Here, we present a protocol to analyze the effectiveness and safety of Angong Niuhuang Pill in the treatment of viral encephalitis.
This meta-analysis aims to evaluate the efficacy of Angong Niuhuang Pill (ANP) as an adjuvant therapy in the treatment of viral encephalitis. Seven databases (PubMed, Cochrane Library, Embase, SinoMed, CNKI, VIP and WanFang) were included for literature retrieval from inception to July 2023. Randomized controlled trials comparing ANP plus conventional therapy with conventional therapy alone were eligible. Pooled effect sizes and 95% confidence intervals (CIs) were calculated for evaluating efficacy and safety. Sensitivity analysis and publication bias assessments were performed for analyzing the inconclusiveness of findings. 13 studies involving 1045 cases were included for meta-analysis. Adjuvant treatment with ANP increased the probability of the total effective rate by 17% compared with conventional treatment (Risk ratios (RR) = 1.17, 95%CI [1.08, 1.27]). The disappearance time of clinical syndromes and signs was significantly decreased after adjuvant treatment with ANP, including the time of defervescence (weighted mean difference (WMD) = -1.59, 95%CI [-2.09, -1.09]), the time of consciousness recovery (WMD = -1.79, 95%CI [-2.06, -1.51]), the time of headache disappearance (WMD = -1.51, 95%CI [-1.93, -1.08]), the time of tic disappearance (WMD = -1.88, 95%CI [-2.39, -1.36]). The adjuvant efficacy of ANP for treating viral encephalitis (VE) appears to improve the total effective rate and shorten the disappearance time of clinical syndromes. More high-quality randomized controlled trials (RCTs) are needed to support our findings.
Encephalitis is defined as the inflammation of the brain parenchyma with high mortality, characterized by neurological pathological signs, fever, abnormal mental status, and altered electroencephalographic (EEG) and neuroimaging1,2. The incidence of encephalitis ranges between 7 and 15 cases per 100,000 people3,4,5. The cause of approximately half of encephalitis cases is unknown, and viral encephalitis (VE) accounts for about 20% to 50% of diagnosed cases5,6. Common viruses include human enterovirus, human herpesvirus, herpes simplex virus, human immunodeficiency virus, and cytomegalovirus7. Patients with VE require intensive monitoring and supportive measures to maintain vital signs and alleviate cerebral inflammation8. Acyclovir is an empiric treatment for all cases of VE9. Evidence for the use of glucocorticoids, immunoglobulins, and interferon alfa are inconsistent8. Angong Niuhuang Pill (ANP), one of the well-known orifice-opening medicines, is widely used in mental and conscious abnormalities due to critical illness. China Medical Education Association recommended ANP as an A-level strength of recommendation and II-level quality of evidence (evidence from non-randomized studies or randomized controlled trials)10.
A systematic review (SR) is a comprehensive collection and rigorous evaluation of all clinical studies that meet the inclusion criteria. Meta-analysis is a methodology for quantitative analysis in SR. Distinguishing from the methods of basic meta-analysis, a trim and fill procedure was adopted to test the robustness of the effect size estimate to the assessment of publication bias11. A small number of studies were removed until a new effect size made the plot symmetrical. Additional studies were added back to the plot based on imputed mirror-image studies. If the difference of the recalculated pooled estimate (PE) before and after the trim and fill procedure was less than 0.05, the effect size estimate of publication bias was robust. TSA 0.9.5.10 software was used to reduce the risk of false-positive results and calculate the required information size (RIS), which was beneficial to make our findings more robust. Given that no systematic review of the data on the improvement of symptoms, signs, biochemical indicators, and adverse reactions of ANP in the treatment of VE, we therefore performed this meta-analysis of the available randomized controlled trials.
1. Protocol registration
2. Software installation
3. Database retrieval
4. Inclusion and exclusion criteria
5. Literature management and screening
6. Quality assessment
7. Data synthesis and analysis
Search results and study characteristics
Systematic retrieval yielded 122 studies and manual searching added 9 relevant literatures. After removing 49 duplicates, 82 studies remained to be screened by abstract and full-text. Ultimately, 13 studies12,13,14,15,16,17,18,19,20,21,22,23,24 involving 1045 cases were included for meta-analysis (Figure 1). The characteristics of eligible studies are summarized in Table 1. These trials were published in China from 1997 to 2023. All cases with VE were children whose basic data had no significant difference. In terms of the risk of bias, all included studies were suboptimal (Table 2). Only 4 studies had a low risk in random sequence generation, and no trial reported allocation concealment. All studies had complete outcome data. Both blinding and selective outcome reporting of included studies were unclear.
Outcome comparison
Total effective rate
The total effective rate was reported as the primary outcome in 11 studies12,13,14,16,17,18,19,20,21,22,24. As shown in Figure 2A, the total effective rate of adjuvant treatment with ANP was 17% higher than conventional treatment (RR = 1.17, 95% CI[1.08,1.27], I2 = 13.3%, P = 0.00). Harbord's test (P = 0.56) suggested that there was no publication bias (Figure 2B). For RIS, the relative risk reduction (RRR) was set as -17% and the relative event rate of the control group was set as 80%. As shown in Figure 2C, the cumulative Z curve crossed the conventional boundary value and RIS, which indicated that the total effective rate of adjuvant treatment with ANP was significantly higher than conventional therapy and met the requirement of sample size.
Time of defervescence
The time of defervescence for evaluation was reported in 11 studies12,13,14,15,16,17,18,19,20,23,24. Forest plot (Figure 3A) showed that the time of defervescence in adjuvant treatment with ANP was significantly shorter than in conventional treatment (WMD = -1.59, 95% CI[-2.09,-1.09], I2 = 92.1%, P = 0.00). The high heterogeneity is possibly attributed to Yu's23 and Fu's16 studies (Figure 3B). The continuous variable adapted Egger's test for publication bias assessment, P=0.03, which indicated that the outcome referred to publication bias (Figure 3C). However, the trim and fill procedure did not process data, implying the pooled WMD of time of defervescence was robust (Figure 3D). The time of defervescence of adjuvant treatment with ANP was significantly less than conventional therapy and met the requirement of sample size(Figure 3E).
Time of consciousness recovery
The time of consciousness recovery was reported in 8 studies12,13,14,15,16,17,19,20. Forest plot (Figure 4A) showed consciousness recovery was 1.79 days faster in adjuvant treatment with ANP than in conventional treatment (WMD = -1.79, 95% CI[-2.06, -1.51], I2 = 61.9%,P = 0.00). Sensitivity analysis revealed that Zhao's13 study was a noteworthy factor causing high heterogeneity (Figure 4B). No publication bias was verified (P = 0.70) (Figure 4C).
Time of headache disappearance
The time of headache disappearance was reported in 6 studies12,13,15,16,17,20. Figure 5A shows as a forest plot, the time of headache disappearance of adjuvant treatment with ANP was significantly shorter than conventional treatment (WMD = -1.51, 95% CI[-1.93, -1.08], I2 = 80.5%, P = 0.00). Wang's12 study could be the possible cause of high heterogeneity via sensitivity analysis (Figure 5B).
Time of tic disappearance
The time of tic disappearance was reported as an outcome measure in 6 studies13,14,15,19,20,23. Figure 6A showed adjuvant treatment with ANP could significantly shorten the time of tic disappearance than conventional treatment (WMD = -1.88, 95% CI[-2.39, -1.36], I2 = 86.6%, P = 0.00). Sensitivity analysis implied high heterogeneity could be due to the studies of Zhao13 and Yu23 (Figure 6B).
Figure 1: Flow chart of meta-analysis. Please click here to view a larger version of this figure.
Figure 2: Total effective rate analysis. (A) Forest plots. (B) Publication bias assessment. (C) Trial sequential analysis. Please click here to view a larger version of this figure.
Figure 3: Analysis of time of defervescence. (A) Forest plots, (B) sensitivity analysis, (C) Publication bias assessment, (D) trim and fill procedure, and (E) trial sequential analysis. Please click here to view a larger version of this figure.
Figure 4: Analysis of time of consciousness recovery analysis. (A) Forest plots, (B) sensitivity analysis, and (C) Publication bias assessment Please click here to view a larger version of this figure.
Figure 5: Analysis of time of headache disappearance. (A) Forest plots, and (B) sensitivity analysis. Please click here to view a larger version of this figure.
Figure 6: Analysis of time of tic disappearance. (A) Forest plots, and (B) sensitivity analysis. Please click here to view a larger version of this figure.
Table 1: Basic characteristics of included studies. T: Treatment;
C: control;
CT: conventional therapy;
IG: immune globulin;
MGS: Monosialotetrahexosyl ganglioside Sodium;
Effective rate;
Disappearance of headache (day);
Time to defervescence (day);
Recovery of consciousness (day);
Disappearance of motor tics (days). Please click here to download this Table.
Table 2: Risk of bias assessment Please click here to download this Table.
Supplementary Figure 1: Search examples in the PubMed database. (A) The main window of literature retrieval builder in PubMed. (B) The main window for downloading all the retrieved literature. Please click here to download this File.
Supplementary Figure 2: Literature management in Endnote. (A) The main window for creating a new group is (B) The main window of importing the retrieved literature. (C) The main window for removing duplicates. Please click here to download this File.
Supplementary Figure 3: Meta-analysis in Stata. (A)The main window for data entry of forest plots of continuous data. (B) The main window for choosing the model and statistics of continuous data. (C) The main window for data entry of forest plots of dichotomous data. (D) The main window for choosing the model and statistics of dichotomous data. (E) The main window for Egger's test. (F) The main window for the trim and fill procedure. Please click here to download this File.
Supplementary Figure 4: TSA analysis. (A) The main window for creating a new meta-analysis file. (B) The main window for setting effect measure, model, and zero event handling. (C) The main window for adding studies. (D) The different requirements of continuous and dichotomous data. (E) The main window for adding parameters in TSA analysis. Please click here to download this File.
This meta-analysis provides evidence that the adjunctive ANP can improve the total effective rate by 17% in patients with VE. Adjuvant therapy with ANP can significantly shorten the disappearance time of positive pathological signs and symptoms. Xingnaojing (XNJ) injection extracted from ANP is widely used for abnormal consciousness associated with encephalopathy. A recent meta-analysis verified the adjunctive effect of XNJ for treating VE, contributing to an increase in the number of cure cases and shortening the time of symptoms relieving25. The components of XNJ are not exactly the same as ANP. According to Pharmacopoeia of the People's Republic of China (2020 version), ANP was composed of Bovisc Alculus (Niuhuang), Moschus (Shexiang), Powerdered Buffalo Horn Extract (Shuiniujiao Nongsuofen), Margarita (Zhenzhu), Cinnabaris (Zhusha), Realgar (Xionghuang), Coptidis Rhizoma (Huanglian), Scutellariae Radix (Huangqin), Gardeniae Fructus (Zhizi), Curcumae Radix (Yujin) and Borneolum (Bingpian). The identification items of active ingredients include bile acid, berberine hydrochloride, baicalin, muscone, borneolum, etc. In this study, the level of neuroinflammation indicators (NSE, S100β, and TNF-ɑ) was significantly decreased after adjuvant treatment with ANP compared with conventional therapy, which enhanced the credibility of the conclusion. Herpes simplex virus (HSV) is the most common cause of VE in America6. Berberine inhibited HSV replication by suppressing the activation of cellular c-Jun N-terminal kinase (JNK) and nuclear factor κB (NF-κB) pathways26. Exacted from scutellariae radix, baicalin inhibits respiratory syncytial virus replication and increases the expression of type I interferons27. With a highly restricted permeability, the blood-brain barrier (BBB) is a physical roadblock between the central nervous system (CNS) and other systems. The virus infects the CNS through three ways of BBB, including the paracellular pathway, the transcellular pathway, and the "trojan horse" mechanism1. Borneol (BO) can promote other components to enter the BBB28, thus synergistically exerting their antiviral effect. At the molecular level, BO can down-regulate the expression of iNOS and TNF-ɑ29; at the cellular level, it improves morphological changes and neuronic functions, microglial cells, and BBB30. Moschus is often used in stroke first aid. In the cerebral ischemic model, the muscone can repair neuronal synaptic connections, which contributes to improving neuronal necrosis and protecting BBB31. Therefore, the mechanism of ANP in the treatment of VE may refer to the effects of anti-inflammation, anti-virus, repairing BBB, and neuroprotection.
In this analysis, 2 studies14,16 reported the comparisons of adverse events in control and treatment groups, including vomiting, rash, and headache. However, the comparisons between the two groups were not significantly different. After adjunct treatment with ANP, epilepsy still has a possible risk of seizure, and the course of the disease has not decreased significantly. ANP includes realgar and cinnabar, which can lead to liver and kidney injury, but its safety is still unclear and should be used with caution.
In this protocol, we detailed how to perform meta-analysis via Stata, which assesses the difference in efficacy between the two treatments and verifies the robustness of the results through sensitivity analysis, publication bias analysis, and trim and fill procedure. By comprehensively analyzing the above results of outcomes, three studies contributed significantly to the heterogeneity of the literature, including the studies of Yu, Wang, and Zhao. After re-reading the full article, we speculated that it might be due to the small sample size of Yu's23 study, only severe cases with VE of Wang's12 study, and poor methodology of Zhao's13 study. Besides, Egger's test suggested that the analysis of the time of defervescence had a possibility of publication bias. The trim and fill procedure did not process any data which verified the conclusiveness of the result. Maybe the suboptimal quality of literature interferes with the test of publication bias. Furthermore, TSA was used to assess the reliability and stability of cumulative evidence32. The cumulative Z-curves crossed the conventional test boundary and RIS, suggesting that there was adequate evidence to conclude the better efficiency of ANP. In addition to basic meta-analysis, the protocol offers a more reliable evaluation of results, which can be used to evaluate the efficacy of two therapies. However, this protocol can only be used to assess the difference in efficacy between the two treatments but cannot quantify the dose and duration of ANP. Notably, the completion of this protocol is based on software operation, which needs to ensure the correct code command and data input.
To enhance the conclusiveness of meta-analysis, some suggestions are put forward as follows: (1) Proper typing of TCM syndrome should be considered in patient's selection for evaluating efficiency adequately; (2) Additional VE-related biochemical indexes could be added into trial protocols for improving the objectivity of curative effect; (3) The baseline information of cases should be collected and described as more possible, such as the type of virus, disease condition, and administration method; (4) Strictly following the randomized controlled principle to improve the quality of evidence.
In summary, the adjuvant efficacy of ANP in the treatment of VE includes improving the total effective rate and shortening the disappearance time of clinical syndrome. However, due to the suboptimal quality of evidence, our findings are not definitive and require more high-quality RCTs to support.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (No. 81973919), the National Natural Science Foundation of Guangdong Province (No. 2019A1515011299), and the Innovation and Strong School project of the Department of Education of Guangdong Province (No. 2016KTSCX015).
PROSPERO | National Institute for Health and Care Research | https://www.crd.york.ac.uk/PROSPERO/ | |
STATA | StataCorp | https://www.stata.com/ | |
TSA | Copenhagen Trial Unit, Centre for Clinical Intervention Research | https://ctu.dk/tsa/downloads/ | |
PubMed | National Library of Medicine | https://pubmed.ncbi.nlm.nih.gov/ | |
Cochrane Library | Cochrane Library | https://www.cochranelibrary.com/ | |
Embase | Elsevier | https://www.embase.com/search/quick | |
SinoMed | SinoMed | http://www.sinomed.ac.cn/index.jsp | |
CNKI | China National Knowledge Infrastructure | https://www.cnki.net/ | |
VIP | Chongqing VIP Information Co., Ltd. | http://www.cqvip.com/ | |
WanFang database | Wanfang Data Knowledge Service Platform | https://www.wanfangdata.com.cn/index.html |