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Background: Association between chronic spontaneous urticaria (CSU) and autoimmunity has been well documented. Autologous serum skin testing could support the autoimmune etiology of CSU, whereas it is difficult to interpret and could not be performed on antihistamine omitted patients. It was found that immunoglobulin G (IgG) autoantibodies (autoAbs) against high-affinity IgE receptor (Fc ε R1) were suggested as a potential trigger in the pathogenesis of CSU. Although many ELISA protocols have been developed to detect these autoAbs, they lacked validation or a reliable cut-off point. We, therefore, aimed to develop a validated ELISA with a reliable cut-off point to quantitate IgG anti-Fc ε R1 α autoAbs for CSU., Methods: We developed an in-house ELISA to quantitate IgG anti-Fc ε R1 α autoAbs. Sera from 233 CSU patients and 25 healthy people were used to test with ELISA. The cut-off point was obtained from the results subjected to analyze with receiver operating characteristic (ROC) analysis. ELISA was validated with 116 CSU patients and 150 healthy donors., Results: ELISA revealed that healthy people had a basal level of IgG anti-Fc ε R1 α autoAbs, whereas their levels were significantly lower than autoAbs levels in CSU patients. ROC analysis of ELISA determined the cut-off point at 936.7 ng/ml. Our ELISA was validated and provided excellent sensitivity and specificity at 98.28% and 92.67%, respectively., Conclusion: Our ELISA could detect significant levels of IgG anti-Fc ε R1 α autoAbs in CSU, supporting that these autoAbs were associated with CSU etiology. Our validated ELISA with the reliable cut-off point provided excellent accuracy at 95.11% (98.28% sensitivity and 92.67% specificity). Our ELISA could be an alternative test benefit for the patient who is unable to omit antihistamine treatment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Chattip Sripatumtong et al.)

Drug hypersensitivity reactions (DHRs) occasionally present with severe cutaneous adverse reactions (SCARs) which result in a high risk of morbidity and mortality. Although SCARs are rare, the occurrence could lead to a significant increase in healthcare and economic burden, especially when more than one possible culprit drug is implicated. Therefore, the accurate identification of the culprit drug(s) is important for correct labeling and subsequent patient education and avoidance. To date, clinical evaluation using causality assessment has limitations because the assessment may be inaccurate due to the overlapping timelines when multiple drugs are initiated/continued. Moreover, drug provocation tests (DPTs) which is the gold standard in diagnosis, are contraindicated, and in vivo skin tests may also be associated with risks of triggering SCAR. The European Network for Drug Allergy recommended that in vitro tests, if available, should be performed before any in vivo tests. Basophil activation tests and lymphocyte transformation tests, could serve as reliable in vitro tests for both immediate and delayed-type DHR. Many academic medical centers with affiliated laboratory services offer these tests in the diagnostic evaluation of SCARs in clinical practice. This not only complements identification of the culprit drug(s), but may also be used to test for potentially non cross-reactive alternatives, hence avoiding DPTs. In this review, we summarize the roles of in vitro tests in identifying the culprit drug(s) in SCARs, issues with utilization and interpretation of test results, and our experience in clinical practice., Competing Interests: Conflict of Interest: The authors have no financial conflicts of interest., (Copyright © 2020. Asia Pacific Association of Allergy, Asthma and Clinical Immunology.)

Background: Pythium insidiosum has been mainly reported to cause morbidity and mortality in thalassemia patients. P. insidiosum zoospores can germinate to be hyphae within a few hours; therefore, it is difficult to study the initial immune response that P. insidiosum zoospores induce. The present study aims to compare immune responses against P. insidiosum zoospore infection by comparing monocytes/macrophages from thalassemia patients with those from non-thalassemia controls., Methods: In order to keepP. insidiosum in the zoospore stage in vitro for inoculation, the P. insidiosum zoospores were preserved without germination by treatment with inorganic hypochlorite solution. CD14+ cells were isolated from peripheral blood mononuclear cells of thalassemia and non-thalassemia donors and then left to transition to macrophages. Monocytes/macrophage culture was infected with P. insidiosum zoospores and culture supernatants were subjected to Th1/Th2 multiplex cytokine detection., Results: Our study of cytokine production revealed that the basal level of GM-CSF produced by thalassemia monocytes/macrophages was lower than that observed in monocytes/macrophages of non-thalassemia individuals. Higher GM-CSF and IFN-γ response was also found when cells from non-thalassemia people were stimulated with P. insidiosum zoospores compared to thalassemia cells. It was also found that TNF-α, GM-CSF and IFN-γ productions from monocytes/macrophages of thalassemia patients who received iron chelator treatment were significantly higher than those produced from thalassemia patients without iron chelator treatment., Conclusion: For the first time, the present study demonstrates defective immune responses in monocytes/macrophages derived from thalassemia patients in response toP. insidiosum zoospore infection. The results also show an inverse correlation between iron overload and cytokine production in monocytes/macrophages of thalassemia patients. This finding could explain why thalassemia patients are susceptible to P. insidiosum infection., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: In developing countries, renal specialists are scarce and physician-to-patient contact time is limited. While conventional hospital-based, physician-oriented approach has been the main focus of chronic kidney disease (CKD) care, a comprehensive multidisciplinary health care program (Integrated CKD Care) has been introduced as an alternate intervention to delay CKD progression in a community population. The main objective is to assess effectiveness of Integrated CKD Care in delaying CKD progression., Methods: We carried out a community-based, cluster randomized controlled trial. Four hundred forty-two stage 3-4 CKD patients were enrolled. In addition to the standard treatments provided to both groups, the patients in the intervention group also received "Integrated CKD Care". This was delivered by a multidisciplinary team of hospital staff in conjunction with a community CKD care network (subdistrict healthcare officers and village health volunteers) to provide group counseling during each hospital visit and quarterly home visits to monitor compliance with the treatment. Duration of the study was 2 years. The primary outcome was difference of mean eGFR between the intervention and the control groups over the study period., Results: The mean difference of eGFR over time in the intervention group was significantly lower than the control group by 2.74 ml/min/1.73 m 2 (95%CI 0.60-4.50, p = 0.009). Seventy composite clinical endpoints were reported during the study period with significantly different incidences between the control and the intervention groups (119.1 versus 69.4 per 1000 person-years; hazard ratio (HR) 0.59, 95% CI 0.4-0.9, p = 0.03)., Conclusion: Integrated CKD Care can delay CKD progression in resource-limited settings., Trial Registration: ( NCT01978951 ). Prospectively registered as of December 8, 2012.

Viral surface proteins, premembrane protein (prM) and envelope (E) protein have been shown to induce a production of antibodies that are involved in both enhancement and neutralization. To explore the feasibility of modifying the relative immune responses to prM and E proteins, four DNA constructs were created and administered into groups of Balb/c mice; pPW01 contains prM and E genes of DENV1, pPW02 contains prM and E genes of DENV2, pPW03 contains DENV1 prM and DENV2 E, and pPW04 contains DENV2 prM and DENV1 E. Exchange of either prM or E from a heterologous serotype does not appear to have an effect on the immunogenicity of the proteins. We have proved that the chimeric pPW03 and pPW04 constructs can produce humoral response in mice. Immunized sera were subjected to neutralization and enhancement assays against DENV2. The results showed that only serotype-specific anti-E antibodies conferred protective function, while the cross-reactive anti-E and anti-prM enhanced infection. In addition, the enhancement of DENV2 infection exhibited a serotype-preference for anti-E antibodies while such response was not observed with anti-prM, reflecting a degree of structural conservation of prM. Taken together, neutralization and enhancement appeared to occur at the same time during the course of infection. Successful prevention of severe symptoms of DENV infection depends on the ability to induce high levels of neutralizing antibodies to subdue the effect of enhancing antibodies.

Lipopolysaccharide (LPS) structural modifications have been shown to specifically affect the pathogenesis of many gram-negative pathogens. In Francisella, modification of the lipid A component of LPS resulted in a molecule with no to low endotoxic activity. The role of the terminal lipid A phosphates in host recognition and pathogenesis was determined using a Francisella novicida mutant that lacked the 4' phosphatase enzyme (LpxF). The lipid A of this strain retained the phosphate moiety at the 4' position and the N-linked fatty acid at the 3' position on the diglucosamine backbone. Studies were undertaken to determine the pathogenesis of this mutant strain via the pulmonary and subcutaneous routes of infection. Mice infected with the lpxF-null F. novicida mutant by either route survived primary infection and subsequently developed protective immunity against a lethal wild-type (WT) F. novicida challenge. To determine the mechanism(s) by which the host controlled primary infection by the lpxF-null mutant, the role of innate immune components, including Toll-like receptor 2 (TLR2), TLR4, caspase-1, MyD88, alpha interferon (IFN-α), and gamma interferon(IFN-γ), was examined using knockout mice. Interestingly, only the IFN-γ knockout mice succumbed to a primary lpxF-null F. novicida mutant infection, highlighting the importance of IFN-γ production. To determine the role of components of the host adaptive immune system that elicit the long-term protective immune response, T- and B-cell deficient RAG1(-/-) mice were examined. All mice survived primary infection; however, RAG1(-/-) mice did not survive WT challenge, highlighting a role for T and B cells in the protective immune response.

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria.

Francisella tularensis (Ft) is a highly infectious gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow-derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88(-/-) mice infected with either strain. MyD88(-/-) mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant.

Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.

A serotyping assay for hepatitis C virus (HCV) was evaluated with samples from Thailand, where the distribution of HCV genotypes was different from that in Western countries where the assay was designed and validated. The sensitivity of the assay was low (58%) for HCV RNA-positive samples compared to that of the genotyping assay (95%, P < 0.01). In addition, only 36% of anti-HCV-positive but HCV RNA-negative samples could be serotyped. The serotypes and genotypes were identical in 96% of the samples that could be typed by both methods. Most of the samples with genotype 6, which was common in Southeast Asia, were nontypeable by this serotyping assay.

The immunoreactivity profiles of plasma samples obtained from patients infected with different hepatitis C virus (HCV) genotypes were studied using immunoblot assay containing multiple HCV antigens. The immunoblot assay was found to be positive in 81.5% of 195 blood donors who had anti-HCV antibodies as detected by second generation enzyme immunoassays. The samples reacted preferentially with the viral core, NS3-1 and NS5 antigens, and these reactivities were not influenced by HCV genotype. However, the reactivities with NS3-2 and NS4 antigens varied depending on HCV genotypes. The samples from patients infected with HCV genotype 1 reacted well with NS3-2 and NS4 antigens whereas those with other genotypes did not. In addition, samples with the unclassified HCV genotype reacted poorly with all antigens, except NS3-1. This study demonstrates the importance of the core, NS3-1 and NS5 antigens in the detection of antibodies against HCV, especially in areas where more than one genotypes of HCV are present. It also demonstrates that there is a need for further improvement of the currently used assays as new HCV genotypes are recently discovered.

The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5' noncoding and core regions of the viral genome were hybridized to genotype- and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.

The prevalences of infections with hepatitis C virus (HCV) and hepatitis B virus (HBV) were determined in 110 Thai patients with liver cancer, of whom 80 and 30 had histological diagnoses of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), respectively. Hepatitis B surface antigen was detected in 63.8% of HCC patients and 16.7% of those with CCA. Antibodies to HCV, detected by a third-generation enzyme immunoassay, were found in 11.3% of HCC patients and in no CCA patient. HCV ribonucleic acid (RNA) was detected by polymerase chain reaction in 6 anti-HCV positive patients, and also in 2 patients who had no detectable anti-HCV antibody. A total of 11 patients had evidence of HCV infection, 8 of whom were infected with HCV alone. HCV genotypes were determined in all 8 patients who had HCV RNA; genotype 3a was the most common (62.5%). These results demonstrate that, in Thailand where both HBV and HCV are endemic, HBV infection is still the most important risk factor for HCC, but HCV also has an important role in those without HBV infection. In addition, the genotypic distribution of HCV in HCC in Thailand is similar to that in the general population. No specific association between genotype 1b and HCC was observed.

An improved system for amplification of hepatitis C virus genome (HCV) was developed based on a multiplex nested polymerase chain reaction format. Two sets of oligonucleotide primers were used simultaneously. One was derived from the conserved sequences in the 5' non-coding region of the viral genome which can bind to the viral genome of all genotypes. The other set of primers was designed from a sequence in the nonstructural-5 region of HCV. HCV genotypes 1 and 3 can be differentiated by the banding patterns of amplified DNA products. All of 39 samples containing the HCV genotype 1 could be amplified with primers in the 5' non-coding region only, whereas 92% of those with genotype 3 could be amplified by both primer sets. In addition, HCV RNA can be detected in 81% of 84 anti-HCV-positive blood donors and in 0% of 34 anti-HCV-negative cases. Of the HCV RNA-positive specimens, 69% showed genotype 1-like patterns while 31% showed genotype 3-like patterns. The detection rate of HCV RNA in this study was much higher than that in our previous report due to the improvement of new primers which can detect all genotypes of the virus. In conclusion, this improved amplification system is a sensitive method for rapid identification of HCV RNA in clinical specimens that can simultaneously differentiate the two most common genotypes of HCV found in Thailand.