Insight into pathogenomics and phylogeography of hypervirulent and highly-lethal Mycobacterium tuberculosis strain cluster.

BACKGROUND: . The Mycobacterium tuberculosis Beijing genotype is globally spread lineage with important medical properties that however vary among its subtypes. M. tuberculosis Beijing 14717-15-cluster was recently discovered as both multidrug-resistant, hypervirulent, and highly-lethal strain circulating in the Far Eastern region of Russia. Here, we aimed to analyze its pathogenomic features and phylogeographic pattern. RESULTS: . The study collection included M. tuberculosis DNA collected between 1996 and 2020 in different world regions. The bacterial DNA was subjected to genotyping and whole genome sequencing followed by bioinformatics and phylogenetic analysis. The PCR-based assay to detect specific SNPs of the Beijing 14717-15-cluster was developed and used for its screening in the global collections. Phylogenomic and phylogeographic analysis confirmed endemic prevalence of the Beijing 14717-15-cluster in the Asian part of Russia, and distant common ancestor with isolates from Korea (> 115 SNPs). The Beijing 14717-15-cluster isolates had two common resistance mutations RpsL Lys88Arg and KatG Ser315Thr and belonged to spoligotype SIT269. The Russian isolates of this cluster were from the Asian Russia while 4 isolates were from the Netherlands and Spain. The cluster-specific SNPs that significantly affect the protein function were identified in silico in genes within different categories (lipid metabolism, regulatory proteins, intermediary metabolism and respiration, PE/PPE, cell wall and cell processes). CONCLUSIONS: . We developed a simple method based on real-time PCR to detect clinically significant MDR and hypervirulent Beijing 14717-15-cluster. Most of the identified cluster-specific mutations were previously unreported and could potentially be associated with increased pathogenic properties of this hypervirulent M. tuberculosis strain. Further experimental study to assess the pathobiological role of these mutations is warranted.

Insight into Population Structure and Drug Resistance of Pediatric Tuberculosis Strains from China and Russia Gained through Whole-Genome Sequencing.

This study aimed to determine phenotypic and genotypic drug resistance patterns of Mycobacterium tuberculosis strains from children with tuberculosis (TB) in China and Russia, two high-burden countries for multi/extensively-drug resistant (MDR/XDR) TB. Whole-genome sequencing data of M. tuberculosis isolates from China (n = 137) and Russia (n = 60) were analyzed for phylogenetic markers and drug-resistance mutations, followed by comparison with phenotypic susceptibility data. The Beijing genotype was detected in 126 Chinese and 50 Russian isolates. The Euro-American lineage was detected in 10 Russian and 11 Chinese isolates. In the Russian collection, the Beijing genotype and Beijing B0/W148-cluster were dominated by MDR strains (68% and 94%, respectively). Ninety percent of B0/W148 strains were phenotypically pre-XDR. In the Chinese collection, neither of the Beijing sublineages was associated with MDR/pre-XDR status. MDR was mostly caused by low fitness cost mutations (rpoB S450L, katG S315T, rpsL K43R). Chinese rifampicin-resistant strains demonstrated a higher diversity of resistance mutations than Russian isolates (p = 0.003). The rifampicin and isoniazid resistance compensatory mutations were detected in some MDR strains, but they were not widespread. The molecular mechanisms of M. tuberculosis adaptation to anti-TB treatment are not unique to the pediatric strains, but they reflect the general situation with TB in Russia and China.

Advances in the regulation of inflammasome activation by GBP family in infectious diseases.

Guanylate-binding proteins (GBPs) are a subfamily of interferon-inducible proteins that undertake distinct roles in the the context of bacteria, virus, chlamydia and parasites infections. These proteins exert a notable influence on the progression and outcomes of infectious diseases. Within the realm of host cell-autonomous immunity against pathogens, GBPs have been identified as the regulators of pyroptosis through canonical and noncanonical inflammasome activation pathways. In this review, we summarize the structure and evolution of GBP family members, the canonical and noncanonical inflammasome activation pathways, the roles of GBPs in regulating inflammasome activation, and the mechanisms of GBPs affecting infections induced by different pathogens. We hope to provide new basic research clues for the pathogenesis and diagnosis and treatment of infectious diseases.

Molecular characterization of multidrug-resistant tuberculosis against levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, and delamanid in southwest of China.

OBJECTIVES: To explore the drug susceptibility of levofloxacin (LFX), moxifloxacin (MFX), bedaquiline (BDQ), linezolid (LZD), clofazimine (CFZ) and delamanid (DLM) against multidrug resistant tuberculosis (MDR-TB) isolates from drug resistance survey of southwest China, and to illustrate the genetic characteristics of MDR-TB isolates with acquired drug resistance. METHODS: A total of 339 strains were collected from smear-positive TB patients in the drug resistance survey of southwest China between January 2014 and December 2016. The MICs for the above mentioned drugs were determined for MDR-TB by conventional drug susceptibility testing. Genes related to drug resistance were amplified with their corresponding pairs of primers. RESULTS: MDR was observed in 88 (26.0%; 88/339) isolates. LFX had the highest resistance rate (50.0%; 44/88), followed by MFX (38.6%; 34/88). The resistance rate to LZD, CFZ, and DLM was 4.5% (4/88), 3.4% (3/88), and 4.5% (4/88), respectively, and the lowest resistance rate was observed in BDQ (2.3%; 2/88). Of the 45 isolates resistant to LFX and MFX, the most prevalent resistance mutation was found in gyrA with the substitution of codon 94 (34/45, 75.6%). Two strains with CFZ - BDQ cross resistance had a mutation in the Rv0678 gene. Of the four LZD resistant isolates, two carried mutations in rplC gene. For the four isolates resistant to DLM, one isolate had mutations in codon 318 of fbiC gene, and two isolates were with mutations in codon 81 of ddn gene. CONCLUSION: This study provided evidence of the usefulness of new anti-TB drugs in the treatment of MDR-TB in China.

[Overview of systematic reviews/Meta-analysis of Xingnaojing Injection in treatment of intracerebral hemorrhage].

To overview of systematic reviews/Meta-analysis of Xingnaojing Injection(XNJ) in the treatment of intracerebral hemorrhage(ICH). The systematic reviews concerning XNJ in the treatment of ICH were retrieved from four Chinese databases, four English databases, Chinese Clinical Trial Registry and ClinicalTrail.gov, with the retrieval time set from their inception to September 2020. Following the independent screening and data extraction by two researchers, a measurement tool to assess systematic evaluation 2(AMSTAR 2) and grades of recommendation, assessment, development and evaluation(GRADE) system were used to evaluate the metho-dological, reporting and evidence qualities of the 10 included systematic reviews. The results showed that XNJ was superior to the wes-tern medicine or conventional treatment in improving the effective rate and National Institutes of Health stroke scale(NIHSS) score, Barthel index(BI), and Glasgow coma scale(GCS) score and Chinese stroke scale(CSS) score, and reducing the mortality and cerebral hematoma volume, without inducing obvious adverse reactions. In general, the methodological, reporting and evidence qualities of the 10 included systematic reviews were poor. The AMSTAR 2 scores showed that key items No. 2 and No. 16 failed to meet the stan-dard, resulting in poor methodological quality. There was only one outcome indicator graded by GRADE as intermediate quality, 43% indicators as low quality, 42% indicators as extremely low quality, and none as high quality. These available evidences have suggested that the methodological, reporting and evidence qualities of the systematic evaluation concerning XNJ for the treatment of ICH need to be improved. Most evidences support that XNJ was better than the western medicine or conventional treatment in the treatment of ICH, but the methodological quality and the reliability of outcome indicators in relevant systematic review were low. More high-quality studies are still required for further verification.

[Systematic review and Meta-analysis on efficacy and safety of Naoxueshu Oral Liquid in treatment of hypertensive intracerebral hemorrhage].

To systematically review the efficacy and safety of Naoxueshu Oral Liquid in treatment of hypertensive intracerebral hemorrhage, four Chinese databases, four English databases, clinical trials registration center(ClinicalTrials.gov) and Chinese clinical trial registry were retrieved. The retrieval time was from the establishment of each database to September 9, 2020. According to the set criteria, the randomized controlled trial(RCT) of Naoxueshu Oral Liquid combined with conventional Western medicine was selected. The "Cochrane bias risk assessment" tool was used to evaluate the quality of the included studies. RevMan 5.4.1 was used to conduct Meta-analysis of the included studies and GRADE system was used to evaluate the evidence quality of the outcome indicators. Eleven studies were finally included, with a total sample size of 1 221 cases, 612 cases in the treatment group and 609 cases in the control group. Meta-analysis showed that Naoxueshu Oral Liquid combined with conventional Western medicine had no significant difference compare with conventional Western medicine in reducing National Institute of health stroke scale(NIHSS) after 2 weeks of treatment for hypertensive intracerebral hemorrhage(MD=-1.59,95%CI[-3.46,0.29],P=0.10), but was superior to conventional Western medicine after 30 d of treatment(MD=-1.16,95%CI [-1.39,-0.94],P<0.000 01). Naoxueshu Oral Liquid combined with conventional Western medicine was superior to conventional Western medicine in improving Glasgow coma scale(MD=1.00,95%CI[0,2.00],P=0.05) and reducing the incidence of secondary brain insults(RR=0.38,95%CI[0.24,0.59],P<0.000 1), but there was no significant difference in increasing Barthel index(MD=1.00,95%CI[-0.30,2.30],P=0.13). In terms of effective rate, studies using Guideline for clinical trials of new patent Chinese medicines, NHISS or Glasgow outcome scale(GOS) had shown that Naoxueshu Oral Liquid combined with conventional Western medicine was superior to conventional Western medicine(RR_(Guideline for clinical trials of new patent Chinese medicines)=1.27,95%CI[1.10,1.46],P=0.001;RR_(NHISS)=1.26,95%CI[1.13,1.40],P<0.000 1;RR_(GOS)=1.54,95%CI[1.22,1.93],P=0.000 2). In reduction of hematoma volume, Naoxueshu Oral Liquid combined with conventional Western medicine was superior to conventional Western medicine after 2 and 4 weeks of treatment(MD_(2 week)=-2.31,95%CI[-3.12,-1.49],P<0.000 01;MD_(4 week)=-2.04,95%CI[-2.41,-1.68],P<0.000 01). GRADE system showed that the evidence level of the above outcome indicators was low and extremely low. In terms of adverse reactions, two of the included studies reported mild adverse reactions, and the rest of studies were not mentioned, so this study was not able to make a positive evaluation of the safety of Naoxueshu Oral Liquid. This study showed that compared with conventional Western medicine, combined Naoxueshu Oral Liquid may be better for hypertensive intracerebral hemorrhage. However, due to the high bias risk in the included studies, more large-sample and high-quality RCTs are still needed in the future.

[Situation analysis of outcome indicators of randomized controlled trials of traditional Chinese medicine in treatment of hypertensive intracerebral hemorrhage in recent three years].

The study aims to analyze the outcome indicators of randomized controlled trial(RCT) of traditional Chinese medicine(TCM) in the treatment of hypertensive intracerebral hemorrhage(HICH) in recent three years, and thus provide suggestions for the future studies in this field. Four English databases, four Chinese databases and two online registration websites of clinical trials were searched. The RCTs published between January 2018 and September 2020 were screened. The risk of bias was assessed and outcome measures were classified. A total of 151 839 articles were retrieved, of which 44 RCTs were included for analysis after screening. The outcome measures of the included RCTs were classified into 7 categories, among which the symptoms/signs category showed the highest reporting rate. National Institute of Health stroke scale(72.73%) was the most frequently reported outcome indicator, while the vo-lume of intracerebral hemorrhage determined by computerized tomography(36.36%) was the most frequently reported lab test outcome. Most studies collect the outcomes at the end of treatment, while 9 studies reported long-term outcomes 3 months or more after onset. Compared with those of international clinical trials, the application of some of the outcomes was reasonable, focusing on patients' symptoms, quality of life and objective outcomes. However, there were still several problems: unclear primary and secondary outcome measures, insufficient attention to long-term prognosis, insufficient attention to social function, few TCM outcomes, lack of measurement blindness and the use of unreasonable composite outcomes. It is recommended that researchers should rationally design the outcome indicators of clinical trials and develop the core outcome set.

[Systematic review and Meta-analysis of efficacy and safety of acupuncture therapy on hypertensive intracerebral hemorrhage].

To systematically review the efficacy and safety of acupuncture combined with minimally invasive surgery or basic the-rapy in treating hypertensive intracerebral hemorrhage(HICH) patients compared with minimally invasive surgery or basic treatment. In this study, the four Chinese databases, the four English databases, Chinese Clinical Trial Registry and ClinicalTrail.gov, all above were systematically and comprehensively retrieved from the time of database establishment to September 10, 2020. Rando-mized controlled trials(RCTs) were screened out according to inclusion criteria and exclusion criteria established in advanced. The methodological quality of included studies was evaluated by the tool named "Cochrane bias risk assessment 6.1". Meta-analysis of the included studies was performed using RevMan 5.4, and the quality of outcome indicators was evaluated by the GRADE system. Finally, 17 studies were included, involving 1 852 patients with HICH, and the overall quality of the included studies was not high. According to Meta-analysis,(1)CSS score of the group of acupuncture combined with minimally invasive surgery or basic therapy was superior to the group of minimally invasive surgery or basic therapy(MD=-3.50,95%CI[-4.39,-2.61],P<0.000 01);(2)NIHSS score of the group of acupuncture combined with minimally invasive surgery or basic therapy was superior to the group of minimally invasive surgery or basic therapy(MD=-4.78,95%CI[-5.55,-4.00],P<0.000 01);(3)the cerebral hematoma volume of the group of acupuncture combined with minimally invasive surgery or basic therapy was superior to the group of minimally invasive surgery or basic therapy(MD=-4.44,95%CI[-5.83,-3.04],P<0.000 01);(4)ADL score of the group of acupuncture combined with minimally invasive surgery or basic therapy was superior to the group of minimally invasive surgery or basic therapy(MD=20.81,95%CI[17.25,24.37],P<0.000 01);(5)the GCS score of the group of acupuncture combined with minimally invasive surgery or basic therapy was superior to the group of minimally invasive surgery or basic therapy(MD=2.41,95%CI[1.90,2.91],P<0.000 01). The GRADE system showed an extremely low level of evidence for the above outcome indicators. Adverse reactions were mentioned only in two literatures, with no adverse reactions reported. The available evidence showed that acupuncture combined with minimally invasive surgery or basic therapy had a certain efficacy in patients of HICH compared with minimally invasive surgery or basic therapy. However, due to the high risk of bias in the included studies, its true efficacy needs to be verified by more high-quality studies in the future.

Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection.

A new nucleic acid detection technique, termed Nano-SAMRS-RPA, is reported which employed carbon nanomaterial (graphene oxide, GO) and self-avoiding molecular recognition systems (SAMRS) to improve the specificity of recombinase polymerase amplification (RPA). In the presence of GO and SAMRS primers, the assay artifacts, including primer-dimers, nonspecific products, off-target hybrids, and non-canonical folds, are completely suppressed and eliminated, which makes the creation of RPA-based methods faster by simplifying the primer design and eliminating the need for primer optimization and complex probe. Moreover, a lateral flow bioassay (LFB) was also devised for simply and rapidly indicating the Nano-SAMRS-RPA results. Particularly, the new detection system only requires a single-labeled primer, eliminating the false-positive result from hybridization (the labeled probe and reverse primer) and the use of real-time instrument, more complex enzymatic solutions, and probes. As a result, GO, SAMRS primers, and LFB convert RPA from a technique suited only for the research laboratory into one that has a practical value in clinical settings, field environments, and at points-of-care testing. Human papillomaviruses (HPV) genotypes 16 and 18 were applied as model analytes to test the assay's availability. The initial data indicated that Nano-SAMRS-RPA could detect down to 10 copies per reaction, and the sensitivity (14/14 samples collected from HPV16 and HPV 18 patients) and specificity (75/75 samples collected from non-HPV patients) for clinical sample detection were 100%. The proof-of-concept technique can be reconfigured to detect various nucleic acid sequences by redesigning the specific RPA primers.Graphical abstract.

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.

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.

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 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.

Progress on mechanism of ethambutol resistance in Mycobacterium Tuberculosis.

The occurance and prevalence of multidrug-resistant tuberculosis poses a serious threat to the global tuberculosis control. Ethambutol (EMB) is one of the first-line anti-tuberculosis drugs, which is usually used in combination with isoniazid and rifampicin for treating pan-sensitive tuberculosis, and it can also be used in drug-resistant tuberculosis. However, the situation of EMB resistance is alarmingly high, especially in multi-drug resistant tuberculosis. In China, EMB resistance rate in the previously treated cases was up to 17.2% and showed an increased tendency. What was worse, 51.3%-66.7% of multidrug-resistant tuberculosis cases were resistant to EMB. Thus, it is important to understand the drug resistance mechanism of EMB, which will help to slow down the drug resistance rate of EMB. In this review, we focus on the current status of EMB resistance, the effects of EMB and the mechanisms of EMB resistance in Mycobacterium tuberculosis.

Real-time PCR assay for rapid detection of epidemiologically and clinically significant Mycobacterium tuberculosis Beijing genotype isolates.

Mycobacterium tuberculosis Beijing genotype strains are rapidly disseminating, frequently hypervirulent, and multidrug resistant. Here, we describe a method for their rapid detection by real-time PCR that targets the specific IS6110 insertion in the dnaA-dnaN genome region. The method was evaluated with a geographically and genetically diverse collection representing areas in East Asia and the former Soviet Union in which the Beijing genotype is endemic and epidemic (i.e., major foci of its global propagation) and with clinical specimens.

IFNG polymorphisms are associated with tuberculosis in Han Chinese pediatric female population.

Host genetic factors play a major role in determining differential susceptibility to human tuberculosis (TB), a re-emerging infectious disease throughout the world. Genetic variations in the IFNG gene coding for interferon gamma (IFN-γ), have been identified in TB patients. To investigate the association of the IFNG polymorphisms with TB susceptibility in Chinese pediatric population. A case-control study of 189 TB patients and 164 controls was performed using single-nucleotide polymorphism (SNP) analysis. Genomic DNA was extracted from leukocytes in peripheral blood. Three SNPs of IFNG, including -1616C/T (rs2069705), +874A/T (rs2430561), and +3234C/T (rs2069718), were selected for genotyping and analysis. The +874A and +3234C alleles were more frequent among TB patients (P = 0.108 and P = 0.088), especially in females (both P = 0.029), although this difference was not significant since Bonferroni corrected significance threshold was 0.025 (two of three SNPs were found to be in linkage disequilibrium). More pronounced differences for the +874 and +3234 polymorphisms were found under the genotype comparison between TB cases and controls in the total population [P = 0.026 (borderline non-significance) and P = 0.020, respectively], and in the female subgroup (P = 0.020 and P = 0.020). The dominant model of inheritance was shown to be significant for +874A and +3234C alleles (both P = 0.019) in the female subgroup. The +874A and +3234C alleles were more frequently found in extrapulmonary TB patients than in controls (P = 0.039). Haplotype analysis carried out on these three SNPs showed the TTT haplotype to be more frequent in controls than in TB cases, and this difference showed a strong significance (P = 0.005). The +874A and +3234C alleles may be related to TB susceptibility in the female subgroup in the Chinese pediatric population of North China. The higher rate of +874A (known to correlate with lower IFN-γ expression) in the extrapulmonary TB subgroup suggests a sufficient IFN-γ expression to be not only an important factor for the onset of TB disease but also for limiting its dissemination to lungs.

Artificially intelligent differential diagnosis of enlarged lymph nodes with random vector functional link network plus.

Differential diagnosis of enlarged lymph nodes (ELNs) is essential for the treatment of related patients. Though multi-modal ultrasound including B-mode, Doppler ultrasound, elastography and contrast-enhanced ultrasound (CEUS) can enhance diagnostic performance for ELNs, the scenario of having only single or dual modal data is often encountered. In this study, an artificially intelligent diagnosis model based on the learning using privileged information was proposed to aid in differential diagnosis of ELNs in the case of single or dual modal images. In our model, B-mode, or combined with another modality, was used as the standard information (SI) and other modalities were used as the privileged information (PI). The model was constructed through the combination of the SI and PI in the training stage. By learning from the training samples, a random vector functional link network with privileged information (RVFL+) was obtained, which was used to classify the testing samples of solely the SI. Results showed that the accuracy, precision and Youden's index of the RVFL+ model, using B-mode with elastography as the SI and CEUS as the PI, reached 78.4%, 92.4% and 54.9%, increased by 14.0%, 8.4% and 24.5% compared with the model using B-mode as the SI without the PI. The method based on the LUPI can improve the diagnostic performance for ELNs.

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies µL-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

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.