Jieqiong Li

Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

ABSTRACT Mycobacterium tuberculosis can acquire resistance to rifampin (RIF) through mutations in the rpoB gene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in the rpoA or rpoC gene. This study aimed to identify rpoA and rpoC mutations in clinical M. tuberculosis isolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found in rpoA or rpoC of the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in the rpoA (n = 6) or rpoC (n = 83) gene. The proportion of rpoC mutations was significantly higher in new cases (P = 0.023) and in strains with the rpoB S531L mutation (P < 0.001). In addition, multidrug-resistant (MDR) strains with rpoC mutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P = 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDR M. tuberculosis strains.

Genetic Contribution of CISH Promoter Polymorphisms to Susceptibility to Tuberculosis in Chinese Children

Tuberculosis (TB) is the leading cause of death due to an infectious disease worldwide, particularly in developing countries. A series of candidate genes have been suggested to be associated with development of TB disease. Among them, the human Cytokine-inducible Src homology 2(SH2) domain protein (CISH) gene has been very recently reported to be involved in T cell activation and differentiation in response to Mycobacterium tuberculosis infection. Here, we studied the association between CISH promoter polymorphisms and pediatric TB. A case-control study enrolled 352 TB patients and 527 healthy controls, who were of Han Chinese ethnicity and aged from 0.2 to 18 years. CISH gene promoter SNPs rs414171, rs622502 and rs809451 were genotyped in all subjects and transcriptional activity, mRNA level, and plasma cytokine level of subjects with different genotypes were further examined. Carriers with rs414171TT homozygotes and rs809451GC heterozygotes had a 1.78-fold (95% CI,1.16–2.74) and 1.86-fold (95% CI, 1.26–2.74) excess risk of developing TB compared to those with wild-type genotypes. A greater risk of TB disease was observed in population carrying C−809451-T−414171-C−622502 haplotype (OR 3.66, 95% CI:2.12–6.32). The G−809451-A−414171-C−622502-containing CISH promoter drove a 5.43-fold increased reporter expression compared to the C−809451-T−414171-C−622502-containing counterpart in Hela cell lines (P = 0.0009). PBMCs carrying rs414171TT homozygotes and rs809451GC heterozygotes showed a reduced CISH mRNA level compared to cells carrying wild type genotypes. Individuals with the rs414171TT genotype had significantly increased IL-12p40 and IL-10 production. In conclusion, CISH promoter rs414171 and rs809451 polymorphisms may play a vital role in mediating individual susceptibility to tuberculosis.

Impaired Function of CD5+CD19+CD1dhi B10 Cells on IgE Secretion in an Atopic Dermatitis-Like Mouse Model

Atopic dermatitis (AD) is a chronic inflammatory pruritic skin disease in which the pathogenic mechanism is complicated and not completely understood. Reports on the role of regulated cells in AD have recently evolved to regulate B cells, which may play a role in allergic inflammation as well. In the present study, we examined the frequency and regulatory function of CD5+CD19+CD1dhi B10 cells in an AD-like mouse model. Our results showed that the percentage of CD5+CD19+CD1dhi B10 cells increased while the frequency of IL-10-producing B cells in CD19+B cells decreased in the mice of AD group. Moreover, no difference in the percentage of B10pro+B10 cells was observed between the AD and control groups. Strikingly, B10 cells from control mice effectively inhibited IgE secretion, whereas the suppressive function of B10 cells from the AD mice was significantly decreased, which was similar to that observed in the group without B10. Altogether, these results suggest that the number of IL-10-producing B cells decreased in the AD group and these cells showed a defective regulatory function on IgE secretion.

Performance of the Interferon Gamma Release Assays in Tuberculosis Disease in Children Five Years Old or Less

Interferon Gamma Release Assays (IGRAs) were developed for the indirect or immunologic diagnosis of tuberculosis infection; however, they have also been used to assist in difficult to diagnose cases of tuberculosis disease in adults, and to a lesser extent, in children, especially in those under 5 years old. We evaluated the utility of using an IGRA in pediatric tuberculosis in younger children in a hospital setting. The diagnostic accuracy of T-SPOT.TB and TST was assessed in 117 children with active tuberculosis and 413 children with respiratory tract infection. Sensitivity and specificity were calculated for the tests used individually and together. Concordance was also calculated. Sensitivity of T-SPOT.TB (82.9%) was higher than TST (78.6% using a 5mm cut-off), especially in children confirmed to have TB. T-SPOT.TB was more specific than TST using a 5mm cut-off (96.1% vs. 70.9%). Combining T-SPOT.TB and TST results improved the sensitivity to 96.6%. In conclusion, the results of the current study indicate that T-SPOT.TB has good sensitivity and specificity, supporting its use among patients of this age. A combination of IGRA and TST would be useful additions to assist in the diagnosis of childhood TB.

Utility of Novel Plasma Metabolic Markers in the Diagnosis of Pediatric Tuberculosis: A Classification and Regression Tree Analysis Approach

Although tuberculosis (TB) has been the greatest killer due to a single infectious disease, pediatric TB is still hard to diagnose because of the lack of sensitive biomarkers. Metabolomics is increasingly being applied in infectious diseases. But little is known regarding metabolic biomarkers in children with TB. A combination of a NMR-based plasma metabolic method and classification and regression tree (CART) analysis was used to provide a broader range of applications in TB diagnosis in our study. Plasma samples obtained from 28 active TB children and 37 non-TB controls (including 21 RTIs and 16 healthy children) were analyzed by an orthogonal partial least-squares discriminant analysis (OPLS-DA) model, and 17 metabolites were identified that can separate children with TB from non-TB controls. CART analysis was then used to choose 3 of the markers, l-valine, pyruvic acid, and betaine, with the least error. The sensitivity, specificity, and area under the curve (AUC) of the 3 metabolites is 85.7% (24/28, 95% CI, 66.4%, 95.3%), 94.6% (35/37, 95% CI, 80.5%, 99.1%), and 0.984(95% CI, 0.917, 1.000), respectively. The 3 metabolites demonstrated sensitivity of 82.4% (14/17, 95% CI, 55.8%, 95.3%) and specificity of 83.9% (26/31, 95% CI, 65.5%, 93.9%), respectively, in 48 blinded subjects in an independent cohort. Taken together, the novel plasma metabolites are potentially useful for diagnosis of pediatric TB and would provide insights into the disease mechanism.

Characterization of plasma proteins in children of different Mycobacterium tuberculosis infection status using label-free quantitative proteomics

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is an infectious disease found worldwide. Children infected with MTB are more likely to progress to active TB (ATB); however, the molecular mechanism behind this process has long been a mystery. We employed the label-free quantitative proteomic technology to identify and characterize differences in plasma proteins between ATB and latent TB infection (LTBI) in children. To detect differences that are indicative of MTB infection, we first selected proteins whose expressions were markedly different between the ATB and LTBI groups and the control groups (inflammatory disease control (IDC) and healthy control (HC) groups). A total of 521 proteins differed (> 1.5-fold or < 0.6-fold) in the LTBI group, and 318 proteins in the ATB group when compared with the control groups. Of these, 49 overlapping proteins were differentially expressed between LTBI and ATB. Gene Ontology (GO) analysis revealed most proteins had a cellular and organelle distribution. The MTB infection status was mainly related to differences in binding, cellular and metabolic processes. XRCC4, PCF11, SEMA4A and ATP11A were selected and further verified by qPCR and western blot. At the mRNA level, the expression of XRCC4, PCF11and SEMA4A presented an increased trend in ATB group compare with LTBI. At the protein level, the expression of all these proteins by western blot in ATB/LTBI was consistent with the trends from proteomic detection. Our results provide important data for future mechanism studies and biomarker selection for MTB infection in children.

Positive epistasis of major low-cost drug resistance mutations rpoB531-TTG and katG315-ACC depends on the phylogenetic background of mycobacterium tuberculosis strains

Mycobacterium tuberculosis Beijing genotype strains increasingly circulate in different world regions, either as historical endemic, e.g. in East Asia, or recently imported, e.g. in South America, and this family is regarded as the most successful lineage of the global tuberculosis (TB) epidemic. Here we analysed the transmission capacity of these strains in the context of their phylogenetic background and drug resistance mutations. The study collection included all multidrug resistant (MDR) strains of Beijing genotype isolated in Beijing Chest Hospital, the largest tertiary TB facility in North China, in 2011-2013 (n = 278). Strains were subjected to NTF/IS6110 and 24-loci MIRU-VNTR analysis. Drug resistance mutations were detected in rpoB, katG, inhA and oxyR-ahpC. A total of 58 and 220 strains were assigned to the ancient and modern Beijing sublineages, respectively. 24-MIRU-VNTR clustering was higher in modern versus ancient Beijing strains (35.9% vs. 12.1%; P <0.001). After taking into consideration the presence of rpoB and katG mutations, clustering decreased to 15.9% in modern and 0% in ancient strains. The most frequent combination of mutations (rpoB531-TTG and katG315-ACC) was more prevalent in clustered versus non-clustered isolates in the modern sublineage (23/35 vs. 47/185; P <0.0001). To conclude, a combination of the known low-fitness-cost rpoB531-TTG and katG315-ACC mutations likely facilitates the increased transmission ability of MDR strains of the modern but not ancient Beijing sublineage. Accordingly, positive epistasis of major low-cost drug resistance-conferring mutations is influenced by the phylogenetic background of M. tuberculosis strains.

Discovery of susceptibility loci associated with tuberculosis in Han Chinese

Genome-wide association studies (GWASs) have revealed the worldwide heterogeneity of genetic factors in tuberculosis (TB) susceptibility. Despite having the third highest global TB burden, no TB-related GWAS has been performed in China. Here, we performed the first three-stage GWAS on TB in the Han Chinese population. In the stage 1 (discovery stage), after quality control, 691 388 SNPs present in 972 TB patients and 1537 controls were retained. After replication on an additional 3460 TB patients and 4862 controls (stages 2 and 3), we identified three significant loci associated with TB, the most significant of which was rs4240897 (logistic regression P = 1.41 × 10-11, odds ratio = 0.79). The aforementioned three SNPs were harbored by MFN2, RGS12 and human leukocyte antigen class II beta chain paralogue encoding genes, all of which are candidate immune genes associated with TB. Our findings provide new insight into the genetic background of TB in the Han Chinese population.

Rs1914663 of SFTPA 1 gene is associated with pediatric tuberculosis in Han Chinese population.

UNLABELLED Surfactant protein A (SP-A), a part of the innate immune system of the lung, performs a vital role in the host defense against Mycobacterium tuberculosis (MTB) infection. In order to investigate the relationship between SFTPA polymorphism variations and Tuberculosis (TB) in a Chinese pediatric group, we conducted a case-control study using single-nucleotide polymorphism (SNP) analysis. Significant difference of the allelic distribution of rs1914663 in SFTPA gene was observed between TB group and control group and, T allele of rs1914663 was associated with increased risk for TB (control vs. TB, OR 1.42, 95% CI: 1.10-1.81, P=0.005). In addition, the TC+TT genotype of rs1914663 was higher in PTB and non-severe TB than that in controls. The haplotype comprising rs17881720-A and rs17879335-G was a resistance factor while the haplotype comprising rs1914663-T and rs1059225-G was found to be a susceptibility factor to TB. Using a case-control study, we identified a genetic polymorphism in the SFTPA that regulates host susceptibility to pediatric TB in the Han Chinese population.

Screening and Identification of APOC1 as a Novel Potential Biomarker for Differentiate of Mycoplasma pneumoniae in Children.

Background: Although Mycoplasma pneumoniae (MP) is a common cause of community-acquired pneumonia (CAP) in children, the currently used diagnostic methods are not optimal. Proteomics is increasingly being used to study the biomarkers of infectious diseases. Methods: Label-free quantitative proteomics and liquid chromatography-mass/mass spectrometry were used to analyze the fold change of protein expression in plasma of children with MP pneumonia (MPP), infectious disease control (IDC), and healthy control (HC) groups. Selected proteins that can distinguish MPP from HC and IDC were further validated by enzyme-linked immunosorbent assay (ELISA). Results: After multivariate analyses, 27 potential plasma biomarkers were identified to be expressed differently among child MPP, HC, and IDC groups. Among these proteins, SERPINA3, APOC1, ANXA6, KNTC1, and CFLAR were selected for ELISA verification. SERPINA3, APOC1, and CFLAR levels were significantly different among the three groups and the ratios were consistent with the trends of proteomics results. A comparison of MPP patients and HC showed APOC1 had the largest area under the curve (AUC) of 0.853, with 77.6% sensitivity and 81.1% specificity. When APOC1 levels were compared between MPP and IDC patients, it also showed a relatively high AUC of 0.882, with 77.6% sensitivity and 85.3% specificity. Conclusion: APOC1 is a potential biomarker for the rapid and noninvasive diagnosis of MPP in children. The present finding may offer new insights into the pathogenesis and biomarker selection of MPP in children.