Intestinal microbiota disruption limits the isoniazid mediated clearance of Mycobacterium tuberculosis in mice.

Tuberculosis (TB) continues to remain a global threat due to the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains and toxicity associated with TB drugs. Intestinal microbiota has been reported to affect the host response to immunotherapy and drugs. However, how it affects the potency of first-line TB drug isoniazid (INH) is largely unknown. Here, we examined the impact of gut microbial dysbiosis on INH efficiency to kill Mtb. In this study, we employed in vivo mouse model, pretreated with broad-spectrum antibiotics (Abx) cocktail to disrupt their intestinal microbial population prior to Mtb infection and subsequent INH therapy. We demonstrated that microbiota disruption results in the impairment of INH-mediated Mtb clearance, and aggravated TB-associated tissue pathology. Further, it suppressed the innate immunity and reduced CD4 T-cell response against Mtb. Interestingly, a distinct shift of gut microbial profile was noted with abundance of Enterococcus and reduction of Lactobacillus and Bifidobacterium population. Our results show that the intestinal microbiota is crucial determinant in efficacy of INH to kill Mtb and impacts the host immune response against infection. This work provides an intriguing insight into the potential links between host gut microbiota and potency of INH.

Drug-specific CD4+ T-cell immune responses are responsible for antituberculosis drug-induced maculopapular exanthema and drug reaction with eosinophilia and systemic symptoms syndrome.

BACKGROUND: A multidrug regimen including isoniazid, rifampicin, pyrazinamide and ethambutol is commonly used as first-line treatment for tuberculosis. However, this regimen can occasionally result in severe adverse drug reactions, such as drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome and drug-induced liver injury. The culprit drug and mechanistic basis for the hypersensitive reaction are unknown. OBJECTIVES: To investigate drug-specific T-cell responses in patients with antituberculosis drug (ATD)-induced cutaneous hypersensitivity and its underlying mechanism. METHODS: We enrolled eight patients with ATD-induced maculopapular exanthema and DRESS and performed a lymphocyte transformation test. Subsequently, drug-specific T-cell clones were generated from four of the patients who showed proliferation in response to ATDs. We measured the drug-specific proliferative responses and counted the drug-specific interferon (IFN)-γ/granzyme B-producing cells after drug stimulation. Antihuman leukocyte antigen (HLA) class I and class II blocking antibodies were used to analyse human leukocyte antigen-restricted T-cell responses. RESULTS: Positive proliferative responses to ATDs were mostly found in patients with cutaneous hypersensitivity. Furthermore, we isolated isoniazid/rifampicin-specific T cells from patients, which consisted primarily of CD4+ T cells. Drug-specific CD4+ T cells proliferated and secreted IFN-γ/granzyme B when stimulated with isoniazid or rifampicin, respectively. Isoniazid-responsive T-cell clones did not proliferate in the presence of rifampicin and vice versa. Drug-specific T-cell responses were blocked in the presence of anti-HLA class II antibodies. CONCLUSIONS: This study identifies the presence of isoniazid/rifampicin-specific T cells in patients with ATD-induced maculopapular exanthema and DRESS. Furthermore, it highlights the important role of drug-specific T-cell immune responses in the pathogenesis of these reactions.

Detection of anti-isoniazid and anti-cytochrome P450 antibodies in patients with isoniazid-induced liver failure.

UNLABELLED: Isoniazid (INH)-induced hepatotoxicity remains one of the most common causes of drug-induced idiosyncratic liver injury and liver failure. This form of liver injury is not believed to be immune-mediated because it is not usually associated with fever or rash, does not recur more rapidly on rechallenge, and previous studies have failed to identify anti-INH antibodies (Abs). In this study, we found Abs present in sera of 15 of 19 cases of INH-induced liver failure. Anti-INH Abs were present in 8 sera; 11 had anti-cytochrome P450 (CYP)2E1 Abs, 14 had Abs against CYP2E1 modified by INH, 14 had anti-CYP3A4 antibodies, and 10 had anti-CYP2C9 Abs. INH was found to form covalent adducts with CYP2E1, CYP3A4, and CYP2C9. None of these Abs were detected in sera from INH-treated controls without significant liver injury. The presence of a range of antidrug and autoAbs has been observed in other drug-induced liver injury that is presumed to be immune mediated. CONCLUSION: These data provide strong evidence that INH induces an immune response that causes INH-induced liver injury.

IgG3 is the dominant subtype of anti-isoniazid antibodies in patients with isoniazid-induced liver failure.

Isoniazid (INH) therapy is associated with a significant incidence of idiosyncratic liver failure. We recently reported eight cases of INH-induced liver failure in which patients had antidrug and anticytochrome P450 antibodies. However, it was unclear what role these antibodies play in the mechanism of INH-induced liver injury. Here, we report that the dominant isotype of anti-INH antibodies was IgG, with IgG3 being the dominant subtype. IgG3 antibodies are associated with a Th1-type immune response and fix complement. IgG3 antibodies have been associated with other forms of liver injury and may play a pathogenic role in INH-induced liver injury.

A case of isoniazid-induced liver injury diagnosed by use of the DLST, and successful reintroduction of isoniazid for pleural tuberculosis.

A 54-year-old woman was admitted for pleural tuberculosis diagnosed by right chest pain and cough. She received combination antituberculosis therapy consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide. However, liver damage was observed 15 days after initiation of therapy (aspartate aminotransferase (AST) 248 IU/l, alanine transaminase (ALT), 132 IU/l). The patient was given glycyrrhizinate intravenously, but liver damage gradually increased (AST 628 IU/l, ALT 467 IU/l) and all tuberculosis drugs were ceased. We diagnosed drug-induced liver damage due to isoniazid according to results of the drug lymphocyte stimulation test. We successfully reintroduced rifampicin and streptomycin, and carried out desensitization therapy for isoniazid without liver injury recurrence. Reintroduction of a drug suspected to cause drug-induced liver injury is generally not recommended; however, our experience suggests that isoniazid, a first-line antituberculosis drug, may be reintroduced after desensitization.

Efficacy and tolerability of desensitization in the treatment of delayed drug hypersensitivities to anti-tuberculosis medications.

BACKGROUND: Delayed drug hypersensitivity to first-line anti-tuberculosis medication is a major challenge in tuberculosis treatment. OBJECTIVE: This study was performed to investigate the efficacy/tolerability of desensitization therapy in treatment of first-line anti-tuberculosis medication hypersensitivity and the usefulness of immunologic evaluation therein. METHODS: This study was conducted as a prospective, observational cohort study. Subjects who experienced hypersensitivity reactions, including maculopapular exanthema (MPE) and drug reaction with eosinophilia and systemic symptoms (DRESS), to first-line anti-tuberculosis medications (isoniazid [INH], ethambutol [EMB], rifampin [RFP], and pyrazinamide [PZA]) were enrolled. Patch, intradermal, lymphocyte transformation, and oral provocation tests were performed to determine culprit drugs, which were desensitized with rapid and graded challenge protocols. Breakthrough reactions (BTRs) during or after desensitization were assessed. RESULTS: In total, 31 desensitization treatments (INH, 8; EMB, 8; RFP, 11; PZA, 4) to 12 patients (8 with MPE and 4 with DRESS) were performed. The overall success rate of desensitization was 80.7%. All the study subjects except one completed the full course of anti-tuberculosis treatment. The overall BTR free rate was 64.5%. Sixteen (80%) treatments for MPE and four (36.4%) for DRESS were BTR free (P = 0.023). Drugs that were positive on any two of three immunologic studies showed significantly high BTR rates (P = 0.014), although this was not correlated with desensitization failure rate. CONCLUSION: Rapid desensitization therapy to multiple anti-tuberculosis medications for delayed drug hypersensitivity was safe and successful. Combination of multiple immunologic evaluations may predict BTR although it needs validation in larger studies.

From the Cover: Characterization of Isoniazid-Specific T-Cell Clones in Patients with anti-Tuberculosis Drug-Related Liver and Skin Injury.

Isoniazid, rifampicin, pyrazinamide, and ethambutol are commonly used for the treatment of tuberculosis. Drug exposure is occasionally associated with liver and/or skin injury. The aim of this study was to determine whether drug-specific T-cells are detectable in patients with adverse reactions and if so characterize the nature of the T-cell response. Peripheral blood mononuclear cells (PBMC) from 6 patients with anti-tuberculosis drug-related adverse reactions (4 liver, 2 skin) were used to detect drug-responsive T-lymphocytes. Positive lymphocyte transformation test and/or ELIspot results were observed with all 6 patients. Over 3400 T-cell clones were generated from isoniazid, rifampicin, pyrazinamide, or ethambutol-treated PBMC. CD4+ clones from all 3 patients were activated to proliferate and secrete cytotoxic mediators (granzyme B, perforin, FasL) and effector (IFN-γ, Il-13) and regulatory (Il-10) cytokines with isoniazid, but not rifampicin, pyrazinamide, or ethambutol. Il-17 was not detected, while only 1 clone secreted Il-22. Isoniazid-responsive clones were not activated with other anti-tuberculosis drugs or isonicotinic acid albumin adducts. Activation of the clones with isoniazid was MHC class II-restricted and dependent on antigen-presenting cells. Most clones were activated rapidly even in the presence of the enzyme inhibitor 1-aminobenzotriazole. However, a time-dependent pathway of activation involving auto-oxidation of isoniazid was also observed. The discovery of isoniazid-specific CD4+ T-cell clones in patients with liver and skin injury suggests that the adaptive immune system is involved in the pathogenesis of both forms of iatrogenic disease.

The role of enzymatic coupling of drugs to proteins in induction of drug specific antibodies.

Isoniazid and hydralazine were covalently crosslinked by epidermal transglutaminase to serum and cell nuclear proteins. Albumin and nucleohistones were excellent acceptors of these drugs in serum and nuclear extracts, respectively. Drug-albumin and drug-histone conjugates were highly antigenic in rabbits within 4 weeks, eliciting drug-specific antibodies in all immunized animals. A radioimmunoassay for antibodies to isoniazid were developed, using a standard drug-albumin conjugate. The methods described here of enzymatic crosslinking of drugs to proteins facilitate studies of drug-induced hypersensitivity in animals and man.

Lymphocyte transformation test for the evaluation of adverse effects of antituberculous drugs.

The usefulness of the lymphocyte transformation test (LTT) for the analysis of adverse reactions to antituberculous drugs was evaluated. - The LTT was performed with isoniazid and rifampicin in 15 tuberculosis and 2 MOTT (Mycobacteria other than tuberculosis)-infection patients who suffered drug reactions, in 23 patients without any adverse reactions, in 7 controls previously exposed to antituberculous drugs, and in 14 controls who had never been exposed. 4/15 of the hepatotoxic reactions only showed a positive LTT with rifampicin, 3/15 only with isoniazid, and in 8/15 the LTT was negative. In an anaphylactoid shock reaction the LTT was extremely exaggerated for both rifampicin and isoniazid. In patients without any side effects only one slightly increased LTT due to isoniazid was observed. Two healthy controls with previous contact to these drugs showed a positive LTT for isoniazid, one of those with both rifampicin and isoniazid. The LTT was negative in all control persons without any former contact to antituberculous medications. In most cases hepatotoxicity seems to be a pure toxic reaction without the participation of cellular immune mechanisms. LTT can be useful for identifying the drug responsible for immunological side effects.

Detection of IgE antibodies specific to isonicotinic acid hydrazide and its metabolite by enzyme-linked immunosorbent assay and the mechanism of sensitization by inhalation or ingestion of this compound.

A female hospital pharmacist and a nurse developed occupational asthma due to the inhalation of isonicotinic acid hydrazide (INH) powder. The mechanism of sensitization was examined in this study. Furthermore, IgE antibodies specific to INH and isonicotinic acid (INA), the major metabolite of INH, were examined by means of enzyme-linked immunosorbent assay (ELISA). IgE anti-INH antibody activity was detected by ELISA in sera from 5 out of 8 hospital workers who inhaled INH powder during the handling of INH, and in sera from 10 out of 142 tuberculosis patients receiving oral INH. IgE anti-INA antibody activity was detected in sera from 3 of the 8 hospital workers, and from 7 of the 142 tuberculosis patients. Neither IgE anti-INH nor anti-INA antibody activity was detected in sera from 45 patients with asthma or from 42 healthy volunteers. The specificity of the IgE antibodies and the cross-antigenicity between INH and INA were confirmed by ELISA inhibition studies. Our results indicate that sensitization to INH can occur not only by inhalation but also by oral administration, and that the asthmatic symptoms of the patients were caused by an IgE antibody specific to INH as a hapten.

Positive allergy study (Intradermal, patch, and lymphocyte transformation tests) in a case of isoniazid-Induced DRESS

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Clinical, laboratory and imaging findings of the patients with disseminated bacilli Calmette–Guerin disease

OBJECTIVES: In the present study, we reviewed 44 cases of disseminated BCG infection during a 10-year period in an Iranian referral children medical centre hospital. MATERIAL AND METHODS: In this study, all of the patients with clinical and laboratory findings that were compatible with a diagnosis of disseminated BCG were included. RESULTS: Through 10 years evaluation, 44 patients were found with disseminated BCG disease. Hepatomegaly and splenomegaly were seen in 68% and 66% of patients, respectively. Osteomyelitis was observed in 9% of our cases. Decrease in blood cells including anaemia, leucopoenia, neutropenia and thrombocytopenia were associated with more severe disease and even deaths. Moreover, 80% and 70% of patients who died had high level of C reactive protein (CRP) and erythrocyte sedimentation rate (ESR). Among the dead patients, 80% had abnormal sonography. Thirty nine percent of patients had immunodeficiency, while more than half of the patients who died had no identified immunodeficiency. CONCLUSION: These findings confirm the need to do sonography as well as bone imaging immediately in all patients with BCGitis. Assessment of the inflammatory factors in order to predict the prognosis of the disease is recommended. Furthermore, complete blood count would provide important information and should perform in all patients with BCGitis

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Isonicotinic acid hydrazide as an antigen.

Detection by in vitro serologic techniques of circulating antibody directed against isonicotinic acid hydrazide (INH, known as isoniazid) in humans has not been reported. In the past few months, however, sera from a patient with a recent history of isoniazid hypersensitivity of the immediate type have been studied, and reaginic antibodies (IgE) specific to INH were detected by means of an enzyme-linked allergosorbent test. Preliminary enzyme-linked allergosorbent tests also demonstrated that INH-specific IgE occurred in the serum of two of 150 patients with tuberculosis who had been treated with INH.

Antibodies specific to isoniazid and isonicotinic acid.

Rabbit antisera to isoniazid (INH) and its major metabolite, isonicotinic acid (INA), were prepared by immunization with conjugates of these compounds with human serum albumin. The antisera were rendered hapten-specific by exhaustive absorption with the immunizing carrier. Purified anti-hapten antibodies were also isolated with appropriate immunosorbents. As demonstrated by inhibition of the quantitative precipitin curves and of precipitating immune complexes in immunodiffusion tests, the antibodies to the two haptens reacted with either INH or INA, and also with isonicotinamide (INC); these three related molecules share the isonicotinyl group. The relative effectiveness of inhibition by free hapten of precipitating immune complexes consisting of either anti-INH or anti-INA antibodies and the related hapten-protein conjugates was INH greater than INC greater than INA.

Evaluation of isoniazid-associated hepatitis by immunological tests.

In a retrospective study of patients developing hepatitis or persistent serum glutamic oxaloacetic transaminase (SGOT) elevations while receiving isoniazid, it was found that the lymphocyte transformation test (LTT) was positive in nineteen cases (95%) in response to stimulation by isoniazid, isonicotinic acid and conjugates of these compounds with human serum albumin. However, no significant amount of antibody against isoniazid was detected in the sera of these patients by a sensitive radioimmunoassay. By contrast, no positive LTT was seen in normal controls or in patients receiving isoniazid without evidence of liver damage, while in patients with transient SGOT abnormalities, the LTT was positive only at the time of liver dysfunction. There was no correlation between the degree of lymphocyte transformation and the severity of liver damage. However, there were differences in the patterns of response to the four stimulatory preparations used. Thus patients with overt hepatitis most frequently responded to isoniazid, while individuals with only SGOT abnormalities showed stimulation in the LTT more often with a conjugate of isonicotinic acid and human serum albumin. It appears, therefore, that the presence of isoniazid-induced liver damage is associated with the presence of cellular hypersensitivity to the drug. The differences in lymphocyte reactivity in the two groups might indicate a potential means of predicting which individuals are at increased risk of developing overt hepatitis when exhibiting evidence of minor liver dysfunction while receiving isoniazid.

Drug-specific immune responses induced by procainamide, hydralazine and isoniazid in guinea-pigs.

The drug-induced graft vs host reaction (GVHR) hypothesis requires, as its first step, specific T-cell immune responses to the drug-modified self. Procainamide, isoniazid and hydralazine are known to provoke various allergic reactions including GVHR-like adverse effects in man. We now report that drug-specific immune responses can easily be induced by these drugs in guinea-pigs. Twenty-five milligrams of each of these drugs and penicillin G, which is known to make covalent bonds with proteins and to also induce drug-specific immune responses, were mixed with complete Freund's adjuvant (CFA) and subcutaneously (s.c.) injected twice at an interval of 2 weeks into female Hartley guinea-pigs. The antibodies to these drugs were assessed by means of an enzyme-linked immunosorbent assay (ELISA). Two weeks after the last injection, all animals treated with isoniazid, hydralazine and penicillin G produced high titers of antibodies to these drugs. Antibodies to procainamide were also detected, although their antibody titers were low. The specificity of the antibodies produced were tested by the inhibition of ELISA and concentration-dependent inhibition was observed. Delayed type hypersensitivity (DTH) reactions were also observed in the animals treated with procainamide, isoniazid and hydralazine 2 weeks after the last injection. These results suggest that the allergic reactions observed in clinical use are related to the inducing potential of drug-specific immune responses in an animal system. Therefore, immunization of guinea-pigs with test drugs and CFA may give useful information for predicting the occurrence of allergic reactions in man.

[The role of phenotypic factors in predicting the effectiveness of pharmacological prevention of tuberculosis in children]. / Rol' fenotipicheskikh faktorov pri prognozirovanii éffektivnosti khimioprofilaktiki tuberkuleza u detei.

The role of genetic markers such as dermatoglyphics, sex and INH inactivation in predicting the efficiency of tuberculosis chemoprophylaxis has been studied in 76 children (41 boys and 35 girls) with a tuberculin reaction reversal. The dermatoglyphic signs and the type of INH inactivation were found to be related to the efficiency of chemoprophylaxis. This makes it possible to use the child's phenotype for prognosis and better individual approach to tuberculosis chemoprophylaxis. An association has been found between the INH inactivation type and dermatoglyphics.