdrawn from the shunt valve over time post-operation, and marker levels were quantified and compared with pre-operative levels. Brain-derived markers slightly increased on post-operative days 1–3 and further increased on days 4–6 . Man-Tf,The correlation coefficients for each marker combination were examined at post-operative days 1–6 . Brain-dCSF specimens were withdrawn at post-operative months 3, 6 and 12. The time course of the marker level changes was examined by comparing pre- and post-operative levels. The brain-derived marker levels increased significantly in the post-operative period, while the Sia-Tf and total protein levels increased only marginally .Correlation analyses revealed that the brain-derived markers correlated well with each other at each time point: Man-Tf vs. GlcNAc-Tf (rs = 0.59–0.76), Man-Tf vs. L-PGDS (rs = 0.64–0.74), and L-PGDS vs. GlcNAc-Tf (rs = 0.62–0.76) . Blood-dWe previously reported that the CSF levels of brain-derived glycoproteins, such as Man-Tf, GlcNAc-Tf, and L-PGDS, are well correlated with each other in neurodegenerative disorders, including AD . The preConcerning the mechanisms that underlie the elevation of brain-derived proteins in SIH, it is known that the disease is caused by CSF leakage, which induces intracranial hypotension and subsequent brain dislocation, leading to orthostatic headache and dizziness, among other symptoms ,17,19. S2O) is much lower than that of the controls (70–180 mm H2O) [Next, in relation to the mechanisms associated with the elevation of blood-derived proteins in SIH, our previous study demonstrated that total protein, albumin, immunoglobulin, and Sia-Tf markedly increased in CSF, but not in serum . The pat mm H2O) . ConsequConcerning the potential mechanisms that underlie marker changes in iNPH, it was previously demonstrated that affected patients show an accumulation of CSF in the ventricles, which is possibly due to a reduced absorption of CSF . A seconLastly, we discuss the diagnostic applications of the markers examined in our work. Mase et al., reported that L-PGDS levels were decreased in the CSF of NPH patients . We alsoThe present results revealed that all markers were elevated in SIH compared with the non-SIH CSF samples. Based on its sensitivity of 97%, specificity of 95%, and AUC of 0.98, our results suggest that Man-Tf could be a good diagnostic marker for SIH. In contrast, the other markers we examined would not be applicable for diagnostic purposes given their low sensitivity values (46~77%).−20), which prevents Fe-dependent cellular toxicity. Indeed, Fe toxicity is suspected to induce neuronal death in AD pathology. Man-Tf, mainly produced by neurons, may play a role as a local protein that protects against iron toxicity, although the mechanisms underlying this have yet to be elucidated.We previously demonstrated that Man-Tf derived from neurons could be used as a diagnostic marker for AD and MCI . Indeed,DSM-5”. SIH was suspected by clinical presentation, particularly by orthostatic headaches, MRI and/or computed tomography (CT), and RI scintigraphy. Patients were diagnosed based on the International Classification of Headache Disorders, 3rd edition (beta-version) [2O), (ii) no recent history of dural puncture, and (iii) not attributable to another disorder. The iNPH was diagnosed according to clinical guidelines issued by the Japanese Society of Normal Pressure Hydrocephalus, Version 3 [Patients were consecutively recruited from Fukushima Medical University (FMU), Juntendo University (JTN), Sanno Hospital (SNO), and the National Center for Neuropsychiatric Disease (NCNP). The number of patients, age (mean ± S.D.), male/female distribution, and hospitals for CSF collection for each disease type are listed in version)  and the version) : (i) morersion 3 . Briefly2 (TBST-CaCl2) at room temperature for 2 h. Plates were washed twice with TBST-CaCl2, and horseradish peroxidase-labeled streptavidin (50 ng/mL)  in TBST-CaCl2 was added and incubated for 2 h at room temperature. The plates were washed twice with TBST and then a TMB Microwell Peroxidase Substrate System  was added. Color development was stopped by adding 1N phosphoric acid. Absorbances were measured at 450 nm by a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific). Assays were performed in triplicate.CSF samples were aliquoted and stored at −80 °C until use. Repeated freeze and thawing (>two times) was avoided. Each assay was performed in triplicate. Man-Tf, GlcNAc-Tf, and Sia-Tf were quantified in lectin-ELISAs according to our previous studies ,31. Lect2 for maximum binding, but other lectins do not. For quantifying Sia-Tf, assays similar to the rBC2L-A lectin-ELISA described above were performed, except that, instead of biotinylated rBC2L-A, biotinylated SSA lectin (1 μg/mL) was used with TBST buffer without CaCl2. For quantifying GlcNAc-Tf, rPVL lectin was immobilized, and captured GlcNAc-Tf was detected with anti-Tf antibody (0.2 μg/mL).Recombinant BC2L-A lectin requires 10 mM CaClTotal Tf was quantified with a Human Transferrin ELISA Quantitation Set . The protein concentration was estimated using a Micro-BCA protein assay kit .Statistical analyses were performed using SPSS software (version 26). Data normality was examined using the Shapiro–Wilk test. Spearman’s rank correlation coefficients (rs) and Pearson’s correlation coefficients (r) were analyzed with and without data normality, respectively. Significant differences among multiple comparisons were assessed by the Kruskal–Wallis method followed by Bonferroni correction. Non-parametric comparisons were assessed by a Mann–Whitney U test.The present study reveals that CSF contains brain-derived glycoproteins such as L-PGDS, Man-Tf, and GlcNAc-Tf, and that their CSF levels were highly correlated with each other in several neurological diseases. INPH, disorders featuring abnormal CSF metabolism, was treated with shunt operation. After the surgical intervention, brain-derived glycoproteins increased over a year and their CSF levels were highly correlated with each other. The present observational and interventional study reveals that the glycoproteins are novel biomarkers for CSF metabolism.


Current diagnostics for meningoencephalitis pose a risk for suboptimal performance characteristics, with a current equipoise surrounding parameters that influence clinical utility of the BioFire FilmArray Meningitis Encephalitis Panel (MEP). The goal of this study is to examine the clinical utility of the MEP when using a cerebrospinal fluid white blood cell count (CSF WBC) threshold of > 50.All MEPs performed at Wake Forest Baptist Hospital from March 2016 to September 2022 were retrospectively analyzed to assess clinical utility of the threshold of CSF WBC > 50. The independent variable was CSF WBC greater or less than 50, with the dependent variable being clinical utility, deemed by the investigator. Logistic regression was performed to assess association of CSF WBC with clinical utility. In a secondary analysis, we repeated the regression using CSF WBC as a continuous predictor.Streptococcus pneumoniae. Among positive MEPs, 17 (15%) had WBC < 50. CSF WBC > 50 was associated with high utility with an odds ratio of 16.1 . When treating CSF WBC as a continuous variable, logistic regression revealed a 25% chance that the MEP would have high utility at this threshold, increasing to 50% at a CSF WBC > 310 .A total of 522 MEPs were analyzed, with 113 (22%) positive and 409 (78%) negative. Positive MEPs were predominantly viruses (65%) followed by bacteria (25%) and fungi (10%). Enterovirus was the most common pathogen detected in 24 (21%) MEPs, seen in Table 1. Among positive MEPs, characteristics associated with high utility were elevated CSF WBC (p=0.038) and older age (p=0.05). Among all MEPs, high utility was seen with increased CSF WBC > 50 (p< 0.001) and positive cultures (p< 0.001). Post hoc analysis of all MEPs revealed a specificity of 0.45, sensitivity of 0.94, PPV of 0.94, and NPV of 0.45 when using culture as the gold standard for bacteria or fungi. 23 (5%) samples were MEP positive and culture negative. Of these, 11 (48%) were MEP positive for Baseline CharacteristicsTable 1Probability of Clinical Utility as a Function of CSF WBCFigure 1Among MEPs ordered for suspicion of meningoencephalitis, the threshold of a CSF WBC > 50 was associated with high utility. Using this threshold to influence pre-test probability may serve as an important parameter to decrease false positive results of the MEP for diagnosis of meningoencephalitis.All Authors: No reported disclosures