In this study, we aimed to build a machine-learning predictive model for the identification of triple negative breast cancer, the most aggressive subtype, using quantitative parameters and radiomics features extracted from tumor lesions on hybrid PET/MRI. The good performance of the model supports the hypothesis that hybrid PET/MRI can provide quantitative data able to non-invasively detect tumor biological characteristics using artificial intelligence software and further encourages the conduction of additional studies for this purpose.18F-FDG PET/MRI is effective in molecular subtyping of breast cancer (BC) and specifically in discriminating triple negative (TN) from other molecular subtypes of BC. Methods: Eighty-six patients with 98 BC lesions  were included and underwent simultaneous 18F-FDG PET/MRI of the breast. A 3D segmentation of BC lesion was performed on T2w, DCE, DWI and PET images. Quantitative diffusion and metabolic parameters were calculated and radiomics features extracted. Data were selected using the LASSO regression and used by a fine gaussian support vector machine (SVM) classifier with a 5-fold cross validation for identification of TNBC lesions. Results: Eight radiomics models were built based on different combinations of quantitative parameters and/or radiomic features. The best performance  was found for the model combining first order, neighborhood gray level dependence matrix and size zone matrix-based radiomics features extracted from ADC and PET images. Conclusion: A ML-based radiomics model applied to 18F-FDG PET/MRI is able to non-invasively discriminate TNBC lesions from other BC molecular subtypes with high accuracy. In a future perspective, a “virtual biopsy” might be performed with radiomics signatures.Purpose: To investigate whether a machine learning (ML)-based radiomics model applied to  Breast cancer (BC) is a heterogeneous disease with a multifactorial etiology  affecting the capability of cells to repair DNA damages ,2,3. In The assessment of molecular subtypes is, therefore, the “sine qua non” for treatment planning of BC. In patients with TNBC, it is especially important as they usually require upfront systemic treatment before surgery. Currently, the assessment of molecular subtypes is performed through invasive tissue sampling using invasive core needle biopsy, an approach inherently limited by sampling bias and providing only a snapshot of the biology of the entire tumor. Indeed, molecular biomarkers are likely to differ within the tumor, a phenomenon known as intratumor heterogeneity . The dev18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) as well as magnetic resonance imaging (MRI) diffusion-weighted imaging (DWI), and dynamic contrast-enhanced (DCE) techniques are the gold standard for the in vivo assessment of tumor metabolism (18F-FDG PET), cellularity (DWI) and neoangiogenesis (DCE) [18F-FDG PET and MRI have shown high sensitivity [18F-FDG PET/MRI has been shown to be promising for the accurate and non-invasive biological characterization of BC [Developments have led to sophisticated imaging techniques that can depict the functional properties of breast tumors and their underlying biology. Currently, is (DCE) ,12,13. Fsitivity ,15. Moreon of BC .18F-FDG PET/MRI for BC phenotyping, specifically for hormone receptor-positive  BC identification has been demonstrated [Initial studies have demonstrated the potential of radiomics analysis coupled with machine learning (ML) based on either PET or MRI (both DCE and DWI) for the comprehensive assessment of tumor phenotypes and for the development of predictive models ,18,19. Mnstrated ; neverth18F-FDG PET/MRI can identify the radiomic signature of TNBC. Therefore, we aimed to build an ML-based predictive model using both quantitative imaging parameters and radiomic features extracted from simultaneous 18F-FDG PET/MRI to distinguish TNBC from other molecular BC subtypes.We hypothesized that AI-enhanced simultaneous 18F-FDG PET/MRI of the breast between June 2016 and June 2020. Inclusion criteria were: >18 years-old subjects; histologically verified BC lesions; and not pregnant or breastfeeding. Exclusion criteria were: patients with standard contraindications for performing MRI examinations ; patients for whom histological proof of malignancy was not available; patients with malignant lesions other than BC; tumor recurrence; incomplete 18F-FDG PET/MRI examinations; and patients with PET, DCE, or DWI images that were not suitable for subsequent multiparametric and radiomics analyses. Patients included in this study have been investigated in a previous study with different purpose and results [This prospective single-institution study was approved by the institutional review board, and written informed consent was obtained from all participants. All patients underwent simultaneous multiparametric  results .18F-FDG PET/MRI was performed as previously described [18F-FDG PET/MRI protocol are reported in Simultaneous escribed , using aA board-certified breast radiologist and a nuclear medicine physician with 7 and 11 years of experience, respectively, independently evaluated all PET/MR images, using a previously described method that proved to be highly reproducible .MR images were analyzed for both DWI and perfusion-weighted imaging (PWI) quantitative parameters using a free, open-source software . In detail, 2D circle ROIs were placed on apparent diffusion coefficient (ADC) maps for ADCmean calculation of tumor lesion and contralateral breast parenchyma. Thereafter, 2D ROIs were drawn over tumor lesions on first post-contrast DCE images and then pasted on perfusion maps DCE maps for the extraction of quantitative perfusion parameters, including mean transit time (MTT), plasma flow (PF), and volume distribution (VD), according to previous evidence . DetailsPET images were analyzed for the quantification of tumor uptake using the Hermes Hybrid Viewer . Maximum, mean, and minimum standardized uptake values  were calculated by placing a 3D volume of interest (VOI) with a fixed threshold at the level of tumor lesions; care was taken to exclude surrounding background parenchymal uptake. The same approach was used for the extraction of SUVmean of the ipsilateral and contralateral normal appearing breast parenchyma, away from the nipple and areola.Whole BC lesions were segmented on T2-weighted, DCE, DWI, and PET images using a dedicated software . DCE (first post-contrast timepoint), DWI, and PET images were annotated using a semi-automated method selecting a lower boundary of signal intensity, while a slice-by-slice approach was used for segmenting BC lesions on T2-weighted images. In all cases, VOIs were placed within the margins of the lesions and care was taken to exclude macroscopic necrosis as well as cystic and hemorrhagic areas or biopsy markers. Prior to the extraction of radiomic features, data for all images were reduced to 16 grey levels. The Computational Environment for Radiological Research (CERR), compatible with the Image Biomarker Standardization Initiative (IBSI), platform was used to extract radiomic features  from DCEAs our study dataset consisted of a limited number of cases relative to a high number of extracted features, radiomic feature selection was performed using Least Absolute Shrinkage and Selection Operator (LASSO) regression , and subMalignant tumor samples from core biopsy and/or surgical specimen were analyzed to define tumor histology, grade, and immunohistochemical status including ER, PgR, Ki-67 expression, and overexpression and/or amplification of HER2 of each breast cancer lesion. The St. Gallen surrogate molecular subtype definitions were used to classify breast lesions .t-test were performed to assess differences in terms of lesion size, quantitative parameters and radiomics parameters between TN and non-TN breast cancer subtypes. McNemar’s test was used to assess differences in terms of diagnostic performance among the different radiomics models. p values ≤ 0.05 were considered statistically significant. Confidence intervals for diagnostic metrics were calculated using a bootstrapping approach. Statistical analysis was conducted using SPSS, Version 25.0. 2017 .The Kolmogorov–Smirnov test was performed to assess whether data were normally distributed. Accordingly, the Mann–Whitney test or independent According to the inclusion and exclusion criteria, 144 patients were initially enrolled in the study. Of these, 86 female patients (mean age 52 ± 13 years) were included in the final study sample, with 98 histologically proven BC lesions (mean size: 28.31 ± 16.8 mm), comprising 25 TN, 10 Luminal A, 51 Luminal B, and 12 HER2+ lesions. In detail, there were eight patients with two BC lesions in the same breast and four patients with bilateral BC lesions. The majority  of histological types was represented by ductal invasive carcinoma. The flowchart of patient inclusion is illustrated in p < 0.001). No differences were observed in terms of lesion size and remaining quantitative parameters between TN and non-TN lesions. Mean values of tumor size and quantitative parameters are reported in TN lesions showed significantly higher SUVmax (9.5 vs. 4.9), SUVmean (5.7 vs. 3.4), and SUVmin (3.1 vs. 2) compared with lesions of other BC subtypes , and DCE-MRI, ADC, and PET (Model 7); and quantitative parameters combined with radiomic features (Model 8). Quantitative parameters and/or radiomic features selected for each model are reported in Eight radiomic models (Models 1–8) were developed for TNBC identification, using the following combinations of quantitative parameters and radiomic features: quantitative parameters alone (Model 1); radiomic features extracted from ADC (Model 2), DCE (Model 3), PET (Model 4) and T2-weighted (Model 5) images; combinations of radiomic features extracted from different Across all models, the area under the receiver operating curve (AUC) ranged from 0.725  to 0.887 . Model 7 showed the best performance in discriminating TNBC from other BC subtypes, with a diagnostic accuracy of 82.8% (95% CI: 78.2–87.1%), sensitivity of 79.7% (95% CI: 71.6–86.5%), and specificity of 86.0% (95% CI: 80.8–90.4%). In detail, Model 7 included size zone matrix-based features extracted from ADC and PET images as well as first-order and neighborhood gray level dependence matrix-based features extracted from PET images. Of note, Model 1, based on quantitative parameters extracted from both primary lesions  and contralateral breast tissue (ADCmean), achieved the second-best AUC of 0.884 (95% CI: 0.867–0.898), followed by Model 8, combining quantitative parameters and radiomic features, which showed an AUC of 0.871 (95% CI: 0.849–0.889).18F-FDG PET/MR images, the best performing one was Model 2, based on features extracted from ADC maps, which yielded an AUC of 0.826 (95% CI: 0.758–0.984). On the other hand, the worst performing one was Model 5, based on features extracted from T2-weighted images, which yielded an AUC of 0.725 (95% CI: 0.679–0.765). Accuracy metrics of all radiomic model are reported in Among the models built using radiomic features extracted from individual p = 0.005). No differences were observed between Model 7 and the remaining radiomic models. Comparisons in terms of diagnostic performance among all models are reported in A statistically significant difference in terms of diagnostic performance was found using McNemar’s test between the best performing model (Model 7) and the worst performing one (Model 5)  specifically included features extracted from functional images i.e., ADC and PET, supporting the hypothesis that functional data such as tumor cellularity and metabolism may better depict biological tumor features compared to morphologic sequences. It is worth noting that no significant differences in terms of diagnostic performance were found between Model 7 and all other models except for Model 5 which was based on solely on radiomic features extracted from T2-weighted images.In the present study, we evaluated AI-enhanced simultaneous 18F-FDG PET and MRI are already indicated for both local and global staging of locally advanced breast cancer as well as for treatment monitoring. As such, it is permissible to imagine that, with a single imaging examination, tumor diagnosis, staging, and phenotyping could be obtained non-invasively at the same time. In this light, “virtual biopsies” could be performed once radiomic profiles specific to molecular subtypes have been defined, aiming at providing genetic and phenotypic alterations which are representative of the whole tumor and comprehensively describe tumor heterogeneity. Furthermore, the extraction of quantitative imaging data from the whole tumor could allow the spatio-longitudinal monitoring of biomarker heterogeneity changes during treatment and the early identification of clonal dynamics and genetic modifications related to the occurrence of drug resistance.Such findings support the expectation that, in the near future, molecular data could be non-invasively obtained by imaging through the application of artificial intelligence tools. This issue is particularly relevant if we consider that PET and MRI represent the most promising imaging modalities for this purpose, due to their ability to non-invasively inform on cancer metabolism, cellularity, and neoangiogenesis. Such properties are reported to be different between BC subtypes which exhibit different biological aggressiveness and behavior . TNBC is18F-FDG PET/MRI for a comprehensive analysis of molecular subtype, Ki67 expression, nodal status, and presence of distant metastasis in a population of 124 BC patients [So far, several attempts have been made to non-invasively define BC molecular subtype through the use of radiomics and machine learning applied to PET and MRI ,18,19. Ppatients . For thepatients . Howeverpatients . Furtherpatients . Five ra18F-FDG PET/MRI was limited due to high demand from clinical needs. Furthermore, TNBC is relatively rare compared to other subtypes, thus larger numbers can only be recruited in a multi-centric setting over a reasonable time period. Due to this limitation, we refrained from using a subset of cases as a held-out test set. Indeed, a five-fold cross-validation was used, as done in previous studies involving the preliminary assessment of the applicability of the model to an unseen population [Our study has several limitations to be acknowledged, the first being the relatively small sample size, as accessibility to simultaneous pulation ,39. Pote18F-FDG PET/MRI can non-invasively identify TNBC, the most aggressive tumor type requiring intensified treatment, with high accuracy. Additional investigations on larger cohorts of patients are necessary to validate our model and fully assess its generalizability.AI-enhanced simultaneous


The impact of public health policies during the COVID-19 pandemic on people who inject drugs (PWID) has varied across regions. In other countries, recent research has shown that PWID access to harm reduction services, despite rapid adaptations, has been negatively impacted. Our study describes these impacts in a rural state.We conducted semi-structured interviews with PWID, community partners, and healthcare providers in the rural state of Maine (USA). We explored how changes made during the pandemic impacted access to harm reduction services, including basic services , syringe service programs, safe drug supply, low barrier treatment, and peer support. Interviews were analyzed using the framework method to apply Penchansky’s model of access, with Saurman’s modification, which includes six dimensions of access—accessibility, availability, acceptability, affordability, accommodation, awareness.N = 9 community partners, N = 9 healthcare providers, N = 18 PWID). Policies such as mobile outreach expansion, mail delivery of equipment, and relaxed telemedicine regulations facilitated accessibility to syringe service programs and low barrier buprenorphine treatment. Public health policies, such as social distancing and screening policies, reduced contact, which subsequently reduced acceptability and awareness of many services. Elimination of the one-for-one needle exchange in some areas increased, acceptability , and affordability for PWID. However, some areas actually began enforcing a one-for-one needle exchange policy, which reduced affordability, acceptability, and awareness of services.We interviewed thirty-six stakeholders (Changes resulting from the COVID-19 pandemic have impacted all dimensions of access to harm reduction services among PWID. While some barriers to harm reduction services were unavoidable during the pandemic, we found that specific policy decisions mitigated service barriers, while other policies exacerbated them. Relaxing needle exchange policies were particularly helpful in facilitating access to harm reduction services by giving community organizations flexibility to adapt to the evolving needs of PWID. These results can inform policies and service delivery to optimally mitigate the negative impacts on PWID during, and beyond, the pandemic.The online version contains supplementary material available at 10.1186/s12954-022-00660-2. The impact of the COVID-19 pandemic on how people who inject drugs (PWID) access harm reduction services, particularly in rural areas of the USA, is poorly understood. Harm reduction services, such as syringe service programs (SSPs), basic services , safe supply, peer support, and low barrier buprenorphine treatment play important roles in mitigating adverse impacts of drug use, such as infections and overdoses . Prior tEvidence is emerging on the impacts of the COVID-19 pandemic on PWID in different regions of the USA. Interviews with people who use drugs (primarily methamphetamine), in rural communities in Oregon revealed limited access to SSPs during the pandemic . AnotherIn Maine, an emergency shut-down order was declared on March 31, 2020, shortly after the first presumptive COVID-19 case was identified . At thatawareness, accessibility, availability, acceptability, affordability, and accommodation. The model, which has been used in prior health service-related studies  is that it doesn't do a good enough job of communicating with drug users.” (CP 2)Changes around the one-for-one needle exchange policy were also confusing for many PWID and community partners. For example, the executive order temporarily eliminated the one-for-one needle exchange policy in the state, but a few months into the pandemic, the city of Portland was instructed to enforce the one-for-one needle exchange policy. Several PWID who accessed the Portland SSP found the policy change both confusing and detrimental to their health. The reported confusion demonstrates that efforts to communicate these various changes were unsuccessful. One community partner suggested these issues predated the pandemic:“Previously, I was just able to go to a jail and work on those values, […] So a lot of that work was being established there. But now, because I'm not able to go in there and as people are coming out, I actually don't know what are the different services that are lacking.” (CP 05)Lacking a robust system of communication to reach people who inject, organizations relied on informal channels and direct relationships between service providers and service users to relay information. Unfortunately, COVID-19 policies that limited visitors to correctional facilities and other spaces also impacted the ability of some community partners do in-person outreach in those locations. As a result, they reported missed opportunities to connect with PWID and link them to harm reduction services.I think we have a lot of recognition that there are people for whom this isn't a great form of connecting which is why we've been really maintaining our in-person outreach in the places that we have those programs to connect with primarily people who are unhoused, who are camping in the woods, who are really living in a way that doesn't facilitate doing a FaceTime with us. (CP 3)Many community partners increased their outreach efforts to be able to connect with more dispersed populations:Community partners attempted to share information when possible, informing clients about other services that were available during brief contact or including contact cards in bags distributed with safe injection supplied. Additionally, because of the temporary executive order that allowed for mail delivery of drug equipment, several mobile SSPs were able to mail flyers advertising their services. SSP clients that requested naloxone for overdose prevention received a flyer in the mail alerting them that they could contact the SSP for other equipment as well. This approach was found to increase awareness and reduce stigma around accessing safer use equipment. As more mobile SSPs set up in outdoor spaces, their presence also increased awareness about accessing safer use equipment.“We made a decision as a program early on that we were going to remain open as long as we could. And be available to support patients in the community...with additional safety measures in place...” (Pr 1).Other facilitators that increased awareness to harm reduction services, specifically low barrier buprenorphine treatment, involved services simply staying open and visible outdoors. Two providers from a low-barrier urban clinic started delivering services outdoors early in the pandemic, which demonstrated to community members their availability:“…in terms of building that relationship for me has been consistency and has been presence. […] And so I think not having that face-to-face, to me has meant a lack of consistency and a lack of physical presence to build that baseline trust with each other, which then causes a lot of gaps in the services that people need.” (CP 5).Some COVID-19 policies made particular harm reduction services less acceptable to PWID. Many service providers emphasized the ways that regular, face-to-face interactions allowed them to establish trusting relationships with their clients. New restrictions often prevented them from being present in the same space:“Yes, they do have them, but once again, sometimes with [PWID], it's all about trust and having that rapport with folks. So, some folks are very hesitant to engage with new providers, even though we try to encourage that and help to make referrals and all those sorts of things. It still can be a barrier.” (CP 6)These relationships between clients and providers are also relevant to understanding difficulties adjusting to changing sites among PWID. Asked whether there are alternative services PWID can use, one community partner explained:While COVID-19 screening , testing, physical distancing policies and quarantine protocols were acceptable to some people, COVID-19 protocols also reduced acceptability to many participants seeking harm reduction services. For several reasons, including fears of COVID-19 testing and possible quarantine, PWID participants also preferred to avoid the shelters. Many stayed in tents, while others stayed in hotels or cars.ten or fewer people, reduced acceptability of services:“They get really frustrated…you have to tell them ‘Hey, there’s 10 people here…you got to wait down the street…a lot of people have gotten so frustrated that they just left and they were like ‘ I don’t got time for this.’ And then, where are they going without their safe using equipment?” (CP 1)Service providers at mobile SSPs reported that guidelines around physical distancing, specifically staying six feet apart and only allowing gatherings of “It wasn’t like this before…It was just like ‘I want you to be safe. Take as many as you need to be safe.’…I feel like, ‘Why are they changing this all of a sudden? Do you want people safe or do you want health [problems]…There’s going to be a rise in infections and HIV, hepatitis C. Why are we regressing in this now?” (PWID 17)Enforcement of the one-for-one syringe policy in Portland impacted access in a variety of ways detailed in each section. One impact was reduced acceptability, as established relationships between a Portland SSP and its clients were undermined:“…people are being given a hard time by the police a lot more…they don't necessarily get arrested if they have more than 10 needles, but they get a ticket or they'll get a summons and they'll have to go to court. Or the police will just confiscate their stuff sometimes…It ties in with this whole criminalization of being homeless, as well as criminalization of using drugs. “ (CP 7).Interactions with law enforcement in the setting of the one-for-one needle exchange policy in Portland specifically reduced access to safer use equipment. At the time of the study, due to regulatory barriers such as the requirement to carry less than 11 hypodermic needles, participants carrying more than the limit of safer use equipment reported issues with law enforcement:“…every time I go, the pharmacies are trying to say they don't have needles, and blah, blah, blah, and it's just like, you all can't be out .” (PWID 12)Though not reported as a barrier exacerbated by the pandemic specifically, several PWID participants also felt that stigma was a barrier to accessing safer use equipment outside of SSPs. For example, PWID in rural areas experienced stigma when buying unused injection equipment and naloxone at community pharmacies:In terms of access to low barrier buprenorphine treatment, lack of face-to-face interactions reduced acceptability for some PWID and provider participants. For those PWID participants who still sought substance use disorder treatment in person, COVID-19 policies, particularly masking requirements, reduced acceptability. One PWID participant reported a history of kidnapping, and that wearing a mask was traumatizing for her, and masking requirements contributed to her decision to abandon a methadone program.“I think we're really just trying to listen to the clients even more than we already have. […] we've always had a great staff, but I think they're really shining even more now. In the slower pace, you're really able to sit with folks more and hear them out and try to do things that you might not have otherwise had time for at one point […] Just let them know that while [this Site] doesn't look the same as it did three months ago, the spirit of [the Site] and our goal is still the same.” (CP 6)In terms of other harm reduction services, trust in peers/community partners generally increased acceptability of peer support and SSP, so services that were able to continue operating were crucial. Some services actually increased outreach, or dedicated greater efforts to building relationships:“It seems there's been this real shift that has been really effective. People have been really accessing services that way. I think partly just because there's more anonymity. You can show up. You're in, you're out.” (CP 3)Mobile SSPs also increased outreach. For some PWID, these new means of accessing safe injection/smoking supplies were more acceptable than previous options because they provided anonymity by operating in less populated, remote areas:The mail delivery of equipment was also appealing to many participants due to perceived anonymity and thus increasing acceptability of services. Regarding MOUD treatment, some providers speculated that the inconsistency of drug supply/contaminated drug supply encouraged more people to seek treatment with MOUD, though we were unable to corroborate this in PWID interviews. Some clients did report that telemedicine visits for MOUD to be more acceptable than in-person visits, largely due to reduced travel time and costs. Finally, while social distancing and screening policies decreased the acceptability of sites for some clients, community partners reported that others appreciated the increased protection from COVID-19.The ability of people to get to desired services was impacted in complicated ways, particularly as certain services transitioned from in-person provision to contact-less alternatives—counseling and treatment provided through telemedicine, and the mailing of injection supplies. Clients were differentially impacted depending on their access to smartphones, internet service, or a permanent address. Participants reported that people who did not have access to a phone and/or video calling, particularly those in rural areas, had difficulty accessing peer support groups. Similarly, lack of cell phone coverage and limited data plans were major barriers to low barrier telemedicine services in rural areas .Mobile units that distributed safe use equipment and naloxone increased SSP accessibility by bringing equipment closer to clients. A shift to telemedicine for MOUD treatment meant that patients with smartphones and access to Wi-Fi were able to more easily access treatment, without the need to physically travel to clinics . The cli“We have had a hard time getting alcohol wipes, still waters, even fresh cotton, sterile cottons have been on back order…” (CP 1)Community partners at SSPs reported experiencing both a lack of personal protective equipment and shortages of safe injection supplies:“[Do they have everything that you do need?] Yes and no. They don't really have a lot of the pipes and stuff right now. […] And when it runs out, it runs out. You never know what they're going to have or how much; it's all about the donations and the volunteers. Sometimes it's clunky, sometimes there's nothing.” (PWID 3)These issues limited what could be offered to clients:Some harm reduction suppliers also experienced staff shortages, especially earlier in the pandemic. Staff and volunteers were sometimes sick or did not want to risk exposure to the coronavirus. To minimize COVID-19 transmission, some on-site SSPs switched to pre-packaging equipment. However, this “one size fits all” change may have resulted in decreased availability of preferred supplies for some clients.“I've certainly have had a number of my patients seem to be doing worse in terms of their substance use, that they're admitting that they're using more as a way to cope with stress.” (Pr 3)In the midst of this restricted supply, providers reported increased demand as some patients were using more drugs during the pandemic, possibly to cope with stress:“People [are] either using substances that they're not familiar with or purchasing substances from somebody who they're not familiar with and getting something that they don't know what it is or they thought it was something else. So we're seeing a lot of that. We're seeing a lot of people…using recklessly because they don't know when they're going to find something else.” (CP 3)Increased substance use meant increased demand for unused equipment in the setting of reduced access. Service providers often expressed concern about unsafe use:“We have a supply distributor that's based out of Washington and certain things were just on back order and there was no getting around it. We're pretty much all getting together as the organizations. Like if one organization got 20 000 boxes of alcohol wipes, they were giving it out between the rest of us. […] Which gave all the organizations access, but limited access.” (CP 1)During national shortages of medical supplies, outreach and community collaborations were important factors in increasing availability of basic services. Service providers reported increased communication between organizations to try and meet the needs of their clients:Furthermore, service providers described leveraging connections among organizations to get access to naloxone and allow more distribution. SSP collaborations and sharing of equipment was thus an important facilitator that increased availability of safer use equipment for many SSP clients in rural areas. Relaxing rules and regulations around buprenorphine prescribing made low barrier buprenorphine treatment more available. These changes particularly allowed providers to prescribe buprenorphine via telemedicine at the first appointment, and thereby increased access .“The first time the cops closed us down, they closed us down saying that, we're not an essential service, you know what I mean? I mean it was a big hit just hearing that, because it would be an essential service if it was a bunch of privileged kids getting girl scout cookies at Walmart.” (CP 1)Some SSPs were instructed to close early in the pandemic, which reduced access to services.“… we only distribute once a week per town…I’ve had so many reports of people saying that they’re using supplies for money to get drugs because money’s harder to come by. And that leaves them without the safe using supply. And then, they end up sharing….” (CP 1).While several mobile SSPs re-opened during the pandemic, these services still had limited hours. According to some participants, the lack of optimal service access led to more unsafe injection practices:Limited hours reduced access to SSPs and other basic services , particu“We also kind of, on the fly, had to introduce a lot of COVID precautions that we really haven't been told to do…things like gloves, keeping people six feet apart from one another, asking folks not to touch the table and the stuff with it... And my coworker… even fashioned a $3 six-foot distance pole, a basket on the end of a six-foot pole.” (CP 2)Community resilience, particularly the commitment of walk-in clinics to remain open despite risks and challenges, allowed service providers to better accommodate clients. The resilience of peer support organizations also increased adequacy of services; community partners reported lack of clear guidance early in the pandemic, however they adapted quickly to COVID-19 social distancing policies.“It would be easier at the end of the day for me to get COVID. But honestly, if we’re not helping people when they really need it the most, what are we even doing?”( CP 1)Despite the risk of potential COVID-19 exposure, several peer services remained open through phone or video calls. Some participants reported some peer support meetings still taking place in person, despite COVID-19 social distancing policies. SSP outreach workers continued to deliver services, despite some limitations such as less face-to-face interactions and physical distancing outdoors. No clients of mobile SSPs in rural areas reported any issues with accessing unused equipment.Stricter adherence to the one-for-one needle exchange policy in the city of Portland reduced access to safer use equipment, as clients who appeared at the SSP without used needles to exchange were unable to acquire them. The enforcement of the one-for-one needle exchange policy also meant that some participants had to purchase equipment from pharmacies, which could also be cost prohibitive.Several PWID participants reported higher drug costs , as well as unemployment issues during the pandemic; if unable to afford travel costs to pharmacy or SSP, or if unable to afford equipment at pharmacies, financial challenges could affect their ability to access unused drug equipment and/or access low barrier treatment services.“The [SSP outreach workers] around here are pretty cool…they’ll give you as much as you need…unless they’re really, really short and they don’t have a lot of supplies that day, but usually they just say take as much as you want, as much as you need, give them to your friends, pass them out. They’re really…awesome.” (PWID 3)A key facilitator that increased affordability, i.e., how clients perceived services, was the elimination of a statewide policy requiring one-for-one needle exchange at SSPs. Most SSPs adopted the change, particularly those operating in non-urban parts of Maine, meaning that people could freely access safe supplies without the need to carry used and therefore “illegal” equipment. While some PWID expressed concerns that needles would not be disposed properly in special containers installed throughout the city, most reported positive impacts of the change:Relaxation of the one-for-one rule and restrictions on sites for SSPs also allowed mobile SSPs more flexibility to supply equipment throughout the state. Being able to access free equipment from mobile SSPs, also mitigated travel costs and equipment costs at pharmacies or other sites. In addition, PWID participants accessing treatment using video calls also reduced travel costs and therefore increased affordability of treatment services.“I think the city, the police in particular, and city hall could do a lot to lift some morale of people that are sleeping in the park and that get kicked out. We woke up at eight this morning to police kicking on our tent, kicking it and saying, "Hey, wake up. You guys got to move. You're not allowed to be in all of Portland with your tent." I mean, what the hell are we supposed to do? Move to the next town? I mean, I just sat down for the whole day, and now we feel hopeless, and alone, and even further down.” (PWID 6)Many participants reported that PWID lack safe housing or shelter options and experienced reduced access to a variety of public services. Changes in operating hours, available supplies and restrictive policies exacerbated the perceived loss of resources. Additionally, enforcement of public health policies throughout cities and towns that prevented people from congregating left many PWID expressing feelings of abandonment:In this study, we found that changes that occurred during the COVID-19 pandemic affected harm reduction services across many dimensions of access. Notably, we identified several facilitators and barriers to accessing harm reduction services during the COVID-19 pandemic that have policy and service delivery implications. Policies that resulted in the elimination of the one-for-one needle exchange, expansion of mobile SSP units, and permission of mail delivery of safer use equipment were particularly important in improving accessibility, availability, acceptability, and affordability of harm reduction services. On the other hand, modifiable policies, such as restrictions on drug, needle/syringe and other unused equipment possession, as well as the implementation of the one-for-one needle exchange policy, restricted harm reduction service availability, acceptability and affordability.Harm reduction service changes that occurred during this study were similar to recent studies; SSPs, for example, adapted quickly, though still faced service disruption , 40. SerOur results suggest that the one-for-one needle exchange policy, which reduced availability to and affordability of safer use equipment in this study, is a policy that should be permanently eliminated. Increased access to safer use equipment can improve injection practices and minimize adverse outcomes, such as injection drug-use associated infections , 43. NotIn our study, the combination of mail delivery of safer use equipment with expansion of mobile SSP units increased service access. These services are particularly important for rural areas where distance is a barrier to SSP use . Mail deWe found that allowing mobile units and mail delivery of equipment described above, as well as supporting policies that reduce stigma, such as drug decriminalization, may facilitate increased trust. Trust in community partners and providers and community resilience were also important facilitators to accessing harm reduction services during the pandemic. These findings are in line with prior work , 48. SevWe also identified several barriers to accessing harm reduction services that are modifiable from a policy perspective. At the time of the study, due to state laws restricting the possession of more than one needle/syringe, as well as possession of drug of small amounts of drug, several participants reported harassment and other adverse outcomes from law enforcement. Lack of regulated, safe drug supply and the presence of new/unknown drug sources was raised as a serious concern due to overdose risk . Given tThere were several limitations to our study. Due to social distancing restrictions around COVID-19, interviews were conducted over the phone. In-person interviews allow more opportunities to build rapport with participants, facilitating additional information. Since this was not a longitudinal study, we did not build a relationship/rapport with PWID first, therefore it is possible some information was withheld due to distrust. Our recruiting methods favored PWID who were accessing harm reduction services or congregating in areas where our flyers were posted. Thus, our findings may not be generalizable to PWID disengaged from community services. Our sample was limited to people who speak English; therefore, we are missing perspectives from non-English speaking PWID. Since Maine is a state with predominantly (94.4%) white people , race/etIn this study, we identified facilitators and barriers to harm reduction service access in a rural state. Advocating for relaxed policies; specifically elimination of the one-for-one exchange policy, and allowance of mobile units and mail delivery of unused equipment, can facilitate increased access to harm reduction services. The use of telemedicine, particularly in rural areas, can also facilitate access to low barrier treatment for some PWID. Finally, strengthening support for SSPs should be a priority; SSPs were not only integral in making unused equipment accessible to PWID so they could use drugs safely, but they were also key in providing non-judgmental, innovative services to a population that is often otherwise neglected by society.Changes resulting from the COVID-19 pandemic have impacted many dimensions of access to harm reduction services among people who use drugs. Our study adds to the existing literature by identifying facilitators and barriers to accessing an array of harm reduction services in a rural context. We have included the perspectives of people who inject drugs, community partners and providers, all of whom have valuable insight on how to improve access to harm reduction services. Our results can inform policies and service delivery to maximally mitigate the negative impacts on people who use drugs, particularly in rural areas during, and beyond, the pandemic.Additional file 1. Interview guides.Additional file 2. Additional study participant information.


Background: Myeloid cells are critical determinants of the sustained inflammation in Crohn’s Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. Methods: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14+ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. Results: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1β, IL6, and TNFα secretion from healthy donor CD14+ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14+ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. Conclusions: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients. Crohn’s disease (CD) is a complex immune-mediated disease presenting as chronic inflammation of the gastrointestinal tract . ImmunomBromodomain and extra terminal (BET) domain-containing proteins are a family of epigenetic readers  that bind acetylated lysine residues of histone proteins to allow for transcriptional complex formation and gene expression 8). Regarding regulation of the immune response, the BET proteins are essential for the transcription of several inflammation-related genes and have therefore been targets of interest in drug development for inflammatory diseases and cancer . Regardi. Small mEsterase sensitive motif (ESM) technology has previously been described to achieve cell specific accumulation of the active drug, targeting mononuclear myeloid cells based on the presence of carboxylesterase-1 (CES1) . This apDetailed information on the materials, methods, and associated references can be found in the GSK3361191 (ESM-iBET) and its non-hydrolysable control GSK3235220 (iBET) were provided by GlaxoSmithKline . GSK3361191 (ESM-iBET) is a BET inhibitor with an esterase sensitive motif (ESM), and GSK3235220 (iBET) is a pan BET-inhibitor. GSK3361191 (ESM-iBET) is similar in its mechanism of action compared to an earlier published compound, GSK3358699 [The following clinical samples were analyzed: intestinal biopsies of inflammatory bowel disease (IBD) patients (CD patients and ulcerative colitis patients), PBMCs of CD patients, and fistula tract tissue of fCD patients. Samples were obtained from the department of gastroenterology and/or surgery at the Amsterdam UMC, University of Amsterdam, under the approval of the accredited Medical Ethics Committee  or the biobank committee of the Amsterdam UMC (178 #A201470). Intestinal biopsies, PBMCs, and fistula samples were cryopreserved and handled according to the methodology published by Konnikova et al. . Detaile2O and supplemented with 10% v/v of EQ Four Element Calibration beads . After acquisition, the data were normalized, and individual files were deconvoluted using the CyTOF software v6.7 functions. Different lineages  were clustered using FlowSOM and subsequent manual annotation [Human clinical samples (see description above) were immunophenotyped using a CyTOF Helios mass cytometer. Staining, barcoding, data acquisition, and mass cytometry analysis are described in the supplementary methods. We made use of three different antibody panels: a biopsy panel, a PBMC panel, and a fistula panel found in . Acquisinotation . Data isnotation ,26.We obtained buffy coats  from Sanquin Blood Bank, Amsterdam, and isolated the peripheral blood mononuclear cells (PBMCs) according to standard Ficoll  density gradient centrifugation protocol . We furtFor the in vitro culture for cytokine analysis, the CD14+ monocytes or PBMCs were pre-treated for 1 h with a concentration range of 0.002, 0.01, 0.04, 0.156, 0.625, 2.5, and 10 µM of either GSK3361191 (ESM-iBET), non-hydrolysable control GSK3235220 (iBET), or DMSO. After 1 h, cells were washed to remove the extracellular compound, stimulated with 10 ng/mL LPS dissolved in RPMI medium , and incubated overnight. Supernatant was collected, and cytokine  analysis was performed using Cytometric Bead Array (CBA) . Intracellular TNFα protein expression was detected by flow cytometry analysis  and analyzed using FlowJo software . Cytokine data is visualized by normalizing the actual measured values to the DMSO control to correct for the biological variation in every individual donor. For in vitro culture for RNA transcriptomics analysis, CD14+ monocytes were pre-treated for 1 h with 40 nM of either GSK3361191 (ESM-iBET), non-hydrolysable control GSK3235220 (iBET), or DMSO. After 1 h, cells were washed and stimulated with 10 ng/mL LPS dissolved in RPMI medium  and incubated for 4 h. CD fistula samples were obtained from fistulizing CD patients undergoing surgery  at the Amsterdam UMC, location AMC. Fistula scrapings were mechanically digested by mashing and flushing through a 100 µm cell strainer  placed on a 50 mL tube , and immune cells were isolated using Ficoll isolation . Immune p-value < 0.05. Gene set enrichment analysis was conducted with the fgsea package (v1.20) [Transcriptomic analyses were performed through RNA sequencing. Briefly, mRNA was isolated using the Isolate RNA mini kit  and converted into cDNA. Subsequently, cDNA was sequenced in a 150 bp paired-ended fashion on the Illumina NovaSeq6000 to a depth of 40 million reads at the Amsterdam UMC Genomics Core Facility. The quality control of the reads was performed with FastQC (v0.11.8) and summarization through MultiQC (v1.0) . The raw (v1.20)  using th (v1.20) . Visuali (v1.20) .p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; SEM is the standard error of the mean.Statistical analysis was performed using GraphPad Prism 7.0 . Statistical testing was performed using a two-way ANOVA test; * In order to explore the potential application of ESM-conjugated molecules in the treatment of IBD, we examined CES1 expression in IBD clinical samples using mass cytometry analysis, with particular emphasis on the inflamed local tissue environment. First, we investigated the immune cell composition in intestinal biopsies collected from IBD patients during endoscopy. Biopsies were taken from six IBD patients and collected from inflamed areas (n = 6) or non-inflamed areas (n = 2). We were able to identify CD27− (naïve) and CD27+ (memory) B cells (CD45+CD45RA+HLA-DR+CD69+CD44+), CD4 T cells (CD45+CD3+CD69+ CD2+CD4+CD5+CD28+), CD8 T cells (CD45+CD3+CD8a+CD5+), mononuclear myeloid cells (CD45+CD14+CES1+HLA-DR+CD11a+CD44+CD11b+), epithelial cells (CD45−EpCAM+CD95+), and NK cells (CD45+CD45RA+CD161+CD2+CD7+) A,C. FurtNext, we aimed to define CES1-expressing immune cell populations in the peripheral blood of CD patients, determining whether CES1 expression, among identified populations, differs between biological therapy-responsive and non-responsive patients. Therefore, we isolated PBMCs from CD patients treated with the biological agent vedolizumab and analyzed them using CyTOF to define CES1 expression among identified immune cell populations. We identified CD4+ T cells, CD8+ T cells, NK cells, and B cells and extended the analysis of myeloid subsets to include classical monocytes (CD14+++CD16−), intermediate monocytes (CD14++CD16+), non-classical monocytes (CD14+CD16++), cDCs (CD16−CD14−HLADR++CD11a+ CD2+), and pDCs (CD16−CD14−HLADR+++CD45RA+ CD2++) A,C, althNext, we explored the immune cell composition within the highly inflamed tissue environment of the fistula tracts of CD patients, using a penetrating phenotype that underwent surgical intervention (n = 13). We analyzed cells retrieved from CD fistula tract curettage material using CyTOF and established the presence of different immune cell subsets, including basophils, B cells, CD4+ T cells, CD8+ T cells, NK cells, and mononuclear myeloid cell subsets A,C. CES1Next, we evaluated the anti-inflammatory efficacy of an ESM-iBET (GSK33611910) and compared this to its non-hydrolysable iBET control (GSK3235220) in LPS stimulated monocytes and PBMCs from healthy donors. Since ESM-iBET specifically accumulates in CES1-positive myeloid cells, we hypothesized a more potent immunosuppressive effect in the monocytes. PBMCs from healthy donors (n = 3) were treated with a concentration range (0.002–10 µM) of ESM-iBET or iBET, and TNFα expression from CD14-expressing cells was determined using flow cytometry A. The frNext, CD14+ monocytes from healthy donors (n = 3) were treated with a concentration range (0.002–10 µM) of ESM-iBET or iBET, and secreted inflammatory cytokines  were quantified with cytometric bead array (CBA). ESM-iBET demonstrated significantly increased potency to inhibit IL1β, IL6, and TNFα secretion when compared to the control iBET, with calculated IC50 of 9.6 nM vs. 257.4 nM (IL1β), 19.1 nM vs. 269.4 nM (IL6), and 14.8 nM vs. 145.6 nM (TNFα) for ESM-iBET or iBET, respectively C. Subsequently, we extended these observations to evaluate ESM-iBET (GSK33611910) or iBET (GSK3235220) anti-inflammatory activity in immune cells retrieved from inflamed fistula tract of CD patients. Ex vivo isolated immune cells were treated with a concentration range (0.002–10 µM) of ESM-iBET or iBET, and inflammatory cytokines , secreted overnight, were quantified. Both inhibitors efficiently reduced secreted inflammatory cytokines at relatively higher concentrations when compared to PBMC and monocyte cultures, with calculated IC50 of 441.3 nM vs. 1185 nM (IL1β), 280.3 nM vs. 358.3 nM (IL6), and 150.4 nM vs. 255.2 nM (TNFα) for ESM-iBET and iBET, respectively. There was no significant difference between the concentration of ESM-iBET and iBET required to inhibit inflammatory cytokine secretions from fCD ex vivo isolated immune cells D.In order to gain more insight into the transcriptome changes mediated by BET inhibition, we compared monocytes pre-treated with ESM-iBET or iBET to the DMSO control treatment. In line with earlier functional assays , we expeWe identified 253 significantly differentially expressed genes (DEGs), of which 163 were upregulated and 90 were downregulated. Interestingly, visualizing the top 20 upregulated or downregulated DEGs in response to BET inhibition suggested that while both BET inhibitors functioned concordantly, ESM-iBET conferred a stronger effect, as opposed to iBET, at equimolar level concentrations B, in linFurthermore, we performed gene set enrichment analysis (GSEA) with functional annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Multiple inflammation-related pathways were among the top significantly negatively enriched pathways in response to ESM-iBET pre-treatment D. We couNext, we aimed to explore the inflammation related cytokines and chemokines ligands and their receptors that are targeted by BET inhibition in the monocytes. The gene set enrichment analysis (GSEA) of the cytokine–cytokine receptor interaction pathway was negatively enriched in response to ESM-iBET treatment when compared to the DMSO-treated monocytes E. MultipNext, we explored whether ESM-iBET affects the transcription of inflammatory pathways and target genes of therapeutic interest in CD. TNFα and JAK-STAT signaling pathways are key players in CD pathogenesis and therefore, are of important therapeutic relevance . GSEA shDysregulated innate immunity plays a fundamental role in the sustained and recurring inflammation in CD. Through epigenetic mechanisms, BET proteins are essential for inflammatory gene expression . In the In intestinal biopsies, CES1 was mainly restricted to the CD14-expressing myeloid cells. An earlier study demonstrated a toxic effect of non-selective BET-inhibitors on intestinal epithelial cells (14) and a worsening of DSS-induced colitis in a mouse model upon BET-inhibition (13), suggesting the toxicity of non-selective BET-inhibition on intestinal epithelial cells. In this context, we demonstrated that the majority of EpCAM+ epithelial cells did not express CES1, which is of particular interest here for the application of ESM-iBET in inflammatory intestinal diseases by sparing intestinal epithelial cells, while targeting inflammatory mononuclear myeloid cells. However, a small fraction of CES1-expressing epithelial cells (median of 8.5%) is noted, which may require further investigation to determine whether this reflects a specific epithelial cell subset and whether it may have a detrimental effect on CES1-assisted drug delivery applications in IBD.In CD peripheral blood PBMCs, CES1 expression is confined to the myeloid compartment, including classical monocytes (CD14+++CD16−), intermediate monocytes (CD14++CD16+), non-classical monocytes (CD14+CD16++), cDCs (CD16−CD14−HLADR++CD11a+CD2+), and pDCs (CD16−CD14−HLADR+++CD45RA+CD2++), similar to the expression pattern demonstrated earlier in healthy donor blood (20). Interestingly, we observe the highest CES1 expression among circulating non-classical (CD14+CD16++) monocytes from CD patients, in contrast to healthy donors, where CES1 expression was the highest among classical monocytes (CD14+++CD16−) (20). This may potentially be reflective of a preferential trafficking of CES1-expressing classical monocytes to the locally inflamed colon in CD patients, as classical monocytes are recruited to fuel the inflammatory process ,49. The Using a non-hydrolysable iBET control (GSK3235220), we demonstrated a higher potency of ESM-iBET (GSK3361191) to inhibit inflammatory cytokines in CES1-expressing monocytes in both PBMCs or purified CD14+ monocyte culture compared to equimolar concentrations of iBET (GSK3235220). This validates the specific CES1 assisted delivery of ESM-iBET and the augmented anti-inflammatory effect. However, despite the inhibition of inflammatory cytokine secretion from ex vivo immune cells retrieved from CD fistula tracts by ESM-iBET, no differential efficacy over similar concentrations of iBET was observed. This can be explained by the presence of other immune cells, such as T cells and B cells mixed with a low yield of CES1-expressing myeloid cells, within these cell preparations compared to PBMCs and purified monocytes; therefore, the contribution of ESM-iBET targeted CES1-expressing cells to overall secreted cytokines is minimal to demonstrate an observed difference. The transcriptomic analysis of ESM-iBET (GSK3361191)-treated monocytes demonstrates a potent inhibitory effect on multiple inflammation related genes and pathways. This is in line with earlier reports using other iBET in human primary monocytes , human aThe monocyte transcriptional analysis detailed herein demonstrates that ESM-iBET can efficiently target key components of multiple inflammatory pathways involved in the pathophysiology of CD. Within the JAK-STAT signaling pathway, ESM-iBET specifically downregulated STAT5A gene expression, in line with earlier reports ,61,62, wOverall, the monocyte transcriptomic analysis demonstrates a potent effect on multiple pathways of potential therapeutic relevance to CD by ESM-iBET. Current biologic therapies are aimed at targeting specific cytokines or pathway components, which can only be beneficial to patients in which this particular pathway is predominantly driving inflammation. The advantage of a CES1-targeted iBET (ESM-iBET) is that it can interfere with multiple CD-relevant inflammatory pathways simultaneously in monocyte/myeloid cells expressing CES1, while minimizing broad iBET effects in non-CES1 expressing cells. Whether specific targeting of mononuclear myeloid cells would demonstrate clinical benefits remains uncertain. In a complex in vivo environment, other cell types, such as intestinal B cells, T cells, epithelial, and stromal cells, all contribute to intestinal inflammation. We demonstrated a specific pattern of CES1 expression in CD patients, which is confined to monocytes, macrophages, and DC populations, across blood and local inflamed tissues of CD patients. We demonstrated the increased potency of ESM-iBET (GSK3361191) in CES1-expressing monocytes compared to the non-targeted control, iBET (GSK3235220). We also defined ESM-iBET targets at the transcriptional level in the peripheral monocytes, which are therapeutically relevant in CD patients.


Determining accurate estimates for the characteristics of the severe acute respiratory syndrome coronavirus 2 in the upper and lower respiratory tracts, by fitting mathematical models to data, is made difficult by the lack of measurements early in the infection. To determine the sensitivity of the parameter estimates to the noise in the data, we developed a novel two-patch within-host mathematical model that considered the infection of both respiratory tracts and assumed that the viral load in the lower respiratory tract decays in a density dependent manner and investigated its ability to match population level data. We proposed several approaches that can improve practical identifiability of parameters, including an optimal experimental approach, and found that availability of viral data early in the infection is of essence for improving the accuracy of the estimates. Our findings can be useful for designing interventions. Similarly, understanding the kinetics of SARS-CoV-2 in the lower respiratory tract (LRT) is important for predicting the potential for severe disease, respiratory failure, and/or death13. Insights into the mechanism of SARS-CoV-2-host interactions and their role in transmission and disease have been found using mathematical models applied to longitudinal data17. While these studies are instrumental in determining important parameters , their predictions are limited by the lack of data early in the infection. As such, with few (if any) samples available before viral titers peak, the early virus kinetics and the mechanisms for these early kinetics are uncertain. In this study, we investigate the sensitivity of the predicted outcomes of a within-host model of SARS-CoV-2 infection to the availability of data during different stages of the infection and use our findings to make recommendation.Understanding the upper respiratory tract (URT) kinetics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for designing public health interventions such as testing, isolation, quarantine, and drug therapies18. The study showed independent virus replication in upper and lower respiratory tracts19 suggesting the possibility that virus kinetics, disease stages, and host involvement in control and pathogenesis are dependent on which area of the respiratory tract is homing SARS-CoV-2 at different stages of the disease22. One shortcoming when evaluating the data in this study comes from the fact that viral RNA was collected only after the patients became symptomatic, with an estimated first data point available on average 5–7 days after infection. Several within-host mathematical models developed and applied to the data set in the Wolfel et al. study have evaluated SARS-CoV-2 parameters, determined the role of innate immune responses, found connections between total RNA and infectious titers, and identified the efficacy of drug therapies7. We are interested in determining how the lack of data early in the infection affects these estimates.A German study by Wolfel et al. collected data from nine patients infected early in the pandemic through contact with the same index case26 that includes both URT and LRT patches. We used the data from Wolfel et al. to estimate pertinent parameters and investigated the sensitivity of the estimated parameters to the presence of data at different stages of infection. To accomplish this, we created virtual data sets that span various stages of the infection and determined how our initial predictions are being influenced by the additional data. Such results may influence our understanding of both viral expansion and the effect of inoculum dose on disease progression.We first developed our own within-host model that does not consider innate immunity explicitly. The model is an expansion of the within-host mathematical models developed for influenza and other respiratory infections18. We model this by developing a two patch within-host model, where the patches are the two respiratory tracts which are linked through virus migration between patches, or viral shedding. Both respiratory tract patches assume interactions between uninfected epithelial cells, j, t. Here, u describing the URT patch and l describing the LRT patch. Target cells in each patch get infected at rates K is the viral load in the LRT where the clearance is half maximal. The two patches are linked via the virus populations, with a proportion SARS-CoV-2 virus infects and replicates in epithelial cells of the upper and lower respiratory tract18.We model the initial conditions of the system Eq.  as follomentclass2pt{minim19. Early detection allowed for rapid contact tracing, testing, and monitoring of the affected community: young healthy professionals in their mid-thirties. A followup study published time series for the post symptoms virus data isolated from oral-and nasopharyngeal throat swabs (in copies per swabs) and from sputum samples (in RNA copies per mL) for the same patient population over their entire course of disease. The patients’ throat swabs and sputum data  were obtained through personal communication with the authors. Since we know the incubation period for each patient19 ,,\documenmentclass2pt{minimSuppose put Eqs.  and 4)  \documenput Eqs.  and (4).mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimThe within-host model Eq. , and of conducting airways . Since the gas exchange region is affected by SARS-Cov-2 only in severe cases20 we ignore it, and restrict the LRT compartment to the conducting airways whose surface area is 36. Therefore, we obtain an initial epithelial cell target population in the LRT of 18. Lastly, the incubation periods were estimated in Ref.19 and are listed in Table We assume that the upper respiratory tract susceptible population is mentclass2pt{minimDuring the data collection process, observations are perturbed with noise. Hence, the URT and LRT viral load deviates from the smooth trajectory of the observations 38. MCMC methods generate a sequence of random samples N. The random walk Metropolis algorithm is one of the most extensively used MCMC algorithms. The Metropolis algorithm starts at position 37) and use the MATLAB toolbox provided by the same authors39. In comparison to other Metropolis algorithms, the Markov chain constructed with DRAM is robust and converges rapidly  to determine the remaining nine parameters of the model Eq. \documentodel Eq.  and the odel Eq. , the likentclass1pt{minimaThe two patch within-host model Eq.  is novelentclass1pt{minima18. We used Bayesian parameter estimation with the viral samples in URT and LRT from a single individual (patient A) and the entire population  and approximated posterior distributions with To study the kinetics of SARS-CoV-2 in the upper and lower respiratory tracts we developed a two patch within-host model Eq.  that ass}pl see “”. The re}pl see “ was valiWe generated prediction graphs of the within-host model Eq.  by samplK where While the viral titers decay to low levels  and the entire population, During MCMC data fitting, we used parameters limits predetermined to range around a single point estimation obtained using the ’fminsearch’ algorithm in Matlab  restrictions on the parameter space and the types of distributions we are imposing on the parameters, or (2) the limited data points early in the infection.To investigate the first hypothesis, we collected samples in the parameter space ofentclass1pt{minimaTo investigate the second hypothesis, we created synthetic data and used it to further examine how the timing of the data collection in the population correlates to the structure of the resulting parameter estimations. We assumed that the real data corresponds to the solution of model Eq.  with parentclass1pt{minimaTo determine whether practical identifiability is lost in each experiment we created parameter histograms for each parameters see Fig. . When da18. We estimated nine unknown parameters using an MCMC Bayesian fitting approach37. To avoid over fitting, we determined best estimates in a single patient (for which we have 26 data points) and in the entire population (for which we have 201 data points). We found shorter virus life-spans in LRT compared to viral URT, 2–3 h compared to 5.7–8.5 h. Our LRT estimates are similar to the fixed (and non-tract specific) virus life-span of 2.4 h used by Ke et al.3 and the estimated (and tract specific) life-span of 1.2 h in Wang et al.6, but longer than the 10 h seen in influenza and used by Hernandez et al.7. The between tracts differences may suggest the presence of additional immune mediated viral clearance in the LRT. We found similar infected cells life-span between the two tracts, with a range of 4.2–8 h, shorter than in other studies7. Lastly, the mean URT basic reproductive number for the entire population, 3. While we assumed two-way viral shedding between tracts, data fitting suggested higher virus shedding from upper and lower respiratory tracts than the other way around, consistent with other studies3.In this study, we developed a within-host mathematical model of SARS-CoV-2 infection that connected the virus kinetics in the upper and lower respiratory tracts of infected individuals and used it to determine the tract specific viral parameters. We removed viral production rates, to ensure structural identifiability, and fitted the rescaled model Eq.  to publiInterestingly, we found that the estimated LRT infectivity rate parameter follows a bimodal distribution when the model was fitted to the entire population data. We attributed this behavior to practical non-identifiability. Practical non-identifiability is observed when the measured data is contaminated with noise. We have inherently accounted for noisy data by combining viral measurements from nine patients with different viral profiles. We investigated several ways for improving practical identifiability of this parameter and found that both estimating the logaritmic value 43. Such a process, known as optimal experimental design, aims to obtain additional information about a system through the addition of new measurements. Since in system Eq. . This simplification may be the leading reason for larger infected cell death rate estimates in our study compared to other studies7. Thirdly, due to the novelty of the model, we have no information on parameter priors. Therefore, we fitted the within-host model to the patient A and population data, and used those estimates as means in the prior distributions. However, since the resulting means fall within ranges observed for other acute infections48, and since we consider large standard deviations around the prior means, we are confident that we are covering a large enough search space that does not exclude viable outcomes.Our study has several limitations. First, we considered a density dependent clearance term for the URT virus that saturates at around 1–2 In conclusion, we have developed a within-host model of SARS-CoV-2 infection in the upper and lower respiratory tracts, used it to determine pertinent viral parameters, and suggested the optimal experimental designs that can help improve the model predictions. These techniques may inform interventions.Supplementary Information.


Shark depredation is a complex social-ecological issue that affects a range of fisheries worldwide. Increasing concern about the impacts of shark depredation, and how it intersects with the broader context of fisheries management, has driven recent research in this area, especially in Australia and the United States. This review synthesises these recent advances and provides strategic guidance for researchers aiming to characterise the occurrence of depredation, identify the shark species responsible, and test deterrent and management approaches to reduce its impacts. Specifically, the review covers the application of social science approaches, as well as advances in video camera and genetic methods for identifying depredating species. The practicalities and considerations for testing magnetic, electrical, and acoustic deterrent devices are discussed in light of recent research. Key concepts for the management of shark depredation are reviewed, with recommendations made to guide future research and policy development. Specific management responses to address shark depredation are lacking, and this review emphasizes that a “silver bullet” approach for mitigating depredation does not yet exist. Rather, future efforts to manage shark depredation must rely on a diverse range of integrated approaches involving those in the fishery , social scientists, educators, and other stakeholders. Depredation, where a predator  completely or partially consumes an animal caught by fishing gear, is an issue in many fisheries around the world and in recent years has received increasing attention from researchers and fishery managers  or interviews  represent the most popular social science strategies to understand shark depredation impacts, including rates of depredation and fishers’ attitudes, perceptions, and behaviours  are behavioural solutions , to more complex examinations of the themes, values, and meanings of the content. This methodology has been used to examine dominant narratives and attitudes in the media reporting on shark bites  to represent individual or group “mental models” of a complex system, such as depredation Fig. . Fuzzy-cDepredation is influenced by a range of factors, including shark behaviour and abundance, spatial and temporal distribution of fishing effort and changes in fishing methods, gear and equipment. However, there remains a lack of empirical data to characterise these issues, and consequently much of the current knowledge is composed of anecdotal accounts and personal opinions. Biological data collection can help to address key questions relating to depredation, such as the impacts of shark depredation on target species, which shark species are involved and how their movement patterns and behaviour influence the occurrence of depredation. Such information can then inform future work to design effective mitigation approaches.Shark depredation can have substantial impacts on the target species of a diverse range of fisheries. In particular, shark depredation can increase the overall level of mortality that occurs, especially in fisheries where fishers are seeking to reach an allowed quota  or bag limit . Depredation of target species in catch-and-release recreational fisheries causes mortality for released fish that would otherwise mostly survive (thus undermining the catch-and-release objective), as well as diminishing the recreational fishing experience and negating the benefits of tagging programs.Scomberomorus commerson) in a commercial trolling fishery in Australia. Two studies in northwest Western Australia presented results for shark depredation rates for common recreational target species, including narrow-barred Spanish mackerel, spangled emperor (Lethrinus nebulosus) and coral trout (Plectropomus spp.)  in the Florida Keys, USA  in the Bahamas (Cooke and Phillip Lutjanus campechanus) is another key target species known to be affected by shark depredation in the southeast USA  were species not often perceived as predators, such as those from the families Aetobatidae and Rhinidae, and that there are a variety of species that simply interact with bait rather than depredating catches  are commonly used to assess depredation. Video footage collected using GoPro™ cameras mounted to crab traps showed that the primary species responsible for depredation of spanner crabs  were depredating and interacting with hooked fish on a longline  to view depredation could also be trialled to see if the combination of greater manoeuvrability and a live camera feed is able to allow additional data to be collected.The use of genetic techniques for the identification of predator species responsible for depredation continues to grow, and shows increasing promise, with several research papers successfully using genetic approaches to identify depredating shark species from trace DNA collected off the remains of depredated species  was achieved by Fotedar et al.  using a Variation in the time taken to swab depredated remains is also believed to affect the success rate of identifying depredating species. Vardon et al.  found thCarcharhinus spp., one of the most common genera responsible for depredation events studied to date  regions, such as COI, ND2, ND4 and CtyB, which provide additional benefits when working with trace DNA due to the multi-copy nature of mtDNA. Several studies have also highlighted the importance of using primers that are specifically designed to amplify DNA of depredating species and block DNA of the species being depredated . Furthermore, the ability to swab residual DNA of different tissue types (e.g. hard exoskeletons of crustaceans or molluscs) remains untested and could be either more or less suitable than swabbing of fish soft tissue. In some circumstances, examining the bite wound on a depredated fish or invertebrate could be used to distinguish between broader groups of depredating taxa  , barium ferrite (BaFe12O19), and neodymium ferrite (NdFeB) have had varied success as shark deterrents and are not always effective, particularly when there is competition among sharks during feeding  calls. While these have been shown to be an effective shark deterrent  the level of statistical power required, (2) the current base-level of shark depredation , (3) the level of decrease in depredation that is required for a deterrent to be considered “effective”, and 4) the change in effectiveness over time since the initial exposure (i.e. whether habituation occurs in the sharks). When planning and carrying out field sampling it is necessary to consider: (1) proximity of sampling sites to one another, (2) adequate numbers of catchable fish and sharks are present, (3) duration of fishing session to enable fish to be caught and sharks to be encountered, (4) randomisation of the order in which a control and each deterrent device are tested while fishing, (5) maintaining the same number of fishing lines in the water throughout the sampling, and (6) maintaining consistency in the fishing equipment/hardware  used throughout the sampling  in Florida  and are thus still subject to depredation. The only study that has investigated this aspect indicated that fish are far less likely to be depredated on descender devices , a known depredating species . Behaviour change may also result in indirect-economic costs, such as reduced enjoyment and reduced catches.Thunnus spp.) and yellowtail kingfish , compared to demersal species . Yet, there are widespread anecdotal reports that shark populations have increased as fishers report seeing many more sharks around their vessels than in the past. While it is possible that some local populations of sharks have increased, particularly species with more productive life history traits and relatively high site fidelity (Pardo et al. The issue represents an extremely challenging space for fishery managers, decision-makers, and politicians. Many stakeholders believe shark depredation is increasing due to increasing shark populations, particularly after the closure of some shark fisheries in the US and Australia. However, there is minimal long-term scientific data to investigate changes in abundance of sharks and there is no clear evidence to link changes in relative abundance of shark to increases in depredation rates. Studies from Australia (Braccini et al. It is also important to disentangle reported changes in relative abundance from the potential influence of changes in shark behaviour when investigating reports of increased depredation. In some areas there has been an increase in recreational fishing activity, such as in remote parts of Western Australia in recent years due to the COVID-19 pandemic (Ryan et al. It is necessary to consider how to manage stakeholders impacted by shark depredation scenarios, as well as the sharks. In several other HWC scenarios, the focus has shifted from one focused solely on the predator responsible, such as where the predominant management approach is to cull or catch and relocate the predator (Reynolds and Tapper Regardless of the methods used to mitigate depredation, it is highly unlikely that this HWC will be eliminated completely (Lennox et al. In the future, we can apply lessons learned from similarly contentious HWCs in the terrestrial realm. Recent meta-analyses indicate that reactionary predator removal programs in terrestrial ecosystems are rarely successful in reducing livestock depredation by apex predators (Eklund et al.