Systematic review and network meta-analysis of efficacy and safety of interventions for preventing anti-tuberculosis drug induced liver injury

Anti-tuberculosis drug induced liver injury (Anti-TB DILI) is the most common adverse events (AEs) necessitating therapy interruption but there is no preventing regimen. This study aimed to examine the efficacy and safety of herbs/alternative medicines for preventing anti-TB DILI. Relevant articles were identified through a systematic search in 5 international databases from inception till March 2022. All randomized controlled trials (RCT) assessing the effects of herbal or alternative medicines against anti-TB DILI were included. The network meta-analysis (NMA) was used to synthesize the evidence for preventing hepatotoxicity using a random-effects model. A total of 3423 patients from 14 RCTs were included. The NMA indicated that supplementation of Turmeric plus Tinospora cordifolia (RR 0.07; 95% CI 0.02 to 0.28), and N-acetyl cysteine (NAC) (RR 0.09; 95% CI 0.01 to 0.75) significantly reduced the incidence of anti-TB DILI compared with placebo. In addition, poly herbal product significantly reduced alkaline phosphatase (ALP) (MD − 21.80; 95% CI − 33.80 to − 9.80) and total bilirubin (Tbil) compared with placebo (MD − 0.51; 95% CI − 0.76 to − 0.26). There was no statistically significant difference in the occurrence of AEs in any intervention. In conclusion, Turmeric plus Tinospora cordifolia, NAC and poly-herbal product may provide benefit for preventing anti-TB DILI in TB patients. However, these findings are based on a small number of studies. Additional studies are warranted to confirm the findings.


Search strategy
We searched PubMed, Scopus, Embase, Web of Science, Cochrane Central Register of Controlled Trials without time and language restrictions from inceptions till March 2022 to identify randomized controlled trials (RCT) for studies assessing the efficacy of herbal or alternative medicines for preventing anti-TB DILI.The following words were used: ("antituberculosis" OR "antituberculous" OR "tuberculosis" OR "isoniazid" OR "rifampicin" OR "pyrazinamide" OR "ethambutol" OR "streptomycin") AND ("hepatoprotectants" OR "hepatoprotective drug" OR "liver protectant" OR "liver aid") AND ("Liver Disease" OR "Liver Dysfunction" OR "hepatic disease" OR "hepatic dysfunction" OR "liver failure" OR "hepatic failure" OR "liver insufficiency" OR "hepatic insufficiency" OR "drug-induced liver injury") AND ("prevention" OR "prophylaxis" OR "preventive therapy").A comprehensive description of the search strategy for each databases is provided in Supplementary Table S1.In addition, we performed hand searches a reference list of relevant articles, reviews, and meta-analyses to avoid missing any relevant literature.

Selection criteria
We included studies based on following criteria: (1) RCTs conducted on new case TB patients, regardless of age, gender, setting or level of blinding; and (2) studies that examined the effects of herb or alternative medicines on preventing anti-TB DILI measured by incidence of anti-TB DILI or liver function tests including AST, ALT, ALP and Tbil.We excluded observational studies, systemic reviews, descriptive reviews, case reports, animal and in-vitro studies.In addition, studies with insufficient data on the selected outcomes and inaccessible studies were also excluded.Full description of inclusion and exclusion criteria was shown in Supplementary Table S2.

Study selection and data extraction
Two authors (PP, PA) independently select the studies and extract data.The study selection was started by screening the potential titles and abstracts following inclusion-exclusion criteria.Full-text articles of the potential studies were assessed independently by two reviewers (PP, PA).Disagreements were resolved by discussions with a third independent researcher (RS, NC, DS).The following information was extracted from eligible studies: name of the first author, year of publication, study design and location, sample size, characteristics of enrolled participants (mean age, sex, TB regimen, baseline liver function test and health status), characteristics of interventions (type, dosage, frequency, duration), follow up time, definition of DILI and study results (number or incidence of patients occurred hepatotoxicity induced by anti-TB drug, mean and standard deviation (SD) of ALT, AST, ALP before and after intervention) and adverse events.

Quality assessment of included studies
The quality of the included studies was assessed using the revised Cochrane Risk of Bias Tool for Randomised Trials (RoB version 2.0) 20 by two independent researcher (PP, PA).Bias is assessed as a judgment (high, low, or unclear) for individual studies from five domains including randomization process, bias due to deviations from intended intervention, missing outcome data, measurement and reporting.Any disagreements between the two reviewers were resolved though consensus by consulting a third author (RS, NC, DS) until consensus was reached.

Outcome measures and statistical analyses
The primary outcome was efficacy of herbal or alternative medicines for preventing anti-TB DILI measured by the incidence of patients having hepatotoxicity assessed based on liver function test and clinical symptoms 20 .Levels of liver function tests including AST, ALT, ALP and Tbil as well as adverse events were analyzed as secondary outcomes.
A pairwise meta-analysis (head-to-head comparisons between individual interventions) was performed for both primary and secondary outcomes.A random-effects model was used for all analysis 21 .For dichotomous and continuous outcomes of the same interventions, pooled direct effect size was calculated as risk ratios (RRs) and mean differences (MD) with corresponding 95% confidence intervals (95% CIs), respectively.Heterogeneity in each direct comparison was assessed using the I 2 statistics and Q-test with a significance level set at P-value < 0.05 22 .Potential sources of heterogeneity were explored using subgroup analyses based on study duration or measurement time and anti-TB DILI criteria.All calculations were based on an intention-to-treat basis, assuming the worst-case scenario, where missing participants were considered non-responders 23 .

Network maps
Network maps of main analyses for primary and secondary outcomes were presented in Fig. 2A and B respectively.For primary outcome, a total of 12 trials with 10 interventions involving 3192 participants examined the effect of each intervention on occurrence of anti-TB DILI (Fig. 2A).Overall, Silymarin and its' derivative were investigated in the highest number of comparisons (5 of 12 studies), followed by vitamin D (2 of 12 studies) and one study for L-carnitine, Bicyclol, Glucuronolactone, Glutathione, Vitamin A, Vitamin A plus Vitamin D, Turmeric plus Tinospora cordifolia extract and Garlic powder.Assessment of study and clinical characteristics of included studies revealed no clinically significant differences among studies included in the network meta-analysis.For secondary outcomes, a total of 10 trials involving 2629 participants examined the effect of interventions on change of AST and ALT levels while 8 trials involving 2269 participants and 7 trials involving 1498 participants examined the effect of interventions on change of ALP and total bilirubin levels (Fig. 2B).There were no inconsistency and no evidence of violation of transitivity assumptions in all networks (Supplementary Table S5).

Primary and secondary outcomes
Treatment effect estimates for pairwise meta-analysis (direct evidence) and network meta-analysis of all primary and secondary outcomes were presented in Table 3.For primary outcome, effects of NAC, Bicyclol, Carnitine, Garlic, Silymarin, turmeric plus Tinospora cordifolia, Vitamin A, Vitamin A plus D, Vitamin C and Vitamin D on occurrence of anti-TB DILI were analyzed.The results from the NMA were generally consistent with those from the pairwise meta-analysis, except for Bicyclol.Pairwise meta-analysis showed that used of Bicyclol had www.nature.com/scientificreports/statistically significant lower occurrence of anti-TB DILI than placebo (RR 0.58; 95% CI 0.39 to 0.86), I 2 = NA, 1 trial with 231 participants) but not significant in NMA (Table 3).NMA of primary outcome showed no evidence of inconsistency and violation of transitivity assumptions (Supplementary tableS5).Pooled estimated effect using NMA indicated that only two interventions including Turmeric plus Tinospora cordifolia extract (RR 0.07; 95% CI 0.02 to 0.28) and NAC (RR 0.09; 95% CI 0.01 to 0.75) had statistically significant lower occurrence of anti-TB DILI compared to placebo (Table 3).Moreover, Turmeric plus Tinospora cordifolia extract also had statistically significant lower occurrence of anti-TB DILI than other interventions except NAC (Table 4).In terms of preventing anti-TB DILI occurrence rankings from the SUCRA analysis, Turmeric plus Tinospora cordifolia extract ranked first, followed by NAC and Garlic tablets (Fig. 3, Supplementary Table S6).However, neither NAC nor Garlic powder demonstrated statistically significant results in any comparison (Table 4).Interestingly, Silymarin, a majority herb used as hepatoprotective effect, did not significantly reduce incidence of anti-TB DILI compared with placebo (RR 0.75; 95% CI 0.39 to 1.44).
For secondary outcomes effects of NAC, Silymarin, Vitamin A, Vitamin A plus D, Vitamin C, Vitamin D, Poly herbal, HuganPian, Glucuronolactone and Glutathione on levels of liver function tests including AST, ALT, ALP and Tbil were analyzed.Pairwise meta-analysis showed that AST levels among those receiving NAC, Silymarin, Vitamin A plus D, and Poly herbal were significantly lowered compared to those in the placebo group but not significant in NMA (Table 3).In addition, pairwise meta-analysis showed that levels of ALT and ALP were statistically reduced by most interventions but not significant in NMA (Table 3).For NMA, there was no evidence of inconsistency and violation of transitivity assumptions (Supplementary Table S5) and pooled estimated effect indicated that there was no any intervention showed significant reduction of AST, ALT level when compared with placebo at the end of study.However, poly-herbal preparation showed statistically significant on reduction of ALP and Tbil compared with placebo.In addition, vitamin D also showed benefit on reduction of total bilirubin (Table 3).

Subgroup analyses
Prespecified subgroup analysis for primary outcome according to difference of measurement times was not performed because of insufficient data.For secondary outcomes, significant results were observed for subgroup analysis at week 4 of measurement.NMA indicated that concomitant used of anti-TB drug with NAC, Vitamin D and Poly-herbal preparation were more efficacious on reduction of AST and ALT levels than placebo whereas poly-herbal preparation and Vitamin D were statistically significant difference on reduction of ALP and Tbil, respectively.The findings were consistent with the main analysis for effect of Poly-herbal preparation and vitamin D on reduction level of ALP and Tbil but not for others (Fig. 4).There was insufficient data to conduct subgroup analyses for studies measured outcome at other measurement times.

Sensitivity analyses and small-study effects assessment
The results from sensitivity analysis using fixed-effect models were comparable with those in main analysis using random-effect model for most interventions (Supplementary Fig. S2).Sensitivity analysis excluding study with high risk of bias was not performed because all included studies were rated as low risk or some concern risk of bias.Comparison-adjusted funnel plots of the main analyses showed no evidence of asymmetry or small-study effect (Supplementary Fig. S3).

Discussion
We conducted a systematic review and network meta-analysis to provide a critical summary of evidence of all available herbs and alternative medicines on prevention the occurrence of anti-TB DILI and levels of liver function tests including AST, ALT, ALP and Tbil.Based on pairwise meta-analysis, our findings demonstrated that several interventions significantly decrease the occurrence of anti-TB-DILI and levels of liver function tests such as NAC, Bicyclol, Silymarin, Turmeric plus Tinospora cordifolia, Vitamin A, Vitamin D or Vitamin A plus D as well as Poly herbal preparation.However, comparing with placebo in NMA, only Turmeric plus Tinospora cordifolia extract and NAC still significantly decrease the occurrence of anti-TB-DILI by 93% and 91%, respectively, when given concomitantly with tuberculosis drugs in new-initiated TB patients.In terms of rankings for preventing anti-TB-DILI occurrence from the SUCRA analysis, Turmeric plus Tinospora cordifolia extract ranked the first, followed by NAC and garlic tablets.In addition, NMA indicated that only two interventions including Poly-herbal preparation and Vitamin D showed statistically significant reduction of ALP and total bilirubin, bilirubin, respectively.However, there were only a few trials of each intervention in this NMA.Therefore, more research is needed to reach more robust conclusions.Some plausible mechanisms may explain the supplementation's beneficial effects of Turmeric plus Tinospora cordifolia extract and NAC on anti-TB DILI.Oxidative stress 36 and systemic or liver inflammation 37 are the two pathological conditions that are implicated in the development and progression of anti-TB DILI.Previous studies indicated that Turmeric plus Tinospora cordifolia extract and NAC had more antioxidant activities 38 and anti-inflammatory properties 39 in both preclinical and clinical studies.Thus, product containing Turmeric plus Tinospora cordifolia extract and NAC may have protective effects on reduction of anti-TB DILI and liver function test parameters.
Considering magnitude of Silymarin benefit, although AST levels from pairwise meta-analysis was significantly less in patients using Silymarin compared with those in the placebo group and this result was consistent with those of previous meta-analyses of RCTs, which indicated that supplementation with Silymarin have www.nature.com/scientificreports/favorable effects on reduction AST levels 40,41 , it should be carefully interpreted because it was unclear whether the observed magnitude of AST reduction was of clinical significance.In fact, there was no evidence of minimal clinically important difference for AST or other liver enzyme reduction and magnitude of reduction depended on baseline levels.In addition, the current evidence from NMA was not support supplementation with Silymarin for both reduction of the occurrence of anti-TB DILI and liver function tests.The challenge of treatment new case TB with standard regimen is a high incidence of hepatitis due to anti-TB drug.In current practice, there is an absence of good evidence supporting the effectiveness of any intervention www.nature.com/scientificreports/for prevention anti-TB DILI.Notably, the outcome measurement differed across studies and few trials were available for this indication.Therefore, there was a big room to develop products from herbal medicines or new drugs for prevention anti-TB DILI in the future.Considering safety issue, herbal or alternative medicine used in all included trials were safe because there was no serious AEs and the number of AEs in intervention group were comparable to those in placebo group.The most common AEs reported in the included studies were gastrointestinal event such as nausea/vomiting, or abdominal pain with mild to moderate degree and no need additional treatment.In addition, drug-herbs and drug-alternative medicine interactions are the crucial issues that physicians should be concerned when herb or alternative medicines were used for preventing anti-TB DILI.However, there was no evidence of drug-herb and drug-alternative medicine interactions for all included interventions.Therefore, herbs and alternative medicines included in this study may be safe in terms of drug interaction with current anti-TB drugs.
The major limitation of this study was that the participants in each study may have different demographic characteristics.Most of included trials did not provided information of external interference factors such as other medicines or other factors related to hepatotoxicity, thus it is difficult to evaluate transitivity of the network due to lack of information.However, inconsistency test using the design-by-treatment interaction model indicated no evidence of inconsistency in all analyzed networks.A difference of outcome measurement in included studies is one of the limitations of this study.Based on clinical view point, evaluating effect of herbal or alternative medicines using occurrence of hepatitis or liver injury following the World Health Organization assessment criteria may be valuable and more represent clinical significant than evaluating only the difference or reduction of liver enzyme.
The strength of this study is the first comprehensive summary of the effects of all herbs and alternative medicines on prevention of anti-TB drug induced hepatotoxicity, undertaken with the high standard of systematic review and network meta-analysis of RCTs regardless of language, and report aligned with PRISMA guideline 2020 42 .The meta-analysis and network meta-analysis of RCTs are at the top in the hierarchy of clinical evidence.These types of research methods are well known and widely acceptable 43 .For this reason, systematic reviews and meta-analyses or network meta-analyses on herbal medicines are increasingly published [43][44][45][46][47][48][49][50] and deemed more The incidence rate of mild AEs were 10.7% (nausea/vomiting; n = 3)

Figure 2 .
Figure 2. Network map for the occurrence of anti-TB-DILI (A) and for changes of liver function test (B).

Table 2 .
Characteristics of patients and interventions.

Table 3 .
Pairwise meta-analysis (MA) and network meta-analysis (NMA) effects of each intervention compared to placebo or no treatment.Significant values are in bold.*Compared with Jian'ganle, anti-TB-DILI Anti-tuberculosis drug induced liver injury, AST Aspartate Aminotransferase, ALT Alanine Transaminase, ALP Alkaline Phosphatase, MA Pairwise meta-analysis, NA Data not available, NMA Network meta-analysis, RR Risk ratio, WMD Weighted mean difference, Tbi Total bilirubin.

Table 5 .
Adverse events.*A total of non-hepatotoxic adverse events (AEs) of all studies are similar between the treatment and control groups and there was no serious adverse event (SAE).non-hepatotoxic adverse events (AEs) of all studies are similar between the treatment and control groups and there was no serious adverse event (SAE) The examples of these AEs were nausea, vomiting, skin rash, epigastric pain and discomfort, malaise, dizziness, arthralgia, peripheral neuropathy, anorexia and insomnia and sickle crisis Chu et al. (2015) 28 Bicyclol Placebo Incidence rate of AEs were 3.4% (n = 4; dizziness + headache (2), rash (2)) Incidence rate of total AEs were 37.71% (n = 69) The examples of AEs were rash, vomiting/nausea, allergy, blurry vision, leucopenia, arthralgia, hearing loss, and peripheral neuropathy Incidence rate of total AEs were 34.76% (n = 65) The examples of AEs were rash, vomiting/ nausea, allergy, blurry vision, leucopenia, arthralgia, hearing loss, and peripheral neuropathy Luangchosiri et al. (2015) 17 Silymarin Placebo The incidence rate of mild AEs were 10.7% (nausea/vomiting; n = 3)