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.

Characteristics and clinical role of bronchoscopy in diagnosis of childhood endobronchial tuberculosis.

BACKGROUND: Endobronchial tuberculosis (EBTB) is the most frequent complication of primary pulmonary tuberculosis (PTB) in children. The aim of the study was to analyze characteristics and clinical role of bronchoscopy in diagnosis of childhood EBTB. METHODS: A retrospective, descriptive study was undertaken in 157 children with EBTB undergone flexible bronchoscopy (FB) between January 2006 and June 2014. RESULTS: The median age of the enrolled patients was 3.4 years, with 73.2% of patients under five years old. The most common subtype was tumorous type (145/157, 92.4%). If only involved bronchus were considered, the common affected sites were right middle lobe bronchus (49/228, 21.5%), left upper lobe bronchus (41/228, 18.0%), right upper lobe bronchus (41/228, 18.0%), right main bronchus (35/228, 15.4%), respectively. Children younger than five years old were at higher risk to have multiple endobronchial lesions (P=0.044), with an odds ratio of 2.313 (95% confidence interval: 1.009-5.299). Before the bronchoscopy, only 16 (10.2%) patients were highly suspected of EBTB, while the others were diagnosed as PTB without EBTB (69.4%), or misdiagnosed as pneumonia or foreign body aspiration (20.4%) on admission. CONCLUSIONS: The patients under five years old are at high risk to progress to EBTB and have multiple endobronchial lesions. The most frequent subtype of EBTB in children is tumorous type. The lesions are seen in the right bronchial system more frequently. FB should be performed to detect the endobronchial lesions in suspected patients as soon as possible.

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.

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.

Russian "successful" clone B0/W148 of Mycobacterium tuberculosis Beijing genotype: a multiplex PCR assay for rapid detection and global screening.

We describe a multiplex PCR assay to detect the Mycobacterium tuberculosis Beijing genotype variant B0/W148, which is considered a "successful" clone of M. tuberculosis, widespread in Russia. Specificity and sensitivity of the assay were 100% based on the analysis of a collection of 516 M. tuberculosis isolates of different genotypes and origins. This assay may be used for accurate and simple detection and surveillance of this clinically and epidemiologically important variant of M. tuberculosis.

[Association of NRAMP1 gene polymorphisms with the susceptibility to tuberculosis in ethnic Han Chinese children].

OBJECTIVE: Tuberculosis is still a public health problem. Host genetic factors, such as polymorphisms in NRAMP1 gene, may play a role in the development of tuberculosis. To clarify the effect of NRAMP1 gene polymorphisms on the development of childhood tuberculosis, the association of NRAMP1 gene polymorphisms with susceptibility to tuberculosis in the ethnic Han Chinese children was investigated. METHODS: From January 2005 to March 2008, 130 ethnic Han children with tuberculosis (TB group) were enrolled. Three hundred and ninety hospitalized ethnic Han children for physical examination in the surgery department were used as the control group. The controls were matched with tuberculosis children by age, sex and area. PCR-RFLP analysis was performed on DNA samples to identify allele genotypes of INT4 and D543N in NRAMP1 gene. Genotype frequency differences between tuberculosis patients and controls were analyzed using x2 test. RESULTS: No statistical difference was found in the genotype frequency of variants G/C and C/C at the INT4 locus between the TB and the control groups. At the D543N locus, the frequency of genotype variants (G/A and A/A) was significantly higher in the TB group (34/130) than that in the control group (66/390) (x2=5.349, P<0.05; OR=1.74, 95%CI=1.08-2.79). When stratified by sex, differences in the genotype distribution were observed only in females at the D543N locus, which the variant genotypes were higher in the TB group (16/52) than in the control group (21/155) (x2=7.866, P<0.05; OR=2.84, 95%CI=1.34-5.99). For males, there was no difference between the TB and the control groups. At the INT4 locus, no difference was observed between the two groups in boys and girls. CONCLUSIONS: Genotypic variation at the D543N locus in NRAMP1 gene may be associated with susceptibility to tuberculosis in ethnic Han Chinese children. Variant genotypes in NRAMP1 gene (G/A and A/A) may be susceptible genotypes to tuberculosis in ethnic Han Chinese children. Girls with variant genotypes were more susceptible to tuberculosis.