Respiratory microbiota imbalance in children with Mycoplasma pneumoniae pneumonia.

Although previous studies have reported the dysregulation of respiratory tract microbiota in infectious diseases, insufficient data exist regarding respiratory microbiota imbalances in the lower respiratory tracts (LRTs) of children with Mycoplasma pneumoniae pneumonia (MPP). Here, we analysed the microbial community using 16S rRNA gene sequencing. Finally, bronchoalveolar lavage fluid (BALF) samples from 158 children with MPP and 29 with bacterial or viral pneumonia (control group) were collected. The diversity of the microbial community was significantly different between the two groups. A significantly increased abundance of Tenericutes and Mycoplasma was detected in the MPP group, exceeding 67% and 65% of the total bacterial population, respectively. Using Mycoplasma abundance as the diagnostic method, the sensitivity and specificity of the model was 97.5% and 96.6%, respectively. Compared to the mild MPP group, lower alpha diversity and significantly increased Mycoplasma abundance were found in the severe MPP group (P < 0.01). The abundance of Mycoplasma was positively correlated with complications and clinical indices in children with severe MPP compared with children with mild MPP. Our study describes the features of the LRT microbiota of children with MPP and uncovered its association with disease severity. This finding may offer insights into the pathogenesis of MPP in children.

Mutations of Mycobacterium tuberculosis induced by anti-tuberculosis treatment result in metabolism changes and elevation of ethambutol resistance.

Selective pressure from antibiotic use is one of the most important risk factors associated with the development of drug resistance in Mycobacterium tuberculosis (MTB). However, the mechanisms underlying drug resistance at the molecular level remain partly unclear. Therefore, the purpose of this study was to investigate the potential functional effect of novel mutations arising from anti-tuberculosis treatment. We analyzed two multidrug-resistant TB (MDR-TB) isolates from the same patient; one collected before and one almost a year after commencing MDR-TB treatment. The post-treatment isolate exhibited elevated ethambutol resistance. We sequenced the whole genomes of the two clinical isolates and detected six novel polymorphisms affecting the genes Rv1026, nc0021, Rv2155c, Rv2437, and Rv3696c, and the intergenic region between Rv2764c and Rv2765. Metabolomics approach was used to reveal the effect of the found variation on the metabolic pathways of MTB. Partial least squares-discriminant analysis showed a clear differentiation between the two isolates, involving a total of 175 metabolites. Pathway analysis showed that these metabolites are mainly involved in amino sugar and nucleotide sugar metabolism, ß-alanine metabolism, sulfur metabolism, and galactose metabolism. The increased ethambutol resistance exhibited by the post-treatment MDR-TB strain could speculatively be linked to the identified genetic variations, which affected the synthesis of a number of metabolites associated with sources of carbon and energy. This may have been the main factor underlying the increased ethambutol resistance of this isolate.

Clinical and Drug Resistance Characteristics of New Pediatric Tuberculosis Cases in Northern China.

AIM: The aim of this study was to evaluate the clinical features and characteristics of drug resistance in newly diagnosed pediatric tuberculosis (TB) patients in northern China. METHODS: Mycobacterium tuberculosis isolates were collected from September 2010 to October 2016 at the Beijing Children's Hospital. Patients were divided into two groups (resistant to at least one drug and pan-susceptible) according to drug susceptibility testing (DST) results. RESULTS: A total of 132 new cases, mainly from northern China (87.9%), were included in the study. The median age was 1.9 years (1 month-15 years). Resistance to at least one drug was detected in Mycobacterium tuberculosis isolates from 33 (25%) cases. Eight cases of multidrug-resistant TB (MDR-TB) (6.1%) were detected. The two groups did not differ in clinical presentations (disease site, fever >2 weeks, and cough >2 weeks) or in chest imaging (lesion location, lymphadenitis [mediastinal], and pleural effusion). CONCLUSIONS: The rate of Mycobacterium tuberculosis drug resistance in new pediatric TB cases was as high as in the new adult patients surveyed in the national drug resistance survey conducted in 2007. No significant difference was observed in clinical features between patients infected with drug-resistant and drug-susceptible strains. Routine DST is important for prescribing effective antituberculosis treatment regimens.

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.

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.

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.

Evolutionary History and Ongoing Transmission of Phylogenetic Sublineages of Mycobacterium tuberculosis Beijing Genotype in China.

Mycobacterium tuberculosis Beijing genotype originated in China and has undergone a dramatic population growth and global spread in the last century. Here, a collection of M. tuberculosis Beijing family isolates from different provinces across all China was genotyped by high-resolution (24-MIRU-VNTR) and low-resolution, high-rank (modern and ancient sublineages) markers. The molecular profiles and global and local phylogenies were compared to the strain phenotype and patient data. The phylogeographic patterns observed in the studied collection demonstrate that large-scale (but not middle/small-scale) distance remains one of the decisive factors of the genetic divergence of M. tuberculosis populations. Analysis of diversity and network topology of the local collections appears to corroborate a recent intriguing hypothesis about Beijing genotype originating in South China. Placing our results within the Eurasian context suggested that important Russian B0/W148 and Asian/Russian A0/94-32 epidemic clones of the Beijing genotype could trace their origins to the northeastern and northwestern regions of China, respectively. The higher clustering of the modern isolates in children and lack of increased MDR rate in any sublineage suggest that not association with drug resistance but other (e.g., speculatively, virulence-related) properties underlie an enhanced dissemination of the evolutionarily recent, modern sublineage of the Beijing genotype in China.

Identification of differentially expressed transcripts targeted by the knockdown of endogenous IFITM3.

Interferon inducible transmembrane protein 3 (IFITM3) is a double transmembrane protein. As a member of the IFITM family, IFITM3 can be upregulated by interferon (IFN) to be involved in various biological processes. In order to determine whether gene expression profiles can be altered by a lack of IFITM3, the present study used shRNAs lentivirus for knocking down the endogenous expression of IFITM3 in human HeLa cells and human whole genome microarrays to obtain gene expression profiles. A total of 1,011 downregulated transcripts and 615 upregulated transcripts were identified using the Agilent expression platform. The identified transcripts were involved in multiple pathways, including the complement pathways, and the antigen processing and presentation pathway. The present study identified the transcripts, which were affected by the downregulation of endogenous IFITM3 and the pathways they were involved in. These findings may lead to an improved understanding of the biological functions of IFITM3.

Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Beijing, China: 2006 versus 2012.

BACKGROUND: As the epidemic of MDR-TB and XDR-TB becomes increasingly severe, it is important to determine the clinical characteristics and molecular epidemiology of MDR-TB and XDR-TB. Recently, many studies have shown that clinical features and molecular characteristics of drug-resistant strains vary in different geographical areas, however, further information is needed to assess the dynamic evolution of drug-resistant TB. Comparative studies between different time periods are necessary to elucidate the development of drug-resistant TB. RESULTS: A total of 255 and 537 strains were collected from Beijing Chest Hospital in 2006 and in 2012, respectively. Drug-resistance rates and mutations associated with resistance to first-line anti-tuberculosis (TB) drugs were compared. The overall rate of drug resistance among strains of TB in 2012 was 54.4 %, significantly higher than that in 2006 (34.9 %, P < 0.001). Rates of resistance to each first-line drug (isoniazid, rifampicin, streptomycin and ethambutol) and to second-line drug ofloxacin increased significantly from 2006 to 2012. The overall MDR rate also increased significantly from 2006 (14.9 %) to 2012 (27.0 %). The rate of MDR increased significantly between these two time periods in previously treated cases (P = 0.023) but not in new cases (P = 0.073), and the rate of XDR was similar in new cases at the two time periods, but was marginally higher in 2012 in previously treated cases (P = 0.056). Previous treatment was found to be a risk factor for drug-resistant TB, especially for MDR-TB. In addition, the proportion of drug resistant isolates in which katG, the mabA-inhA promoter, oxyR-ahpC intergenic region, rpoB, rpsL, and embB were mutated was similar in 2006 and 2012, however patterns of mutation in these loci were more diverse in 2012 compared to 2006. CONCLUSIONS: Our data suggests that the prevalence of drug resistant TB remains high in Beijing, China, and that increasing rates of resistance in M. tuberculosis to all anti-TB drugs should be considered when choosing an optimal anti-TB regimen. Moreover, acquired multi-drug resistance may play a primary role in the MDR-TB epidemic in Beijing, China. Consequently, this highlights the importance of an earlier start to effective and supervised treatment in order to reduce the burden of retreatment.

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

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.

Prevalence of drug resistant Mycobacterium tuberculosis among children in China.

The available data on the epidemic of drug resistant tuberculosis (TB) among children in China is limited. This study attempted to clarify the drug resistance profiles of clinical strains isolated from children and estimate risk factors related to acquisition of drug resistance. All Mycobacterium tuberculosis strains from children (age <15 years) and adolescent (age 15-18 years) TB patients received in the strain library of Chinese Center for Disease Control and Prevention between January 2005 and December 2012 were included in the study. A study collection included 450 clinical isolates (100 from children, 159 from adolescents, and 191 from adults) from all over China. Drug susceptibility testing was performed by a proportion method. As a result, the drug resistance and multi-drug resistance (MDR) rates in children were 55% (55/100) and 22% (22/100), respectively. In children with MDR-TB, new cases accounted for 40.9% (9/22). Compared with adults, the drug resistance rates were similar in all subgroups (new cases, previously treated cases and all cases) of children (P > 0.05), except for the lower resistance rate to isoniazid in total cases of children (P = 0.011). Patient related information was included in the MDR-TB association analysis. The treatment history was found to be strongly associated with MDR-TB in all three age groups (P < 0.05). Our results demonstrate that the prevalence of drug resistant TB in children in China is alarmingly high and similar to that seen in adults. In contrast, in adolescents, the drug resistance rate to most tested drugs was lower than in adults. Primary transmission and inadequate treatment are two equally important factors for the high MDR-TB rate in children. Thus, major efforts in the TB control in children should focus on decreasing the transmission of drug resistant TB and early testing of drug resistance.

Rapid diagnosis of childhood pulmonary tuberculosis by Xpert MTB/RIF assay using bronchoalveolar lavage fluid.

In order to evaluate the diagnostic accuracy of the Xpert MTB/RIF assay on childhood pulmonary tuberculosis (PTB) using bronchoalveolar lavage fluid (BALF), we evaluated the sensitivity, specificity, positive predictive value, and negative predictive value of Xpert MTB/RIF assay using BALF in comparison with acid-fast bacilli (AFB) microscopy and Mycobacterium tuberculosis (MTB) culture for diagnosing childhood PTB using Chinese "composite clinical reference standard" (CCRS) as reference standard. Two hundred fifty-five children with suspected PTB were enrolled at Beijing Children's Hospital from September 2010 to July 2013. Compared with Chinese CCRS, the sensitivity of AFB microscopy, MTB culture, and Xpert MTB/RIF assay was 8.4%, 28.9%, and 53.0%, respectively. The specificity of three assays was all 100%. Xpert MTB/RIF assay could detect 33.9% of cases with negative MTB culture, and 48.7% of cases with negative AFB microscopy. Younger age (<3 years), absence of BCG scar, and contact with TB patient were found significantly associated with a positive result of Xpert MTB/RIF assay. In conclusion, Xpert MTB/RIF assay using BALF can assist in diagnosing childhood PTB much faster when fiberoptic bronchoscopy is necessary according to the chest radiograph.

A country-wide study of spoligotype and drug resistance characteristics of Mycobacterium tuberculosis isolates from children in China.

BACKGROUND: Tuberculosis (TB) is still a big threat to human health, especially in children. However, an isolation of Mycobacterium tuberculosis culture from pediatric cases remains a challenge. In order to provide some scientific basis for children TB control, we investigated the genotyping and drug resistance characteristics of M. tuberculosis isolates from pediatric cases in China. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a total of 440 strains including 90 from children (<15 years), 159 from adolescents (15-18 years) and 191 from adults (>18 years) isolated in 25 provinces across China were subjected to spoligotyping and drug susceptibility testing. As a result, Beijing family strains were shown to remain predominant in China (85.6%, 81.1% and 75.4% in three above groups, respectively), especially among new children cases (91.0% vs. 69.6% in previously treated cases, P=0.03). The prevalence of the Beijing genotype isolates was higher in northern and central China in the total collection (85.1% in northern and 83.9% in central vs. 61.6% in southern China, P<0.001) and a similar trend was seen in all three age groups (P=0.708, <0.001 and 0.025, respectively). In adolescents, the frequencies of isoniazid (INH)-resistant and ethambutol (EMB)-resistant isolates were significantly higher among Beijing strains compared to non-Beijing genotype strains (P=0.028 for INH and P=0.027 for EMB). Furthermore, strong association was observed between resistance to rifampicine (RIF), streptomycin (STR) and multidrug resistance (MDR) among Beijing compared to non-Beijing strains in previously treated cases of children (P=0.01, 0.01 and 0.025, respectively). CONCLUSION/SIGNIFICANCE: Beijing family was more prevalent in northern and central China compared to southern China and these strains were predominant in all age groups. The genetic diversity of M. tuberculosis isolates from children was similar to that found in adolescents and adults. Beijing genotype was associated with RIF, STR and MDR resistance in previously treated children.