Correlation of ovalbumin of egg white components with allergic diseases in children.

BACKGROUND: Immunoglobulin E (IgE)-mediated food allergy, such as egg white allergy, is common in young children (<3 years old), but not all young children sensitive to egg white present with allergic symptoms. This study investigated the relationship between sensitization to egg white component allergens and clinical manifestations of allergic diseases in young children. METHODS: From March to December 2010, 2256 children with physician-diagnosed allergic diseases were tested for serum levels of egg white, ovalbumin, and ovomucoid-specific IgE in the Pediatric Allergy and Asthma Center of Chang Gung Memorial Hospital. Serum was analyzed for specific IgE antibodies to egg white, ovalbumin, and ovomucoid by ImmunoCAP (Phadia, Uppsala, Sweden). Allergen-specific IgE levels ≥0.35 kUA/L were defined as positive. RESULTS: There was a significantly higher sensitization rate to egg white and its components in children aged 2-4 years old. The sensitization rate to egg white, ovalbumin, and ovomucoid in this age group was 53.5%, 48.3%, and 37.2%, respectively, and the trend of the sensitization decreased with age (p < 0.001). After adjusting for age, sensitization to egg white and ovalbumin was associated with children with dermatitis [egg white: odds ratio (OR) = 1.28, 95% confidence intervals (CI) = 1.03-1.58, p < 0.05; ovalbumin: OR = 1.30, 95% CI = 1.04-1.62, p < 0.05]. Children with ovomucoid sensitization had no statistically significant risk among different groups in the current study. CONCLUSION: Children aged 2-4 years old have higher sensitivity to egg white, ovalbumin, and ovomucoid. Children with egg white and ovalbumin sensitization have a higher risk for atopic dermatitis, and ovalbumin has a more important contribution. Furthermore, we suggested that in children with atopic dermatitis, if they are aged 2-4 years old and are having egg white and ovalbumin sensitization, avoiding eating raw or slightly heated eggs might have a beneficial effect.

Total serum IgE in a population-based study of Asian children in Taiwan: reference value and significance in the diagnosis of allergy.

BACKGROUND: Total serum immunoglobulin (IgE) test is usually performed to aid in the diagnosis of allergic diseases, but its reference values may vary among people of different ethnic backgrounds. OBJECTIVES: To establish reference values of total IgE in Asian children and to assess their significance in the diagnosis of atopy and allergic diseases. STUDY DESIGN: 1321 Asian children aged 5-18 years in the Prediction of Allergies in Taiwanese CHildren (PATCH) study, a population-based cohort, were evaluated for total and specific IgE by ImmunoCAP and Phadiatop Infant, respectively. RESULTS: Male, atopy, allergic diseases, recent symptoms of upper respiratory infection, and lower FEV1/FVC, were associated with higher total IgE levels in univariate analyses. Multivariate analysis revealed that atopy was the single most important determinant explaining 66.1% of the variability of total IgE levels in this population. The area under the receiver-operator characteristic (ROC) curve of total IgE for diagnosing atopy, asthma, rhinitis, and eczema were 0.92, 0.72, 0.70, and 0.70, respectively. The sensitivity, specificity, and positive and negative predictive values of total IgE at the optimal cutoff of 77.7 kU/L on the ROC curve for diagnosing atopy were 82.3%, 87.1%, 89.5%, and 78.6%, respectively. The corresponding values using the upper 95% CI of total IgE (164.3 kU/L) in non-atopic children were 61.2%, 95.0%, 94.3%, and 64.6%, respectively; whereas a customary cutoff (100 kU/L) provided accuracy between that of the aforementioned two cutoffs. Total IgE at the cutoff of 77.7 kU/L provided modest sensitivity and specificity (49.0%-78.3%) for diagnosing allergic diseases, but had high negative predictive values (84.2%-97.9%). CONCLUSIONS: Total serum IgE discriminates Asian children with and without atopy independent of allergic symptoms, with an optimal cutoff of 77.7 kU/L. The study confirms the insufficient diagnostic accuracy of total IgE alone to detect allergic diseases, but low total IgE levels may help exclude allergic diseases.

Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen.

SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye. Preferential binding of SG during PCR and inhibition of PCR often result in failure to detect multiple amplicons in multiplex reactions. In the present study, a novel single-tube, multiplex real-time PCR with EvaGreen dye (EG) was developed and evaluated for simultaneous detection of pathogenic targets by using five potato viruses as models. The PCR products obtained using five sets of specific primers were analyzed by melting curve analysis. The assay could specifically detect and differentiate the five potato viruses by producing a distinct peak for each amplification product and exhibited a high reproducibility with coefficients of variation from 0.01 to 0.25 %. Detection sensitivity of the assay ranged from 100 to 500 copies/µL for each virus. The results of this study demonstrate that multiplex real-time PCR and melting-curve analysis with EG is a sensitive, specific and inexpensive method for simultaneous detection of multiple pathogens.

Patterns of sensitization to peanut allergen components in Taiwanese Preschool children.

BACKGROUND/PURPOSE: Peanut allergy is very common in Western countries, although it is seldom encountered in Eastern countries. Peanuts are comprised of at least 11 components, but the contribution to clinical symptoms by each component in each individual is not known. This study investigated the distributions of sensitivity to peanut allergen components among Taiwanese children who were sensitized to peanuts and followed the evolution of sensitization patterns to these components. METHODS: We enrolled 29 preschool children (age=2.11±1.36 years) who were sensitized to peanuts above class 3. Serum was analyzed for specific immunoglobulin E (IgE) antibodies to recombinant Ara h 1, Ara h 2, Ara h 3, Ara h 8, and Ara h 9. Allergen component-specific IgE ≥0.35 kU(A)/L was defined as positive. Eighteen children were retested 22.64±5.1 months later. Peanut allergy symptoms were recorded from detailed questionnaires. RESULTS: The percentages of children sensitized to Ara h 1, 2, 3, 8, and 9 were, respectively, 51.8%, 65.5%, 62.1%, 13.8%, and 24.1%. Regarding changing patterns of peanut component sensitization at follow-up, children with clinical symptoms to peanuts had persistent elevations of Ara h 2-specific IgE: 12.6±1.01 up to 34.15±19.4 kU(A)/L; p=0.144. In contrast, Ara h 2 concentrations decreased significantly in children without clinical symptoms. Ara h 8 and 9 were nonspecific for children with or without symptoms. CONCLUSION: Ara h 1, Ara h 2, and Ara h 3 were major components contributing to peanut sensitization in Taiwanese children. Ara h 2 was probably the most important component that contributed to clinical symptoms and remained steady in children who had peanut allergy.

A novel diagnostic platform based on multiplex ligase detection-PCR and microarray for simultaneous detection of swine viruses.

Simultaneous detection and identification of multiple pathogens is required in many diagnostic fields. In this study a novel method based on a multiplex ligase detection (LD)-polymerase chain reaction (PCR) and microarray (MLPM) is described to detect simultaneously several swine viruses involved in reproductive and/or respiratory problems. The multiplex diagnostic system was validated using standard plasmids, and clinical samples. Using this strategy as few as 10 copies of target plasmids were detected successfully. Each probe pair yielded specific positive signal only in its target site. In addition, when six target plasmids were present simultaneously sufficient robust signals were generated in their corresponding sites of six plasmid templates and no obvious signals were detected in non-target sites. Compared to real-time PCR, the MLPM showed specificities and sensitivities of 95.7-100% and 100% for 47 clinical samples tested, respectively. The results demonstrate that this novel assay is a specific, sensitive, and multiplex diagnostic method for detection of multiple pathogens and can also be adapted easily for diagnostic purposes.

Development of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for diagnostic platform.

A multiplex reversal transcription-ligase detection reaction-polymerase chain reaction (MRLP) based universal microarray for the simultaneous pathogens detection was established by using potato viruses as a model. The proposed procedure integrated LDR for multiplicity and specificity, PCR amplification by universal primers for sensitivity, which required design of upstream and downstream LDR probes specific for each virus, and subsequent Zip-code microarray for multiplex and specific identification. Each MRLP fragments carried a unique sequence (complementary Zip-code sequence, cZip-code) which identified a virus by addressed to the location on the microarray where the Zip-code sequence has been spotted. Such Zip-code microarray and universal primers are therefore "universal" being unrelated to a specific molecular analyte. With this technique, target RNAs of ten potato viruses were reversal transcribed by random primer in a single reaction, then subjected to LDR and asymmetric labeling PCR as template, finally, the MRLP amplicons were analyzed by microarray hybridization and subsequent scanning. The technique platform was optimized and evaluated by using reference samples and artificial samples, which can specifically detect down to 3 copies of single or mixed plasmid templates. Due to its universality, multiplexing, specificity and sensitivity, this method can be recommended for simultaneously detecting a large number of different target types in related fields.