The risk of complications following surgical procedures is significantly increased in patients with SARS-CoV-2 infection. However, the mechanisms underlying these correlations are not fully known. Spinal cord injury (SCI) patients who underwent reconstructive surgery for pressure ulcers (PUs) before and during the COVID-19 pandemic were included in this study. The patient’s postoperative progression was registered, and the subcutaneous white adipose tissue (s-WAT) surrounding the ulcers was analyzed by proteomic and immunohistochemical assays to identify the molecular/cellular signatures of impaired recovery. Patients with SCI and a COVID-19-positive diagnosis showed worse recovery and severe postoperative complications, requiring reintervention. Several proteins were upregulated in the adipose tissue of these patients. Among them, CKMT2 and CKM stood out, and CKM increased for up to 60 days after the COVID-19 diagnosis. Moreover, CKMT2 and CKM were largely found in MGCs within the s-WAT of COVID patients. Some of these proteins presented post-translational modifications and were targeted by autoantibodies in the serum of COVID patients. Overall, our results indicate that CKMT2, CKM, and the presence of MGCs in the adipose tissue surrounding PUs in post-COVID patients could be predictive biomarkers of postsurgical complications. These results suggest that the inflammatory response in adipose tissue may underlie the defective repair seen after surgery. Pressure ulcers (PUs) are one of the most frequent complications of spinal cord injury (SCI) . While cThe COVID-19 pandemic has had a strong impact on surgical operations. Not only has the number of interventions significantly decreased ,4, but tThe presence of angiotensin-converting enzyme 2 (ACE2), a SARS-CoV-2 receptor widely expressed in adipose tissue , has proIn the current study, we aimed to determine the particularities of the adipose tissue surrounding ulcers in patients with SCI who suffered from SARS-CoV-2 infection in the early postoperative period, as well as to identify features that may help to understand the underlying processes and prognostic biomarkers to anticipate complications.Patients who underwent PU reconstructive surgery before and during the COVID-19 pandemic, from January 2019 to July 2021, at the Paraplegic National Hospital  were included in this study. The study was approved by the local institutional review board  and followed the principles of the Declaration of Helsinki. In addition, personal data protection was ensured, as required by Spanish Law (Organic Law on Data Protection 3/2018). All patients provided informed consent before inclusion in the study.Patients of any sex with SCI and PUs (grade III or IV) who required reconstructive surgery between 2019 and 2021 were selected by screening our medical records. The patient’s available data included sex, age, SCI type, ulcer characteristics, comorbidities, SARS-CoV-2 infection status (if recruitment was performed after the onset of the COVID-19 pandemic) , clinicaGroups were defined as follows:Group A (n = 7), pre-pandemic (2019): patients who required PU reconstructive surgery between 1 January and 30 December 2019, defined as the non-COVID group.Group B (n = 10), during the pandemic period: patients who required PU reconstructive surgery from 1 January 2020 to 30 July 2021. This group was stratified by SARS-CoV-2 infection status  between COVID-19-negative (n = 4) and COVID-19-positive (n = 3) patients, defined as post-COVID. Patients without PCR test results were excluded from the study (n = 3). The final number of patients in Group B was 7. During the initial surgical debridement, the unhealthy skin edge of the ulcer was marked, and the ulcer was excised to a healthy bleeding level. Immediately after reconstruction, patients were treated according to a postoperative protocol based on the criteria followed at the Paraplegic National Hospital , a national reference center for the treatment of SCI and PU, which includes their accommodation in therapeutic postural beds. Photographs of the PUs were taken 1–2 days before surgery and 15–25 days after surgery. COVID patients waited for surgery until confirmation of a negative SARS-CoV-2 RT-PCR nasopharyngeal swab result. All surgical interventions were performed by the same surgeon and his team.Open biopsy specimens were collected from the ulcerated tissue area and from an adjacent sample consisting of subcutaneous white adipose tissue (s-WAT) peripheral to the ulcer. To ensure the collection of the purest s-WAT, tissue was collected and resected at least 2 cm away from the ulcerated area. Immediately after removal, each tissue sample was divided into two pieces: one was fixed in 10% formalin for histological examination, and the other was snap-frozen in liquid nitrogen for further mRNA and protein extraction.g for 10 min at 4 °C, and serum was collected, aliquoted, and stored at −80 °C until use.Peripheral blood was collected in anticoagulant-free tubes by venipuncture of the medial cubital vein. Blood clots were formed by maintaining the tubes at room temperature (RT) for 30 min. Blood was centrifuged at 1500× w/v) SDC; 10 mM TCEP; 100 mM TRIS; 40 mM chloroacetamide; protease and phosphatase inhibitors pH: 8.5), overnight at 4 °C with rotation. The samples were centrifuged at 13,000× g for 15 min at 2–8 °C. The top layer (adipocytes) was then discarded. The resulting liquid layer was isolated and the remaining pellets were discarded.Frozen adipose tissue was homogenized in 0.5 mL of lysis buffer (1 % (Frozen adipose tissue (50–100 mg) was homogenized with a power homogenizer in 1 mL of QIAzol lysis reagent , and total RNA was obtained by chloroform extraction and precipitation with isopropanol.A detailed description of all of these processes can be found in p = 0.75), as was the degree of ulceration . From the total number of individuals, 11 out 14 were paraplegic (79%), while the rest suffered from tetraplegia. In addition, half of them  had cardiovascular problems. The complete patient clinical and demographic variables are shown in The mean age of patients was similar in both groups  and post-COVID-19 patients (n = 2) by 2D-WB . Our resA comprehensive analysis of the identified peptides showed the presence of post-translational modifications (PTMs) in lysine and serine residues C. The deFinally, due to postsurgical healing problems and the need for reintervention, additional samples were extracted from these patients after the initial surgery. It was observed that CKM and CKMT2 expression was transitory, as both isoforms were not further expressed after the second month E.Immunohistochemistry assays confirmed the higher expression of CKM and CKMT2 in post-COVID patients . AlthougFinally, the potential immunogenicity of these post-translationally modified proteins was evaluated . For thiAfter incubating the PVDF membranes containing proteins from the s-WAT of post-COVID patients with the serum of control subjects (without PU or SCI), serum IgGs and IgMs targeting the 2D spots at the intermediate isoelectric point were detected A. HoweveTo elucidate whether these autoantibodies were caused by COVID infection, s-WAT proteins from COVID patients were exposed to sera from non-COVID patients with SCI and PU . Remarkably, the non-COVID sera showed immunoreactivity only against the same intermediate spot targeted by the control sera, suggesting that autoantibodies binding to the other spots were specifically detected only after COVID infection.Neither of these signals was observed in membranes containing s-WAT proteins from non-COVID patients incubated with sera from non-COVID controls or post-COVID patients.The COVID pandemic has been a significant setback in the field of surgery. Beyond the large number of interventions that have been suspended and rescheduled , those tProteomic approaches revealed a significant increase in three CK isoforms in the s-WAT of post-COVID patients. CK is a ubiquitous enzyme with several isoforms . IncreasFurthermore, CKM presented several PTMs, such as phosphorylation of Lys-156 and Ser-158, which were only found in patients with SCI and COVID-19. Previous studies have shown that CK phosphorylation regulates its activity and affects cellular concentrations of ATP, thereby affecting energy flux within cells ,24,25. FHistological analysis of s-WAT from COVID patients revealed the presence of MGCs. MGCs are polykaryons of monocytic origin that arise in pathological lesions of widely varying etiology . MGCs haFinally, previous studies have reported a correlation between the presence of autoantibodies and a worse prognosis of the disease in COVID patients ,40. In oOverall, our results confirmed that patients with SCI who tested COVID-19-positive prior to surgery had worse recovery and severe postoperative complications, requiring reintervention. Moreover, the presence of MGCs, together with the overexpression of different CK isoforms found in the s-WAT of post-COVID patients, may be associated with the impaired wound healing observed. Given the limited number of samples that could be obtained for this study, future studies are required to confirm the results presented here. Nevertheless, our results suggest that both MGCs and high levels of CK isoforms might be considered potential signatures of adverse prognosis in SCI/PU patients infected with coronavirus.


Throughout the COVID-19 pandemic, numerous non-human species were shown to be susceptible to natural infection by SARS-CoV-2, including farmed American mink. Once infected, American mink can transfer the virus from mink to human and mink to mink, resulting in a high rate of viral mutation. Therefore, outbreak surveillance on American mink farms is imperative for both mink and human health. Historically, disease surveillance on mink farms has consisted of a combination of mortality and live animal sampling; however, these methodologies have significant limitations. This study compared PCR testing of both deceased and live animal samples to environmental samples on an active outbreak premise, to determine the utility of environmental sampling. Environmental sampling mirrored trends in both deceased and live animal sampling in terms of percent positivity and appeared more sensitive in some low-prevalence instances. PCR CT values of environmental samples were significantly different from live animal samples’ CT values and were consistently high (mean CT = 36.2), likely indicating a low amount of viral RNA in the samples. There is compelling evidence in favour of environmental sampling for the purpose of disease surveillance, specifically as an early warning tool for SARS-CoV-2; however, further work is needed to ultimately determine whether environmental samples are viable sources for molecular epidemiology investigations. The SARS-CoV-2 pandemic has shown the usefulness of environmental sampling in disease surveillance and outbreak response ,2. EnvirNeovison vison) are one of the most highly susceptible non-human species, with evidence of human to mink, mink to human and mink to mink transmission [Throughout the COVID-19 pandemic, numerous non-human species were shown to be susceptible to natural infection with SARS-CoV-2, including many domesticated species . Farmed smission ,6,7,8. Osmission ,6,9,10. smission . Due to smission .SARS-CoV-2 surveillance within mink farms is historically conducted through the testing of recent mortalities and/or sampling of live mink . HoweverHow do results from environmental samples compare to those obtained from live and dead mink in terms of percent positivity and PCR CT values?In terms of environmental sampling methods, how do manure trough swabs or cage swabs compare regarding detection of SARS-CoV-2?Can environmental samples pick up sufficient viral RNA for successful genomic sequencing to be used in molecular epidemiological studies ?The purpose of this study was to continue the evaluation of the utility of environmental sampling for SARS-CoV-2 surveillance in mink. Specifically, we aimed to address the following research questions:This study took place during an outbreak of SARS-CoV-2 on a mink farm in British Columbia, Canada. The outbreak was detected on 2 May 2021, and continued until April 2022, at which point the farm was mandated to be depopulated by the BC Ministry of Agriculture and Food. The outbreak was detected during a mandatory passive surveillance of all dead mink on BC farms—a program that was initiated after the first outbreak of SARS-CoV-2 on a different farm on 21 April 2021.All mink that died on-farm were frozen and submitted weekly to the Animal Health Centre (AHC), BC Ministry of Agriculture and Food. Mink carcasses were tested once per week. Carcasses were thawed and a cross-sectional cut was made at the level of the cartilaginous nasal septum. A sterile 1.5 mL swab was inserted into the exposed nasal canal to the level of the notched indicator on the swab so that the tip of the swab reached the back of the nasopharynx. The swab was swirled several times and then placed in a tube containing viral transport media (VTM).Approximately bi-weekly, a stratified random selection of 65 live mink was tested in accordance with BC Ministry of Agriculture and Food protocols to detect the presence of SARS-CoV-2 at a ≥5% prevalence. Each mink was selected at random with approximately evenly distributed locations through the sheds to represent the spatial extent of the farm and the approximate age–sex distribution of the farm. For instance, if there were 13 active mink sheds, then 1 mink was selected at random from five approximately evenly spaced regions within each shed. If 33% of the mink herd was adult female, then every third mink selected would be an adult female. Oropharyngeal samples were collected from each selected mink by the herd veterinarian. The mink was manually restrained, and a sterile polyester swab was inserted into the mink’s mouth through a gag . The swab was twirled several times as far back in the oral cavity as possible and then placed into a pre-labelled tube containing VTM.Environmental samples were taken at the same time as live animal samples. While the mink was removed from its cage for an oral sample, a paired cage sample was also taken. The cage was sampled by rubbing a sterile polyester swab along an approximately 10 cm × 10 cm area of the wire mesh surface of both the cage ceiling and floor. Mink feed is placed on top of the enclosures and their manure drops into a collection system through the mesh flooring. Therefore, these areas of the cage were expected to have a higher likelihood of exposure to bodily fluids  that contain SARS-CoV-2.Manure troughs are an automated manure collection system with one trough collecting all urine and faeces, under a row of multiple mink cages. These troughs provide a convenient sample encompassing excreta from many individuals. The manure troughs (n = 12) were sampled by inserting a sterile polyester swab into the outflow end of the trough, swirling the swab several times, and then placing the swab into a pre-labelled tube containing VTM.Environmental sampling continued every two weeks for a total of 6 weeks after the mink were depopulated, at which time the farm was decommissioned.thermofisher.com). Primers and a probe that target the E gene to create a 113-base pair (bp) amplicon were used to detect SARS-CoV-2. Forward primer 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′; probe 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3′, reverse primer 5′-ATATTGCAGCAGTACGCACACA-3′. Reaction concentrations of the SARS-CoV-2 primers and probe were 800 nM and 200 nM, respectively. An enterovirus exogenous PCR control  was spiked in the RNA isolation step and the 61 bp amplicon was detected with the following primers and probe. Forward primer 5′-ATGCGGCTAATCCCAACCT-3′, probe 5′-VIC-CAGGTGGTCACAAAC-MGBNFQ-3′, and reverse primer 5′ CGTTACGACAGGCCAATCACT-3′ .): 5 μL of extracted RNA template was added to the master mix. Real-time PCR (RT-PCR) was performed on the Applied Biosystems 7500 Fast Real-Time PCR System thermocycler using the following amplification profile: one cycle of 50 °C, 30 min; one cycle of 95 °C, one min; 40 cycles of 95 °C, 15 s; and 60 °C, one min. Change in fluorescence was recorded at the elongation step of each cycle. Swabs with CT values ≥ 35.9 were considered negative. Samples above the cut-off point that did exhibit amplification were either retested at the Animal Health Center or sent to the National Center for Foreign Animal Disease in Winnipeg for confirmatory testing. CT values were not recorded at the AHC.The majority of mortality samples were tested at the Animal Health Center using qPCR. Viral RNA was isolated using the Applied Biosystems Incorporated (ABI) MagMax-96 Express magnetic particle processor  with the MagMax™-96 Viral RNA Isolation Kit , as per kit instructions. The MagMax program (“AM1836_DW_v50”) was available on the ThermoFisher website  were refrigerated prior to shipment to the BC Centre for Disease Control (BC CDC) Public Health Laboratory, where they were tested for SARS-CoV-2 using qPCR. The BCCDC Public Health Laboratory (PHL) developed a real-time, multiplex reverse transcription PCR assay targeting the envelope (E) and the RdRP genes of SARS-CoV-2. The E gene primers and probes are as previously described above; the primer and probe details for RdRp are as follows: F-TGCCGATAAGTATGTCCGCA, R-CAGCATCGTCAGAGAGTATCATCATT, FAM-MGB-Probe-TTGACACAGACTTTGTGAATG. Briefly, total nucleic acids were extracted from 200 μL of specimen  using the Applied BioSystems MagMax™ Express 96 Nucleic Acid Extractor and the MagMax Viral/Pathogen Nucleic Acid Isolation Kit  according to the manufacturer’s recommendations. Real-time PCR was performed using the TaqMan Fast Virus Master Mix (Thermo Fisher Scientific) on the Applied Biosystems 7500 FAST real-time PCR system (Thermo Fisher Scientific). PCR set-up was performed using a volume of 5 µL of specimen extract, for a final reaction volume of 20 µL. Sample swabs with Ct values < 40 in both the E gene and RdRp were considered positive; those with Ct values < 40 in only one assay were considered indeterminant; and those with Ct values ≥ 40 were considered negative. For the purpose of this study, indeterminants were classified as negatives to improve specificity within our comparisons.t-test. CT values were not compared between environmental samples and mortality samples because the majority of deceased mink were tested at the AHC where CT values were not recorded.Results were analysed with R, RStudio , and Microsoft Excel (2020) software ,19,20. TIn total, 1166 environmental samples , 413 mortality swab samples, and 720 live swab samples were collected. There were 15 environmental sampling events, 12 live sampling events, and 42 mortality sampling events.p-value = 0.031).At the level of the farm, when comparing PCR results (positive vs. negative) between live and environmental samples (including both cage and manure swabs) over time, trends in percent positivity of environmental samples generally mirrored that of live animal samples . HoweverAt the level of the farm, when comparing PCR results (positive vs. negative) between mortality and environmental samples (including both cage and manure trough swabs) over time, trends in percent positivity of environmental samples closely aligned with that of mortality samples . The nump-value = 0.153).At the level of the farm, when comparing PCR results (positive vs. negative) between cage and manure trough swabs over time, trends in percent positivity of cage swab samples generally mirrored that of manure swabs . HoweverThe results of this study suggest that environmental samples could be used for the surveillance of SARS-CoV-2 outbreaks on American mink farms. At the level of the farm, trends in PCR results for environmental samples (positive vs. negative) mirrored those for both live animal and mortality samples over time. Furthermore, the percent positivity for environmental samples was generally greater than that of live animal samples, and there were two instances (2 September and 28 January) during the outbreak where live animal samples were negative while environmental samples remained positive. This may suggest that environmental samples are more ‘sensitive’ for the detection of SARS-CoV-2 infection circulating at a low prevalence in mink. Specifically, it is unlikely that the farm was truly negative on 2 September and 28 January, given that the subsequent sampling events found percent positivity values of 3.08% and 1.54%, respectively. The live animal sampling was performed in accordance with determining disease presence at a ≥5% prevalence, which does speak to the utility of using environmental sampling in parallel with live animal sampling. If live animal sampling was used alone, the farm may have been falsely declared negative in these two instances. Conversely, because viral cultures were not performed, it is possible that RNA detected were not viable virions, but instead persistent RNA contamination. However, environmental samples can detect disease at low prevalence. Wastewater surveillance had a detection probability of 87% when infection prevalence in the community was 0.01% for SARS-CoV-2 in humans .Alternatively, in one Danish study where 100% of animals sampled on farm were positive, SARS-CoV-2 was only detected at low levels through air and water sampling, with no positives detected in the feed . SimilarThe high CT values of cage swab samples suggests that inactive viral RNA, rather than viable virions, was being detected. Previous research into the stability of SARS-CoV-2 in the environment found that infectious virus could remain on non-porous surfaces such as stainless steel for 7 days, whereas non-infectious viral RNA could be recovered for longer periods (up to 57 days) ,23,24. TOne of the most notable findings was the persistence of positive cage samples for at least 6 weeks after the mink had been depopulated. Less porous surfaces, such as steel, are known to absorb droplets less easily, preserving active virus for a period of time, which could explain this persistence . The perNotably, cage swabs remained positive when the manure trough swabs became negative immediately after depopulation. This was unexpected given that wastewater studies have indicated that infectious particles have a relatively long persistence within sewage . It may Mink housing units have previously been used for sampling SARS-CoV-2, including testing food residues from the tops of cages, bedding material, water bowls and faecal material . SARS-CoWhen comparing both environmental sampling modalities, manure trough swabs had a consistently higher percent positivity compared with cage swabs, although trends in percent positivity over time were similar. This is consistent with the findings of a Danish study where 54% (51/95) of faecal material samples were positive compared with 10% (9/90) of the food residues swabs (similar to our cage swabs) . This coOur study suggests that environmental samples are not the preferred sample type for sequencing SARS-CoV-2 genomes obtained from mink farms. Cage and manure trough swabs had mean CT values of 36.99 (IQR = 0.90) and 36.29 (IQR = 1.65), respectively, which was significantly less than the CT values for animal samples . For the assay used in this study, samples with CT values < 33 produced reliable sequencing results, with decreasing success as CT values increased. High CT values can be a result of low concentrations of RNA, and alternative sequencing methods should be investigated if phylogenetic trees are used in the surveillance for SARS-CoV-2 on mink farms. This is similar to what has been observed for the environmental surveillance of avian influenza virus, where researchers have turned to more advanced genomic techniques, such as targeted resequencing, to obtain AIV sequences from environmental samples [An important limitation of this study is the fact that only one outbreak was included, which may limit the generalizability of our findings. Furthermore, no viral cultures were performed; thus, we could not determine whether positive PCR results reflected the presence of live virus vs. inactivated virus. Additionally, the AHC and BC CDC had different cut-off points for CT values to determine positive vs. negative. The lower CT value cut-off point at the AHC may have caused underestimates in mortalities that were positive compared with the live and environmental samples that were tested at BC CDC. However, this is unlikely to have altered the relative trends in positives overall because many of the mortalities were retested at the National Center for Foreign Animal Disease if amplification was detected above the cut-off point. Finally, because individual mink and cages were not re-sampled at subsequent time points, we could not determine how viral shedding or cage contamination in individual mink varied over time.These data suggest that environmental samples could be reliable and sensitive for detecting and monitoring SARS-CoV-2 outbreaks in mink at the level of the farm, although their utility for the detection of infection in individual animals appears less consistent. Environmental samples may be best suited as a convenient method to survey seemingly negative farms and act as an early detection tool for SARS-CoV-2 outbreaks. Once an outbreak is determined, it would be advisable to continue environmental sampling in tandem with more conventional modalities to aid in the detection of low-prevalence infection within the farm. Environmental samples should not be used as the sole modality in determining disease freedom, given the questionable persistence post depopulation seen in our study. Additionally, it may be challenging to obtain whole genome sequences from environmental samples; however, this may be possible with different sequencing methods. Further research is needed to better understand SARS-CoV-2 persistence on different surfaces and substrates within the farm environment and to develop methods for obtaining sequences from environmental samples.


Cognitive practice effects (PEs) can delay detection of progression from cognitively unimpaired to mild cognitive impairment (MCI). They also reduce diagnostic accuracy as suggested by biomarker positivity data. Even among those who decline, PEs can mask steeper declines by inflating cognitive scores. Within MCI samples, PEs may increase reversion rates and thus impede detection of further impairment. Within an MCI sample at baseline, we evaluated how PEs impact prevalence, reversion rates, and dementia progression after 1 year.SD = 7.4). We identified test-naïve participants who were demographically matched to returnees at their 1-year follow-up. Since the only major difference between groups was that one completed testing once and the other twice, comparison of scores in each group yielded PEs. PEs were subtracted from each test to yield PE-adjusted scores. Biomarkers included cerebrospinal fluid phosphorylated tau and amyloid beta. Cox proportional models predicted time until first dementia diagnosis using PE-unadjusted and PE-adjusted diagnoses.We examined 329 baseline Alzheimer’s Disease Neuroimaging Initiative MCI participants , and reduced reversion to normal by 28.8% (57 vs. 80 reverters). PEs also increased stability of single-domain MCI by 12.0% (164 vs. 147). Compared to PE-unadjusted diagnoses, use of PE-adjusted follow-up diagnoses led to a twofold increase in hazard ratios for incident dementia. We classified individuals as false reverters if they reverted to cognitively unimpaired status based on PE-unadjusted scores, but remained classified as MCI cases after accounting for PEs. When amyloid and tau positivity were examined together, 72.2% of these false reverters were positive for at least one biomarker.Even when PEs are small, they can meaningfully change whether some individuals with MCI retain the diagnosis at a 1-year follow-up. Accounting for PEs resulted in increased MCI prevalence and altered stability/reversion rates. This improved diagnostic accuracy also increased the dementia-predicting ability of MCI diagnoses. Mild cognitive impairment (MCI) is characterized by cognitive deficits in the presence of minimal to no impairment in functional activities . MCI is Nearly all AD clinical trials have focused on treating individuals with dementia in an effort to mitigate or reverse the disease. Unfortunately, the failure rate for these trials is greater than 99% . As a reThere is concern regarding stability of MCI diagnosis that limits its use in clinical and research settings. Although 10–12% of those with MCI are expected to convert to AD per year, 20–50% of individuals revert from MCI to CN status within 2–5 years . Over a Much of the MCI reversion rate literature was published prior to 2016 and was summarized by three articles . These aPractice effects (PEs) on cognitive tests used to diagnose MCI are a likely contributor to MCI reversion rates. They mask cognitive decline by increasing scores at follow-up testing relative to how an individual would have performed if they were naïve to the test. PEs are due to familiarity with specific test items , and/or increased comfort and familiarity with the general assessment process  . PEs in The MCI classification methods, particularly in research, almost always rely on use of cut-off scores to define cognitive impairment . The samvia PE-unadjusted scores but MCI via PE-adjusted scores. By retaining these participants in the MCI pool, we expected the PE-adjusted classifications to result in improved diagnostic stability and decreased reversion rates. Also, we expected the biomarker profile and the time until first dementia diagnosis of the false reverters to be more similar to the stable MCI participants than to true reverters . Finally, in a post hoc analysis, we modeled the impact of PE adjustment on studies concerned with progression to dementia, a common outcome in clinical drug trials and research studies.In the present analyses, we utilized a sample of Alzheimer’s Disease Neuroimaging Initiative (ADNI) participants who were diagnosed as MCI at baseline. We sought to (1) calculate 1-year follow-up cognitive classifications using PE-unadjusted and PE-adjusted scores, (2) compare reversion rates and diagnostic stability between PE-unadjusted and PE-adjusted classifications, and (3) provide criterion validity for the PE-adjusted classifications through baseline biomarker data and time until first dementia diagnosis. We hypothesized that the PE-adjusted scores would reveal false reverters, i.e., participants at follow-up who were classified as CN 1. The ADNI, led by Principal Investigator Michael W. Weiner, MD, was launched in 2003 as a public-private partnership. The primary goal of ADNI has been to test whether serial magnetic resonance imaging, positron emission tomography, other biological markers, and clinical and neuropsychological assessment can be combined to measure the progression of MCI and early AD. For up-to-date information, see www.adni-info.org. Participants from the ADNI-1, ADNI-GO, and ADNI-2 cohorts were included.Data used in the preparation of this article were obtained from ADNIMild cognitive impairment was diagnosed using the Jak-Bondi approach . ParticiSD = 2.9), 61.4% (n = 202) were female, and mean baseline age was 73.1 years (SD = 7.4).We identified 344 individuals who were classified as MCI at baseline. Of those 344, 329 returned for a 12-month follow-up visit and also completed all cognitive measures at both assessments. Mean educational level of returnees was 16.4 years  delayed recall. Language tasks included the Boston Naming Test and Animal Fluency. Attention-executive function tasks were Trails A and Trails B. The American National Adult Reading Test provided an estimate of premorbid IQ. Only participants who had complete test data and completed the same version of tests at the baseline and 12-month visits were included.Z-scores were calculated for the PE-adjusted and -unadjusted scores based on independent external norms that accounted for age, sex, and education for all tests except the AVLT (z-scored based on the ADNI participants who were CN at baseline (n = 889) because we were unable to find appropriate external norms for this sample that also accounted for age, sex, and education. AVLT demographic corrections were based on a regression model that followed the same approach as the other normative adjustments. Beta values were multiplied by an individual’s corresponding age, sex, and education. The products were then removed from the AVLT raw scores. These adjusted AVLT scores were then z-scored.the AVLT . The AVL2 : Aβ+: Aβ < 977 pg/mL; p-tau+: p-tau > 21.8 pg/mL; t-tau+: t-tau > 270 pg/mL , phosphorylated tau (p-tau), and total tau (t-tau). The ADNI biomarker core (University of Pennsylvania) used the fully automated Elecsys immunoassay (Roche Diagnostics). Sample collection and processing have been described previously . Cutoffs70 pg/mL . There wDementia was diagnosed according to ADNI criteria: (1) Memory complaint by subject or study partner that is verified by a study partner; (2) Mini-Mental State Examination score between 20–26 (inclusive); (3) Clinical Dementia Rating score of either 0.5 or 1; (4) An impaired delayed memory score on the Logical memory test: ≤ to 8 for 16 or more years of education; ≤ to 4 for 8–15 years of education; or ≤to 2 for 0–7 more years of education; (5) National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association criteria for probable AD . No partAlthough review papers have noted that PEs can exist even when there is longitudinal decline in observed performance, as expected within a sample at risk for AD , few havThe goal of the replacement method is to obtain follow-up scores at retest that are free of PEs and comparable to normative data (which assume no presence of PEs). Some researchers have used PEs in other ways, such in short-term retest paradigms . The goat-tests and chi-squared tests ensured that returnees and pseudo-replacements were matched at a group level (ps > 0.8). Thus, this sample of pseudo-replacement participants was demographically identical to the returnee subsample. In a traditional replacement participants method of PE-adjustment returnees and non-returnees are combined into a “baseline” subsample that excludes replacements. In this method, we used a “proportional baseline” subsample that included the baseline scores for the returnees chosen at that iteration as well as all other subjects not chosen to be pseudo-replacements (approximately 75% of sample). However, the removal of the pseudo-replacements from the sample led to an artificially high portion of lower-performing baseline participants since the pseudo-replacements perform at a similar level to returnees at baseline. To correct for this issue, we calculated the retention and attrition rates for that visit in the overall sample. Because the PE for each test was calculated individually, we used test-specific retention and attrition rates, which resulted in a slight variation in rates; the average retention rate was 66% (65–70%) and the average attrition rate was 34% (30–35%). We then used these rates in the creation of the proportional baseline mean (see below). Of note, due to the bootstrapping and matching procedure, the number of participants in each group  varied but was always greater than 80 participants.Because replacement participants were not part of the original ADNI study design, we created what we refer to as the pseudo-replacement method of PE adjustment. We have fully described this method previously in an examination of individuals who were cognitively normal at baseline . BrieflyThe equations below were used to calculate the PE:T2 represents the mean score of the returnee sample at their second assessment, Pseudo-replacementsT1 represents the mean score of the pseudo-replacement sample , and ReturneesT1 represents the mean score of returnees at their first assessment. The Proportional BaselineT1 was a weighted mean calculated by multiplying the returnee baseline scores by the test-specific retention rate (65–75%) and the remaining portion of the subsample by the test-specific attrition rate (30–35%%). The difference score represents the sum of the PE and the attrition effect. The attrition effect accounts for the fact that individuals who return for follow-up are typically higher-performing or healthier than those who drop out. Subtracting the attrition effect from the difference score prevents over-estimation of the PE  follow-up test score to provide PE-adjusted raw scores. Cohen’s Time until first dementia diagnosis in months from baseline was also used to validate PE-adjusted diagnoses. Cognitive data used to diagnose dementia by ADNI were not adjusted for PEs. Wilcoxon rank sum tests were used to compare groups due to the non-normal distribution of months until first dementia diagnosis. It was expected that those who reverted to CN status at follow-up would progress to dementia more slowly than those who remained classified as having MCI. As such, if PE adjustment improved diagnostic accuracy by correctly relabeling some false reverter (based on PE-unadjusted scores) as MCI, then a comparison between MCI and CN groups should show a larger and more statistically significant difference when using PE-adjusted scores than when using PE-unadjusted scores. PE-adjustment should also alter a comparison between those who truly revert and the false reverters, with false reverters progressing faster than true reverters. The following four time-until-dementia comparisons were tested: PE-adjusted MCI versus PE-adjusted CN; PE-unadjusted MCI versus PE-unadjusted CN; False reverters versus PE-unadjusted MCI; and False reverters versus PE-adjusted CN.We also expected that the false reverters (based on PE-unadjusted scores) would have a biomarker profile more similar to the stable MCI participants than the true reverters. Thus, we calculated rates of biomarker positivity for diagnostic groups (Stable MCI and reverters) first using PE-unadjusted scores and then with PE-adjusted scores.post hoc analyses, Cox proportional hazard models compared progression to dementia between those who were diagnosed as MCI at follow-up and those who reverted to CN. All models used classification (Stable MCI vs. reverters) as the independent variable of interest and months from baseline until first dementia diagnosis as the dependent variable. Covariates were age and education. Models were completed first with PE-unadjusted scores and then with PE-adjusted scores.In Time-to-dementia analyses included a full model and three timeframe-restricted models: 16–150 months (full sample data), 16–24, 16–36, and 16–48 months. The models with restricted timeframes attempted to demonstrate how predictive the classification was for studies with shorter follow-up periods. Because, in these hypothetical studies, we could not know if a participant progressed to dementia past the specified timeframe, each model was right-censored with time to event defined as time to first dementia diagnosis or time to last follow-up within the restricted time period. As this project utilized existing data, the maximum follow-up period was set to 150 months because that was the longest available timeframe within ADNI.d = 0.06–0.26). PE-adjustment resulted in 23 more participants (+9%) classified as MCI at 1-year follow-up than when using PE-unadjusted scores (272 vs. 249). Of the 23, 16 (+9%) were classified as single-domain MCI and 7 participants classified as multi-domain MCI (+9%). Regarding specific cognitive domains, PE-adjustment resulted in 24 more participants (+11%) classified with memory impairment (233 vs. 209), 6 more participants (+9%) classified with attention-executive impairments (73 vs. 67), and 5 more participants (+7%) classified with language impairments (72 vs. 67). Full results are presented in PEs were non-zero for 5 of the 6 measures  and rangThe overall 1-year stability of MCI (lack of reversion to CN) was raised by 7% when adjusting for PEs . Across groups  and within each cognitive domain , PE adjustment increased the number of participants who retained their baseline diagnosis of MCI (Range: +2 [+3%] to +22 [+11%]). In particular, there were significantly more participants who remained in the impaired range at follow-up on memory when using PE-adjusted data versus PE-unadjusted data . A similar significant result was also found when considering stability of single-domain MCI . n = 80) using PE-unadjusted scores and 17.3% (n = 57) using PE-adjusted scores. This indicates that adjusting for PEs resulted in a 28.8% reduction in the overall reversion rate. The overall reversion rate  was 24.3% . However, in the same comparison based on PE-adjusted scores, those in the stable MCI group progressed significantly faster than those who reverted to CN .We also compared how PE-adjusted and PE-unadjusted classification affected rate of progression to dementia. Of the 329 returnees, 159 progressed to dementia (48% of sample). As shown in via PE-unadjusted scores (median = 30.03 months). The false reverters progressed to dementia more quickly than those who were classified as CN based on PE-adjusted scores at follow-up. There was not a significantly different rate of progression to dementia between false reverters and PE-adjusted CNs, or between false reverters and PE-unadjusted MCI based on Mann–Whitney U tests (ps > 0.17).Ten of the false reverters (6.2%) progressed to dementia. These participants progressed to dementia in a similar time frame as the those diagnosed with MCI post hoc Cox proportional hazard models to compare progression to dementia from 12-month follow-up between those who were diagnosed as MCI at follow-up and those who reverted to CN. Across all models, the hazard ratio associated with increased risk of dementia progression among stable MCI participants was nearly twice as large when adjusted for PEs compared to PE-unadjusted diagnoses . When false reverters were removed by adjusting for PEs, the median time until first dementia diagnosis was increased +23 months). To further investigate this finding, we performed 3 months.There were 226 participants with baseline biomarker data. As shown in The validity and utility of MCI criteria are weakened by high reversion rates, which have been a longstanding problem for MCI as a construct . As a rePathologically, AD is characterized by a progressive change in amyloid beta and tau protein levels in the brain . AlthougUse of a robust normal sample partially addresses PEs as the cut-off for MCI diagnosis varies at each timepoint based on the distribution of scores among participants who remain CN across all visits , 2019. IProponents of MCI as a diagnostic entity note that individuals with the diagnosis are more likely to progress to AD, and do so at a faster rate than CN individuals . Those cAlthough adjusting for PEs slightly altered the median time until first dementia diagnosis, statistical comparisons between groups were non-significant. To further investigate these findings, we completed Cox proportional hazard models. Using PE-unadjusted data, we found that the stable MCI group converted to dementia significantly faster than the  reverter group, as expected. When models were completed with PE-adjusted data, we found that the hazard ratios sharply increased, suggesting that the PE-adjusted classifications improved differentiation between the (true) reverters and the stable MCI participants. Not accounting for PEs may thus obscure true effects or push significance above threshold, influencing subsequent interpretation.Interestingly, hazard ratios were less different between PE-adjusted and PE-unadjusted models when analyses were completed over the full 150-month timeframe (HRs: 6.0. versus 3.7) compared to shorter time frames . These results are consistent with the idea that PE adjustment leads to earlier detection of at-risk participants, which would be particularly important for studies with shorter follow-up periods. Importantly, clinical drug trials for AD typically involve shorter follow-up periods, so increasing the number of individuals expected to progress to dementia during the trial period will increase sensitivity to treatment effects. Therefore, failure to account for PEs may have a large impact on the design of treatment studies and interpretation of their results. Earlier detection of at-risk individuals is also of obvious importance for clinical care.All participants completed the logical memory test at a screening assessment, baseline, and 12-month visit; all other tests were completed only twice. Therefore, it is possible that the PE for logical memory is misestimated. However, as the effect size of the logical memory PE is similar to that of the other memory task (AVLT), it seems likely that our estimate is still valid.n = 8; 24% of deaths). Importantly, all participants who passed away were diagnosed as stable MCI (impaired at baseline and follow-up) by both the PE-adjusted and PE-unadjusted datasets. As such, although mortality may have impacted results, this effect was equal within the PE-adjusted and PE-unadjusted analyses.Our time until dementia analyses did not account for death. Of the 329 participants included in these analyses, 33 passed away before study completion (10.0%). The modal time until death was 48-months past baseline visit . The grantee organization is the Northern California Institute for Research and Education, and the study is coordinated by the Alzheimer’s Therapeutic Research Institute at the University of Southern California. ADNI data are disseminated by the Laboratory for Neuroimaging at the University of Southern California.Data collection and sharing for this project was funded by the Alzheimer’s Disease Neuroimaging Initiative (ADNI)  and DOD ADNI (Department of Defense award number W81XWH-12-2-0012). ADNI was funded by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, and through generous contributions from the following: AbbVie, Alzheimer’s Association; Alzheimer’s Drug Discovery Foundation; Araclon Biotech; BioClinica, Inc.; Biogen; Bristol-Myers Squibb Company; CereSpir, Inc.; Cogstate; Eisai Inc.; Elan Pharmaceuticals, Inc.; Eli Lilly and Company; EuroImmun; F. Hoffmann-La Roche Ltd. And its affiliated company Genentech, Inc.; Fujirebio; GE Healthcare; IXICO Ltd.; Janssen Alzheimer Immunotherapy Research & Development, LLC.; Johnson & Johnson Pharmaceutical Research & Development LLC.; Lumosity; Lundbeck; Merck & Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx Research; Neurotrack Technologies; Novartis Pharmaceuticals Corporation; Pfizer Inc.; Piramal Imaging; Servier; Takeda Pharmaceutical Company; and Transition Therapeutics. The Canadian Institutes of Health Research is providing funds to support ADNI clinical sites in Canada. Private sector contributions are facilitated by the Foundation for the National Institutes of Health (http://adni.loni.usc.edu/.Publicly available datasets were analyzed in this study. This data can be found here: The studies involving human participants were reviewed and approved by University of California, San Diego. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.MS-C and WK conceived the study. XT and AG provided guidance on statistical analysis. EE, MB, JSE, and KT made determination of MCI diagnoses. MS-C, WK, JAE, MP, and DG contributed to the practice effects methodology. WK, CF, ML, and MS-C obtained primary funding to support this work. All authors provided critical review and commentary on the manuscript.MB receives royalties from Oxford University Press. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gastric cancer is the fifth most common tumor worldwide. In the past couple of decades, there have been many advancements toward earlier detection and better treatments for this aggressive disease. There are currently many serological tests and biomarkers being investigated to allow for the non-invasive early diagnosis of gastric cancer. The treatment options for this tumor are always rapidly evolving, and we will emphasize the medical treatment options available. We hope to outline and explain some of these latest advancements to allow clinicians and future researchers to have a better understanding of this rapidly changing field and allow them to make informed decisions for the care of their patients.Gastric cancer is an enigmatic malignancy that has recently been shown to be increasing in incidence globally. There has been recent progress in emerging technologies for the diagnosis and treatment of the disease. Improvements in non-invasive diagnostic techniques with serological tests and biomarkers have led to decreased use of invasive procedures such as endoscopy. A multidisciplinary approach is used to treat gastric cancer, with recent significant advancements in systemic therapies used in combination with cytotoxic chemotherapies. New therapeutic targets have been identified and clinical trials are taking place to assess their efficacy and safety. In this review, we provide an overview of the current and emerging treatment strategies and diagnostic techniques for gastric cancer. Other red foods .Given its high incidence, it is imperative that further research is done to better understand this disease. In this review, we summarize the current status of diagnostic modalities and treatments for gastric cancer.In the United States, about one-third of patients diagnosed with gastric cancer have a distant metastasis at the time of diagnosis . Upper GCurrently, the most widely used biomarkers for the diagnosis and monitoring of gastric cancer are carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9. Both CEA and CA19-9 have been shown to correlate with tumor burden and depth of tumor invasion [Pretreatment CA-125 is another classical prognostic marker for gastric cancer that has been associated with gastric cancer recurrence, rendering it a diagnostic tool to predict poor prognosis. Furthermore, many studies have demonstrated that elevated levels of serum CA-125 correlate with peritoneal dissemination of the primary gastric tumor. Although the sensitivity of this biomarker is poor, ranging from 19.78%  in some p < 0.05) [Another tumor marker that has been gaining significant traction for the purpose of gastric cancer screening and monitoring is CA72-4 . Althoug < 0.05) . HoweverPepsinogen is another protein biomarker that can be used for the screening of gastric cancer. Pepsinogen, a precursor of pepsin, is secreted by the chief cells of the stomach and can be present in two forms, pepsinogen I (PG I), which is secreted from the fundus, and pepsinogen II (PGII), which is secreted from the pylorus and Brunner’s glands of the duodenum ,14. In np < 0.001), muscularis propria invasion , and worse TNM stage  [Another promising biomarker is trefoil factor 3 (TFF3). This minute peptide is secreted from the goblet cells of the small and large intestine and can also be secreted from the gastric mucosa that has undergone intestinal metaplasia. Studies have shown high serum TFF3 levels to have a sensitivity of 80.9% and specificity of 81% for gastric cancer . A meta-< 0.001) . Further< 0.001) ,21. DespAlpha-fetoprotein (AFP) is another biomarker for gastric cancer that is most commonly seen in a rare subset of AFP-producing gastric carcinomas . AFP is In 1869, a liquid biopsy was performed from the peripheral blood and provided the first known evidence for the presence of circulating tumor cells (CTCs) . Liquid Studies have shown that the clinical utility of CTCs is mainly limited to monitoring gastric tumor treatment response and prognosis rather than the early diagnosis of the tumor . For exap < 0.001) and DFS (p < 0.001) [Circulating tumor DNA (ctDNA) is another biomarker that can be extracted from liquid biopsies for the diagnosis and monitoring of gastric cancer. CtDNA can be produced from primary tumor cells, CTCs, or a distant metastasis and it can give a wide range of information on the malignancy, such as methylation status and other genetic alterations . Fang et< 0.001) . The stu< 0.001) .Non-coding RNAs (ncRNAs) are types of RNA that do not encode protein and can be classified into two subgroups: small ncRNAs (sncRNAs) and long ncRNAs (lncRNAs). The sncRNAs can be further subclassified into microRNAs (miRNAs), small nuclear RNAs (snRNAs), and piwi-interacting RNAs (piRNAs). These various non-coding RNAs can also be used in the detection and monitoring of gastric cancer.p < 0.01), with a sensitivity of 81.3% and a specificity of 73.4% [miRNAs play a crucial role in various cellular functions through regulating epigenetic mechanisms; these functions include cellular growth, apoptosis, differentiation, and even gastric tumor carcinogenesis. Wu et al. studied 50 GC patients and 50 patients without GC and found an increase in levels of miRNA-21 in patients with GC compared to those who did not have it  has been shown to be increased in the serum of GC patients compared to normal controls . LikewisExtracellular vesicles (EVs), also known as exosomes, are small spherical structures with an outer lipid bilayer that are secreted from cells into the extracellular space, and participate in inter-cellular communication through the transfer of functional molecules scavenged and secreted into EVs ,49. Exosp < 0.0001), suggesting it to be another potential biomarker for GC. Heat shock proteins (HSP) 60 and 70 have been found in higher concentration within exosomes derived from malignant ascites in GC patients compared with exosomes derived from ascites in non-GC patients [Some exosomal proteins are involved in the development of GC. TGF-β1 is an immunosuppressive cytokine that has been detected in exosomes of GC patients and was found to be correlated with lymphatic metastasis ,53. Trippatients . HSP-60 patients . These eBARHL2, LINE1, SOX17, and miRNA-34b/c gene. Gastric juice-derived exosomal BARHL2 gene methylation was suggested to have promising potential as a biomarker with GC patients being more likely to have BARHL2 methylation compared to non-GC controls (90% sensitivity and 100% specificity) [LINE1 methylation whereas SOX17 gene methylation was detected in both early and advanced gastric cancer of both intestinal and diffuse type [The use of exosomal DNA for the diagnosis and prognosis of GC is an area of research rarely targeted in the literature. There have been only three studies investigating this up until now with only four exosomal genes identified in relation with GC so far: ificity) . Anotheruse type . These fIt has been proposed that exosomal miRNAs have promising potential as diagnostic molecules for GC tumors. Ren et al. extracted exosomes from GC cell lines and non-GC cell lines and found that the exosomes of GC cell lines contained higher levels of miRNA-21-5p and miRNA-30-p compared to the non-GC cell lines . AnotherAlthough there is a wide range of therapies available for the management of gastric cancer, the molecular and clinical heterogeneity associated with the disease has led to newer classifications of GC patients which provide therapeutic approaches based on the genome and clinical evidence. Current guidelines recommend all patients eligible for systemic treatment undergo molecular profiling to determine the appropriate therapy and treatment strategy.The human epidermal growth factor receptor (ErbB or EGFR) family is composed of four types of tyrosine kinase receptors (TKRs): EGFR (ErbB-1 or HER-1), HER-2 (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4). These receptors play a critical role in cell growth, proliferation, and migration of tumors . It has Normally, when a ligand  binds EGFR, it induces tyrosine phosphorylation, which stimulates multiple downstream signaling cascades that in turn promote cell proliferation, angiogenesis, migration, survival, and adhesion. Deregulation of EGFR signaling can occur through multiple mechanisms such as receptor overexpression, activating mutations, and gene copy numbers (GCNs) .Although EGFR amplification has been shown to occur in a significant proportion of gastric cancers, there is no general consensus on its prognostic value. Some studies suggest that a higher overexpression is associated with poorer outcomes ,94 whilep > 0.05). However, two larger phase III randomized trials, REAL3 and EXPAND, demonstrated that there was no improvement in survival for patients with advanced gastric cancer treated with anti-EGFR therapy [p = 0.013) worse mOS in the panitumumab subgroup and the EXPAND trial showed a statistically significant (p = 0.032) worse mPFS in the cetuximab subgroup. Hence the evaluation of EGFR inhibition was abruptly abandoned for gastric cancer.Early phase II clinical trials have suggested a potential benefit for the use of EGFR inhibitors in patients with gastric cancer. For example, Richards et al.  demonstr therapy ,75. In fHowever, one caveat to the phase III trials was that there was no patient selection performed on the basis of EGFR amplification/overexpression which rendered the results as questionable due to the heterogeneity in the expression of EGFR in gastric cancer . To addrHER-2 is a proto-oncogene that encodes the transmembrane receptor-like HER2 protein. When activated, it initiates signaling pathways that lead to cell proliferation, differentiation, and vascular and lymphatic angiogenesis . HER-2 oThe anti-HER-2 monoclonal antibody, Trastuzumab, has been the only well-established cornerstone management for many years for advanced HER-2 positive gastric cancer. The landmark phase III ToGA trial  conducteAnother monoclonal antibody investigated for use in AGC is pertuzumab. It has a similar mechanism of action as trastuzumab except that pertuzumab binds to the dimerization domain of HER-2, which prevents HER-2 heterodimerization with other HER family receptors, whereas trastuzumab binds to the transmembrane domain, which prevents HER-2 dimerization . The phap = 0.01) and ORR  compared to patients on chemotherapy alone, in patients with AGC as a third-line or later therapy. Following this study, the FDA approved T-DXd for use in AGC after failure with a trastuzumab-containing regimen. RC48 is another ADC that linked humanized anti-HER-2 IgG1, a valine–citrulline linker, and MMAE (a microtubule inhibitor) together, and a phase II study [Antibody–drug conjugates (ADC) are another therapeutic strategy currently being investigated for AGC. Trastuzumab emtansine (T-DM1) is an ADC consisting of the anti-HER-2 monoclonal antibody trastuzumab with the tubulin inhibitor emtansine. Emtansine is released into HER-2-positive tumor cells to cause mitotic arrest and apoptosis . HoweverII study  found anp = 0.91), although the PFS  and ORR  were significant [p = 0.1044) or mPFS , despite a significant ORR  [TKIs are another category of drugs used to target HER-2 in AGC. Lapatinib is a small molecule TKI that inhibits both EGFR and HER-2, which results in reduced intracellular signaling and hence suppressed tumor proliferation . The phanificant . The TyT< 0.001) .The process of angiogenesis is modulated by the interaction of VEGF with its TKRs, known as VEGFRs. There are four types of VEGF  that have been identified along with three types of VEGFRs  .Therapies targeting this pathway can be either monoclonal antibodies or TKIs. Even though there is no measurable predictive factor to determine which patients respond better to VEGF pathway inhibition, expression of VEGF has been seen to occur in almost 48% of gastric cancers and is associated with poorer prognosis . SeveralBevacizumab is an anti-VEGF-A monoclonal antibody that inhibits circulating VEGF-A activity . AlthougMany phase II trials ,139,140 Evasion of the immune system is an established hallmark of cancer . ProgramIn the “era of revolution” in cancer management with immunotherapy, there have been attempts to integrate immune checkpoint inhibitors in the therapeutic algorithm for AGC. Gastric tumors that are Epstein–Barr virus (EBV) positive and microsatellite-unstable (MSI) have been shown to be potentially most responsive to immunotherapy drugs . EBV-posBlocking the PD-1/PD-L1 interaction can enhance the immune response against tumors. Pembrolizumab is a humanized IgG4 monoclonal anti-PD-1 antibody. The phase II KEYNOTE-059  trial shAvelumab is an anti-PD-L1 monoclonal antibody that was investigated in the phase III JAVELIN Gastric 100 study  as a maip = 0.00006). The results of the KEYNOTE-811 study led the FDA to grant accelerated approval on pembrolizumab plus trastuzumab and chemotherapy for first-line treatment of HER-2-positive gastric cancer in May 2021 [Immune checkpoint inhibitors have also been combined with other targeted therapies to produce promising results. For AGC, studies have mainly investigated the combination of antu-HER-2 monoclonal antibodies and VEGF/VEGFR inhibitors with immunotherapy. The phase II PANTHERA  study inMay 2021 .Margetuximab is a novel monoclonal antibody that binds to the same HER-2 dimerization domain as trastuzumab but, in contrast to trastuzumab, it has increased binding to the activating Fcy receptor IIIa and decreased binding to the Fcy receptor IIb, which results in enhanced anti-tumor activity compared to trastuzumab . FurtherNivoRAM was a phase I/II study that investigated the efficacy of combining nivolumab with paclitaxel and ramucirumab as a second-line treatment of AGC and the results showed patients to have an ORR of 26.7% . The EPOCombinations of different immune checkpoint inhibitor medications, mainly PD-1/PD-L1 inhibitors with CTLA-4 inhibitors, have also been explored for gastric cancer. The rationale behind this combination could be due to the fact that one of the causes of resistance to PD-1/PD-L1 blockade is the presence of immune suppression through other immune checkpoints, such as CTLA-4, which is a key negative regulator of anti-tumor T-cell response . The phaTAS-102 is an oral cytotoxic drug composed of trifluridine (TFD), an analog of the thymidine-based nucleoside which inhibits tumor cell growth by being incorporated into DNA during DNA synthesis, and tipiracil (TPI), a molecule which inhibits the metabolism of TFD, thereby prolonging its ability to exert effect . Table 5The mesenchymal-epithelial transition factor receptor (c-MET) is a proto-oncogenic receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF). Activation of c-MET receptor promotes tumor formation through increased mitosis and inhibition of apoptosis. C-MET overexpression and gene amplification is a marker of poor prognosis in gastric cancer . RilotumAlthough phase II studies  have sugSome tyrosine kinase inhibitors of the c-Met/HGF pathway, such as foretinib  and tivaThe fibroblast growth factor receptor (FGFR) has four family members: FGFR-1, FGFR-2, FGFR-3, and FGFR-4. Of these, FGFR-2 has been shown to be the most frequently amplified and altered in gastric cancer, being overexpressed in around 2–30% of GCs . Table 7Poly (ADP-ribose) polymerase (PARP) is important for DNA single-strand break repairs. In tumor cells that possess homologous recombination deficiency, inhibition of PARP can lead to the formation of single-strand breaks which are then transformed into DNA double-strand breaks (that are unable to be repaired through homologous recombination), ultimately leading to genomic instability and tumor cell death . Table 8Matrix metalloproteinase-9 (MMP-9) is known to promote wound healing through collagen deposition as well as activation of cytokines and growth factors. MMP-9-mediated cleavage of cytokines such as interleukin (IL)-8 and IL-1β can induce tumor growth. MMP-9 also cleaves and activates growth factors such as VEGF and FGF-2. Inhibition of MMP-9 can suppress the tumor micro-environment and reduce tumor growth . HoweverThe phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) is activated in 30% and 60% of GCs, respectively, and is associated with tumor progression . EveroliIn conclusion, there have been significant recent developments in the detection and treatment of GC. Although there has been improvement, there are still numerous challenges. The amount of clinical data is growing every day but despite this, there are currently not enough high-quality, well-designed multi-center prospective trials available. Furthermore, the enormous inter-tumor and intra-tumor heterogeneity of GC across individuals and populations results in a lag in transitioning the current molecular research into clinical practice for patient benefit . The dia


Paediatric cervical spine trauma, though rare, is difficult to detect as the injuries are often soft-tissue injuries and thus not visible using conventional radiography. A 6-and-a-half-year-old child presented with neck pain following a fall. A thorough radiological workup over several days demonstrated soft-tissue injuries, undetected by initial cervical X-rays, requiring MRI to definitively prove. The patient recovered with conservative treatment.Teaching Point: Paediatric cervical spine injuries often present with soft tissue injuries, which can missed on X-rays and require further imaging to detect. Paediatric cervical spine trauma is rare, with an incidence only half of that of the adult population 1–1.3%), generally due to motor vehicle crashes (MVCs) (57.51%), followed by falls and sports –1.3%, ge. PaediatImportantly, children are more susceptible to spinal cord injury without radiographic abnormality than adults, though by age 8 to 10 the injury pattern resembles that of the adult population as the spine has matured .A 6-and-a-half-year-old boy presented to the emergency department with neck pain after falling from a bouncy castle. Initially, a cervical X-ray was ordered where a C3-C4 retrolisthesis was described . The patMost paediatric vertebral injuries involve the cervical spine, as opposed to lumbar and thoracic spine injuries in adults . They al1Eight predictors of cervical spine injuries, the PERCARN (Pediatric Emergency Care Applied Research Network) criteria, allowing detection of up to 98% of cervical spine injuries, are altered mental status, focal neurological deficit, torticollis, substantial torso injury, neck pain, predisposing medical conditions, diving, and high-risk MVCs 3.The Easter Association for the Surgery of Trauma (EAST) deems CT the best initial modality regardless, in contrast to the American College of Radiology’s recommendations, which prefer X-rays . For hig2Lateral radiography is the most sensitive view for paediatric spine injury, though only 74–85% sensitive for fractures 3. The od222The use of CT as a screening tool is debated, with some studies showing superior sensitivity whilst others finding no differences for significant injuries when compared to standard radiographs . It shou2Finally, MRI is the most specific and sensitive method for paediatric spine trauma but has limited availability and is subject to other technical barriers like the need for anaesthesia . HoweverIn the paediatric population soft tissue and ligamentous injuries are more common and may be the only signs of injury 3. These Of the types of injuries than can occur AOSpine B injuries (distraction) occur most frequently in children under 3 and decrease with age. These tend to be injuries of the posterior tension band, seen as widening of the interspinous distance or uncovering of the facets [The clinical significance of findings such as soft tissue contusion, oedema in the interspinous ligaments with intact anterior and middle pillars and isolated disruption of the nuchal ligament is unclear . When a Paediatric spine injuries are relatively rare, can be difficult to detect, and are an important entity due to their high morbidity, mortality, and the frequent necessity of surgery 3. The im


Secondary infections were significantly higher in patients who were on invasive mechanical ventilation (p < 0.001), had central venous access (p = 0.023), and urinary catheters (p < 0.001). The mortality rate was significantly higher in those with secondary infections . Our study concluded that COVID-19 patients admitted to ICUs have a high prevalence of HAIs associated with greater mortality. Key factors need to be addressed to reduce HAIs.Healthcare-associated infections (HAIs) have a considerable impact on morbidity, mortality and costs. The COVID-19 pandemic resulted in an appreciable number of hospitalized patients being admitted to intensive care units (ICUs) globally with a greater risk of HAIs. Consequently, there is a need to evaluate predictors and outcomes of HAIs among COVID-19 patients admitted to ICUs. A retrospective study of patients with COVID-19 admitted to ICUs of three tertiary care hospitals in the Punjab province over a five-month period in 2021 was undertaken to ascertain predictors and outcomes of HAIs. Of the 4534 hospitalized COVID-19 patients, 678 were admitted to ICUs, of which 636 patients fulfilled the inclusion criteria. Overall, 67 HAIs were identified among the admitted patients. Ventilator-associated lower respiratory tract infections and catheter-related urinary tract infections were the most frequent HAIs. A significantly higher number of patients who developed HAIs were on anticoagulants ( They represent a significant public health problem in terms of increasing hospital stay, morbidity and mortality as well as associated costs, including patients with coronavirus disease of 2019 (COVID-19) ,4,5,6,7.tobacter . Overalltobacter . In Chin$20,377–$8,235 forLower rates of HAIs were recently reported in Scotland at approximately 1% of acute care patients pre-COVID-19 . OverallGenerally, the burden of HAIs is higher in low- and middle-income countries (LMICs) compared with high-income countries. This arises because of typically suboptimal adherence to infection prevention and control guidelines, lack of effective policies to prevent HAIs, scarcity of strict regulations and their monitoring, inadequate hand hygiene protocols and a lack of antimicrobial stewardship programs (ASPs) ,20,21,22HAIs can develop in a hospital setting in patients receiving health care after 48 h or more after hospital admission ,24. InvaThe COVID-19 pandemic considerably impacted on healthcare delivery systems, appreciably increasing hospitalizations, especially in ICUs ,35,36,37Pakistan is a low-middle income South Asian country where the COVID-19 pandemic has appreciably impacted morbidity and mortality, with five different disease waves reported since the first reported case on 26 February 2020 ,49. A prTo date, there appears to be no study conducted in Pakistan to ascertain the prevalence of HAIs among COVID-19 patients admitted to ICUs. This is important given the increased morbidity and mortality among such patients ,25,52. CA total of 8740 suspected (results waiting) and/or confirmed (by RT-PCR) COVID-19 patients visited or were brought to the designated hospitals during the study period, with 4534 COVID-19 positive patients subsequently hospitalized. Of these, 678 patients were admitted to the ICUs. A total of 636 cases were subsequently included in the final analysis .p = 0.039) and patients who presented with critical diseases. The demographic details and clinical features of the COVID-19 patients admitted to the ICUs with or without HAIs are shown in A total of 67 HAIs from different microorganisms developed among the 62 patients with HAIs on the ICU giving a prevalence of 9.7%. Out of the 67 HAIs, 23 were ventilator-associated lower respiratory tract infections, 16 catheter-related urinary tract infections, 14 catheter-related blood stream infections, 12 blood infections of unknown origin and two were fungal infections .Staphylococcus aureus, pseudomonas aeruginosa and Klebsiella pneumoniae were identified in 18, 17 and 13 patients, respectively.Details of the pathogens causing these HAIs are presented in p = 0.003), antithrombotic agents (p < 0.001), antivirals (p < 0.001) and IL-6 inhibiting agents (p < 0.001) compared with the non-HAI group. In the case of anticoagulants, it was not possible to differentiate their prescribing for either prophylaxis or treatment. The rate of secondary infection was significantly higher in patients who were on invasive mechanical ventilation (p < 0.001), had central venous access (p = 0.023), or had a urinary catheter placed (p < 0.001).The use of antipyretics, corticosteroids and antibiotics was universal (100%) in the studied cases . Nearly The predictors of HAI development are shown in p < 0.001). Overall, 96% of patients admitted to the ICU with COVID-19 had a complete recovery and were discharged from the hospital, whereas 3.6% (n = 23) died. The mortality rate though was significantly higher in patients who developed HAIs .As shown in p < 0.001) and heart disease  were the independent predictors of mortality.In our multivariable-adjusted model using covariates that were significantly associated with mortality in the univariate analysis, e.g., COVID-19 severity , underlying heart disease, invasive mechanical ventilation, presence of a urinary catheter, and HAI development, HAIs  just after the emergence of the COVID-19 pandemic, who indicated the prevalence of HAIs among all patients admitted with COVID-19 was 7.1% in a single centre in Wuhan, China . In a stOur study highlighted that the most common HAIs reported among patients with COVID-19 admitted to the ICU were ventilator-associated lower respiratory tract infections, i.e., ventilator-associated pneumonia, followed by catheter-associated urinary tract infections and catheter-related blood stream infections. These findings are similar to those of Grasselli et al. (2021) , with hiStaphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae, which is similar to other studies; however, others have reported differences [Acinetobacter baumannii were most frequent cause of HAIs among critically ill hospitalized COVID-19 patients [The most frequently isolated microorganisms associated with HAIs in our study were ferences ,72,73. Oferences , with a patients . DiffereOur study also revealed that HAIs among ICU patients developed more often among those aged above 50 years and with comorbidities including diabetes mellitus and hypertension, as well as with COVID-19 severity, abnormal chest X-rays and laboratory investigations including C-reactive proteins and white blood cells. This is similar to studies from Belgium, China, Italy and Spain as well as among a number of LMICs ,61,64,65Similar to other studies ,45,52, wFinally, we are aware that 100% of COVID-19 patients in the ICU were prescribed antibiotics irrespective of whether they had an HAI or not. This likely reflects clinical uncertainty and concern for bacterial co-infection in patients who are severely unwell and vulnerable to acute deterioration. High antibiotic prescribing among patients admitted to hospital with COVID-19 has been observed widely in Pakistan and across countries and is in direct contrast with the low rates of bacterial co-infection observed in numerous studies ,76,77,78We are aware that there are a number of limitations with our study. Firstly, we collected data from only three public sector tertiary care hospitals. Consequently, we cannot generalize our findings to the whole of Pakistan. Secondly, we did not collect information from private sector hospitals. However, this was deliberate as patients admitted to ICUs in tertiary public hospitals are likely to be more critically ill than those in private or lower-level public hospitals. Despite these limitations, we believe our findings will facilitate clinicians, public health experts and policy makers regarding potential measures to reduce HAIs among hospitalized patients particularly those with COVID-19. This information can subsequently lead to the development of possible quality improvement programmes as part of ASPs among ICUs in Pakistan to reduce future HAI rates.A retrospective review of medical records was conducted among patients admitted to the ICU due to COVID-19, in three purposely selected public sector tertiary care hospitals, designated for COVID-19 in the Punjab province. Data was collected for all COVID-19 ICU patients over a five-month period from April to August 2021, which was during the third and fourth COVID-19 waves in Pakistan. This methodology involving a retrospective review of patient records is similar to other studies involving the co-authors ,81,82,83All three hospitals were equipped with the necessary equipment and devices, including mechanical ventilators, laboratory facilities and medicines to treat patients with COVID-19.A data collection form was designed based on published studies, as well as current guidelines ,26,84,85The draft data collection form was tested in a pilot study involving 30 patients to see if the developed forms were able to capture the necessary data sets for the study. These patients were subsequently excluded from the full study. Based on the pilot study results, no modification to the data collection form was necessary.The team of investigators  visited the three hospitals after permission was granted by the hospital administration. They were provided with full training by the principal investigator (ZUM) before commencing the data collection process. The medical records of patients were accessed and reviewed thoroughly to obtain the required information. All the records were in a paper-based format and in case of any uncertainties, healthcare professions were requested for subsequent interpretation.The inclusion criteria were any patient with confirmed COVID-19 (confirmed via RT-PCR testing) admitted to the ICU between April and August 2021, among the three selected hospitals, who had a complete medical record available in the record room. All other patients admitted to the hospitals with COVID-19, but who were not admitted to the ICU, those admitted to the ICU with COVID-19 but outside the time period for the study, and those with incomplete medical records, were excluded from this study.® sheet, cleaned and after coding imported into the Statistical Package for the Social Sciences . The mean and standard deviation were calculated for continuous variables whereas frequency/number (N) and percentage (%) were calculated for categorical variables. Demographic data, clinical features of COVID-19 patients, and outcomes were compared between patients with HAIs and those with no HAI using χ2 test and Fischer’s exact test as appropriate. We performed binary logistic regression to determine the factors associated with the development of HAIs among COVID-19 patients as well as for the predictors of mortality. A p < 0.05 (two-sided) was considered statistically significant.The data were entered onto a Microsoft ExcelEthical approval was obtained from the Office of Research, Innovation and Commercialization (ORIC), Lahore College for Women University (LCWU), Lahore, Pakistan (Ref. no. ORIC/LCWU/395). Ethical approval of this study was also obtained from the ethics committees of the participating hospitals.® sheet accessible only to the researchers in order to ensure patient confidentiality.Since all data was obtained from medical records and drug prescription charts without patient contact, written consent was not required. This is similar to other retrospective and point prevalence surveys conducted by the co-authors ,93,94,95There is a high prevalence of HAIs among COVID-19 patients admitted to ICUs in Pakistan. This is associated with higher mortality, although mortality rates are lower than seen in a number of other countries potentially reflecting lower rates of mechanical ventilation and central venous catheters in the cohort studied. Independent factors associated with an increase in HAIs were the use of urinary catheters and mechanical ventilation. Tocilizumab was also independently associated with higher risk of HAI; however, this may reflect disease severity and longer hospital stay and requires further study. By adopting core elements of infection prevention and control, the prevalence as well as the mortality associated with HAIs can be minimized. This includes reviewing key issues and approaches surrounding the use of catheters alongside mechanical ventilation. Moreover, a multisectoral approach under a targeted antimicrobial stewardship programme (ASP) can help address inappropriate antimicrobial use among critically ill patients admitted to the ICU. We will now be working with key personnel in these hospitals to seek ways to reduce HAIs among ICUs in Pakistan through instigating appropriate ASPs and will be following this up in future studies.


While vaccination against COVID-19 is still ongoing, some rare adverse events temporally related to vaccinations have been reported, particularly with ChAdOx1 nCoV-19. Here, a 77-year-old male presented to our outpatient department with persistent ptosis of his left eye for 1 month. He initially received vaccination with ChAdOx1 nCoV-19 and developed symptoms of Bell's palsy 3 days later. He received a 14-day course of prednisolone, but the ptosis persisted. Marin-Amat syndrome was compatible with his symptoms of ptosis exacerbation during orbicularis oris exertion. A temporal correlation between ChAdOx1 nCoV-19 vaccination and Bell's palsy without infectious or autoimmune diseases was delineated. Further studies are needed to clarify the possible relationship between these two events. The coronavirus disease 2019 (COVID-19) global pandemic started in late 2019 and has recently been getting under control owing to the development of COVID-19 vaccines. One COVID-19 vaccine, ChAdOx1 nCoV-19, is an adenovirus-vectored vaccine and is nearly 100% effective in preventing serious events following COVID-19 infection  for 14 days; however, the left droopy eyelid showed little improvement. He then visited our ophthalmology clinic for persistent droopy eyelid despite a course of two-week prednisolone. His corrected visual acuity was 6/7.5 and 6/6 on his right and left eye, respectively. The intraocular pressure of both eyes was 18 mmHg. Slit-lamp examination was unremarkable except for puffy eyelids of his left eye with intermittent myokymic motion of his left upper and lower lids. Marginal reflex distance 1 (MRD1) of his left eye was 1 mm, in contrast to 3 mm on the right. His left eye was closed tightly as we asked him to open his mouth or smile  and started noticing that his left eyelid gradually became ptotic 3 days later. Eleven days after the initial vaccination, he experienced weakness of the left facial muscle. Physical examination revealed left-sided blink reflex impairment, ptosis, and weakness of left side facial muscle including orbicularis oris. Extraocular movement and light reflex were intact on both eyes. There was no vesicle/blistering noted on his face and his extraocular movement as well as light reflex were intact on both sides. Lab tests including thyroid function, vitamin Bor smile . Note thor smile . Marin-AThis paper reported a case of Bell's palsy in a patient who received the ChA-dOx1 nCoV-19 vaccine. The diagnosis of Bell's palsy presented after excluding the known etiology resulting in facial palsy. Although few studies have reported facial weakness after ChAdOx1 nCoV-19 vaccination, none have reported idiopathic Bell's palsy after ChAdOx1 nCoV-19 vaccination, to our knowledge. Diogo et al.  reportedThe etiology of Bell's palsy is speculated to be multifactorial. Virus reactivation and inflammation may play a major role in the pathogenesis of Bell's palsy . Howevervia exploiting the information in the WHO pharmacovigilance database, VigiBase. Notably, cerebral venous thrombosis was found to have a high association with COVID-19 vaccines  after an initial insult to the facial nerve . The patWhether the aberrant facial regeneration is related to vaccination is unknown. The diagnosis of Marin-Amat syndrome is often overseen, and prudent evaluation for patients with ptosis, especially those with persistent ptosis after facial nerve injury, is needed. This case exhibits features of Marin-Amat syndrome, including intermittent orbicularis oculi myokymia, which was exacerbated by jaw movement. This feature implies a previous facial nerve insult, which is thought to be Bell's palsy, in our case.This is the first case report delineating an episode of Bell's palsy complicated with Marin-Amat syndrome following ChAdOx1 nCoV-19 vaccination. Further studies are warranted to clarify the relationship between Bell's palsy and ChAdOx1 nCoV-19 vaccination.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Institutional Review Board of Tri-Service General Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Conceptualization: P-FT and Y-JC. Methodology, software, validation, formal analysis, investigation, resources, writing—review and editing, visualization, supervision, and project administration: Y-JC. Data curation and writing—original draft preparation: P-FT. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The double-positive disease is the co-occurrence of antiglomerular basement membrane (anti-GBM) disease and antineutrophil cytoplasmic antibodies (ANCAs) and is an uncommon cause of renal failure. Our case of triple-positive disease is an even rarer cause of isolated renal failure, as it includes anti-GBM, antimyeloperoxidase (MPO), and antiproteinase 3 (PR3). We present a case of a 62-year-old Caucasian male with a history of multiple comorbidities, who presented to the emergency department (ED) with worsening dyspnea on exertion that started about one month prior to admission. He was found to be in renal failure secondary to triple-positive disease. We believe that the likely mechanism of our patient's triple-positive disease was a drug-induced ANCA vasculitis overlapping with Goodpasture's syndrome. We believe our case to be a valuable addition to the literature as it is a rare overlap syndrome without a previously established disease course or etiology. Rapidly progressive glomerulonephritis resulting from the coexistence of antiglomerular basement membrane (anti-GBM) disease and antineutrophil cytoplasmic antibodies (ANCAs) is an unusual cause of renal failure and is regarded as a “double-positive” disease (DP) . DPD is A 62-year-old Caucasian male with a past medical history of hypothyroidism, vitamin D deficiency, hyperlipidemia, prediabetes, coronary artery disease status postright coronary artery stent placement, and subarachnoid hematoma presented to the emergency department (ED) with worsening dyspnea on exertion that started about one month prior to admission. Symptoms started a few days after flu-like symptoms. It was associated with persistent productive cough, fatigue, nausea, vomiting, diarrhea, generalized weakness, decreased appetite, change in the color of his urine, and myalgia. He denied any chest pain, dizziness, headache, lower extremity edema, recent travel history, or sick contacts. He worked as a supervisor in a construction company. He drank about 1-2 bottles of beer daily. He smoked 2 packs of tobacco per day . He denied any use of illicit drugs. Family history was remarkable for gastric carcinoma in the mother. He received 2 doses of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Of note, he endorsed taking daily high doses (unspecified) of ibuprofen and naproxen about 3–4 times a day for 30 days, due to persistent severe myalgia. His other home medications included alprazolam, atorvastatin, oxycodone-acetaminophen, levothyroxine, aspirin, and cyclobenzaprine.2. The cardiopulmonary examination was unremarkable except for tachycardia. There were no murmurs, rubs, or gallops. Lung sounds were audible on auscultation. There was no jugular venous distention or lower extremity edema. Bilateral involuntary rhythmic tremors in his hands at rest and with the movement were noted. Laboratory studies  was 25.54 kg/m studies  revealedThe electrocardiogram and chest X-ray was unremarkable. Computed tomography of the chest and abdomen showed nonobstructing right renal calculi with bilateral perinephric stranding, confirmed with renal ultrasound. There was no hydronephrosis. The patient had been oliguric and was admitted for the management of acute kidney injury (AKI). Within 12 hours of the presentation, he was placed on a bicarbonate drip and then underwent urgent hemodialysis (HD). 16 hours later, he had a fever of 102°F with leukocytosis and was started on empiric antibiotics. Blood culture grew Enterococcus faecium only in 1 bottle. Despite undergoing 3 rounds of scheduled HD sessions , there was no improvement in his Cr level or urine output, prompting further workup. Autoimmune panel results are shown in the following :A diagnosis of double-positiveanti-GBM and ANCA was made. There was a collective management approach involving nephrology, infectious disease, and hematology. The patient was started on plasmapheresis in addition to steroids, with recommendations to obtain a renal biopsy to help determine the duration of therapy (7 days or 2–3 weeks). On day 6, a right renal biopsy was obtained, and the result confirmed severe anti-GBM (Goodpasture syndrome) with 100% glomerular involvement , exhibiting antiglomerular basement (anti-GBM) and antineutrophil cytoplasmic antibodies (ANCAs) are auto-antibody mediated small vessel vasculitis with ideal preference to renal and pulmonary systems . SeveralM in GPS . ContrasM in GPS . ANCA-asM in GPS , 7, 8.DAPD has a reported incidence of 0.00006% worldwide and accounts for about 25% of cases of GPS , 9. In aα3 chain of type IV collagen) to the development of antibodies against anti-GBM (GPS) [The pathophysiology of this disease has been speculated because of an initial injury to the BM triggered by ANCAs, exposing the antigens (NC1 domain of BM (GPS) . Also, cBM (GPS) , 12, 13.Diagnostic criteria for GPS include 1: the presence of clinical symptoms, 2: positivity serology tests, and 3: histological findings from the kidney or lung biopsy. These findings include linear staining with IgG under immunofluorescence, using light microscopy . In addiDAPD is one of the 10 indications for therapeutic plasmapheresis (TP). A combination of this treatment with immunosuppressive therapy like steroids and cyclophosphamide has shown good benefits in several reported retrospective studies and case reports , 15. TheOur patient is a Caucasian male who presented to the ED with complaints of dyspnea on exertion a few days after flu-like symptoms. We initially speculated that his acute kidney injury was a result of his use of high doses of ibuprofen & naproxen use plus the rhabdomyolysis as was evident with elevated creatinine kinase level. Renal failure was refractory to maintenance fluid and continuous renal replacement therapy, prompting further workup. Serology tests with renal biopsy-proven results confirmed a “triple positive” (anti-GBM + MPO/p-ANCA + PR3/c-ANCA) GBM disease. Only about 2.9% of this group of patients have been reported .A possible hypothesis is that this was the rare co-occurrence of drug-induced ANCA vasculitis and Goodpasture syndrome in the same patient. Drug-inducedANCA-associated vasculitis (AAV) does not yet have a well-established pathogenesis , so it iDual ANCA is extremely rare outside the setting of drug-induced ANCA and is in fact, only seen every now and then, even due to drugs . Such caThere are no established markers in either laboratory or clinical findings to differentiate primary AAV from drug-induced AAV with certainty. However, some retrospective studies comparing ATD-induced AAV (arguably the most common drug-induced AAV) and primary AAV did reveal certain patterns. Serum-positive ANA was seen more frequently in ATD-induced AAV, as well as a tendency to have multiple target antigens including MPO and PR3. In primary AAV, ANCAs were generally recognized as either MPO or PR3 . Such fiOur patient as mentioned of course, also had a significant increase in CK levels >4000 on presentation. It is also of interest to note a seemingly insignificant, however, possibly relevant detail in the presentation of our patient, where he stated that his symptoms all began after a flu-like illness. A history of upper respiratory infections has been noted in other patients with ANCA-associated glomerulonephritis as well. The mechanism suggested explaining this observation in individuals with a genetic predisposition and infections that could trigger NET production and MPO exposure .Thus, we believe that the etiology of our patient's triple positive condition was likely multifactorial, with statin use as the culminating factor in the cascade of contributing events. This case also emphasizes the importance of keeping in mind a broad array of contributing factors while taking patient history. Only through a thorough review of each unique patient presentation can we uncover new patterns that could then be studied to help us in prevention.Multiple mechanisms have been hypothesized when it comes to drug-induced AAV, but it is certain that all the mechanisms involved are not yet known. The importance of unearthing more cases of drug-induced AAV becomes even more significant in such a scenario where studying new drugs causing these cases could lead to the discovery of novel mechanisms that have yet not occurred to us. Additionally, the matter of what predisposed this individual to the development of such an autoimmune condition is certainly a matter of interest, especially given that Goodpasture syndrome has been notorious for developing with an abrupt onset and no prior indicators. If we were able to unearth certain patterns in the presentation of these patients, perhaps we could be more vigilant in identifying patients at risk for both Goodpasture syndrome as well as AAV. In cases such as these, where timely treatment is of the essence, such a discovery would indeed make a true difference in the disease course of these patients.