Identification of T-cell epitopes from benzylpenicillin conjugated to human serum albumin and implication in penicillin allergy.

BACKGROUND: There is in vitro evidence that T cells from allergic patients react to benzylpenicillin-human serum albumin (BP-HSA) bioconjugates. Our group has recently shown the existence of naïve CD4+ T cells recognizing BP-HSA in healthy donors. However, BP-haptenated peptides from HSA participating in the immunization of allergic patients have never been identified. The purpose of the present study is to identify immunodominant BP-haptenated peptides from HSA involved in immunization of patients to BP and to refine the frequency calculation of naïve CD4+ T cells recognizing BP. METHODS: Co-cultures were established with CD4+ T cells from non-allergic donors and mature autologous dendritic cells (DCs) loaded with BP-HSA or BP-haptenated peptides from HSA. The CD4+ T-cell response specific for BP-HSA or for individual BP-haptenated peptides was measured using an interferon-γ (IFN-γ) ELISpot assay. The frequency of BP-specific CD4+ T cells was then calculated using the Poisson distribution. BP-HSA and BP-haptenated peptides recognition by allergic patients was evaluated on peripheral blood mononuclear cells (PBMCs) using a lymphocyte transformation test (LTT). RESULTS: Results showed that BP-HSA and BP-haptenated peptides were recognized by naïve T cells from 15/16 and 13/14 tested healthy donors, respectively. Most donors responded to 3 peptides with BP covalently bound on lysines 159, 212, and 525. Two of these benzylpenicilloylated peptides (lysines 159 and 525) were also found to induce PBMCs proliferation in patients with allergic reaction to penicillins. CONCLUSION: This study identifies and characterizes for the first time the BP-haptenated peptides from HSA involved in the immunization of patients to penicillins.

Decline in 2 Serial Postmortem Tryptase Measurements Beyond 72 Hours After Death in an Antibiotic-Related Anaphylactic Death.

Anaphylaxis can be difficult to diagnose in the postmortem setting. Postmortem tryptase is a widely used ancillary test in aiding the diagnosis in which an elevation supports a death from anaphylaxis. Postmortem tryptase can be difficult to interpret, and the effects of postmortem kinetics are not fully understood. Clinically, mast cell tryptase returns to baseline 24 to 72 hours after an anaphylactic stimulus. We report another anaphylactic death from antibiotic administration in which 2 serial postmortem total tryptase measurements at 3 days (72 hours) and 6 days (144 hours) after death declined from 522 µg/L to 300 µg/L (baseline, 5.6 µg/L). The declination appears to be slower than what is expected in the clinical setting. This case highlights yet another example of the difficult and complex interaction of postmortem interval on postmortem tryptase, especially in an anaphylactic death. We suggest that early blood sampling and serial tests be performed if possible in suspected anaphylactic death.

Life-Threatening Atypical Case of Acute Generalized Exanthematous Pustulosis.

Antibiotics are known to cause severe cutaneous adverse reactions, such as the rare acute generalized exanthematous pustulosis (AGEP). Unlike Stevens-Johnson syndrome or toxic epidermal necrolysis, AGEP is rarely life-threatening. Systemic involvement is not typical, and if present usually coincides with a mild elevation of the hepatic enzymes and a decrease in renal function. Hence, AGEP is known to have a good prognosis and to be life-threatening only in elderly patients or patients with chronic diseases. Herein, we report a case of AGEP in a young healthy male leading to systemic inflammatory response syndrome and to treatment in an intensive care unit after being treated with 5 different antibiotics. Initial symptoms were not indicative for AGEP and the patient's course of disease led promptly to critical cardiorespiratory symptoms and systemic inflammatory response syndrome. We assume that the administration of the 5 different antibiotics resulted in type IV allergy as well as secondary infection with Enterococcus faecium and Staphylococcus aureus, while the underlying periodontitis also contributed to the severity of this case.

Selective immediate responders to amoxicillin and clavulanic acid tolerate penicillin derivative administration after confirming the diagnosis.

BACKGROUND: An increasing number of patients show immediate selective hypersensitivity reactions to clavulanic acid (CLV) and amoxicillin (AX), probably due to their increased prescription. The maintenance of this response should be established. OBJECTIVE: To assess that the immediate hypersensitivity selective response to AX or to CLV is maintained after repeated administration of penicillin G (PG)/penicillin V (PV) and AX. METHODS: Patients with proven immediate hypersensitivity to AX (Group A) or CLV (Group B) were included. Diagnosis was performed using skin tests with major and minor determinants of PG (PPL/MDM), AX and CLV and by drug provocation test (DPT) if required. Selectivity was established by confirming tolerance to PG/PV (Group A) and to PG/PV and AX (Group B). The maintenance of the selective response was verified by repeating DPT, 15 days after the initial investigation, with the same procedure. RESULTS: Of 51 patients, 78% belonged to Group A and 22% to Group B. Most had anaphylaxis. In Group A, 72% were skin test positive; 28% required DPT. In Group B, 63% were skin test positive; 37% required DPT. Only two AX-selective cases developed positive responses after re-provocation with PG/PV. No cases selective for CLV developed a positive response to PG, PV or AX. DISCUSSION: The selective response to AX appears consistent, and a response to penicillin determinants only develops in a minority of cases. For the case of CLV, the selective response appears not to be modified by exposure to penicillin determinants, meaning that patients with CLV allergy can take penicillin derivatives safely.

Recognition of multiepitope dendrimeric antigens by human immunoglobulin E.

In vitro drug allergy tests have limited sensitivity, partly due to a poor understanding of the immunological recognition of in vitro drug-protein conjugates. We have designed and synthesized multivalent mono- and bi-epitope dendrimeric antigen (DeAn) conjugates and studied their chemical and tridimensional structures. We describe differences in the spatial distribution and conformation of these conjugated epitopes for the first time: a partially hidden benzylpenicilloyl and a more exposed amoxicilloyl. Our data suggest that DeAn conjugates provide a useful model for studying IgE recognition in patients who suffer from an allergic reaction to benzylpenicillin and/or amoxicillin. 1D and 2D NMR, MDS and immunochemical studies provide evidence that both antigen composition and tridimensional distribution play key roles in IgE-antigen recognition. Bi-epitope DeAn conjugates could potentially allow the diagnosis of patients allergic to any of these two drugs with a single test and represent the basis for a broadly-applicable in vitro assay. From the clinical editor: The prevalence of allergic drug reactions is rising and there is an imperative need to identify patients at risk. In this interesting and important article, the authors developed a novel method for detecting drug specific IgE antibodies, responsible for allergic reactions, by using multivalent mono- and bi-epitope Dendrimeric Antigen (DeAn) conjugates. The continued success of this research may pave way of eventual development of a simple diagnostic test.

Skin testing only with penicillin G in children with a history of penicillin allergy.

BACKGROUND: The absence of commercially available penicilloyl-polylysine (PPL) for most of the last decade severely hampered the practice of penicillin allergy evaluation because skin testing without PPL is reported to have a poor negative predictive value (NPV). OBJECTIVE: To determine the safety and NPV of skin testing without PPL using only penicillin G followed by a 3-dose graded challenge to the incriminated penicillin in children with a history of penicillin allergy. METHODS: Patients evaluated for a history of penicillin allergy at the CHU Sainte-Justine Allergy Clinic between December 2006 and December 2009 were skin tested only with penicillin G and underwent a 3-dose graded challenge to the culprit penicillin if the skin test result was negative. RESULTS: Among 563 patients skin tested to penicillin G, 185 (33%) had a positive skin test result. These patients had a shorter interval between the initial reaction and skin testing compared with patients with a negative skin test result (P = .03). A total of 375 of 378 patients (99%) with a negative skin test result were challenged and 18 (4.8%) reacted, translating into a NPV of 95.2% (95% confidence interval [CI], 92.5%-97.1%). Three of 17 patients with a history of anaphylaxis and a negative skin test result reacted to challenge (NPV, 82.4%; 95% CI, 59.0-93.8%). All challenge reactions were mild and resolved promptly with treatment. CONCLUSION: Among children with a history of penicillin allergy, skin testing only with penicillin G followed by a 3-dose graded challenge to the incriminated penicillin is safe and yields a good NPV. This approach could be useful when PPL is unavailable.

Identification and frequency of circulating CD4(+) T lymphocytes specific to Benzylpenicillin in healthy donors.

BACKGROUND: Drug hypersensitivity is known to rely on a drug-specific T-cell response. Amplitude of antigen-specific T-cell response is partly controlled by the size of the antigen-specific naïve CD4(+) T-cell repertoire, but estimate of this repertoire has never been investigated for allergenic drugs. The purpose of this study was to evaluate the frequency of benzylpenicillin-specific CD4(+) T lymphocytes in healthy donors. METHODS: Co-cultures were established with CD4(+) T lymphocytes from healthy donors and mature autologous dendritic cells loaded with benzylpenicillin coupled to human serum albumin. CD4(+) T lymphocytes were stimulated once a week for 4 weeks with benzylpenicillin coupled to human serum albumin. The CD4(+) T-cell response was measured using an interferon-γ ELISPOT assay. Frequency of benzylpenicillin-specific naive CD4(+) T lymphocytes was then calculated using the Poisson distribution law. RESULTS: Results showed the presence of benzylpenicillin-specific CD4(+) T lymphocytes in 9 of 10 tested healthy donors irrespective of their HLA typing, with a mean frequency of 0.29 cells per million of CD4(+) T cells. Experiments performed on naive (CD45RA(+) ) and on memory (CD45RO(+) ) CD4(+) T lymphocytes showed that these benzylpenicillin-specific CD4(+) T lymphocytes belonged to the naive T-cell subpopulation. CONCLUSION: This study showed for the first time the existence of naive CD4(+) T lymphocytes specific to benzylpenicillin in healthy donors.

Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs.

All available therapies for human allergic disease target IgE mediated pathologic responses after IgE has been produced. We are developing tetracyclines as anti-allergy drugs to prevent IgE production, based on our findings that minocycline or doxycycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses (ELISA, mast cell mediated cutaneous late phase responses); the tetracyclines also strongly suppress peak IgE responses of BPO-KLH sensitized mice (ELISPOT assay, ELISA, skin tests). The antibiotic activity of the tetracyclines is not required for suppression of IgE responses; inclusion of minocycline or doxycycline in sterile culture prevents anti-CD40/IL-4 mediated induction of memory IgE responses by PBMC of allergic asthmatic patients (ELISA), and induction of specific memory IgE responses by spleen cells of BPO-KLH sensitized mice (ELISPOT assay, ELISA). The tetracyclines affect an epsilon specific pathway because IgM, IgG and IgA responses did not decrease. Further, in humans, DTH responses to recall antigens did not decrease. In related studies, we found that two distinct T cell subsets: CD4+CD60 negative and CD8+CD60+ (CD60 is a ganglioside) (humans) and CD4+ Asialo GM1 ganglioside negative and CD8+Asialo GM1 ganglioside+ (mice), both are required for induction of memory IgE responses. Phosphorylated (phos) p38 MAP kinase, but not phos ERK or phos JNK expression by CD4+ and CD8+, including CD8+CD60+, T cells is increased in allergic asthmatic humans, as is IL-4 and IL-10 production. The tetracyclines appear to target T cell pathways to induce suppression of IgE responses because they suppress phos p38 MAP kinase expression by both CD4+ and CD8+, including CD8+CD60+, T cell subsets, and IL-4 and IL-10, while upregulating IL-2 and IFN gamma, and suppressing IgE responses. Our finding that tetracyclines do not require antibiotic activity to suppress IgE responses opens the door to development of new tetracycline-based and other therapeutics for human allergic disease.

Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. II. Selective inactivation of hapten-reactive suppressor T cells by hapten-nonimmunogenic copolymers of D-amino acids, and its application to the study of suppressor T-cell effect on helper T-cell development.

An experimental condition was established in vivo for selectively eliminating hapten-reactive suppressor T-cell activity generated in mice primed with a para-azobenzoate (PAB)-mouse gamma globulin (MGG)-conjugate and treated with PAB-nonimmunogenic copolymer of D-amino acids (D- glutamic acid and D-lysine; D-GL). The elimination of suppressor T-cell activity with PAB-D-GL treatment from the mixed populations of hapten- reactive suppressor and helper T cells substantially increased apparent helper T-cell activity. Moreover, the inhibition of PAB-reactive suppressor T-cell generation by the pretreatment with PAB-D-GL before the PAB-MGG-priming increased the development of PAB-reactive helper T-cell activity. The analysis of hapten-specificity of helper T cells revealed that the reactivity of helper cells developed in the absence of suppressor T cells was more specific for primed PAB-determinants and their cross-reactivities to structurally related determinants such as meta-azobenzoate (MAB) significantly decreased, as compared with the helper T-cell population developed in the presence of suppressor T lymphocytes. In addition, those helper T cells generated in the absence of suppressor T cells were highly susceptible to tolerogenesis by PAB-D- GL. Similarly, the elimination of suppressor T lymphocytes also enhanced helper T-cell activity in a polyclonal fashion in the T-T cell interactions between benzylpenicilloyl (BPO)-reactive T cells and PAB- reactive T cells after immunization of mice with BPO-MGG-PAB. Thus inhibition of BPO-reactive suppressor T-cell development by the BPO-v-GL- pretreatment resulted in augmented generation of PAB-reactive helper T cells with higher susceptibility of tolerogenesis to PAB-D-GL. Thus, these results support the notion that suppressor T cells eventually suppress helper T-cell activity and indicate that the function of suppressor T cells related to helper T-cell development is to inhibit the increase in the specificity and apparent affinity of helper T cells in the primary immune response. The hapten-reactive suppressor and helper T lymphocytes are considered as a model system of T cells that regulate the immune response, and the potential applicability of this system to manipulating various T cell-mediated immune responses is discussed in this context.

Benzylpenicillin skin testing is still important in diagnosing immediate hypersensitivity reactions to penicillins.

BACKGROUND: The fact that both Hollister-Stier and Allergopharma ceased the production of penicilloyl-polylysine (PPL) and minor determinant mixture (MDM) in 2004 is severely hampering the diagnosis of beta-lactam hypersensitivity and may produce negative consequences. OBJECTIVE: To assess the contribution of skin testing with benzylpenicillin to the diagnosis of immunoglobulin E-mediated hypersensitivity to penicillins, in order to determine how much such testing could compensate for PPL and MDM unavailability. METHODS: We selected patients with histories of immediate reactions to penicillins and positive results to skin tests for one or more penicillin reagents (PPL, MDM, or benzylpenicillin), one or more semi-synthetic penicillins (ampicillin, amoxicillin, or piperacillin), or both. RESULTS: A total of 300 patients were selected, 105 in the French center and 195 in the Italian centers. Amoxicillin and ampicillin were the main responsible drugs. The most common clinical manifestation was anaphylaxis. The reagents most frequently positive to skin tests were amoxicillin (188, 62.7%), ampicillin (151, 50.3%), and benzylpenicillin (111, 37.0%). Among the 300 subjects, 113 (37.7%) were positive only to semi-synthetic penicillins, 109 (36.3%) to both semi-synthetic penicillins and the classic penicillin reagents, and 78 (26.0%) only to the latter. In the last group, 64 (21.3% of the 300 subjects) were positive only to PPL and/or MDM and 14 (4.7%) to benzylpenicillin, of whom 8 (2.7%) were positive only to the latter. CONCLUSIONS: Skin testing with benzylpenicillin can partially compensate for PPL and MDM unavailability. Moreover, it can slightly increase the allergologic workup's sensitivity and therefore reduce the number of potentially dangerous challenges.

Hypersensitivity reactions to penicillins: studies in a group of patients with negative benzylpenicillin G skin test.

BACKGROUND: Although skin tests are usually employed to evaluate current penicillin allergy status, a negative result does not exclude hypersensitivity. There is a need for accurate in vitro tests to exclude hypersensitivity. A radioallergosorbent test (RAST) is a potentially good supplementary approach, but there is little information on the suitability of this method to diagnose penicillin hypersensitivity in subjects with a negative skin test to benzylpenicillin. METHODS: A total of 133 patients with a negative skin test to benzylpenicillin G (PG) and all of whom developed allergic reactions to PG were studied. RAST was used to detect eight kinds of specific IgE antibodies to penicillins in serum, which included four kinds of major and minor antigenic determinants to four penicillin drugs. The combination sites for the specific IgE antibodies were studied by RAST inhibition test. RESULTS: The rate of positive reactions for the specific IgE antibodies was 59.40% (79/133). Of the eight kinds of antigenic determinants, the positive rates for specific IgE against the major and minor determinants were 39.10% (52) and 42.86% (57) respectively. Of the four drugs, positive cases only to PG were 10 (7.5%), were significantly fewer than the cross-reacting positive cases (36) to PG (P < 0.01). In the RAST inhibition studies all drugs exhibited good inhibitory potencies, and in some instances the side-chain of the penicillins could induce specific responses with a variable degree of cross-reactivity among the different penicillins. CONCLUSION: Radioallergosorbent test is a good complementary test in persons who are skin-test negative with PG, and the sensitivity of RAST increases with increasing specificity of IgE antibodies to be detected. 6-APA and the groups, making part of the different side-chains on penicillins, all contributed to the cross-reactivity.

The specificity of tests for anti-beta-lactam IgE antibodies declines progressively with increase of total serum IgE.

BACKGROUND: Immediate allergic reactions to beta-lactam antibiotics are mediated by specific IgE antibodies. The Phadia CAP System FEIA is a commercial method for quantification of specific IgE. We wished to determine anti-beta-lactam IgE antibodies in patients without penicillin allergy but with high levels of total IgE. METHODS: Sera from 41 patients (31 with high total IgE, 10 with low total IgE) were analyzed for IgE antibodies specific to penicilloyl G, penicilloyl V, amoxicilloyl and ampicilloyl using the CAP FEIA((R)) method that was available up to 2006. Seven sera that tested positive were rechecked in a new improved system available after 2006. RESULTS: In patients without a history of penicillin allergy, the specificities of commercial tests for anti-beta-lactam IgE antibodies were 100%, 60%, 27% and 20% at total IgE levels of 8-263 kU/l, 500-664 kU/l, 1000-2000 kU/l and > 2000 kU/l, respectively. In seven retested sera, only 2 (28%) were still positive for penicillin-specific IgE antibody. CONCLUSION: Before 2006, tests for anti-beta-lactam IgE antibody in patients with total IgE > 500 kU/l were probably often false positive. Patients who were diagnosed as penicillin allergic before 2006 solely on the basis of a positive CAP FEIA test for specific IgE should be considered for diagnostic reevaluation.

The anti-penicillin antibodies levels in sensitive and insensitive people to intradermal skin test.

BACKGROUND: The hypersensitivity reaction to penicillin is a public health problem. Immunological responses to penicillin and other beta-lactam antibiotics can be classified into immediate and non-immediate responses. The immediate hypersensitivity is mediated by IgE; however, the non-immediate sensitivity is facilitated by other isotypes of antibody or T lymphocytes. OBJECTIVE: This research detected the non-IgE antibody value against penicillin in allergic and normal people. METHODS: Thirty-eight samples from patients with positive or negative intradermal skin testing results of penicillin allergy were included in this study. The total antibody and IgM levels against penicillin G were defined by in-house ELISA test. RESULTS: The results showed a significant (P< 0.05) elevation in total immunoglobulin and non-IgM anti-penicillin antibody of sensitive groups; however, the anti-penicillin IgM was significantly greater in non-sensitive peoples. CONCLUSIONS: Although the sensitized people to penicillin cannot be certainly detected with the total antibody, specific IgG and IgM value against penicillin, these values are good indicators for prediction of immediate and late response of the immune system to penicillin.

Serological studies of piperacillin antibodies.

BACKGROUND: Penicillin-induced immune hemolytic anemia (IHA) is associated with immunoglobulin G antipenicillin detected by testing penicillin-coated red blood cells (RBCs). Antibodies to piperacillin, a semisynthetic penicillin, would be expected to react similarly; however, antipiperacillin can be detected by testing in the presence of the drug. Piperacillin is commonly used in combination with tazobactam, which causes nonimmunologic protein adsorption onto RBCs. In six cases of piperacillin-induced IHA, reactivity with piperacillin-coated RBCs was not similar to reactivity of antipenicillin with penicillin-coated RBCs. STUDY DESIGN AND METHODS: Antipiperacillin was tested against piperacillin-coated RBCs prepared using different pH buffers. Plasma from blood donors and sera/plasma from patients were tested with piperacillin-coated, penicillin-coated, and uncoated RBCs. Hapten inhibition studies were performed using different concentrations of piperacillin. Donors' plasma were tested in the presence of piperacillin; sera from patients with IHA were tested in the presence of tazobactam. RESULTS: Piperacillin required high pH for binding to RBCs. Agglutination of piperacillin-coated RBCs was observed in 91 percent of donors' and 49 percent of patients' plasma and was inhibited by piperacillin. In contrast to patients with IHA due to piperacillin, donors' plasma tested in the presence of piperacillin did not react. Tazobactam antibodies were not detected. CONCLUSION: A high percentage of donors' and patients' plasma contain an antibody to piperacillin or a chemically related structure detected by testing with piperacillin-coated RBCs. A diagnosis of piperacillin-induced IHA should not be made solely on the reactivity of a patient's plasma/serum with piperacillin- or piperacillin/tazobactam-coated RBCs; testing in the presence of piperacillin is more reliable.

Fixation of penicilloyl groups to albumin and appearance of anti-penicilloyl antibodies in penicillin-treated patients.

Penicilloyl groups, which have been connected to penicillin allergy, are derived from penicillin by cleavage of the beta lactam ring and bind covalently to proteins. Fixation of penicilloyl groups was studied in seven patients given large amounts of penicillin. Penicilloyl groups were found essentially on the albumin molecule at sites not accessible to anti-penicilloyl antibodies, except after pronase digestion. The amount of penicilloyl groups was proportional to the cumulated doses of penicillin. The decline of penicilloyl groups with time after treatment interruption was exponential. The half-life of penicilloylated albumin was lower than or equal to that of normal albumin. The presence of anti-penicilloyl antibodies was demonstrated in 19 out of 34 penicillin-treated patients (including the seven mentioned above). The relative scarcity of penicillin allergy as compared with the frequent occurrence of anti-penicilloyl antibodies may be partly related to unavailable sites of penicilloyl groups within the albumin molecule.

Hapten-specific T-cell unresponsiveness induced bybenzylpenicilloyl autologous gamma globulin conjugates in human lymphocyes in vitro.

The aim of these studies was to determine whether unresponsiveness to the main determinant of penicillin, benzylpenicilloyl, can be induced in human peripheral lymphocytes in vitro by conjugates of benzylpenicilloyl (BPO) autologous gamma globulin (HGG). Initially it was shown that conjugates of BPO-keyhole limpet hemocyanin (KLH) elicited lymphocyte proliferation in the peripheral blood lymphocytes of six out of nine adult individuals in vitro. In contrast, conjugates of dinitrophenylated KLH and of BPO-HGG and the carriers HGG and KLH alone failed to do so. Similarly, release of the non-specific helper factor, lymphocyte mitogenic factor (LMF) occurred only after BPO-KLH stimulation. LMF activity was measured by B-cell proliferation and incorporation of radioactive amino acids into secreted immunoglobulin. Treatment with BPO-HGG for 24 h in vitro inhibited BPO-KLH-induced lymphocyte proliferation and LMF release. Treatment with either HGG, dinitrophenylated HGG, BPO-KLH, or BPO-human serum albumin failed to abrogate T-cell lymphocyte proliferation of human lymphocytes in vitro. The antigen specificity of the reduced immunologic responsiveness was further demonstrated by the observation that lymphocytes treated with BPO-HGG for 24 h in vitro responded normally to tetanus toxoid antigen.The data suggest that conjugates of BPO-HGG induce hapten-specific helper T-cell unresponsiveness in vitro.

Assessment of a new brand of determinants for skin testing in a large group of patients with suspected beta-lactam allergy.

BACKGROUND: Skin testing with major and minor determinants of benzylpenicillin is recommended standard practice for the evaluation of patients with immediate hypersensitivity reactions to beta-lactams. However, commercial reagents for this purpose were recently dropped from the European market. OBJECTIVE: In the present study, we assessed a new brand of reagents for use in skin testing in patients with suspected penicillin allergy. METHODS: Prick tests and intradermal tests were performed with benzylpenicilloyl polylysine (PPL) and minor determinant mixture (MDM). Penicillin G, amoxicillin, and the culprit beta-lactam were also tested. If skin tests were negative, a single-blind oral challenge test was performed with the culprit active principle or penicillin. If both skin tests and challenge tests were negative, the same procedure was repeated between 2 and 4 weeks later. RESULTS: A total of 636 patients were assessed. The allergy study was positive in 69 patients. Skin tests with PPL were positive in 30 patients (46.8%) and with MDM in 28 (43.7%). Sixteen patients displayed a positive reaction to both PPL and MDM (25%), while 42 patients (65.6%) had a positive reaction to either PPL or MDM alone. Thirty-two patients had positive skin test reactions to penicillin G or another p-lactam antibiotic. Five patients in whom a negative result was obtained in skin tests had a positive reaction to oral challenge. CONCLUSIONS: Our results indicate that a new brand of determinants that is commercially available in Europe is a reliable and useful tool for the diagnosis of beta-lactam allergy. The new reagents are a safe alternative to the previously available brand.