Procedures

Randomization lists, stratified by study sites and indicating the randomization number and corresponding treatment arm, were provided prior to the start of the study by a statistician of the London School of Hygiene & Tropical Medicine. The codes were put in sealed envelopes and assigned to patients as they were included in the study.

Each patient received first-line antituberculosis treatment (Isoniazid, Rifampicin, Ethambutol, and Pyrazinamide) followed by initiation of ARVs (Antiretroviral) therapy (2NRTI + EFV) (Table 1).

Patients in arm B had received first-line antituberculosis treatment followed by ARVs two months after initiation of antituberculosis treatment [18]. Patients in arm C received antituberculosis treatment with a high dose of Rifampicin (15 mg/kg/day instead of 10 mg/kg/day) during the intensive phase of TB treatment followed by ARV therapy initiated two months after the antituberculosis treatment.

The duration of TB treatment was six months during which all the patients received a medical examination at each visit and performed blood tests for liver function assessment (ALT, AST, γ-GT, ALP, and total bilirubin). In all, the patients attended 10 visits scheduled as follows: first visit (Visit 1), one week before inclusion, second visit (Visit 2) on the day of inclusion, visits three to five (Visits 3-5); two, four, six weeks after inclusion and the remaining visits (Visits 6-10); three, four, five, and six months after inclusion.

Outcomes

The primary outcome was an elevation of ALT at least 5 times the Upper Limit of Normal (ULN) corresponding to Grade 3 (5.0 ≤ ALT < 10.0 ULN) or 4 (ALT ≥ 10.0 ULN) of the Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events (Version 2.0, November 2014) [19].

The secondary outcomes were an isolated elevation Grade 4 of AST, γ-GT, ALP, and/or total bilirubin.

No case of treatment discontinuation has been recorded among the patients during the follow-up.

Statistical Analysis

The endpoints were analyzed per protocol. Quantitative variables were presented using means or medians with their standard deviation or interquartile range. The qualitative variables were presented as proportions. Statistical analyzes were performed with R software Version 4.1.0 and MedCalc software version 20.0.5.

The chi-square test and Fisher's exact test were used to compare the proportions two by two. The significance level of the tests was set at 5%.

RESULTS AND DISCUSSION

Characteristics of Patients

A total of 780 patients were randomized into three treatment arms (Table 2). They were 212 from Benin, 470 from Guinea, 98 from Senegal and 53.97 % (421 out of 780) were men, 2.44% (19 out of 780) co-infected with HBV or HCV.

n: Number of study participants, SD: Standard Deviation, ml: millimeter, CD4: Cluster of Differentiation 4, IQR: Interquartile range, BMI: Body Mass Index

From the additional statistical analysis performed, no confounding factors were identified among the demographic, co-medications, and comorbidities available variables

Few cases of Grade 4 toxicity for ALP and total bilirubin were identified among the patients at baseline (Table 3).

ALT: Alanine aminotransferase; ASAT: Aspartate aminotransferase; ɤ-GT: Gamma-glutamyl transpeptidase; ALP: Alkaline phosphatase

Hepatic Safety

During the patients’ follow-up, there were no significant differences between the ALT mean values in the study arms (Table 4).

There were no significant differences between arms B and C patients during the follow-up at Visit 6.

The Grades 3 or 4 of γ-GT, ALP, and total bilirubin were more frequent within the study arms. However, there was no significant difference.

To the best of our knowledge, this study is the first conducted in the three West African countries to assess the hepatic tolerability of a high-dose Rifampicin antituberculosis regimen in TB/HIV co-infected patients. This was a randomized clinical trial with patients' characteristics at baseline well distributed among the three arms. The participants from Guinea were more represented probably due to the higher TB/HIV co-infection prevalence in the country [20]. The number of patients per country and gender ratio were identical for all the randomization arms.

The lymphocytes T CD4 count of the patients included in this study was between 50 and 350 cells/mm³. These rates are compatible with high survival in TB/HIV co-infected patients. In addition, patients with a CD4 count < 50 have a higher risk of death requiring immediate start (during the first two weeks after diagnosis) of ART. They are also at risk for Immune Reconstitution Inflammatory Syndrome (IRIS) due to anti-TB treatment. On the other hand, in patients with T cells CD4 count of > 350, antiretroviral treatment may be postponed beyond two months. In the study by Petros et al., patients who had presented liver injury despite their average levels of CD4 count (69.3 ± 48.7 cells/mm³) between 50 and 350 cells/mm³ had the HLA-B*57 (Human Leukocyte Antigen B subtype 57) phenotype [21]. It is therefore obvious that independently of the Rifampicin doses, an immuno-allergic mechanism would also contribute to the onset of liver injury induced by antituberculosis drugs.

Many other studies such as those of Milstein et al. and Boeree et al. confirmed in TB patients the safety and efficacy of different treatment regimens with high doses of Rifampicin ranging from 20 to 35 mg/kg/day [9, 11]. TB/HIV co-infection being a risk factor for liver injury, high dose Rifampicin regimens (From 20 to 35 mg/kg/day) are risky. In this study, the use of Rifampicin at the dose of 15 mg/kg/day was reasonable and safe for TB/HIV co-infected patients.

Patients in arm C had shown acute and mild toxicity that disappeared during the continuation phase of TB treatment. They did not show any significant increase in ɤ-GT and ALP after two months of treatment (Sixth visit). The increase in ɤ-GT and ALP is frequent in the first trimester of TB treatment. This is said to be the consequence of an immuno-allergic mechanism in which intensity varies from one patient to another [22].

Rare cases of hepatic toxicity corresponding to Grade 3 elevation in ALT had been recorded among patients in arm C. However, no case of toxicity had been recorded in patients in arm B (Reference arm). These were cases of transient toxicity that disappeared with the treatment continuation. However, there was no significant difference between the mean ALT values ​​of the three randomization arms.

Concomitant treatment, low Body Mass Index (BMI), and oxidative stress are well-known risk factors of liver injury in TB/HIV co-infected patients [23, 24]. During the follow-up of the patients, no concomitant treatment had been recorded. Despite the risk of hepatotoxicity linked to BMI less than 18.5 kg/m², the patients in the three study arms did not present any serious signs of hepatotoxicity [25]. Oxidative stress was not assessed in this study. Its assessment would have made it possible to know the effects of anti-TB drugs and ARVs.

Among the first-line antituberculosis drugs, Isoniazid is the most hepatotoxic [26-30]. In this study, all patients received the same dose (5 mg/kg/day) that is only hepatotoxic for patients with a mutation of the N-Acetyltransferase 2 (NAT2), Glutathione S-Transferase (GST), or Cytochrome P4502E1 (CYP2E1) gene [30-33]. Some patients may have two or more mutations of these genes leading to a higher risk of liver injury. In order to secure the use of Isoniazid, it is important to determine the genotype of the patients before using the drug. Under treatment, it is also important to frequently check the levels of liver enzymes. 

PZA could be hepatotoxic for TB patients co-infected by HCV or HIV. In 2020, two studies published by Oscanoa et al. and Kwon et al. have shown that liver toxicity induced by PZA is most frequent than thought [34, 35]. Therefore, the incidence of liver toxicity due to PZA seems to be underestimated as shown by the great number of publications ranking INH as the first liver toxic drug.

Apart from liver toxicity, there are other kinds of toxicities (kidney toxicity, blood toxicity, etc.) that could be assessed in order to improve TB treatment among TB/HIV co-infected patients.

CONCLUSION

This study showed that a high-dose Rifampicin regimen for TB treatment in TB/HIV co-infected patients was as safe as a standard regimen associated with late initiation of ARV therapy. There were no significant differences between the mean values of ALT, AST, γ-GT, ALP, and total bilirubin of the standard regimens and the Rifampicin high-dose regimen. However, a few cases of liver injury corresponding to Grade 3 or 4 of hepatic parameters elevations were recorded in the study arms.

Additional studies are needed to assess the renal and hematological safety of the high-dose Rifampicin regimen to ensure its safety.

Study Protocol  

Available in the supporting information and at dx.doi.org/10.17504/protocols.io.btipnkdn.

Trial Registration

PACTR201105000291300, RAFA

ACKNOWLEDGMENTS: We thank the patients and collaborators of all the countries for their participation and contributions to this study.

CONFLICT OF INTEREST: None

FINANCIAL SUPPORT: The RAFA Trial was funded by EDCTP under the reference number IP.2009.33011.009.

ETHICS STATEMENT: The RAFA trial has been approved by the National Ethics Committees of Benin (Reference 004 31 March 2011), Guinea (Reference 02/CNERS/11) and Senegal (Reference 000117MSP/DS/CNERS).