A cascade amplification strategy based on rolling circle amplification and hybridization chain reaction for ultrasensitive detection of pathogens.

Rapid and accurate detection of a variety of pathogens is very important for the prevention, control, and diagnosis of infectious diseases. Herein, an ultrasensitive nucleic acid isothermal cascade amplification technique based on rolling circle amplification (RCA) coupled with hybridization chain reaction (HCR) was developed for ORF1ab (opening reading frame 1a/b) for SARS-CoV-2 detection. In this scheme, the ORF1ab sequence hybridized with a padlock probe to trigger RCA reaction. Specifically, the recognition site for a unique nicking enzyme was incorporated into the padlock probe to cut the RCA products into short intermediate amplicons, which contain dual HCR initiation sites and can be directly used as primers for HCR. HCR probes, H1 and H2, labeled with FAM (FAM-H1 and FAM-H2) spontaneously participated in the HCR and formed a long nicked dsDNA. Additional probes were quenched by graphene oxide (GO) via π-stacking to decrease the background signal. Meanwhile, the fluorescence signal can be strongly amplified by the synergistic effect of FAM and SYBR green I. The proposed RCA-HCR method can be used to detect ORF1ab at concentrations as low as 7.65 fM. Moreover, the reliability of the RCA-HCR method in serum samples has also been validated. Satisfactory recoveries ranging from 85% to 113% for ORF1ab can be obtained. Therefore, this facile and ultrasensitive RCA-HCR assay provides a new promising tool for ORF1ab analysis and can be extended to the detection of various kinds of pathogens and genetic biomarkers.

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection.

Nucleic acid amplification techniques have always been one of the hot spots of research, especially in the outbreak of COVID-19. From the initial polymerase chain reaction (PCR) to the current popular isothermal amplification, each new amplification techniques provides new ideas and methods for nucleic acid detection. However, limited by thermostable DNA polymerase and expensive thermal cycler, PCR is difficult to achieve point of care testing (POCT). Although isothermal amplification techniques overcome the defects of temperature control, single isothermal amplification is also limited by false positives, nucleic acid sequence compatibility, and signal amplification capability to some extent. Fortunately, efforts to integrating different enzymes or amplification techniques that enable to achieve intercatalyst communication and cascaded biotransformations may overcome the corner of single isothermal amplification. In this review, we systematically summarized the design fundamentals, signal generation, evolution, and application of cascade amplification. More importantly, the challenges and trends of cascade amplification were discussed in depth.

Breaking out of the pandemic: How can firms match internal competence with external resources to shape operational resilience?

This study explores how firms sought to effectively match their internal competence with external resources from the supply chain network to improve operational resilience (OR) during the COVID‐19 pandemic. Drawing upon matching theory, this study provides an internal–external matching perspective based on flexibility–stability features of OR to explain the operational mechanisms underlying the different matchings between internal flexibility (i.e., product diversity)/stability (i.e., operational efficiency) and external flexibility (i.e., structural holes)/stability (i.e., network centrality). We find that more heterogeneous matchings between internal (external) flexibility and external (internal) stability have a complementary effect that enhances OR, whereas more homogeneous matchings between internal flexibility (or stability) and external flexibility (or stability) have a substitutive effect that reduces OR. This study provides valuable contributions to research focusing on the supply chain, organizational resilience, and operations management.

Epidemiology of respiratory syncytial virus in hospitalized children with community-acquired pneumonia in Guangzhou: a 10-year study.

Background: Respiratory syncytial virus (RSV) is one of the most common virus causing community-acquired pneumonia (CAP) in children. To guide the prevention, diagnosis and treatment of RSV, we aimed to analyze the epidemiology of RSV in hospitalized children with CAP. Methods: A total of 9,837 hospitalized children (≤14 years old) with CAP from January 2010 to December 2019 were reviewed. Using the real-time polymerase chain reaction (RT-PCR), the oropharyngeal swab specimens were collected and tested for RSV, influenza virus A (INFA), influenza virus B (INFB), parainfluenza virus (PIV), enterovirus (EV), coronavirus (CoV), human metapneumovirus (HMPV), human bocavirus (HBoV), human rhinovirus (HRV), and adenovirus (ADV) for each patient. Results: The detection rate of RSV was 15.3% (1,507/9,837). From 2010 to 2019, the RSV detection rate showed a wavy change (χ2=166.982, P<0.001), with the highest detection rate in 2011 (158/636, 24.8%). RSV can be detected throughout the year, with the highest detection rate in February (123/482, 25.5%). Children younger than 0.5 years old had the highest detection rate (410/1,671, 24.5%). The detection rate of RSV in male children (1,024/6,226, 16.4%) was higher than that in female children (483/3,611, 13.4%) (P<0.001). A proportion of 17.7% (266/1,507) of RSV positive cases were also co-infected with other viruses, and INFA (41/266, 15.4%) was the most common coinfection virus. After adjusting for potential confounders, the RSV-positive children were associated with increased risk of severe pneumonia [odds ratio (OR) 1.26, 95% confidence interval (CI): 1.04 to 1.53, P=0.019]. Moreover, children with severe pneumonia had significantly lower cycle threshold (CT) values of RSV than those without severe pneumonia (28.88±3.89 vs. 30.42±3.33, P<0.01). Patients with coinfection (38/266, 14.3%) had a higher risk of severe pneumonia than those without coinfection (142/1,241, 11.4%), but the difference was not statistically significant (OR 1.39, 95% CI: 0.94 to 2.05, P=0.101). Conclusions: The detection rate of RSV in CAP hospitalized children changed by years, months, ages, and sexes. CAP hospitalized children with RSV are more likely to develop severe pneumonia than those without RSV. Policy makers and doctors should make timely adjustments to prevention measures, medical resources and treatment options based on these epidemiological characteristics.

CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

Evaluation on the inclusion behavior of ß-cyclodextrins with lycorine and its hydrochloride.

Lycorine (Lyc) and its hydrochloride (Lyc∙HCl) as effective drugs can fight against many diseases including novel coronavirus (COVID-19) based on their antiviral and antitumor mechanism. Beta-cyclodextrin (ß-CD) is considered a promising carrier in improving its efficacy while minimizing cytotoxicity due to the good spatial compatibility with Lyc. However, the detailed mechanism of inclusion interaction still remains to be further evaluated. In this paper, six inclusion complexes based on ß-CDs, Lyc and Lyc∙HCl were processed through ultrasound in the mixed solvent of ethanol and water, and their inclusion behavior was characterized after lyophilization. It was found that the inclusion complexes based on sulfobutyl-beta-cyclodextrin (SBE-ß-CD) and Lyc∙HCl had the best encapsulation effect among prepared inclusion complexes, which may be attributed to the electrostatic interaction between sulfonic group of SBE-ß-CD and quaternary amino group of Lyc∙HCl. Moreover, the complexes based on SBE-ß-CD displayed pH-sensitive drug release property, good solubilization, stability and blood compatibility, indicating their potential as suitable drug carriers for Lyc and Lyc∙HCl.

Tocilizumab, an IL6-receptor antibody, proved effective as adjuvant therapy for cytokine storm induced by severe infection in patients with hematologic malignancy.

Patients with hematological malignancies who experience severe infections are at risk of developing dangerous complications due to excessive inflammatory cytokines. To improve the prognosis, it is crucial to identify better ways to manage the systemic inflammatory storm after infection. In this study, we evaluated four patients with hematological malignancies who developed severe bloodstream infections during the agranulocytosis phase. Despite receiving antibiotics, all four patients presented elevated serum IL-6 levels as well as persistent hypotension or organ injury. Adjuvant therapy with tocilizumab, an IL-6-receptor antibody, was administered, and three of the four patients showed significant improvement. Unfortunately, the fourth patient died due to multiple organ failure caused by antibiotic resistance. Our preliminary experience suggests that tocilizumab, as an adjuvant therapy, may help alleviate systemic inflammation and reduce risk of organ injury in patients with elevated IL-6 levels and severe infection. Further randomized controlled trials are needed to confirm the effectiveness of this IL-6 targeting approach.

Dynamic Link Prediction for Discovery of New Impactful COVID-19 Research Approaches.

In fighting the COVID-19 pandemic, the main challenges include the lack of prior research and the urgency to find effective solutions. It is essential to accurately and rapidly summarize the relevant research work and explore potential solutions for diagnosis, treatment and prevention of COVID-19. It is a daunting task to summarize the numerous existing research works and to assess their effectiveness. This paper explores the discovery of new COVID-19 research approaches based on dynamic link prediction, which analyze the dynamic topological network of keywords to predict possible connections of research concepts. A dynamic link prediction method based on multi-granularity feature fusion is proposed. Firstly, a multi-granularity temporal feature fusion method is adopted to extract the temporal evolution of different order subgraphs. Secondly, a hierarchical feature weighting method is proposed to emphasize actively evolving nodes. Thirdly, a semantic repetition sampling mechanism is designed to avoid the negative effect of semantically equivalent medical entities on the real structure of the graph, and to capture the real topological structure features. Experiments are performed on the COVID-19 Open Research Dataset to assess the performance of the model. The results show that the proposed model performs significantly better than existing state-of-the-art models, thereby confirming the effectiveness of the proposed method for the discovery of new COVID-19 research approaches.

Unintended consequences of COVID-19 public policy responses on renewable energy power: evidence from OECD countries in the EU.

Since 2020, governments around the world have implemented many types of public policies in response to the outbreak of COVID-19. These dramatic public policies have substantially changed production and consumption activities, thereby temporarily lowering electricity use and greenhouse gas emissions. This study argues that pandemic-induced public policies have unintentionally slowed the transition to renewable energy use in the EU since the decline in greenhouse gas emissions due to the lockdowns helped countries temporarily reduce their total emissions. We employ a fixed-effect model to investigate the effects of different types of COVID-19 public policy responses on electricity production, consumption, and net imports in 12 OECD countries in the EU, and we mainly focus on the share of electricity production from renewable energy sources. Among several public policy responses, stringent lockdown policies, such as workplace closures, stay-at-home requirements, and restrictions on gathering size, have negative and statistically significant impacts on electricity generation and consumption. Furthermore, countries with stringent lockdown policies are more likely to import electricity from other countries to mitigate the electricity shortages in their domestic markets. Importantly, we find that lockdown events have negative and statistically significant effects on the share of renewables in electricity production while increasing the share of fossil fuels in electricity production. In contrast, economic support policies such as income support, debt relief, and economic stimulus programs help reduce the share of fossil fuels in electricity production and decrease the net import of electricity from other countries. Our results indicate that the public policies implemented in response to the outbreak of COVID-19 have mixed effects on the transition to renewable energy sources in the EU, suggesting that the current decline in greenhouse gas emissions comes from the reduction in electricity use due to lockdown events instead of the adoption of renewable energy use and discouraging the transition to renewable energy sources.

Identification and semisynthesis of (-)-anisomelic acid as oral agent against SARS-CoV-2 in mice.

(-)-Anisomelic acid, isolated from Anisomeles indica (L.) Kuntze (Labiatae) leaves, is a macrocyclic cembranolide with a trans-fused α-methylene-γ-lactone motif. Anisomelic acid effectively inhibits SARS-CoV-2 replication and viral-induced cytopathic effects with an EC50 of 1.1 and 4.3 µM, respectively. Challenge studies of SARS-CoV-2-infected K18-hACE2 mice showed that oral administration of anisomelic acid and subcutaneous dosing of remdesivir can both reduce the viral titers in the lung tissue at the same level. To facilitate drug discovery, we used a semisynthetic approach to shorten the project timelines. The enantioselective semisynthesis of anisomelic acid from the naturally enriched and commercially available starting material (+)-costunolide was achieved in five steps with a 27% overall yield. The developed chemistry provides opportunities for developing anisomelic-acid-based novel ligands for selectively targeting proteins involved in viral infections.

Sodium Reduction in Restaurant Food: A Randomized Controlled Trial in China.

Restaurant food is one of the important sources of sodium intake in China. We aimed to determine whether a restaurant-based comprehensive intervention program may induce lower sodium content in restaurant food. A randomized controlled trial was implemented between 2019 and 2020 in 192 restaurants in China. After baseline assessment, the restaurants were randomly assigned to either an intervention or a control group (1:1). Comprehensive activities designed for intervention restaurants were conducted for one year. The primary outcome was the difference in change of sodium content estimated by the mean values of five best-selling dishes for each restaurant, from baseline to the end of the trial between groups. In total, 66 control restaurants and 80 intervention restaurants completed the follow-up assessment. The average sodium content of dishes at baseline was 540.9 ± 176.8 mg/100 g in control and 551.9 ± 149.0 mg/100 g in intervention restaurants. The mean effect of intervention after adjusting for confounding factors was -43.63 mg/100 g (95% CI: from -92.94 to 5.66, p = 0.08), representing an 8% reduction in sodium content. The restaurant-based intervention led to a modest but not significant reduction in the sodium content of restaurant food. There is great urgency for implementing effective and sustainable salt reduction programs, due to the rapid increase in the consumption of restaurant food in China.

The effect of COVID-19 vaccines on sperm parameters: a systematic review and meta-analysis.

Published data were gathered for a meta-analysis to determine the difference in sperm parameters before and after administration of different types of coronavirus disease 2019 (COVID-19) vaccines, because the reproductive toxicity of COVID-19 vaccines has not yet been evaluated in clinical trials and COVID-19 has been associated with decreases in sperm quality. The preferred procedures for systematic reviews and meta-analyses were followed in the conduct and reporting of this study. The average sperm parameters of all sperm donors' multiple sperm donations were compared before and after receiving various COVID-19 vaccinations. Semen volume, total sperm motility, total sperm count, morphological change, and sperm concentration were the primary outcome measures. We compiled and analyzed the results of six studies on total sperm motility, six studies on semen volume, six studies on sperm concentration, two studies on morphological change, and two studies on total sperm count. Parameter comparisons with patients who had and had not been vaccinated were only reported in one of the included studies. When different types of COVID-19 vaccine injections were compared, no discernible differences in parameters were observed. According to the available data, the parameters of semen are unaffected by inactivated or messenger RNA (mRNA) COVID-19 vaccinations. To support these findings, additional prospectively designed research is required.

A study on level of mental health and influencing factors of medical staff in the makeshift hospital during COVID-19 in Shanghai

Objective　To analyze the mental health status of medical staff in the Fourth Branch of National Convention and Exhibition Center Makeshift Hospital during the COVID-19 epidemic in Shanghai to lay a theoretical foundation for the mental health and psychological intervention of medical staff in COVID-19 and other public health emergencies. Methods　An online questionnaire survey was conducted with the generalized anxiety disorder scale (GAD-7), patient health questionnaire (PHQ-9), and Athens insomnia scale (AIS) before medical staff entering the makeshift hospital and one month later. Results　The detection rates of anxiety, depression and insomnia were 18.4%, 22.1% and 27.0% respectively before entering the makeshift hospital, and 28.8%, 59.3% and 64.2% respectively during the follow-up period one month later. The GAD-7, PHQ-9 and AIS scores of medical staff after working in the makeshift hospital for one month increased significantly compared with those at the baseline period (P<0.01). Female and previous history of using sedative and hypnotic drugs were risk factors for increased depression level among medical staff in the makeshift hospital. Conclusions　The anxiety, depression and insomnia levels of the medical staff in Shanghai increased after working in the makeshift hospital for one month. It is of great significance for the front-line support work to identify the medical staff with serious psychological problems and carry out psychological intervention in the early stage.

Association between acute stress response and peer bullying behaviors among middle school students

Objective: To explore the association between acute stress response during the outbreak of COVID-19 and peer bullying behaviors during the normalized management of COVID-19 among middle school students, and to provide a basis for developing relevant measures for peer bullying prevention.

Impact of the COVID-19 on electricity consumption of open university campus buildings - The case of Twente University in the Netherlands.

Since the COVID-19 outbreak, the restrictive policies enacted by countries in response to the epidemic have led to changes in the movement of people in public places, which has had a direct impact on the use and energy consumption of various public buildings. This study was based on electricity consumption data for 25 on-campus public buildings at 1-hour intervals between January 2020 and June 2022 at Tewnte University in the Netherlands, and after the data were climate-corrected by multiple regression analysis, the changes in EU and EUI for various types of buildings were compared for different restriction periods using ANOVA, LSD and t-tests. And additionally, further analyzed the changes and reasons for the electricity consumption of various public buildings on campus and customers' electricity consumption behavior in a period of time after the lifting of the epidemic restriction policy. The results of ANOVA analysis show that the restriction policy has a significant effect on teaching, sports, and cultural buildings, and the electricity intensity of the three types of buildings is reduced by 0.28, 0.09, and 0.07 kwh/m2/day respectively under the strict restriction policy; The t-test results show that during the restriction period, all building types, except for living and academic buildings, show a significant decreasing trend, with the teaching buildings having the greatest energy saving potential, with an average daily EU reduction of 1088kwh/day and an EUI reduction of 0.075kwh/ m2/day. The above findings provide a case study of a complete cycle of energy consumption changes in university buildings under similar epidemic restriction policies before and after the epidemic restriction, and inform the electricity allocation policies of university and government energy management authorities.

Would You Accept Virtual Tourism? The Impact of COVID-19 Risk Perception on Technology Acceptance from a Comparative Perspective

Due to the COVID-19 pandemic, the tourism industry and its stakeholders have tried to develop a new virtual tourism market, but its effectiveness remains to be tested. We proposed and tested a new measurement scale composed of ease of use, usefulness, autonomy, enjoyment, perceived risk of COVID-19, and attitude. In total, 274 questionnaires were collected by the purposive sampling method and 239 of them were valid, with 57 potential virtual tourists (who knew of but had not used VR in tourism) and 182 actual virtual tourists (who had experienced virtual tourism). Then, we used path analysis to test the hypothetical model and compared the results of two groups. The results show that (1) the popularity of virtual tourism is limited, (2) ease of use significantly affects usefulness and enjoyment for the two groups, (3) usefulness significantly affects autonomy and enjoyment for the two groups, (4) perceived risk of COVID-19 has a direct impact on the attitude towards virtual tourism for the two groups rather than a moderating role, and (5) expected ease of use has a significant effect on autonomy, and autonomy further influences enjoyment for potential tourists. This paper is an explorative attempt to explore virtual technology applied in tourism during the COVID-19 pandemic. The results provide theoretical contributions and practical implications for technology improvement, tourism marketing, and virtual tourism development.

Safety of inactivated SARS-CoV-2 vaccines in myasthenia gravis: A survey-based study.

Background: Vaccination remains the most effective measure to prevent SARS-CoV-2 infection and worse outcomes. However, many myasthenia gravis (MG) patients are hesitant to receive vaccine due to fear of worsening. Methods: MG patients were consecutively enrolled in two MG centers in North China. The "worsening" after vaccination was self-reported by MG patients, and severity was measured with a single simple question. The general characteristics and disease status immediately prior to the first dose were compared between the worsening and non-worsening groups. Independent factors associated with worsening were explored with multivariate regression analysis. Results: One hundred and seven patients were included. Eleven patients (10.3%) reported worsening after vaccination, including eight patients with mild or moderate worsening and three patients with severe worsening. Only one of them (0.9%) needed an escalation of immunosuppressive treatments. There were significant differences between the worsening and non-worsening groups in terms of Myasthenia Gravis Foundation of America classes immediately before the first dose and intervals since the last aggravation. Precipitating factors might contribute to the worsening in some patients. Logistic regression revealed that only interval since the last aggravation ≤6 months was associated with worsening after SARS-CoV-2 vaccination (P = 0.01, OR = 8.62, 95% CI: 1.93-38.46). Conclusion: SARS-CoV-2 vaccines (an overwhelming majority were inactivated vaccines) were found safe in milder Chinese MG patients who finished two doses. Worsening after vaccination was more frequently seen in patients who were presumed as potentially unstable (intervals since last aggravation ≤6 months). However, mild worsening did occur in patients who were presumed to be stable. Precipitating factors should still be sought and treated for better outcome.

Bioinformatics-based SARS-CoV-2 epitopes design and the impact of Spike protein mutants on epitope humoral immunities

Graphical Background Epitope selection is the key to peptide vaccines development. Bioinformatics tools can efficiently improve the screening of antigenic epitopes and help to choose the right ones. Objective To predict, synthesize and testify peptide epitopes at spike protein, assess the effect of mutations on epitope humoral immunity, thus provide clues for the design and development of epitope peptide vaccines against SARS-CoV-2. Methods Bioinformatics servers and immunological tools were used to identify the helper T lymphocyte, cytotoxic T lymphocyte, and linear B lymphocyte epitopes on the S protein of SARS-CoV-2. Physicochemical properties of candidate epitopes were analyzed using IEDB, VaxiJen, and AllerTOP online software. Three candidate epitopes were synthesized and their antigenic responses were evaluated by binding antibody detection. Results A total of 20 antigenic, non-toxic and non-allergenic candidate epitopes were identified from 1502 epitopes, including 6 helper T-cell epitopes, 13 cytotoxic T-cell epitopes, and 1 linear B cell epitope. After immunization with antigen containing candidate epitopes S206-221, S403-425, and S1157-1170 in rabbits, the binding titers of serum antibody to the corresponding peptide, S protein, receptor-binding domain protein were (415044, 2582, 209.3), (852819, 45238, 457767) and (357897, 10528, 13.79), respectively. The binding titers to Omicron S protein were 642, 12878 and 7750, respectively, showing that N211L, DEL212 and K417N mutations cause the reduction of the antibody binding activity. Conclusions Bioinformatic methods are effective in peptide epitopes design. Certain mutations of the Omicron would lead to the loss of antibody affinity to Omicron S protein.

Safety of inactivated SARS-CoV-2 vaccines in myasthenia gravis: A survey-based study

Background Vaccination remains the most effective measure to prevent SARS-CoV-2 infection and worse outcomes. However, many myasthenia gravis (MG) patients are hesitant to receive vaccine due to fear of worsening. Methods MG patients were consecutively enrolled in two MG centers in North China. The “worsening” after vaccination was self-reported by MG patients, and severity was measured with a single simple question. The general characteristics and disease status immediately prior to the first dose were compared between the worsening and non-worsening groups. Independent factors associated with worsening were explored with multivariate regression analysis. Results One hundred and seven patients were included. Eleven patients (10.3%) reported worsening after vaccination, including eight patients with mild or moderate worsening and three patients with severe worsening. Only one of them (0.9%) needed an escalation of immunosuppressive treatments. There were significant differences between the worsening and non-worsening groups in terms of Myasthenia Gravis Foundation of America classes immediately before the first dose and intervals since the last aggravation. Precipitating factors might contribute to the worsening in some patients. Logistic regression revealed that only interval since the last aggravation ≤6 months was associated with worsening after SARS-CoV-2 vaccination (P = 0.01, OR = 8.62, 95% CI: 1.93–38.46). Conclusion SARS-CoV-2 vaccines (an overwhelming majority were inactivated vaccines) were found safe in milder Chinese MG patients who finished two doses. Worsening after vaccination was more frequently seen in patients who were presumed as potentially unstable (intervals since last aggravation ≤6 months). However, mild worsening did occur in patients who were presumed to be stable. Precipitating factors should still be sought and treated for better outcome.