AxoDetect: an automated nerve image segmentation and quantification workflow for computational nerve modeling.

Objective.Bioelectronic treatments targeting near-organ innervation have unprecedented clinical applications. Particularly in the spleen, the inhibition of the cholinergic inflammatory response by near-organ nerve stimulation has potential to replace pharmacological treatments in chronic and autoimmune diseases. A caveat is that the optimization of therapeutic stimulation parameters relies onin vivoexperimentation, which becomes challenging due to the small nerve diameters (2 µm), complex anatomy, and mixed axon type composition of the autonomic nerves. Effective development ofin silicomodels requires tools which allow for fast and efficient quantification of axonal composition of specific nerves. Current approaches to generate such information rely on manual image segmentation and quantification.Approach.We developed a combined image-segmentation and model-generation software called AxoDetect: a target- and format-agnostic computer vision algorithm which can segment myelin, endo/epineurium, and both myelinated and unmyelinated fibers from a nerve image without training.Main results.AxoDetect is over 10 times faster on average when compared with current automatic methods while maintaining flexibility through the use of tunable pixel threshold filters to detect different types of tissue. When compared to a distribution-based and a manually segmented model of the splenic nerve terminal branch 1, the model generated with AxoDetect had comparable threshold prediction and was able to accurately detect an increase in activation threshold caused by the addition of surrounding fat tissue to the modeled nerve.Significance.AxoDetect contributes to the acceleration of neuromodulation treatment development through faster model design and iteration without requiring training. Furthermore, the computer vision approach and tunable nature of the filters in our method allow for its use in a variety of histological applications. Our approach will impact not only the study of nerves but also the design of implantable neural interfaces to enhance bioelectronic therapeutic options.

Bioelectronic Medicine: a multidisciplinary roadmap from biophysics to precision therapies.

Bioelectronic Medicine stands as an emerging field that rapidly evolves and offers distinctive clinical benefits, alongside unique challenges. It consists of the modulation of the nervous system by precise delivery of electrical current for the treatment of clinical conditions, such as post-stroke movement recovery or drug-resistant disorders. The unquestionable clinical impact of Bioelectronic Medicine is underscored by the successful translation to humans in the last decades, and the long list of preclinical studies. Given the emergency of accelerating the progress in new neuromodulation treatments (i.e., drug-resistant hypertension, autoimmune and degenerative diseases), collaboration between multiple fields is imperative. This work intends to foster multidisciplinary work and bring together different fields to provide the fundamental basis underlying Bioelectronic Medicine. In this review we will go from the biophysics of the cell membrane, which we consider the inner core of neuromodulation, to patient care. We will discuss the recently discovered mechanism of neurotransmission switching and how it will impact neuromodulation design, and we will provide an update on neuronal and glial basis in health and disease. The advances in biomedical technology have facilitated the collection of large amounts of data, thereby introducing new challenges in data analysis. We will discuss the current approaches and challenges in high throughput data analysis, encompassing big data, networks, artificial intelligence, and internet of things. Emphasis will be placed on understanding the electrochemical properties of neural interfaces, along with the integration of biocompatible and reliable materials and compliance with biomedical regulations for translational applications. Preclinical validation is foundational to the translational process, and we will discuss the critical aspects of such animal studies. Finally, we will focus on the patient point-of-care and challenges in neuromodulation as the ultimate goal of bioelectronic medicine. This review is a call to scientists from different fields to work together with a common endeavor: accelerate the decoding and modulation of the nervous system in a new era of therapeutic possibilities.

Trends in Parkinson's mortality in Mexico 2000-2020.

OBJECTIVE: To describe the recent trends in Parkinson's disease mortality in Mexico during 2000-2020. METHOD: The adjusted mortality rate per 100,000 inhabitants was calculated using the direct method and the world standard population. Trend analysis was performed with the Joinpoint software. RESULTS: The average mortality rate was 1.26/100,000 inhabitants (SD: 0.09), and males showed higher mortality than females (M/F ratio=1.60). Older individuals ≥70 years old showed higher mortality rates than the rest of the age groups. During the period of study, a significant increase in mortality was observed from 2000 to 2005, while from 2005 to 2020 no significant trend was observed in all the studied groups. CONCLUSIONS: In Mexico, males and older individuals showed the highest mortality rates. The socioeconomic regions with high levels of wellness showed the highest mortality rates levels. Parkinson's mortality rate has remained constant since 2005 in Mexico.

Biomechanics Characterization of Autonomic and Somatic Nerves by High Dynamic Closed-Loop MEMS force sensing.

The biomechanics of peripheral nerves are determined by the blood-nerve barrier (BNB), together with the epineural barrier, extracellular matrix, and axonal composition, which maintain structural and functional stability. These elements are often ignored in the fabrication of penetrating devices, and the implant process is traumatic due to the mechanical distress, compromising the function of neuroprosthesis for sensory-motor restoration in amputees. Miniaturization of penetrating interfaces offers the unique opportunity of decoding individual nerve fibers associated to specific functions, however, a main issue for their implant is the lack of high-precision standardization of insertion forces. Current automatized electromechanical force sensors are available; however, their sensitivity and range amplitude are limited (i.e. mN), and have been tested only in-vitro. We previously developed a high-precision bi-directional micro-electromechanical force sensor, with a closed-loop mechanism (MEMS-CLFS), that while measuring with high-precision (-211.7µN to 211.5µN with a resolution of 4.74nN), can be used in alive animal. Our technology has an on-chip electrothermal displacement sensor with a shuttle beam displacement amplification mechanism, for large range and high-frequency resolution (dynamic range of 92.9 dB), which eliminates the adverse effect of flexural nonlinearity measurements, observed with other systems, and reduces the mechanical impact on delicate biological tissue. In this work, we use the MEMS-CLFS for in-vivo bidirectional measurement of biomechanics in somatic and autonomic nerves. Furthermore we define the mechanical implications of irrigation and collagen VI in the BNB, which is different for both autonomic and somatic nerves (~ 8.5-8.6 fold density of collagen VI and vasculature CD31+ in the VN vs ScN). This study allowed us to create a mathematical approach to predict insertion forces. Our data highlights the necessity of nerve-customization forces to prevent injury when implanting interfaces, and describes a high precision MEMS technology and mathematical model for their measurements.

Exploring High-Throughput Immunoassays for Biomarker Validation in Rheumatic Diseases in the Context of the Human Proteome Project.

Rheumatic diseases are high prevalence pathologies with different etiology and evolution and low sensitivity in clinical diagnosis. Therefore, it is necessary to develop an early diagnosis method which allows personalized treatment, depending on the specific pathology. The biology/disease initiative, at Human Proteome Project, is an integrative approach to identify relevant proteins in the human proteome associated with pathologies. A previously reported literature data mining analysis, which identified proteins related to osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PSA) was used to establish a systematic prioritization of potential biomarkers candidates for further evaluation by functional proteomics studies. The aim was to study the protein profile of serum samples from patients with rheumatic diseases such as OA, RA, and PSA. To achieve this goal, customized antibody microarrays (containing 151 antibodies targeting 121 specific proteins) were used to identify biomarkers related to early and specific diagnosis in a screening of 960 serum samples (nondepleted) (OA, n = 480; RA, n = 192; PSA, n = 288). This functional proteomics screening has allowed the determination of a panel (30 serum proteins) as potential biomarkers for these rheumatic diseases, displaying receiver operating characteristics curves with area under the curve values of 80-90%.

Frontal aslant tract: Anatomy and tractography description in the Mexican population.

Background: The aim of the study was to describe the origin, course, and termination of frontal aslant tract (FAT) in the Mexican population of neurosurgical referral centers. Methods: From January 2018 to May 2019, we analyzed 50 magnetic resonance imaging (MRI) studies in diffusion tensor imaging sequences of patients of the National Institute of Neurology and Neurosurgery "Manuel Velasco Suárez." Five brains were fixed by the Klingler method and dissected in the neurosurgery laboratory of the Hospital Civil de Guadalajara to identify the origin, trajectory, and ending of the FAT. Results: FAT was identified in 100% of the MRI and brain dissections. The origin of the FAT was observed in 63% from the supplementary premotor area, 24% from the supplementary motor area, and 13% in both areas. Its ending was observed in the pars opercularis in 81%, pars triangularis in 9%, and in both pars opercularis and ventral premotor area in 10% in the magnetic resonance images, with a left side predominance. In the hemispheres dissections, the origin of FAT was identified in 60% from the supplementary premotor area, 20% from the supplementary motor area, and 20% in both areas. Its ending was observed in the pars opercularis in 80% and the pars triangularis in 20%. It was not identified as an individual fascicle connected with the contralateral FAT. Conclusion: In the Mexican population, FAT has a left predominance; it is originated more frequently in the supplementary premotor area, passes dorsal to the superior longitudinal fascicle II and the superior periinsular sulcus, and ends more commonly in the pars opercularis.

Renal Nerve Activity and Arterial Depressor Responses Induced by Neuromodulation of the Deep Peroneal Nerve in Spontaneously Hypertensive Rats.

Hypertension is a main cause of death in the United States with more than 103 million adults affected. While pharmacological treatments are effective, blood pressure (BP) remains uncontrolled in 50-60% of resistant hypertensive subjects. Using a custom-wired miniature electrode, we previously reported that deep peroneal nerve stimulation (DPNS) elicited acute cardiovascular depressor responses in anesthetized spontaneously hypertensive rats (SHRs). Here, we further study this effect by implementing a wireless system and exploring different stimulation parameters to achieve a maximum depressor response. Our results indicate that DPNS consistently induces a reduction in BP and suggests that renal sympathetic nerve activity (RSNA) is altered by this bioelectronic treatment. To test the acute effect of DPNS in awake animals, we developed a novel miniaturized wireless microchannel electrode (w-µCE), with a Z-shaped microchannel through which the target nerves slide and lock into the recording/stimulation chamber. Animals implanted with w-µCE and BP telemetry systems for 3 weeks showed an average BP of 150 ± 14 mmHg, which was reduced significantly by an active DPNS session to 135 ± 8 mmHg (p < 0.04), but not in sham-treated animals. The depressor response in animals with an active w-µCE was progressively returned to baseline levels 14 min later (164 ± 26 mmHg). This depressor response was confirmed in restrained fully awake animals that received DPNS for 10 days, where tail-cuff BP measurements showed that systolic BP in SHR lowered 10% at 1 h and 16% 2 h after the DPNS when compared to the post-implantation baseline. Together, these results support the use of DPN neuromodulation as a possible strategy to lower BP in drug-resistant hypertension.

CD34+CD19-CD22+ B-cell progenitors may underlie phenotypic escape in patients treated with CD19-directed therapies.

CD19-directed immunotherapies have revolutionized the treatment of advanced B-cell acute lymphoblastic leukemia (B-ALL). Despite initial impressive rates of complete remission (CR) many patients ultimately relapse. Patients with B-ALL successfully treated with CD19-directed T cells eventually relapse, which, coupled with the early onset of CD22 expression during B-cell development, suggests that preexisting CD34+CD22+CD19- (pre)-leukemic cells represent an "early progenitor origin-related" mechanism underlying phenotypic escape to CD19-directed immunotherapies. We demonstrate that CD22 expression precedes CD19 expression during B-cell development. CD34+CD19-CD22+ cells are found in diagnostic and relapsed bone marrow samples of ∼70% of patients with B-ALL, and their frequency increases twofold in patients with B-ALL in CR after CD19 CAR T-cell therapy. The median of CD34+CD19-CD22+ cells before treatment was threefold higher in patients in whom B-ALL relapsed after CD19-directed immunotherapy (median follow-up, 24 months). Fluorescence in situ hybridization analysis in flow-sorted cell populations and xenograft modeling revealed that CD34+CD19-CD22+ cells harbor the genetic abnormalities present at diagnosis and initiate leukemogenesis in vivo. Our data suggest that preleukemic CD34+CD19-CD22+ progenitors underlie phenotypic escape after CD19-directed immunotherapies and reinforce ongoing clinical studies aimed at CD19/CD22 dual targeting as a strategy for reducing CD19- relapses. The implementation of CD34/CD19/CD22 immunophenotyping in clinical laboratories for initial diagnosis and subsequent monitoring of patients with B-ALL during CD19-targeted therapy is encouraged.

Novel CHRDL1 mutation causing X-linked megalocornea in a family with mild anterior segment manifestations in carrier females.

PURPOSE: X-linked megalocornea (XMC) is a rare anterior segment malformation characterized by a nonprogressive enlargement of the cornea to 13 mm or greater in the setting of normal intraocular pressure. XMC is caused by mutations in the CHRDL1 gene and it is inherited as an X-linked recessive trait affecting only males. Here, we describe the results of phenotypic and genetic assessment in a novel XMC pedigree. METHODS: Three subjects (a father and his two daughters) underwent a complete clinical and imaging ocular examination including biomicroscopy, fundoscopy, tonometry, visual acuity, Pentacam Scheimpflug imaging, anterior segment Swept Source OCT, and ultrabiomicroscopy. Genetic analysis was performed through whole exome sequencing in 3 family members. Candidate variants were validated by sanger sequencing. RESULTS: The affected father exhibited megalocornea, very deep anterior chambers, retrocorneal pigmentation, iris atrophy, queer iris configuration, extremely open iridocorneal angles, and cataracts. Notably, both daughters showed queer iris configuration and abnormally widely open iridocorneal angles in both eyes. Genetic analysis identified a novel hemizygous c.207+1G>A splicing variant in CHRDL1 in the affected father. Both mildly affected daughters were heterozygous for the pathogenic variant. CONCLUSIONS: Here, we report an additional XMC family due to a novel mutation in the CHRDL1 gene. Mild anterior segment anomalies were observed in two heterozygous carriers demonstrating for the first time a CHRDL1-linked phenotype in females. A detailed comparison of the clinical and genetic features of this pedigree with those observed in previously published XMC cases is also presented.

Health-related quality of life and multidimensional fitness profile in polio survivors.

PURPOSE: To assess and compare the HRQoL and physical fitness of polio survivors with healthy individuals. METHODS: Thirty-seven polio survivors and 40 healthy individuals were recruited in this cross-sectional study. The Fatigue Severity Scale (FSS) was used to evaluate participant's level of fatigue; Short Form 36 (SF-36) was used for evaluating HRQoL, and hand-grip strength, lumbar trunk muscle endurance, flexibility, balance, and aerobic endurance were used as measures for physical fitness. RESULTS: Polio survivors had lower HRQoL in most of the dimensions of the SF-36, and they showed a lower level of physical fitness than controls. In particular, mobility-related variables were 20-40% lower in people with PP. CONCLUSIONS: Subjects with PP had lower scores for the physical component of the HRQoL questionnaire, reduced physical fitness, increased fatigue, less mobility, and a higher fall risk than controls. The outcomes of the study can be useful to the design of future programs tailored specifically to improve the assessment of the physical fitness of subjects with paralytic polio and to facilitate interventions based on appropriate physical exercise regimens.Implications for rehabilitationPost-polio syndrome is a disabling disease that impacts in fitness, physical and psychological health-related quality of life of polio survivors.Polio survivors should undergo physical activity programs that focus on improving mainly their mobility and physical functioning (walking, self-care, and climbing stairs), thereby reducing fall risk and fatigue.Individual tailored physical exercise programs should be promoted in order to improve HRQoL in this population.

Platinized graphene fiber electrodes uncover direct spleen-vagus communication.

Neural interfacing nerve fascicles along the splenic neurovascular plexus (SNVP) is needed to better understand the spleen physiology, and for selective neuromodulation of this major organ. However, their small size and anatomical location have proven to be a significant challenge. Here, we use a reduced liquid crystalline graphene oxide (rGO) fiber coated with platinum (Pt) as a super-flexible suture-like electrode to interface multiple SNVP. The Pt-rGO fibers work as a handover knot electrodes over the small SNVP, allowing sensitive recording from four splenic nerve terminal branches (SN 1-4), to uncover differential activity and axon composition among them. Here, the asymmetric defasciculation of the SN branches is revealed by electron microscopy, and the functional compartmentalization in spleen innervation is evidenced in response to hypoxia and pharmacological modulation of mean arterial pressure. We demonstrate that electrical stimulation of cervical and sub-diaphragmatic vagus nerve (VN), evokes activity in a subset of SN terminal branches, providing evidence for a direct VN control over the spleen. This notion is supported by adenoviral tract-tracing of SN branches, revealing an unconventional direct brain-spleen projection. High-performance Pt-rGO fiber electrodes, may be used for the fine neural modulation of other small neurovascular plexus at the point of entry of major organs as a bioelectronic medical alternative.

Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays.

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

Enhanced in vitro anticancer activity of yeast expressed recombinant glucose oxidase versus commercial enzyme.

Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H2O2, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H2O2-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment. KEY POINTS: • Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications. • Extracellular expression facilitates protein purification. • Glucose oxidase has potential application in cancer treatment.

Genomic profiling of sporadic liver metastatic colorectal cancer.

Sporadic colorectal cancer (sCRC) is the third leading cause of cancer death in the Western world. Approximately, a quarter of sCRC patients present metastatic dissemination at the moment of diagnosis, the liver being the most frequently affected organ. Additionally, this group of CRC patients is characterized by a worse prognosis. In the last decades, significant technological developments for genome analysis have fostered the identification and characterization of genetic alterations involved in the pathogenesis of sCRC. However, genetic alterations involved in the metastatic process through which tumor cells are able to colonize other tissues with a different microenvironment, still remain to be fully identified. Here, we review current knowledge about the most relevant genomic alterations involved in the liver metastatic process of sCRC, including detailed information about the genetic profile of primary colorectal tumors vs. their paired liver metastases.

Mortality From Alcohol-Related Liver Cirrhosis in Mexico (2000-2017).

Background: Alcohol is the main cause of liver cirrhosis. The objective of this study was to analyze the mortality rates of alcohol-related cirrhosis in Mexico from 2000 to 2017. Methods: Mortality data from alcohol-related cirrhosis were obtained from the National Institute of Statistics and Geography. Rates were adjusted to the World Standard Population and were calculated with a direct method. The differences between genders were evaluated with Student's t-test, while the ANOVA test was used for differences among age groups. A trend analysis was performed with an ln regression of adjusted mortality rates and analyzed with Student's t-test. Results: The mean age-adjusted mortality rate during the study period was 13.28 per 100,000 inhabitants. A significant decrease in mortality rates was observed, from 20.55 to 10.62 per 100,000 inhabitants. All age groups studied showed a significant decrease in mortality. The mortality rate was higher in males than in females. Conclusions: Mortality from alcohol-related cirrhosis decreased in Mexico. Males still have the highest mortality rate.

Intraneural ultramicroelectrode arrays for function-specific interfacing to the vagus nerve.

Selective interfacing to small multifunctional nerves such as the vagus nerve (VN) which is the main multimodal autonomic nerve that provides a major communication pathway from vital peripheral organs to the brain, can have significant potential in treating and diagnosing diseases as well as enhancing our understanding of peripheral nerve circuits. Here we describe the fabrication of a 16-channel intraneural electrode array with ultramicro-dimensioned electrodes to achieve improved functionally selective recording. We demonstrate that the amorphous silicon carbide ultramicroelectrode arrays (a-SiC UMEAs) provide selectivity in the detection of neural activity in the cVN related to changes in systemic oxygenation and blood pressure. We will also demonstrate spatially selective recording of micro-compound action potentials (µCAPs) by electrical stimulation of the subdiaphragmatic branches of the VN. Distinct neural activity was recorded on electrodes separated by less than about 100 µm. This is the first time that this level of spatially selectivity recording has been demonstrated in the cVN with an intraneural multielectrode array.

Mechanically Robust, Softening Shape Memory Polymer Probes for Intracortical Recording.

While intracortical microelectrode arrays (MEAs) may be useful in a variety of basic and clinical scenarios, their implementation is hindered by a variety of factors, many of which are related to the stiff material composition of the device. MEAs are often fabricated from high modulus materials such as silicon, leaving devices vulnerable to brittle fracture and thus complicating device fabrication and handling. For this reason, polymer-based devices are being heavily investigated; however, their implementation is often difficult due to mechanical instability that requires insertion aids during implantation. In this study, we design and fabricate intracortical MEAs from a shape memory polymer (SMP) substrate that remains stiff at room temperature but softens to 20 MPa after implantation, therefore allowing the device to be implanted without aids. We demonstrate chronic recordings and electrochemical measurements for 16 weeks in rat cortex and show that the devices are robust to physical deformation, therefore making them advantageous for surgical implementation.

Response to SARS-CoV-2 Pandemic in a Non-COVID-19 Designated Latin-American Neurosurgery Department.

Background: Mexico declared the first case of novel coronavirus disease (COVID-19) in February 2020. At the time we write this article, our country is facing a community spread phase, expecting a rapid increase in the number of cases and fatalities. The Fray Antonio Alcalde Civil Hospital of Guadalajara has been declared a non-COVID-19 hospital with the mission of providing care to patients already registered and also those transferred from neurosurgical departments of neighboring centers, which have been converted into COVID-19 only hospitals. Methods: An organized response regarding personnel, surgical case selection, operating room behavior, and facility reorganization were designed to prevent an internal coronavirus outbreak in the neurosurgery department at the Fray Antonio Alcalde Civil Hospital of Guadalajara. Results: Distancing actions by the staff and residents, including ward case discussions, neurosurgery rounds, and classes, will be carried out virtually. We classified neurosurgical patients into 4 groups depending on whether their condition demands care in 0-6 hours, 6-48 hours, 48 hours to 14 days, and >14 days. Subsequently, a questionnaire with epidemiologic, radiologic, clinical, and serologic criteria will be applied to determine the risk of COVID-19 infection to define to which area they are going to be transferred according to the different risk zones in our facility. Conclusions: Despite not being a COVID-19 center, we consider all patients at the neurosurgical ward and staff members as asymptomatic carriers or infected in the preclinical period. Specific measures must be taken to ensure the safety and care of neurosurgical patients and medical staff during the community spread phase.