Yuttana Srinoulprasert

Antigen Detection Assay for Identification of Penicillium marneffei Infection

ABSTRACT Two recently produced monoclonal antibodies were used to develop an antigen capture enzyme-linked immunosorbent assay (ELISA) for rapid diagnosis of Penicillium marneffei. The method was evaluated with 53 patients with culture-confirmed penicilliosis and 240 controls. The diagnostic sensitivity, specificity, and accuracy of the ELISA were 92.45, 97.5, and 96.59%, respectively.

Enhancement of drug-specific lymphocyte proliferation using CD25(hi)-depleted CD3(+) effector cells.

Background: The lymphocyte transformation test (LTT) is used for in vitro diagnosis of drug hypersensitivity reactions. While its specificity is over 90%, sensitivity is limited and depends on the type of reaction, drug and possibly time interval between the event and analysis. Removal of regulatory T cells (Treg/CD25hi) from in vitro stimulated cell cultures was previously reported to be a promising method to increase the sensitivity of proliferation tests. Objective: The aim of this investigation is to evaluate the effect of removal of regulatory T cells on the sensitivity of the LTT. Methods: Patients with well-documented drug hypersensitivity were recruited. Peripheral blood mononuclear cells, isolated CD3+ and CD3+ T cells depleted of the CD25hi fraction were used as effector cells in the LTT. Irrelevant drugs were also included to determine specificity. 3H-thymidine incorporation was utilized as the detection system and results were expressed as a stimulation index (SI). Results: SIs of 7/11 LTTs were reduced after a mean time interval of 10.5 months (LTT 1 vs. LTT 2). Removal of the CD25hi fraction, which was FOXP3+ and had a suppressive effect on drug-induced proliferation, resulted in an increased response to the relevant drugs. Sensitivity was increased from 25 to 82.35% with dramatically enhanced SI (2.05 to 6.02). Specificity was not affected. Conclusion: Removal of Treg/CD25hi cells can increase the frequency and strengths of drug-specific proliferation without affecting specificity. This approach might be useful in certain drug hypersensitivity reactions with borderline responses or long time interval since the hypersensitivity reaction.

In Vitro Test to Confirm Diagnosis of Allopurinol‐Induced Severe Cutaneous Adverse Reactions

Allopurinol is a frequent cause of severe cutaneous adverse reactions (SCARs), such as drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). The reactions can potentially be fatal. As drug rechallenge in patients with a history of drug‐induced SCARs is contraindicated, in vitro testing may have a diagnostic role as a confirmation test.

Multiple Drug Hypersensitivity

Multiple drug hypersensitivity (MDH) is a syndrome that develops as a consequence of massive T-cell stimulations and is characterized by long-lasting drug hypersensitivity reactions (DHR) to different drugs. The initial symptoms are mostly severe exanthems or drug rash with eosinophilia and systemic symptoms (DRESS). Subsequent symptoms due to another drug often appear in the following weeks, overlapping with the first DHR, or months to years later after resolution of the initial presentation. The second DHR includes exanthema, erythroderma, DRESS, Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), hepatitis, and agranulocytosis. The eliciting drugs can be identified by positive skin or in vitro tests. The drugs involved in starting the MDH are the same as for DRESS, and they are usually given in rather high doses. Fixed drug combination therapies like sulfamethoxazole/trimethoprim or piperacillin/tazobactam are frequently involved in MDH, and 30-40% of patients with severe DHR to combination therapy show T-cell reactions to both components. The drug-induced T-cell stimulation appears to be due to the p-i mechanism. Importantly, a permanent T-cell activation characterized by PD-1+/CD38+ expression on CD4+/CD25low T cells can be found in the circulation of patients with MDH for many years. In conclusion, MDH is a drug-elicited syndrome characterized by a long-lasting hyperresponsiveness to multiple, structurally unrelated drugs with clinically diverse symptoms.

Engagement of Penicillium marneffei conidia with multiple pattern recognition receptors on human monocytes

P. marneffei is a thermal dimorphic fungus which causes penicilliosis, an opportunistic infection in immunocompromised patients in South and Southeast Asia. Little is known about the innate immune response to P. marneffei infection. Therefore, the initial response of macrophages to P. marneffei conidia was evaluated by us. Adhesion between monocytes from healthy humans and fungal conidia was examined and found to be specifically inhibited by MAbs against PRR, such as MR, (TLR)1, TLR2, TLR4, TLR6, CD14, CD11a, CD11b, and CD18. To study the consequences of these interactions, cytokines were also examined by ELISA. Binding of P. marneffei conidia to monocytes was significantly inhibited, in a dose‐dependent manner, by MAbs against MR, TLR1, TLR2, TLR4, TLR6, CD14, CD11b and CD18. When monocytes were co‐cultured with the conidia, there was an increase in the amount of surface CD40 and CD86 expression, together with TNF‐α and IL‐1β production, compared to unstimulated controls. In assays containing anti‐TLR4 or anti‐CD14 antibody, reduction in the amount of TNF‐α released by monocytes stimulated with P. marneffei conidia was detected. In addition, it was found that production of TNF‐α and IL‐1β from adherent peripheral blood monocytes was partially impaired when heat‐inactivated autologous serum, in place of untreated autologous serum, was added to the assay. These results demonstrate that various PRR on human monocytes participate in the initial recognition of P. marneffei conidia, and the engagement of PRR could partly initiate proinflammatory cytokine production.

Reliability and validity of the Thai Drug Hypersensitivity Quality of Life Questionnaire: a multi-center study

Objective To adapted the Drug Hypersensitivity Quality of Life (DrHy-Q) Questionnaire from Italian into Thai and assessed its validity and reliability. Design Prospectively recruited during January 2012-May 2017. Setting Multicenter; six Thai tertiary university hospitals. Study Participants Total of 306 patients with physician-diagnosed drug hypersensitivity. Interventions Internal consistency and test-retest reliability were evaluated among 68 participants using Cronbachs ɑ and intra-class correlation coefficient (ICC). The validity of Thai DrHy-Q was assessed among 306 participants who completed World Health Organization Quality of Life-BREF (WHOQOL-BREF-THAI). Construct and divergent validities were assessed for Thai DrHy-Q. Known-groups validity assessing discriminating ability was conducted in Thai DrHy-Q and WHOQOL-BREF-THAI. Main outcome measures Validity; reliability; single vs. multiple drug allergy; non-severe cutaneous adverse reactions (SCAR) vs. SCAR. Results Thai DrHy-Q showed good reliability (Cronbachs ɑ = 0.94 and ICC = 0.8). Unidimensional factor structure was established by confirmatory factor analysis (CFI&TLI = 0.999, RMSEA = 0.02). Divergent validity was confirmed by weak correlation between Thai DrHy-Q and WHOQOL-BREF-THAI domains (Pearsons r = -0.41 to -0.19). Known-groups validity of Thai DrHy-Q was confirmed with significant difference between patients with and without life-threatening SCAR (P = 0.02) and patients with multiple implicated drug classes vs. those with one class (P < 0.01); while WHOQOL-BREF-THAI could differentiate presence of life-threatening SCAR (P < 0.01) but not multiple-drug allergy. Conclusions Thai DrHy-Q was reliable and valid in evaluating quality of life among patients with drug hypersensitivity. Thai DrHy-Q was able to discriminate serious drug allergy phenotypes from non-serious manifestations in clinical practice and capture more specific drug-hypersensitivity aspects than WHOQOL-BREF-THAI.

Evaluation of a lymphocyte transformation test and cytokine detection assay to identify phenytoin and carbamazepine provoked DRESS or SJS/TEN in epilepsy patients

&NA; Phenytoin (PHE) and carbamazepine (CBZ) are first rank causative drugs that can induce drug rash with eosinophilia and systemic symptoms (DRESS) and Stevens‐Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Identification of anti‐epileptic drugs as a culprit drug has been problematic; hence, in vitro tests could be promising methods to define causative drugs without clinical risk. The aim of this study is to evaluate the efficacy of lymphocyte transformation tests (LTT) and cytokine detection assays in identifying PHE and CBZ as culprit drugs. Peripheral blood mononuclear cells were collected from normal, PHE/CBZ tolerance and PHE/CBZ hypersensitivity cohorts and utilized for cell‐culture assays. LTT was performed and culture supernatants were subjected to multiple cytokine detection assays. Our study showed that LTT correlated with outcomes of Naranjos assessment with statistical significance (r = 0.614). Various sensitivities of LTT and cytokine detection assays were demonstrated. Although both assays provided excellent specificity, LTT yielded higher sensitivity as compared to those of cytokine detection assays in both DRESS and SJS/TEN. Regardless whether specificity is, this is the first report to demonstrate that IL‐4 detection assay could enhance sensitivity to identify culprit drug when it was combined with LTT for SJS/TEN patients or it was combined with IL‐2/IFN‐&ggr; detection assays for DRESS ones. HighlightsLymphocyte transformation test has a significant correlation with outcomes of Naranjos assessment.Lymphocyte transformation test yielded higher sensitivity than those of cytokine detection assays in both DRESS and SJS/TEN.IL‐4 could be promising marker to identify culprit drug in epilepsy patients