This systematic review focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients that had detected SARS-CoV-2 virus in cerebrospinal fluid (CSF). A systematic literature search was carried out in PubMed, Embase, Scopus, Web of Science, Medrxiv, and Biorxiv databases from inception to 19 December 2021. Case reports or case series involving patients with proved SARS-CoV-2 presence in CSF by polymerize chain reaction were included. Our search strategy produced 23 articles documenting a total of 23 patients with positive SARS-CoV-2 in the CSF. Fever (55%) was the most common symptom, followed by headaches (41%), cough (32%), and vomiting/nausea (32%). The majority of the cases included was encephalitis (57%), 8 of which were confirmed by magnetic resonance imaging. The second most prevalent presentation was meningitis. The cerebral spinal fluid analysis found disparities in protein levels and normal glucose levels in most cases. This study demonstrates that SARS-CoV-2 can enter the nervous system via various routes and cause CNS infection symptoms. SARS-CoV-2 has been shown to infect the CNS even when no respiratory symptoms are present and nasopharyngeal swabs are negative. As a result, SARS-CoV-2 should be considered as a possible cause of CNS infection and tested for in the CSF. In December 2019, the first cases of coronavirus disease 2019 (COVID-19), a disease related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were identified in China . PatientA preliminary retrospective study of 214 hospitalized patients found that 36% of them had neurological symptoms . AccordiThe main objective of this systematic review of case reports is to define the change in CSF composition, presentations and outcomes in clinical scenarios of patients with confirmed PCR SARS-CoV-2 viral ribonucleic acid (RNA) presence in CSF.The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline was used to record this systematic review see  14]. Th. Th14]. We developed our search strategy in PubMed using Medical Subject Headings (MeSH) terms for COVID-19 and central nervous system infections in case reports and case series study types. Then, Polyglot translator was used to transfer the developed search strategy to Embase, Scopus, and Web of Science . SimilarWe included case reports, case series, or letters that met the following criteria: (1) cases with CNS inflammation (2) with confirmed presence of SARS-CoV-2 RNA in the CSF using PCR (not SARS-CoV-2 antibodies in CSF) (3) without a confirmed co-infection with another pathogen in the CSF (4) and in a clinical scenario . We accepted any diagnostic criteria presented in the included article to characterize CNS disorders because various studies had varying criteria for defining CNS disorders. Encephalitis should be detected using magnetic resonance imaging (MRI), an electroencephalogram (EEG), or CSF analysis while a computed tomography (CT) scan is only accepted if there is clear evidence and MRI was not performed, whereas encephalopathy can only be diagnosed using physical signs, according to the included article. Meningitis diagnosis must be confirmed either by CSF analysis or MRI. Included studies were not restricted to patient demographics, and there were no language or publication date restrictions. We excluded cases where CNS inflammation was due to any SARS-CoV-2 vaccines. Google Translator was used to translate all articles written in a foreign language into English when needed.The records obtained from the literature search were further evaluated using the Rayyan platform to screen titles and abstracts . Titles We extracted data from each eligible study on patient demographics , country, presenting symptoms, physical examination, method of SARS-CoV-2 diagnosis, CSF analysis , other lab investigations, diagnosis, and outcome. Data extracted from each study was conducted by two reviewers independently, with any inconsistencies referred to a third reviewer. All of the data were summarized and compiled into an online Excel spreadsheet that was accessible to all of the authors.Case reports are by their nature biased. In systematic reviews of case reports, however, standardized tools have been created to assess their methodological quality. To assess the quality of the case series and reports included in the study, we used Murad et al.’s modified Newcastle–Ottawa Scale (NOS) . SelectiData extracted from each article were summarized and presented in a table. The cases were then described narratively in the text to combine and highlight the similarities between them and, where possible, to draw conclusions. Due to the descriptive nature of this systematic review and the small number of cases, we employed descriptive statistics to present demographics and clinical characteristics. Means were used to report continuous variables, while using frequencies and percentages were used for dichotomous variables. The 23 articles that met our inclusion criteria reported on 23 individual patients from 12 different countries. Information about COVID-19 presenting signs and symptoms in children was available for all the cases. Of the 23 cases, 14 had fever (61%), 7 cases had headaches (39%), 7 patients had cough (30%), 5 cases had vomiting/nausea (22%), 3 had fatigue (13%), 2 cases had myalgia (9%), 2 had shortness of breath (9%), and no one had upper respiratory symptoms, rhinorrhea, sneezing, or nasal congestion. No studies reported data points on the presence or absence of pediatric anosmia or dysgeusia. Thirteen of the 23 (57%) patients were identified with SARS-CoV-2-associated encephalitis. Three instances were diagnosed as meningoencephalitis, one case as acute necrotizing encephalopathy, one case as anti-N-methyl-D-aspartate (NMDAR) encephalitis, one case as limbic encephalitis, one case as encephalomyelitis, and the other six cases as encephalitis without a defined classification. Eight of the 13 instances were identified using brain MRI, one using brain CT, two using CSF analysis, one using EEG, and one using both CSF analysis and EEG. There were two cases of encephalopathy without evidence of encephalitis. One of them showed normal neuroimaging, while in the other case, MRI was not done. There were four single occurrences of meningitis, one of which had meningeosis carcinomatosis. Two patients were diagnosed using CSF analysis, one through MRI, and one through combined MRI and CSF analysis. The remaining four cases were acute transverse myelitis confirmed by Spinal MRI, acute cerebellitis diagnosed by brain MRI, brainstem encephalitis diagnosed by brain MRI, and demyelinating syndrome with normal neuroimaging. More details on the diagnoses and neuroimaging findings can be found in Using Murad et al.’s standardized tool for assessing case reports and case series quality , 6  , which cMultiple case reports in the literature have reported that lumbar puncture is contraindicated, and therefore CSF could not be obtained for SARS-CoV-2 testing ,52. In aTo give prompt and effective care to patients with COVID-19 CNS-related infection, guidelines for early imaging and CSF analysis should be developed. Lumbar puncture must be done when the risk of CNS-related COVID-19 is high as indicated by severe signs and symptoms of CNS infections and the presence of CNS hyperintense lesions or leptomeningeal enhancement in imaging ,68. MoreTo our knowledge, this is the first study that focuses mainly on the presence of SARS-CoV-2 in the CSF and that has attempted to combine all available evidence in the literature in a systematic methodology with a standardized appraisal of quality in included cases. Therefore, our review study is an essential first step toward a better understanding of the CSF changes and consequences accompanied by SARS-CoV-2 CSF infiltration. To conduct more rigorous research, epidemiological studies can expand on the findings presented here. This narrative synthesis of case reports has a number of drawbacks. Firstly, case reports are inherently subjective, provide a non-random sample, and in many cases do not allow for causality claims . In manyThis study shows that SARS-CoV-2 can be present in the nervous system via different routes, and it can present with CNS infection symptoms. SARS-CoV-2 has shown the ability to infect the CNS even when there are no respiratory symptoms and nasopharyngeal swabs are negative. Hence, SARS-CoV-2 should be considered as a possible cause of CNS infection, and testing for it in the CSF should be conducted.


Over 6.37 million people have died from COVID-19 worldwide, but factors influencing COVID-19-related mortality remain understudied. We aimed to describe and identify risk factors for COVID-19 mortality in the Colorado Center for Personalized Medicine (CCPM) Biobank using integrated data sources, including Electronic Health Records (EHRs). We calculated cause-specific mortality and case-fatality rates for COVID-19 and common pre-existing health conditions defined by diagnostic phecodes and encounters in EHRs. We performed multivariable logistic regression analyses of the association between each pre-existing condition and COVID-19 mortality. Of the 155,859 Biobank participants enrolled as of July 2022, 20,797 had been diagnosed with COVID-19. Of 5334 Biobank participants who had died, 190 were attributed to COVID-19. The case-fatality rate was 0.91% and the COVID-19 mortality rate was 122 per 100,000 persons. The odds of dying from COVID-19 were significantly increased among older men, and those with 14 of the 61 pre-existing conditions tested, including hypertensive chronic kidney disease  and type 2 diabetes with renal manifestations . Male patients who are older and have pre-existing kidney diseases may be at higher risk for death from COVID-19 and may require special care. Since its appearance in 2019, COVID-19 has caused 668 million reported infections and 6.73 million deaths worldwide, as of 18 January 2023 [Prior studies have identified several demographic factors such as age, sex, and race-ethnicity that influence COVID-19 mortality. Men are dying at higher rates than women, suggesting sex may be an important factor. According to Global Health 50/50 data, men make up the largest percentage of deaths from COVID-19 in the majority of countries, even in countries predominantly occupied by women . Age hasOther studies have identified pre-existing health conditions as risk factors for COVID-19 mortality, though study findings are inconsistent. According to an early study from the Chinese Center for Disease Control and Prevention, case-fatality rates for COVID-19 were higher in patients with pre-existing health conditions, with the highest rates for cardiovascular disease 10.5%), closely followed by diabetes 7.3%) and COPD (6.3%) .3% and C; however.5%, closIn addition to pre-existing conditions, previous research has investigated the possible link between COVID-19 and the gene that determines blood type, ABO, but the study results vary. Several studies have suggested there is a correlation between blood type and risk of transmission or severity ,13. HoweWhile vaccination adoption has helped to reduce mortality rates, the continued presence and changing nature of COVID-19 make it crucial to understand who is most affected by COVID-19 mortality and what other factors could affect the risk of death. This study expands knowledge on the determinants of COVID-19 mortality in Colorado using a unique population that integrates genetics with electronic health records (EHR) data. Using the Colorado Center for Personalized Medicine (CCPM) Biobank as an integrated data source, we were able to comprehensively evaluate pre-existing risk factors for COVID-19 mortality. In addition to characterizing case fatality and mortality rates for COVID-19, pre-existing health conditions based on phecodes as well as blood type were analyzed as risk factors for COVID-19 mortality while controlling for sociodemographic factors.We conducted a retrospective cohort study of participants of the CCPM Biobank. The full cohort selection is shown in De-identified data for studies of COVID-19 were provided by the Health Data Compass data warehouse, using unique masked IDs to connect electronic health records (EHR) information with genetic data and mortality for CCPM Biobank participants. This study population included only Biobank participants (enrolled as of 27 July 2022) who were active in the UCHealth system prior to COVID-19—where active was defined as having at least three face-to-face encounters with the UCHealth system—in any capacity which could range from routine physicals to chronic care, between January 2015 and January 2020.Phecodes, a high-throughput phenotyping tool to define disease status and our main exposure of interest, were constructed from ICD-9 and ICD-10 billing codes using the PheWAS R package . A persoThe main explanatory variables investigated were pre-existing cardiovascular diseases, diabetes, and respiratory diseases. These were selected a priori based on prior literature. The specific phecodes from these three categories were selected for analysis if they had at least 10 deaths from COVID-19. In addition, 12 phecodes that had at least 10 deaths from COVID-19 and were not previously reported in the COVID-19 literature were examined as “control” phecodes. These control phecodes were included to establish a benchmark in which to compare the outcomes and would support the validity of any correlations between the explanatory phecodes and COVID-19 mortality. Using these unrelated pre-existing conditions ensures there is not a selection bias for pre-existing conditions as a whole. A list of the 289 phecodes compiled  can be found in the p < 1 × 10−12 were removed prior to imputation using the TOPMed imputation server [A subset of the CCPM Biobank has genetic data currently available as described in detail in Wiley et al. Briefly, genotyping was performed for 34,435 Biobank samples on the Illumina MEGA-EX and MEGA-Plus platforms. Sample QC was performed by plate checking for sex discordance, excess heterozygosity, high missingness, genetic duplicates, and control sample concordance. SNP QC checking was performed for high missingness, high differential in allele frequency by sex, high differential in allele frequency by plate, and high discordance across 5 control samples consistent across all plates. Strand ambiguous, monomorphic, and SNPs with ancestry-specific Hardy–Weinberg equilibrium n server ,20. GenoGenetically predicted ABO blood types were derived from all Biobank participants with genotype data based on haplotypes of three SNPs, following the methodology of McLachlan et al. . The comThe Health Data Compass clinical data warehouse captures the mortality of UCHealth patients through routine linkage with vital records (death certificates) data provided by the Colorado Department of Public Health and Environment (CDPHE). Available mortality information included cause of death as certified by a physician or coroner/medical examiner, corresponding ICD-10 codes generated by the Centers for Disease Control and Prevention, and age at death. The response variable, death from COVID-19, was classified as a patient either having the ICD-10 code for COVID-19 (U07.1) coded as a cause of death or having “COVID-19” or its synonymous names  as contributing factors of death. COVID-19 infection was defined as those with a diagnostic ICD-10 code U07.1 in the EHR or who died from COVID-19. Due to ~3 month lag in death certificate registration, cause of death coding, and clinical data updates, mortality is expected to be ~95% complete. The study period included COVID-19 cases and deaths between January 2020 and July 2022.The covariates included as possible confounders included age, sex, healthcare utilization (HCU), and race/ethnicity. Given the de-identified nature of the dataset, all analytical variables were categorized as needed to maintain >10 subjects in each level to protect patient privacy. Age, defined by years since birth at the July 2022 data freeze, was taken from the EHR and categorized as follows: 18–49, 50–64, 65–79, and 80+. Sex  was also obtained from the EHR. HCU was defined by the number of face-to-face visits between January 2015 and January 2020, with three being the minimum number based on selection criteria. HCU was modeled in quintiles due to the strong right skew of the distribution, with categories (and cut points) at HCU-1 (3–10 visits), HCU-2 (11–22 visits), HCU-3 (23–42 visits), HCU-4 (43–85 visits), and HCU-5 (>85 visits). For the pre-existing health condition analyses, race and ethnicity were taken from the EHR and combined into a single race/ethnicity variable  due to the low-frequency sample sizes among racial-ethnic minority Biobank participants. For the blood type analyses that were conducted on participants with genetic information, ancestry principal components (PCs) were used as a better approximation for race/ethnicity than what was self-reported in the EHR. We were unable to include education and income level in statistical analyses as they were only available at aggregate levels based on a patient’s 3-digit zip code of residence.t-tests for categorical and continuous variables, respectively.All data analyses and figures were produced using SAS 9.4, R Studio, and R version 4.2.0. Case fatality rates for COVID-19 were calculated as the deaths due to COVID-19 in the Biobank divided by the total number of people in the Biobank who were diagnosed with COVID-19. Case fatality for COVID-19 was also calculated among those with cardiovascular disease, diabetes, and respiratory disease as sub-groups. The period mortality rate for COVID-19 was calculated as the deaths due to COVID-19 in the Biobank during the study period divided by the total Biobank population. These values were multiplied by 100,000 to obtain the number of dead per 100,000 people. This was repeated to calculate the mortality rates for cardiovascular disease, diabetes, and respiratory disease. Descriptive statistics  were calculated to characterize explanatory variables overall and by outcome group. Significance in outcome (death from COVID-19 versus survived COVID-19) was tested using chi-square and n = 61) at an alpha of 0.05 (p < 0.0008). This was repeated for the control phecode analyses (n = 12) at an alpha of 0.05 (p < 0.004).Unadjusted multiple logistic regressions were performed to identify associations between each of the pre-existing health conditions and the risk of death from COVID-19. These models were then adjusted for age, sex, HCU, and race/ethnicity. For these associations, odds ratios and 95% confidence intervals were obtained. Odds ratios were used as a measure of risk for death from COVID-19. Phecodes were tested in aggregate categories  and in smaller classifications for the 61 phecodes that had at least 10 deaths from COVID-19. This included 39 cardiovascular disease phecodes, 7 diabetes phecodes, and 15 respiratory disease phecodes. p < 0.05) by COVID-19 outcome. Compared with those who survived COVID-19, those who died from COVID-19 were older , enriched for a higher proportion of male participants , participants with pre-existing cardiovascular disease , pre-existing diabetes , and pre-existing respiratory disease . Due to the low number of participants with genotyped B and AB blood types, the number of COVID-19 cases and deaths for these groups were not able to be reported in order to maintain anonymity, however, the Fisher’s exact test suggested significance (p < 0.001).A total of 155,859 participants met the inclusion criteria, and 31,482 had genetic data available. Descriptive statistics for the explanatory variables and covariates are shown in The mortality rates calculated are shown in The results for the case fatality rates are shown in p < 0.0008. The phecodes with the largest odds ratios were hypertensive heart and/or renal disease  and hypertensive chronic kidney disorder . Of the seven diabetes phecodes, three were positively associated with COVID-19 mortality. The diabetes phecode with the largest increase in risk for death from COVID-19 was type 2 diabetes with renal manifestations . Of the 15 respiratory disease phecodes tested, four were positively associated with COVID-19 mortality. The respiratory phecodes with the largest effect sizes were dependence on a respirator, ventilator, or supplemental oxygen  and respiratory failure .The results of the adjusted logistic regression models for the 61 individual phecodes with at least 10 COVID-19 deaths are shown in p < 0.004  died from COVID-19. This case fatality rate was higher among those with pre-existing cardiovascular disease (1.55%), diabetes (2.13%), and respiratory disease (1.25%). Those who died from COVID-19 were more likely to be older and men compared with those who survived COVID-19. After adjustment for demographic factors, we identified 14 pre-existing conditions (phecodes) significantly associated with death from COVID-19, including several related to renal and kidney dysfunction.Our findings support what has been observed in other COVID-19 studies. Sex appears to be a relevant factor for COVID-19 outcomes, as seen by the higher proportion of men who died, despite making up a minority of the study population. The difference in outcomes based on sex has been apparent since the beginning of the pandemic. Previous studies suggest that this difference could be due to a combination of biological and social factors. Cardiovascular disease predominately occurs in men, specifically in those over 50 years, a comorbidity already linked to COVID-19 mortality ,10,11. IThe COVID-19 mortality rate of 122 deaths per 100,000 persons in the study population was greater than the Colorado COVID-19 mortality rate as of April 2022, which was 88 deaths per 100,000 . The morThe percentage of those who died from COVID-19 and also had pre-existing cardiovascular disease, diabetes, and respiratory disease  were double to triple the percentage of those in the Biobank with these conditions as a whole . This supported previous research that found that these preexisting conditions were more prevalent among those who died from COVID-19 . This isThere was not a significant association between aggregate categories of cardiovascular disease and respiratory disease and COVID-19 outcomes, in contrast to research at the start of the pandemic. It is possible that the level of significant risk for COVID-19 mortality among those with one of the two pre-existing conditions has changed from the beginning of 2020 to 2022 due to the introduction of COVID-19 vaccines. Recent studies have shown that despite the emergence of new variants, those who received the COVID-19 vaccine have a decreased risk of death as compared with those who did not receive the vaccine . TherefoThe high percentage of individual phecodes that were significantly associated with a negative outcome reflects previous research that looked more closely at specific diseases such as coronary heart disease , rather The retrospective design is a strength of the study, as the pre-existing conditions occurred in EHR prior to the appearance of COVID-19. For each patient, extra care was used to confirm that they had the pre-existing health condition listed on their EHR, requiring at least three separate records. By limiting these records to the five years preceding the COVID-19 pandemic, it confirmed that none of these conditions were COVID-19 related. By only including those who have had three face-to-face visits with the UCHealth system, we ensured that these were patients that used the UCHealth system as their main medical provider. A limitation of this project was that due to a small number of COVID-19 cases among those with genetic information, more informative analyses were not able to be performed for blood type, and no firm conclusions could be made. However, the use of genetic information in addition to health records may inform future studies. These procedures should be repeated as more genetic data is generated in the Biobank or larger studies with available genotyping and quality mortality data.This project adds to the continuously growing pool of information on the epidemiology of COVID-19 mortality using integrated data sources  to provide a more complete picture. Patients with pre-existing cardiovascular diseases and diabetes, especially those involving renal complications, and prior severe respiratory dysfunction who present to health systems with COVID-19 might be at higher risk for mortality and should be closely monitored.


In humans, an acutely traumatic experience can lead to post-traumatic stress disorder (PTSD), which is often characterized by changes in anxiety and motivation months after trauma. There are few demonstrations of the persistent motivational effects of an acute stressor in rodent approaches to PTSD. In two experiments, we evaluated the persistent effects of a battery of footshocks in one context on appetitive Pavlovian conditioning, instrumental learning, and Pavlovian-to-instrumental transfer (PIT) in a different context. In Experiment 1, a battery of footshocks before appetitive training caused deficits in single-outcome PIT (SO-PIT) in male Long Evans rats. The same battery of footshocks after appetitive training, but before testing had little effect on SO-PIT overall, but there were some deficits in within-stimulus expression of SO-PIT. In Experiment 2, the battery of footshocks had no effect on sensory-specific PIT in male or female rats, but two sex differences emerged: males showed more generalized fear from the aversive to the appetitive context compared to females, and females showed less evidence for sensory-specific PIT compared to males. Males showed robust sensory-specific PIT, with clear extinction and spontaneous recovery of the sensory-specific PIT effect across test sessions. These findings show that (a) an acute trauma can have persistent effects on general motivational processes and (b) in sensory-specific PIT, females may show transfer through generalized motivational processes, whereas males may rely on specific features of the cues and outcomes to augment instrumental responding selectively. We discuss implications for current approaches to stress and motivation in preclinical approaches to PTSD. Intensely stressful events can cause long-term aberrations in learning, memory, and motivation. In humans, the experience of either intense acute or chronic stress can lead to post-traumatic stress disorder (PTSD) which is characterized by persistent intrusive memories of the trauma, avoidance of reminders of the trauma, negative changes in cognition and mood, and alterations in arousal/stress reactivity, all which persist 30 days beyond trauma . A majorOne approach to evaluating persistent effects of acute stress is stress-enhanced fear learning (SEFL), which is based on years of work on the cellular, molecular, and behavioral mechanisms of fear conditioning. This approach results in a persistent hyperresponsivity to a mild stressor in one context following an exposure to a battery of intense footshocks in a different context . ChronicStress-enhanced fear learning has been adapted to study the persistent alterations of trauma on appetitive processes by following the shock battery session with appetitive training and testing in a novel context . Meyer eThe Pavlovian-to-instrumental transfer (PIT) procedure allows the influence of Pavlovian stimuli on instrumental behaviors to be assessed. This approach may capture aspects of general affective motivation for reward, as well as sensory-specific memories involving associations among stimuli, responses, and predicted outcomes . In singBecause PIT separates Pavlovian and instrumental memory processes, it is a powerful tool for investigating how trauma alters specific memory and motivational processes . Much ofN = 89); group sizes, and sexes are outlined below in individual methods sections. Rats were pair and triplicate housed and maintained on a reverse light-dark schedule. Experimental procedures were conducted during the dark phase of the cycle. For all experiments rats were food restricted to between 85 and 90% of their ad libitum weights and maintained at this weight for the duration. Weights were maintained by providing daily rations following each training session; for females and males, approximately 10 and 15 g per rat was needed to hold weights steady.Long Evan rats were used in all the experiments in this study (Two distinct contexts housed in separate rooms were used for fear conditioning (Ctx A) and for appetitive training and testing (Ctx B). Chambers differed in dimensions, patterned backdrops, olfactory cues, floor textures, and other internal features. All chambers were standard Med Associates operant chambers housed in sound attenuating cabinets. Ctx A was 29.53 cm × 24.84 cm × 18.67 cm (LxWxH) with metal rod grid floors , a horizontal zig zagged pattern back drop, and a single house light. For olfactory distinctiveness gauze pads infused with clover leaf essential oil (Crafter’s Choice™ Clove Leaf EO- Certified 100% Pure 1050) were placed into the scat collection trays at the base of the chambers. Ctx B was 29.53 cm × 23.5 cm × 27.31 cm with wire mesh inserts  placed over metal rod grid floors and a backdrop with randomly distributed differently sized squares or circles. For olfactory distinctiveness, citrus cilantro fragrance oil infused gauze pads were placed in the scat trays at the start of each session (Crafter’s Choice™ Citrus Cilantro Fragrance Oil 548). Internal features of Ctx B chambers included two retractable levers flanking a recessed food magazine, a single speaker above the magazine, and two flat lights above the levers on the front wall of the chamber. On the opposing wall, a single hooded houselight was situated at the top center. Food hoppers and syringe pumps were externally housed and connected to the magazine for delivery of pellet and liquid reinforcement.Rats were placed in Ctx A, where they received 15, 1 mA footshocks delivered through the metal rods of chamber’s grid floor. The session lasted 90 min and shocks were delivered on variable-time 360 s (VT 360″) schedule. Control rats were exposed to the same context for 90 min without any shocks. All sessions were video recorded for subsequent quantification of freezing behavior.All appetitive training was conducted in Ctx B. First, rats received magazine training to learn to collect pellet reinforcements  from the recessed magazine. Each session lasted 20 min, in which 20 pellets were delivered on a VT 60″ schedule. The number of magazine sessions differed across experiments; see individual experimental timelines for more details. Rats then learned to press an Active lever to earn pellets while responses on a simultaneously available Inactive lever had no consequence . Initially, rats were trained on a continuous reinforcement schedule (CRF) in which every press earned a single pellet until reaching the acquisition criterion of 50 pellets in a 40 min single session. Next, rats were trained on variable interval (VI) schedules of reinforcement in 20 min sessions where the schedule was thinned across sessions as follows: VI 10″, VI 30″, and VI 60″ . At the start of each instrumental session the houselights turned on with simultaneous insertion of the levers and the session terminated with the light turning off and retraction of the levers. After instrumental training, rats received eight 55-min Pavlovian discrimination sessions, in which one conditional stimulus  was reinforced (CS+) and one CS was non-reinforced (CS-). On each CS+ trial, four pellets were delivered on a VT 30″ schedule. The first trial began 2 min after the session start and subsequent trials were presented after a variable 5 min intertrial interval (ITI). Each CS was presented four times in a quasi-random order. Each session began with the house light turning on and ended 2 min after the last trial with the house light turning off. Following training, rats were tested for Pavlovian-to-instrumental transfer in 41 min sessions under extinction conditions. Each test began with the house light turning on and insertion of both levers, then 10 min into the session the first CS trial began, and subsequent trials were presented on a fixed 2-min ITI. Each CS was presented four times in a quasi-random order and the session ended 1 min after termination of the last trial. Rats were tested in two separate sessions on two consecutive days.ad libitum SS-PIT test.As with SO-PIT, all training and testing was conducted in Ctx B. Two unconditional stimulus outcomes were used for reinforcement, Bioserv Dustless Precision Pellets (45 mg) and 20% sucrose (0.1 ml). Magazine training was conducted separately for each outcome using the same parameters as described above. Next rats were trained to press one lever for pellets and one for sucrose in separate sessions. Instrumental training proceeded as described for SO PIT starting with CRF, ending with VI 60″ . Next rats underwent Pavlovian conditioning to associate one CS with pellets and the other with sucrose; a white noise and flashing lights  were used as distinct CSs . Each CS was presented for 2 min per trial during which four US deliveries were made on a VT 30″ schedule. Trials were separated by variable 5-min ITIs. The first trial was presented 2 min into the session and the session ended 2 min following termination of the last trial. Each CS-O pair was trained in separate 27 min sessions . Rats were given 20-min instrumental reminder sessions (VI 60″) for each R-O association 1 day before each test. Test sessions were identical to that described in SO-PIT. Rats were tested in three sessions with instrumental reminder sessions between each test. After session 3, rats were given instrumental reminder training on the following day, then food restriction was lifted and rats were tested 1 day later for a final via automated response measures recorded by the Med Associates software controlling the operant chamber machinery. For statistical analyses, ANOVAs, unpaired t-tests, and Holm-Sidak’s (HS) multiple comparisons were used planned and post hoc comparisons and are specified in more detail in the section “Results.”For analysis of freezing behavior, videos were scored manually by visually sampling behavior for 2 s every 10 s during the 3-min immediately prior to each shock delivery (or yoked time period for controls). One video in Experiment 2 containing three females was corrupted during the recording and therefore data from shock conditioning were lost for these subjects. For appetitive training and testing, data were collected N = 32) approximately 70 days old at the start of the experiment were purchased from Charles River Laboratories and shipped to the OHSU animal facility 1 week before the start of the experiment. Rats were food restricted to 85–90% of their ad libitum bodyweights for the duration of the experiment. An illustration of the experimental timeline for Experiment 1 is shown in Male Long Evans rats (N = 22) and male (N = 35) Long Evans rats were bred at OHSU from breeders purchased from Charles River Laboratories. Rats were weaned at postnatal (PN) day 23. At PN 70 or older, rats were food restricted and the following day given either control treatment or massive shock in Ctx A. The following day appetitive training for SS-PIT began in Ctx B as described above. An illustration of the experimental timeline for Experiment 2 is shown in Female  = 361.1, p < 0.01; main effect of time, F = 21.41, p < 0.01; time by treatment interaction, F = 21.41, p < 0.01).Freezing behavior in the Pre-Shock group was evident by the second shock and reached asymptotic levels by the third shock, whereas freezing behavior was expectedly absent in the Pre-Ctrl group . Two-wayp > 0.05), these groups were collapsed . Magazine training began the day after fear conditioning. As can be seen in F = 4.38, p < 0.05; HS post hoc comparison: Pre-Ctrl vs. Pre-Shock, t(28) = 2.98, p = 0.01]. During CRF training, the time to reach the acquisition criterion was delayed in the Pre-Shock group compared to the Collapsed Ctrl group . Active lever pressing increased across VI sessions in both groups and despite the ordinal difference in the groups there was no significant difference in Active lever responding between the groups . Inactive lever pressing was very low throughout training, but systematically increased without any group differences during the last 4 VI 60″ sessions . In both the Collapsed Ctrl and Pre-Shock groups responding on the Active lever was significantly higher than the Inactive lever and this difference increased over sessions . As an additional evaluation of the effects of pre-training shock on instrumental responding, we restricted comparison of responding to the Pre-Ctrl and Pre-Shock groups and found no significant difference in response rates, though there was modest ordinal difference with the Pre-Shock group performing lower in some sessions than Pre-Ctrl .For all training data, when Pre-Ctrl, Post-Ctrl, and Post-Shock groups did not differ  = 52.85, p < 0.01; Pre-Shock: F = 22.51, p < 0.01; CS by session interaction: Collapsed Ctrl: F = 11.90, p < 0.01; Pre Shock: F = 4.59, p < 0.01]. As expected, magazine responding to the CS+ increased systematically across sessions and response rates between the Collapsed Ctrl and Pre-Shock groups did not differ . Magazine responding during the ITI and CS- did not differ between the Collapsed Ctrl and Pre-Shock groups and was expectedly low across training . ITI responding significantly decreased across sessions, whereas CS- responding varied across days, but not systematically . As an additional evaluation of the effects of pre-training shock on responding during Pavlovian conditioning, we directly compared Pre-Ctrl and Pre-Shock groups, finding no group differences in responding .During the eight sessions of appetitive Pavlovian conditioning, all groups responded more to the CS+ than to the CS- .For the Post-Ctrl and Post-Shock groups, rats were given context exposure or received massive shock in Ctx A the day following the final appetitive Pavlovian conditioning session (the Pre-groups were left undisturbed in their home cages on this day). The Post-Ctrl and Post-Shock were counterbalanced to ensure even distribution of CS-O assignments and to match levels of appetitive Pavlovian conditional responding . During testing, we evaluated magazine entries and lever pressing during the Pavlovian CSs. Both groups retained the CS+/CS- discrimination, without notable group differences . Each group showed greater magazine responding during the CS+ compared to the ITI . Furthermore, CS+ magazine responding was significantly greater than CS- magazine responding (t(58) = 5.02, p < 0.01; Pre-Shock: t(58) = 2.95, p < 0.01; Post-Shock: t(58) = 4.14, p < 0.01).On the following 2 days all groups were tested for SO-PIT in Ctx B (one session per day). Although overall responding was lower during Test 2, there were no interactions with test session, so the two tests were combined for analysis. There also were no differences between Pre- and Post-Ctrl groups, so those data were combined for analysis  = 10.07, p < 0.01; no effect of group, p = 0.30; no group by phase interaction, p = 0.51]. The critical determinant of SO-PIT expression is whether Active lever responding is significantly greater during CS+ presentations compared to CS- presentations. Therefore, to determine if each individual group expressed reliable SO-PIT we conducted planned comparisons to evaluate this. Both the Ctrl and the Post-Shock groups showed SO-PIT with CS+ presentations evoking greater Active lever responding compared to CS- presentations, which did not augment responding above ITI levels . This SO-PIT effect, however, was absent in the Pre-Shock group [p = 0.62]. Furthermore, in the Ctrl and Post-Shock groups Active lever responding was greater during CS+ vs. ITI periods, but this augmentation by the CS+ was absent in the Pre-Shock group .Next, we examined Active lever responding to evaluate PIT . In the Pre-Shock group, SO-PIT was entirely absent across the CS window and did not change systematically over time . Although SO-PIT was evident across the session in the Post-Shock group when comparing CS+ to CS-, when pre-CS response rates were incorporated, PIT was not evident in every CS time bin and this was enough variability to weaken the overall main effect of CS .We also evaluated how SO-PIT manifested across the CS presentation window by looking at the mean time course of responding in 30 s bins from 60 s before to 60 s post CS presentations. These data are presented as elevation scores, which were calculated for each individual by subtracting the mean response during the pre-CS window (Bins 1 and 2) from the response rate in a given bin during the CS. In the Ctrl group, SO-PIT was sustained and strengthened across the entire 2-min CS window .As a final measure of PIT, we examined the latency to lever press following CS+ and CS- onsets. Consistent with the rate data, the Ctrl and Post-Shock groups showed a shorter latency to press the Active lever following CS+ vs. CS- presentations, whereas the Pre-Shock group did not show any difference in the latency to press following CS+ vs. CS- presentations . To further explore early sex effects on freezing, we focused on freezing behavior in the first four Pre-Shock periods. Here we found that females froze significantly less in these early Pre-Shock periods compared to males . Freezing did not emerge in Ctrl Female or Male rats (ps > 0.99).Ctx A treatment was conducted the day before the start of appetitive conditioning. In the Shock groups freezing during the Pre-Shock periods emerged rapidly in both males and females without pronounced sex differences  = 7.15, p < 0.01] with no reliable main effect of sex or interaction, ps = 0.19 (p = 0.40), but shocked males made fewer entries than did control males . Next rats went through CRF training and data were analyzed by taking the mean time to acquire both instrumental responses. The time to reach the acquisition criteria did not differ between treatment groups or sex [ps > 0.20]. In the next phase, rats were trained on VI reinforcement schedules for eight sessions. Data were analyzed as the mean rate of responding across both levers. In both sexes, response rates increased across sessions, were similar between Ctrl and Shock groups, and this did not interact with session . We also compared responding between sexes for each treatment group. In Ctrl, responding did not differ between sexes, but in the Shock groups, response rates in Females were lower than in Males overall .Appetitive training in Ctx B began with magazine training; data were analyzed as the mean rate of responding across all sessions . There ws = 0.19 . PlannedF = 17.10, p < 0.01; Males: F = 29.46, p < 0.01; no effects of treatment groups, Females, p = 0.80; Males: p = 0.87]. In both sexes, response rates during the ITI were low and did not differ between treatment groups . Finally, we compared response rates between sexes for each treatment group and found no sex differences in either Ctrl or Shock groups .In the last appetitive training phase rats underwent sensory-specific Pavlovian conditioning. These data were analyzed by averaging response rates across both CSs. In both sexes, magazine entries during the CSs increased across training sessions and did not differ between treatment groups .Rats were tested in three separate tests with instrumental reminder sessions on the days between testing. Initial analysis was conducted by averaging performance in these tests. First, to evaluate the possibility of any notable sex differences or interactions between sex and trauma on the magnitude of SS-PIT, we calculated a mean score for SS-PIT magnitude by subtracting the Diff lever response rate from the Same lever rate on each test and then averaging these discrimination scores. We found no effect of shock treatment, but we did find a sex difference with overall lower SS-PIT in females compared to males and this did not interact with shock treatment . In addition to sensory-specific transfer (Same > Diff), both Ctrl and Shock females exhibited significant General transfer on the Different lever such that CS presentations significantly elevated responding on the Different lever over ITI response rates, though this effect was marginal in the Ctrl group . The pattern was the same in males, such that both Ctrl and Shock groups showed sensory specific transfer with greater Same lever responding than Different with no differences between the Ctrl and Shock groups . As in the females, both the Ctrl and Shock groups showed significant General transfer with presentation of CS evoking a significant increase in responses on the Different lever from ITI responding . In summary, Shock did not disrupt SS-PIT in either female or male rats.Next, to directly examine SS-PIT we looked at the means lever response rates for ITI, Same lever, and Different lever responding averaged across all tests. In females, both Ctrl and Shock groups exhibited sensory-specific PIT with a given CS, preferentially increasing responding on the lever associated with the same outcome as the CS and there were no differences between the Ctrl and Shock groups . In contrast in males, the trial analysis revealed robust SS-PIT on the first trial of all three tests in both Ctrl and Shock groups; SS-PIT was absent on the final trials of each test  = 10.24, p < 0.01; HS planned comparison: Same vs. Diff: Ctrl, p = 0.28; Shock, p = 0.17; no effect of treatment, p = 0.95]. In contrast, in both male Ctrl and Shock groups SS-PIT was evident and with CS presentations evoking greater Same lever responses than Different lever responses .Given the mild deficits in SS-PIT in females, we conducted an additional SS-PIT test, but under  satiety . Thus, tIn this series of experiments, we show that an acute intensely stressful experience prior to appetitive training persistently reduces single-outcome Pavlovian-to-instrumental transfer (SO-PIT) in male rats, but this same experience does not alter the expression of Sensory-Specific PIT (SS-PIT) in male or female rats. These data show an acute highly stressful experience produces long lasting changes in general affective motivation for natural reward, as captured here with SO-PIT without overtly altering sensory-specific memory processes.The effects of stress on learning, memory, and motivation are well documented in the literature. These studies have used acute stressors  to evaluate the effects of stress on appetitive responding during or soon after a stressful experience . TypicalOur approach showing persistent changes of stress in appetitive responding is derived from the stress-enhanced fear learning (SEFL) procedure, in which a battery of footshocks in one context causes increased reactivity to a single footshock in a different context. SEFL occurs in contexts that have a long history of drug self-administration  and the These findings parallel two important aspects of PTSD as it often develops in humans: (1) a triggering traumatic event and (2) behavioral aberrations that persist long after the trauma. The results of work on drug-seeking in the context of SEFL parallel an important comorbidity seen in humans where PTSD is a major risk factor for substance use disorder. However, drugs of abuse alone trigger profound and persistent changes in appetitive motivation and the underlying circuitry, which makes it difficult to isolate the persistent impact of the acute trauma alone on appetitive processes. Furthermore, while many individuals suffering from PTSD experience substance use problems many do not, and understanding the impact of trauma on motivation for natural reward is important to understanding how PTSD manifests in such individuals and how to treat these comorbid symptoms.In Experiment 1 we demonstrated that in male rats a battery of footshocks before, but not after appetitive training blocks expression of SO-PIT. This effect was specific to the test phase; there were no differences in acquisition of the Pavlovian CS+ vs. CS- discrimination or in acquisition of instrumental responding. During the PIT test, animals shocked prior to appetitive training did not show augmentation of instrumental responding during the CS+. The absence of SO-PIT was also apparent in the average time course of responding per trial and in the latency to press the levers following CS+ vs. CS- presentations. Despite the failure for the excitatory properties of the CS+ to transfer to instrumental responding, these rats still showed reliable Pavlovian conditional responding, preferentially entering the food cup during CS+ vs. CS- presentations. Thus, the failure for SO-PIT to manifest could not be explained by a failure to encode the Pavlovian discrimination. With all these considerations in mind, our data suggest that trauma can induce lasting deficits in the ability for Pavlovian cues associated with natural reward to acquire general motivational properties in males.Our finding here that shock prior to training induces deficits in SO-PIT are consistent with a study in humans showing that participants scoring high on anxiety and stress metrics did not show general PIT, which is thought to rely on the same mechanisms as SO-PIT, whereas those with low stress and anxiety did . AlthougAlthough SO-PIT was disrupted by shock trauma administered prior to training, this same treatment after training, but before testing, had less impact on SO-PIT, with no effect overall but some subtle within-trial deficits. This result is consistent with a study in rats showing that both single and multiple stressors administer either in the light or the dark phase of the circadian cycle did not block SO-PIT in Lister Hooded male rats . It is nSO-PIT taps into basic motivational properties of Pavlovian cues, however it does not provide a clean read out of sensory-specific learning, memory, and motivation. In Experiment 1, all groups showed reliable conditional stimulus discrimination (CS+ vs. CS-) regardless of shock history, arguing against trauma-induced problems with sensory memory encoding; however, the possibility remained that sensory-specific learning may be altered by a history of acute trauma. We therefore tested whether shock before training would alter the expression of SS-PIT, a clean measure of sensory-specific encoding. Here we used both female and male rats. Shock prior to training did not prevent the expression of SS-PIT in either male or female rats. Specifically, CS presentation selectively invigorated responding on the lever that previously shared the same outcome as the CS in presentation vs. the lever that previously delivered a different outcome. This finding confirms that a history of trauma does not prevent the formation of distinct sensory-specific memories in either males or females. This finding is consistent with a previous study that used chronic unpredictable stress to examine the effects of stress on SS-PIT in male Wistar rats . In the In Experiment 2, there were several sex differences during the course of fear conditioning and PIT training and testing. First, during fear conditioning, females showed delayed acquisition relative to males early in the conditioning session. This delay may reflect early differences in expression of different behaviors  was more consistent across testing in males, with females showing SS-PIT on only one of the 12 test trials. Moreover, when tested in the task . The losthe task .via exposure to the outcomes  effect was strongest in early trials growing weaker across trials and then re-emerging without any notable degradation upon retesting. Given that PIT testing is conducted under extinction conditions, the Pavlovian CSs are essentially given extinction training and while rats are retrained on the instrumental associations between tests, they never received additional CS-US pairings. Thus, the robust revival of the PIT effect on each subsequent test is quite similar to classic spontaneous recovery. It is also consistent with the intervening instrumental retraining establishing a reinstatement of selective associations outcomes . The resoutcomes . Howeveroutcomes . To our Another possibility is that our data reflect a stress-induced insensitivity to the motivational aspects of natural reward, which may render individuals more vulnerable to the motivational properties of drug reward. The suppressive effects of stress and depression on consumption and instrumental motivation for natural rewards has been well documented . Thus ouSO- and SS-PIT are dissociable both as psychological constructs and also rely on different brain circuits . Lesion The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the OHSU IACUC.RD and KL designed the experiments and wrote the manuscript. RD conducted the experimental work and performed the statistical analyses. Both authors contributed to the article and approved the submitted version.


Primary cultures of retinal pigment epithelium (RPE) from human adult donors (haRPE) and induced pluripotent stem cell derived-RPE (iPSC-RPE) are valuable model systems for gaining mechanistic insight and for testing potential therapies for age-related macular degeneration (AMD). This study evaluated the treatment response of haRPE and iPSC-RPE to oxidative stress and potential therapeutics addressing mitochondrial defects. haRPE and iSPC-RPE were derived from donors with or without AMD. Mitochondrial function was measured after treatment with menadione, AICAR, or trehalose and the response to treatment was compared between cell models and by disease status. In a subset of samples, haRPE and iPSC-RPE were generated from the same human donor to make a side-by-side comparison of the two cell models’ response to treatment. Disease-specific responses to all three treatments was observed in the haRPE. In contrast, iPSC-RPE had a similar response to all treatments irrespective of disease status. Analysis of haRPE and iPSC-RPE generated from the same human donor showed a similar response for donors without AMD, but there were significant differences in treatment response between cell models generated from AMD donors. These results support the use of iPSC-RPE and haRPE when investigating AMD mechanisms and new therapeutics but indicates that attention to experimental conditions is required. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with an estimated 288 million cases of AMD worldwide by 2040 . There aChronic oxidative stress is a well-recognized factor in AMD pathobiology . A majorDue to their role in retinal health and their eventual death with AMD progression, the RPE is a key target for AMD treatments. There are a number of in vitro human RPE cell models, including human adult primary RPE (haRPE) and RPE derived from induced pluripotent stem cells (iPSC-RPE). haRPE cultures from deceased human donors have provided valuable insight into AMD phenotypes and disease mechanisms and have been used to investigate the efficacy of potential therapeutics ,6,7,8. HWith each experimental system, it is essential to understand how closely the model system replicates the target cell in vivo. Our group and others have shown that haRPE and iPSC-RPE cultures exhibit many cardinal features of native RPE, including the formation of a pigmented epithelial layer with tight junctions, and the expression of RPE signature genes and proteins ,9,10,14.In prior investigations using haRPE, we observed reduced mitochondrial function, increased resistance to oxidative stress, and a greater response to mitochondrial-targeted drugs in RPE cultured from donors with AMD ,7. BasedIn the aggregate data, disease-specific differences were observed at baseline and in response to all three treatments in the haRPE. However, irrespective of disease status, iPSC-RPE had a similar response to all three treatments and matched the treatment response of haRPE from donors without AMD. Data from haRPE and iPSC-RPE generated from the same human donor showed a similar response to treatment when generated from donors without AMD. Similar to the aggregate data, iPSC-RPE and haRPE generated from donors with AMD had a significantly different response to treatments.Donor eyes were obtained with the informed consent of the donor or donor’s family for use in medical research in accordance with the Declaration of Helsinki. The Minnesota Grading System (MGS) was used to classify donor eyes into No AMD (MGS1) and AMD (MGS2 and MGS3) . EvaluatBoth cell models were cultured using the same conditions and culture media beginning at passage 3 and throughout each assay, with media changes twice a week. Confluent cell layers were used in the experiments, with seeding density and plate type provided in each experimental method. The demographics for donors used to generate haRPE and iPSC-RPE are provided in For drug treatments, cells were cultured in media containing 1% FBS. The doses and timing of all treatments are indicated in their respective figures. The treatment conditions for the final experiments were 25 μM menadione for 24 h, 500 μM AICAR for 48 h, and 100 mM trehalose for 48 h.Genomic DNA was extracted from graded donor retinal tissue as described previously . SamplesApproximately 300,000 cells were plated and grown to confluence in 12-well plates. Cells were treated with varying concentrations  of menadione for 24 h. RNA was collected, and cDNA was synthesized and quantified as previously described . Real-tihaRPE and iPSC-RPE  were plated into clear-bottom black-sided 96-well plates (Costar) and incubated for up to 48 h before staining and imaging. Cells were stained with 100 nM Lysotracker Red DND-99 (Fisher Scientific) and NucBlue Live Cell Stain (Invitrogen) and imaged as previously described .Mitochondrial function in treated and untreated RPE was measured using the XFe96 Extracellular Flux Analyzer  and the Cell Mito Stress Test (CMST) assay. In XFe96-well plates, 40,000 cells were plated and incubated for up to 2 days before treatment and CMST assays. The CMST assay protocol was performed according to the manufacturer’s instructions (Agilent Technologies) and our previous publication ,7,16. OxSub-confluent (5000 cells/well) were plated into a ½ area 96-well clear-bottom, black-sided plates. After treatment, cell death was determined using CyQuant (Thermo Fisher) following the manufacturer’s protocol. Data were calculated relative to no treatment and lysis control wells. Cell death from confluent cells  was calculated from Seahorse plate cell counts after CMST assay.haRPE and iPSC-RPE  were plated in 6.5 mm transwell inserts and cultured for 5 weeks. Media was collected from apical and basal chambers and ELISA for vascular endothelial growth factor A  and pigment epithelium-derived factor  were performed as described ,16.haRPE and iPSC-RPE  were plated into 12 well-plates and whole cell lysates were collected using RIPA buffer after treatment . Protein concentrations were determined with BCA assay  using albumin as the standard. 10 μg of protein was loaded for each sample and Western blots were performed as described [t-test was used to compare treatment effects to no treatment controls as well as to compare haRPE and iPSC-RPE . A paired t-test was used to compare haRPE and iPSC-RPE generated from the same human donor . Statistical analysis was performed using Graphpad Prism 9. p ≤ 0.05 was considered statistically significant.The data shown were calculated relative to no treatment controls for each donor (fold change relative to no treatment). The student’s n = 12; aged 71 ± 7.5) and with AMD . iPSC-RPE were generated from donors without AMD  and with AMD . There was no significant difference between age ranges of haRPE and iPSC-RPE for No AMD (p = 0.82) and AMD (p > 0.99). Both cell models had a nearly balanced gender distribution, with the ratio of males and females 16:12 for haRPE and 9:11 for iPSC-RPE.Donor demographics are provided in Prior to treatment, we first assessed haRPE and iPSC-RPE under basal conditions . To veriThe mitochondrial function in haRPE and iPSC-RPE was compared under basal conditions by measuring the oxygen consumption rate (OCR) using an XF Extracellular Flux Analyzer and the Cell Mito Stress Test (CMST). A comparison of mitochondrial function by disease status found that haRPE from AMD donors have lower mitochondrial function, whereas iPSC-RPE had no significant differences based on disease E,F. ThesTreatment with menadione was used to determine the cell’s response to mitochondrial oxidative stress. To establish the optimal dose, cells were treated with increasing concentrations of menadione for 24 h and then assayed for antioxidant gene expression, cell death, mitochondrial protein content, and mitochondrial function. The expression of the antioxidant genes HO-1 and SOD2, whose proteins are localized to the cytosol and mitochondria, respectively, was used to gauge the response to oxidative stress. Elevated expression of both HO-1 and SOD2 was observed in haRPE but was limited to only SOD2 in iPSC-RPE . Since cIn measuring mitochondrial function, menadione caused a dose-dependent decrease in both haRPE and iPSC-RPE. haRPE had a significant decrease in BR, MR, SRC, and ATP at 50 µM menadione B. iPSC-RBased on these data, we chose 25 µM menadione, which caused an approximate 35% and 45% decrease in mitochondrial function in haRPE and iPSC-RPE, respectively. Our aggregate cohort of haRPE and iPSC-RPE were treated with 25 µM menadione for 24 h before measuring mitochondrial function. To understand how disease status effects treatment response, we grouped both haRPE and iPSC-RPE by the donor’s disease state. Following treatment, haRPE from No AMD donors had substantial decreases in every parameter, with significant decreases to MR and SRC E,F. In aAICAR, an analog of AMP, acts as a direct activator of adenosine-monophosphate-activated protein kinase (AMPK). Upon activation, AMPK phosphorylates a number of transcription factors that stimulate mitochondrial biogenesis and regulate metabolism . Cells wWe subsequently used 500 µM AICAR for 48 h to test its effect on mitochondrial function, grouping the aggregate cohort of haRPE and iPSC-RPE by the donor’s disease state. In haRPE and iPSC-RPE from No AMD donors, AICAR treatment significantly decreased BR and ATP C,D,I,J. Trehalose is a naturally occurring sugar that increases autophagy via AKT and the transcription factor TFEB, leading to the upregulation of genes associated with the Coordinated Lysosomal Expression and Regulation (CLEAR) network . We utilOur laboratory and others have shown that the genetic background of an individual can influence both the mitochondrial function and response to treatments ,17,29,30t-test to determine if the response of haRPE and iPSC-RPE were significantly different. Bar graphs show the average response to each treatment with data points from individual donors superimposed over each bar, along with the results of the paired t-test . Other in vitro manipulations that replicate the diseased environment include growing iPSC-RPE on nitrited membranes , exposinWe found consistent results between groups of cells used in previous studies. For instance, the results within the haRPE group agree with results from previous reports of mitochondrial dysfunction, resistance to oxidative stress, and better responses to drug treatments in haRPE from AMD donors ,6,7. ResA number of factors could contribute to the different results and conclusions between studies. Most notably, the growth conditions of the iPSC-RPE vary from study to study. For example, cells grown on different types of matrixes can respond differently due to changes in the cellular environment ,16,19,41Our work and others have also found that the individual’s risk alleles can have a significant impact on RPE function. Two polymorphisms associated with AMD are complement factor H  and age-related maculopathy susceptibility 2 . Comparing the cells by genotype may reveal differences that are not observed when grouping the cells by donor disease state. Previous studies have reported that iPSC-RPE from donors with the high-risk allele for CFH have reduced mitochondrial function, increased inflammatory markers, and increased accumulation of lipid droplets ,30,42. SWhile this study reveals that iPSC-RPE do not replicate the treatment response of diseased haRPE under current culture conditions, it does show the benefits of this valuable model system. We found that irrespective of disease state, iPSC-RPE match the response to treatment of haRPE from donors without AMD. This phenotype may benefit iPSC-RPE when transplanted into patient eyes, as they may better replenish and maintain the RPE layer upon transplant. Additionally, we observed an increased secretion of PEDF in the iPSC-RPE, which may prove beneficial in transplants as PEDF promotes development and survival of photoreceptors ,44.Other benefits of iPSC-RPE include their ability to produce large numbers of cells, and their generation from a number of cell types and living patients . These bIn conclusion, haRPE and iPSC-RPE are both important model systems for studying AMD. The current study highlights important differences and limitations of each cell culture system. We found that iPSC-RPE do not recapitulate the treatment response of haRPE from human donors with AMD but do replicate the response of haRPE from donors without AMD. The differences observed may be due to loss of epigenetic markers during iPSC-RPE differentiation or their growth in a non-diseased environment. The results of our study support the continued use of iPSC-RPE and haRPE when investigating AMD mechanisms and new therapeutics, but indicates that careful attention to the experimental conditions and donor genotype is required.


The relationship between Multidrug Resistant-Gram Negative Bacteria (MDR-GNB) infection and colonization in critically ill COVID-19 patients has been observed, however, it is still poorly understood. This study evaluated the risk factors for acquiring MDR-GNB in patients with severe COVID-19 in Intensive Care Units (ICU).This is a nested case-control study in a cohort of 400 adult patients (≥ 18 years old) with COVID-19, hospitalized in the ICU of 4 hospitals in the city of Curitiba, Brazil. Cases were critical COVID-19 patients with one or more MDR GNB from any surveillance and/or clinical cultures were taken during their ICU stay. Controls were patients from the same units with negative cultures for MDR-GNB. Bivariate and multivariate analyses were done.Sixty-seven cases and 143 controls were included. Independent risk factors for MDR bacteria were: male gender ; the hospital of admission ; mechanical ventilation ; and desaturation on admission .Male gender, desaturation, mechanical ventilation, and the hospital of admission were the independent factors associated with MDR-GNB in patients in the ICU with COVID-19. The only modifiable factor was the hospital of admission, where a newly opened hospital posed a higher risk. Therefore, coordinated actions toward a better quality of care for critically ill COVID-19 patients are essential. Bacterial Antimicrobial Resistance (AMR) is one of the main threats to global health, limiting therapeutic options for treating infectious diseases. This leads to longer hospital stays, higher medical costs, and increased mortality.Mainly during the first and second waves of the COVID-19 pandemic, there was excessive use of antimicrobials due to the severity of the patients and the lack of therapeutic options. Overuse of antibiotics has been related to antimicrobial resistance.Studies have shown that mainly critically ill patients with COVID-19 have secondary infections,Although MDR-GNB infections and colonizations have been linked to prolonged length of stay in the ICU, longer invasive mechanical ventilation, and steroid therapy,st March and 31st December 2020.This is a nested case-control study in a cohort of 400 adult patients (≥ 18 years old) with COVID-19 hospitalized in the Intensive Care Units (ICU) of 4 hospitals in the city of Curitiba, Brazil:Cases were defined as severe COVID-19 patients with one or more MDR GNB from any surveillance and/or clinical cultures taken during their ICU stay.Controls were severe COVID-19 patients in which no MDR GNB and/or MDR gram-positive bacteria were isolated  during their ICU stay. Patients who did not undergo surveillance cultures were excluded from the control group.COVID-19 was defined as an infection confirmed with a positive Real-Time reverse transcriptase Polymerase Chain Reaction (RT-PCR) for SARS-CoV-2 from a nasopharyngeal swab, associated with suggestive signs, symptoms, and/or compatible radiological findings.2) ≤94% in ambient air; or requiring supplemental oxygen; requiring mechanical ventilation; or requiring extracorporeal membrane oxygenation.Patients with severe disease were defined as patients with Oxygen Saturation .The WHO Clinical Progression ScaleEnterobacteriales resistant from first to third/fourth generation cephalosporins;Carbapenem-Resistant Enterobacteriales (CRE);Acinetobacter spp. (CRA);Carbapenem-Resistant Pseudomonas aeruginosa (CRP).Carbapenem-Resistant The following GNBs were considered to be Multidrug Resistant (MDR):For bacterial identification, MICROSCAN WalkAway®  automated system was used. Sensitivity to antimicrobial agents was also determined by MICROSCAN WalkAway® system according to the Brazilian Committee on Antimicrobial Susceptibility (BRCast) criteria. The phenotypic confirmation of ESBL production was done using the double-disk synergy test with cefotaxime, ceftazidime, and cefepime with and without clavulanic acid. Combined disk diffusion using phenylboronic acid as an inhibitor with cefoxitin was used for phenotypic confirmation of AmpC phenotype. Carbapenemase production was demonstrated by the modified Hodge test and by the Modified Carbapenem Inactivation Method (mCIM) and EDTA-mCIM (eCIM).Staphylococcus Aureus (MSSA) and gram-negative bacteria such as Enterobacteriales and Pseudomonas. These included piperacillin-tazobactam, meropenem, cefepime, ceftazidime, amikacin, gentamicin, and fluoroquinolones.Broad-spectrum antibiotics were defined as any agent with activity against gram-positive bacteria such as Methicillin-Susceptible 2) to Fraction of Inspired Oxygen (FiO2). Lung radiologic alterations were defined based on the medical report of the chest radiograph or computed tomography. Duration of the disease from the onset of symptoms, and length of stay in the hospital and in the ICU was also documented.Data were extracted from electronic medical records using a data collection form. Demographic characteristics included age, sex, weight, and Body Mass Index (BMI). Clinical information included signs, symptoms, comorbidities, and treatment measures . Laboratory assessment consisted of laboratory tests on admission to the ICU and at the moment of diagnosis of a secondary infection: C-reactive protein, total leukocyte count, number of lymphocytes, creatinine, glucose, D-dimer, troponin, and the ratio of Arterial Oxygen Partial pressure .The authors compared all critical COVID-19 patients with one or more MDR GNB from any surveillance and/or clinical cultures taken during their ICU admission (cases), with the controls.The possible association of demographic and clinical variables with each dependent variable was initially tested in a bivariate analysis calculating the odds ratios and 95% Confidence Interval for each variable. Variables potentially associated with the development of each dependent variable in bivariable analysis (p < 0.20) were included in a multivariable logistic regression model in order to determine the adjusted odds ratios. Mechanical ventilation was chosen to represent invasive devices in multivariable analysis.2/FiO2 ratio, absolute lymphocyte count, and absolute leukocyte count were transformed from continuous variables to categorical variables. For CRP, the cutoff point used was the value of 108 mg/L, based on the study 2/FiO2 four categories of severity of acute respiratory syndrome were used, according to Villar et al.3 cells/µL, based on Wagner et al.,3 cells/µL.The variables C-reactive Protein (CRP), PaOStatistical analyses were performed using EPI Info 7 Software ; p-values < 0.05 were considered to be statistically significant.From a cohort of 400 adult patients, 67 cases  and 143 controls were included.2/FiO2 ratio on ICU admission; pronation; and use of devices in the ICU , while the hospital of admission increased the risk by 3 fold . Mechanical ventilation increased the risk of MDR by 26 fold  while for desaturation the risk was 2.6 fold .Acinetobacter baumannii was the most frequent bacteria (22 cases – 65%) followed by CR-Enterobacterales (10 cases – 29%) and CR-Pseudomonas (2 cases – 6%). In clinical cultures, there was also a predominance of CR-Acinetobacter baumannii. In respiratory samples, 15 were CRA; 11 were Enterobacteriales resistant to 3rd and 4th generation cephalosporins; 5 were CRE, and 2 were CRP. In blood cultures, 6 were CRA; 5 were CRE; 3 were Enterobacteriales resistant to 3rd and 4th generation cephalosporins, and 2 were CRP. Finally, in urine cultures, 2 of each were CRA; CRE; and Enterobacteriales resistant to 3rd and 4th generation cephalosporins; and 1 was CRP.In surveillance cultures, CR-38 cases had healthcare-associated infections caused by MDR-GNB: 28 were lower tract respiratory ; 7 central venous Catheter-related Bloodstream Infections [CLABSI], and 3 Catheter-Associated Urinary Tract Infection (CAUTI)].Death was higher among the cases (72%) than controls (37%) .In this nested case-control study in a cohort of 400 adult patients with COVID-19 admitted to the ICU of 4 hospitals, the authors found that the independent factors associated with MDR-GNB were the hospital of admission, the use of mechanical ventilation, desaturation, and male gender.Among these four factors, the hospital of admission is the only modifiable variable. This highlights the fact that Infection Prevention and Control (IPC) measures play an essential role in health facilities, making safer places to care for COVID-19 patients, and avoiding colonization, cross-transmission, and infection by MDR bacteria. Even more so because in severe cases of COVID-19 bacterial infection occurs in approximately 13%, much lower than in influenza, which is > 34%.The characteristics of the work environment affect the quality of care both directly and indirectly. In the present study, the hospital with the highest risk for MDR-GNB had been closed and was reopened as a field hospital for COVID-19 patients. The structure of the hospital, as well as the ICU, was adapted to receive COVID-19 patients. In addition, there was a mixture of experienced and inexperienced healthcare workers due to a scarcity of specialized personnel. The processes of work were being organized while the patients were being hospitalized, as well as the training of medical and multidisciplinary teams on infection prevention and control: hand hygiene, the use of personal protective equipment, equipment disinfection, and environmental cleaning. Furthermore, the overcrowding of patients may also have affected the implementation of isolation measures for patients colonized with MDR, leading to potential outbreaks.Acinetobacter baumannii was the most frequent MDR-GNB. This bacterium has the capacity to survive for prolonged periods on inanimate objects and even in hand sanitizers.Acinetobacter.A. baumannii (CRAB) increased from 7.9% in 2019 to 12.4% in 2020. The incidence of CRAB per 1000 patient days increased significantly after April 2020 and correlated strongly with the incidence of COVID-19.In fact, in the present study Many studies have shown the correlation between MDR and mechanical ventilation and/or the use of invasive devices in patients with COVID-19.In the present study, patients with desaturation on hospital admission had a higher risk for MDR bacteria. Patients with severe COVID-19 have a dysfunctional immune response, which triggers a cytokine storm that mediates widespread lung inflammation that in itself mediates damage to the lung through excessive secretion of proteases and reactive oxygen species, in addition to the direct damage caused by the virus. Together, these result in diffuse alveolar damage, including desquamation of alveolar cells, hyaline membrane formation, and pulmonary edema. This limits the efficiency of gas exchange in the lung, causing difficulty in breathing and low blood oxygen levels. The lung also becomes more vulnerable to secondary infections, among them MDR-GNB, mainly when adequate environmental cleaning and hand hygiene are lacking.Finally, it is not clear why MDR-GNB is more prevalent in men.The strength of this study is its multicenter design, although only hospitals in one city were included, and the relatively large number of cases and controls. However, many patients could not be included as controls due to the absence of surveillance cultures to ascertain that they were not casesIn conclusion, in the present study, male gender, desaturation, mechanical ventilation, and the hospital of admission were the independent factors associated with MDR-GNB in patients in ICU with COVID-19. The only modifiable factor was the hospital of admission, as the newly opened hospital posed a higher risk. Therefore, efforts are needed to improve the quality of care to patients with severe COVID-19 in order to avoid or reduce colonization and infection by MDR-GNB bacteria. These findings also suggest that better public health preparedness for emergencies in Brazil is necessary. Good quality of health care should be the focus of robust actions in order to mitigate the transmission of MDR-GNB bacteria.The authors declare no conflicts of interest.


Dear Editor,The COVID-19 pandemic has negatively impacted TB control globally.Following the Correspondence by Louie et al,Mycobacterium tuberculosis) during the period January 1, 2019 to December 31, 2020. Chest imaging studies were independently reviewed by a radiologist (LF). Disease severity on chest imaging was determined by the extent of the thoracic cavity involvement; with less than 25%, 25–50%, and >50% of thoracic cavity involvement, which corresponded to mild, moderate, and extensive disease, respectively.® MTB/RIF ; and CXR. Chest CT scans were also available for 23/41 (56%) patients diagnosed with M. tuberculosis culture-positive pulmonary TB during 2019 and for 18/28 (64%) TB patients during 2020. Xpert is routinely performed on all first and second sputum samples received for AFB culture. In contrast, there is no maximum number of AFB cultures which can be performed. Our primary outcomes were to measure: 1) the number of pulmonary TB tests (sputum samples for AFB smears and cultures) per year, 2) the number of culture-positive pulmonary TB diagnoses per year, 3) sputum AFB smear microscopy positivity rate per year, and 4) severity grading of chest imaging studies. Using a two-sample test for proportions, we analyzed the distribution of these four primary outcomes in two time periods, pre-COVID-19  and then during the COVID-19 pandemic .Our primary aim was to evaluate the impact of COVID-19 on pulmonary TB diagnosis by reviewing whether there would be a meaningful change in the frequency of sputum collection for acid-fast bacilli (AFB) and/or AFB smear positivity during the COVID-19 pandemic (2020) compared to the prior period (2019). We hypothesized that there would be a decrease in TB diagnosis during the COVID-19 period, which could be secondary to either a decrease in providers ordering TB tests, or a delay in patient presentation for care due to stay at home ordinance and attempts to maintain social distancing. We reasoned that a delay in patient presentation would lead to a shift towards more severe TB disease at diagnosis, with a higher proportion and grading of AFB smear positivity and/or more advanced disease on chest radiograph (CXR). This is a non-interventional retrospective study for which we reviewed microbiology data, as well as CXR and computed tomography (CT) scans for patients diagnosed with pulmonary TB  compared to pre-COVID-19  . CXR evaluation for moderate and extensive TB disease did show statistical significance as a measure of disease severity, which suggests patients had more extensive TB disease at the time of diagnosis during the COVID-19 study period  . The absy period . SeveralThis study has some limitations. Although our sample size was large, the number of patients diagnosed with pulmonary TB was relatively small, which may have impacted our conclusions. Additionally, the time periods used in this study may have led to misclassification: we started the 2020 analysis in January as opposed to March when COVID-19 cases were first detected, so may have attributed some of the pre-COVID-19 cases to the COVID-19 period. Finally, we did not include clinical variables, which may have been helpful in interpreting the results.In conclusion, we found a 37% decrease in TB tests (sputum samples sent for AFB smears and cultures) during COVID-19 (2020) compared to the period immediately prior (2019). The decrease in number of reported cases of TB raises concerns for potentially undiagnosed TB in a community where approximately 80% of TB disease results from the reactivation of TB infection. Although the proportion of patients diagnosed with pulmonary TB did not change, chest imaging indicated patients who presented in 2020 had more severe disease. These patients could have delayed seeking medical care during the pandemic leading to more severe disease state on presentation. These patients would therefore be infectious for longer, with increased possibility of transmitting TB to close contacts prior to diagnosis. In this relatively small study, the differences in the other measured variables are not significant, but at a country-wide level these relatively small differences may have a large impact on public health.


Objective: Pregnant women with autoimmune rheumatic diseases are considered to have a high risk of obstetric complications with the emergence of the Coronavirus disease (COVID-19) pandemic. Therefore, we aimed to assess the impact of COVID-19 on this high-risk group.Methods: This cross-sectional cohort study (March to December 2020) was conducted at the largest tertiary center in Qatar . Eighty consecutive patients following up at the center during pregnancy were surveyed through telephonic interviews. Data on COVID-19 and pregnancy outcomes were extracted from electronic hospital records.Results:Eighty pregnant women with autoimmune rheumatic diseases were included. Among them, 17 (21.3%)  were diagnosed with COVID-19, five were hospitalized, and only one required intensive care unit admission. The proportion of adverse obstetric outcomes in the cohort was 29.5% . Prematurity  and caesarean section  were the most prevalent adverse events. There was no statistical difference in adverse pregnancy outcomes between women with and without COVID-19.Conclusion: COVID-19 did not affect pregnancy outcomes in women with autoimmune rheumatic diseases. Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the coronavirus disease COVID-19) pandemic, pregnant women have received particular attention for being at high risk for increased morbidity and mortality. A systematic review analysed 198 studies (December 2019 to June 2020) and found that pregnant women are less likely to develop clinical symptoms of COVID-19 compared to non-pregnant women but have higher odds for intensive care admissions  and invasive ventilation . The most prominent perinatal adverse event in women with COVID-19 is preterm delivery  [9 pandemiPregnancy in women with autoimmune rheumatic diseases such as systemic lupus erythematosus (SLE), Sjögren syndrome, and inflammatory arthropathies is associated with a risk of adverse outcomes, including miscarriage, intrauterine foetal death, pre-eclampsia, prematurity, and neonatal morbidities . In addiA web-based survey study from a single centre in New York evaluated the impact of COVID-19 on pregnant patients with rheumatic diseases and reported a similar prevalence among pregnant and non-pregnant women reporting to the centre. It also found that prenatal care was impacted in the majority of these patients .Bermas et al. examined data from the COVID-19 Global Rheumatology Alliance registry and assessed pregnancy outcomes in 22 patients with rheumatic diseases who were pregnant when they encountered the infection. Only 10 pregnant women needed hospitalisation, and all recovered from the infection. They also reported favourable pregnancy outcomes with 16/22 term births, one miscarriage, and one termination at nine weeks [At present, there is a paucity of data regarding the impact of COVID-19 on pregnant women with autoimmune rheumatic diseases . Thus, wThis cross-sectional study was conducted at the largest tertiary health care centre in Qatar  in compliance with the Helsinki Declaration and was approved by the Medical Research Centre and Institutional Review Board at Hamad Medical Corporation (MRC-01-20-617).We screened 80 pregnant women who visited a specialised "Pregnancy and Rheumatic Disease Clinic" at least once, from the start of the COVID-19 pandemic in Qatar until the end of the recruitment period . Pregnant patients diagnosed with autoimmune rheumatic disease according to international criteria (ACR/EULAR) by a rheumatologist and with a singleton pregnancy were followed up once in each trimester and once postpartum. Follow-up data included baseline demographics , diagnosis, medical history, comorbidities, medication during pregnancy, obstetric and perinatal outcomes, gestational age (weeks), birth weight (kilograms), neonatal intensive care unit (NICU) admission, mode of delivery, and congenital malformations. Obstetric morbidities including pre-eclampsia, gestational diabetes, premature rupture of the membranes (PROM), stillbirth, and miscarriage were diagnosed based on the ACOG (American College of Obstetricians and Gynecologists) definitions. Prematurity was defined as delivery before 37 weeks of gestation, and low birth weight was defined as birth weight less than 2.5 kg. The treating physicians documented the standard data on disease activity, pregnancy, and peripartum outcomes from the hospital's electronic records.Disease activity was measured using the following scores: (a) for rheumatoid arthritis, the Clinical Disease Activity Index score of ≤3 for remission and >3 for active disease; (b) for lupus, the SLE Disease Activity Index score was adjusted for pregnancy, and a score of >3 was used to stratify "active disease"; (c) for the other connective tissue/inflammatory diseases , patient and physician global assessment were used. Remission was defined as patient global assessment ≤1 using a 0-10 visual analogue scale. Independent from transient flares, a disease was recorded as "active" if it required therapy escalation and remission was not achieved by the end of pregnancy.Eighty consecutive pregnant patients were approached and informed about this COVID-19 study. Eligibility criteria include any woman previously diagnosed with an autoimmune rheumatic disease and who is currently pregnant with a singleton. All patients were eligible and agreed to participate. After obtaining verbal consent, the participants completed a telephonic survey questionnaire that included baseline characteristics, medical history, comorbidities, medications used during pregnancy, history of travel, and contact with confirmed COVID-19 cases. They were provided with a hotline number to report whether they had been diagnosed or had been in close contact with any household or co-worker at the same office with COVID-19 positivity or infection. The COVID-19 diagnosis was only confirmed based on a positive nasopharyngeal polymerase chain reaction (PCR). The survey data has been updated with the most recent information.At the completion of the recruitment period, two investigators (NH and HA) systematically extracted prespecified clinical variables, including disease activity measures in the third trimester, medications during pregnancy, pregnancy morbidities, peripartum outcomes, and the course of COVID-19 infection, from the electronic records of all participants. Data from the surveys and the electronic records were then linked and analysed.Statistical methodsSample characteristics are presented as frequency distributions for categorical variables using the chi-square test and means and standard deviations for continuous variables using the t-test. Statistical significance was defined as p < 0.05. All analyses were performed using IBM SPSS Statistics for Windows, version 26.0 .Eighty pregnant women with rheumatic diseases, who completed their pregnancy during the study period, were included in this analysis. Table Pregnancy and neonatal outcomesResults of the univariate analysis for pregnancy and neonatal outcomes are presented in Table COVID-19 during pregnancyThe prevalence of COVID-19 among this study's sample was 21.3% (95% CI = 12.9-31.8%). At the time of the SARS-CoV-2 PCR, most patients were asymptomatic (90%); however, all patients developed mild symptoms later during the illness.Fever was the most reported symptom (11 patients), followed by cough and diarrhoea . Only two patients experienced dyspnoea, and two had anosmia. Five patients were hospitalised, and only one required oxygen therapy and intensive care admission. All patients continued using their antirheumatic medication during the infection period until full recovery.This study is one of the first few studies to investigate the pregnancy outcomes in pregnant women with rheumatic diseases who had COVID-19 infection and those without COVID-19 infection. We identified a higher prevalence of COVID-19 among our sample (21.3%) than the previously reported 10% in pregnant and peripartum women . The infPatients with rheumatic diseases, in general, have an increased risk of intensive care unit (ICU) admission and mechanical ventilation . Among pThe Society for Obstetric Anaesthesia and Perinatology (SOAP) COVID-19 Registry studied the peripartum outcomes in women who had COVID-19 infections and those without COVID-19 and found that most patients with the infection were asymptomatic 61.1%). Patients with symptomatic infection carried an increased risk for caesarean section, increased postpartum length of stay, and premature delivery [1.1%. PatIn our cohort, six of the very premature children required NICU admission but recovered completely. Neonates born to mothers who were and were not previously diagnosed with COVID-19 were not routinely tested using PCR. Thus, the COVID-19 status of the newborns remained unknown. One infant was diagnosed with COVID-19 at two months of age after being in close contact with his grandmother and mother, both of whom were diagnosed with the disease. He remained asymptomatic while his mother continued breastfeeding and using her antirheumatic medications. Nevertheless, a previous study has shown that vertical transmission of COVID-19 is extremely low .Although some DMARDs have been promising in the treatment of COVID-19 infection cases and could have a role in the prevention of cytokine storms , it is sThe limitations of this study include the dependence on the patients’ ability to recall symptoms of infection and a history of close contact, which makes the findings related to these two parameters relatively inaccurate in spite of our attempt to provide patients with hotline access to report this information. The remaining data related to pregnancy outcomes were confirmed by hospital electronic records and were not sensitive to recall bias. Further limitations include the small sample size, lack of a control group of healthy pregnant women, and the discrepancy in sample size between the two groups, rendering it challenging to deduce the impact of the underlying autoimmune rheumatic disease, independent of the impact of COVID-19, on the pregnancy outcome.This is the first study in Qatar to report the outcome of pregnancy in patients with autoimmune rheumatic diseases during the COVID-19 pandemic. Despite the high proportion of COVID-19 among these patients, neither the presence of symptoms nor the underlying rheumatic disease or its treatment resulted in increased adverse maternal or pregnancy outcomes. This observation may reassure pregnant patients with rheumatic diseases during the pandemic. Nevertheless, larger prospective cohorts and matched non-rheumatic disease  controls are required to further assess the impact of antirheumatic medications and disease activity on pregnancy outcomes in patients with COVID-19.