Background: Data on the prevalence of bacterial co-infections among COVID-19 patients are limited, especially in our country, Indonesia. We aimed to assess the rate of bacterial co-infections in hospitalized COVID-19 patients and report the most common microorganisms involved and the antibiotic use in these patients.Methods: This study is a retrospective cohort study,among COVID-19 adult patients admitted to Universitas Airlangga Hospital Surabaya from 14 March-30 September 2020. The bacterial infection is defined based on clinical assessment, laboratory parameters, and microbiology results.Results: A total of 218 patients with moderate to critical illness and confirmed COVID-19 were included in this study. Bacterial infection was confirmed in 43 patients (19.7%). COVID-19 patients with bacterial infections had longer hospital length of stay (17.6 6.62 vs 13.317.12), a higher proportion of respiratory failure, intensive care treatment, and ventilator use. COVID-19 patients with bacterial infection had a worse prognosis than those without bacterial infection (p<0.04). The empirical antibiotic was given to 75.2% of the patients. Gram-negative bacteria were commonly found as causative agents in this study .Conclusion: COVID-19 patients with bacterial infection have a longer length of stay and worse outcomes. Healthcare-associated infections during intensive care treatment for COVID-19 patients must be carefully prevented. In Indonesia, until November 2020, there were 522,581 confirmed cases of COVID-19 with 68,604 active cases, 437,456 recovered cases, and 16,521 (3%) deaths. According to national data, among the total number of cases, East Java is the second-highest prevalence of 60,190 (11.6%). As of November 26, 2020, the cumulative data on confirmed COVID-19 patients in Surabaya were 16,763 with 1204 (7.03%) deaths1.Coronavirus disease (COVID)-19 has experienced an increase in 2,995,758 positive cases and 204,987 deaths in distribution areas of more than 213 countries2. COVID-19 pneumonia itself is also related to increasing ICU care, secondary infection rate, and higher invasive treatment3. Co-infection of SARS-CoV-2 and other microorganisms such as viruses, bacteria, and fungi is an essential factor in COVID-19 treatment since this condition may raise the difficulty in diagnosis, treatment, and the prognosis, also increasing the mortality5. Upper respiratory tract infection, a typical manifestation of COVID-19, is challenging to differentiate from other causes of pneumonia.Data regarding secondary respiratory infection in COVID-19 patients are still limited in Indonesia even though the cases distribution is still worldwide. Several reports suggest that secondary infection will impact the patients survival and increase intensive care unit (ICU) treatment6.There are various types of co-infection in COVID-19; such as: 1) secondary SARS-CoV-2 following bacterial infection or colonization; 2) mixed infection between viral and bacterial pneumonia infection; 3) secondary bacterial superinfection following SARS-CoV-2 infection. The mechanisms underlying those types of infection are very dependent on the onset and involving complex interactions between three different agents . Immune response towards SARS-CoV-2 infection only differs with mixed infection to bacteria or viral pneumonia. Therefore, it could be hypothesized that any co-infection will worsen the outcome and severity of COVID-197. This study aims to describe bacterial co-infection and antibiotic use among patients who confirmed SARS-CoV-2 infection from moderate to critically ill manifestation in Universitas Airlangga hospital Surabaya.Although several studies have investigated the epidemiological and clinical characteristic of COVID-19, information regarding SARS-CoV-2 infection with secondary infection are limited8 and the Indonesian Ministry of Health guidelines9. Confirmed COVID-19 patients were proven by oropharyngeal and nasopharyngeal swabs SARS-CoV-2 PCR (polymerase chain reaction). Bacterial infection was defined based on clinical assessment, laboratory parameters, and inflammatory parameters (C-reactive protein (CRP) and procalcitonin). Bacterial co-infection of SARS-CoV-2 defines if the culture samples were taken at patient presentation to the hospital or < 48 hours admission, while secondary bacterial infection defined if the culture samples were taken > 48 hours of admission. The bacterial causative agents were extracted from data of microbiology, that were identified by Microbiology automated machine Vitek-2 compact, as a routine procedure in this hospital.This study is a retrospective cohort study, total sampling, among COVID-19 adult patients admitted to Universitas Airlangga Hospital Surabaya. This hospital is an academic hospital and also a referral hospital for COVID-19 management in Surabaya, East Java Region. We included cases of moderate to critically ill COVID-19 patients between 14 March and 30 September 2020 that were admitted in the intensive care unit or high-care unit. COVID-19 diagnosis was made based on World Health Organization (WHO) guidelinesThis study was approved by the Ethical Committee of Universitas Airlangga Hospital (171/KEP/2020). Written informed consent was obtained from all participants prior to the start of the study.10. Culture examination was performed when there was suspicion of bacterial infection or sepsis. The sample for culture was taken from blood, urine, and respiratory tract.Data were taken from medical records and microbiology reports from the laboratory. Incomplete medical records were excluded. Clinical characteristics were divided according to the severity of the disease. Moderate case definitions are: 1) clinically sign of pneumonia ; 2) Oxygen saturation 93% free air. Severe case definitions are if there were clinically sign of pneumonia, and one of the following: 1) respiration rate >30 times per minute, or 2) severe respiratory distress, or 3) oxygen saturation < 93% free air. Critically ill cases defined if there were acute respiratory distress syndrome (ARDS), sepsis, and septic shock11. Descriptive statistics included categorical variables reported as number (percentage) and continuous variables as mean (standard deviation). For missing data, we used listwise deletion or univariable and multivariable analysis. Chi-square test and Mann-Whitney test were used accordingly to the type of variable. Categorical variables were shown as number (%) and continuous variables as mean (standard deviation) or median (range) depending on whether the data are normally distributed or not. Statistical significance was assessed by means of chi-squared for dichotomous variables, or by means of the two independent sample t-test or the Mann-Whitney U test for continuous variable depending on whether the data are normally distributed or not.Data were analyzed with SPSS version 24.0 From March 14 until September 30, 2020, a total of 218 patients confirmed for SARS-CoV-2 infection were admitted to Universitas Airlangga Hospital Surabaya from moderate to critically ill condition. Patients characteristic symptoms were defined according to their severity. Clinical characteristics and main comorbidities are detailed in2)  respectively. Some symptoms vary in duration between groups. Severe patients complained of cough symptoms for an average of 10.6 days, longer than those in the moderate or critically ill group. The duration of fever was almost similar between groups, namely 68.8 days.Characteristic symptoms and laboratory result are shown in2: FiO2 ratio (p = 0.005) in either severe or critically ill COVID-19 patients than moderate cases. The procalcitonin level significantly increased in severe and critical illness conditions. The CRP level also increased but was not statistically significant. The majority of patients (82.9%) were recovered and discharged from the hospital, while 9.6% of the patient died. Most of the patients who died were in critically ill condition at presentation to hospital. From the laboratory findings, the mean leucocyte count (p = 0.014), neutrophil count (p = 0.001), procalcitonin (p = 0.033), basal urea nitrogen (BUN) (p = 0.007), alanine aminotransferase (ALT) levels (p = 0.01) were significantly higher in either severe or critically ill COVID-19 patients than moderate cases. We also found a lower lymphocyte count (p = 0.001) and PaOBacterial infection was confirmed in 43 patients (19.7%) see. There wMortality occurs in 16.28% of COVID-19 patients with bacterial infections, higher than those without bacterial infection (8%). Overall, COVID-19 patients with bacterial infection had a worse prognosis than those without bacterial infection (p<0.04).The empirical antibiotic was given to 75.2% of the patients. Antibiotics used in these patients were quinolones (60.1%), cephalosporins (28.44%), carbapenem (23.85%), and aminoglycosides (4.59%). Quinolone used in these patients was mostly levofloxacin (79.39%), and the others were moxifloxacin (20.61%). The carbapenem used in this study was meropenem. Cephalosporins used were ceftriaxone, ceftazidime, cefotaxime, cefoperazone-sulbactam, and cefuroxime see.Klebsiella pneumoniae (2),Pseudomonas fluorescens (1),Pseudomonas putida (1),Staphylococcus epidermidis (2),Staphylococcus haemolyticus (1),Staphylococcus hominis (2). Bacteria found in urine were ESBL-producingEscherichia coli (2),Enterococcus faecalis (1), andPseudomonas putida (1). There were 47 isolates found in sputum cultures, in which six of them were fungi (Candida spp). The most frequent bacteria found wasAcinetobacter baumannii (9), followed byKlebsiella pneumoniae (5),Pseudomonas aeruginosa (4),Escherichia Coli (2),Enterobacter cloacae complex (3), andStaphylococcus haemolyticus (3)  and the others from endotracheal tube aspirate (n=10). Gram-negative bacteria dominate the culture result (70.37%). Bacteria found in the blood were extended-spectrum -lactamase (ESBL)-producing (3) see.et al. reported that the median age of the 191 patients was 56.0 years (1887 years), and most patients were male12. Lvet al. reported 354 hospitalized patients, 175 (49.44%) were male, and the median age was 62 years (2390 years)13. A study conducted in Saudi Arabia reported among the 99 hospitalized patients, the median age was 44 years (range 1987), and the majority were men (66%)14. We found that diabetes and hypertension were the most common comorbidity, similar to these other studies1215.Our study focuses on bacterial infection results in COVID-19 patients and evaluates their clinical and microbiological features. Clinical characteristics of the study group were described according to disease severity. According to disease severity, the number of patients with moderate, severe, and critically ill manifestations were 126 (57.8%), 40 (18.3%), and 52 (23.9%), respectively. The average age was 52.45 (14.44) years, and 55.05% of patients were male. Other studies regarding the characteristic of hospitalized COVID-19 patients showed various results. Zhou16. Neutrophil and lymphocyte ratio (NLR) has been proposed as a prognostic marker of severity in various chronic inflammatory diseases, including cardiovascular diseases and oncological processes17.In this study, inflammation signs such as leucocyte count, neutrophil count, procalcitonin in the critically ill group were higher than the others. Lymphopenia in COVID-19 patients occurs through various mechanisms such as direct virus invasion lymphocytes, lymphatic organ destruction, altered inflammatory cytokines production leading to lymphocyte apoptosis, and inhibiting lymphocytes function by metabolic molecules produced by metabolic disorders, such as hyperlactic academiaet al. in elderly COVID-19 patients. They also concluded that bacterial infection was also considered as a predictor of mortality in these patients18. Increased procalcitonin level in COVID-19 patients may be associated with the release of some cytokines, especially IL-6. It is established that procalcitonin is a better marker to predict severity, prognosis, or the sepsis course and is also helpful to guide antibiotic usage. Increased procalcitonin in critically ill patients with COVID-19 can represent a bacterial co-infection, and blood cultures for bacteria detection are needed to a prompt response19.Our result showed a significant increase of procalcitonin level and a higher proportion of bacterial infection in severe and critically ill COVID-19 patients. This finding is consistent with the study result from Wang21. In Fuet al. study, 13.9% (5 of 36) of the patients in the ICU were diagnosed with COVID-19 and secondary bacterial infection. In another report that was published from a UK secondary care setting, 27 among 836 patients (3.2%) had early confirmed bacterial isolates identified (05 days post-admission), rising to 51 cases (6.1%) during the admission. In a study conducted in Shiraz, Iran, in 2009, Hassanzadehet al. suggested that ICU-acquired infections were documented in 51.7% of ICU patients, with a mortality rate of 10.9% (5 patients)23. Our finding was relatively higher among other studies. The possible explanation is that most of the patients had been treated in hospital for more than two weeks (average 1217 days). Secondary infections usually correspond with nosocomial or healthcare-associated infections. Nosocomial infections are most commonly correlated with invasive medical devices or surgical procedures. Lower respiratory tract and bloodstream infections have the highest mortality, while urinary tract infections are the most common24.In this study, we found that bacterial infection was confirmed in 43 patients . The bacterial infection that was detected at patient admission  was 23%, while bacterial infection that occurs late during hospital stay (secondary infection) was 77%. The prevalence of bacterial co-infections in patients admitted to the ICU for acute respiratory failure related to COVID-19 pneumonia is poorly studied25. Sharifpouret al. reported that the median length of stay is around 15 days . A study on respiratory co-infection in patients with pandemic 2009 influenza A (H1N1) virus infection showed that ICU length of stay was three days longer among patients with co-infection. Another study by Zhouet al. reported a longer length of stay of 8.0 days (4.012.0) of all patients with COVID-19 admitted to their ICU12. These findings suggest that the length of ICU stay can be prolonged if patients become co-infected.The median length of stay among patients in our study was higher in the bacterial co-infection group rather than non co-infection, with an average of 17.6 and 13.31 days, respectively. This finding is consistent with the result of several studieset al. (50%), although they also included virus and fungal cultures3. Gram-negative bacteria were reported responsible for more than 30% of healthcare-associated infections, and these bacteria predominate in cases of ventilator-associated pneumonia (47%) and urinary tract infections (45%)24. In intensive care units (ICUs), gram-negative bacteria account for about 70% of these types of infections26. Gram-negative bacteria are highly efficient at up-regulating or acquiring genes that code for antimicrobial resistance mechanisms, especially in the presence of antibiotic selection pressure. Among gram-negative bacteria, theEnterobacteriaceae family being the most commonly found and multidrug-resistant organisms, includingPseudomonas aeruginosa,Acinetobacter baumannii, and extended-spectrum -lactamase (ESBL)producing or carbapenemase-producingEnterobacteriaceae, are increasingly being reported worldwide24. The identification of bacterial infection with gram-negative organisms is more reflected as a complication of ICU care and is not suggested as a specific predilection for co-infections in COVID-1923.The most common bacterial infection in this study was from respiratory tract infection, followed by bloodstream infection and urinary tract infection. The culture result was dominated by gram-negative bacteria (75.68%). This finding is similar to the result from ZhangAcinetobacter baumannii is the most common bacteria found in this study. Several reports regarding bacterial infection in COVID-19 also found a similar result24.Acinetobacter baumannii is the common cause of respiratory tract infection, especially in ICU settings where patients often received mechanical ventilation. Environmental contamination also plays a role in this phenomenon. Our ICUs for COVID-19 patients were set as a large ward consist of 16 beds. Although we already managed the distance between beds for more than 1.5 meters, the inter-patient transmission is hard to avoid. It is crucial to maintain hand hygiene compliance of the staff, strict cleanliness, and disinfection of the hospital environment in the hospital, especially in high-care or ICU setting.2. Secondary infection and nosocomial infection are also other considerations in the use of antibiotics. In this study, 75.3% of patients included were given antibiotics. A meta-analysis conducted about bacterial co-infection and secondary infection in COVID-19 patients reported that the majority of patients with COVID-19 received antibiotics 27. Another meta-analysis reported that >90% of COVID-19 patients were given an empirical antibiotic, while the bacterial infection was detected only in 7% of hospitalized patients and 14% of ICU patients28. Quinolones (levofloxacin and moxifloxacin) were frequently prescribed, followed by cephalosporins and carbapenems. Indonesian national guidelines for COVID-19 treatment recommend using antibiotics in severe and critically ill patients, especially if there was suspicion of bacterial infection. The empirical antibiotic choice was intravenous azithromycin or levofloxacin10. In our hospital, levofloxacin is more feasible than azithromycin, so clinicians prefer to prescribe this drug. In our hospital, antibiotic use usually was decided based on clinical signs and laboratory parameters . Culture examinations were not routinely performed, preferably in ICU/high-care settings and in patients showing bacterial infection.Antibiotic is often given to COVID-19 patients for various reasons. The clinical manifestation on presentation was sometimes challenging to distinguish with bacterial infection6. Although bacterial infection in COVID-19 patients has already been reported in various results, evidence supports the restrictive use of antibiotics. Clinical guidelines and standard testing to diagnose bacterial infection in COVID-19 are not clearly available. The microbiological examination is an important strategy to confirm bacterial infection and decide the antibiotic choice4. Sputum, blood culture samples, and also pneumococcal urinary antigen testing should also be performed. Antibiotics should be stopped in patients who represent cultures, and urinary antigen tests show no signs of bacterial pathogens after 48 hours29. The widespread use of antibiotics in this pandemic era raises concern about antimicrobial resistanceThere are some limitations to our study. First, we only included COVID-19 patients in high-care and intensive care units. In low-care settings, bacterial infection cannot be confirmed due to limited data. Second, we only reported bacterial infection from microbiology results. Virus culture or gene sequencing to detect pathogens is not available in our hospital. Bacterial culture was not performed in all COVID-19 patients, so in a condition where the sign of infection is absent such as in elderly or in immunocompromised patients, the bacterial infection might be underdiagnosed.Acinetobacter baumannii that yielded from sputum. Increased antibiotic use and multi-drug resistant organism is an emerging problem in this pandemic situation. Infection control practice need to strictly conducted to reduced secondary or healthcare-associated infectionBacterial infection was detected in 19.7% of COVID-19 patients admitted in high-care and intensive care units, predominantly secondary infections. COVID-19 patients who suffered from bacterial infection have a longer length of stay and have higher mortality. The pathogen commonly found in this study washttps://doi.org/10.5061/dryad.sxksn032830.Dryad: The clinical impact of bacterial co-infection among moderate, severe and critically ill COVID-19 patients in the second referral hospital in Surabaya,This project contains the following underlying data:-Baseline characteristics for all patients-Lab data for all participantsCreative Commons Zero No rights reserved data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the "in our country Indonesia"the sentence can be improved as: While the prevalence of COVID in Indonesia is high, data on the prevalence of...are limited.Abstract, background, removeThe information for the antibiotic selection is already in the heading of "pathogens associated with bacterial infection", I wonder if the last paragraph on the "bacterial infection" can be shifted under the "pathogens"heading?Discussion: what are suggestions for further research raised from this paper? There are some uncertainties mentioned by the authors regarding the use/guideline of antibiotics in COVID. It can be one of them.I can see that there are some improvements in the manuscript that the authors have provided. However, I believe the quality can be further improved by accomodating these minor suggestions:Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Health service research in LMICsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. A well written article and proper data collection to support the conclusion. From a LMIC like Indonesia, this article shares important findings, since the co-bacterial infection is not more than 20%, higher than reported in IDSA data but lower compared to what had been expected in clinical practice among Indonesian doctors. Authors might add value by putting a highlight on what is the clinical appearance of the co-bacterial infected vs. the non co-bacterial infected for readers to apply in daily practice.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:tropical medicine and infectious diseaseI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you very much for the opportunity for reviewing this manuscript. This manuscript has presented interesting findings of bacterial coinfection in COVID-19 patients. Data collected for the research were from patients' medical records in Univ Airlangga hospital in Indonesia.Introduction - Line 1. I would recommend the authors to improve the first sentence of the first and second paragraph to improve clarity. The presentations of Methods, Results, and Discussion in the manuscript are appropriate. However, there are some minor revisions needed, which I believe are beneficial for improving the manuscript:Methods - Just to clarify, so the infection was examined if there is any suspicion of bacterial infection and not a routine examination? - Are there any prior investigations related to this topic in Indonesia? Please write/present if in the introduction there is any.Results - Patient characteristic and laboratory findings, is there any table/summary table associated with the patients' leucocyte count, procalcitonin? Please cite/involve the summary table in the main manuscript.Discussion - Are there any things that still unknown/need further investigation arising from this research? - Statistical analysis: SPSS version 24.0 (citation?). - What are the study implications for further research and practice? - I would recommend a language proofreading for this manuscript. Best wishes to the authors for the manuscript revision.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Health service research in LMICsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We firstly would like to thank you for the kindly comprehensive review of our manuscript. Here's to answer and respond to your questions:We believe that there is no prior investigation linked to this topic ever done before in Indonesia 2. MethodsWe clarify that the bacterial culture examination was done on the suspicious patients only. 3. ResultsWe have enclosed the leucocyte and procalcitonin data of the patients on the table 1. 4. DiscussionIn the study to come, further investigation can probably be done on the low health-care setting, also bigger concerns on under-diagnosed bacterial co-infection of elderly and immunocompromised patients can be taken into consideration.The implication of the study is that we find the bacterial culture examination necessary in the COVID-19 patients management since the bacterial co-infection might worsen the clinical outcome. 1. Introduction Furthermore, we also appreciate your suggestions in order to improve the final quality of this manuscript. We are currently working on the minor revisions, proof-reading, and going to submit the latest version promptly. We have just process revised several points on the manuscript as what the reviewer, named Fitriana Murriya Ekawati, had recommended a couple days ago. We include the latest version of our manuscript down below along with highlighted changes. We also insert our reply to the reviewer, which said: We firstly would like to thank you for the kindly comprehensive review of our manuscript. Here's to answer and respond to your questions: 1. Introduction We believe that there is no prior investigation linked to this topic ever done before in Indonesia. We added this information in the second and last paragraphs of Introduction 2. Methods Yes, the bacterial culture examination was done on the suspicious patients only. Please refer to the last sentence of the paragraph 8 in the Discussion section. 3. Results We have enclosed the leucocyte and procalcitonin data of the patients on the table 1 on Result section 4. Discussion In the study to come, further investigation can probably be done in the low health-care setting, also bigger concerns on under-diagnosed bacterial co-infection of elderly and immunocompromised patients can be taken into consideration. we added this sentence on the last paragraph of the discussion. The implication of the study is that we find the bacterial culture examination necessary in the COVID-19 patients management since the bacterial co-infection might worsen the clinical outcome. Therefore this examination should be conducted when the patient shows signs of infection during the intensive care. We added this sentence in the second paragraph of the introduction. Furthermore, we also appreciate your suggestions in order to improve the final quality of this manuscript. We are currently working on the minor revisions, proof-reading, and going to submit the latest version it promptly Hope this will meet all the requirement and we will receive the final pdf proof soon. Thanks in advance, Tri Pudy Asmarawati and research team


To date, more than 100 million people worldwide have recovered from COVID-19. Unfortunately, although the virus is eradicated in such patients, fibrotic irreversible interstitial lung disease  is clinically evident. Given the vast numbers of individuals affected, it is urgent to design a strategy to prevent a second wave of late mortality associated with COVID-19 PF as a long-term consequence of such a devastating pandemic. Available antifibrotic therapies, namely nintedanib and pirfenidone, might have a role in attenuating profibrotic pathways in SARS-CoV-2 infection but are not economically sustainable by national health systems and have critical adverse effects. It is our opinion that the mesenchymal stem cell secretome could offer a new therapeutic approach in treating COVID-19 fibrotic lungs through its anti-inflammatory and antifibrotic factors. The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 117 million global cases and 2.6 million global deaths in the first year of the pandemic . TherefoTo date, more than 100 million people worldwide have recovered from COVID-19. Unfortunately, although the virus is eradicated in such patients, fibrotic interstitial lung disease  may become clinically evident in a large percentage of patients.Generally speaking, PF can develop either following chronic inflammation or as a primary, genetically influenced and age-related fibroproliferative process, as in idiopathic pulmonary fibrosis (IPF). Previous coronavirus outbreaks have been associated with substantial post-viral fibrosis and physiological impairment; therefore, given the pandemic’s scale for SARS-CoV-2 infection, the global burden of fibrotic lung disease will probably increase considerably in the next years . Indeed,http://www.clinicaltrials.gov (accessed on 9 March 2021)). However, therapy with nintedanib or pirfenidone is not economically sustainable by national health systems (NHS) for the many patients expected in the coming years  every three months vs. placebo. There were no significant adverse effects after the administration of MSCs in any patients. With MSC therapy, the 6-min walk distance significantly improved in 13 weeks, DLCO in 26 weeks and FVC in 39 weeks vs. placebo. FVC for 12 months with MSCs increased by 7.8% from baseline, whereas it declined by 5.9% with placebo [After many successful preclinical studies, treatment with mesenchymal stem cells (MSCs) is considered a potential new therapy for PF . A recen placebo . The nonAlthough those findings might move future trials toward a new step in stem cells transplantation, MSC therapy is still linked to logistical problems  and to the need to have a laboratory available for cell manipulation where the infusion is performed. In addition, MSCs effectiveness may be hampered by low-engraftment or by a hard-to-control batch-to-batch variability . In thisIt is worth mentioning that the use of secretome is linked to advantages: (i) the availability of an on-the-shelf product with the same effectiveness as MSCs; (ii) ease of storage/transport/use in outpatient settings; and (iii) safer than replicating live cells . Specifically for safety, a recent review of the literature reveals that the administration of secretome or EVs by intravenous injection is safe, as no adverse effects have been reported . ConcernDespite no clinical trials currently investigating MSCs or their secretome in the treatment of COVID-19 PF, it is our opinion that they may represent a well-suited approach for treating patients with COVID-19 PF. Preliminary results and the scientific background encourage further investigation on MSC secretome treatment for COVID-19 PF with well-designed clinical trials under regulatory authorities’ control.


Abdominal aortic aneurysms (AAAs) have posed a great threat to human life, and the necessity of its monitoring and treatment is decided by symptomatology and/or the aneurysm size. Accumulating evidence suggests that circular RNAs (circRNAs) contribute a part to the pathogenesis of AAAs. circRNAs are novel single-stranded RNAs with a closed loop structure and high stability, having become the candidate biomarkers for numerous kinds of human disorders. Besides, circRNAs act as molecular “sponge” in organisms, capable of regulating the transcription level. Here, we characterize that the molecular mechanisms underlying the role of circRNAs in AAA development were further elucidated. In the present work, studies on the biosynthesis, bibliometrics, and mechanisms of action of circRNAs were aims comprehensively reviewed, the role of circRNAs in the AAA pathogenic mechanism was illustrated, and their potential in diagnosing AAAs was examined. Moreover, the current evidence about the effects of circRNAs on AAA development through modulating endothelial cells (ECs), macrophages, and vascular smooth muscle cells (VSMCs) was summarized. Through thorough investigation, the molecular mechanisms underlying the role of circRNAs in AAA development were further elucidated. The results demonstrated that circRNAs had the application potential in the diagnosis and prevention of AAAs in clinical practice. The study of circRNA regulatory pathways would be of great assistance to the etiologic research of AAAs. Abdominal aortic aneurysms (AAAs) frequently induce cardiovascular death among the elderly male population within various European and Asian countries –3. AAAs Conventionally, AAAs are mostly treated with elective retroperitoneal or open transperitoneal surgery , but nowThe pathogenic mechanism of AAAs is complicated and involves multiple factors. Previous studies suggest that AAAs are associated with the weakened defective adventitial/medial arterial layers, like fibroblasts and smooth muscle cells (SMCs) –22. ReceDue to strong gene expression regulation effects, epigenetic alterations, such as histone modification, DNA methylation, and noncoding RNA (ncRNA) modification –27, haveIn particular, many articles have demonstrated the important roles of ncRNAs in cardiovascular diseases (CVDs), such as aortic dissection and AAAs. Meanwhile, circRNAs are also considered to exhibit certain effects on some CVDs, but their expression levels and roles in AAAs still remain unclarified.In the present work, the roles of circRNAs in the occurrence and development of AAAs were illustrated, and their significance in treating AAAs was discussed. In addition, the regulation effect of circRNAs and their corresponding target genes on AAAs and the underlying mechanisms were also investigated. The ncRNAs that were previously recognized to be involved in several processes leading in AAAs were highlighted. The in-depth investigation on such circRNAs will shed lights on the prevention and treatment of AAAs, and these circRNAs may be used as candidate prognostic biomarkers and therapeutic targets for the prediction of the AAA incidence and the evaluation of patient prognosis.βR1, TGFβR2, and TGFβR3 gene mutations promote the production of matrix metalloproteinases (MMPs) and eventually lead to medial degradation [A Mendelian factor that causes AAAs often refers to single-gene mutations . Marfan radation . Other sradation . AAAs maradation .There are various non-Mendelian factors leading to AAAs, including the ECM, endothelial cell (EC) specification, SMCs differentiation, inflammation, and cell adhesion.The ECM is a complex network of macromolecular substances that supports and connects tissue as well as mediates tissue generation and cell physiological activities. The proteins of the Matrix Metalloproteinases (MMP) family can regulate the ECM turnover, which is related to the formation of genes that encode MMP and ECM components in AAAs. Besides, upregulated MMP-2 and MMP-9 levels can be detected within human AAA tissue , 34. MorAAAs are related to rs1795061 localized at around 40 kb upstream of the SMYD2 transcription start site . SMYD prIn the study of Jones et al., the SNP rs2836411, an AAA risk variation localized at 21q22.2 in the intrinsic region of ERG gene, was detected . ERG can+T cells are found to accumulate on the aneurysm wall, and AAA-related SNPs are detected in some human genes encoding such critical inflammatory components. AAA patients show an increased circulating IL-6 level. IL-6 is secreted by the aneurysm, and its expression level is related to the aneurysm surface area. Angiotensin II can affect AAAs by acting on the IL-6 signaling pathway in mice.CD4Epigenetic modifications are induced by developmental or environmental factors. They do not change the genetic code but can regulate the tissue- or context-specific expression of information encoded in DNA , 44. Tra+ CD25+ regulatory T cells, induces AAA occurrence [DNA methylation, one of the potent epigenetic mechanisms, is crucial in preserving the DNA structure, deactivating the X chromosome and maintaining chromosome stability. Besides, DNA methylation also modulates components (retrotransposons and transposons) and regulates gene levels . It is ccurrence . Apart fcurrence . The stucurrence .−/−AAA mice infused with Angiotensin (Ang) II are higher than those in normal controls [Gene activation and inactivation depend on specific signatures of histone modifications in critical enhancer or promoter gene regions. Such modifications are harbored in histone deacetylases, histone acetylases, together with methyltransferases . The fincontrols . As a clcontrols .It has been established that over 97% of the genome can encode the noncoding transcripts, most of which can be processed into short ncRNAs (such as miRNAs) and lncRNAs. There are various factors leading to AAAs, and the pathology and pathogenic mechanisms of AAAs at the molecular level remain largely unclear. Therefore, it will become a novel research direction to investigate the AAA etiology at the epigenetic level. Studies have shown that ncRNAs take part in the AAA genesis. Many ncRNAs are differentially expressed in AAA patients and regulate gene expression at transcriptional and posttranscriptional levels. The regulatory pathways of some ncRNAs have also been confirmed, which is conducive to the study of other molecular mechanisms , 53–56.MiRNAs are short (21-23-nt-long) RNA molecules existing in eukaryotes, capable of regulating the expression of other genes . They ar2 and Ang II-induced aneurysm formation in mice [The most typical type of miRNAs is miRNA-29 family members, which promote the AAA formation by regulating fibrosis and the ECM. miRNA-29 inhibits the expression of several ECM proteins and antiapoptotic factors in SMCs, thereby boosting the aneurysm generation , 67. Stu in mice . In conclncRNAs are a type of ncRNAs longer than 200 nt without the protein encoding function, and they are involved in regulating numerous processes . In receLike lncRNAs, circRNA transcripts have been a recent addition to the functionally relevant ncRNAs in our genomic landscape. They have long been disregarded since their discovery in the 1990s due in part to a limitation in their detection method, but the emerging novel bioinformatics and deep RNA sequencing approaches provide the foundation for circRNA research . Recent Firstly, we explored the relationship between publication volume and time in Then, we also explored the relationship between publication volume and time for ncRNA in Keyword analysis is relatively essential to the research of the entire paper. It can help us to figure out the research direction. Q value) greater than >3 generally means a significant clustering structure. In the figure, modularity Q = 0.684. The classification method is to name the index terms in the search keywords. We can get ten clusters with an order from #0 to #10. The smaller the number # is, the more keywords the cluster contains. Each cluster is composed of multiple closely related words. The largest cluster is “aortic aneurysm” (#0), followed by “abdominal aortic aneurysm formation” (#1). A module clustering value  of these circRNAs in AAA development in animal models and their therapeutic potential are yet to be elucidated.Zhou et al. from China observed differential expression of circRNAs in AAA samples and controls, so they subsequently conducted high-throughput sequencing to determine the circRNA expression patterns in four pairs of aortic samples, including four consecutive AAA cases undergoing open surgery and four brain-dead heart-beating organ samples . FinallyAs indicated in the interaction networks of circRNAs/miRNAs, hsa_circ_0002168 and hsa_circ_0005360 contain one binding site for miR-15a and miR-181b, respectively. It is evident that overexpressed miR-181b in AAA patients downregulates elastin and the MMP-3 tissue inhibitor, thereby promoting AAA development . As for Wang et al. collected two samples from each of the AAA and control groups and analyzed the circRNA expression profiles. The AAA samples of C57BL/6J male mice were treated with Ang II and 3,4-benzopyrene (BAP), and the circRNA expression in these AAA samples was compared with that in the control group . The qRTTo prove the role of circRNA CCDC66 in the pathogenesis of AAAs and identify its corresponding pathway, Yang et al. carried out a study and found that circCCDC66 played a role in the proliferation of VSMCs . ThroughFindings of this study reveal that miR-28-5p, circCBFB, GRIA4, and LYPD3 are involved in AAAs. circCBFB serves as the miR-28-5p sponge, capable of regulating the proliferation and apoptosis of VSMCs in a LYPD3/GRIA4-dependent manner . With reThere are over 70 miR-7 binding sites in the cerebellar degeneration-related protein 1 antisense RNA (CDR1as), and these sites regulate the effect of CDR1as on the target gene expression . AccordiThrough screening circRNAs in tissue specimens from AAA patients, Zheng et al. observed increased expression of hsa-circ-000595 in diseased specimens [At present, the pathogenic mechanism of AAAs is mainly explored through studying aortic tissue from AAA patients undergoing open surgery. Several subtypes of vascular cells  are involved in AAA genesis  Figure .Disordered ECs may induce adventitia and media inflammation . The chrHowever, these physiological characteristics facilitate the formation of vulnerable atherosclerotic plaques in the aneurysmal wall, which illustrates that AS is a vital risk factor for AAAs . Age, smNumerous pathophysiological stimuli can result in EC disorders in AAAs, including hypoxia , advanceα axis [A study has revealed the potential role of hypoxia-mediated new vessel formation in the pathogenic mechanism of AAAs . Dang etα axis . Recent α axis . Furtherα axis . AccordiκB/NOD as the miR-197 sponge [High glucose (HG) affects the formation and development of AAAs , 111 thr7 sponge . Previou7 sponge . In addi7 sponge .2O2. OxLDLs promote inflammatory cell infiltration into the aortic wall through enhancing the activities of MMP2 [βR2 and the corresponding downstream phosph-SMAD3 [OxLDLs affect the O2•- concentration in several ways. Increasing the O2•- concentration causes the NOS to dephosphorize to produce O2•- , 118, in of MMP2 , chemoki of MMP2 . Researcph-SMAD3 . A recenph-SMAD3 . Wang etph-SMAD3 . It is aph-SMAD3 .VSMC phenotypic transformation within AAAs is ascribed to pathophysiological incentives as diverse as ox-LDLs, Ang II, proinflammatory factors, and hyperglycemia .The circ_0010283 level in ox LDL-challenged VSMCs is remarkably higher than that in control VSMCs. The circ_0010283 target is miR-370-3p, which targets HMGB1. As a result, circ_0010283 modulates the level of HMGB1 through miR-370-3p . The higAng II has been suggested to enhance VSMC proliferation in the process of vascular remodeling and play a vital part in AAA formation. According to circRNA microarray analysis, mmu-circRNA-42742 is conspicuously downregulated in Ang II-treated mouse aortic smooth muscle cells (MASMCs). The parental gene of mmu-circRNA-42742 is NRG-1, which modulates vascular remodeling via the ErbB signal transduction pathway. Besides, the circNRG-1/miR-193b-5p/NRG-1 axis can be potentially used as the Ang II target for inhibiting VSMC apoptosis and facilitating vascular remodeling .The analysis of circRNA expression in the HG-induced VSMCs shows that circWDR77 is upregulated. The predicted results are miR-124 and fibroblast growth factor 2 (FGF-2), whose interaction is verified by the RNA pull-down experiment and luciferase reporter gene assay. More importantly, Wang et al. found that exosomes from HG-induced HUVECs caused VSMC senescence via the circRNA-0077930/miR622/Kras ceRNA axis, while exosomes with circRNA-0077930 depletion had no implications for VSMC senescence .Furthermore, Zheng et al. noticed the upregulation of hsa_circ_000595 in the hypoxia-induced human aortic smooth muscle cells (HASMCs). They concluded that hsa_circ_000595 silencing acted as the miR-19a sponge to suppress cell apoptosis [α-treated VSMCs, and circ-Sirt1 overexpression impedes the NF-κB p65 nuclear translocation and lowers the levels of adhesion molecules and proinflammatory cytokines  [κB p65 induced by TNF-α through reversing the inhibition effect of miR-132/212 on SIRT1. The study results of Kong et al. suggest that circ-Sirt1 may mitigate the NF-κB p65-induced inflammation via direct and indirect approaches. After treating the human aortic vascular smooth muscle cells (HA-VSMCs) with PDGF-BB, Peng et al. found that circDHCR24 deletion served as the miR-149-5p sponge and diminished the cell proliferation [AAAs are a type of chronic inflammatory disorders, so the in vitro AS model can be constructed by inflammatory stimulation. circRNA-0044073 is upregulated in the LPS-treated human umbilical vein smooth muscle cells (HUVSMCs) but downregulated in LPS-treated HUVECs. Further exploration indicates that circRNA-0044073 overexpression evidently promotes HUVSMC and HUVEC proliferation and invasion through the miR-107/Janus kinase (JAK)/STAT signal transduction pathway . As demo ICAM-1) . Moreoveferation .Additionally, the levels of circ_RUSC2 and circ_SATB2 decrease within the proliferating VSMCs. It is disclosed by Mao et al. that circ-SATB2 promotes STIM1, which can be explained by the role of miR-939 in proliferative VSMCs. Mechanically, circ-SATB2 overexpression inhibits the expression of contractile VSMC marker SM22a, while miR-939 promotes its expression, indicating an association between circ-SATB2 and VSMC phenotypic differentiation . circ_RUκB signaling pathway. When Raw264.7 cells are transfected with the circ_1639 mimic, the level of miR-122 gene is significantly reduced, while the expression of its target gene TNFRSF13C increases. Hence, it can be concluded that in Raw264.7 cells, circ_1639 affects the inflammatory response through the miR-122/TNFRSF13C regulatory axis [CircRNAs are proven to be essential for the modulation of macrophages. Studies have shown that in LPS-induced macrophages Raw264.7, circ_1639 promotes the proinflammatory response in Raw264.7 cells. The study of Lu et al. demonstrates that circ_1639 expression is tremendously upregulated in LPS-treated Raw264.7 cells. The level of p-P65 increases in circ_1639 mock-transfected cells, but declines in cells transfected with circ_1639 inhibitor plasmid. Therefore, circ_1639 regulates inflammatory response through the NF-ory axis .In Raw264.7 macrophages stimulated by the calcitonin gene-related peptide (CGRP), increased expression of mmu_circRNA_007893 and decreased expression of miR-485-5p are detected. The coimmunoprecipitation reaction further confirms the interaction among mmu_circRNA_007893, miR-485-5p, and IL-6. In Raw264.7 cells, the mmu_circRNA_007893/miR-485-5p/IL-6 regulatory pathway regulates the inflammatory response by controlling the expression of the cytokine IL-6 and ultimately affects the cell function .α and IL-6) and anti-inflammatory cytokines. There is a lot of evidence that circRNAs play a vital role in macrophages, and it will assist with further research on the diseases caused by macrophages and circRNAs.The above results indicate that the function of macrophages can be affected by the expression level of the circRNA regulatory axis, such as the proinflammatory cytokines  patients undergoing surgery . As obseThrough carrying out RNA-Seq on the affected ascending aortic samples from patients with acute Stanford type A aortic dissection (AAAD), Tian et al. have identified 506 evidently differentially expressed circRNAs . BesidesOne recent effort identifies the hsa_circ_0021001 in peripheral blood of patients with intracranial aneurysms, but its potential contribution to aneurysmal expansion is not expounded . Hsa_cirIn a study, the circRNA sequencing on IA patients recognizes two novel circRNAs in the peripheral blood samples from IA cases, and their expression in peripheral blood of normal subjects are also analyzed. The results suggest that hsa_circ_0008433 and hsa_circ_0072309 are new and critical circRNAs associated with IAs. This study may provide novel prognostic biomarkers and therapeutic targets for IAs .Wang et al. acquired arterial wall tissue samples from the aneurysm site in 12 cases and discovered that circRNA_0020397 was downregulated in IAs. The decreased circRNA_0020397 expression possibly suppressed the proliferation of VSMCs through upregulating miR-138 levels and downregulating KDR levels .AAAs are one of the major causes leading to cardiovascular death among the senile male population, and their etiology is complex, including apoptosis of SMCs and inflammatory reaction. It is of urgent need to develop new pharmacological approaches or gene therapy strategies for delaying aneurysm development or lowering the risk of acute rupture. Over the last few decades, ncRNAs are increasingly identified as critical regulators for AAA occurrence and development. Hence, it is necessary to identify abnormally expressed ncRNAs and validate them in relevant human AAA tissue and animal models, so as to better explore the pathophysiological mechanisms related to AAA genesis and development.Different from traditional linear RNAs, circRNAs are a novel class of RNAs with a closed loop structure that can be detected in the eukaryotic genome. Besides, they show higher stability and greater resistance to RNase degradation, so they are widely used as biomarkers. However, there is no evidence that peripheral blood circRNAs are effective biomarkers for the diagnosis of AAAs, and further investigation is required to confirm the relationship between circulating circRNAs and AAAs.It is reported that circRNAs function as the molecular “sponge,” capable of regulating transcription and posttranscriptional gene expression by binding to and blocking microRNA regulatory factors. The regulatory pathway of circRNAs is circRNA-miRNA-mRNA. A circRNA specifically binds to an miRNA and inhibits its expression, thereby regulating the expression level of the template RNA.In this paper, the relationship between circRNAs and AAAs is briefly described. It is proved that several circRNAs affect the formation of AAAs by regulating the proliferation and apoptosis of VSMCs. Meanwhile, the changes of aneurysmal wall cells  in the AAA development process can be well analyzed based on the circRNAs extracted from these cells. When the differential expression of these circRNAs is verified, the molecular mechanisms of circRNAs in regulating AAA occurrence are investigated both in vitro and in vivo. Meanwhile, the mechanism of action is further elaborated.Because of the above-mentioned characteristics, circRNAs have been listed as a biomarker and therapeutic target for AAAs. The expression and regulation of circRNAs will directly affect the development of AAAs.


The World Health Organization (WHO) has declared the pandemic of COVID-19 infection in March 2020, most of cases presented with mild symptoms, and a significant number of cases showed variable neurological pictures. Vocal cord paralysis with no clear cause is termed as idiopathic vocal cord paralysis and supposed to be caused by viral infection. This is a case series study; data were collected prospectively from patients presented to the ENT clinic of Kasr Alainy (Cairo university) and Railway hospitals. Patients presented with defective vocal cord movement with concurrently or recently passed COVID-19 infection were reported from March 2020 to April 2021.Authors have reported 6 cases of vocal cord paralysis mainly unilateral due to COVID-19 infection as an only clear cause within 14 months. Age ranges from 39 to 69 years, 2 males and 4 females. Patients presented with different clinical scenarios. Follow-up of the cases showed spontaneous recovery in 5 cases and one case underwent cord medialization.Viral infection could be an underlying cause of idiopathic laryngeal cord paralysis; in the new era of the COVID-19 pandemic, physicians all over the world noticed variable neurological pictures; in this study, we presented 6 cases of vocal cord paralysis mainly unilateral supposed to be due to COVID-19 infection; all cases showed spontaneous recovery apart from one case that needed medialization of the cord. The World Health Organization (WHO) has declared the pandemic of COVID-19 infection in March 2020. The disease has been arisen in Wuhan, China, and caused by respiratory droplets that carry SARS-CoV-2 , and a sParalysis of the vocal cords is a serious problem; most of the causes are well known apart from some cases that presented with cord paralysis with no clear cause , 10. AutIn this study, authors have spotted the light on a number of cases of laryngeal cord paralysis mainly unilateral as a result of COVID-19 infection.A case series study, data were collected prospectively from patients presented to the ENT clinic of Kasr Alainy (Cairo university) and Railway hospitals. Patients who presented with defective vocal cord movement with concurrently or recently passed COVID-19 infection were reported from March 2020 to April 2021.Authors have reported 6 cases of vocal cord paralysis mainly unilateral due to COVID-19 infection as an only clear cause , D-dimer, serum ferritin, and lactate dehydrogenase (LDH). A new CT scan carried out on the skull, neck, and upper half chest was clearly free from any masses (Fig. Another lady, 39 years old, coming with a history of a 5-day duration of fever, bony aches, anosmia, hypogeusia, dry cough, and change of voice. Nasopharyngeal swab and PCR for COVID-19 and CT scan on the chest were carried out and the results were confirmatory. She was isolated and hospitalized for treatment. Flexible fibro-optic laryngoscopic examination was performed after 3 weeks when the swab turned negative and it was diagnostic for right cord paralysis. A new CT carried out on the skull, neck, and upper half chest was clearly free from any masses Fig. .Fig. 2RA gentleman, 41 years old, past history of COVID-19 infection 5 weeks ago and hoarseness of voice 2 weeks ago. Left vocal cord paralysis was diagnosed by a fibro-optic laryngoscope Fig. .Fig. 3LA 51-year-old lady presented with just a mild degree of noisy difficult breathing of 5 days with no other symptoms; examination showed left vocal cord paralysis and right cord paresis. No history of neck surgery was found; a CT scan on the chest, neck, and skull base was carried out as a routine in cord paralysis cases but it showed COVID-19 opacities by accident. Then, she referred for nasopharyngeal swab and PCR that gave a positive result for COVID-19 Fig. .Fig. 4LA lady, 44-year-old patient, coming to the clinic with fever, anosmia, mild abdominal dyspepsia, nausea, cough, and change of voice 3 days ago. Diagnosis confirmed as COVID-19; after treatment in an isolation hospital and being negative by PCR, the larynx was examined and left vocal cord paralysis was diagnosed Fig. .Fig. 5LA 69-year-old man already referred from an isolation hospital after finishing his treatment from COVID-19. The cause of referral was the persistent cough and change of voice despite a clear recent CT scan on the chest and negative post-treatment PCR. Laryngeal examination showed right vocal cord paralysis (Fig. All cases started speech therapy and were monitored by regular visits every 2 weeks. Five cases showed variable degrees of spontaneous recovery within 2–5 months; vocal cord medialization by hyaluronic acid was carried out in one case who showed no spontaneous improvement.This study suggests an association between the COVID-19 infection and the laryngeal vocal cord paralysis as there is no other clear cause of vocal cord paralysis in this study’s cases; none of the study cases was diabetic; also, all cases showed nearly the same onset of COVID-19 symptoms and laryngeal paralysis. Bhatt et al. have suggested the same theory of association between cord paralysis and viral upper respiratory tract infection .Some authors have noticed that the incidence of idiopathic vocal cord paralysis is higher in winter due to the higher spread of droplet infections of viral origin –16.Previous publications have diagnosed specific virus infections like herpes simplex, Epstein-Barr, West Nile, and varicella-zoster viruses clinically and laboratory in cases with idiopathic cord paralysis , 17–21.There are many examples of viral-induced neuropathy in the field of otolaryngology other than the larynx. Anosmia and ageusia became very popular with COVID-19 infection , 8, alsoThe incidence of post-COVID-19 vocal fold paralysis is relatively low; however, mild paresis may be discovered accidentally. Furthermore, laryngeal examination has been limited by the pandemic. Thus, the prevalence, severity, and consequence of vocal cord insult during the COVID-19 pandemic remain to be determined . In a stFinally, literature has focused on anosmia and ageusia as a recently common picture of COVID-19 infection and it lacks studies that entail the issue of COVID-19 impact on vocal cord innervation and mobility.Viral infection could be an underlying cause of idiopathic laryngeal cord paralysis; in the new era of the COVID-19 pandemic, physicians all over the world noticed variable neurological pictures; in this study, we presented 6 cases of vocal cord paralysis mainly unilateral supposed to be due to COVID-19 infection; all cases showed spontaneous recovery apart from one case that needed medialization of the cord.


Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigen (Ag) tests have been widely employed to identify patients for a rapid diagnosis and pandemic control. Rapid lateral-flow techniques are currently the most used, but automated technologies have emerged as another viable alternative to molecular methods. We aimed to evaluate the analytical performance of the DiaSorin Liaison SARS-CoV-2 Ag test in asymptomatic population and close contacts, for its use as a tool in pandemic control efforts.Material and Methods: A retrospective study was conducted. A total of 861 samples were included, 291 (34%) were positive for SARS-CoV-2 with cycle threshold (Ct) <40, and 570 (66%) were negative.Results: A strong correlation was observed between reverse transcriptase-PCR (RT-PCR) Ct and Ag 50% Tissue Culture Infectious Dose per milliliter  and all RT-PCR negative samples tested negative for the 200 TCID50/ml SARS-Cov-2 Ag cutoff, i.e., a specificity of 100% was reached (95% CI: 99.4–100.0%). Samples with <25 Ct and/or >106 extrapolated copies/ml were reached a sensitivity of 100% (95% IC 97.0–100.0%). For intermediate viral loads (>105 extrapolated copies/ml or <30 Ct), the sensitivity value still exceeded 80%. As with other Ag methods, samples between 30 and 40 Ct could not be detected with a reliable sensitivity.Conclusions: The LIAISON® SARS-CoV-2 Ag assay displays an acceptable sensitivity and a very high specificity that is useful for detecting the presence of SARS-CoV-2 in nasal swabs (NPS) of asymptomatic population or to regular monitoring of risk groups in controlled settings. Additionally, the flexibility in processing different samples and in the sampling preparation process makes this test an option for its use in high throughput laboratories. Automated tests may facilitate result reporting and yield consistent data, while avoiding some of the pitfalls of rapid lateral-flow techniques, such as observer variability. Since the Coronavirus Disease-2019 (COVID-19) pandemic has emerged, efforts in its control have been focused on the development of high-sensitivity diagnostic tools and rapid systems for more afford able strategies.The European Center for Disease Control (ECDC) in its document of November 19, 2020 “Options for the use of rapid antigen (Ag) tests for COVID-19 in the EU/EEA and the UK” indicates the need to implement policies and rapid systems of detection in certain settings where there is a high risk of transmission, such as social healthcare centers or hospital settings. The use of rapid Ag tests is appropriate in high-prevalence settings when a positive result is likely to indicate true infection and in low-prevalence settings to quickly identify highly infectious cases  ≥80% and specificity (E) ≥97% and that have undergone independent validation studies  should bAntigen tests identify the presence of the nucleocapsid Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Ag, which is usually detectable in samples from the upper respiratory tract during the acute phase of infection. These tests do not have an amplification step, so their analytical detection limits are higher than PCR. Nonetheless, SARS-CoV-2 has an exponential growth in infected patients, so even with initial low viral loads; it is possible that in just a few hours, the viral levels reach the detection thresholds of Ag tests. This pattern of viral load kinetics could explain why repeated population screenings with Ag tests may lead to effective detection policies. Taking into account these criteria, some authors state that Ag-detection tests used frequently can still have a high sensitivity to detect carriers or infected patients without the need to meet the analytical limit of detection of the PCR as the reference test .Several authors have evaluated Ag-detection tests as a screening method for a symptomatic population obtaining very good results in terms of sensitivity and specificity . Also, wThe option to test asymptomatic individuals has been considered to ensure a safe environment in certain settings, such as hospitals or schools , due to Recently, the DiaSorin company has launched a SARS-CoV-2 Ag sandwich-type direct chemiluminescence immunoassay (CLIA) for a quantitative determination of SARS-CoV-2 nucleocapsid protein Ag in nasal (NS) or nasopharyngeal swabs (NPS). The final reaction consists of the emission of a light signal, giving rise to relative light units (RLU) that are directly proportional to the concentration of SARS-CoV-2 viral Ag present in the samples. Automation allows greater performance and control of the entire analytical and post-analytical process of the sample. This technology could be applied in those settings with low incidence even in an asymptomatic population or to trace COVID-19 contacts for a faster result than using PCR.We aimed to evaluate the capacity of the LIAISON® SARS-CoV-2 Ag assay in comparison with the reference technique used for the diagnosis of SARS-CoV-2 infection .This retrospective study was approved by the Ethics Committee of the Germans Trias i Pujol Hospital, Badalona, Spain: PI-21-096. The study enrolled 861 asymptomatic individuals from primary healthcare centers of the Metro Nord health administrative region of Catalonia during the month of January 2021. NS specimens from these 861 individuals were collected in 3 ml Universal Transport Media (UTM) or Viral Transport Media (VTM).The aim of this study was to assess the analytical performance of the SARS-CoV-2 Ag-detection test in asymptomatic individuals who were contacts of positive patients and in healthcare workers undergoing repeated screenings.Asymptomatic population was defined as patients with a definitive molecular diagnosis of COVID-19 positive without presenting symptoms, such as fever, cough, myalgia, or other symptoms related to COVID-19.Close contacts were defined as a person who has spent more than 15 consecutive minutes with the positive case at a distance closer than 2 m, within 48 h of the person getting symptoms or testing positive for SARS-CoV-2.50/ml). The Liaison XL instrument directly calculates SARS-CoV-2 viral concentrations from 22 up to 105 TCID50/ml. The specimens with ≥200 TCID50/ml values are considered positive, results <200 TCID50/ml are considered negative. In a previous iteration of the test, results between ≥100 and <200 TCID50/ml were classified as equivocal. To facilitate comparisons with other authors, results have been presented with both cutoffs.Nasal specimens were processed by reverse transcription-PCR  according to the instructions of the manufacturer. A total of 1,000 μl of these samples were pipetted into a tube containing 1 ml of Liaison SARS-CoV-2 sample inactivation buffer for virus inactivation. The tubes were kept at room temperature for 120 min before testing. This procedure was performed on a type II biological safety cabinet. The quantification of SARS-CoV-2 Ag was determined using the Liaison SARS-CoV-2 Ag assay on the Liaison XL platform according to the instructions of the manufacturer. SARS-CoV-2 concentrations are expressed as 50% tissue culture infectious doses per milliliter (TCIDFor absolute quantification based on RT-PCR cycle threshold (Ct) values, a standard curve was built using 1/2 serial dilutions of a SARS-CoV-2 RNA  in a range of concentrations from 1,400,000 copies/ml to 684 copies/ml. The measurements were made in different CFX instruments, by different technicians, and on the same day to account for the inherent variability of PCRs. The resulting standard curve was used to extrapolate the viral load of each sample (in copies/ml) from their respective Ct.Concordance between results obtained for Liaison SARS-CoV-2 Ag assay and RT-PCR was established using the Spearman correlation index. In addition, NPV, positive predictive value (PPV), and positive and negative likelihood ratio  were calculated. These data were obtained with an estimated incidence of 1% according to Government Data supplied in January 2021 with RT-PCR results  from female individuals, with an age distribution ranging from 1 to 94 years old (median: 44 years old). There were a total of 87 samples from patients younger than 15 years old that were included in the analysis. Two hundred and ninety-one samples (34%) were positive for SARS-CoV-2, with Ct < 40 . If we consider the gray zone cutoff of 100–199 TCID50/ml previously proposed by the manufacturer, the specificity was 99.3% (95% CI: 98.2–99.8%). Classification differences between both cutoffs were observed only in the 25–35 Ct range. Both thresholds detected all samples with Ct < 25 and were not able to detect any sample with Ct > 35. Adjusted sensitivities for different Ct and extrapolated copies/ml are listed in 14·e(−0.709Ct).All RT-PCR negative samples were tested negative for the 200 TCIDOne of the goals of clinical microbiology is to develop new tools for accurate diagnostic. In this sense, the COVID pandemic was a cornerstone for the introduction of molecular and new approaches for the diagnosis of infectious diseases in most clinical microbiology laboratories. The rapidity in results, such as the one offered by rapid Ag devices, is mandatory to manage the isolation of patients and to create a safe environment for healthcare workers. However, a shorter diagnosis time must not significantly reduce the ability to perform a correct diagnosis.Rapid Ag devices, such as lateral flow tests, have many advantages but sometimes fail to detect viral Ags in specimens from which the virus was isolated and therefore pose a risk of misdiagnosing infected people. The limit of detection of Ag-RDTs could be 10,000-fold lower than those of nucleic acid amplification tests (NAATs)  but coul6 extrapolated copies per ml, the Liaison Ag test displays a 100% of sensitivity and specificity with the currently recommended cutoff (200 TCID50/ml). Even with lower viral loads (>105 extrapolated copies or <30 Ct), the sensitivity value is still >80% (84.08%). If the previously recommended undetermined cutoff of 100–200 TCID50/ml is used, a slightly better sensitivity is obtained (93.63%) for samples >105 copies or <30 (91.36%) Ct, with a still high specificity of 99.3%. These data are similar to other groups who also study the performance of this technology  or E (Especificity) because it is less likely to change with the prevalence of the disorder. With a 200 TCID50/ml cutoff, the LR+ is not calculable because it would have to be divided by zero, since we have not had false positives. In any case, it can be seen that the LR+ is very good in all cases and excellent at the 200 cutoff. LR– at Ct <40 is poor with both cutoffs. On the other hand, if we take 30 Ct as a cutoff point, the LR– becomes good or very good at 200 and 100 .The PPV with a 200 cutoff is 100%, but on the other hand, the PPV with a cutoff of 100 is 43% or 56% depending on the Ct. Therefore, it seems clear that 200 cutoff is better for the accuracy of the test and is now the cutoff established by DiaSorin as the reference cutoff. The 100 cutoff still could be useful in populations with high suspicion of COVID-19 or high incidence rates, maintaining an indeterminate zone as a marker of follow-up.50/ml, which could be considered equivocal, must be carefully regarded depending on clinical symptoms and the timing of possible exposure. It would be recommended that patients with equivocal results underwent several follow-up determinations to definitively rule out SARS-CoV-2 infection. As NS, oropharyngeal swabs, or even saliva samples (Based on our results, values between 100 and 199 TCIDChemiluminescence assays are commonly used in highly automated instruments and are known for their high sensitivity and practicability compared to other immunological assays . So, CLIThis study has several limitations. Firstly, our study populations are asymptomatic patients in whom the evolution of the infection is unknown, and it might be a problem because we do not work with homogeneous specimens. Additionally, some subjects develop symptoms a few days after the test, and we were not able to record this information, so some information about symptoms is not well-documented. In addition, we used frozen samples and freeze-thaw cycles may affect the integrity  of the NIn addition, the pediatric population was included in the analysis despite it was suggested that children could be facilitators in the spread of SARS-CoV2 infection because many affected children might be asymptomatic  but seveLastly, the study was performed in January, so we do not know the accuracy of this test with new variants of concern; despite data demonstrate that the SARS-CoV-2 proteome is slowly accumulating mutations, fortunately .In conclusion, LIAISON® SARS-CoV-2 Ag is a good alternative for diagnostic purposes for symptomatic individuals but also in close contact and asymptomatic patients. The good agreement with RT-PCR and its high specificity makes it an ideal choice as a diagnostic tool in controlled low-incidence settings. The easiness in sample management and processing is a strength for choosing LIAISON® SARS-CoV-2 Ag in the COVID-19 diagnostic algorithms.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Comitè d'Etica de la Investigació amb medicaments Hospital Universitari Germans Trias i Pujol. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.GF-R, JB, VG, and SM: conceptualization and writing—original draft. GF-R, JB, VG, SM, and LJ: data curation. JB, VG, SM, and LJ: formal analysis. GF-R, JB, VG, SM, LJ, AB, and CC: methodology. GF-R and IB: resources. GF-R, JB, and IB: supervision. GF-R, JB, VG, SM, LJ, AB, CC, and IB: writing—review and editing. All authors contributed to the article and approved the submitted version.Diasorin Iberia supports this project by granting part of the funds for reagents. However, this commercial sponsor had no involvement in study design, collection, analysis, or interpretation of data, writing the manuscript, and the decision to submit the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


While it is now apparent clinical sequelae (long COVID) may persist after acute COVID-19, their nature, frequency and aetiology are poorly characterised. This study aims to regularly synthesise evidence on long COVID characteristics, to help inform clinical management, rehabilitation strategies and interventional studies to improve long-term outcomes.et al. Results were analysed using descriptive statistics and meta-analyses to estimate prevalence.A living systematic review. Medline, CINAHL (EBSCO), Global Health (Ovid), WHO Global Research on COVID-19 database, LitCovid and Google Scholar were searched till 17 March 2021. Studies including at least 100 people with confirmed or clinically suspected COVID-19 at 12 weeks or more post onset were included. Risk of bias was assessed using the tool produced by Hoy A total of 39 studies were included: 32 cohort, 6 cross-sectional and 1 case–control. Most showed high or moderate risk of bias. None were set in low-income countries and few included children. Studies reported on 10 951 people  in 12 countries. Most included previously hospitalised people . The longest mean follow-up time was 221.7 (SD: 10.9) days post COVID-19 onset. Over 60 physical and psychological signs and symptoms with wide prevalence were reported, most commonly weakness , general malaise , fatigue , concentration impairment  and breathlessness . 37% (95% CI 18% to 60%) of patients reported reduced quality of life; 26% (10/39) of studies presented evidence of reduced pulmonary function.Long COVID is a complex condition with prolonged heterogeneous symptoms. The nature of studies precludes a precise case definition or risk evaluation. There is an urgent need for prospective, robust, standardised, controlled studies into aetiology, risk factors and biomarkers to characterise long COVID in different at-risk populations and settings.CRD42020211131. A significant number of people continue to describe ongoing symptoms long after the acute phase of COVID-19, often referred to as long COVID.Long COVID is a heterogeneous condition with an uncertain prevalence, for which there is currently no precise case definition.The breadth of reported symptoms suggests a complex, heterogeneous condition affecting both those who were hospitalised and those managed in the community.Our review identifies weakness , general malaise , fatigue , concentration impairment  and breathlessness  as the most common symptoms reported.The current evidence base of the clinical spectrum of long COVID is limited, based on heterogenous data, and vulnerable to biases, hence caution should be used when interpreting or generalising the results.Our review identifies areas where further long COVID research is critically needed to help characterise long COVID in different populations and define its aetiology, risk factors and biomarkers, as well as the impact on variants of concern and vaccination on long-term outcomes.SARS-CoV-2 first emerged in December 2019 causing a widespread pandemic. Most people experience asymptomatic or mild-to-moderate acute COVID-19 symptoms, while around 15% of people are estimated to progress to more severe disease requiring hospitalisation and approximately 5% become critically ill.1While the acute phase of the disease was characterised early, there are still limited data on long-term outcomes.6The number of people living with long COVID is unknown. Attempts to quantify the prevalence of long COVID use different methods, including national surveys and patient-led studies, making it difficult to compare across studies. The UK’s Office for National Statistics has estimated that on average 1 in 5 people have symptoms beyond 5 weeks, while 1 in 10 have symptoms persisting over 12 weeks.9The symptoms of long COVID are equally ill-defined, with patients describing it as a fluctuating illness of disparate symptoms.Our early understanding of long COVID has been accumulated from case reports and cross-sectional online survey studies as the pandemic global research focus has largely been on studies of hospitalised patients during the acute phase. As the pandemic progresses, emerging studies have followed up patients to present the fluctuating multiorgan sequelae of acute COVID-19, yet evidence is still scarce. There continues to be a call to further understand and acknowledge this condition by incorporating patient knowledge and experiences, together with standardised studies, exploring underlying aetiologies behind different syndromes.12 13Given the enormous number of people worldwide who have suffered from COVID-19, it is essential to establish a precise categorisation of long COVID. Such categorisation will not only help people better understand their symptoms but also direct research into prevention, treatment and support, ultimately allowing us to understand and prepare to respond to the long-term consequences inflicted by the COVID-19 pandemic. Our review seeks to synthesise and continually update the evidence on the character and prevalence of long COVID.BMJ Global Health and on a dedicated webpage with infographics and a brief summary for lay people and professionals.Systematic reviews conducted early during the COVID-19 pandemic soon became redundant due to the rapidity with which new research was released. In recognition of this, many reviewers have moved towards the concept of a ‘living systematic review’ (LSR), which compared with traditional systematic reviews has in-built mechanisms for regular update and renewal.10.1136/bmjgh-2021-005427.supp1Supplementary dataThis report was structured according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement guidelines.17The following databases were searched: Medline and CINAHL (EBSCO), Global Health (Ovid), WHO Global Research Database on COVID-19 and LitCovid from 1 January 2020 to 17 March 2021. Additionally, we searched Google Scholar on 17 March 2021, screening the first 500 titles. A ‘backwards’ snowball search was conducted of the references of systematic reviews. Full search terms are included in Peer-reviewed studies were considered eligible if they included at least 100 people with laboratory confirmed and/or clinically diagnosed COVID-19. Without a clear, internationally agreed case definition, we included studies that reported symptoms or outcomes assessed at 12 or more weeks post COVID-19 onset.Screening was performed independently by two systematic reviewers. Any disagreements were resolved via consensus or a third reviewer. Non-English articles were translated using Google Translate and assessed by a systematic reviewer with good knowledge of the language. The data were managed using the review software Rayyan.19Data extraction was performed using Microsoft Excel. A data extraction template informed by a previous reviewet alThe included studies were assessed for risk of bias using the tool produced by Hoy 2 statistic.We undertook individual descriptive analysis for each study. We presented symptom proportions by different settings, as presented in the individual studies: hospitalised, non-hospitalised or a mix of both populations if no subset data were available. Symptoms were broadly grouped into physiological clusters through discussion with clinicians. Proportion of symptoms and its 95% CIs were estimated using the exact method.The study team includes members who have been affected by long-term COVID-19 sequalae, including members of Long COVID Support,They actively contributed to the development of the study protocol, to inform the research questions and interpretation and presentation of the findings and to communicate the results to different audiences. The results of this LSR will be disseminated to long COVID patient forums for discussion and feedback to inform research priorities and updates.We identified 6459 studies, of which 39 met the inclusion criteria , all of Most studies were set in Europe , followed by Asia , North America  and the Middle East 8% (3/39) . There wThe map shows the global distribution of the studies identified and the shading shows the combined studies population size by country.Most studies included adults, while 10% (4/39) also included children.Most studies  were cohorts of hospitalised patients post discharge, 10% (4/39) followed up people who were not hospitalised, while 23% (9/39) included both . Of the inclusions in this review, 78% (8520/10 951) were previously hospitalised during the acute COVID-19 phase. Twenty-two studies included people requiring ICU admission during the acute phase.31 33–35 37 38 40–55The longest follow-up period in any study was a mean of 221.7 (SD: 10.9) days post onset. Only 56% (22/39) of studies specified COVID-19 severity,33 35–57 59–63 65 67–69Overall, 12 studies were assessed as high risk of bias, 22 as moderate risk of bias and 5 as low risk of bias. Most studies had a high risk of bias with regard to the generalisability of their results to the wider population with COVID-19. High risk of bias ratings were most common for external validity, with item 1 (representation of target population) and item 3 (random selection) having the most high risk of bias ratings . Further35 36 43 44Patients suffering from long COVID report a wide range of new or persistent symptoms, in both the hospitalised and non-hospitalised populations. Symptoms were broadly organised into physiological ‘clusters’ for the purpose of presentation and interpretation of this review .The focus of each study included in our analysis varied. Some authors focused solely on a specialty, such as dentistry, or a specific symptom, such as cognition, making comparative analysis difficult. Even among those studies which took a broad approach, the prevalence of symptoms was diverse. Similarly, the prevalence of the more commonly reported symptoms varied markedly.2 values (>80%) were observed, they resulted from narrow dispersions in the estimates and well-separated estimates and CIs between studies  were weakness , general malaise , fatigue , concentration impairment  and breathlessness . Across studies, 37% (95% CI 18.43 to 59.93) of patients reported reduced quality of life. Although high I studies . The difPatients also reported a diverse array of less prevalent symptoms and signs, including sweating, chest pain, sore throat, anxiety and headaches, among others. The prevalence of these symptoms was lower, usually less than 20%. Multiple studies assessed lung sequelae and respiratory performance through outpatient visits follow-up .66Pulmonary function tests were reported in 26% (10/39) of studies,One study assessed kidney function in people with COVID-19-associated acute kidney injury (AKI) compared with people with non-COVID-19-associated AKI, found that COVID-19-related AKI was associated with decreased kidney recovery during outpatient follow-up.36Exploring the literature, we sought to produce a meta-analysis of risk factors for long COVID. We found a considerable diversity of reported risk factors, including age, sex, comorbidities, ethnicity and severity of the acute phase.Several cohorts  assessed whether there was an association between the severity of initial COVID-19, including symptom load, level of hospital care, need for mechanical ventilation and the risk of persisting sequelae. An association between female gender and long COVID risk has also been noted in longitudinal studies , as has the association between presence of comorbidity,The limitations of the existing evidence base and inconsistency of reported findings preclude confident conclusions at this time. Instead, we have summarised the reported significant associations to date  and suggOur work represents the most comprehensive review of evidence regarding long COVID yet produced. Accurate to 17 March 2021, this LSR captures the breadth of persistent symptoms reported in 39 studies, including over 10 000 people. These data suggest long COVID is a syndrome affecting both previously hospitalised and non-hospitalised people, characterised by marked fatigue, weakness, general malaise, breathlessness and concentration impairment lasting for a prolonged period of time. Besides these common symptoms, there is a diverse array of secondary symptoms. The findings in this review show symptoms and prevalence aligned to current knowledge on long COVID. The Office for National Statistics (ONS) Cohort Study, including control participants, reports the most common symptoms persisting for 12 or more weeks included fatigue (8.3%), headache (7.2%), cough (7%) and myalgia (5.6%).7A deeper understanding of long COVID is currently prevented by the limitations of the published literature. The studies included in our review were highly heterogeneous due to differences in their study designs, settings, populations, follow-up time and symptom ascertainment methods. In addition, studies used inconsistent terminology describing symptoms and limited details and stratification on pre-existing comorbidities, the severity of COVID-19 and treatment methods. This inconsistency and limited reporting partly explain the high degree of variability observed. The lack of case–control studies prevent a direct attribution of symptoms solely to COVID-19; larger prospective studies with matched control groups are needed. We note that there are large, robust prospective cohort studies of hospitalised patients73The findings have identified several research gaps and priorities. The majority of long COVID cohorts were conducted in Western Europe on patients recently discharged from hospital. There is a paucity of evidence on the long-term effects of COVID-19 in low-to-middle income countries and in people who were not hospitalised. Similarly, there were no studies identified focusing on children, despite evidence showing that children and young people are also affected by long COVID.Our review also highlights a need for standardised and validated COVID-19 research tools to harmonise data collection, improve quality and reduce reporting variability. For instance, fatigue is one of the most commonly reported symptoms of long COVID. However, the symptom alone is not clearly defined and it is open to different interpretations, hence it requires a validated tool such as the Visual Analogue Scale, graded fatigue scale for robust, objective and comparative analysis. ISARIC has developed open access research tools available to sites globally to facilitate standardisation of data collection, analysis and interpretation for adults and children of an age.Similarly, our study highlights the need for further research to refine the many circulating interim case definitions and precisely characterise long COVID, including the potential impacts of variants of concern and vaccination on long COVID.As this is an LSR, emerging themes from this first version will inform future updates. The LSR will be updated periodically, as new research is published internationally, in order to provide relevant up to date information for clinicians, patients, researchers, policy-makers and health-service commissioners. Version changes will be identified and previous reports will be archived.This LSR summarises published evidence on the spectrum of long-term COVID-19-associated symptoms and sequelae (as of 17 March 2021). It is clear that long COVID affects different populations, with a wide range of symptomatology. Our findings suggest this multiorgan syndrome is characterised by fatigue, weakness, malaise, breathlessness and concentration impairment, among other less frequent symptoms. Currently, the strength of the available evidence is limited and prone to bias. The long-term effects of COVID-19, in both hospitalised and non-hospitalised individuals, including children and at-risk populations, should be a priority for future research using standardised and controlled study designs. Robust research is needed to characterise and define long COVID and identify risk factors and underlying aetiology, in order to inform prevention, rehabilitation, clinical and public health management to improve recovery and long-term COVID-19 outcomes. This LSR will be updated approximately every 6 months as new evidence emerges for up to 2 years.


The intrathecal drug delivery system (IDDS) is successfully utilized for the treatment of chronic pain conditions; however, they are associated with complications related to human error and system failure. A case report is presented of a patient with opioid withdrawal (OW) secondary to assumed catheter microfracture. Interrogation of the IDDS allowed for the collection of pre- and post-treatment/stabilization cerebrospinal fluid (CSF), which is used to investigate the possible physiological determinants of OW.A 46-year-old female with a history of low back pain after traumatic low back injury status post-IDDS placement for failed back surgery syndrome presented with signs and symptoms concerning for OW. After every other possible explanation was ruled out, it was hypothesized that there may be IDDS catheter microfracture(s), and catheter replacement led to symptom resolution. There were no significant differences in cytokine levels tested in pre-CSF versus post-CSF samples. Whole-cell patch-clamp electrophysiology analysis of human-induced pluripotent stem cell-derived nociceptors after treatment with pre- and post-CSF samples demonstrated modulation of action potential waveform.In patients presenting with acute OW attribution IDDS malfunction, catheter microfracture must be in the differential, and non-conventional interrogation of the IDDS catheter should be considered. The possible differences in pre-CSF and post-CSF may be more complicated than previously postulated, as there were no significant differences in cytokine profiles; however, treatment of in vitro neurons with pre- and post-CSF resulted in differential neuronal excitability, which may account for some of the symptoms of OW. Chronic pain conditions, including lower back pain (LBP), considerably impact the lives of patients and account for significant health care expenditure . LBP in In this case, troubleshooting the IDDS permitted the collection of cerebrospinal fluid (CSF) prior to and after treatment of a patient with acute opioid withdrawal (OW), allowing for the first-in-human testing of the hypothesis of a “memory trace” in the CSF, which can induce a form of long-term potentiation at C-fiber synapses . Upon acTo date, this is the first reported case to postulate OW that may be due to catheter microfracture not detected using conventional interrogation of the IDDS components. It is also the first-in-human investigation of the effects of CSF pre- and post-treatment of OW to determine possible physiological determinants of OW related to the CSF.Participant recruitmentThe patient provided verbal and written informed consent. Verbal approval was attained at the time of presentation from the patient to report her case and results in a case report format.Implanted intrathecal drug delivery systemThe products used were Medtronic SynchroMed II Pump , Medtronic Intrathecal Catheter Spinal Segment Revision Kit, and Medtronic Indura 1PC Intrathecal Catheter.Cell culture methodTM Matrix 3 (Anatomic Corp). Cells were grown in ChronoTM Senso-MM complete growth medium (Anatomic Corp). Cells were plated at 105 cells/cm2 and incubated at 37°C, 5% CO2, and 95% humidity. Culture media were replaced the day after plating and then every two days thereafter. Cells appeared dorsal root ganglia-like and fired action potentials shooting over +20 mV by day 7 in vitro.The hiPSCs  were recWhole-cell patch-clamp electrophysiology analysis methodHiPSC-derived nociceptors were identified by infrared-differential interference contrast (IR-DIC) microscopy using an Olympus digital camera. Current clamp recordings were performed as previously described .Cytokine analysis methodA total of 200 µL/sample was analyzed using the Human Cytokine Array Kit  and imaged using an Odyssey Fc Imaging System  with an exposure time of 2 minutes for optimal contrast. Fluorescence intensity values measured using ImageJ.Statistical analysis methodStatistical analysis was performed with GraphPad Prism 8.0 . Statistical significance attributed results yielding p-value < 0.05.Patient presentationA 46-year-old female post-IDDS placement for failed back surgery syndrome presented for signs and symptoms concerning for OW. The patient’s pain had increased from her baseline of 2/10 to 9/10. She complained of approximately 24 hours of insomnia, anxiety, severe tremors, bilateral lower extremity myalgias and arthralgia, nausea, non-bloody emesis, diarrhea, and sensation of being flushed. She denied any new focal neurological symptoms. Her Clinical Opiate Withdrawal Scale score was 27 (moderately severe) . Vital sShe reported only taking hydrochlorothiazide for chronic hypertension at home. The pump was programmed to bolus 60 mcg fentanyl/0.75 mg bupivacaine over two minutes without relief. She received 4 mg intravenous (IV) hydromorphone followed by 50 mcg IV fentanyl in the emergency room. She reported that her symptoms somewhat improved with the IV medications, but she continued to be in visible distress.The patient was taken to the operating room on the night of presentation for catheter dye study Figure , pump roWhile waiting to return to the operating room, her scheduled and PRN (pro re nata) medications included IV hydromorphone patient-controlled analgesia 0.5 mg, IV hydromorphone 0.5 mg, clonidine 0.1 mg PO , IV diphenhydramine 12.5 mg, and IV Ativan 1 mg. The following day, OW symptoms had resolved. The patient underwent a pocket revision of ITP and catheter reimplantation , serine protease inhibitor E1 (serpin-E1), C-X-C motif chemokine ligand 12 (CXCL12), and interleukin-18 (IL-18) were detected by the assay Figure . We did MIF levels were significantly higher than serpin-E1, CXCL12, and IL-18 . These data suggest that MIF may be significantly upregulated in patients with IDDS to control chronic pain.In this case study, we present the first case report of a patient undergoing acute OW secondary to assumed catheter microfracture. We suspect microfracture of the catheter, as live fluoroscopic interrogation of the IDDS revealed no malfunction of the pump (as the pump motor was interrogated and observed to turn appropriately), no issues with catheter patency (as CSF was aspirated from the access port), no contrast leakage at the catheter connection site as well as no contrast leakage along the length of the catheter , and no subdural extravasation at the catheter tip. Moreover, the contrast pattern at the tip of the catheter ruled out the possibility of occlusion at the tip by granuloma formation. The most convincing evidence excluding the Medtronic SynchroMed II Pump and implicating catheter malfunction was the lasting resolution of symptoms upon catheter replacement. The authors of this case report agree with Dawes et al. that the presence of catheter microfractures may be intermittently patent, leading to unremarkable routine investigations . It may This case highlights the importance of keeping catheter microfracture in the differential, with unremarkable IDDS interrogation. As previously proposed by Dawes et al. “careful search of the catheter under magnification [with appropriate magnification under scanning electron microscopy] should also be done to look for small fractures when revising a catheter, particularly when the symptomatology is confusing and a microfracture suspected” . The autThe differential effects of pre- and post-treatment CSF indicate altered ion channel activity controlling AP shape that may lead to increased neuronal firing affecting reported pain in the patient . MacrophIn patients presenting with acute OW attribution IDDS malfunction, catheter microfracture must be in the differential, and non-conventional interrogation of the IDDS catheter should be considered. The possible differences in pre-CSF and post-CSF may be more complicated than previously postulated as there were no significant differences in cytokine profiles; however, treatment of in vitro neurons with pre- and post-CSF resulted in differential neuronal excitability, which may account for some of the symptoms of OW.