Broad-spectrum monoclonal antibody and a sensitive multi-residue indirect competitive enzyme-linked immunosorbent assay for the antibacterial synergists in samples of animal origin.

To monitor the abuse of antibacterial synergists, a hapten, trimethoprim carboxylic derivative (TMPCOOH), was designed by using molecular modelling technology. A broad-spectrum monoclonal antibody (mAb) TMP/2G1 was prepared, for which the IC50 values of trimethoprim, diaveridine, aditoprim, baquiloprim, ormetoprim, and brodimoprim were 0.232, 0.527, 1.479, 4.354, 0.965, and 0.119 µg L-1, respectively. Based on the broad spectrum mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to determine the residues of antibacterial synergists. The limit of detection regarding the developed ic-ELISA for antibacterial synergists ranged from 0.025 to 1.126 µg L-1 in milk, honey and edible animal tissues. The recoveries ranged from 81.4% to 107.7%, with a coefficient of variation less than 20%. A good correlation (R2 = 0.994) between the ic-ELISA and HPLC-MS/MS showed the reliability of the developed ic-ELISA.

Trimethoprim stimulates T-cells through metabolism-dependent and -independent pathways.

Pathways of drug-specific T-cell stimulation have not been fully defined. The aim of this study was to use T-cell clones from a patient hypersensitive to the drug trimethoprim to characterize the involvement of drug metabolism and processing in antigen presentation and cross-reactivity patterns. The MHC-restricted CD4+ and CD8+ T-cell response was dependent on the presence of antigen-presenting cells, and both processing-dependent and -independent pathways of antigen presentation were detected. Stimulation of certain clones was blocked through inhibition of drug-metabolizing enzyme activity. Trimethoprim clones were additionally stimulated with diaveridine and pyrimethamine but not other closely related structures.

Haemolytic anaemia and renal failure associated with antibodies to trimethoprim and sulfamethoxazole.

AIM: The aim of this study was to support a clinical diagnosis of drug-induced immune haemolytic anaemia (DIIHA). BACKGROUND: DIIHA is rare and has only been described twice with the antibiotic combination of trimethoprim (TMP) and sulfamethoxazole (SMX). METHODS/MATERIALS: Serologic tests for drug antibodies were performed using methods previously published by our laboratory. RESULTS: A 44-year-old woman experienced body aches, chills, chest pressure, nausea and a rash while receiving TMP-SMX; a week later her haemoglobin was low and she was in renal failure. At the hospital, the direct antiglobulin test (DAT) was positive (C3 only) and the serum reacted with all red blood cells (RBCs) by the gel method only (TMP-SMX is present in the RBC diluent used for the gel method). At the Red Cross immunohaematology laboratory, the patient's serum was reactive in the presence of TMP-SMX (haemolysis and positive antiglobulin test), pure TMP (positive antiglobulin test using anti-IgG only) and pure SMX (haemolysis and positive antiglobulin test using both anti-IgG and anti-C3). The patient was treated with transfusions and haemodialysis and was discharged after a week in stable condition. CONCLUSION: We describe a patient who appeared to have haemolytic anaemia and renal failure associated with antibodies to both TMP and SMX.

Epitope mapping of ligand-receptor interactions by diffusion NMR.

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation. Therefore, this diffusion artifact can be used to characterize the ligand binding epitope. The concept was investigated using dihydrofolate reductase (DHFR) and its ligand trimethoprim (TMP), and the epitope map of TMP on DHFR generated with this method is in excellent agreement with the structural and dynamic studies by crystallography and NMR, as well as the medicinal chemistry results.

Efficacy and safety of desensitization with sulfamethoxazole and trimethoprim in 48 previously hypersensitive patients infected with human immunodeficiency virus.

OBJECTIVE: To study the safety and efficacy of desensitization with the use of a combination product of sulfamethoxazole and trimethoprim in previously hypersensitive patients infected with the human immunodeficiency virus. DESIGN: Prospective survey, with a median follow-up of 16 months (range, 5-24 months). SETTING: Day-care hospital in a referral center. PATIENTS: All human immunodeficiency virus-infected patients who had a history of allergic reactions (eg, rash) to sulfamethoxazole-trimethoprim and who required sulfamethoxazole-trimethoprim prophylaxis. INTERVENTION: The desensitization procedure took 2 days. The full dose (sulfamethoxazole-trimethoprim, 400-80 mg) was reached on the third day according to the following schedule: day 1--4-0.8 mg at 9 AM, 8-1.6 mg at 11 AM, 20-4 mg at 1 PM, and 40-8 mg at 5 PM; day 2--80-16 mg at 9 AM, 160-32 mg at 3 PM, and 200-40 mg at 9 PM; and day 3--400-80 mg at 9 AM. MAIN OUTCOME MEASURE: The onset of cutaneous adverse effects attributable to sulfamethoxazole-trimethoprim therapy within 3 months after desensitization. RESULTS: Of the 48 evaluable patients, 37 (77%) tolerated sulfamethoxazole-trimethoprim desensitization without toxic effects and continued to take sulfamethoxazole-trimethoprim daily. Desensitization failed in 11 cases (5 on day 1, 3 on day 2, and 1 each on days 9, 11, and 90). Acute hypotension and a nonfatal myocardial infarction developed in 1 of these patients. The factors that were predictive of failure were a relatively high CD4+ cell percentage (11% vs 8%; P = .008) and a relatively high CD4+/CD8+ ratio (0.27 vs 0.12; P = .02). CONCLUSIONS: The efficacy of desensitization with sulfamethoxazole-trimethoprim was confirmed; this desensitization procedure was more often successful in patients with lower CD4+ cell percentages and CD4+/CD8+ ratios. However, sulfamethoxazole-trimethoprim therapy should be reintroduced carefully.

Fine structural specificity differences of trimethoprim allergenic determinants.

BACKGROUND: Adverse reactions, including immediate hypersensitivity, to the widely used antibacterial agent trimethoprim occur quite frequently. In recent years some progress has been made in developing an immunoassay to aid diagnosis of type 1 allergic reactions to trimethoprim and to define the basis of IgE antibody recognition of the drug. OBJECTIVES: The molecular basis of IgE binding to trimethoprim was examined more closely with a view to defining the fine structural recognition differences between patient's sera. Utilization of such information may lead to immunoassays that are more specific and sensitive and of greater diagnostic value. METHODS: Immunoassays for specific IgE antibodies and quantitative hapten inhibition studies with trimethoprim and selected structural analogues were employed, together with sera from eight subjects clearly defined clinically as allergic to trimethoprim. RESULTS: Three different allergenic determinant structures have been identified on the trimethoprim molecule. Identification of the 3,4-dimethoxybenzyl group as a determinant was achieved on the basis of inhibitory activities of diaveridine, 3,4-dimethoxyphenylethylamine, 3,4-dimethoxybenzoic acid and 3,4,5-trimethoxycinnamic acid. Evidence that the opposite end of the trimethoprim molecule was not being recognized was obtained from results with some pyrimidine derivatives, each of which showed no activity. Identification of the second determinant, the 2,4-diamino-5-(3',4'-dimethoxybenzyl) pyrimidine group, rested mainly on the superior inhibitory potency of diaveridine, which differs from trimethoprim by just one methoxy group. With sera from some trimethoprim-allergic subjects, only trimethoprim was active, suggesting that the entire molecule was a third IgE-binding determinant structure. CONCLUSION: As with other drug allergenic determinants defined so far, heterogeneity of trimethoprim IgE-binding determinants exists, and fine structural differences between determinants may be as small as a single methoxy group. Identification of the 2,4-diamino-5-(3',4'-dimethoxybenzyl) pyrimidine group as an allergenic determinant increases the number of known trimethoprim determinants to three, and suggests that the number and heterogeneity of determinants will be a reflection of the number of allergic subjects studied.

Drugs as allergens. The molecular basis of IgE binding to trimethoprim.

The combining site specificities of IgE antibodies that react with the oral antibacterial agent trimethoprim and found in the sera of two subjects who experienced anaphylaxis after taking the drug, were investigated. Hapten inhibition studies with some close analogues of trimethoprim and a range of other structurally related compounds showed that the allergenic determinant complementary to the IgE antibodies in the serum of one of the subjects was the 3,4-dimethoxybenzyl group. The complementary allergenic structure recognized by the IgE antibodies in the serum from the second subject comprised both the trimethoxybenzyl and diaminopyrimidine rings of trimethoprim. Thus, as with thiopentone, but unlike the neuromuscular blocking drugs, the trimethoprim molecule has more than one determinant each with the capacity to provoke IgE formation, interact with the antibody combining site and provoke drug-induced allergic reactions. The general approach set out here employing carefully selected structural analogues in hapten inhibition studies should be invaluable for confirming specificity and identifying allergenic determinants in IgE antibody-mediated allergic drug reactions.

An immunoassay for the detection of IgE antibodies to trimethoprim in the sera of allergic patients.

An immunoassay was developed to detect IgE antibodies to the widely used antibacterial drug trimethoprim. Significant levels of trimethoprim-reactive IgE antibodies were found in the sera of two patients who had experienced life-threatening allergic reactions following administration of a combination of trimethoprim and sulphamethoxazole. No IgE antibodies reactive with sulphamethoxazole were found in the sera of either patient. Inhibition experiments revealed that a high degree of cross-reactivity occurs between the drug-reactive IgE antibodies and two structural analogues of trimethoprim, 6-hydroxy- and 6-chlorotrimethoprim. These experiments also indicated that the combining sites of the trimethoprim-reactive IgE antibodies in the two sera were probably complementary to different parts of the trimethoprim molecule. The assay should supplement skin testing in determining the offending drug in patients with suspected allergic sensitivity to trimethoprim-sulphamethoxazole complex.

Metabolite-specific (IgG) and drug-specific antibodies (IgG, IgM) in two cases of trimethoprim-sulfamethoxazole-induced immune thrombocytopenia.

Two cases of trimethoprim-sulfamethoxazole (TMP-SMX)-induced immune thrombocytopenia are reported in which unusual drug-dependent platelet antibodies were demonstrated by immunofluorescence and enzyme-linked immunosorbent assay. Whereas two distinct sulfamethoxazole-dependent antibodies of the IgG and IgM class were detectable in the serum of one patient, the serum of the other patient contained a platelet antibody exclusively reactive with N-4-acetyl-sulfamethoxazole, a metabolite of sulfamethoxazole. Urine from a healthy volunteer collected after administration of therapeutic doses of TMP-SMX proved to be an appropriate source of ex vivo metabolites for antibody testing. The results of this study stress the role of metabolite-specific antibodies in drug-dependent immune thrombocytopenia and underscore the necessity of including metabolite preparations of drugs in serologic analyses.

[Immuno-allergic reactions to cotrimoxazole in a hospital population]. / Reazioni immuno-allergiche a cotrimoxazolo in una popolazione ospedalizzata.

By a study of 87 oncologic hospitalized patients, affected by serious infectious complications and treated with high-dose antibiotic therapy including co-trimoxazole, the authors evaluate the allergic and immunologic reactions to the drug on clinical and serological basis and try to outline the pathogenic implicated mechanisms.

Oral desensitization to trimethoprim-sulfamethoxazole in a patient with acquired immunodeficiency syndrome.

Intravenous administration of trimethoprim-sulfamethoxazole (TMS) on three occasions resulted in fever, rash, and wheezing in a 30-year-old man with acquired immunodeficiency syndrome with Pneumocytis carinii pneumonia. Pentamidine administration produced fever, severe myositis, and clinical deterioration, and therefore, desensitization to TMS was attempted. This was done with low doses of oral suspension and then intravenous administration during a period of 60 hours. The patient reacted with fevers, arthralgia, and erythema that cleared 4 days after the desensitization was completed. This single case of successful desensitization to TMS in a patient with acquired immunodeficiency syndrome suggests that the oral route may be useful in similar situations when this drug is urgently needed.

[Radioimmunologic detection of IgE and IgG antibodies against drugs. Conclusions after experience with over 1200 patients]. / Radioimmunologische Erfassung von IgEund IgG-Antikörpern gegen Medikamente. Standortbestimmung nach Erfahrungen mit über 1200 Patienten.

Based on the radioallergosorbent test (RAST), the authors have developed a series of assays to detect IgE and IgG antibodies against a number of frequently used drugs. In this system drugs bound covalently to cellulose paper are incubated with serum and washed; the hapten-specific IgE and IgG antibodies are then qualified and quantified by means of 125I-labelled anti-human IgE and IgG respectively. Thus far the sera of 1,228 patients have been analyzed following therapy with betalactam antibiotics, co-trimoxazole, salicylates, pyrazolones, flavonoids and tetrahydroisoquinoline. The induction of IgG antibodies is a frequent occurrence and that of IgE rare. Isolated high titers of IgE are associated mainly with anaphylactic reactions; in the presence of simultaneously raised IgG titers such side reactions are often absent. Highest IgG titers were found in patients with immune hemolysis after betalactam antibiotics, flavonoids and tetrahydroisoquinoline. In the other types of side reaction specific IgG titers were not significantly higher than in patients without side reactions. The estimation of circulating antibodies against drugs cannot yet be utilized diagnostically except in the rare cases of anaphylactic side reactions. However, the method described permits specific and sensitive detection of sensitization and is suited for scientific purposes.

Bullous esophageal lesions due to cotrimoxazole: an immune-mediated process?

A 78-yr-old man experienced a generalized bullous eruption of the skin (a Stevens-Johnson variant of erythema multiforme) with simultaneous involvement of the esophagus due to co-trimoxazole. Immunologic tests revealed specific antibodies of the immunoglobulin G class but not of the immunoglobulin E class against sulfamethoxazole, and in particular against trimethoprim. Lymphocyte transformation tests demonstrated sensitized lymphocytes against trimethoprim but not sulfamethoxazole. The esophageal mucosa showed intraepithelial vesicle formation with diffuse cytoplasmic deposits of immunoglobulin G. This adverse drug reaction involving both the skin and the esophagus appears to be immune-mediated.

Lymphocyte transformation test in drug-induced toxic epidermal necrolysis.

Lymphocyte transformation tests (LTT) to drugs remain widely used in drug reactions, despite controversies about their real usefulness. We tested the lymphocytes of 12 patients recovering from a drug-induced Toxic epidermal necrolysis (TEN). There was no difference between the amounts of thymidine incorporated when patients' lymphocytes were cultivated with culprit or innocent drugs. In both situations the lymphocytes from patients reacted like the lymphocytes from controls cultivated with the same panel of drugs. These negative results do not exclude that a hypersensitivity reaction may play a role in the physiopathology of TEN. Anyhow, they clearly indicate that testing lymphocyte transformation to drugs has no practical value in the diagnosis of TEN.

Lymphocyte transformation studies in suspected hypersensitivity to trimethoprim-sulphamethoxazole.

In a group of patients with suspected and probable adverse skin reactions to trimethoprim-sulphamethoxazole, the lymphocyte transformation test (LTT) was positive in approximately 60% of subjects tested. Levels of stimulation in the majority of cases were low, probably reflecting either use of inappropriate stimulatory forms of the drug or lack of marked reactivity. Since there appeared to be a correlation between peak stimulatory index and duration of the rash, the latter explanation appeared more likely.