This study examined associations between different sources of chronic perceived stress and deleterious behaviors  among first-year college students.The study used data on 885 first-year students (ages 18–20) at a large public university in North Carolina. The prevalence of deleterious behaviors was evaluated. Associations were estimated between different sources of chronic perceived stress  and health behaviors after controlling for psychosocial supports and demographics. Moderating effects of gender and moderate-severe anxiety/depression symptoms were also tested.19% of first-year student reported symptoms of eating disorders, 42% insufficient sleep, and 43% insufficient vigorous physical activity. Perceived chronic stress increased the odds of reporting these deleterious behaviors. These effects were not moderated by gender or moderate-severe anxiety/depression symptoms. Appearance- and health-related stress were associated with eating disorder symptoms; health- and romantic-related stress were associated with insufficient sleep; and health-related stress was associated with insufficient vigorous physical activity.Outcomes were survey-based. The study was based on cross-sectional data from a single university, so the direction of causality cannot be determined, and more work needs to be done to determine whether this would extend to other populations. Young adulthood is a critical period for establishing healthy behaviors that have lasting effects over the life course . This maCommon chronic stressors can lead to poor health behaviors during the transition to college. First-year students experience myriad new stressors, including transitioning to a new academic environment with new academic challenges, navigating new social environments, and new responsibilities regarding finances and time management. Many are living away from home for the first time and without the supports that they relied on prior to college. As a result, they may be particularly vulnerable to adopting deleterious behaviors in response to stress –11. The Associations between overall stress and disordered eating, insufficient sleep, and insufficient vigorous physical activity (VPA) can arise both because students adapt their behaviors to cope with stress and because students have less time and energy to engage in healthy eating, sleep, and physical activity when they are overwhelmed by other activities  and stress. Though many students may be exposed to multiple stressors, students who experience high perceived stress may also be more vulnerable to engaging in deleterious behaviors than students who experience lower perceived stress. While these associations have been well established –15, littFurthermore, little is known about what sources of stress are associated with deleterious health behaviors during the transition to college. Addressing this gap would enable policy makers to respond more effectively by targeting interventions to the source of stress. For instance, some types of stress  may be addressed through college policies and practices ; whereas other types of stress  may be better addressed through coaching and mentorship. While existing studies have examined academic ,17, sociFew studies consider determinants of multiple health behaviors in combination among college students. One study focused on first-year students in an Australian university found that stress was associated with sleep, but not physical inactivity or poor eating habits , whereasGender and mental health may moderate the effects of stress on health behaviors. Both may influence exposure to stress and the degree of stress experienced by college students. However, few studies have evaluated these moderating effects. Higher prevalence of eating disorders and insufficient sleep have been found in women than in men ,15, but psychosocial resources) may help protect students from the negative consequences of stress [In this paper, we use survey data collected on first-year students at a large public university in North Carolina (NC) to analyze whether different sources of chronic perceived stress are associated with certain deleterious behaviors—eating disorder symptoms, insufficient sleep, and insufficient VPA—during the challenging transition to university. Research shows that psychological resources and social support . Our overall response rate of 30% (N = 1124) was consistent with many online surveys . After dWe measured the prevalence of eating disorders using the Eating disorder Screen for Primary care (ESP) scale, which consists of the following questions: (1) “Are you satisfied with your eating patterns?”, (2) “Do you ever eat in secret?”, (3) “Does your weight affect the way you feel about yourself?”, (4) Do you currently suffer with or have you ever suffered in the past with an eating disorder?.” We created an indicator  of eating disorder symptoms for individuals with two or more symptoms . CronbacWe measured insufficient sleep by asking participants to report the number of hours of sleep they get on a typical night during college. Following recommendations of the American Academy of Sleep Medicine and Sleep Research Society , we creaWe asked participants to report the number of days over the past 7 days in which they had exercised or participated in physical activity for at least 20 minutes that made them sweat and breathe hard, such as basketball, soccer, running, swimming laps, fast bike riding, fast dancing, or similar aerobic activities. Consistent with the recommendation for aerobic activity from the American College of Sports Medicine and the American Heart Association [overall perceived stress by extracting a principal component score from these items based on a polychoric principal component analysis [We measured perceived stress based on questions adapted from the College Chronic Life Stress Survey . Studentanalysis . The expmoderate-severe anxiety or depression symptoms. Cronbach alphas were .86 for PHQ-8 and .90 for GAD-7.We measured anxiety and depression symptoms using the PHQ-8 and GAD-7, respectively. The PHQ-8 is an 8-item, clinically-validated scale with scores summed to range from 0 to 24 . IndividWe measured four different types of psychosocial controls that have been shown to matter for how students cope with stress . We measWe classified students who reported Hispanic ethnicity as Hispanic regardless of their race, and non-Hispanic (NH) students according to their race, including NH Black, NH White, NH Asian, and NH Other, where other includes students who reported more than one race. Gender was classified as woman or man based on student’s self-reported gender identity. The small number of students reporting non-binary (n = 3), other (n = 4) or missing (n = 16) gender identities values were classified based on their self-reported sex assigned at birth. We defined a sexual or gender minority as a student who reported having a gender identity other than their sex assigned at birth or a sexual orientation other than heterosexual. As an indicator of low-income status, we identified whether students reported that they were recipients of free or reduced-price lunch in high school (1 = yes) or not (0 = no). We defined first-generation college students as those who reported that neither parent/guardian had completed a 4-year post-secondary degree. In addition, we control for the month in which the survey was conducted and an indicator (0/1) for whether the student responded when the survey incentive was offered.We calculated the prevalence of eating disorder symptoms, insufficient sleep, and insufficient VPA; the prevalence of overall perceived stress and different sources of stress; the prevalence of moderate-severe anxiety or depression symptoms; and the means and standard errors of our psychosocial and demographic control variables. Next, we evaluated differences in means of all variables by gender and by moderate-severe anxiety/depression symptoms using two-sided t-tests. We then estimated logistic regressions of each deleterious behavior as a function of perceived stress controlling for demographic characteristics (Model 1) and progressively added psychosocial controls (Model 2), moderate-severe anxiety/depression symptoms (Model 3), gender x stress interactions (Model 4) and moderate-severe anxiety/depression x stress interactions (Model 5). Lastly, controlling for demographics and psychosocial resources, we estimated logistic regressions of each behavior as a function of the six different sources of moderate-severe perceived stress—academic, future, peers, friendships, romantic, appearance, health, chronic illness, financial, work, family—and moderate-severe anxiety/depression symptoms (Model 1). We used mean imputation with missing indicators (0/1) for a small number (<5%) of observations with missing data on control variables.Nineteen percent of first-year college students had symptoms of eating disorders, 42% obtained insufficient sleep, and 43% engaged in insufficient VPA in our sample . The mosOur estimation sample was 9% Hispanic, 15% NH Asian, 6% NH Black, 64% NH White, and 17% first-generation college students. This is comparable to the demographics of the entering first-year college student population in Fall 2019: 9% Hispanic, 12% NH Asian, 9% NH Black, 56% NH White, and 19% first-generation college students .Compared to men, women were significantly more likely to suffer from eating disorder symptoms (24% vs. 10%) and insufficient VPA (45% vs. 38%) . Gender Compared to those without anxiety/depression symptoms, students with moderate-severe anxiety/depression symptoms exhibited significantly higher eating disorder symptoms (31% vs. 14%), insufficient sleep (51% vs. 38%), and insufficient VPA (51% vs. 39%). They also reported significantly higher perceived stress overall and significantly higher prevalence of each source of perceived stress except for future- and romantic-related stress compared to those without anxiety/depression symptoms. Between these two groups, the largest differences were in the prevalence of health- and family-related stress, 23 and 22 percentage points, respectively.Students who experienced higher perceived stress were significantly more likely to engage in deleterious behaviors . A one sCompared to men, women had 153% higher odds of reporting eating disorders, but not significantly higher odds of insufficient sleep or VPA (Model 1). Compared to students with no or low anxiety/depression symptoms, students with moderate-severe anxiety/depression symptoms also had 53% higher odds of reporting eating disorder symptoms and 56% higher odds of insufficient sleep, but did not have significantly higher odds of insufficient VPA (Model 3). We found no significant interactions between stress and gender or stress and moderate-severe anxiety/depression symptoms for any of our outcomes  to deve


Anti-cancer therapy often causes premature ovarian insufficiency and infertility as the ovarian follicle reserve is extremely sensitive to chemotherapy drugs, such as cisplatin. Various fertility preservation methods have been explored for women, especially prepubertal girls undergoing radiotherapy and chemotherapy due to cancer. In recent years, mesenchymal stem cell-derived exosomes (MSC-exos) have been reported to play an important role in tissue repair and the treatment of various diseases. In the current study, we observed that human umbilical cord-derived MSC-exos (hucMSC-exos) after short-term culture improved follicular survival and development while receiving cisplatin treatment. Moreover, intravenous injection of hucMSC-exos improved ovarian function and ameliorated inflammatory environment within the ovary. The underlying mechanism of hucMSC-exos on fertility preservation was associated with the down-regulation of p53-related apoptosis and their anti-inflammatory function. Based on these findings, we propose that hucMSC-exos may be a potential approach to improve fertility in women diagnosed with cancer. There is now ample evidence that cancer treatment can deplete the ovarian reserve, resulting in early menopause. Chemotherapy-induced premature ovarian failure (POF) is a major long-term adverse effect of anti-cancer therapy. Therefore, the development of new approaches to preserve fertility for female patients, especially for those younger patients with a strong desire for childbearing who require chemotherapy drugs, is critical.The effects of chemotherapy drugs on female reproduction were reported since the 1970s, which were initially associated with cyclophosphamide (CPM) treatmentcis-diamminedichloroplatinum (Ⅱ) (CDDP) is a widely used and effective metal-based chemotherapeutic drug for many types of cancer, including cancers of testicule, ovary, breast, and lung. It is often used in combination with other cytotoxic drugs. Chemotherapeutic drugs, such as CDDP, usually cause double-strand DNA damage, which may induce apoptosis in granulosa cells and oocytes. Females are born with a finite number of primordial follicles, most of which will remain dormant for decades until they are activated into the growing follicular pool. Primordial follicles are very sensitive to the toxicity of chemotherapeutic agents, which may activate the PI3K/AKT signaling pathway that leads to the loss of primordial follicles–7. Therefore, it is crucial to explore new approaches of protecting the reproductive reserve, while using chemotherapeutic drugs.. To date, hucMSCs have been studied in clinical therapeutic trials. The stem cell therapy has also emerged as a new treatment for female infertility in recent years. Successful pregnancies have been reported in premature ovarian insufficiency (POI) patients who received hucMSC transplantation. hucMSCs have also been shown to improve the reserve function of perimenopausal ovaries through a paracrine mechanism. However, more research is needed to explore the underlying molecular mechanisms of hucMSCs and how they protect the ovarian reserve.In the past decade, mesenchymal stem cells (MSCs) have attracted a great attention due to their reparative function and board therapeutic potential. Human umbilical cord-derived MSCs (hucMSCs) are a type of multipotent stem cell with many advantages, including wide availability, easy accessibility, low immunogenicity and high self-renewal ability. Similar to their cell source, stem cell-derived exosomes have shown therapeutic effectsvia their reparative and regenerative abilities. It has been shown that MSCs-derived exosomes (MSC-exos) have the same efficacy in tissue repair as MSCs. Recent studies have indicated the potential of hucMSC-derived exosomes (hucMSC-exos) in improving ovarian function in aged mice. However, the role of hucMSC-exos in protecting ovarian reserve and restoring ovarian function after chemotherapy remains unclear. Therefore, we aims to explore whether hucMSC-exos have an ameliorative effect on the damage to ovarian reserve after CDDP chemotherapy. Based on the therapeutic potential of exosomes, we believe that stem cell exosome therapy might represent a new strategy for protecting ovarian reserve during chemotherapy.Many investigators have proved that MSCs secrete products, especially exosomes, to play key roles in tissue repair and healing. Exosomes are extracellular vesicles with a size rage of 30 to 150 nm, which are important in intercellular communicationMice from the Institute of Cancer Research (ICR) were obtained from Nanjing Medical University and housed in the Animal Core Facility of Nanjing Medical University. All mice were maintained under a 12 h light/dark cycle at 22 ℃ with free access to food and water. Female ICR mice at day three were used for ovarian culture. All animal work was approved by the Committee on the Ethics of Animal Experiments at Nanjing Medical University. Superovulation was performed with one intraperitoneal injection of pregnant mare serum gonadotropin  and followed by another injection of human chorionic gonadotropin  48 h later.2 for capacitation. Metaphase Ⅱ oocytes were then placed into 250 μL of media with sperm (2 × 105/mL to 3 × 105/mL) for fertilization. Six hours later, zygotes with clear pronuclei were transferred into fresh G-1 media (Vitrolife) overnight until the two-cell embryonic. The fertilized embryos were cultured in G-1 media for five days to the blastocyst stage.Donor sperm were first collected from male mice into G-IVF media  and incubated under oil for 1 h at 37 ℃ in 5% CO3 small pieces and washed with phosphate buffered saline  containing 100 U/mL penicillin and 100 mg/mL streptomycin  for three times. Then tissues were cultured in DMEM/F-12 that contained 10% fetal bovine serum  and 1% penicillin and streptomycin at 37 ℃ and 5% CO2.Fresh umbilical cords were collected from informed and voluntary mothers at Sir Run Run Hospital of Nanjing Medical University. The experiment protocol was approved by the Ethics of Committee of Nanjing Medical University. Briefly, umbilical cords were dissected into 1 mmg for 10 min at 4 ℃ and centrifuged at 2000g for 10 min to remove dead cells. Then, supernatants were centrifuged at 10 000g for 30 min to remove cell debris and supernatant was then centrifuged at 150 000g for 90 min to acquire exosome pellets. The concentration and particle size were measured by nanoparticle tracking analysis . All procedures were performed at 4 ℃. Exosomes were incubated in 2% paraformaldehyde solution overnight for transmission electron microscopy analysis. Two microliters of the exosome solution were transferred onto a carbon-coated copper grid. Then, the grid was observed using a transmission electron microscope .Briefly, the cultured medium was removed and washed twice with PBS when hucMSC reached 60%–70% confluence. MSCs were then cultured with de-exosome medium for two days. The supernatants were collected and centrifuged at 300The ovarian culture-conditioned medium consists of MEM-α (Gibco) supplemented with 0.23 mmol/L pyruvic acid, 50 mg/L streptomycin sulfate, 75 mg/L penicillin G, 0.03 U/mL follicle-stimulating hormone, and 3 mg/mL bovine serum albumin. Cell culture medium of 0.4 mL was added to the bottom of each well, and the postnatal day-three (PD3) ovaries were isolated and cultured in inserts . Five ovaries were randomly distributed to the control and treated groups. Intact ovaries from PD3 female ICR mice were treated with PBS or CDDP  combined with or without hucMSC-exos (20 μg/mL) for 24 h, and then transferred to the conditional medium for additional 96-h culture before collection for further analysis.g for 90 min to remove un-conjugated dye. Labeled exosomes were incubated with PD3 ovaries for 24 h. After incubation, ovaries were collected and fixed in 4% formaldehyde for frozen sections. After staining nuclei with Hoechst 33342 , slides were observed and calculated by using a confocal laser scanning microscope .hucMSC-exos were labeled with PKH67 dye (green)  at room temperature for 2 h and ultracentrifugated at 120 000, ovaries were collected and fixed in 10% formalin overnight. Then, the tissues were cut into serial sections (5 μm thick) and stained by hematoxylin and eosin. To evaluate follicular development, follicles were counted every five sections using the fractionator and nucleator principles. The number of follicles of newborn ovaries was counted from mid-ovary sections. All sections were counted by two individuals.For follicle countingFor immunohistochemistry, sections were deparaffinized and rehydrated, endogenous peroxidase activity was further blocked in 3% hydrogen peroxide in methanol for 15 min. The antigen of ovarian sections were retrieved by high temperature (95 ℃) for 16 min in sodium citrate buffer (pH 6.0). Then the sections were blocked with goat serum for 60 min at room temperature , and incubated with primary antibodies at 4 ℃ overnight. The sections were colored with diaminobenzidine (DAB) reagent. For immunofluorescence staining, the sections were incubated with secondary antibody  at 37 ℃ for 60 min followed by the incubation with Hoechst 33342 (Invitrogen).Total protein was extracted from ovaries using RIPA buffer  containing 1% protease inhibitor (MCE). Proteins were separated by 10% SDS-PAGE and transferred into polyvinylidene fluoride membranes . The membranes were incubated with primary antibodies overnight at 4 ℃ after being blocked with Tris-buffered saline with Tween 20 (TBST) containing 5% nonfat dry milk for 60 min. The following primary antibodies were used: Alix , TSG101 , CD9 (Proteintech), β-tubulin , γH2AX , BAX , p53 (CST), Cleaved caspase-3 (CST), Bcl-2 . Then, the membranes were washed three times with TBST for 10 min and incubated with secondary antibodies for 60 min. The signals were obtained through enhanced chemiluminescence  on the Tanon 5200 analysis system .Supplementary Table 1 (available online). The specificity of PCR products was assessed by melting curve analyses.Total RNAs were isolated from ovaries using TRIzol reagent (Invitrogen). Total RNAs (500–1000 ng per reaction) were reverse transcribed into cDNAs by FastQuant RT Kit . Real-time reverse transcription PCR (qRT-PCR) was then performed by SYBR Green Mix  on an ABI StepOnePlus platform (Thermo Fisher Scientific). All primer sequences are listed inCells were fixed in 4% paraformaldehyde with 0.25% Triton X-100 in PBS, and then incubated with primary antibodies at 4 ℃ for 2 h. Primary antibodies purchased from BD Verse  were used: PE Mouse Anti-Human CD90, APC Mouse Anti-Human CD105, FITC Mouse Anti-Human CD44, FITC Mouse Anti-Human CD34, FITC Mouse Anti-Human 45, and PE Mouse Anti-Human CD11b. After washing with PBST, cells were analyzed by flow cytometry (BD Verse). The data were further analyzed by FlowJo 7.0 software .t-test for two treatment groups and one-way ANOVA followed by Bonferroni's post-hoc multiple comparison correction for multiple treatment groups.P< 0.05 was considered statistically significant.GraphPad Prism 9.0 and SPSS 20.0 were used to perform the Chi-square test. All data were expressed as mean ± standard deviation and analyzed using unpaired two-tailed Student'sSupplementary Fig. 1, available online). Then the exosome-specific markers Alix, TSG101, and CD9 in hucMSC-exos, isolated from culture supernatant of three to seven passages of hucMSC, were examined by Western blotting, and the results showed that Alix, TSG101, and CD9 were highly expressed in hucMSC-exos . Transmission electron microscopy analysis showed that the obtained hucMSCs-exos were in a round structure with double-layer membranes , and the particles had a typical size of 81 to 109 nm revealed by nanoparticle tracking analysis . These results suggested that exosomes were successfully isolated from hucMSC.Flow cytometry was performed to identify surface markers of hucMSCs, and the results showed that CD44, CD90, and CD105 were highly expressed in hucMSCs, while CD34, CD45, and CD11b were negatively expressed .To observe the uptake of hucMSC-exos in ovaries). The number of follicles was counted from mid-ovary sections after treatments. We found a significant decrease in the number of both primordial and growing follicles in the ovaries of the CDDP group, compared with that of the control group, but hucMSC-exos treatment reduced CDDP-induced loss of primordial and growing follicles in ovaries . Immunohistochemistry staining of the proliferating cell nuclear antigen (PCNA) in ovaries showed a decreased PCNA expression and more abnormal oocytes in the CDDP-treated group, compared with the control group as well as the increased PCNA expression and less abnormal oocytes in the CDDP + hucMSC-exos group, compared with the CDDP group ( and). These results indicated that CDDP treatment directly destroyed ovarian reserve and hucMSC-derived exosomes could protect primordial follicles to a certain extent.To assess follicular development, immunofluorescent staining of DEAD-box polypeptide 4 (DDX4) was used to localize oocytes. The results showed that the DDX4 staining could be clearly observed in primordial follicle cytoplasm (–15. Immunofluorescence staining of phosphorylated histone H2AX (γH2AX), a marker of DNA damage, revealed a significant increase in the number of double-strand breaks (DSBs) in CDDP treated ovaries, which was reversed in ovaries of the CDDP + hucMSC-exos group ( and). Moreover, γH2AX expression detected by Western blotting was lower in the CDDP + hucMSC-exos group than in the CDDP group . These results suggested that hucMSC-exos could repair ovarian DNA double-strand damage.Previous studies have reported that CDDP treatment leads to follicle apoptosis and DNA damage and). The geneBcl2 andBax play important roles in apoptosis. The effects of CDDP and hucMSC-exos on Bcl-2/Bax apoptotic pathway in ovaries were analyzed by Western blotting. The results showed that Bcl-2/Bax ratio was significantly down-regulated in the CDDP group, compared with the control group, and up-regulated in the CDDP + hucMSC-exos group, compared with the CDDP group . These results suggested that hucMSC-exos could preserve follicles by inhibiting oocyte DNA breakage and apoptosis in ovaries.The apoptosis level in ovaries was detected by TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay, and the results showed a higher level of apoptosis of granulosa cells and stromal cells in CDDP-treated ovaries, compared with the control group, but the apoptosis level was returned to that similar to the control group after hucMSC-exos treatment following CDDP challenge  signal was detectable in the ovarian medulla and surrounding follicles . Two weeks after PBS/exosomes injection, the ovarian volume of the CDDP + hucMSC-exos group was greater than that of the CDDP group . Total follicle counting showed a significant loss of follicle in the CDDP group and a retention of follicles in the CDDP + hucMSC-exos group . Follicles counting results revealed that, compared with controlled ovaries, the CDDP-treated ones revealed extreme damage in terms of follicular development, including a decreased follicle count at developmental stages and an increase in atretic follicles . In contrast, ovaries received hucMSC-exos treatment following CDDP challenge showed an obvious improvement in the number of growing follicles at different developmental stages compared with the CDDP group .A diagram scheme for). We then examined the function of hucMSC-exos for ovulation and mature egg production. After superovulation, an increased number of ovulated oocytes were harvested from the mice of the CDDP + hucMSC-exos group, compared with the CDDP group . These results suggested that thein vivo hucMSC-exos treatment improved oocyte retrieval.The concentration of serum estradiol (E2) was measured using an enzyme-linked immunosorbent assay. Compared with the control group, CDDP treatment induced a significant down regulation of E2 in the serum, which was attenuated by hucMSC-exos treatment (in vitro fertilization (IVF) ability of oocytes from each group. After IVF with the donor sperm, the rates of two-cell embryos to blastocysts in the CDDP group were reduced, but they were rescued by hucMSC-exos transplantation (–). These results indicated that hucMSC-exos played a protection role in cisplatin-induced follicle damage and restored follicular developmentin vivo to enhance fertility.Then, we evaluated thei.e.,Kitl andAmhr) in ovaries were detected by qRT-PCR. The results showed that the expression levels ofKitl andAmhr were significantly upregulated in ovaries of the CDDP + hucMSC-exos group, compared with the CDDP group . Immunostaining of PCNA showed that more positive signals were detected in granulosa cells in the CDDP + hucMSC-exos group, compared with the CDDP group . These results indicated that hucMSC-exos promoted granulosa cell proliferation and improved granulosa functions.In the neonatal mouse ovary culture model, we observed an increased level of apoptosis in early follicular granulosa cells after CDDP treatment. Therefore, we explored whether hucMSC-exos could improve follicle development by restoring granulosa cell function. The expression levels of granulosa cell-associated genes ( and). The apoptosis level in early granulosa cells was increased in the CDDP group. To demonstrate the protective role of hucMSC-exos against cells damage in the ovaries, we dissociated primary ovarian granulosa cells from ovaries and cultured themin vitro. The mRNA expression levels of anti-apoptosis geneBcl2 and pro-apoptosis geneBax in granulosa cells were detected by qRT-PCR. The results showed thatBcl2 expression was significantly increased in the CDDP + hucMSC-exos group, compared with the CDDP group, whileBax expression was significantly reduced in the CDDP + hucMSC-exos group, compared with the CDDP group ( and). Western blotting was performed to detect the expression of apoptosis-related proteins in granulosa cells. Compared with the CDDP group, the expression levels of p53 and cleaved caspase-3 were down-regulated, and the Bcl-2 level was also up-regulated in the CDDP + hucMSC-exos group . These results indicated a decrease in the level of apoptosis in the ovary after hucMSC-exos treatment. Similarly, immunohistochemistry staining of cleaved caspase-3 showed that hucMSC-exos treatment reduced cell apoptosis in the ovaries induced by CDDP . These results consistently showed that hucMSC-exos had a protective effect against CDDP-induced granulosa damage.Results of the TUNEL assay further demonstrated that apoptosis in the ovaries was increased in the CDDP group, compared with the control group, and decreased in CDDP + hucMSC-exos group, compared with the CDDP group . The results showed the decreased positive signals for M1-like macrophages in the hucMSC-exos group, compared with the CDDP group ( and). The results of immunofluorescence staining of F4/80 showed that macrophage infiltration in the ovaries was increased in the CDDP group, compared with the control group and decreased in the CDDP + hucMSC-exos group, compared with the CDDP group ( and). The results of qRT-PCR showed that hucMSC-exos treatment could significantly suppress CDDP-induced increase in the expression of pro-inflammatory genes, includingIl1b andIl6 ( and). Taken together, the results suggested that hucMSC-exos treatment could alter the inflammatory environment in CDDP-treated ovaries.CDDP has been reported to induce pro-inflammatory signaling, which is associated with persisting markers of inflammation, melatonin, and AS101–21. In the current study, we proposed and proved that hucMSC-exos had a protective effect on follicular protection after chemotherapy  treatment. Additionally, in hucMSC-Exos-treated ovaries, we found follicle developmental restoration. hucMSC-exos significantly increased the number of eggs that ovulated from superovulated ovaries, as well as blastocyst formation. These findings are consistent with our previous reports about the fertility restoration in aged ovaries after hucMSC-exos administration. Our findings suggest that hucMSC-exos may be another candidate to preserve ovarian reserve and improve ovarian function after CDDP chemotherapy.Women of childbearing age often experience POF and infertility due to the damaging effect of chemotherapy drugs, which are regular side effects of anti-cancer therapies. How to prevent POF and protect follicle pools among this population with fertility aspirations have received increasing attention. At present, the clinical application to prevent ovarian function damage by chemotherapy is not very extensive. The main current approaches to fertility preservation after chemotherapy include administration of sphingosine-1-phosphate. The proposed mechanism of CDDP to damage follicles may be through the increase of DNA damage and the activation of apoptosis related genes, likeP53 family, especially in immature follicles. In the current study, the significant increase in γH2AX signals in CDDP-cultured ovaries indicates that cisplatin induces DSBs in oocytes, leading to ovarian reserve damage. Notably, a decrease in the number of DSB positive-labeled oocytes was observed after hucMSC-exos treatment. These findings suggest that hucMSC-exos treatment may repair DNA DSBs in oocytes. The observation likely explains why more primordial follicles preservation detected in newborn ovaries.As the most common chemotherapy drug, cisplatin is still the first-line chemotherapeutic drug for the treatment of several cancers, including breast cancer, and widely used in clinical treatment of hematologic and solid tumors in children and women of childbearing age. However, it is still unclear how is the mechanism of CDDP exposure in ovarian failure. There is a growing evidence that exposure to CDDP leads to follicular excessive consumption and loss. p53 is a tumor suppressor protein that regulates cell growth by modulating its target genesP27,Bcl2,Bax, andPuma (the gene encoding p53 up-regulated modulator of apoptosis). In the current study, we observed that the apoptosis of granulosa cells and DSBs in oocytes increased after CDDP treatment, which is consistent with the effects of CDDP on the ovaries in a rat model. Investigators have pointed out that the activation of p53 can induce apoptosis and cell migration. Here, we found that hucMSC-exos up-regulated Bcl-2 protein expression and down-regulated cleaved caspase-3 protein expression to protect granulosa cells from CDDP injury. Moreover, hucMSC-exos reduced the apoptotic sign in ovarian follicles, which suggests hucMSC-exos may improve ovarian function. In our current study, we found that the expression of p53 was decreased in granulosa cell cultured by CDDP. After hucMSC-exos treatment, we were surprised to find that embryonic development after IVF was improved. It also indirectly demonstrates that hucMSC-exos improve follicular development by the restoration of granulosa function.Studies have reported a pro-apoptosis function induced by CDDPvia experimental animal models–28. Compared with other sources of stem cells, hucMSCs are the first choice for cell therapy due to their non-invasive isolation, low immunogenicity and the ability of fast self-renewal. However, hucMSCs have relative limitations in the quantification of bioactive substances and the logistics for delivery in clinical therapies. Several investigators suggested that the culture medium of MSCs had therapeutic effects in POI murine models. Compared with MSCs, exosome, a member of extracellular vesicles may be a better choice in the clinic, because of their low immunogenicity, high clinical safety and stable bioactivity. Therefore, the application of MSC-exos is more effective and safer than the MSC therapy.With the developments in stem cell therapy, many investigators focus on the use of adipose-derived MSCs or bone marrow-derived MSCs in the regeneration and functional recoveryThe successful maturation of oocytes is associated with systemic hormone regulation, granulosa cell function, and ovarian microenvironment. Similarly, the homeostasis of the perifollicular environment depends on the maintenance of multiple cells, including immune cells and stromal cells. CDDP induces a myriad of inflammatory cytokines and chemokines, including translocation factors, which lead to the production of tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine that is actively involved in CDDP-induced inflammation. Therefore, we examined the expression of inflammatory factors in the ovaries after CDDP treatment. The expression of major proinflammatory cytokines IL-1β and IL-6 increased in CDDP-treated ovaries. The increased number of macrophages found in the ovary after CDDP treatment indicates that CDDP is correlated with a proinflammatory environment in ovaries. To be noted, hucMSC-exos improved ovarian follicular development, meanwhile the anti-inflammatory effects of hucMSC-exos were required for the amelioration of ovarian microenvironment and improved ovarian function in the CDDP-treated mice.The molecular mechanism, involved in hucMSC-exos repair of the ovarian function, needs to be further explored. The active components in hucMSC-exos may regulate ovarian microenvironment. We will focus on what components of hucMSC-exos are involved in the recovery of ovarian function in future studies.None.


Globally, two out of five children living with HIV (CLHIV) are unaware of their HIV status, and a little more than 50% are receiving antiretroviral therapy (ART). This paper describes case-finding strategies and their contribution to identifying CLHIV and linking them to ART in Nigeria.This before-after study used program data abstracted during the implementation of different paediatric-focused strategies , community-driven EID, and community-based testing) delivered in health facilities and in communities to improve HIV case identification. Data were abstracted for children (0 to 14 years) who received HIV testing services and were initiated on ART in Akwa Ibom State, Nigeria during the pre-implementation period (April–June 2021) and during the implementation period (July–September 2021). Descriptive statistics were used to describe the testing coverage, positivity rate (proportion of tests that were positive for HIV), linkage to ART, and ART coverage, by age, sex, and testing modality. Interrupted time series analysis (ITSA) on STATA 14 was used to estimate the effect of the implementation of these strategies on HIV testing uptake and positivity rate at a 0.05 significance level.A total of 70,210 children were tested for HIV within the six-month period, and 1,012 CLHIV were identified. A total of 78%  of the tests and 83.4% (n = 844) CLHIV were diagnosed during the implementation period. During implementation, the HIV positivity rate increased from 1.09%  to 1.54% , while linkage to ART increased from 99.4% (167/168) to 99.8% (842/844). The contribution from community-based modalities to CLHIV identified increased from 63% (106/168) to 84% (709/844) during the implementation, with the majority, 60.8% (431/709), from community-based index testing. Overall, ART coverage increased from 39.7 to 55.6% at the end of the intervention period.The findings show that expanding differentiated HIV testing approaches provided mostly in the community significantly increased pediatric case identification. However, ART coverage remains low, especially for younger age groups, and requires further efforts. Globally, an estimated 2.8 million children and adolescents ages 0–19 years) are living with HIV, with only 54% receiving antiretroviral therapy (ART) at the end of 2020. About 310,000 new infections and 120,000 AIDS-related deaths were reported among children younger than 15 years in 2020 [9 years aNigeria has the third-largest burden of HIV in children  behind only South Africa and Mozambique . ApproxiDifferent strategies have been used in isolation to improve treatment coverage among children –11. The This was a before-after study conducted in 21 local government areas (LGAs) in Akwa Ibom State, Nigeria. We reviewed program data for children (0–14 years) who received HIV testing services between April and September 2021. The data were compared for two time periods: the pre-implementation period, from April to June 2021, and the implementation period, from July to September 2021. During the pre-implementation period, facility-based HIV testing services were largely used to identify CLHIV, while a mix of facility and community-based strategies was employed during the implementation period.Akwa Ibom State, located in the southern part of Nigeria has an estimated population of 6,497,967 , 14. ChiTo improve treatment coverage among CLHIV, a paediatric task team was constituted to review and identify efficient HIV case-finding strategies. The team was composed of a consultant Paediatrician , community team physicians (one per LGA), dedicated paediatric counsellor testers, and case managers. The team held key informant interviews with their caregivers of CLHIV on treatment and to understand contextual issues influencing the uptake of services. Brainstorming sessions were held with healthcare workers, PLHIV groups, civil society groups, community leaders, caregivers, and program staff to review program data and identify feasible models and evidence-based practices targeted at children that would be scalable within the operating environment . SeveralFive of the six strategies were used in the pre-implementation period, but expanded in scope and reach with the addition of a sixth strategy (community-driven EID) during the implementation period. While the pre-implementation strategies were largely facility based with minimal community supplementation, the implementation strategies were largely community-based. For provider-initiated testing and counselling (PITC), healthcare providers offered to test children at risk of HIV who attend health facility clinics . This was expanded during the implementation period into the community and offered at traditional birth attendant (TBA) homes and non-ART health facilities. For orphans and vulnerable children (OVC) testing, households enrolled on the OVC program were offered HIV testing and counselling in collaboration with other USAID partners. During family-based index testing (FBIT), biological children and siblings (younger than 15 years) of index HIV-positive clients were enumerated, and testing was extended into the community by dedicated testers who followed up with elicited contacts. Early infant diagnosis (EID) using dried blood spot (DBS) for HIV-exposed children younger than 18 months was scaled up, with testing conducted at a central laboratory. EID using DBS was expanded beyond the health facilities into community locations (community-driven EID) to cover the many out-of-facility births (up to 78%) occurring in these locations . Point-oSome testing strategies, such as FBIT, were used across multiple age groups, while others, such as EID, only applied to the youngest age group. Testing at the immunization clinics was focused on identifying missed opportunities among index children and their older siblings. Community ART management teams comprised of community mobilizers (for community entry and mapping), counsellor testers for testing, clinicians  and case managers (for case management and follow-up).Routine program data from patient records were reported daily into service registers and entered into the Lafiya Management Information System (LAMIS)  by trainData on the number of HIV tests conducted among children aged 0–14 years of age; and the number of positives identified were extracted from the DHIS2; while the information on children started on antiretroviral therapy, were extracted from the LAMIS. Quality checks were done to remove entries due to invalid, or duplicate data prior to analysis. Where there were missing data elements, such information was abstracted from the source document or patient care folders.Frequencies were used to summarize key outcomes across gender and other demographic characteristics. LGAs were categorized as urban, peri-urban, or rural.The outcomes considered were case identification (number of HIV-positive tests), positivity rate (proportion of HIV tests that were positive), ART initiation (number of newly identified HIV-positive children initiated on ART) and linkage rate (proportion of newly identified HIV-positive children initiated on ART) for the pre-implementation and implementation periods. Children were considered HIV-positive if they had two positive nucleic acid tests on independent blood samples (in children less than 18 of age) and two positive antibody test results on independent blood samples in children greater than 18 months of age.We conducted an interrupted time series analysis (ITSA) on STATA 14 to estimate the effect of the implementation of these strategies on HIV testing uptake and positivity rate. The model, however, was fitted to weekly level data to account for variations that occurred with the change in strategy implementation. T-test statistic was conducted to compare the difference in average testing within the two periods.Kaplan Meier’s log-rank test was used to compare time to ART for both periods. Also, ART coverage for both periods was compared using the Mann-Whitney U test with a 0.05 significance level. The baseline for ART coverage was computed from the HIV spectrum estimate data of the CLHIV burden of the 21 supported LGAs.A total of 70,210 children were tested for HIV tests over six months: 15,389 were tested during the pre-implementation period and 168 CLHIV identified, 54,821 were tested during the implementation period and 844 CLHIV identified  . The number of children tested for HIV and received test results increased by 1,390  in the first week of intervention and was followed by significant weekly increases in HIV tests conducted (relative to the pre-implementation phase)  .Similar to testing, there was no significant improvement in HIV-positive cases identified every week pre-implementation  . However, the number of HIV-positive children identified increased by 36  in the first week of the intervention, followed by significant weekly increases in the number of positive children identified (relative to the pre-implementation phase)  .The positivity rate increased from 1.1%  to 1.5%  between the two periods. This increase was seen across all age groups except among children less than one year where the positivity rate marginally reduced from 5.9 to 5.8%.The relative increase in the absolute number of children tested and those diagnosed with HIV across both periods was higher among males  and in the community setting  , while community index testing had the highest contribution to CLHIV identified n = 431, 51.07%)  in the pre-implementation period to 99.8% 842/844) during the implementation period (p = 0.142)  from community HIV testing was higher in this study (1.7%) than in other settings where community testing has been conducted . Using mIndex testing is a well-recognized strategy for efficient HIV case finding, especially in low-prevalence settings and subpopulations , 37. In Although significant improvements in case finding were seen in all age groups, the largest proportion (47%) of CLHIV identified were older children (10–14 years). Similar studies from Kenya and Zimbabwe identified slightly younger children, with median ages of 8 and 11 years, respectively –42. ThisThe lack of timely EID has been documented as a reason for poor uptake of treatment among HIV-exposed infants . ExtendiThe results showed that linkage to ART was optimal across all age groups, unlike in Lesotho, where Sindelar, K. and colleagues reported a linkage rate of 82% after 12 months from community-based interventions . In our The study had some limitations. The main weakness of before and after designs is the lack of a control group. This limits the value of information obtained on the intervention outcome link. Without a control group, it is difficult to establish the cause-and-effect relationship between the intervention and the outcome and to account for possible confounders. Furthermore, the analysis covered three months and did not account for how these strategies would affect results in the long term, including the cost-effectiveness of these strategies. However, these interventions have been implemented beyond the study period. Lastly, HIV testing data for EID services were documented at the facility level for both community- and facility-driven EID efforts; thus, it was difficult to attribute contributions from the community and facility. However, the difference between periods in the total number of samples collected emphasizes the value of community-driven EID.Despite these limitations, this study is the first community-wide intervention that used multiple testing strategies in Nigeria, thus, providing insights to policymakers and program implementers on the use of differentiated HIV testing for case identification among children. As one of the countries with the highest burden of HIV, scaling up these interventions could help Nigeria end AIDS in children by 2030. The results from differentiated testing strategies provides evidence for managers, policymakers, and other stakeholders to inform decision-making and improve implementation of HIV testing programs from implementation design to resource allocation.The study showed that the combination of multiple strategies in facility and community setting rapidly improved access to HIV diagnosis among children. Although the overall yield from HIV testing for children is low and requires an enormous amount of resources, index testing remained the most efficient strategy for HIV case finding. Treatment programs designed for children living with HIV should incorporate community-based services to facility-based services to achieve rapid results. More strategies can be explored to reach this younger age group.


Introduction: As in other chronic conditions, medication adherence is important in the treatment of peripheral arterial disease (PAD). Our study aimed at a) analysing non-adherence to angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) in groups of older ACEI and ARB users with PAD, and b) identifying characteristics associated with non-adherence.Methods: We focused on the implementation phase of adherence . The study cohort included ACEI/ARB users aged ≥65 years in whom PAD was newly diagnosed during 2012. Non-adherence was defined as Proportion of Days Covered (PDC) < 80%.Results: Among 7,080 ACEI/ARB users , there was no significant difference in the overall proportion of non-adherent patients between ACEI and ARB users . There were differences in factors associated with non-adherence between the groups of persistent and non-persistent  ACEI and ARB users. Increasing age, dementia and bronchial asthma were associated with non-adherence in persistent ACEI users. General practitioner as index prescriber was associated with adherence in the groups of non-persistent ACEI users and persistent ARB users.Conclusion: Identified factors associated with non-adherence may help in determining the groups of patients who require increased attention. Peripheral arterial disease (PAD) represents the third most important cause of atherosclerotic morbidity following stroke and coronary heart disease . In 2010PAD is defined as a progressive disorder accompanied with stenosis and/or occlusion of large and medium-sized arteries, other than those that supply the heart or brain . This diAdherence to medications represents a basic precondition of successful treatment of PAD. Adherence consists of three phases: initiation, implementation, and persistence. Initiation represents taking the first dose of a prescribed medication. Implementation reflects the extent to which a patient´s dosing regimen corresponds to that recommended by the physician. Persistence represents the time between initiation and the last dose before discontinuation (stopping treatment) .The data for our retrospective register-based study were collected from the database of the General Health Insurance Company, the largest health insurance provider in Slovakia which covers approximately 63% of the population. From this database, patients in whom PAD was newly diagnosed between 1 January and 31 December 2012 were identified. From them, patients aged ≥65 years treated with ACEIs or ARBs were selected into the study cohort. The derivation of the study cohort is described in our previous manuscript .Our study focused on the implementation phase of medication adherence. Proportion of Days Covered (PDC) represents a method to evaluate this phase in this type of register-based data . PDC wasFactors associated with the likelihood of non-adherence were analysed separately in persistent and non-persistent patients. The same patient- and medication-related characteristics as those included in our previous study on non-persistence  were anaContinuous variables were characterised as means ± standard deviations (SD) and categorical variables as frequencies and percentages.2-test. The Fisher exact test was applied in the case when the expected count was less than five in ≥20% of cells of the contingency table. The Mann-Whitney U test was applied to compare continuous variables between the two groups. The reason for the use of this non-parametric test was the non-Gaussian distribution of evaluated variables. The normality of the distribution was analysed with the Kolmogorov-Smirnov test.Categorical variables were compared between the two groups using the χThe most important characteristics associated with the probability of non-adherence were identified with binary logistic regression. The method of forward conditional was applied in this model. Results are presented as odds ratios with 95% confidence intervals .α = 0.05. The statistical software IBM SPSS for Windows, version 29, was used .All statistical tests were carried out at the significance level of Since the 5-year follow-up period is relatively long, we identified factors associated with non-adherence in the model with a shorter 3-year follow-up period.p = 0.136 according to the χ2-test). We also did not find any significant difference in the overall proportions of non-adherent patients between ACEI and ARB users .The baseline characteristics of the study cohort are described in p < 0.001 according to the χ2-test). In the group of ARB users, 372 (74.1%) patients were persistent and 130 (25.9%) non-persistent with ARB treatment. Among persistent patients, non-adherence was found in 50 (13.4%) patients, whereas in the group of non-persistent patients, 27 (20.8%) patients were defined as non-adherent (p = 0.046 according to the χ2-test). In the group of ACEI users, 5,066 (77.0%) patients were persistent and 1,512 (23.0%) non-persistent with ACEI treatment. Within the group of persistent patients, non-adherence (PDC<80%) was observed in 617 (12.2%) patients, while in the group of non-persistent patients, non-adherence was identified in 299 (19.8%) patients  persistent and 1,350 (20.5%) non-persistent patients of which 13.6% and 18.7% were non-adherent, respectively. Of ARB users, 390 (77.7%) were persistent and 112 (22.3%) non-persistent of which 15.6% and 18.8% were non-adherent, respectively. versus persistent patients among both ACEI and ARB users. These results indicate that discontinuation is commonly preceded by decreased adherence (poor implementation). More factors associated with the likelihood of non-adherence were found in the groups of ACEI users compared to ARB users.No significant difference was found in the overall proportions of non-adherent patients between ACEI and ARB users. Non-adherence was significantly more common among non-persistent patients Increasing age was associated with an increased likelihood of non-adherence among persistent ACEI users. In our previous manuscript , increasAmong comorbid conditions, dementia and bronchial asthma/chronic obstructive pulmonary disease were associated with non-adherence in the group of persistent ACEI users. Dementia decreased the likelihood of non-persistence with ACEI/ARB treatment in our previous study . Poor imAnxiety disorders were associated with adherence among non-persistent ACEI users. This result may be associated with meticulous medication-taking behaviour in anxious patients who had regularly taken their medication before discontinuing it. In contrast to our finding, Increasing number of medications was associated with adherence in the group of persistent ACEI users. This factor was associated with persistence with ACEI/ARB treatment in our previous study . This reAmong CV co-medication, administration of cardiac glycosides was associated with an increased likelihood of non-adherence among persistent ARB users, while mineralocorticoid receptor antagonists increased the probability of non-adherence among persistent ACEI users. In addition, administration of beta-blockers was associated with adherence among non-persistent ACEI users, and administration of calcium channel blockers was associated with adherence among persistent ACEI users and persistent ARB users. In our previous study , adminisAmong non-persistent ACEI users, being a new user of ACEIs was associated with adherence to treatment. In our previous study , being aversus family medicine or had some other specialisation. Authors of that study did not explain their finding.General practitioner as index prescriber was associated with adherence among non-persistent ACEI users and persistent ARB users. In our previous study , generalSome ACEIs used in our study were associated with the likelihood of non-adherence compared to perindopril. Lisinopril and ramipril were associated with an increased likelihood of non-adherence among non-persistent ACEI users, while trandolapril and quinapril were associated with adherence among persistent ACEI users. In our previous study, administration of imidapril, fosinopril and valsartan was associated with non-persistence, while enalapril was associated with persistence with ACEI/ARB treatment . The desOur study has some limitations which should be considered when interpreting the study results. The database of the General Health Insurance Company is created for insurance purposes and not for research. It is impossible to identify whether medications were taken as prescribed and to determine who was responsible for treatment discontinuation (patient or physician). Data on adverse effects were not available in this database. Other limitation consists in the small number of ARB users compared to ACEI users. This significant difference may be explained by the preference of ACEIs as the first-choice drugs and the use of ARBs in the case of intolerance of ACEIs. On the other hand, the large sample size which covers all regions of the Slovak Republic as well as detailed and precise data on patients’ comorbid conditions and medications represent the strengths of our study.In our study, no differences in the proportions of non-adherent patients were found between older ACEI and ARB users with PAD. Significantly higher proportions of non-adherent patients were found among non-persistent patients in comparison with persistent patients among both ACEI and ARB users. This result indicates worsening of adherence before discontinuation of treatment among non-persistent patients. Factors associated with non-adherence identified in our study may give indications for identifying patients at an increased probability of non-adherence in whom special attention should be paid to improving their adherence so as to ensure effective secondary prevention of PAD.


Several vaccines have been developed against SARS-CoV-2 and subsequently approved by national/international regulators. Detecting specific antibodies after vaccination enables us to evaluate the vaccine’s effectiveness. We conducted a prospective longitudinal study among members of Tarbiat Modares University of Tehran, Iran, from 4 September 2021 until 29 December 2021. We aimed to compare the humoral immunogenicity of 3 vaccine types. Participants consisted of 462 adults. Anti-SARS-CoV-2 receptor-binding domain [RBD] IgG titer was compared in 3 groups, each vaccinated by available vaccines in Iran at the time: Oxford/AstraZeneca, COVIran Barekat, and Sinopharm. The median IgG titer was: 91.2, 105.6, 224.0 BAU/ml for Sinopharm, COVIran Barekat and Oxford/AstraZeneca respectively after the first dose; 195.2, 192.0, 337.6 BAU/ml after the second one. We also analyzed the frequency of antibody presence in each vaccine group, in the same order the results were 59.0%, 62.6% and 89.4% after the first dose and 92.1%,89.5% and 98.9% after the second. The comparison of results demonstrated that AstraZeneca vaccine is a superior candidate vaccine for COVID-19 vaccination out of the three. Our data also demonstrated statistically significant higher antibody titer among recipients with an infection history. According to World Health Organization (WHO) report Sars-Cov-2 virus had already caused 771,549,718 confirmed cases of COVID-19 and 6,974,473 deaths, by October 25th 2023. As of October 21th 2023, a total of 13,533,465,652 vaccine doses were administered worldwide. meanwhile in Iran, morbidity and mortality rates were 7,619,981 and 146,480 respectively. A total of 155,445,801 vaccine doses were administered by 14 August 20232. Meanwhile, vaccine distribution in the developing countries were slow, and they were not close to optimal vaccination coverage; As a result, vast groups of unvaccinated people may play a critical role in COVID-19 circulation and lead to new variants evolution which can potentially be vaccine-resistant. So, swift worldwide mass vaccination with efficient vaccines is invaluable3. During SARS-COV2 pandemic, mutations in spike protein potentially increased transmission of the virus and its likelihood of escape from the antibodies raised against the parental sequence used in the administrated vaccines5. Although mutations can diminish the efficacy of antibodies produced against the original spike sequence, vaccines are recommended for public use in order to increase the global herd immunity6.During this time, safe and effective vaccines were added to WHO’s “Emergency Use List”, and multiple vaccines received authorization for emergency use by their national and/or international regulators7. Spike protein is expressed on the surface of virus particle, triggering both antibody and T cell responses that can be protective against COVID-19. A safer alternative platform is utilizing inactivated vaccines. Sinopharm and COVIran Barekat vaccines were both produced based on the killed/inactivated virus strategy. The principal advantage of these vaccines is their similarity to the natural infection, which may induce a stronger and long-lasting immune response9. A meta-analysis containing 54 studies from 18 different countries, comprising about 12 million individuals, observed that 6–8 months after recovery the prevalence of SARS-CoV-2 specific immunological memory remained high. In fact, nearly 90% of recovered individuals had evidence of immunological memory of SARS-CoV-2 at 6–8 months after recovery10.AstraZeneca vaccine was developed through the cooperation of Oxford University and AstraZeneca (ChAdOx1). It is based on genetic modifications to replication-defective adenoviruses that are inactivated due to deletion of the E1 gene, which is replaced with the spike gene of SARS-COV-212. At the time of this study, health-related challenges of COVID-19 include lack of access to vaccines in several countries and rejection of those who do have access to it3, the lack of an effective specific antiviral treatment13, and the possibility of transmission of the virus to pets and farm animals15.It is necessary to update the current vaccines to match the circulating SARS-CoV-2 variants. We believe that an imperative instrument to tracing infected individuals and saving their lives is the development of sensitive and specific detection methods for mass screening and testing as well as using effective vaccines16.Today, scientists and governments have realized the need for effective methods for diagnosing viruses such as SARS-CoV-2 at the early stages of their spread. Besides, to control the SARS-CoV-2 spread, it is important that vaccines with high efficacy and accessibility be available worldwide18.Several factors may affect the protection offered by vaccines, including vaccine platforms type, SARS-CoV-2 variants, age and immunity status of the vaccine recipient, infection history, dosing intervals, vaccine storage and transportation capacity20. Antibody testing for SARS-CoV-2 allows us to compare vaccine efficacy among participants with or without past exposure to the virus, even after it has been cleared by the immune system. A study indicated that individuals with pre-exposure to COVID-19 produced more antibodies against COVID-19 after vaccination. Furthermore, extensive exposure to the virus and prolonged duration of the illness increases the chance of producing antibodies against all strains of SARS- CoV-221.Several serum-based evaluations were performed to reflect humoral immunity status which is often correlated with poor or strong responses obtained during infection or vaccine administration. The receptor binding domain (RBD) is responsible for binding to the ACE2 receptor on the cellular membrane, and the target protein for evaluating humoral and cellular immunity for vaccine efficacy evaluationThe exact effectiveness of vaccines against SARS-CoV-2 remains unknown. This study evaluates SARS-CoV-2 antibody surveillance in a normal academic population in Iran. It was designed to compare the efficacy of two international COVID-19 vaccines against one that is produced in Iran, by measuring levels of serum antibody produced by them. All of these vaccines were commonly used for mass vaccination in Iran. RBD domain of spike protein was used in ELISA assays to detect antibody titer of SARS-CoV-2.In this study, distribution of age, sex, and history of infection of SARS-CoV-2 was compared in participants receiving the 3 vaccines types, and their potential effects on the produced antibody levels were assessed.The efficacy of adenovector-based vaccines such as AstraZeneca and killed whole virion vaccines such as Sinopharm and COVIran Barekat was evaluated by serological assessment in individuals with or without prior infection by SARS-CoV-2. The humoral immunogenicity of these vaccines was compared by measuring the serum anti-SARS-CoV-2 anti-RBD antibodies levels.A total of 462 participants were included in this study, who received 3 different types of approved and available SARS-CoV-2 vaccines in Iran. The participant's ages ranged from 20 to 70 years old with a median of 37 (IQR 30–45), 44.2% of them were female, and 10.6% of all of the participants had priorSARS-CoV-2 infection history. In terms of age, sex, or infection history distribution, there were no statistically significant differences observed among the participants in the 3 vaccine groups. The demographic characteristics of participants are demonstrated in Table Serum antibody titers were measured one month after each of the first and second vaccine inoculation. The median of antibody titers and IQR are demonstrated in Table The frequency and percentage of participants who tested positive for anti-SARS-CoV-2 anti-S1-RBD antibody in each vaccine group are shown in Table We also assessed the impact of infection history on antibody titer through the Mann–Whitney U test for each vaccine group. After the first vaccination, distribution of antibody titer was significantly different based on infection history in AstraZeneca, Sinopharm, and the overall groups. Antibody titers were higher in participants with prior covid-19 infection. On the contrary, it was the same across categories of infection history for COVIran Barekat. After the second dose, compared to non-infected participants, our data demonstrated significant differences in antibody titers among recipients with an infection history, only in AstraZeneca and overall groups Table . Spearma23.Humoral immunity is a major factor which plays a critical role in vaccines effectiveness. Despite the fact that the exact neutralizing response is investigated thorough cVNT, which requires biosafety level 3 equipment, Anti-SARS-CoV-2 anti RBD titer can be measured to evaluate humoral responses after infection or vaccination. The sVNT is practical in numerous studies on features of COVID-19, including vaccine efficacy during preclinical and clinical trials of different vaccine candidates and monitoring neutralizing antibody titers in vaccine recipients after mass vaccinationThis was a prospective longitudinal study performed on 462 adult participants each receiving 2 doses of either AstraZeneca, COVIran Barekat or Sinopharm vaccines. One month after the injection offirst dose, a significant statistical difference was shown when comparing inactivated vaccine recipients (COVIran Barekat or Sinopharm) with participants who received AstraZeneca vaccine, both in the antibody presence  and the median antibody titer (AstraZeneca 224.0 (IQR 68.8–396.8) BAU/ml, COVIran Barekat 105.6 (IQR 16.0–284.8) BAU/ml, and Sinopharm 91.2 (IQR 16.0–276.8) BAU/ml, P-Value < 0.001). Also, the same results were obtained after the injection of the second dose, antibody presence were as follows: AstraZeneca 98.9%, COVIran Barekat 89.5%, and Sinopharm 92.1% . Also, the median antibody titer was significantly higher in AstraZeneca recipients (AstraZeneca 377.6 (IQR 275.2–416.0) BAU/ml, COVIran Barekat 192.0 (IQR 72.0–297.6) BAU/ml, and Sinopharm 195.2 (IQR73.6–302.4) BAU/ml, P-Value < 0.001). Inevitably, the protection offered by neutralizing antibodies through vaccination can be varied due to the differences in vaccine platforms and the interval of doses. Our findings demonstrated that Astra Zeneca has led to higher immunogenicity compared to other available vaccines in Iran.24. These results are in line with our findings about the higher anti-SARS-CoV-2 anti RBD IgG detection rate and titer in AstraZeneca recipients.An Iranian cohort monitoring study has revealed that when comparing AstraZeneca recipients with inactivated vaccines recipients, although the former has a significantly higher immunity during the interval of the first and second dose, after the second dosage no significant difference was found among different vaccine platforms. They concluded that SARS-CoV-2 vaccines, while unable to prevent the infection, can significantly reduce mortality rate25.It is obvious that antibody titers, post-infection or vaccination, cause a range of protective effects specifically by reducing the mortality rate. A study estimated that the required neutralizing antibody titer to completely prevent symptomatic viral infection is approximately sixfold higher compared to what is required to prevent severe outcomes of SARS-CoV-2 infection26. The antibody response is a crucial aspect of adaptive immunity against viral infections. Based on the predominant isotypes and profiles of somatic hypermutations of the antibodies, the humoral immune response can be divided into two phases. In the extrafollicular (EF) phase, B cells are activated and quickly differentiate into plasma cells in foci outside of the follicle within a few days after the infection. They produce antibodies that contain few somatic hypermutations but can still have reasonably high affinities and neutralize the virus27. Another study revealed the chances of reinfection are significantly lower in individuals who tested positive for antibodies. Furthermore, getting vaccinated after a SARS-CoV-2 infection enhances the impact of this response. Research has found that neutralizing antibodies are present in 99% of people infected with SARS-CoV-2 before. It is believed that the antibodies could provide strong protection against infection and reinfection in individuals without COVID-19 infection history, but were vaccinated previously28.In this study, we assessed antibodies in participants infected or not infected early in the pandemic during alfa and delta variants circulation time. The assessment of serum antibody activity after the first dose of SARS-CoV-2 vaccination (in September 2020) showed that participants who had previously confirmed infection in each vaccine group, or overall had a significantly higher amount of antibody titer as demonstrated in the RBD-based ELISA assay, except for COVIran Barekat group, which might be due to the low number of infected persons in this group: 11 out of 155 (7.09%). Moreover 2 of 11 COVIran Barekat recipients who had prior infection history, did not produce antibodies after the first vaccine administration. Considering that both of them were young , they might be non-responders. The impact of these two samples was magnified in such a small fraction. Therefore, when we used Whitney U test for each vaccine group in order to investigate the impact of infection history, it led to a non-significant p-value for COVIran barekat group. These findings confirmed that additional antigenic exposure further improves antibody efficacy against SARS-CoV-2 variants. Protective antibodies from prior infection, with or without vaccination, induced antibodies against the spike protein to interfere with its function29.Many scientists are of the same concern about the waning of neutralizing antibody titers induced through prior infection or vaccination. Besides, the continuous emerge of new Variants Of Concern (VOC) of SARS-CoV-2 due to its broad spread and frequent mutation, has caused several neutralizing antibodies to lose their antiviral activities. However, neutralizing antibodies can recognize different epitopes in spike protein and some of them can neutralize new VOCs. As a result of that, pervious immunity induced by infection or vaccine might alleviate infection. It was suggested that, in order to decrease the VOC’s chance of escape from vaccine induced neutralizing antibodies, future vaccines must consider different groups of epitopes of RBD30. Mucosal vaccines have been successful in protecting against diseases, but there are still limitations and risks associated with their clinical development. One concern is that mucosal surfaces are frequently exposed to harmless foreign substances and commensals, which can lead to tolerogenic immune programs that may result in weak induction of pro-inflammatory responses. Effective adjuvants can help break tolerance, however, there are limited mucosal adjuvants that are safe for human use and many require further testing to demonstrate their safety and efficacy. Additionally, antigen dilution can hinder vaccine delivery at mucosal surfaces, where ciliated cells quickly clear away debris and pathogens in a protective mucous layer31.The next step is to develop vaccines which are able to prevent infection completely. Mucosal immunity inducing vaccines might be effective in preventing infection and restricting viral transmission, however, there are still a number of obstacles to overcome to reach their full potential33.In this study, previous infection history was significantly related to higher anti SARS-CoV-2 anti RBD IgG titers . Many studies subscribe to this theory, which states infection history can boost vaccine effectiveness and lead to an increase in antibody titer and furthermore can shorten the interval of vaccine injection and rises in antibody titer34. However, another meta-analysis including approximately 7 million participants from 18 studies, has found that vaccine effectiveness in prevention of severe SARS-CoV-2 infection was significantly lower among participants older than 65 years old35. It might be due to the fact that different vaccine types were investigated in that study; since it investigated mRNA-based vaccines and Johnson and Johnson vaccine. Moreover, our study’s aim was to evaluate antibody titer among university staff and students, leading to 84% of our participants being 50 years old or younger, which may result in limitations.After categorizing participants based on their sex or age, no significant difference was observed in the median antibody titer. AstraZeneca clinical trial phase 2/3 data has not shown any significant difference in neutralizing antibody titer among different age groups, after the second dose injection. The data also demonstrated that neutralizing antibody has been detected among 99% of participants 14 days after the second dose injectionThe limitations of this study are: it did not consider children and elderly citizens (> 70), cell-mediated immune responses were not assessed, and last but not least, 34% of the participants in the first sampling did not return to partake in the second round of it.Although, the emergence of new VOCs and waning neutralizing activity of vaccines has faced researchers with a dilemma, how often should we administer SARS-CoV-2 vaccines and more importantly how safe are these repeated injections? Further studies need to address the problems of safety and efficacy of the developed and the under-development vaccines against SARS-CoV-2, which are achievable through real-world surveys.This study demonstrated that AstraZeneca vaccine induced higher humoral immunogenicity when compared to the inactivated vaccines , since it had more positive cases of antibody presence and higher antibody. Also, results of this study, agreeing with several other studies, indicated that infection-induced immunity combined with vaccination results in higher levels of protection against COVID-19 which may prevent severe illness, hospitalization and mortality. Therefore, our findings suggested that vaccination may be more crucial for people who haven’t been infected by SARS-CoV2.Delta variant of SARS-CoV-2 was the predominant strain during this period.This research was a prospective longitudinal study, performed among 462 staff and students of Tarbiat Modares University, Tehran, Iran. It was conducted from 4 September 2021 until 29 December 2021. The All university staff and students who attended the health center of Tarbiat Modares University to get their second dose of vaccination with Oxford/AstraZeneca, COVIran Barekat or Sinopharm, and were willing to participate in the study were enrolled. 2 ml of peripheral venous blood sample was collected for serological survey. The participants were recalled 1 month after the second injection for the second round of blood sample collection.This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Tarbiat Modares University of Tehran (code number: IR.MODARES.REC.1400.138).The researchers explained the method and aim of the study. The participants signed the formal informed consent form and were able to withdraw from the study at any time. They were also able to communicate with the research team for more information about the tests and results, or ask any questions related to this study.At first, demographic data of participants was gathered via filling out an online questionnaire . The participants were put through an online survey one-week after each round of vaccination to self-report any experienced side effects.For each participant, peripheral venous blood samples (2 ml) were collected and transferred to the virology laboratory of Tarbiat Modares University of Tehran. Sera were separated from whole blood by centrifuging samples for 10 min and were stored at − 20 ℃ prior to detecting SARS-CoV-2 antibody level.36) had 6 calibrators with different dilutions , each equal to a particular dilution of antibody in sera. The kit included negative and positive controls. The negative and positive controls included inactivated human serum diluted via diluting buffer, devoid of Anti-SARS-CoV-2 RBD Antibody for the negative and containing it for the positive control.Since our aim was to survey the antibody response in vaccinated individuals, Enzyme-Linked Immunosorbent Assay (ELISA) was used for serological assessment, which detects Anti-SARS-CoV-2 Spike Protein RBD Antibody. In this research Chemobind Anti-SARS-CoV-2 Spike Protein RBD Antibody detection kit was used. We used RBD-based kits representing frequently used antigens for their reactivity to human IgG antibodies using ELISA assay in serum from COVID-19 vaccinated individuals. Besides, suspected results of the ELISA kit  were checked again they were also compared against the EUROIMMUN IgG ELISA test. The homemade kit (Chemobind developed by a knowledge-based enterprise supervised by Tehran University of Medical Sciences. It has been used in other studies and its results has been compared with well-known kits such as EuroimmunThe serum samples were thawed, vortexed, and diluted before testing. In each run, every calibrator along with the positive and the negative controls was used duplicated. The kit was used in accordance with the manufacturer's protocol. Based on the optical density of calibrators and their dilutions a standard curve was plotted in each run. Anti-SARS-CoV-2 RBD antibody titer was calculated for each participant in accordance with the OD measurements for samples and the plotted standard curve of its run using the Curve Expert application. Samples containing higher antibody titer than the most concentrated calibrator > 120 were diluted and tested again.Data entry was done using Microsoft Excel and statistical analysis was performed through IBM SPSS version 28. The collected data were analyzed using Kruskal–Wallis test with Bonferroni correction, Chi-square test, Mann Whitney U test, and Pearson correlation analysis test.


Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in acute-care settings affects patients, healthcare workers, and the healthcare system. We conducted an analysis of risk factors associated with outbreak severity to inform prevention strategies.This cross-sectional analysis of COVID-19 outbreaks was conducted at Fraser Health acute-care sites between March 2020 and March 2021. Outbreak severity measures included COVID-19 attack rate, outbreak duration, and 30-day case mortality. Generalized linear models with generalized estimating equations were used for all outcome measures. A P value of 0.05 indicated statistical significance. Analyses were performed using SAS version 3.8 software, R version 4.1.0 software, and Stata version 16.0 software.Between March 2020 and March 2021, 54 COVID-19 outbreaks were declared in Fraser Health acute-care sites. Overall, a 10% increase in the hand hygiene rate during the outbreak resulted in an 18% decrease in the attack rate (P < .01), 1 fewer death (P = .03), and shorter outbreaks (P < .01). A 10-year increase in unit age was associated with 2.2 additional days of outbreak (P < 0.01) and increases in the attack rate (P < .05) and the number of deaths (P < .01).We observed an inverse relationship between increased hand hygiene compliance during outbreaks and all 3 severity measures. Increased unit age was also associated with increases in each of the severity measures.This study highlights the importance of hand hygiene practices during an outbreak and the difficulties faced by older facilities, many of which have infrastructural challenges. The latter reinforces the need to incorporate infection control standards into healthcare planning and construction. The primary acquisition source for much of these infections is patient-to-patient exposures. An important tenet of hospital infection prevention and control (IPC) is applying additional precaution measures to those with suspected communicable diseases based on symptoms. However, transmission of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) often occurs from asymptomatic or presymptomatic cases who would not have the additional precaution measures applied, therefore resulting in transmission to roommates or other contacts. Additionally, in the context of the COVID-19 pandemic, many hospitals often operated at or above capacity, resulting in crowding, further facilitating transmission.The coronavirus disease 2019 (COVID-19) pandemic has largely affected hospitals with increased numbers of patients and increased risk of transmission in a vulnerable population. Commonly used strategies include universal masking, expanded test criteria that include increased testing of symptomatic patients and healthcare workers (HCWs), universal testing of all patients on admission, and designated COVID-19 units. Other traditional IPC measures, such as standard precautions for all patients, transmission-based precautions for suspected and confirmed cases, and hand hygiene have also been widely practiced. Because these measures are often implemented simultaneously, determining the efficacy of any one measure is challenging.Several strategies have been implemented in hospitals to limit the transmission of SARS-CoV-2 and reduce the impact on unit management. comorbidities of patients involved, and increased staff absence or sickness, extensive examination of the variety of risk factors that may increase COVID-19 outbreak severity is lacking. Understanding the factors associated with increased outbreak severity can help inform outbreak management. We sought to bridge this gap in knowledge by identifying factors associated with COVID-19 outbreak severity, as measured by attack rate, mortality, and outbreak duration, at hospitals in a large health region of British Columbia, Canada.Despite best efforts, nosocomial COVID-19 outbreaks occur. Although studies have identified several factors associated with these outbreaks, including shared bathrooms and sinks, It provides publicly funded healthcare services to more than 1.9 million people in a geographical area. The services provided include primary healthcare, community home care, mental health and substance use services, long-term care, and acute medical and surgical services. The setting for this study comprised the 12 hospitals within Fraser Health, with a total of 3,619 acute-care beds across 3 regions . During the study period, Fraser Health applied multiple strategies to control COVID-19 transmission in hospitals. At the start of the pandemic, Fraser Health implemented universal masking for all HCWs, and in November 2020 Fraser Health implemented universal admission testing of patients. In December 2020, COVID-19 vaccinations became available for HCWs and other eligible populations. During the study period, SARS-CoV-2–positive patients were cohorted on droplet precautions on dedicated units with dedicated staffing. In addition, airborne precautions were applied for all aerosol-generating procedures. All exposed patients were placed on droplet precautions. This study was conducted to support public health and infection control surveillance and was exempt from ethics approval.Fraser Health is the largest regional health authority in British Columbia, Canada.This study was a retrospective analysis of COVID-19 outbreaks from the 10 hospitals that experienced outbreaks during the study period, March 1, 2020, to March 1, 2021. Data were gathered from various sources at the patient, unit, outbreak, and facility levels. At the patient level, SARS-CoV-2 test results, admission, bed moves, and demographic data were extracted from electronic medical records. Comorbidities, total comorbidity factor and resource intensity weights were obtained from data submitted to the Canadian Institute for Health Information (CIHI). Deaths of outbreak cases were obtained from the public health electronic database.Facilities management and nursing staff collected facility-level data. Nursing hours were obtained from the employee scheduling system. Each unit’s hand hygiene rates were collected from audit reports for 2 intervals: (1) before the pandemic  and (2) during each outbreak. IPC outbreak reports were used to determine outbreak-related measures, including outbreak duration, number of cases, and whether outbreak units were closed, partially opened, or transferred to another unit (moved).A confirmed COVID-19 case was a person with laboratory confirmation of SARS-CoV-2 by polymerase chain reaction (PCR). A healthcare-associated (HCA) COVID-19 case was laboratory-confirmed COVID-19 with either (1) symptom onset (or specimen collection date) 5 days or more after admission to a healthcare facility or (2) symptom onset (or specimen collection date) 10 days or less following discharge from a healthcare facility. These criteria were health authority guidelines during the study period. For consistency, the specimen collection date of the first positive test was used as a proxy for symptom onset. Following identification of the index case, all exposed patients were placed on droplet precautions and placed in cohorts separately from any new admissions to the unit, limiting the potential for ongoing transmission from the initial cohort. Patients who tested positive for SARS-CoV-2 in the 60 days before the outbreak were excluded from the exposed patient population for this analysis, according to the health authority’s reinfection definition during the study period.Patients were considered exposed if they had been on the unit in the 2 days preceding the identification of the index case, based on published evidence for COVID-19 infectious period.A COVID-19 outbreak was declared by IPC at the hospital when there was evidence of transmission involving a patient on a unit . Outbreaks were declared in real time as cases were identified. The end of an outbreak was declared 14 days after the last identified exposure to a confirmed case.Healthcare worker cases and outbreaks occurring on Mental Health and Substance Use (MHSU) units (n = 2) were excluded from this analysis.Outbreak severity was measured for 3 variables: (1) COVID-19 attack rate, defined as the number of new cases divided by the number of exposed patients; (2) outbreak duration, defined as the number of days between outbreak declaration and when the outbreak was declared over; and (3) all-cause 30-day case mortality, defined as the number of outbreak cases who died within 30 days of testing positive for SARS-CoV-2.Independent variables were categorized as patient-level factors (summarized at the outbreak level), unit-level, hospital-level, and outbreak-specific factors. The median of patient-level factors for each outbreak was used for the analysis. A list of all variables and their definitions is included in the Supplementary Material (online).The number of patient bed moves during the outbreak was included to assess whether patient movement was associated with any outcome variables. Nursing workload and resourcing were measured by the proportion of nursing hours coded as overtime hours and the number of nursing hours per patient day during the outbreak.Hand hygiene compliance in healthcare workers was assessed by direct observational audits conducted by trained and validated observers. Compliance data were collected electronically using a standardized data collection tool. Audits were conducted monthly in non-outbreak units and daily for units with an outbreak.P values were used to identify significant independent risk factors. Multivariate Gaussian regression analysis was conducted for attack rate, and multivariate negative binomial analysis was used for the number of deaths and outbreak duration. Generalized estimating equations (GEE) were added as an extension to the models to account for clustering. Goodness of fit was assessed by analyzing residual plots and deviance residuals. All models included age and sex to control for confounding. A P value of .05 was considered statistically significant. Statistical analyses were performed with SAS Studio version 3.8 software, R Studio version 4.1.0 software, and Stata/SE version 17.0 software.Statistical analysis was conducted at the outbreak level. Each risk factor was compared to each of the 3 severity measures using simple linear regression. For model selection, backward elimination and stepwise regression were performed using Akaike information criteria (AIC), and During the study period, 54 outbreaks Table  met the The crude analysis identified 4 patient-level factors that led to increased attack rates: resource intensity weight , acute length of stay , total comorbidity factor , and bed moves   observed for every 20-year increase in unit age Table . SimilarOutbreak duration was increased for units with patients who had multiple comorbidities, and each addition to the median number of comorbidities increased outbreak duration by 3.7 days . Unit age was also an important factor in outbreak duration; each decade was associated with 2.2 additional days of outbreak . Finally, overtime nursing hours were important for duration in the crude models; a 10% increase in overtime hours was associated with 8 additional days of outbreak .P < .01). Conversely, a 10-point increase in the median total comorbidity score was associated with a 14% increase in the attack rate (P < .01). A 1.4% increase in the attack rate was observed for every 3 bed moves per patient (P < .01). Regional hospitals were associated with a lower attack rate than community hospitals even after adjusting for confounders (P = .02). Although not statistically significant in the crude analysis, hospitals in the North region appeared to be associated with a lower attack rate than hospitals in the East region (P = .01).After adjusting for age, sex, and other covariates, a 10% increase in the hand hygiene rate during the outbreak resulted in an 18% decrease in the attack rate (P = .03). The North region was protective against severity, with 1.3 fewer deaths occurring on average compared to the East region (P < .01).Hand hygiene rates during the outbreak were strongly associated with fewer deaths, and each 10% increase in rates was associated with 1 fewer death (P < .01). Paradoxically, hand hygiene rates in the year prior to the pandemic showed the opposite effect, with increased hand hygiene rates associated with longer outbreaks. Facility factors were once again important, with regional hospitals reducing outbreak duration by ∼8 hours (P = .02) and North hospitals reducing outbreak duration by roughly the same (P < .01). Patient-level factors played a role in outbreak duration, with each additional comorbidity to the median number increasing duration by 0.11 days (P = .02). Length of stay played a role in duration, but the effect was minimal; every 62 days in acute care was associated with an increase of 1 additional day (P < .01).The importance of hand hygiene during the outbreak was demonstrated in the outbreak duration, where each 20% increase was associated with a reduction of 1 day . This finding was expected because patients with more complex medical conditions are more susceptible to acquiring infections.The total comorbidity factor is “the cumulative percentage increase on the patient cost associated with all comorbidity codes of the case.” In the context of COVID-19 transmission, similar findings were observed in long-term care settings. However, in this study, as the ratio of nursing hours to patient days increased, a marginal increase in the attack rate was observed which is inconsistent with studies from non–acute-care settings. Notably, differences in metrics used to quantify hospital staffing, making comparisons between studies challenging. Additionally, we did not measure individual nursing experience, adherence to infection prevention and control practices, or staff skills and abilities that could affect the observed outcome. Future research could examine the impact of these contextual factors to better understand the role of staffing on healthcare-associated COVID-19 transmission. Finally, in the crude analysis, an increase in the proportion of nursing overtime hours was associated with increased outbreak duration and 30-day case mortality. However, these findings were not significant after controlling for covariates. The study’s small sample size may have contributed to the lack of statistical significance.Our findings support an inverse correlation between staffing levels and healthcare-associated infections.This study had several other limitations. Summary measures were utilized for patient-level data; thus, the findings cannot be inferred to determine an individual patient’s risk for COVID-19 acquisition in healthcare. Additionally, the study did not include HCW cases because it was not possible to identify all HCW cases associated with each outbreak due to the constraints of self-reporting. The findings of this study may not be easily extrapolated to other hospitals due to differences in practices  and surveillance definitions. In addition, this study only applies to the SARS-CoV-2 variants observed during the study period. Other variants may have different infection dynamics and transmissibility. it is the only method that considers appropriate hand hygiene before, during, and after patient care. Additionally, direct observations utilizing standardized data collection tools performed by trained and validated observers, as in this study, are considered the gold standard for hand hygiene compliance measurement.Increased hand hygiene compliance was observed as one of the major predictors of decreased outbreak severity. Although there are inherent biases within this measure due to the Hawthorne effect, Subsequently, by excluding MHSU units, the findings are not generalizable to that context. The use of specimen collection date as a proxy for symptom onset may have resulted in fewer patients designated as exposed to the index case, which subsequently could lead to an overestimation of the attack rate. Because the proxy was used for all outbreaks, it is unlikely to have biased the results of the analysis.Outbreaks on Mental Health and Substance Use (MHSU) units during the study period were excluded as infection control measures and outbreak management strategies may differ in such settings.In this study, we classified patients as susceptible to reinfection after 60 days from their first SARS-CoV-2–positive test, whereas most studies have used 90 days. This feature of our study may have introduced misclassification errors; however, 60 days was applied consistently, minimizing the potential for bias in the results. there was the potential for misclassification errors dependent on community incidence. During the study period, the average community incidence in the region was 8.57 cases per 100,000, resulting in minimal risk of misclassification as healthcare associated.Although cases of patients who tested positive within 10 days of discharge from the hospital were considered healthcare associated, given the lengthy incubation period for SARS-CoV-2 (2–14 days),Finally, patients discharged home from an outbreak unit may have acquired SARS-CoV-2 due to exposure on the unit but were not tested in the community due to clinical presentation or test availability. These unidentified cases may have resulted in underestimation of the attack rate and case mortality but were unlikely to bias the study results in any direction.The findings of this study, along with the limitations, have identified opportunities for future research, including a similar analysis at the patient level to better understand the individual factors that could lead to excess mortality. Additional risk factors that were not identified at a summary level may be identified by focusing the analysis at an individual level.