Plasma Metabolic Profiling of Pediatric Sepsis in a Chinese Cohort.

Sepsis represents one of the most pressing problems in pediatrics, characterized by pathogenic bacteria invading the blood, growing and multiplying in the blood circulation, and ultimately causing severe infections. Most children with sepsis have a rapid disease onset and frequently exhibit sudden high fever or first chills. Here we performed comprehensive metabolomic profiling of plasma samples collected from pediatric sepsis patients to identify specific metabolic alterations associated with these patients (n = 84, designated as case subjects) as compared to healthy cohorts (n = 59, designated as control subjects). Diagnostic models were constructed using MetaboAnalyst, R packages, and multiple statistical methods, such as orthogonal partial least squares-discriminant analysis, principal component analysis, volcano plotting, and one-way ANOVA. Our study revealed a panel of metabolites responsible for the discrimination between case and control subjects with a high predictive value of prognosis. Moreover, significantly altered metabolites in sepsis survivors versus deceased patients (non-survivors) were identified as those involved in amino acids, fatty acids, and carbohydrates metabolism. Nine metabolites including organic acids and fatty acids were also identified with significantly higher abundance in sepsis patients with related microbes, implicating greater potentials to distinguish bacterial species using metabolomic analysis than blood culture. Pathway enrichment analysis further revealed that fatty acid metabolism might play an important role in the pathogenesis of sepsis.

Novel OCRL1 gene mutations in six Chinese families with Lowe syndrome.

BACKGROUND: Lowe syndrome, an X-linked, inheritable disease with clinical symptoms of congenital cataracts, incomplete Fanconi syndrome, and mental retardation, has an approximate incidence of 1 in 500 000. Nearly 200 OCRL mutations related to Lowe syndrome have been found worldwide, with only ten mutations among the Chinese population. Since more mutations may exist in Chinese patients, we sequenced and analyzed the OCRL genes of six children with Lowe syndrome in a medical center in China. METHODS: Peripheral blood was collected from six children with Lowe syndrome and their relatives, and ten healthy adults. Genomic DNA was extracted from the blood and applied to amplify the twenty-four exons and flanking introns of the OCRL gene. The mutations were identified by sequencing. RESULTS: Five mutations (c.1528C>T, c.2187insG, c.1366C>T, c.1499G>A, and c.2581G>A) of the OCRL gene were found in five families; c.2187insG and c.1366C>T were novel mutations. None of the five mutations were detected in 20 normal chromosomes. No mutation was found in the sixth family. CONCLUSION: Two novel mutations of the OCRL gene, c.2187insG and c.1366C>T, were found in Chinese patients with Lowe syndrome, which will provide new clues for the etiology of Lowe syndrome and could be beneficial to genetic diagnosis of the condition.

Phylogenetic analysis of the VP1 gene of Enterovirus 71 in Guangzhou during the high occurrence period of 2008.

An outbreak of hand, foot, and mouth disease (HFMD) in Guangzhou in 2008 affected over 10,000 children and resulted in high hospital admission rates. To investigate the molecular epidemiological pattern of EV71 infections in Guangzhou, throat swab samples were collected from 102 children clinically diagnosed with HFMD from May to July of 2008 in Guangzhou. Partial VP1 (virus protein 1) fragments of Enterovirus 71 (EV71) isolates were sequenced, and used alongside EV71 sequences entered in GenBank to construct a phylogenetic tree using MEGA5.0. Blast and phylogenetic analyses showed that all 21 sequences belonged to subgenogroup C4 of EV71. In early May, diverse strains were circulating in Guangzhou, but by July, only a small number of these strains could be detected. These results could indicate that geographic and climatic features may affect the epidemic characteristics of EV71, and that some C4 strains might retain their infectivity at higher temperatures.

[Relation between insulin resistance and insulin receptor gene methylation in the endometrium of patients with polycystic ovary syndrome].

OBJECTIVE: To study the relationship between insulin resistance and methylation of insulin receptor (INSR) gene in the endometrium of women with polycystic ovary syndrome (PCOS). METHODS: Based on the HOMA index, 35 patients with PCOS were divided into insulin resistant group (IR group, n=18) and non-resistant group (NIR group, n=18). The patients age, serum estriol, testosterone, FSH and LH, fasting insulin and fasting blood glucose were compared between the two groups. The endometrial samples were obtained from the patients to examine DNA methylation status of INSR gene in the endometrial cells using methylation-specific PCR. RESULTS: The BMI, WHR, fasting glucose, fasting insulin, and HOMA index differed significantly between the two groups (P<0.05). PCR analysis showed partial methylation in the promoter region of INSR gene in 13 samples in IR group and 11 samples in NIR group, without detection of full methylation of the INSR gene in either group. The methylation status showed no significant difference between the two groups (P=0.328). CONCLUSION: Partial methylation of the INSR gene occurs in the endometria of PCOS patients, but this study does not provide a strong evidence supporting the relationship between insulin resistance and INSR gene methylation in women with PCOS.

[Effects of allergen and intranasal glucocorticoid on Th17 and RORgamma t in peripheral blood in patients with allergic rhinitis.].

OBJECTIVE: To study the effect of specific immunotherapy and intranasal glucocorticoid on T help 17 (Th17) cells and RORgammat in peripheral blood in patients with allergic rhinitis (AR). METHODS: Forty patients with allergic rhinitis (group A) were divided randomly into two subgroups (group A1 and A2), and each subgroup had 20 patients. The patients in group A1 were treated with intranasal glucocorticoid (INGS) for one-year. The patients in group A2 were treated with special immunotherapy (SIT) for 4 weeks. Blood samples were respectively taken from 10 healthy individuals (group B), 20 AR patients (group A1) before and after SIT with specific standardized allergen and 20 AR patients (group A2) before and after INGS. The ratio of Th17 cells in peripheral blood monouclear cells (PBMC) were analysed by flow cytometry. The expression of RORgammat mRNA were detected by real-time polymerase chain reaction and the interleukin-23(IL-23), IL-17, IL-6 were detected by enzyme-linked immunosorbent assay. RESULTS: The ratio of Th17 cells in PBMC and the expression of RORgammat mRNA in group A [(18.97 +/- 1.05)% and (0.604 +/- 0.027)] were respectively higher than those in group B [(15.12 +/- 1.09)% and (0.447 +/- 0.024)] and the difference reached statistical significance (t were respectively -10.056 and -17.986, each P < 0.01). The level of IL-6, IL-17 and IL-23 in group A were respectively higher than those in group B and the difference reached statistical significance (t were respectively -41.149, -17.618 and -26.824, all P < 0.01). The ratio of Th17 cells in PBMC, the expression of RORgammat mRNA, the level of IL-6, IL-17 and IL-23 before INGS did not show significant difference from those of after INGS in group A1 (t were respectively 0.298, 0.240, -1.136, 0.283 and -1.670, all P > 0.05). The ratio of Th17 cells in PBMC and the expression of RORgammat mRNA were respectively (18.99 +/- 1.14)% and (0.603 +/- 0.027) before SIT and were respectively (16.30 +/- 1.63)% and (0.429 +/- 0.023) after SIT in group A2, and the difference reached statistical significance (t were respectively 6.035 and 22.015, all P < 0.01). The level of IL-6, IL-17 and IL-23 before SIT were lower respectively than those of after SIT in group A2 and the difference reached statistical significance (t were respectively 9.235, 11.289, 7.267, all P < 0.01). CONCLUSIONS: The ratio of Th17 cells in PBMC, the expression of RORgammat mRNA, the level of IL-6, IL-17 and IL-23 were up-regulated in patients with AR. The treatment of SIT could get the 5 items down and the treatment of INGS couldn't.

Retrospective study of adenovirus in autopsied pulmonary tissue of pediatric fatal pneumonia in South China.

BACKGROUND: Adenovirus are the important pathogen of pediatric severe pneumonia. The aim of this study is to analyze the infection, subtype and distribution of adenovirus in autopsied pulmonary tissue of fatal pneumonia in infants and children, and the relationships between adenovirus infection and respiratory illness in South China. METHODS: Nested PCR was performed on DNA extracted from autopsied lung tissue from patients who died of severe pneumonia, and the positive nested PCR products were cloned and sequenced. The adenovirus in autopsied pulmonary tissue was also analyzed by immunohistochemistry assay in a blind way. RESULTS: In the 175 autopsied pulmonary tissues, the positive percentage of adenovirus was 9.14% (16/175) and 2.29% (4/175) detected with nested PCR and immunohistochemistry, respectively. There are three cases of adenovirus serotype 3, twelve cases of adenovirus serotype 4 and one case of serotype 41 determined by sequencing of the cloned positive nested PCR products. CONCLUSION: Adenovirus is an important cause of severe pneumonia, and these data suggest that adenovirus serotype 4 might be an important pathogen responsible for the fatal pneumonia in Guangzhou, South China.

Retrospective analysis of Mycoplasma pneumoniae infection in pediatric fatal pneumonia in Guangzhou, South China.

The objective of this study is to investigate the infection and distribution of Mycoplasma pneumoniae in autopsied pulmonary tissue of pediatric severe pneumonia. Mycoplasma pneumoniae nested polymerase chain reaction and immunohistochemistry were done on autopsy pulmonary tissue from 173 patients who died of severe pneumonia. Mycoplasma pneumoniae was identified in 135/173 (78.03%) and 114/173 (65.89%) samples of autopsied pulmonary tissue of lethal severe pneumonia via nested polymerase chain reaction and immunohistochemistry, respectively. The coincidence of both assays was 92.4%. Mycoplasma pneumoniae associated fatal pneumonia has showed an increasing trend from 1988 to 2005 in South China, and the fatality rate of Mycoplasma pneumoniae associated fatal pneumonia in infants, 1 to 12 months, has risen to 66.9% (97/145). Mycoplasma pneumoniae is a significant cause of severe pneumonia, it is a universal event in infants, and children have died of severe pneumonia in South China. Mycoplasma pneumoniae might be an important pathogen responsible for fatal pneumonia in Guangzhou area, South China.

Determination of hepatitis B virus genotype by flow-through reverse dot blot.

BACKGROUND: The clinical outcome and response to therapy of hepatitis B virus infection differ depending upon viral genotype. Most methods of determining the viral genotype are relatively time-consuming and costly. Moreover, the results of some methods are influenced by single nucleotide mutations. OBJECTIVES: To develop a novel HBV genotyping process insensitive to single nucleotide mutations using an improved reverse dot blot method employing the principle of "flow-through hybridization". STUDY DESIGN: The flow through reverse dot blot (FT-RDB) method was developed using DNA from different HBV genotypes. HBV sequences from Genebank were used to design primers and probes. Specificity and sensitivity of the method were evaluated with clinical samples in which the HBV viral load was quantified by real-time PCR. Results were compared to those of multiplex PCR and sequencing. Another 59 clinical samples were used to test the clinical applicability of the method. RESULTS: We showed that FT-RDB could be made insensitive to single nucleotide mismatch by adjusting the hybridization temperature. All HBV-negative samples showed no signals in the assay. The detection sensitivity of the method was found to be between 10(3) and 10(4) DNA copies/ml. The results of FT-RDB were 84% concordant with those of multiplex PCR, and 96% concordant with sequencing results in 101 cases. The genotype all 59 clinical samples was accurately identified. CONCLUSIONS: We demonstrated that the FT-RDB method was rapid, reliable, accurate and inexpensive. It appears to be useful for routine clinical HBV genotyping even in non-specialized hospital laboratories.

Application of an oligonucleotide array assay for rapid detecting and genotyping of Chlamydia trachomatis from urogenital specimens.

An oligonucleotide array technology was established for rapidly detecting and genotyping Chlamydia trachomatis in urogenital infections. The VS1-VS2 region of the omp1 gene was used to design oligonucleotide probes. Eleven serovar-specific probes to serovars A, B, C, D, E, F, G, H, I, J, and K, and 3 group-specific probes to group B (B, Ba, D, E, L1, and L2), group C (A, C, H, I, J, K, and L3), and an intermediate group (F and G) were synthesized and spotted onto the nylon membrane. Two pairs of universal primers were designed for the nested polymerase chain reaction (PCR) amplification of the VS1-VS2 gene. Digoxigenin-labeled amplicons of the VS1-VS2 gene of C. trachomatis were hybridized to the membrane array. Hybridization signals were read by the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate color development. The assay developed was tested with reference strains of C. trachomatis serovars and clinical samples. The sensitivity was evaluated for 57 samples previously found to be positive for C. trachomatis by using plasmid PCR, and 98.2% (56/57) concordance was obtained. Fourteen oligonucleotide probes were optimized by trying different reaction conditions, showing specific hybridization with the corresponding reference strains, but no cross-reactions with other urogenital microorganisms. Using this procedure, a total of 59 strains were detected from 56 chlamydial samples. Eight genotypes were found, and type D, E, F, and H were the most frequently observed types (77.9%). Three cases (5.4%) had multiple infections with serovars: 1.D/E, 2.D/F, and 3.F/K. To validate the reference strains and confirm the genotype identity as determined by the oligonucleotide array technology, we sequenced all reference strains and 10 selected specimens across variable sequence VS1 and VS2. No discrepancies were found between the array typing and the genotype identity confirmed by nucleotide sequencing of the PCR product. The findings from this study indicated that the oligonucleotide array is a simple, fast, and specific assay for directly detecting and genotyping C. trachomatis from clinical samples.

Rapid and accurate genotyping of YMDD motif variants in the hepatitis B virus genome by an improved reverse dot blot method.

By using the "flow-through hybridization" principle, we developed a new, rapid and accurate reverse dot blot (RDB) method to detect lamivudine resistance-associated YMDD motif variants in hepatitis B virus (HBV) genome. The improved RDB method was very fast at simultaneously detecting HBV YMDD wild-type and mutant motifs. In a blind analysis, 100 samples previously genotyped by DNA clonal sequencing analysis were used to evaluate the sensitivity and specificity of this assay. Conventional restriction fragment length polymorphism (RFLP) data were also used to test our method. In blind experiments, our improved RDB method had an accuracy and specificity of 100%, which was much higher than RFLP, which had an accuracy and specificity of only 83.0%. In clinical detection practice, 49 patients highly suspected of lamivudine resistance were successfully diagnosed by this method. Our improved RDB assay is a simple, rapid, cheap, semiautomatic, accurate, sensitive, and contamination-proof method of detecting lamivudine resistance-associated mutants in the human hepatitis B virus genome.

[Expression of T-bet and its relation with IgE and eosinophil cationic protein in patients with allergic rhinitis].

OBJECTIVE: To investigate the expression of T-bet mRNA in peripheral blood mononuclear cells (PBMC) as well as its relations with total IgE (TIgE), eosinophil cationic protein (ECP) in serum and nasal allergic symptoms in patients with allergic rhinitis (AR). METHODS: The allergen, TIgE and ECP in serum of patients with AR were detected by Unicap CAP system. Blood sample was taken from 8 healthy individuals and 22 patients with allergic rhinitis. PBMC was isolated by density gradient centrifugation and one part of them was cultured with 50 microg/ml mite allergen. PBMC was subjected to analysis of T-bet mRNA expression using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The ratio of T-bet to beta-actin mRNA levels was 0.381 +/- 0.099 in patients and 0.750 +/- 0.067 in normal individuals, the difference was significantly (P <0.01). The expression intensity of T-bet mRNA had no relation to varying severity of allergic symptoms and concentration of ECP and the correlation coefficient was 0.187 and -0.165 (all P > 0.05). However, there was an inverse correlation between expression intensity of T-bet mRNA and TIgE concentration (r = -0.525, P < 0.05). Mean mRNA level (x +/- s) of T-bet expression before and after being stimulated by allergen was 0.381 +/- 0.099 and 0.365 +/- 0.104 respectively, which indicated no significant differences (P > 0.05). CONCLUSIONS: Among allergic patients whose allergen was mite, there was a down-regulated expression of T-bet mRNA, which had no relation to ECP concentration and allergic symptoms, but was one of important links in mechanisms of imbalance of Th1/Th2 in AR. There was no effect of specific allergen on T-bet mRNA in patients with AR T-bet was one of indirect factors that affected the level of IgE.