Immunology Highlights of Four Major Idiosyncratic DILI Subtypes Verified by the RUCAM: A New Evidence-Based Classification

Abstract

Conventionally, drug-induced liver injury (DILI) exists in two types: idiosyncratic and intrinsic. Both types are classified as non-immune disorders, thereby ignoring that some iDILI cases may have an immune or autoimmune background that requires a different therapeutic approach because steroids may be helpful. The purpose of this analysis was to analyze and classify the subtypes of iDILI which, indeed, show autoimmune or immune features among four cohorts, namely idiosyncratic DILI type 1: idiosyncratic drug-induced autoimmune hepatitis (DIAIH), to be differentiated from the classic drug-unrelated idiosyncratic autoimmune hepatitis (AIH); idiosyncratic DILI type 2: human leucocyte antigen-based idiosyncratic drug-induced autoimmune hepatitis; idiosyncratic DILI type 3: anti-cytochrome P450-based idiosyncratic drug-induced autoimmune hepatitis; and idiosyncratic DILI type 4: immune-based idiosyncratic drug-induced liver injury associated with Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). In conclusion, the traditional non-immune and non-autoimmune iDILI, as well as the four immune or autoimmune iDILI subtypes, are now well classified and clinically characterized by the broadly applied Roussel Uclaf Causality Assessment Method (RUCAM), facilitating additional immunology and therapy studies for the four subtypes, all of which could benefit from steroid treatment.

1. Introduction

Drugs used as therapeutic agents may trigger different liver injuries. Idiosyncratic drug-induced liver injury (iDILI) is commonly described as a non-immune and complex disease that requires a robust causality assessment to provide evidence-based feature characteristics of this disruptive disorder [1,2,3,4,5,6]. Using validated diagnostic approaches, substantial information was gathered on cases of iDILI by new drugs like androgenic anabolic steroids [7], antidepressants [8], antituberculosis drugs [9], atezolizumab [10], certolizumab [11], gemcitabine [12], memantine [13], pazopanib [14], sintilimab [15], sunitinib [14], teriflunomide [16], and tigecycline [17]. Similarly, additional descriptions of the features and clinical aspects of iDILI were also provided in other studies focusing on newborn and pediatric patients [18,19,20], geriatric patients [21,22], patients with cystic fibrosis [23], the utility of lymphocyte transformation tests [24], and epidemiology [2,6,25,26]. DILI covers a range of clinical facets with various complexities. It unquestionably has a strong global impact with major clinical significance in view of the risks of liver transplantation as the ultimate therapy approach to prevent death. These clinical outcomes warrant further studies on DILI, which is a worldwide challenge.

By convention, the iDILI pattern is preferentially characterized as a hepatocellular injury, cholestatic injury, or mixed liver injury, resulting from conclusions of international consensus meetings published as the Roussel Uclaf Causality Assessment Method (RUCAM) of 1993 [27,28] and now preferentially used as the updated RUCAM of 2016 [29]. Similarly, drugs may rarely cause RUCAM-based metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH) [30], RUCAM-based drug-induced vanishing bile duct syndrome [31], or even RUCAM-based hepatic sinusoidal obstructive syndrome [32]. However, the diagnosis of iDILI may be confounded by alternative causes, a major clinical issue emerging in cases of erroneously suspected but not verified DILI due to causality assessment by the RUCAM [33,34,35,36]. If alternatives are not recognized in time, published clinical features and mechanistic steps may be incorrectly attributed to liver diseases that have nothing to do with drug treatment.

Common clinical features of the classic non-immune iDILI with diagnosis evidenced by RUCAM include the following: (1) idiosyncrasy, which reflects the fact that the liver injury develops unexpectedly at normal drug doses in a genetically predisposed individual, as opposed to the dose-dependent intrinsic DILI occurring in an individual, which is unrelated to their genetic background and clearly defined by RUCAM [29]; (2) liver injury, with serum activities of alanine aminotransferase (ALT) being at least five times the upper limit of normal (ULN), or with those of alkaline phosphatase (ALP) being at least two times the ULN is required, as defined by the RUCAM [29] and ascertained recently [5]; (3) a pattern of liver injury, as defined by serum ALT and ALP activities being hepatocellular in 42.2%, cholestatic in 40.6%, and mixed in 17.2% of the RUCAM-based iDILI cases [5]; (4) a time to onset of 5–90 days in 53.1%, <5 or <90 days, and ≤30 days from the cessation of the drug in 21.9% of the RUCAM-based iDILI cases [5]; (5) causality, the assessment of which requires the prospective use of the updated RUCAM in future cases [29]; (6) only iDILI cases with a RUCAM score of ≥6, which illustrates that a probable or highly probable causality grading should be used [29]; (7) cohorts that are homogenous and limited to drugs as culprits, excluding all non-drugs, like herbs, that cause herb-induced liver injury (HILI) and not iDILI [29]; (8) incidence, which classically considers the number of new cases of patients with a specific disease in a group of inhabitants within a pre-defined period of time. When incidences of RUCAM-based iDILI were calculated per 100,000 individuals, the results for different countries were as follows [3]: France (13.9) [37], Nigeria (18.2) [38], and Sweden (19.1) [39]. For future studies assessing details on the incidence and prevalence of iDILI, the use of the updated RUCAM has strongly been recommended [1]; (9) prevalence—a parameter reflecting the proportion of a particular population found to be affected by a medical condition at a specific time was assumed as typically low with ≤1%, and often with as low as ≤0.01% in iDILI, but these data remain uncertain as cases were not assessed by the RUCAM [40]; (10) age, which was 55.00 ± 18.35 years in RUCAM-based iDILI cases [5]; (11) gender, where the proportion of males was 73.4% in RUCAM-based iDILI cases [5]; and (12) mortality, which was high in a single RUCAM-based cohort, with 26.4%, but this substantially outnumbered the published results of other studies [41]. The classic RUCAM-based iDILI, with the features described above, clearly characterizes the non-immune liver injury cohort by their lack of serum immune and autoimmune parameters, and this was also verified in 81,856 iDILI cases evaluated by the RUCAM and reported before mid-2020 [42] to be differentiated from autoimmune and immune iDILI cases [43,44].

In this review article, actual new developments within the broad spectrum of immunology concepts in RUCAM-based iDILI were discussed as opposed to the classic non-immune iDILI cases described above. Therefore, the current focus is on autoimmune and immune iDILI, presenting four major types: type 1, which is drug-induced autoimmune hepatitis (DIAIH); type 2, a human leucocyte antigen-based iDILI autoimmune hepatitis; type 3, the anti-cytochrome P450-based idiosyncratic drug-induced autoimmune hepatitis; and type 4, the immune-based iDILI concomitantly with the Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The current literature shows that not all previous iDILI cases have been classified into the four subtypes, a problem easily to be solved in future studies by using appropriate diagnostic algorithms. The aim was to characterize these four different types and formulate the results of the analysis as a practical proposal for clinical use because patients with iDILI and an immune or autoimmune background could benefit from steroid therapy.

2. Literature Search and Search Terms

The literature search strategy involved the PubMed database and Google Science, using the following terms: idiosyncratic drug-induced liver injury; autoimmune drug-induced liver injury; HLA; Stevens–Johnson syndrome; toxic epidermal necrolysis; RUCAM; Roussel Uclaf Causality Assessment Method; immunology; and immune cells. The search was finalized on 25 November 2024.

3. RUCAM

RUCAM was inaugurated with the understanding to help verify causality in iDILI, based on the final recommendations resulting from international consensus meetings with the following participating expert DILI peers [27]: J. P. Benhamou (France), J. Bircher (Germany), G. Danan (France), W. C. Maddrey (USA), J. Neuberger (UK), F. Orlandi (Italy), N. Tygstrup (Denmark), and H. J. Zimmerman (USA) [27,28]. Using cohorts of iDILI cases with established positive reexposure tests, commonly viewed as a gold standard, RUCAM was well validated in the course of the internal validation process [28] and also benefited from external validation [45,46,47,48,49], including interrater reliability [45,46,47]. RUCAM is recognized as a valuable structured clinical method for objective, standardized, and quantitative causality assessment in iDILI cases [27,29], achieved through assigning individual points for the most relevant clinical, biochemical, reexposure, and serologic elements and by excluding obvious non-drug causes. Similarly, conditions relating to concomitant use of other medications or drug use prior to reexposure are well considered as scores in the updated RUCAM. Summarizing the individual points assigned to each key element results in the following final RUCAM causality gradings: score ≤0, excluded causality; 1–2, unlikely; 3–5, possible; 6–8, probable; and ≥9, highly probable, which reflects the likelihood that the hepatic injury is due to a specific medication [27,29]. RUCAM is applied up to now throughout the world in almost 100,000 iDILI cases [42] and outperforms any other method operationally [29,44] and with respect to both method quality and case numbers [44]. It is valued as a user-friendly method, a resources-saving approach that provides results within a short time, circumventing so-called expert rounds with their questionable, untransparent, arbitrary opinions, leaving much room for discussion [29,44]. Convincing evidence is lacking that other methods can replace RUCAM. As an example, both the Naranjo method and the WHO/UMC method were not created specifically for drug-related hepatic injuries but are used in cases of general adverse drug reactions described in various organs or organ systems [44]. Consequently, both methods lacked appropriate validation for iDILI. Missing validation is also a discouraging characteristic feature of the US DILIN method that is confined to its homeland but did not receive worldwide appreciation due to its methodology flaws like lack of validation, missing of individual scores for elements important for iDILI, and providing clouded percentage ranges of probability based on opinions that lack reproducibility, transparency, and objectivity. Assessing the DILI part of the four subtypes should be done by the updated RUCAM alone and not be linked with other unsuitable tools, just to provide clarity.

RUCAM is privileged and applicable without any limitations to assess causality for drugs in all immune and autoimmune iDILI cases. More specifically, there is currently no practical need to incorporate new immunological markers in the updated RUCAM because this would not improve its overall clinical performance. Indeed, such an incorporation is not warranted, considering also the need for a cumbersome new validation of the RUCAM.

4. Definition of Four Major Autoimmune and Immune Drug-Induced Liver Injury Types

The traditional idiosyncratic and intrinsic drug-induced liver injury lacks, by convention, overt immune features [27,29,42]. However, if serum immune parameters are detected in patients with iDILI, their cases may be identified as a new subtype among separate autoimmune or immune liver injury cohorts [50], for which four major types are now known with iDILI diagnoses, verified by RUCAM.

4.1. Type 1: Idiosyncratic Autoimmune DILI

Type 1 corresponds to drug-induced autoimmune hepatitis (DIAIH), as described by many authors in their reports [2,3,4,25,43,44,50,51,52]. DIAIH is best described as the classic RUCAM-based idiosyncratic DILI [27,29] combined with the classic autoimmune hepatitis (AIH) based on criteria of the simplified AIH scoring method [53], requiring, therefore, both tools to verify the DIAIH diagnosis, but this has not been done by all authors. Regarding its autoimmune features, type 1 is caused by a drug and characterized by serum autoimmune parameters preferentially with anti-nuclear antibodies (ANA), the parameter most frequently detected, and increased serum levels of IgG [44]. It has to be differentiated from the classic idiopathic autoimmune hepatitis (AIH) that develops in the absence of any drug treatment or any known culprit [52,53].

4.2. Type 2: HLA-Based Drug-Induced Autoimmune Hepatitis

Type 2 characterizes a group of patients who experienced HLA-based drug-induced autoimmune hepatitis [44,54,55]. Genetically mediated, this type has features of idiosyncrasy that explain its sporadic occurrence. Many drugs are implicated as culprit medications [44,54].

4.3. Type 3: Idiosyncratic Anti-CYP Autoimmune DILI

Type 3 refers to a group of patients who experienced anti-CYP drug-induced autoimmune hepatitis, as evidenced by positive serum anti-CYP antibodies following drug treatment [44]. This type may be seen in connection with drug metabolism via CYPs [44,56,57].

4.4. Type 4: Idiosyncratic Immune Rather than Autoimmune DILI

Type 4 includes cohorts of iDILI patients, in whom typical serum autoimmune parameters like ANA or anti-CYP antibodies are not detected, but instead, innate and adaptive immune mechanisms are discussed based on circumstantial evidence. This applies, for instance, to the iDILI associated with SJS and TEN [58] or to the iDILI due to immune checkpoint inhibitors (ICIs) [43,59].

5. Clinical Characteristics of Four Idiosyncratic Autoimmune and Immune DILI Types

5.1. Type 1: Drug-Induced Autoimmune Hepatitis (DIAIH)

Among the four types of autoimmune and immune iDILI, type 1 is the most abundant one. Clinical features of type 1 are based on iDILI combined with AIH criteria in connection with the seropositivity of serum autoimmune parameters applied in combination or as a single constellation: anti-liver kidney microsomes antibodies (ALKMA), anti-mitochondrial antibodies (AMA), anti-nuclear antibodies (ANA), anti-smooth muscle antibodies (ASMA), and anti-soluble liver antigen antibodies (ASLA) (

Table 1. Type 1: Autoimmune features in selected DIAIH cases.

Serum Autoimmune Parameter	RUCAM Used	Clinical Details of DIAIH Cases and Comments	Drugs and Drug Groups	References
AMA ANA ASMA	YES	At the start of the hospital stay, jaundice was a common initial finding. Portal inflammation and infiltration with lymphoplasmacytic cells were evident in the liver histology specimen.	Antimicrobials	Licata, 2014 [60]
ANA ASMA	YES	This excellent study provided results of serum autoantibodies in cohorts with careful exclusion of confounding cases that had nothing to do with DIAIH but had to be diagnosed as common, non-immune DILI caused, for instance, by antibiotics.	Adalimumab Certolizumab Infliximab	Shelton, 2015 [61]
ANA	YES	In Japan, DIAIH was rarely attributed to chemical drugs as opposed predominantly to herbal TCMs, making the term DIAIH incorrect because herbal products are in no way chemical drugs.	Clarithromycin NSAIDs	Hisamochu, 2016 [62]
ANA ASMA	YES	Initial therapy started with prednisone given as a sole drug, while later, sometimes, azathioprine was provided alone or combined with prednisone. Under these treatment regimes, there was no lethality due to liver disease and no necessity for liver transplantation.	Nitrofurantoin NSAIDs	Yeong, 2016 [63]
ANA	YES	The female gender prevailed with 93% of cases. Improvement was seen in 40% after drug cessation.	Imatinib Infliximab Nitrofurantoin	Björnsson, 2017 [64]
ANA ASMA	NO	With 91%, females were in the majority. Patients were symptomatic with icterus, pruritus, exanthema, and increased body temperature. Eosinophilia was a typical laboratory abnormality in the affected patients. The liver pattern was mostly of the hepatocellular injury type.	Hydralazine Methyldopa Minocycline Nitrofurantoin	de Boer 2017 [65]
ANA	YES	Menotrophin was given as a hormonal fertility medication.	Menotrophin	Alqrinawi, 2019 [66]
AMA ANA ASMA	YES	Steroids and immunomodulators provided an efficient therapy. This was an excellent clinical study on the DIAIH study from Latin America.	Nitrofurantoin NSAIDs	Martinez-Casas, 2018 [67]
ALKMA AMA ANA ASMA	YES	The decline of serum ALT activities one week following starting the treatment with steroid therapy was more evident in the DIAIH cases than in patients with genuine AIH, seen as another diagnostic parameter that could help differentiate DIAIH from AIH.	Atorvastatin Dabigatran Diclofenac Ezetimibe Metamizole Rivaroxaban	Weber, 2019 [68]
SMA	YES	In line with previous reports, the treatment with immunosuppressive agents was efficient.	Infliximab	Valgareisson 2019 [69]
ANA ASMA	YES	Normal IgG levels were reported as well as serum ANA and ASMA positivity in 32% of patients.	ICIs	Riveiro-Barciela, 2020 [70]
AMA ANA ASMA	YES	RUCAM-based liver injury pattern was hepatocellular in 92% of cases. Females predominated with 62%.	Antibacterials Statins	Stephens, 2021 [71]
ALKMA ANA ASLA ASMA	YES	This immuno-allergic phenotype was based on a rash, facial edema, lymphadenopathy, fever, and eosinophilia in a range of 11–27% and of 70–75% in cases with jaundice. An elevated serum IgG level was a common finding. Specimens of the liver showed histological signs of portal inflammation, plasma cell infiltrates, rosettes, and focal necrosis.	Atorvastatin Diclofenac Etanercept Infliximab Methyldopa Minocycline Nitrofurantoin Rosuvastatin	Tan, 2022 [72]
ANA ASMA	YES	In 18% of patients, seronegativity was observed, and cirrhosis in 25% of cases.	Infliximab Minocycline Nitrofurantoin	Chung, 2023 [73]
ANA SMA	YES	At baseline, all patients had jaundice and elevated serum ALT and AST activities.	Ibuprofen Trazodone	Hassoun, 2023 [74]
AMA ANA	YES	The cohort was inhomogeneous due to mixing with non-drugs such as herbs and undetermined classification.	Antibiotics Hormones	Kakisaka 2023 [75]
ANA	YES	A positive test was reported after unintentional reexposure to the same drug.	Atorvastatin	Tse, 2023 [76]
ANA	YES	After drug cessation, a decline in ALT and AST was observed, with an increase on day 10.	Ornidazole	Memiş, 2024 [77]

Data were actualized after being taken from a previous open-access publication [44]. A few data are provisionally due to a lack of appropriate diagnostic tools of RUCAM and/or the simplified AIH score. Abbreviations: ALKMA, anti-liver kidney microsomes antibodies; AMA, anti-mitochondrial antibodies; ANA, anti-nuclear antibodies; ASMA, anti-smooth muscle antibodies; ASLA, anti-soluble liver antigen antibodies; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DIAIH, drug-induced autoimmune hepatitis; NSAIDs, non-steroidal anti-inflammatory drugs; RUCAM, Roussel Uclaf Causality Assessment Method; TCM, traditional Chinese medicines.

) [60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77].

The following clinical characteristics of RUCAM-based DIAIH are well described in the listed reports (Table 1) [60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78]: (1) age was in the range from 15 to 86 years [67,71,73,75]; (2) gender (M:F) was 11:18 [75], with confirmed female predominance (92%) [67], (76%) [73], and (58%) [60]; (3) symptoms included jaundice and pruritus (71%), fatigue and malaise (25%), abdominal pain (11%), and arthralgia (4%) [73]; (4) medium values for total bilirubin were 13.7 mg/dL, ALT 487 U/L, AST 717 U/L, and ALP 193 U/L [67]; (5) serum antibody ANA (71%), ASMA (29%), SLA/LP (0%), LKM (0%), and seronegative (18%) [73]; (6) RUCAM-based liver injury pattern was hepatocellular (92%), cholestatic (4%), and mixed (4%) [70]; (7) liver histology with an Ishak score included periportal interface hepatitis (2.4), confluent necrosis (2.5), focal lytic necrosis (2.3), portal inflammation (2.1), as well as plasma cell aggregates (61%) and eosinophile aggregates (18%) [73]. (8) For most patients (76%), the RUCAM score was 6–8, in line with a probable causality grading for DIAIH by drugs excluding herbs, while 23% scored 3–5, suggesting a possible grading [73], and with a RUCAM score of 7.3, it was viewed as a probable or highly probable causality grading [74]; (9) the drug group most commonly implicated in DIAIH consisted of antibiotics with nitrofurantoin as the drug on top, followed by the tetracyclines minocycline and lymecycline [73]; (10) treatment of patients with DIAIH was accomplished by steroids (79%), azathioprine (43%), mycophenolate mofetil (MMF) (4%), and tacrolimus (4%) [73]; (11) biochemical remission was at 1 month (32%), 3 months (46%), 6 months (50%), and 12 months (57%, death occurred in 7% and liver transplantation was performed in 4% [70] or 14% [73]; and (12) among 362 patients initially diagnosed as AIH, 12/362 cases (6.3%) were finally diagnosed as DIAIH, with 178/362 cases (93.7%) finally being diagnosed as genuine AIH [67].

5.2. Type 2: Idiosyncratic Autoimmune HLA-Based DILI

An association of variable HLA alleles was often observed with RUCAM-based iDILI attributed to drugs, although causality gradings were not always specified (

Table 2. HLA association with selected drugs triggering iDILI was assessed for causality by RUCAM.

Drug	HLA Allele	RUCAM-Based iDILI Cases (n)	References
Amoxicillin	A*01:01 C*03:02 B*58:01 DPB1*01:01	15	Nicoletti, 2019 [78]
Amoxicillin-clavulanate	A*02:01 DQB1*06:02	201	Lucena, 2011 [79]
Amoxicillin-clavulanate	A*30:02 B*18:01 DRB1*15:01 DQB1*06:02	75	Stephens, 2013 [80]
Amoxicillin-clavulanate	DRB1*15:01	14	O’Donohue, 2000 [81]
Antituberculotics + antiretrovirals	B*57:02 B*57:03	46	Petros, 2017 [82]
Carbamazepine	A*31:01	29	Nicoletti, 2019 [83]
Dapsone	B*13:01	4	Devarbhavi, 2022 [84]
Enalapril	A*33:01	4	Nicoletti, 2017 [85]
Erythromycin	A*33:01	10	Nicoletti, 2017 [85]
Fenofibrate	A*33:01	7	Nicoletti, 2017 [85]
Flucloxacillin	B*5701	51	Daly, 2009 [86]
Flucloxacillin	B*57:01	6	Monshi, 2013 [87]
Flucloxacillin	B*57:01 B*57:03	197	Nicoletti, 2019 [78]
Flucloxacillin	B*57:01	1	Teixera, 2020 [88]
Flupirtine	DRB1*16:01-DQB*05:02	11	Nicoletti, 2016 [89]
Infliximab	B*39:01	18	Bruno, 2020 [90]
Isoxazolyl penicillins	C*07:04 DQB1*06:09	6	Nicoletti, 2019 [78]
Methimazole	C*03:02	40	Li, 2019 [91]
Methyldopa	A*33:01	4	Nicoletti, 2017 [85]
Minocycline	B*35:02	25	Urban, 2017 [92]
Nitrofurantoin	A*33:01 DQB1*02:02 A*30:02 DQA1*02:01 DRB1*07:01 DPB1*16:01 C*06:02	26	Daly, 2023 [93]
Sertaline	A*33:01	5	Nicoletti, 2017 [85]
Terbinafine	A*33:01	14	Nicoletti, 2017 [85]
Ticlopidine	A*33:01	5	Nicoletti, 2017 [85]
Trimethoprim-sulfamethoxazole	B*14:01 B*14:02 B*35:01	86	Li, 2021 [94]

Data were reproduced from a previous open-access publication [55]. The diagnosis was affirmed by the authors of the reports using RUCAM. Abbreviations: iDILI, idiosyncratic drug-induced liver injury; HLA, human leucocyte antigen; RUCAM, Roussel Uclaf Causality assessment method.

) [78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94].

Clinical features of RUCAM-based iDILI that were associated with several forms of HLA were variably described in the listed reports but cannot firmly be summarized due to the inhomogeneity of the studied cohorts (Table 2) [78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94]. What is especially valuable for a quick overview are the reports of a single case or publications confined to a single causative drug with a large case number and, thereby, presenting a homogeneous study cohort rather than reports that included many different drugs incriminated in liver injury [78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94]. It is obvious that flucloxacillin is the drug most studied in the evaluated cohort (Table 2). This warrants a detailed analysis of this special iDILI cohort (

Table 3. Flucloxacillin, as a suspected drug of iDILI assessed for HLA association and causality by RUCAM.

DRUG	HLA Allele	RUCAM-Based iDILI Cases (n)	RUCAM-Based Causality	References
Flucloxacillin	B*57:01	51	4/51 patients had a possible causality, 18 a probable one, and 29 a highly probable causality one	Daly, 2009 [86]
Flucloxacillin	B*57:01	6	2/6 patients had a possible causality, 2 a probable, and 2 a highly probable causality	Monshi, 2013 [87]
Flucloxacillin	B*57:01 B*57:03	197	22/197 of the patients had a possible causality, 90 a probable, and 85 a highly probable causality grading	Nicoletti, 2019 [83]
Flucloxacillin	B*57:01	1	RUCAM score of 8, equivalent to a probable causality grading	Teixera, 2020 [88]

The table was retrieved from a recent paper published in an open-access journal [95]. Abbreviations: iDILI, idiosyncratic drug-induced liver injury; HLA, human leucocyte antigen; RUCAM, Roussel Uclaf Causality Assessment Method.

) [78,86,87,88].

Flucloxacillin leads to a rare RUCAM-based iDILI associated with HLA B*57:01 and provides the following typical characteristics [95,96]: (1) The study cohorts, as listed (Table 3), are homogenous, as all patients used in conjunction with flucloxacillin an identical drug and were followed for causality assessment using RUCAM [78,86,87,88], which asks among others for thresholds of ALT and ALP [27,28,29] and thereby provided firm case details. (2) RUCAM-based scoring was highly probable with a score of ≥8 in 72/163 cases (44%), probable with a score of 6–8 in 73/163 cases (45%), and possible with a score of 3–5 in 18/163 cases (11%) [78]. (3) Clinical features included maculopapular exanthem, arthralgia, and eosinophilia [87]. (4) Gender (F/M) presented with 133/64, demonstrating a female predominance [78]. (5) The mean age was 62 ± 13 (SD) years [78]. (6) The mean time to onset was 24 ± 18 days [78]. (7) The total days on a drug were 10 ± 6 days [78]. (8) The pattern of liver injury was hepatocellular in 20%, mixed in 43%, and cholestatic in 38% of cases [78]. (9) ALT was 646 U/L (normal 4–43) and AST 349 U/L (normal 4–43) [88]. (10) Invasive liver biopsy to acquire a liver specimen for histology is not part of the RUCAM algorithm but liver histology was provided in one study [88] out of four reports [78,86,87,88], and as evaluated during hospitalization, there was a slight multifocal, microvesicular steatosis, sinusoidal dilatation, marked congestion, hemorrhage, a multifocal collapse of hepatocytes, and inflammatory infiltrate of the mononuclear type in one single large study [88]. (11) In only 163/197 patients (83%), HLA B*57 positivity was firmly established vs. 34/197 cases (17%) with confirmed HLA B*57 negativity [78]; and (12) the risk of iDILI by flucloxacillin was shown as increased with ages >70 years, female gender, and HLA B*57:01 [78].

Another study on RUCAM-based iDILI by flucloxacillin, but without evaluating the HLA B*57:01 status, provided the following additional features of iDILI by flucloxacillin [96]: (1) An increased risk for iDILI by flucloxacillin use was described in patients aged >70 years, with reexposure to flucloxacillin, and co-medication with other drugs; (2) considering a prevalence of 8.5 iDILI cases by flucloxacillin per 100,000 individuals, approaches of predictive genetic testing for a reaction would be unrealistic because as many as 13,513 people would require screening to avoid one case; and (3) other parameters such as the type of gender and age at onset, which specifically affect the liver injury pattern, as well as RUCAM-based causality gradings [96], were similar to the RUCAM-based HLA B*57:01 cohort study [78].

5.3. Type 3: Idiosyncratic Autoimmune Anti-CYP-Based DILI

Serum anti-CYP antibodies were detected in patients with iDILI attributable to a few drugs [44,57,97,98]. This is expected because 58.3% of drugs implicated in iDILI with verified diagnosis by RUCAM are known substrates of CYP isoforms [56,99]. Among these drugs causing serum anti-CYP antibodies in association with iDILI were halothane (causing anti-CYP 2E1 antibodies) [57,98,99,100,101,102,103], isoflurane (anti-CYP 2E1) [103], isoniazid (anti-CYP 2C9) [99], as well as dihydralazine (anti-CYP 1A2), tienilic acid (anti-CYP 2C9), and antiepileptics (anti-CYP 3A) [98]. For all these drugs, iDILI, as a firm diagnosis, was assumed but not verified using RUCAM [57,97,98,99,100,101,102,103].

In opposition, better-validated results were obtained from case studies of iDILI that RUCAM evaluated to verify the diagnosis [47,104]. Accordingly, serum anti-CYP 2E1 antibodies were detected after anesthesia with the volatile anesthetic sevoflurane in four patients with iDILI and a verified diagnosis by using the RUCAM, resulting in a highly probable causality [104]. These data were similar to those of another report that focused on sevoflurane and desflurane, again modern volatile anesthetics, whereby sevoflurane was used in most cases as a single anesthetic and rarely combined with desflurane [47]. For five patients with iDILI, the RUCAM score was ≥6, and serum anti-CYP 2E1 antibodies were found in three patients with RUCAM scores of 12, 7, and 6, whereas antibodies remained undetected in two DILI patients with a RUCAM score of 12 and 7 [47]. Detailed information on the anesthetic cohorts is available (

Table 4. Serum antibodies represent immune features of iDILI in patients with RUCAM-based iDILI following the use of volatile anesthetics.

Autoimmune Parameter	Details of RUCAM-Based iDILI Cases	Drug	References
Serum anti-CYP 2E1	Patients experiencing iDILI attributed to the volatile anesthetic sevoflurane use exhibited positive serum titers of anti-CYP 2E1 in cases with highly probable causalities and well-described clinical features. Among these were flu-like symptoms, fever, jaundice, vomiting, right upper quadrant abdominal pain, reduced appetite, rash, and myalgias, all described after the second anesthesia. In the liver histology, centrilobular necrosis with hemorrhage and rosetting of liver cells prevailed.	Sevoflurane	Nicoll, 2012 [104]
Serum anti-CYP 2E1	Clinical case details of the RUCAM-based iDILI were published as a result of volatile anesthetic use in combination. Considering alternative causes such as hypotension and DILI by antibiotics or paracetamol was highly appreciated for reasons of clarity and transparency.	Sevoflurane + Desflurane	Bishop, 2019 [47]
Serum anti-TFA	Aditional valuable insight into the mechanistic steps involved in iDILI with a verified diagnosis by RUCAM was evident in patients with detected toxic intermediates in the form of trifluoroacetate (TFA) halide, produced during the drug metabolism via CYP 2E1. This provided clues of irreversible protein adduct formation and free radical generation, resulting in detectable anti-TFA antibodies as a sign of an immune process.	Sevoflurane + Desflurane	Nicoll, 2012 [104] Bishop, 2019 [47]

Data of this table were retrieved from a previous open access publication [44]. Abbreviations: CYP, Cytochrome P450; DILI, drug-induced liver injury; RUCAM, Roussel Uclaf Causality Assessment Method.

) [47,104].

Most informative was the note that sevoflurane caused not only acute but also chronic liver injury that evolved into cirrhosis with portal hypertension in one reported patient [104]. The association of iDILI due to sevoflurane and desflurane with the occurrence of anti-CYP 2E1 as well as anti-trifluoroacetate (TFA) antibodies support the concept that autoimmune processes participate in these iDILI cases (Table 3) [47,104].

5.4. Type 4: Idiosyncratic Immune DILI Combined with SJS and TEN

Liver disease in patients with a dermatological disorder is a rare condition [105]. More specifically, drugs causing liver injury associated with the skin entities of SJS and TEN represent a substantial clinical challenge [106,107,108,109,110,111,112,113,114] because it requires a firm diagnosis of both the liver injury by RUCAM [29] and the cutaneous disorders SJS/TEN by the Algorithm for Drug Causality for Epidermal Necrolysis (ALDEN) [115]. Similar to the updated RUCAM [29], ALDEN is a quantitative tool to assess causality for culprit drugs in suspected SJS/TEN [115].

ALDEN is appreciated as a validated, specific, reproducible, structured, and quantitative method using key items as individually scored elements, whereby the sum of these scores reaches from −12 to 10 and provides a final causality grading of very unlikely, unlikely, possible, probable, and very probable [114]. The six elements specifically include the following: (1) a delay from the initial drug intake to an onset of a reaction (index day); (2) the drug present in the body on the index day; (3) prechallenge/rechallenge; (4) dechallenge; (5) type of drug (notoriety); and finally (6), other cause [115]. As a result, to describe the robust clinical characteristics of this special cohort, patients are preferred with valid diagnoses verified by RUCAM and ALDEN. DILI experts commonly applied RUCAM but occasionally forgot to use ALDEN, which inevitably confounded the results. Selected cases are presented as a listing delineating suspected drugs in alphabetical order and whether an appropriate causality algorithm was applied (

Table 5. Selected drugs implicated in RUCAM-based iDILI and ALDEN-based SJS/TEN.

Drugs/Drug Classes	Cases (n)	Causality Algorithm Use	Outcome	References
Allopurinol	2	RUCAM + ALDEN +	All survived	Devarbhavi, 2016 [58]
Allopurinol	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Amoxicillin	N.A.	RUCAM + ALDEN −	Cases of acute liver failure, outcome unknown	Ortega-Alonso, 2017 [117]
Ampicillin	N.A.	RUCAM + ALDEN −	Cases of acute liver failure, outcome unknown	Ortega-Alonso, 2017 [117]
Aspirin	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Carbamazepine	2	RUCAM + ALDEN +	All died	Devarbhavi, 2016 [58]
Carbamazepine	8	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Carbamazepine	36	RUCAM + ALDEN +	4/36 died	Devarbhavi, 2023 [118]
Ceftazidime	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Ceftriaxone	1	RUCAM + ALDEN +	Lethal outcome	Devarbhavi, 2016 [58]
Ceftriaxone	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Celecoxib	N.A.	RUCAM + ALDEN −	No cases of acute liver failure	Ortega-Alonso, 2017 [117]
Clobazam	2	RUCAM + ALDEN +	1/3 died	Devarbhavi, 2023 [118]
Clonazepam	2	RUCAM + ALDEN +	All survived	Devarbhavi, 2023 [118]
Cotrimoxazole	3	RUCAM + ALDEN +	All survived	Devarbhavi, 2016 [58]
Dapsone	5	RUCAM + ALDEN +	3/5 died	Devarbhavi, 2016 [58]
Fluoxetine	1	RUCAM + ALDEN +	Survived	Agrawal, 2019 [119]
Gabapentin	1	RUCAM + ALDEN +	Survived	Devarbhavi, 2023 [118]
Ibuprofen	N.A.	RUCAM + ALDEN −	Cases of acute liver failure, outcome unknown	Ortega-Alonso, 2017 [117]
Lamotrigine	1	RUCAM + ALDEN +	Lethal outcome	Devarbhavi, 2016 [58]
Lamotrigine	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Lamotrigine	3	RUCAM + ALDEN +	1/3 died	Devarbhavi, 2023 [118]
Leflunomide	3	RUCAM + ALDEN +	All died	Devarbhavi, 2016 [58]
Leflunomide	2	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Levitericetam	1	RUCAM + ALDEN +	Lethal outcome	Devarbhavi, 2016 [58]
Levitericetam	3	RUCAM + ALDEN +	All survived	Devarbhavi, 2023 [118]
Levofloxacin	1	RUCAM + ALDEN +	Survived	Devarbhavi, 2016 [58]
Nevirapine	6	RUCAM + ALDEN +	All survived	Devarbhavi, 2016 [58]
Omeprazole	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Oxacarbazepine	1	RUCAM + ALDEN +	Survived	Devarbhavi, 2016 [58]
Oxacarbazepine	2	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Oxacarbazepine	2	RUCAM + ALDEN +	All survived	Devarbhavi, 2023 [118]
Paracetamol	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Penicillin	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Phenobarbitone	2	RUCAM + ALDEN +	1/2 died	Devarbhavi, 2016 [58]
Phenobarbitone	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Phenobarbitone	8	RUCAM + ALDEN +	2/8 died	Devarbhavi, 2023 [118]
Phenylbutazone	2	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Phenytoin	2	RUCAM + ALDEN +	1/2 died	Devarbhavi, 2016 [58]
Phenytoin	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Phenytoin	71	RUCAM + ALDEN +	4/71 died	Devarbhavi 2023 [118]
Tegafur	1	RUCAM + ALDEN +	N.A.	Zhang, 2020 [116]
Terbinafine	N.A.	RUCAM + ALDEN −	Cases of acute liver failure, outcome unknown	Ortega-Alonso, 2017 [117]
Topiramate	1	RUCAM + ALDEN +	Survived	Devarbhavi, 2023 [118]
Valproate	14	RUCAM + ALDEN +	1/14 died	Devarbhavi, 2023 [118]
Warfarin	1	RUCAM + ALDEN +	Survived	Xiong 2021 [120]
Zonisamide	1	RUCAM + ALDEN +	Survived	Devarbhavi, 2023 [118]

Data were actualized and in detail discussed in and taken from a recent open-access publication [121]. Compilation of selected drugs implicated in causing iDILI in patients with SJS/TEN. In all listed drugs causality of DILI for the culprit drug was verified using the scoring RUCAM algorithm. For most of the drugs, the diagnosis of SJS/TEN was verified by the scoring ALDEN algorithm. Listing was confined to conventional drugs, excluding herbal medicines like herbal TCM because these non-drugs cause herb-induced liver injury (HILI) rather than DILI. The + sign indicated that the specific diagnostic algorithm was used to verify the diagnosis as opposed to the − sign, which signified that the specific algorithm was not applied. Abbreviations: ALDEN, Algorithm for Drug Causality for Epidermal Necrolysis; N.A., Not available; RUCAM, Roussel Uclaf Causality Assessment Method; TCM, traditional Chinese medicines.

) [58,116,117,118,119,120].

For many SJS/TEN patients, iDILI was substantiated by liver tests (LTs) above the normal range, as evidenced by increased serum activities of ALT, AST, or ALP, in addition to RUCAM [58,116,117,118,119,120,121]. Differential dermatology diseases have to be excluded in all three types: SJS, SJS/TEN overlap, and TEN, known as multifaceted disease entities that need a diagnostic verification by ALDEN, associated with a careful exclusion of differential diagnoses and primary focus on alternative dermatology disorders. The exclusion process must be in the hands of an experienced dermatologist to define the correct diagnosis and treatment option. Selected alternative dermatology diseases are provided in the literature [113,114,115,119,120,121,122,123,124,125,126,127,128]. They include [121]: (1) Acute generalized exanthematous pustulosis (AGEP) [113,122]; (2) acute or subacute cutaneous lupus with epidermal necrosis (Rowell syndrome) [113,123]; (3) bullous pemphigoid [113,124]; (4) drug rashes [114]; (5) erythema multiforme major (EMM) [113,114]; (6) exfoliative erythroderma [114]; (7) generalized bullous fixed drug eruption (GBFDE), also termed generalized fixed drug eruption [113,115]; (8) linear IgA bullous dermatosis [113,125]; (9) paraneoplastic pemphigus [113,114]; (10) pemphigus vulgaris (PV) [113,126]; (11) phototoxic eruptions [113,127]; and finally (12) staphylococcal scalded skin syndrome [113,128].

Irritating was the fact that if the SJS/TEN studies used partial cohorts assessed by ALDEN alone and not combined with RUCAM, or if they used non-RUCAM approaches like the Naranjo method, WHO-UMC method, or Liverpool method [121], all tools were not specific for liver toxicity [29], or they applied even the DILI network method [121] that is based on arbitrary subjective opinion in the sense of global introspection. This method never received a correct internal or external validation [55]. As a cautionary note, SJS/TEN are caused by drugs in 85–89% of cases [115,121], but non-drug culprits are also known and have to be considered separately [112,121,129,130,131,132,133,134,135,136]. For this cohort, specific diagnostic and therapeutic approaches may be indicated differently from those of the common iDILI.

6. Perspectives

The novel categorization of immune and autoimmune iDILI subtypes invites other physicians and scientists to add more information for these four subtypes. It will be of interest to see whether blood parameters such as cytokines, chemokines, or specific immune cells such as T cells, B cells, or dendritic cells can contribute to further differentiation of the four subtypes. The results from these studies will expand our knowledge of the immune mechanisms that lead to liver injury, as well as perhaps the role of the innate and adaptive immune systems.

As it presently stands, the therapeutic options are similar for all immune and autoimmune iDILI subtypes, consisting primarily of the cessation of the suspected drug already at the time when iDILI is first considered. The clinical course becomes serious if cessation does not lead to the desired decline of LTs and when clinical signs such as jaundice remain. This calls for a re-evaluation of whether the diagnosis of iDILI is still correct and whether the liver injury must be attributed to a non-drug cause as an alternative condition that may afford another diagnostic and therapeutic approach. Clinical deterioration, despite drug cassation, requires the decision for a steroid therapy of the immune or autoimmune iDILI in all cases belonging to one of the four subtypes. In comparison, a case-by-case decision regarding a steroid treatment is recommended for patients who are not clearly attributable to any of the four subtypes. In a worst-case scenario, liver transplantation must be considered to circumvent lethal outcomes due to untreatable acute liver failure.

7. Conclusions

The new categorization of four major autoimmune and autoimmune idiosyncratic DILI subtypes with disruptive properties of drugs paves the scientific avenue for the universal characterization of these different disorders and fosters comparability studies from countries around the world. The advantage of this new classification is the uniform use of RUCAM as the major diagnostic algorithm to ascertain that the iDILI in question is really a liver injury caused by drugs and not due to non-drug causatives—a general issue often recognized in published DILI reports. In addition, and specifically for the DIAIH subtype, more clarification is needed in the future that a suspected DIAIH case needs causality assessment not only by RUCAM but also by the simplified AIH score to ascertain the AIH part as the correct diagnostic part of the disorder. This will reduce publications of DIAIH cases that were classified erroneously as DIAIH despite lacking the appropriate causality assessment approaches. Finally, the new classification will help in searches for the mechanistic steps involved in the immunology process, as evidenced by elevations of serum cytokines and chemokines, which reflect the actions of hepatic immune processes triggered by the suspect drugs or their metabolites, converting the silent liver to a transparent organ. As parameters that can easily be analyzed in the blood of iDILI patients, these cytokine and chemokine mediators can be used by physicians and scientists for additional research, which can amplify our understanding of the mechanistic steps in homogeneous cohorts of patients with these iDILI subtypes, indicating that dysregulation and cross-talk of mediators may play a role. It is expected that future case reports of iDILI will use this new classification to refine the quality of their cases. Depending on the suspected subtype, the use of validated causality assessment methods like the updated RUCAM, the simplified AIH scoring method, and the ALDEN method is mandatory.