BackgroundWith rising trends of multi-drug organism infections and the limited availability of new antimicrobials, management of such cases has become a hassle for the clinician. Ceftazidime-Avibactam (CEF-AVI) is evolving as an effective alternative to polymyxins in the management of Carbapenem-Resistant Organisms (CRO) infections. The Food and Drug Administration (FDA) has approved CEF-AVI in a restricted group of clinical syndromes where the drug could have potential use.ObjectiveThe goal of this study was to evaluate the clinical outcome in terms of 14-day all-cause mortality and clinical cure at seven days in patients on CEF-AVI.MethodologyA retrospective study was conducted on patients who received CEF-AVI in a period of one year in our hospital. Patients were included in the study if they have received CEF-AVI for more than one day of therapy (DOT) and samples from relevant sites have been sent for culture and sensitivity. Variables and outcomes were collected from the hospital information system and medical records.ResultsA total of 78 patients were included, 52 (66.7%) were started empirically on CEF-AVI while 26 (33.3%) were on targeted therapy. Out of the 78 patients, 43 patients had positive cultures among which 32 patients had Carbapenem-Resistant Enterobacteriaceae (CRE)/Carbapenem-Resistant Pseudomonas aeruginosa (CRPA) infection. The most common clinical syndrome in which the drug was used was occult sepsis  followed by primary bacteremia  and neutropenic sepsis . The clinical efficacy which was primarily assessed in terms of clinical cure was met for 55 (70.5%) patients. The 14-day mortality for the studies group was found to be 18 (23%).ConclusionThe analysis of results shows encouraging clinical cure rates and 14-day mortality rates in a subset of severe infections which has limited treatment options. Avibactam is a beta-lactamase inhibitor that has an action against class A, class C beta-lactamases, and some class D enzymes such as Oxa-48. The Infectious Disease Society of America (IDSA) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) declared the use of ceftazidime-avibactam (CEF-AVI) be restricted to confirmed Carbapenem-Resistant Enterobacteriaceae (CRE) or Carbapenem-Resistant Pseudomonas Aeruginosa (CRPA) infections and the ESCMID guidelines additionally included treatment of multidrug-resistant organism with limited treatment options (LTOs) ,2. In InStudy designThis was a single-center retrospective study conducted on in-patients who received CEF-AVI from January 2021 to December 2021. The protocol was approved by the ethics committee (IHEC no. 285). Patients were included in the study if (i) they received CEF-AVI alone or in combination with any other antibiotic including aztreonam for at least one day of therapy (DOT) and (ii) samples from relevant sites were sent for cultures before the start of antibiotic. Patients who were suspected or culture-confirmed to have urinary tract infections, intra-abdominal infections, or nosocomial pneumonia were excluded from the study. Also, patients were excluded if they could not be followed up for a minimum period of 14 days and those who did not receive one DOT of CEF-AVI.Parameters and data collectionCEF-AVI was given as a 2.5g intravenous infusion over two hours thrice a day in a patient with normal creatinine clearance, dose adjustments for renally impaired patients were based on manufacturer recommendations, and the clinical condition of the patient, for example, a higher than recommended dosage was used in critically ill patients on hemodialysis/renal replacement therapy ,8. IndicData were collected from electronic records and reviewed by study coordinators which included the patient’s demographic profiles, clinical and microbiological profiles, antimicrobial therapy characteristics, and patient outcomes. Combination therapy is the use of another antimicrobial along with CEF-AVI with or without aztreonam except for antimicrobials used for additional gram-positive coverage.Outcomes and statistical analysisThe outcomes from this study were all-cause mortality at 14 days from the start of CEF-AVI and clinical response at seven days from initiation of the antibiotic. Clinical response was categorized as improved/not improved in terms of clinical/radiological/laboratory parameters or death within seven days of initiation of the drug. Secondary objectives were time to initiation of CEF-AVI, average DOT on CEF-AVI, and average dose per day of CEF-AVI.Continuous variables - median with interquartile ranges or mean and categorical variables as frequencies. Comparison between all-cause mortality and clinical response with variables like clinical syndrome or combination therapy was done using chi-square tests/Fischer’s exact.2 with seven patients on hemodialysis during the duration of drug therapy.Eighty-three patients had received CEF-AVI within the study period and fit the study criteria. Five out of the 83 patients were lost to follow-up and discharged against medical advice and the remaining 78 patients were included in the study. The study population had a median age of 55.5 years and mostly comprised males . Diabetes followed by systemic hypertension were the most common co-morbidities seen among the population and there was a likewise distribution of subjects based on the Charlson comorbidity index (CCI) as shown in Table The most common clinical syndrome for which CEF-AVI was used was occult sepsis (34.6%) and followed by primary bacteremia (25.6%). Post-transplant patients included four post-bone marrow transplants and three post-liver transplant recipients. Miscellaneous syndromes include osteomyelitis, liver abscess, otitis media, and suspected meningitis constituting 5.13%, i.e., four out of 78 of the patients. The severity of infection ranged from no sepsis to septic shock .Antimicrobial therapy characteristics that were collected for the study included indication for initiation of CEF-AVI, microbiological characteristics, and combination with aztreonam or other drugs. Sixty-seven percent (52 out of 78) of the indications for initiating CEF-AVI were empiric and 33.3% (26 out of 78) were for targeted reasons. Forty-three 55.12%) patients out of 78 were culture positive. As few patients had multiple isolates totally there were 49 isolates in 43 patients. Thirty-five out of forty-nine isolates (71.4%) were CRO. Most are CRE  and CRPA . One patient had an infection with both CRE and CRPA. Molecular resistance genes were analyzed in only 16 isolates and Oxa-48 was the predominant resistance gene detected (12 out of 16 isolates). CEF-AVI was resistant in nine (28.1%) out of 25 isolates in whom it was tested by the E-strip method. Table 2% patienCEF-AVI was resistant in nine out of 27 isolates that were tested and synergy testing between CEF-AVI and aztreonam was done in 25 out of 35 CRE/CRPA isolates which revealed that 76% of isolates had synergy between the two drugs. Aztreonam was given along with CEF-AVI in 44 patients (56.4%) out of the total 78 patients in the study. Other drugs given in combination included minocycline, polymyxins, metronidazole, levofloxacin, and Fosfomycin which formed 23.1% (18 out of 78) out of the total (Table OutcomesEighteen out of 78 patients 23%) in the study expired within 14 days of initiation of CEF-AVI and 55 patients  improved clinically in seven days. Among the clinical indications treated with CEF-AVI, the highest proportion of death was observed among patients diagnosed with occult sepsis and secondary bacteremia (33%) then neutropenic sepsis (27.3%) -approved clinical syndromes, i.e., urinary tract infections, intra-abdominal infections, or nosocomial pneumonia as other studies have already been conducted among this group including one from our center . Sixty-snt study . Their snt study .et al. demonstrated good clinical efficacy even when the majority of the study patients had septic shock [Most of the study population had sepsis/septic shock forming 65.3% of the total and CCI scoring was distributed evenly between mild (26.9%), moderate (26.9%), and severe (39.8%). Septic shock is associated with high mortality and the pharmacokinetics of most drugs cannot be relied upon in this situation. Other studies depicted good efficacy with varying percentages of patients with sepsis or septic shock and a study by Vena ic shock ,21,22.2. The time to initiation for CEF-AVI was lower in patients with septic shock (less than a day) and 50% (10 out of 20 patients) of these patients died within 14 days. The initiation of CEF-AVI within 48 hours of infection onset was found to be protective in a US study which was not observed here [The average dose per day as well as DOT of CEF-AVI in sepsis and septic shock remained comparable, also among 19 patients who had a creatinine clearance of less than 50 ml/min/1.73 mved here . The medet al., lower clinical cure rates were observed in the participants of the CEF-AVI arm with moderate creatinine clearance, this led to modifications in the dosing recommendations for renally impaired cases [Following the phase 3 trial of CEF-AVI in complicated intraabdominal infection by Mazuski ed cases -26. The The study did not reveal any significant difference in mortality or clinical cure among patients with or without combination therapy with other antibiotics, which included minocycline, polymyxins, metronidazole, levofloxacin, and fosfomycin. This is in concordance with other studies where there was no reduction in mortality with combination therapy whereas, a study on critically ill patients with CRE infections showed a lower 30-day mortality rate ,27,28.Out of the 43 patients who were culture positive, 32 patients had CRE or CRPA infections. Thirty isolates from the study were CRE and five were CRPA. CEF-AVI testing was done in 27 isolates (25 patients), out of them nine showed resistance. Two of these nine isolates harbored both NDM and Oxa-48 genes, in whom synergy with aztreonam was present and all received a combination with aztreonam with no deaths. Synergy testing between CEF-AVI and aztreonam revealed six (24%) isolates having no synergy, with only the above two isolates resistant to CEF-AVI. Oxa-48 gene was the most commonly detected gene followed by NDM, this reflects the predominant resistance gene in the Indian population .Limitations and further researchClostridium difficile-associated diarrhea in the patients. Even though over 40% of isolates from these patients were CRE or CRPA, data on CEF-AVI sensitivity or synergy testing with aztreonam was limited. We also did not repeat cultures in culture-positive patients or assess for relapses/reinfections in them to watch for resistance development.Being a retrospective study there were a lot of limitations with various variables not analyzed. The study did not evaluate adverse drug events or Further research needs to be carried out in our country for various clinical and microbiological indications for CEF-AVI. Studies on the evaluation of drug resistance development in strains where Oxa-48 and NDM resistance genes are prevalent need to be conducted. Even in this study, molecular analysis was not done on the CEF-AVI-resistant strains.The study revealed that in the use of CEF-AVI among patients with clinical indications that have limited antimicrobial treatment options, CEF-AVI has a good clinical cure rate and survival rate. Hence, CEF-AVI can be a promising option in treating various clinical syndromes which are at present not indicated by the FDA, where there is high suspicion of or confirmed multidrug organism infection with LTOs.


SARS-CoV-2 is the causative agent of COVID-19 that has infected over 642 million and killed over 6.6 million people around the globe. Underlying a wide range of clinical manifestations of this disease, from moderate to extremely severe systemic conditions, could be genes or pathways differentially expressing in the hosts. It is therefore important to gain insights into pathways involved in COVID-19 pathogenesis and host defense and thus understand the host response to this pathogen at the physiological and molecular level. To uncover genes and pathways involved in the differential clinical manifestations of this disease, we developed a novel gene co-expression network based pipeline that uses gene expression obtained from different SARS-CoV-2 infected human tissues. We leveraged the network to identify novel genes or pathways that likely differentially express and could be physiologically significant in the COVID-19 pathogenesis and progression but were deemed statistically non-significant and therefore not further investigated in the original studies. Our network-based approach aided in the identification of co-expression modules enriched in differentially expressing genes (DEGs) during different stages of COVID-19 and enabled discovery of novel genes involved in the COVID-19 pathogenesis, by virtue of their transcript abundance and association with genes expressing differentially in modules enriched in DEGs. We further prioritized by considering only those enriched gene modules that have most of their genes differentially expressed, inferred by the original studies or this study, and document here 7 novel genes potentially involved in moderate, 2 in severe, 48 in extremely severe COVID-19, and 96 novel genes involved in the progression of COVID-19 from severe to extremely severe conditions. Our study shines a new light on genes and their networks (modules) that drive the progression of COVID-19 from moderate to extremely severe condition. These findings could aid development of new therapeutics to combat COVID-19. This viral pandemic has affected millions of people around the globe. Viruses being obligate parasites evade the host cell immunity by producing antigenic peptides as well as other proteins using the host cell’s machinery. To enable the functioning of these processes, an intricate series of interactions between the host and virus is required3. To gain insights into the genes and pathways involved in the COVID-19 pathogenesis and host defense, the host response against this pathogen is being studied by scientists worldwide. Although some of the genes and pathways identified in hosts during pathogenesis are common across pathogens, each viral infection generates a unique transcriptional profile4 with the expression of several genes or biological pathways during an infection phase.A novel coronavirus, SARS-CoV-2, the most recent of Sarbecoviruses, was first identified in Wuhan, China in December 201911, these analyses are based mainly on the criteria of expression fold-change and statistical significance for identifying. It is recognized that many of the lowly expressed genes may also be biologically or medically significant but may get filtered out due to their not meeting the preset criteria. Identifying false negatives is a non-trivial problem; it could be tempting to relax the criteria to identify even more true positives but there is an inherent risk of incurring false positives by this approach. In disease conditions such as COVID-19, it is important to reliably infer genes that are playing certain roles in the conditions. We posit that this is possible by invoking multiple complementary approaches, including the classical “gene-by-gene” approach and systems-level approach that could emphasize on gene interactions. Clearly, there is a pressing need of a systems-level approach to investigate networks of host genes regulated during different stages of COVID-19. To address this, a systems-level differential gene expression analysis was performed to decipher genetic factors implicated in various severity conditions of the SARS-CoV-2 infection. The overarching goal of this study is to augment our understanding of genes, as well as pathways, that play diverse roles during various clinical manifestations of COVID-19 pathogenesis, ranging from moderate to extremely severe infections including conditions that require patients to be admitted to intensive care units (ICUs) or put on life support.At present, the host response to the SARS-CoV-2 infection is not well-understood. Advances in high-throughput sequencing technologies have enabled gaining insights into the perturbations in the expression of different genes and biological pathways that result in a wide range of clinical manifestations in hosts. Although several studies have focused on differential gene expression during the SARS-CoV-2 infection16. Thus, the information on genetic factors involved during the progression of COVID-19 from moderate to severe and then to extremely severe infection is lacking. To gain new insights into the progression of COVID-19 severity at the mechanistic and regulatory levels, we developed a novel gene co-expression network based pipeline . The accession numbers of RNA-Seq datasets used for gene co-expression network analysis are GSE154613 and GSE152418. The transcripts from RNA-Seq data were derived using Salmon18 and transcript abundance estimates were obtained using the tximport19 package of R. Samples with reads covering greater than 65% of the human transcriptome in the alignment were used from further analysis  adjusted p-value ≤ 0.05.We obtained the expression profile datasets for SARS-CoV-2 infected tissues, including lung cell lines A549, bulk peripheral blood mononuclear cells (PBMC), and pancreatic organoids created from human embryonic stem cells (hESCs) from National Center for Biotechnology Information (NCBI) GEO database  and pathway enrichment analyses on DEG sets to understand their functional significance using DAVID (https://david.ncifcrf.gov/). GO terms providing information about cellular localization, molecular function, and biological process were obtained. GO terms and biological pathways that were significantly overrepresented  in a DEG set were identified as enriched.As functional annotation enrichment analysis was not performed in the original study21 package and a gene co-expression network was constructed using blockwiseModules function to generate a signed network under a soft-thresholding power of 10, minimum module size of 20, merge cut height of 0.15, and maximum block size of 40,000. Following network construction, gene modules were generated based on a hierarchical clustering approach using WGCNA. The gene modules are comprised of genes that are highly correlated in their expression under many different conditions, as represented in the different RNA-Seq datasets used. The co-expression network is comprised of 21,170 nodes and 7,574,232 edges. Each node represents a gene, and each edge represents a connection or association between the nodes (genes). The association between nodes is quantified based on the topological overlap value, ranging from 0 to 1, taking into consideration both the expression profile similarity, and the similarity of relationships each node has with all other nodes. A WGCNA network is fully interconnected, but each connection is weighted differently. Only genes that fall into module 0 are disconnected from other genes in the network. The large size of the network didn’t allow visualization using Cytoscape22 and therefore, we removed weak edges based on their weights. Edges with weights < 0.19 were removed to enable visualization using Cytoscape. The resultant network that could be visualized with Cytoscape consists of 15,696 nodes and 4,319,901 edges. Note that the aforementioned steps were performed to trim the network to enable visualization. All downstream analyses were performed with the complete (untrimmed) network.We constructed a matrix  of normalized transcript expression values, with rows representing transcripts and columns representing experiments. This expression matrix was then imported to the Weighted Gene Correlation Network Analysis (WGCNA)DEGs from an RNA-Seq dataset representing a SARS-CoV-2 infected tissue or organ were mapped onto the gene network. This provided information about DEG containment of each module in the network. The DEG containment of some modules range from none to a few DEGs, while other modules consist of a large number of DEGs.Modules enriched in differentially expressing genes were determined by assessing the statistical significance of fold enrichment of DEGs in modules with fold enrichment > 1. This was accomplished by performing the Fisher test and the modules with p-value ≤ 0.05 were deemed significantly enriched. We did not use higher threshold for fold enrichment to avoid losing any modules that could be potentially important under certain severity condition of COVID-19.We considered DEG enriched modules and because of the enrichment, we deemed these modules that could likely be representing pathways or subnetworks of pathways to be differentially expressing. However, these modules also harbor genes that were deemed non-DEG by the statistical test. Because of their residing within the DEG enriched modules, it is plausible that many of these genes may be participating in the same biological processes as the DEGs, and thus could be on same pathways as DEGs. Based on this premise, we posit that among these genes, those with fold-change of two or more are likely differentially expressing genes (LDEGs) but were missed as they lie below the detection threshold used by the standard statistical test. We further selected those DEG enriched modules that have a large majority (over 65%) of the genes DEGs or LDEGs. We investigated LDEGs in these modules for their potential roles in COVID-19 pathogenesis, specifically in the progression of COVID-19 severity.LDEGs were annotated for pathways they are part of using the KEGG pathway database as well as based on literature studies. This analysis sheds light on the functions of LDEGs and help us decipher if LDEGs are parts of some known pathways or play roles in yet unknown pathways.To summarize, we first performed differential gene expression analysis to catalog genes that express differentially during different severity conditions of COVID-19. Although, this provided a list of genes of interest, information regarding pathways or networks of pathways in which these genes function was elusive. Mapping differentially expressing genes onto known pathways will identify differentially expressing pathways but will miss yet unknown pathways that differentially express during different COVID-19 states or conditions. We therefore constructed a gene co-expression network using transcriptomic data from SARS-CoV-2 infected tissues, namely, lung, blood, and pancreatic organoids, and identified modules that contain genes that are densely linked to each other because of their co-expression. Genes within the modules that represent pathways or networks of pathways co-express under many different conditions or across different tissues in COVID-19. Mapping of differentially expressing genes from a COVID-19 expression dataset onto the modules allowed to decipher pathways, both known and unknown, that associate with a certain state of COVID-19 represented by the dataset. Specifically, following mapping of DEGs, modules enriched in DEGs were identified and further analyses were performed to identify LDEGs that are prone to be missed by standard protocols of expression analysis. This enabled identification of differentially expressing pathways or networks of pathways, represented by modules in the network, during various stages of COVID-19. Functional annotation (GO and KEGG pathway) enrichment analysis of modules enriched in DEGs was performed for identification of biological processes and pathways involved in various clinical manifestations of COVID-19.12 assessed the relative contributions of multiple factors  to the variance in gene expression between COVID-19 patients and healthy individuals, here we focused on assessing different biological pathways regulated by the DEGs in patients with moderate COVID-19 symptoms , severe COVID-19 symptoms , and extremely severe (ICU) symptoms . Using our approach for differential gene expression analysis (see Methods), we identified even more DEGs than reported by the previous study in all the three stages of COVID-1912. We found 1,540 DEGs in Moderate vs. Healthy, 1,496 DEGs in Severe vs. Healthy, and 3030 DEGs in ICU vs. Healthy  as shown in Fig. CSF1R, CR1, CSF3R, IL1B, IL3RA, CD1D in moderate, KIT, IL5RA, TNF, IL1A, IL1B, ITGA3 in severe and TNF, IL1A, IL1B, ITGA3, HLA-DOA, IL9R, CR2, CD8B, FCER2 in ICU patients. Unlike studies that found perturbation of hematopoietic system to be a marker of severe and fatal cases of COVID-1932, we found dysregulation of 6 genes involved in hematopoietic cell lineage in both moderate and severe infection, while 9 genes were perturbed in ICU indicating that hematopoietic system is dysregulated across different stages of COVID-19 with higher number of genes downregulated during the extremely severe infection.To understand the host response to the SARS-CoV-2 infection, we analyzed transcriptomic data from PBMCs of patients infected with SARS-CoV-2 and healthy controls retrieved from NCBI SRA (SRP267176) or NCBI GEO (GSE152418). The SARS-CoV-2 infected patients showed a wide range of clinical manifestations including moderate, severe and extremely severe (ICU) symptoms of COVID-19. As the authors in the original studyhy Table . To unde12. This analysis was performed to gain insights into genes or pathways differentially regulated during the progression of this disease from the moderate to extremely severe condition. We identified 685 DEGs in Severe vs. Moderate and 344 DEGs in ICU vs. Severe comparisons , and in extremely severe (ICU) versus severe patients (ICU vs. Severe), which were not investigated previously using RNA-Seq of PBMCs representing different COVID-19 stagesns Table . We idenns Table . Some ofIL-6, which encodes a cytokine, and was previously identified as an important marker for progression of COVID-19 by some of the studies36, was not found to be either differentially expressing or likely differentially expressing in our study, which could possibly indicate therapeutic interventions.41, we posit that module 15 is critical in enhancement of disease severity leading to extremely severe COVID-19. In module 53, we identified enrichment of amino acid  metabolism. A recent study has revealed deregulation of amino acids involved in one-carbon metabolism such as glycine, serine and threonine during COVID-1942. As module 53 is conserved across all stages of COVID-19 and is involved in a pathway associated with COVID-19, it is highly likely that this module is critically involved in COVID-19 and needs to be investigated further to understand its role in augmenting the severity of this disease.To uncover gene networks and pathways that play important roles during the SARS-CoV-2 infection, we took a systems approach, namely, performed weighted gene co-expression network analysis (WGCNA) of gene expression data obtained from the COVID-19 patients. First, a gene co-expression network for the human host was constructed Fig.  using RN37, and antigen processing and presentation43 during COVID-19, therefore, it is highly likely that module 70 in involved in moderate COVID-19. In modules 17 and 41, we identified enrichment of olfactory transduction. In module 20, we found enrichment of olfactory and taste transduction pathways. As impairment of olfactory and taste transduction pathways were previously reported in COVID-19 patients45, we posit that pathways represented by modules 17, 41 and 20 are dysregulated during extremely severe COVID-19.Interestingly, we found one module in moderate COVID-19  does not include information about any pre-existing conditions or comorbidities with COVID-19. Several studies have reported that COVID-19 patients with certain pre-existing diseases or conditions, such as cardiovascular conditions (including hypertension), cancer, diabetes, immunosuppression, lung disease, and neurological diseases, are at a greater risk of developing severe infection as well as at a heightened risk of dying due to the SARS-CoV-2 infection12 when the dominating strain was the initial SARS-CoV-2 identified in China, before the emergence of the alpha variant of SARS-CoV-2, which was first reported in September 2020 in Great Britain by the World Health Organization (WHO)80. Following the emergence of the alpha variant, various other variants of SARS-CoV-2, including beta, gamma, delta and omicron, have been reported by WHO80. With the emergence of these new variants and availability of relevant expression data, future studies could focus on identifying genes and pathways involved in pathogenesis and progression of COVID caused by these variants using network based methodologies, similar to the systems-level approach used in this study. Comparison of differentially regulated genes and pathways in humans afflicted by COVID caused by different variants will shed further light on molecular processes that are differentially impacted by SARS-CoV-2 variants.The transcriptomic data used for differential gene expression analysis was taken from a previous study which was published in August 2020Conventional protocols for differential gene expression analysis use expression fold-change and statistical significance test to identify genes that are expressing differentially. Our study highlights the importance of a novel gene co-expression network-based approach to decipher biologically significant entities such as differentially regulated genes or modules that could fall below the detection threshold established in the traditional protocols for analyzing differential gene expression. Our network-based pipeline could be used in tandem with a conventional differential gene expression analysis pipeline to comprehensively identify genes or pathways differentially expressing under certain conditions, e.g. COVID-19 pathogenesis and progression of the disease.Gene co-expression network analysis was performed to identify pathways and networks of pathways represented by modules, which play important roles in different stages of COVID-19, as well as in the progression of the disease from moderate to extremely severe infection. This systems-level analysis also helped to discover novel genes involved in the host defense or COVID-19 pathogenesis, which couldn’t be detected using standard protocols for differential gene expression analysis. Some of these genes encode hypothetical or yet uncharacterized proteins that have not yet been associated with known pathways but could be involved in COVID-19 pathogenesis and progression or potentially aiding the host defense. Our pipeline to identify modules enriched in genes differentially expressing during different stages of COVID-19 provides insights into key genes and pathways that might be playing important roles in COVID-19 pathogenesis and the host defense. Future studies could prioritize genes or pathways deemed significant in COVID-19, which may lead to new interventions to mitigate the symptoms or towards much needed novel therapeutics.Supplementary Tables.


Contrast-associated acute kidney injury (CA-AKI) is a fairly frequent complication of cardiovascular angiography and percutaneous coronary intervention (PCI). The risk is significantly higher in patients with advanced chronic kidney disease (CKD). Prevention is the only option for avoiding the significant morbidity and mortality associated with CA-AKI. This review provides a concise and clinically directed appraisal of the latest pre-procedural and peri-procedural strategies to minimize the risk of CA-AKI in all patients undergoing PCI. By broadly implementing these evidence-based care bundles, we can dramatically improve outcomes in this vulnerable patient population. As the glomerular filtration rate (GFR) declines to < 60 mL/min/1.73m2, the probability of developing CAD increases linearly.4 In fact, patients with stage 3 and 4 CKD have a mortality risk from cardiovascular disease that is approximately two to three times higher, respectively, than patients without CKD.5The prevalence of chronic kidney disease (CKD) in patients with coronary artery disease (CAD) is high, with estimates of its prevalence ranging from 23% to 46%.As significant advances in percutaneous coronary intervention (PCI) have occurred, we now can perform increasingly complex cardiac interventional procedures that previously were not possible. Unfortunately, by the very nature of their complexity, such interventions frequently require large volumes of contrast and often occur in patients who already have established CKD.In this manuscript, we aim to provide a concise and clinically useful appraisal of the latest pre-procedural and peri-procedural strategies to minimize the risk of CA-AKI in all patients undergoing PCI, with the hope that wide and more consistent implementation of these strategies can dramatically improve outcomes in this vulnerable patient population.6 In recent decades, the term “contrast-induced nephropathy” has been used to denote the development of acute kidney injury (AKI) following contrast administration in the absence of a plausible alternative etiology.7 Several recent revelations have indicated that this association is less robust than was previously accepted. For example, a study published in 2014 of 12,508 patients undergoing contrast-enhanced versus non–contrast-enhanced computed tomographic scans observed that the rate of AKI was no different between patients who did or did not receive contrast.8 Additionally, the investigators of AKI-MATRIX, a sub-study of the MATRIX  trial, which compared radial versus femoral access for angiography and PCI in patients with acute coronary syndromes, reported a lower frequency of AKI among patients randomized to undergo radial catheterization despite similar contrast exposure to both groups.9 More recently, the term “contrast-associated acute kidney injury” (CA-AKI) has been adopted by some radiology and nephrology communities to refer to AKI that occurs shortly after administration of contrast.The association between contrast media and kidney dysfunction has been recognized for more than 80 years.10 These criteria also have been adopted by the National Cardiovascular Data Registry (NCDR).There are several definitions for CA-AKI . The mos14 A study of 985,737 patients enrolled in the NCDR reported that CA-AKI occurred in 7.1% of patients undergoing PCI.15 Often, estimates have ranged from 2% in patients with normal baseline kidney function to as high as 20% to 30% in patients with underlying advanced CKD.14 It should also be noted that as same-day discharge after PCI becomes more common, the frequency with which acute kidney injury is reported is likely to decrease since the rise in serum creatinine often occurs several days after contrast exposure. Fortunately, the incidence of CA-AKI requiring dialysis following PCI is rare.16 Importantly, both transient and persistent post-procedural renal dysfunction are independent predictors of mortality.17 Multiple reports have indicated that 1-year and 5-year estimated mortality rates are significantly higher in patients with CA-AKI14 and even more if dialysis was required.13CA-AKI is an underdiagnosed complication of coronary angiography and PCI that has been associated with increased in-hospital morbidity and mortality, increased length of stay, and health care expenditure.2) who went on to develop CA-AKI.18 Importantly, even in patients with normal renal function, development of CA-AKI increased the risk of net adverse clinical event (NACE) and mortality.Recently, Mohebi et al. sought to investigate the determinants and outcomes of patients with CA-AKI from the ADAPT-DES  registry. They included 7,412 patients with creatinine measured before and within 3 days after PCI. CA-AKI was defined as an absolute increase in creatinine > 0.5 mg/dL or 25% increase over baseline. As in other studies, multivariate analysis confirmed that patients developing CA-AKI were older, more acutely ill, more likely to be female, and to have diabetes and underlying CKD. Procedural variables associated with CA-AKI included hypotension during PCI, PCI for ST elevation myocardial infarction (STEMI), radial access, greater number of stents (a surrogate for contrast volume), and use of an intra-aortic balloon pump. At 2 years, CA-AKI was associated with a 2-fold increase in cardiac death and an increased risk of myocardial infarction, stent thrombosis, and bleeding, with the highest risk seen in the subgroup of patients with underlying CKD  and the tubular epithelium.22 Medullary hypoxia can also trigger the release of reactive oxygen species, which cause additional tissue damage through oxidative stress.22In addition, iodinated contrast also has direct toxic effects on the tubular epithelium. The increases in osmotic load and viscosity associated with high osmolarity contrast impairs blood oxygen delivery in the tubular epithelium, increasing hypoxia in the renal medulla.Finally, there are several peri-procedural factors, including hypotension, bleeding, and the inadvertent embolization of atheromatous debris, that amplify the deleterious effects of contrast media and compound the development of AKI.15 These risk factors are best classified into modifiable and nonmodifiable risk factors. Modifiable risk factors may be patient or procedure related.There are multiple independent predictors of CA-AKI and dialysis, with each risk factor having varying contributions.24 The cause is likely to be multifactorial and includes age-related reduction in tubular function and GFR, renal atherosclerosis, and reduced regenerative capacity of the renal parenchyma following acute injury.Advanced age is a risk factor for CA-AKI. Some studies have found age ≥ 75 years to be an independent risk predictor of CA-AKI.15 Similarly, patients with advanced CKD (estimated GFR [eGFR] < 30 mL/min/1.73m2) have more than a 3-fold increase in the adjusted risk of CA-AKI when compared to patients with normal renal function.15Preexisting CKD is the most important risk factor for the development of CA-AKI following PCI. Elevated serum creatinine at baseline is an independent predictor of CA-AKI.25 Diabetes and CKD also appear to act synergistically. In patients who have both diabetes and CKD, the incidence of CA-AKI increases an additional 2-fold (compared to those with diabetes but without CKD) and can be as high as 33%.27Diabetes is another independent risk factor for the development of CA-AKI and the subsequent need for dialysis, independent of preexisting renal dysfunction.24 The overlying etiology here is related to renal hypoperfusion.Acute decompensated heart failure , acute coronary syndrome, and cardiogenic shock have all been associated with an increased risk of CA-AKI.15The risk of CA-AKI increases dramatically when the patient has reduced effective circulatory volume. This holds true regardless of the cause of hypovolemia, such as over diuresis, sepsis, or liver failure. Hypotension from cardiogenic shock and acute congestive heart failure also predispose patients to increased risk of CA-AKI.27 Li et al. demonstrated that in patients undergoing PCI, the incidence of CA-AKI in those who are anemic is higher than that of non-anemic patents . As GFR decreases < 60 mL/min, the incidence of CA-AKI in anemic patients may increase to 2-fold higher compared to that of non-anemic patients.28 The pathophysiology is likely related to exacerbation of local renal hypoxia following contrast media exposure in anemic patients.Patients with CKD are more likely to have anemia (defined as hemoglobin < 12.0 g/dL in women and < 13.0 g/dL in men), which also worsens with declining renal function. Anemia itself has been shown to be an independent predictor of CA-AKI.29 The latest data from the CAPTAIN (Angiotensin-Converting Enzyme Inhibitors and Contrast-Induced Nephropathy in Patients Receiving a Cardiac Catheterization) trial demonstrated that withholding ACEI/ARB ≥ 24 hours pre-procedure resulted in a nonsignificant reduction in CA-AKI and a significant reduction in post-procedure creatinine increase in patients who had moderate renal insufficiency (creatinine ≥ 1.7 mg/dL within 3 months) undergoing cardiac catheterization.30 Given the above, the most prudent approach would be to withhold ACEI/ARB preoperatively in patients with moderate CKD and/or deemed to be at high risk of CA-AKI, and to reinstitute therapy when appropriate. This is a low-cost intervention with significant potential benefits.Concomitant use of nephrotoxic agents such as nonsteroidal anti-inflammatory drugs, cyclosporine-A, tacrolimus, and diuretics increase the risk of CA-AKI by interfering with the autoregulatory responses of the renal circulation. Of note, the data regarding the need to discontinue angiotensin-converting enzyme inhibitors (ACEIs) prior to a procedure are conflicting, and data concerning angiotensin receptor blockers (ARBs) are even more limited.31The timing of cumulative renal injury related to contrast media exposure plays an important role in the development of CA-AKI, even if PCI is not performed. In fact, patients undergoing coronary artery bypass graft surgery within 1 day of coronary angiography have been found to have an approximate two-fold increase in the risk of major adverse renal and cardiac events compared to those who waited ≥ 5 days.33 This is likely due to a combination of impaired systemic perfusion from left ventricular dysfunction, possible use of larger volumes of contrast, and the lack of opportunity for CA-AKI prophylaxis given the emergent nature of the procedure.The risk of CA-AKI is also significantly higher in patients undergoing emergency PCI, for example primary PCI in STEMI, than among patients with chronic coronary syndrome undergoing elective PCI.2. In these patients, staged PCI was associated with a 2.6-fold greater decline in renal function 4 to 12 weeks after the procedure. This adverse effect of staging the procedures might occur because staged PCI exposes patients to greater cumulative contrast loads or because patients at higher risk for kidney injury may be more likely to have the procedures staged. Additionally, the temporal separation between procedures (within 30 days) may not be adequate, leading to recurrent hits to the nephrons and hence added superimposed acute kidney injury.34However, when it comes to staged versus ad-hoc PCI, the benefit of staging is not as clear. Interestingly, Shah et al. showed that across all patients along the entire spectrum of GFR, CA-AKI was not less frequent when PCI was staged compared to when PCI was performed ad-hoc. While the decline in renal function observed in both groups did not differ significantly, worse renal outcomes at 30 days were observed in the staged PCI group with a baseline GFR < 60 mL/min/1.73m35 A contrast volume to creatinine clearance (CV/CrCl) ratio > 2 is an independent predictor of CA-AKI in patients with an eGFR < 30 mL/min/1.73m2.36 The risk of CA-AKI is minimal in patients receiving less than 100 mL of contrast during procedures or if the volume of contrast used is less than 5 mL/kg/serum Cr.25Volume of contrast used is the primary modifiable risk factor. Ample evidence from multiple studies indicates that higher volume contrast use significantly increases the risk of CA-AKI and that its detrimental effects are more pronounced with more progressive CKD.37 As a result, we have now gravitated to using iso-osmolal or low-osmolal contrast media as a standard because iso-osmolal contrast is less arrhythmogenic and is associated with a lower risk of CA-AKI, particularly in patients with existing CKD.Although common parlance categorizes iodinated contrast agents as being high, iso, or low osmolar, they are technically categorized according to their osmolality rather than osmolarity. The risk of CA-AKI is increased with high-osmolal contrast use, particularly in patients with CKD.There continues to be uncertainty about the value of mechanical circulatory support (MCS) in reducing the risk of CA-AKI in patents who are hemodynamically unstable. However, encouraging data indicate that the use of left ventricular assist devices, mainly Impella (Abiomed), to maintain hemodynamic stability and end-organ perfusion in high-risk PCI patients has a role in reducing the incidence of CA-AKI.32 the Blue Cross Blue Shield of Michigan Cardiovascular Collaborative model,38 and the National Cardiovascular Data Registry Cath-PCI registry AKI prediction model.15In patients with CKD, every effort should be made to perform an appropriate risk assessment prior to PCI so both the physician and patient understand the benefits and risks of the procedure. Multiple validated scores can be used to facilitate this risk assessment. These scores all work on the same principle using a combination of the risk factors that predispose patients to CA-AKI after PCI. The three major scoring systems include that proposed by Mehran et al.,16 An increasing risk score is strongly associated with CA-AKI, ranging from 8.4% to 55.9% for a low- to high-risk score, respectively. This score has been validated in groups undergoing both elective and emergent procedures for acute coronary syndrome .39The Mehran scoring system, originally published in 2000, is the most commonly used system for CA-AKI risk stratification and has been validated in a cohort of 8,357 patients undergoing PCI. Eight variables—including hypotension, intra-aortic balloon pump, congestive heart failure NYHA Class III/IV, age > 75 years, anemia, diabetes, contrast media volume, and eGFR—are weighted individually and then combined to create a score that stratifies patients into low (≤ 5) and high risk (≥ 16) for CA-AKI.P = .002).40Mehran et al. recently demonstrated a more contemporary scoring system (the Mehran-2 score) that includes two models. Model 1 consists of the following clinical variables: clinical presentation, eGFR, left ventricular ejection fraction, diabetes, hemoglobin, basal glucose, congestive heart failure, and age. Model 2 goes a step further to include procedural variables: contrast volume, peri-procedural bleeding, no flow or slow flow post procedure, and complex PCI anatomy. The Mehran-2 score was able to discriminate the risk of CA-AKI accurately, although model 2 only slightly improved the discrimination of the risk score . More importantly, this study also demonstrated that patients with CA-AKI had significantly increased risk of death at 1 year . CA-AKI was seen in 6.7% of the LVEDP-based hydration group compared with 16.3% of the standard hydration group .43 The 6-month rate of MACE  was also lower in the LVEDP-guided group . Similar effectiveness of pressure-guided hydration also can be accomplished using right atrial pressures.44In patients without significant comorbidities, intravascular volume expansion with intravenous normal saline is safe and effective. This issue becomes more complex in patients with congestive heart failure. Fortunately, the Prevention of Contrast Renal Injury with Different Hydration Strategies (POSEIDON) trial demonstrated the feasibility of tailoring hydration to reduce the risk of CA-AKI without compromising a patient’s euvolemic status. This study included 396 patients with CKD randomized to either hydration based on left ventricular end-diastolic pressure (LVEDP)  or standard hydration (fluid at 1.5 mL/kg/h). Intravenous saline was administered before contrast exposure and continued for 4 hours after the procedure. The total volume of normal saline administered was higher in the LVEDP-guided hydration arm  was thought to be protective by decreasing endothelial dysfunction and scavenging oxygen free radicals, hence reducing direct oxidative damage to the renal parenchyma. Initial trials, including APART  and RAPPID , showed promising results with a reduction in CA-AKI in patients treated with NAC.48 The incidence of CA-AKI was 9.5% in the sodium bicarbonate group and 8.3% in the sodium chloride group . Similarly, the incidence of CA-AKI was 9.1% in the N-acetylcysteine group and 8.7% in the placebo group .48 Accordingly, the recommended hydration protocol is the intravenous administration of normal saline at a rate of 1 to 1.5 mL/kg/hr for 3 to 12 hours pre-procedure, and 12 to 24 hours post-procedure—as per the Society for Cardiovascular Angiography and Interventions (SCAI) and European Society of Cardiology (ESC) guidelines50—with the option to tailor the aggressiveness of hydration to LVEDP or right atrial pressure where appropriate (This has led to the Prevention of Serious Adverse Events Following Angiography (RESERVE) randomized trial, which provided conclusive evidence that there was no benefit of sodium bicarbonate and/or NAC over placebo among patients undergoing angiography who are at risk for CA-AKI.ropriate .More recently, devices like the RenalGuard system  show promise in further reducing the risk of CA-AKI after PCI. This device makes use of urine flow rate-guided hydration infusion to prevent CA-AKI. One hour before the procedure, an intravenous bolus of normal saline and a dose of furosemide are administered. PCI is started when a urine output of 300 mL/h is achieved. Similarly, matched hydration is continued for another 4 hours following PCI. The idea is that the high urine output dilutes the contrast media  and allows its rapid elimination of contrast from the body, reducing its contact time within the nephrons and its deleterious effects on the kidney.51 and REMEDIAL III (compared to LVEDP-guided hydration)52 trials have shown the system to be superior in preventing CA-AKI compared to current therapies. In REMEDIAL III, CA-AKI for urine flow rate-guided versus LVEDP-guided angiography/PCI was 5.7% versus 10.3% (P = .036). Further clinical trial data are necessary to provide firm recommendations on this approach, but its use should be considered in patients deemed to be at high risk for CA-AKI if it is available.The REMEDIAL II (compared to standard hydration with sodium bicarbonate solution and NAC)47 compared to hydration with half normal saline alone. However, significant weight loss was observed in the patients treated with furosemide, suggesting that the potentially deleterious effect of furosemide was the result of a negative fluid balance. The algorithm used with the RenalGuard system changes this paradigm by maintaining a constant intravascular volume to prevent hypovolemia while allowing a much higher total hydration volume when it is used . This would account for the drastically different results demonstrated by Solomon et al.Of note, Solomon et al. had demonstrated in 1994 that a forced diuresis regimen achieved by high-dose furosemide may be deleterious53 Patti et al. also demonstrated similar benefits in the Atorvastatin for Reduction of MYocardial Damage during Angioplasty–Contrast-Induced Nephropathy (ARMYDA-CA-AKI) trial with the use of short-term, high-dose atorvastatin pretreatment.54 While there remains a need for further evidence in support of the prophylactic use of statins to prevent CA-AKI, the majority of the patients undergoing PCI already have an indication for a high-intensity statin. Therefore, the recommendation is to start a high-intensity statin in statin-naïve patients, and in those who are already taking a statin, continue (or switch to) high-intensity statin therapy.HMG-Co A reductase inhibitors (statins) are believed to have beneficial pleiotropic effects that act to stabilize the renal vascular endothelium through their antioxidant and anti-inflammatory properties. The Patients with Renal Insufficiency Undergoing Coronary Angiography (PROMISS) trial showed no protective effect of simvastatin versus placebo in preventing CA-AKI. In contrast, the Rosuvastatin and Antiplatelet Therapy on Contrast-Induced Acute Kidney Injury and Myocardial Damage in Patients with Acute Coronary Syndrome (PRATO-ACS) trial demonstrated that high-dose rosuvastatin (40 mg loading dose followed by a 20 mg maintenance dose) when compared with placebo significantly reduced the risk of CA-AKI in statin-naive patients presenting with NSTEMI (6.7% versus 15.1%).55 There is no role for dopamine, calcium channel blockers, fenoldopam, or L-arginine in the prevention of CA-AKI, while diuretics and mannitol could further worsen renal function. When in doubt, or in patients with severe CKD (≥ Stage 4), consultation with a nephrologist is recommended to assist with optimization of renal function prior to PCI.Finally, any nephrotoxic medications should be discontinued at least 24 to 48 hours prior to PCI. It is acceptable to continue ACEIs in patients with mild-to-moderate CKD.56Minimizing contrast volume is the single most important procedural strategy to reduce CA-AKI after PCI. This strategy starts at procedure “time-out” with the cath lab team involved in the case recognizing the patients’ baseline creatinine, eGFR, and maximum allowable contrast dose (MACD). MACD can be easily calculated using the formula 5 mL x body weight (kg)/serum creatinine (mg/dL), with a maximum dose of 300 mL. Incremental use of contrast beyond the MACD is associated with a significantly increased risk  of CA-AKI.Increasing complexity of PCI has translated to higher contrast volumes used per procedure. At the same time, it is often not feasible to stop in the middle of a procedure because the MACD has been exceeded. Fortunately, more advanced techniques and technologies are available that can be leveraged to help offset this. See 57 “Zero contrast” PCI was performed 1 week after diagnostic angiography, and this approach resulted in successful PCI with no MACE and preservation of renal function within a median follow-up time of 79 days. The average creatinine was 4.2 g/dL with an eGFR of 16 ± 8 mL/min/1.73 m2. Since then, larger studies have been conducted by Rahim et al. and Shibata et al., which both consistently showed that “zero contrast” PCI may be safely performed even in complex lesions with high procedural success and minimal complications, with satisfactory acute and long-term renal and cardiovascular outcomes.59For patients with advanced CKD who require revascularization, Ali et al. have taken a further step to demonstrate the feasibility and safety of “zero contrast” PCI. This is achieved using a combination of the strategies above but with an extra emphasis on intravascular ultrasound (IVUS) guidance and pre- and post-PCI measurements of fractional flow reserve or iFR to confirm physiological improvement and success of the procedure.60 Similar lack of benefit was seen in a large observational study by Azzalini et al.61With regard to contrast media type, high-osmolar contrast media should always be avoided. However, at present, there is no evidence to recommend iso-osmolar over low-osmolar contrast media for the prevention of AKI. A recent meta-analysis by Pandya et al. from 10 randomized trials that included patients with CKD stage 3 undergoing coronary angiography demonstrated no significant difference in the incidence of CA-AKI between the use of iso-osmolar or low-osmolar contrast media.P < .001) compared with manual injection while also significantly reducing the incidence of CA-AKI by up to 15% .62Dedicated devices to reduce contrast volume administration are slowly becoming standard of care. The most prevalent are automated contrast injectors (ACIs), which can deliver less contrast volume while still maintaining optimal image quality. A recent meta-analysis by Minisinger et al. that included 79,694 patients from 10 studies showed that, on average, ACIs significantly reduced the volume of contrast delivered by up to 45 mL/case  by diverting excess contrast in the aortic root that would not contribute to coronary opacification. However, the trial was unable to show a meaningful difference in the incidence of CA-AKI.63 More research is needed to determine if a newer generation of this device would translate into a clinically meaningful benefit.The DyeVert PLUS system  also is able to reduce the volume of contrast delivered by up to 15.5%  compared with angiography guidance alone.64 While there was no appreciable difference in the incidence of CA-AKI between both groups, this was a pilot trial with a small sample size. The ongoing MOZART II trial (NCT02743156) has been designed to study this further and will likely cement the use of IVUS during PCI to further reduce contrast volume in patients at high risk for CA-AKI.Intravascular imaging is the stalwart of contrast-sparing PCI. This goal is best achieved with IVUS, which can help localize coronary lesions, identify stent landing zones, and confirm adequate stent expansion and apposition with little to no contrast at all. In the Minimizing Contrast Utilization with IVUS Guidance in Coronary Angioplasty (MOZART) trial, Mariani et al. demonstrated that IVUS-guided PCI is safe and markedly reduced contrast utilization . Similar findings also have been demonstrated in the MATRIX-Access trial, which randomized 8,210 patients with ACS undergoing PCI to radial versus femoral access. Radial access was superior to femoral access in decreasing the risk of CA-AKI .The majority of available evidence supports the use of radial access over femoral access in patients undergoing PCI to reduce the risk of CA-AKI. It is postulated that the lower major bleeding rates seen with radial access reduce hemodynamic disturbances that would further compromise renal perfusion and lead to subsequent kidney injury. The radial approach also may reduce atheromatous embolization to the renal arteries by completely avoiding manipulation of the abdominal aorta when advancing catheters. The benefit of radial access in decreasing the risk of CA-AKI has been demonstrated in a meta-analysis of six observational studies , and this is especially true in those patients who had existing CKD.66 Additionally, in Impella-supported patients, there was no association between the severity of CKD and the incidence of CA-AKI. By contrast, the incidence of CA-AKI was greater in unsupported patients with CKD, which was also correlated with the severity of CKD. While further exploration of these findings is warranted to validate this renal protection strategy, these data support a new renal protective strategy to reduce CA-AKI during high-risk PCI.Hemodynamic support with an Impella, especially in patients with hemodynamic instability, cardiogenic shock, acute heart failure, and/or CKD, is a promising strategy to reduce CA-AKI in patients undergoing high-risk PCI. The Impella can reduce the episodes of transient hypotension associated with prolonged balloon inflation times  or no reflow following atherectomy that decrease renal perfusion and cause repetitive insults to the kidneys, which can trigger AKI. In a retrospective study, Flaherty et al. demonstrated that Impella support is protective against AKI during high-risk PCI. The incidence of AKI following high-risk PCI in patients with an ejection fraction < 35% regardless of baseline kidney function is lower with an Impella device than without  and procedural strategies (namely contrast-sparing strategies) to minimize the risk of CA-AKI in all patients undergoing PCI. There is evidence to promote the use of automated contrast injectors, intravascular imaging, radial access, and—in the highest-risk patients—hemodynamic support (with an Impella). While future ongoing studies will validate these strategies, we should continue to implement best practices with what is known today, including the implementation of systemwide evidence-based case bundles to reduce CA-AKI and improve outcomes in this vulnerable patient population.Contrast-associated kidney injury is associated with a high rate of death.Scoring systems exist to predict an individual’s risk of developing contrast-associated kidney injury.Published protocols guide hydration to prevent kidney injury in patients at risk for contrast-associated kidney injury.The use of automated injection devices should be considered to limit the amount of contrast used on coronary angiography.Houston Methodist is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians.1 AMA PRA Category 1 Credit™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.Houston Methodist designates this Journal-based CME activity for a maximum of learn.houstonmethodist.org/MDCVJ-18.4.Click to earn CME credit:


COVID-19 manifests a spectrum of respiratory symptoms, with the more severe often requiring hospitalization. To identify markers for disease progression, we analyzed longitudinal gene expression data from patients with confirmed SARS-CoV-2 infection admitted to the intensive care unit (ICU) for acute hypoxic respiratory failure (AHRF) as well as other ICU patients with or without AHRF and correlated results of gene set enrichment analysis with clinical features. The results were then compared with a second dataset of COVID-19 patients separated by disease stage and severity. Transcriptomic analysis revealed that enrichment of plasma cells (PCs) was characteristic of all COVID-19 patients whereas enrichment of interferon (IFN) and neutrophil gene signatures was specific to patients requiring hospitalization. Furthermore, gene expression results were used to divide AHRF COVID-19 patients into 2 groups with differences in immune profiles and clinical features indicative of severe disease. Thus, transcriptomic analysis reveals gene signatures unique to COVID-19 patients and provides opportunities for identification of the most at-risk individuals. Coronavirus Disease 2019 (COVID-19) is caused by the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which mediates respiratory infections and lung pathology of varying severity . InfecteIn addition to demographic statistics, the immune response of COVID-19 patients has been linked to disease severity and presents an opportunity for utilizing immune profiles to predict patient outcomes. To date, several groups have used a combination of flow cytometry, transcriptome data, cytokine levels, and clinical data to categorize COVID-19 patients and to associate particular immune profiles with disease severity –30. ImmuWe previously utilized publicly available gene expression data to characterize the trajectory of the host immune response to SARS-CoV-2 in the blood, postmortem lung tissue, and bronchoalveolar lavage fluid (BALF) of COVID-19 patients . We haveWe included patients who consented to donate blood to the University of Virginia (UVA) Intensive Care Unit (ICU) Biorepository. We then selected patients with confirmed COVID-19 respiratory failure, viral, non-COVID-19 respiratory failure, and patients with non-viral causes of respiratory failure  in the UVA ICU Biorepository to serve as a comparison cohort. Control patients were patients admitted on mechanical ventilation without respiratory failure . We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines  and our Serum cytokine and chemokine levels were measured by Merck Millipore MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A (HCYTA-60K) and Panel II (HCYP2MAG) assays.Blood was collected into PAXgene® Blood RNA tubes upon admission to the ICU, and after 24 and 72 hours. RNA was prepared and sequenced by Genewiz (Azenta Life Sciences). RNA was isolated using a Qiagen total RNA isolation kit, followed by RNA-seq library preparation using standard Illumina protocols with rRNA and globin depletion. RNA was sequenced on an Illumina HiSeq 4000. Three independent whole blood datasets were analyzed. In the first dataset of patients from the UVA ICU Biorepository, RNA-seq data was obtained from 13 COVID-19 AHRF patients, 8 viral AHRF, 5 non-viral AHRF, and 5 control ICU patients. COVID-19 patients were subdivided into 2 groups (COVID Group 1 and COVID Group 2) based on separation by PCA of the top 500 variable genes. Additional publicly available RNA-seq datasets , were obtained and analyzed as confirmation studies. Additional dataset details can be found in 2 transformed gene expression values were generated using the plotPCA function from the DESeq2 package.For RNA-seq analysis, the quality of raw FASTQ reads was analyzed using FASTQC  to identwww.bioconductor.org/packages/release/bioc/html/GSVA.html). In brief, a matrix of log2 transformed gene expression values for each sample and pre-defined gene sets were used as inputs for the GSVA algorithm. Then enrichment scores (GSVA scores) for each gene set were calculated using a Kolmogorov Smirnoff (KS)-like random walk statistic. GSVA scores for each patient and control were calculated and normalized to scores between -1 (no enrichment) and +1 (enriched). Significance of gene set enrichment between cohorts was calculated using a Welch’s t-test and p-value < 0.05 was considered significant.The R/Bioconductor package GSVA  v1.25.0 . For each analysis, the r2 value indicating the Goodness of Fit and the p-value testing the significance of the slope are displayed.Additional linear regression analyses for individual patient cohorts and between PC GSVA scores and logPatient demographic data from COVID Group 1 and Group 2 were compared using an unpaired t-test with Welch’s correction for continuous variables. Pearson’s chi-squared tests were used to analyze count data between groups. Cytokine data for COVID Group 1, COVID Group 2, and control ICU patients was compared using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 test for multiple comparisons. Significant differences in gene set enrichment between COVID-19 patients and non-COVID-19 patient cohorts were calculated using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 test for multiple comparisons.www.graphpad.com) and a two-tailed p-value of 0.05 was used for statistical significance.All statistical analyses were performed in GraphPad Prism  under SRA project number PRJNA777938. The confirmation study datasets are publicly available under accessions GSE161731 and GSE172114.To identify differences in immune pathology among severe COVID-19 patients, we analyzed whole blood transcriptomes from 13 individuals with COVID-19-induced AHRF, 6 with other viral and 7 with non-viral-induced AHRF, and 5 controls, who were patients admitted to the ICU on mechanical ventilation but without evidence of AHRF –49.To examine differences in inflammatory pathways between COVID-19 patients and control ICU patients, we carried out Gene Set Variation Analysis (GSVA)  using a 2 transformed gene expression values differentiating COVID-19 and control ICU patients indicated a division among COVID-19 patients into two groups  using the logWe then utilized GSVA to examine inflammatory pathways in the two gene expression-derived COVID-19 patient groups in greater detail . This was not found with COVID group 2  as compared to healthy controls. To do this, we analyzed a second publicly available COVID-19 transcriptomic dataset , with conserved and differential enrichment of immune cell and pathway gene signatures. COVID Group 1 was characterized by a lack of activated T cells, increased LDGs, increased CD40-activated B cells, and a general increase in cell proliferation and metabolism pathways. COVID Group 2 was characterized by increased expression of neutrophil subsets, markedly increased IFN gene signatures, and the absence of IgA1 expressing PCs. Aggregated clinical feature data and cytokine profiles for each COVID-19 patient cohort revealed that COVID Group 2 appeared to have more severe disease outcomes and indicated that patients with a similar immune profile would warrant a more targeted and aggressive therapeutic approach to mitigate risk of mortality.Both COVID Group 1 and 2 shared enrichment of the PC signature, which consistently differentiated COVID-19 patients from control ICU patients as well as patients exhibiting respiratory failure from other viral or non-viral sources. However, it is possible that the IgH heavy chain isotype of PCs is indicative of whether a robust and early PC expansion is beneficial to COVID-19 patients and certain PC isotypes have been linked with severe disease . FurtherCOVID Group1 patients appeared to have less severe disease as compared to COVID Group 2. Whereas all presented with AHRF, all Group 1 patients recovered, whereas 2 of Group 2 patients died during their hospitalization. Although our data set is limited by the number of patients analyzed, it suggests that the Group 2 gene signature could serve as prognostic marker and warrant individualized intervention. Lymphopenia is an established feature of COVID-19 and, in particular, a lack of T cell responses has been associated with worse clinical outcome , 59, 60.COVID Group 1 patients also exhibited an increase in genes associated with LDGs, neutrophil-like granulocytes with enhanced capacity for production of Type I IFNs and formation of neutrophil extracellular traps (NETs) that have been identified in severe COVID-19 patients , 62. In In contrast to COVID Group 1, the immune response of COVID Group 2 patients appeared to be associated with increased risk of mortality. The primary immune signatures enriched in COVID Group 2 resembled a dysregulated antiviral innate immune response. In particular, Group 2 exhibited enrichment of neutrophil populations expressing pro-inflammatory and suppressive genes that were previously identified in blood from severe COVID-19 patients , 18. FurIn addition to IFNs, a number of pro-inflammatory cytokines, including members of the IL-1 family, IL-6, IL-8, and TNF have been implicated in COVID-19 pathogenesis and linked to severe disease , 24, 35.Longitudinal gene expression analysis over 72 hours after admission to the ICU revealed that the immune profiles of COVID and non-COVID AHRF patients remained largely unchanged over time. However, among the COVID-19-induced AHRF patients, profiles of COVID Group 1 patients appeared to exhibit greater changes than COVID Group 2. Strikingly, gene signatures of COVID-associated neutrophil subsets and IFN were decreased, whereas the LDG gene signature was increased in COVID Group 1 patients, further supporting the conclusion that the innate immune response in Group 1 patients contributes to viral clearance whereas the response in Group 2 patients contributes to enhanced inflammation and fatal disease.We have applied a combination of bioinformatics approaches to characterize COVID-19 patients based on disease stage and severity using gene expression data, but must acknowledge the limitations of the data. Our initial dataset of ICU patients contained up to 13 patients per cohort and COVID-19 patients were also sub-divided into groups, which reduced the statistical power of our analyses. To mitigate this, we designed the study to solely include patients in the ICU, including non-AHRF controls, and thus reduce patient heterogeneity. In addition, a larger publicly available dataset including both hospitalized and non-hospitalized COVID-19 patients with healthy controls was utilized for validation. However, additional studies comparing ICU patients with more patients per cohort and more uniform inclusion criteria are warranted.Overall, we have identified immune profiles of severe COVID-19 patients associated with full recovery from disease or increased risk of mortality. We propose that these differences in COVID Group 1 and Group 2 patients could be employed to better allocate healthcare resources and design targeted treatment plans to better care for individuals who are at the greatest risk of worse outcomes. Whereas optimal medical care and appropriate ventilator management may be sufficient in patients with immune profiles similar to COVID Group 1, patients with immune profiles similar to COVID Group 2 could benefit from more aggressive therapeutic intervention targeting the dysregulated innate immune response. In particular, drugs targeting Type I IFNs, cytokines, such as IL6 or TNF, or myeloid chemokines such as IP-10 or MCP1 could be effective treatments for these individuals. Our work highlights the heterogeneity among severe cases of COVID-19 and the need for better characterization of hospitalized individuals to determine effective strategies to mitigate pathogenic immune processes that are dysregulated in the most at-risk patients. Furthermore, infected individuals with the potential to progress to severe disease should be identified as early as possible to allow for better resource allocation and early individualized therapies.Data deposited in NCBI under BioProject accession PRJNA777938.The studies involving human participants were reviewed and approved by UVA Institutional Review Board for Health Sciences Research. The patients/participants provided their written informed consent to participate in this study.Conceptualization: CB, LS, JS, AG, AK, and PL. Methodology: CB, LS, JS, AK, and PL. Software: PB. Formal Analysis: AD and PB. Data Curation: PB, CB, LS, JS, and AK. Writing – Original Draft: AD. Writing – Review & Editing, AD, CB, LS, JS, AK, and PL. Visualization: AD. Supervision: AD, AG, AK, and PL. Project Administration: AG, AK, and PL. Funding Acquisition: AK and AG. All authors contributed to the article and approved the submitted version.NIH NHLBI K23 HL143135 (CB). NIH NIAID 1R21AI160334 (CB). COVID-19 Rapid Response Initiative by the Global Infectious Disease Institute, University of Virginia . The RILITE Foundation .We thank the patients who provided samples for this study as well as the authors of previous studies who made their data publicly available and facilitated our analyses.Authors AD, PB, AG and PL were employed by company AMPEL BioSolutions LLC.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


It is estimated that 77.0% of suicide cases occurred in low-and-middle-income countries (LMICs), which would increase because of the COVID-19 pandemic and socioeconomic inequity. However, there is lack of reports on this topic from LMICs, especially during the pandemic. Therefore, this nationwide study aimed to explore self-harm and suicide ideation and its predictive variables during the pandemic in Indonesia as a MIC with the highest COVID-19 fatality rate in Asia.Non-random sampling online survey was conducted nationwide between 25 May and 16 June 2021. The collected data were demographic variables (i.e. age group), loneliness from social isolation using The UCLA Loneliness Scale Six Items (ULS-6), and self-harm and suicide ideation using item 9 of The Patient Health Questionnaire-9 (PHQ-9). Predictive model was analyzed using hierarchical logistic regression.x2 [ (15)] = 1803.46, p < .001), RN2 = .40.A total of 5211 participants from all 34 provinces in Indonesia completed the survey. Among 39.3% of them reported self-harm and suicide ideation during the pandemic, which significantly correlated with loneliness. The predictive variables associated with the likelihood of self-harm and suicide ideation were age, residence, job, religion, sex-gender, sexual orientation, HIV status, disability status, and loneliness. The predictive model showed a significant goodness-of-fit to the observed data (Four out of 10 Indonesians experienced self-harm and suicide ideation during the COVID-19 pandemic, particularly people within the age range of 18–24, living in the Java Island, unemployed/student/retired and freelancer, women, members of minority and marginalized communities, and experience of loneliness during the pandemic.The online version contains supplementary material available at 10.1186/s12888-022-03944-w. • Four in 10 Indonesians experienced self-harm and suicide ideation during the pandemic.• Loneliness from social isolation correlated with self-harm and suicide ideation.• Socioeconomic inequity may contribute to self-harm and suicide ideation.The online version contains supplementary material available at 10.1186/s12888-022-03944-w. Approximately 800,000 people worldwide died by suicide in 2019, 77.0% of whom occurred in low-and-middle-income countries (LMICs) . A systePrevious nationwide study on anxiety conducted early in the pandemic in Indonesia was found about 20% of participants reported clinical anxiety symptoms, mostly among younger people, unemployed, and women . This fiThere are studies on suicide behavior in certain age groups and specific geographical locations in Indonesia in pre-pandemic –20. HoweChange.org IndonesiaChange.org. To improve the survey inclusivity, personalized messages were sent to social media accounts of the marginalized communities . This study was approved by the Research Ethics Committee of Research and Community Service Center (LPPM)-Universitas Katolik Indonesia Atma Jaya (0519A/III/LPPM-PM.10.05/05/2021).The participant inclusion criteria were Indonesian, aged 18 or older, and living in Indonesia. The survey was set up using Typeform online survey software. The software recorded 15,184 view times (only at the cover page) and 10,430 attempts to fill it in. At the end, a total of 5211 participants completed the survey data collection and incomplete responses were automatically deleted by the software. The response and completion rates could not be calculated as view times and attempts to fill in the online survey were not representing the real number of people. For example, one person could visit the cover page multiple times then filled in the survey but discontinued or paused it. When the person tried to fill it in again, previous data was deleted and the software counted it as a new attempt. The participants were predominantly aged 18–24 (45.9%), had a diploma or higher educational background (54.4%), were Muslims (70.9%), and identified as women (67.3%). Details of the participants were provided in the Results and total participants in each province is provided in Supplement All items were presented in Bahasa Indonesia, including the demographic information: age groups, current province of residence, ethnicity, educational background, job status/type, income category, religion/belief, and sex-gender. There were also three questions that asked participants about their minority and marginalized status, namely sexual orientation, HIV status, and disability status that covered both mental and physical disabilities. The demographic variables were grouped in analysis by considering the sociocultural situation in Indonesia. For example, 34 provinces were categorized into two groups, namely “Outside Java Island” and “Within Java Island” because more than half of total population resided in six provinces  in Java Island .α of .81 in this study. The ULS-6 is a cross-cultural measure of loneliness, and it primarily assesses the perception of objective social isolation. Four intensity options were provided for each item from Never (1) to Often (4). The total score ranged from 6 to 24, where higher score indicated more intense loneliness in the last 4 weeks. Participants who answered ‘Never’ on all six items were categorized as “never felt loneliness”, while participants who gave any other responses were grouped in “felt loneliness” for the descriptive analysis. The loneliness intensity was used for the correlational test with self-harm and suicide ideation intensity in the logistic regression analysis.Loneliness from social isolation was measured using The UCLA Loneliness Scale Six Items (ULS-6)-Indonesian version , which hSelf-harm and suicide ideation was measured using item number 9 of The Patient Health Questionnaire-9 (PHQ-9)-Indonesian version , which ip) was set at .05 (two-sided).Data analyses were conducted in Statistical Package for the Social Sciences (SPSS) version 26. Descriptive analysis was performed, followed by a Pearson’s test to analyze the correlation between loneliness intensity and self-harm and suicide ideation. A hierarchical logistic regression analysis was conducted to determine variables that predict self-harm and suicide ideation. Three blocks were built with the demographic variables in Block 1, minority conditions  in Block 2, and loneliness intensity in Block 3. In order to explore the best model, all variables in three blocks were entered using the “Backward-Stepwise” method and the first category of each categorical variable was set as the indicator reference. Adjusted odds ratios with the associated 95% confidence intervals were reported. The significance value .More than one-third of the total participants (39.3%) reported self-harm and suicide ideation during the pandemic, which was relatively higher in some groups, including participants with disability (64.5%), participants with HIV positive (62.8%), and non-heterosexual participants (56.8%). Conversely, the percentage was relatively lower in some other groups, such as participants who were aged 35 or older (14.2%), worked as civil servants/police/military (17.9%), and had monthly income of USD 843 or higher (21.5%). In general, participants reported having self-harm and suicide ideation intensity nearly several days in the last 2 weeks , which was relatively more intense among participants who identified themselves as intersex, transgender, or non-binary and participants with disability compared to other groups. Correlational test showed loneliness intensity was significantly and positively correlated with self-harm and suicide ideation intensity (x2 (15) = 1803.46, p < .001). Nagelkerke’s pseudo R2 indicated the significant variables in the third model explained approximately 40% of the variability. Variables and groups that were significantly associated with higher odds of experiencing self-harm and suicide ideation were age (18–24 years old), current residence (provinces in Java Island), job status/type (unemployed/student/retired and freelancer), religion (non-Islam), sex-gender , sexual orientation , disability status (with disability), and loneliness intensity  showed a significant goodness-of-fit to the observed data , but it was higher than previous study that used the same item among female Indonesian migrant workers in Macau  [This exploratory study aimed to investigate self-harm and suicide ideation and its predictive variables during the COVID-19 pandemic in Indonesia. Findings from this study complemented previous studies on suicidal behavior in specific age groups and limited geographical locations in Indonesia before the pandemic –20. It wD = 0.3) . This diD = 0.3) .The final predictive model showed that the younger age group (18–24 years old) had significantly 2.56 times higher chance of experiencing self-harm and suicide ideation than the older group (35 years or older). Our findings supported another representative population study among Indonesian but it was on anxiety, which found older age participants have lower risk of developing the disorder during the pandemic . We alsoParticipants within the six provinces in Java Island, where more than 56% of total Indonesians resided , were prUnemployed and freelancer groups showed greater chance of having self-harm and suicide ideation, which was up to 1.89 times higher when compared with members of civil servant, police, or military group. This finding mirrored previous nationwide studies that also found unemployed participants were 4.78 times more likely to experience anxiety than health workers in Indonesia . The preAdzan) is considered as an extension of a mosque in practicing the Islamic teachings (dakwah) and for religious purposes like a call to prayer and preaching (khutbah) [gotong royong) events.People with a minority-religion background significantly had 1.33 times greater odds of experiencing self-harm and suicide ideation than Muslim participants, which has the largest population in Indonesia (86.9%) . Movemenkhutbah) . MoreoveCompared to men, women had 1.55 times higher likelihood for experiencing self-harm and suicide ideation. This trend was also found in an anxiety predictive model in Indonesia during the pandemic, where women had significantly 1.61 times higher odds of developing clinically significant anxiety than men . The reaAn alternative explanation is men in the current study might be less open in expressing their feelings and mental problems because they worry about being perceived as weak and less masculine if they admit their struggles , 40. PreRN2 increased from .14 in Model 1 to .40 when participant’s minority conditions  and loneliness intensity were included in Model 3, which became the novelty of this study by discovering the contribution of minority conditions and loneliness intensity to individual’s self-harm and suicide ideation. It was found in the final model that non-heterosexual participants  were significantly more likely to experience self-harm and suicide ideation, 1.72 times than heterosexual people. On the other hand, intersex/transgender/non-binary people also had higher odds in all three predictive models , but the odds were statistically non-significant in the final model. This might be because this group was under-represented in the sample .Three predictive models were developed where the These findings were consistent with a study among Indonesian transgender individuals and MSM that found 69.1% of the participants experienced moderate to high level of psychological distress during the pandemic that increased the risk of suicide behavior . MoreovePeople with positive HIV status who are also part of minority and marginalized communities had 1.90 times greater odds in the final model for experiencing self-harm and suicide ideation. However, this finding was statistically non-significant that might be due to the low representation in the overall participants (0.8%). Despite the non-significant ratio, the current finding reinforced previous studies that highlighted the very limited access to mental health services for LGBTIQ and people living with HIV worldwide, particularly in LMICs, which has worsened during the pandemic , 14.People with disability were significantly predicted to have 2.18 times higher chance of experiencing self-harm and suicide ideation than people without disability. This could be because the pandemic had adversely impacted social interactions, ability to study, and opportunity to earn an income for people with disability, more severely than people without disability. For example, mask-wearing policy as part of COVID-19 health protocols meant people with deaf and hard of hearing were unable to read lip movements and communicate well with others . MoreoveM = 14.0, SD = 4.6) compared with the ULS-6 validation study in Indonesia, Germany, and the US, where Indonesian participants reported the lowest score  [Intensity of loneliness from social isolation among participants in this study was higher ( = 3.83) . In the  = 3.83) .The current study also found that prevalence of loneliness due to social isolation among younger participants was higher than the older group. These young participants might be dominated by students who studied away from their hometown and lived alone, which would amplify loneliness experience. Furthermore, chronic loneliness was consistently found to be associated with psychological disorders, particularly depression, among children, adolescents, and young adults worldwide . HoweverThe important strengths in this study were the large nationwide participants sampled from all provinces in Indonesia with diverse backgrounds and the use of validated instruments. However, there was an over-representation of participants who lived in Java Island that might have different experiences from people in other provinces outside Java Island. Also, despite the data being collected from members of minority and marginalized communities, their numbers were relatively small and might be insufficient for statistical analyses. The question of sex and gender was also mixed due to the public negative sensitivity towards gender nonconformity. Next, this online cross-sectional study does not allow for causal interpretations. The results were also limited to the data collection period and may have the risk of self-selection bias. Lastly, despite the PHQ-9 has been validated among Indonesians this instrument was originally designed to screen for depression; and used a single item (Item 9) to screen self-harm and suicide ideation in non-primary care situations might be less accurate compared with other instruments that are specially designed for this purpose. Therefore, future study should reach participants from outside of Java Island and members of minority and marginalized communities and use a measure with higher sensitivity and specificity for screening suicide ideation in a general setting. Findings from this quantitative study should also be complemented by qualitative investigation, particularly the experiences of minority and marginalized communities that might not be suitable to be analyzed statistically.Four in ten people in Indonesia may experience self-harm and suicide ideation during the COVID-19 pandemic. Variables that significantly contributed to predicting self-harm ideation were age, province, job status, religion, sex, sexual orientation, disability status, and intensity of loneliness from social isolation. The likelihood of experiencing self-harm and suicide ideation were higher among people aged 18–24, lived in Java Island, unemployed/student/retired and freelancer, women, members of minority and marginalized communities , and those experiencing loneliness from social isolation during the pandemic. Mental health treatment and suicide prevention programs during and post-pandemic should prioritize these high-risk groups and be tailored by involving the group members in the planning processes.Additional file 1.