CircularRNA Hsa_circ_0093335 promotes hepatocellular carcinoma progression via sponging miR-338-5p.

Circular RNAs play an important role in the development of various malignancies, including hepatocellular carcinoma (HCC). Nevertheless, the role of Hsa_circ_0093335 (circ0093335) in HCC has not yet been explored. To investigate the biological effects and molecular mechanisms of circ0093335 on HCC. Circ0093335 expression was detected in HCC cells and clinical specimens using qRT-PCR. The association between circ0093335 expression and HCC patients' clinical characteristics was determined using SPSS. The role of circ0093335 in HCC was estimated by overexpression and knockdown experiments in vitro and in vivo. qRT-PCR, nucleoplasma separation assay, FISH assay, RIP, dual luciferase reporter assay and rescue assay were used to validate the regulatory effect of circ0093335 on miR-338-5p. The study findings showed that circ0093335 was upregulated in HCC. High circ0093335 expression was linked with the tumour-node-metastasis stage and microvascular tumour invasion. circ0093335 is greatly involved in HCC cell proliferation, aggressive ability and mouse tumour growth, according to many in vitro and in vivo tests. Mechanistically, circ0093335 downregulated miR-338-5p expression by sponging, consequently promoting HCC progression. Our research indicated that circ0093335 might be a target for HCC therapy since it promotes tumour progression by acting as a miR-338-5p 'sponge'.

Expression Pattern of Cytokines in Patients with Chronic Hepatitis B Receiving PEGinterferon Therapy.

Purpose: Chronic hepatitis B virus (CHB) infection is a worldwide health problem. Polyethylene glycol (PEG)ylated interferon (PEG-IFN) is an available therapy for CHB that has antiviral and immunomodulatory effects. However, PEG-IFN therapy is limited by the fact that only a subset of patients show a sustained response, its severe side effects, and high cost. The aim of this study was to explore novel biomarkers for the early prediction of PEG-IFN treatment response and to uncover its underlying mechanism. Patients and Methods: We enrolled 10 paired patients with Hepatitis B e antigen (HBeAg)-positive CHB who received PEG-IFN-α2a monotherapy. Patient serum samples were collected at 0, 4, 12, 24, and 48 weeks and serum samples were collected from eight healthy people as healthy controls. For confirmation, we enrolled 27 patients with HBeAg-positive CHB receiving PEG-IFN therapy and serum samples at 0 and 12 weeks were obtained. Serum samples were analyzed using Luminex technology. Results: Among 27 assessed cytokines, 10 cytokines were identified to have high expression levels. Among them, six cytokines had significant differences in their levels between the patients with HBeAg-positive CHB and the healthy controls (P < 0.05). Potentially, treatment response could be predicted using the early time points of 4, 12, and 24 weeks. Moreover, after 12 weeks of PEG-IFN treatment, increased levels of pro-inflammatory cytokines and decreased levels of anti-inflammatory cytokines were observed. The fold change of IP-10 between 12 weeks and 0 weeks correlated with the decrease in ALT levels from 0 to 12 weeks (r = 0.2675, P = 0.0024). Conclusion: In patients with CHB, we observed a certain pattern in the levels of cytokines during treatment with PEG-IFN, and the cytokine IP-10 might be a potential biomarker for treatment response.

Effect of adjusted cut-offs of interferon-γ release assays on diagnosis of tuberculosis in patients with fever of unknown origin.

BACKGROUND: Tuberculosis (TB) is a leading cause of fever of unknown origin (FUO). In recent years, interferon-γ release assays (IGRAs) have been widely utilized and the cut-off values given by the manufacturers are set in countries where rates of TB are not as high. METHODS: A prospective cohort study was conducted in a Chinese general hospital to evaluate the diagnostic performance of T-SPOT.TB (T-SPOT) and QuantiFERON-TB Gold (QFT) in detecting active TB (ATB) in a high TB endemic area. Test results were compared with the culture and clinically confirmed diagnosis. Further, we explored an alternative method of interpreting IGRAs by increasing the cut-off values. RESULTS: The sensitivity and specificity of T-SPOT in detecting ATB were 85.3% (95% CI 81.6-94.0%) and 71.8% (95% CI 67.3-76.0%), respectively. The sensitivity and specificity of QFT were 72.3% (95% CI 62.8-80.1%) and 77.0% (95% CI 72.7-80.8%), respectively. Receiver operating characteristic analysis was used for evaluation of different cut-off values. When the cut-off values were adjusted as 125 spot-forming cells (SFCs)/ 2.5*105 cells for T-SPOT and 4.0 IU/ml for QFT, the specificity could be improved to > 90.0% (90.3% and 94.1%, respectively), and the sensitivity were 43.1% and 41.6%, respectively. The new adjusted cut-off values were validated in another independent validation cohort. CONCLUSION: The adjusted cut-off values of the two assays considerably improved the diagnostic value when applied to FUO patients in clinical settings.

Recurrent pulmonary tuberculosis after treatment success: a population-based retrospective study in China.

OBJECTIVES: Post-treatment recurrence remains a challenge for the global control of tuberculosis (TB). This study investigated longitudinal data on pulmonary TB recurrence rates and risk factors for recurrence among successfully treated smear-positive tuberculosis cases in China. METHODS: Between 1st January 2009 and 31st December 2016 we evaluated 33 441 treatment-naïve patients diagnosed with sputum-smear-positive, non-multidrug-resistant TB in Hangzhou, China. We included the data of 9828 patients with TB who were treated successfully. RESULTS: A total of 4.9% of the cases were recurrent (479/9828), identified within a median observation period lasting 1565 days. Altogether, 51.1% (245/479) of the recurrences occurred within 1 year. The cumulative 2- and 5-year recurrence rates were 3.90% (95% confidence interval (CI) 3.3-4.5%) and 5.4% (95%CI 4.8-6.0%), respectively. Prolonged treatment (over 7 months) occurred in 64.7% (6363/9828), with a median treatment duration of 242 days (interquartile range 195-348 days). Male sex (adjusted hazard ratio (aHR) (95%CI) 1.61 (1.30-2.00), p < 0.001), age 60 years old or older (aHR (95%CI) 2.03 (1.70-2.44), p < 0.001), pulmonary cavity (aHR (95%CI) 1.51 (1.25-1.82), p < 0.001) and sputum positivity at 2 months (aHR (95%CI) 1.39 (1.05-1.81), p 0.02) all increased the risk of TB recurrence. Prolonged treatment was associated with reduced TB recurrence (aHR (95%CI) 0.73 (0.61-0.88), p 0.001). CONCLUSIONS: Recurrence remains a problem for successfully treated patients with sputum-smear-positive pulmonary TB, especially those with independent risk factors. Further analysis of prolonged treatment is required.

Efficacy and safety of weekly rifapentine and isoniazid for tuberculosis prevention in Chinese silicosis patients: a randomized controlled trial.

OBJECTIVE: The aim was to evaluate the efficacy, safety and completion rate of 3-month, once-weekly rifapentine and isoniazid for tuberculosis (TB) prevention among Chinese silicosis patients. METHODS: Male silicosis patients without human immunodeficiency virus infection, aged 18 years to 65 years, with or without latent TB infection, were randomized 1:1 to receive rifapentine/isoniazid under direct observation (3RPT/INH group) or were untreated (observation group). Active TB incidence was compared between the two groups with 37 months of follow-up. Safety profile and complete rates were evaluated. RESULTS: A total of 1227 adults with silicosis were screened; 513 eligible participants were enrolled and assigned to 3RPT/INH (n = 254) vs. observation (n = 259). Twenty-eight participants were diagnosed with active TB, and 9 and 19 in the 3RPT/INH group and observation groups, respectively. In the intention-to-treat analysis, the cumulative active TB rate was 3.5% (9/254) in the 3RPT/INH group and 7.3% (19/259) in the observation group (log rank p 0.055). On per protocol analysis, the cumulative active TB rates were 0.7% (1/139) and 7.3% (19/259), respectively (log rank p 0.01). Owing to an unexpected high frequency of adverse events (70.4%) and Grade 3 or 4 AEs (7.9%), the completion rate of the 3RPT/INH regimen was 54.7% (139/254). Twenty-six (10.8%) participants had flu-like systemic drug reactions; five (2.1%) experienced hepatotoxicity. DISCUSSION: Weekly rifapentine/isoniazid prophylaxis prevented active TB among Chinese people with silicosis when taken, irrespective of LTBI screening; efficacy was reduced by lack of compliance. The regimen must be used with caution because of the high rates of adverse effects. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov number: NCT02430259.

Accumulation mechanism of biofilm under different water shear forces along the networked pipelines in a drip irrigation system.

The behavior of clogging has a close relationship with the biofilm attached on inner surface of the pipeline in a drip irrigation system using reclaimed water. Therefore, inhibiting biofilm growth is the key to completely addressing the clogging problem. Water shear forces play a vital role in the formation, development and detachment of biofilm. In order to find out the accumulation mechanism of biofilm under different water shear forces, this paper considered 8 different shear forces with a range of [0, 0.7]Pa on the inner surface of pipelines in drip irrigation systems using three kinds of reclaimed water. The results indicate that dry weight (DW), phospholipid fatty acids (PLFAs) and extracellular polymeric substance (EPS) of biofilms show a S-type trend, the maximum contents were observed when τ was 0.2 Pa or 0. 35 Pa. Besides, the influence of water shear forces on biofilms is dual. The formation of biofilm is a dynamic stabilization process. When there is a relatively large shear force, it is favorable to the transport and renewal of microorganisms and nutrients. Meantime, the renewal speed of biofilms is also relatively fast. It is easy to form the biofilms with large surface and small thickness due to relatively high possibility of detachment. When the shear force is small, the transport speed of microorganisms and nutrients are limited, and the ability of microorganisms to secrete polysaccharides is reduced, which makes the nutrients needed for microbial growth insufficient and the adhesion between particles is also reduced, resulting in loose, unstable and an easily removed biofilm structure. After a comprehensive consideration of the dual influence, the critical controlling threshold of internal water shear force was obtained as [0, 0.20] ∪ [0.35, +∞] Pa. In addition, the growth model established in this paper can well describe the growth kinetics of attached biofilms, and provide theoretical reference for monitoring the occurrence of bio-clogging process in drip irrigation systems.

Preventive tuberculosis treatment effect on QuantiFERON TB-Gold in-tube testing in a high tuberculosis-endemic country: A clinical trial.

BACKGROUND: Whether T-cell interferon-γ responses to Mycobacterium tuberculosis-specific antigens can be influenced by tuberculosis preventive treatment in a high-endemic country is uncertain. METHODS: In this prospective, open-label, controlled study, 513 individuals with silicosis were randomly selected for TB preventive treatment with rifapentine and isoniazid or for observation. QuantiFERON-TB Gold in-tube (QFT-GIT) assay was used to measure IFN-γ response to M. tuberculosis antigens at baseline (T0) and at 6 (T1) and 33 (T2) months after completion of therapy. RESULTS: A total of 220 subjects were included in the final analysis: 105 and 115 in the prevention and observation arms, respectively. The proportions of QFT-GIT reversion from baseline to T1 were similar in the prevention and observation arms (18.4% vs 12.8%, P=0.566). However, reversion from baseline to T2 was more frequent in the prevention arm than in the observation arm, but the difference was not significant (24.2% vs 6.3%, P=0.881). No significant difference was observed in the quantitative responses of QFT-GIT between the two arms during follow-up at T1 (P=0.648) and T2 (P=0.918). CONCLUSIONS: Preventive tuberculosis treatment has no effect on interferon-γ responses measured by serial QFT-GIT assays in a high tuberculosis-endemic country. CLINICAL TRIALS REGISTRATION: http://www.clinicaltrials.gov NCT02430259.

Clinical Evaluation of Diagnosis Efficacy of Active Mycobacterium tuberculosis Complex Infection via Metagenomic Next-Generation Sequencing of Direct Clinical Samples.

Background: Tuberculosis (TB) is now the leading cause of death from infectious disease. Rapid screening and diagnostic methods for TB are urgently required. Rapid development of metagenomics next-generation sequencing (mNGS) in recent years showed promising and satisfying application of mNGS in several kinds of infectious diseases. However, research directly evaluating the ability of mNGS in TB infection is still scarce. Methods: We conducted an adult prospective study in mainland China to evaluate the diagnostic performance of mNGS for detection of Mycobacterium tuberculosis complex (MTB) in multiple forms of direct clinical samples compared with GeneXpert MTB/RIF assay (Xpert), traditional diagnostic methods, and the clinical final diagnosis. Results: Of 123 patients presenting with suspected active TB infection between June 1, 2017, and May 21, 2018, 105 patients underwent synchronous tuberculous testing with culture, Xpert, and mNGS on direct clinical samples including sputum, cerebrospinal fluids, pus, etc. During follow-up, 45 of 105 participants had clinical final diagnosis of active TB infection, including 13 pulmonary TB cases and 32 extrapulmonary TB cases. Compared to clinical final diagnosis, mNGS produced a sensitivity of 44% for all active TB cases, which was similar to Xpert (42%) but much higher than conventional methods (29%). With only one false-positive result, mNGS had a specificity of 98% in our study. mNGS yielded significantly much higher sensitivity in pre-treatment samples (76%) than post-treatment ones (31%) (P = 0.005), which was also true for Xpert and conventional methods. Combining Xpert and mNGS together, the study identified 27 of 45 active TB cases (60%), including all 13 conventional method-identified cases, and the result reached statistical significance compared to conventional methods (McNemar-test P < 0.001). Conclusions: mNGS had a similar diagnostic ability of MTB compared with Xpert and showed potential for a variety of clinical samples. Combined mNGS and Xpert showed an overall superior advantage over conventional methods and significantly improved the etiology diagnosis of both MTB and other pathogens. The result that anti-TB treatment significantly reduced diagnostic efficacy of culture, Xpert, and mNGS highlighted the importance of collecting samples before empirical treatment.

Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis.

The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans. In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay. Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p<0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively. In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB.

Assessing and screening for T-cell epitopes from Mycobacterium tuberculosis RD2 proteins for the diagnosis of active tuberculosis

ABSTRACT The Region of D eletion 2 (RD2) of Mycobacterium tuberculosis encodes reserved antigens that contribute to bacterial virulence. Among these antigens, Rv1983, Rv1986, Rv1987, and Rv1989c have been shown to be immunodominant in infected cattle; however, their diagnostic utility has not been evaluated in humans.In this study, we screened 87 overlapping synthetic peptides encoded by five RD2 proteins for diagnosing tuberculosis epitopes in 50 active tuberculosis (TB) cases, 31 non-tuberculosis patients and 36 healthy individuals. A pool of promising epitopes was then assessed for their diagnostic value in 233 suspected TB patients using a whole blood IFN-γ release assay.Only 10 peptides were recognized by more than 10% of active tuberculosis patients. The IFN-γ release responses to Rv1986-P9, P15, P16, Rv1988-P4, P11, and Rv1987-P11 were significantly higher in the active TB group than in the control groups (p < 0.05). The whole blood IFN-γ release assay based on these epitopes yielded a sensitivity of 51% and a specificity of 85% in diagnosing active tuberculosis, and the corresponding results using the T-SPOT.TB assay were 76% and 75%, respectively.In conclusion, these results suggest that the six epitopes from the RD2 of M. tuberculosis have potential diagnostic value in TB.

Screening and identification of a six-cytokine biosignature for detecting TB infection and discriminating active from latent TB.

BACKGROUND: The early and accurate diagnosis of tuberculosis (TB) is critical for controlling the global TB epidemic. Although early studies have supported the potential role of cytokine biomarkers in blood for the diagnosis of TB, this method requires further investigation and validation in different populations. A set of biomarkers that can discriminate between active TB (ATB) and latent TB infection (LTBI) remains elusive. METHODS: In the current study, we organized two retrospective cohorts and one prospective cohort to investigate the immune responses at different clinical stages of TB infection, as determined by candidate cytokine biomarkers detected with a multiplex cytokine platform. Using a pre-established diagnostic algorithm, participants were classified as ATB, LTBI, and TB uninfected controls (CON). Based on our multiplex cytokine assay, a multi-cytokine biosignature was modelled for the optimal recognition of the different TB infection status. RESULTS: Our analysis identified a six-cytokine biosignature of TB-antigen stimulated IFN-γ, IP-10, and IL-1Ra, and unstimulated IP-10, VEGF, and IL-12 (p70) for a biomarker screening group (n = 88). The diagnostic performance of the biosignature was then validated using a biomarker validation cohort (n = 216) and resulted in a sensitivity of 88.2% and a specificity of 92.1%. In a prospectively recruited clinical validation cohort (n = 194), the six-cytokine biosignature was further evaluated, and displayed a sensitivity of 85.7%, a specificity of 91.3% and an overall accuracy of 88.7%. CONCLUSIONS: We have identified a six-cytokine biosignature for accurately differentiating ATB patients from subjects with LTBI and CON. This approach holds promise as an early and rapid diagnostic test for ATB.

In Vitro Susceptibility of Mycobacterium abscessus and Mycobacterium fortuitum Isolates to 30 Antibiotics.

OBJECTIVE: Nontuberculous mycobacteria (NTM) cause various diseases in humans and animals. Recently, the prevalence of NTM-related disease has been on the rise, becoming an emerging public health problem. The aim of this study was to determine the antibiotic susceptibility profiles of clinical isolates of Mycobacterium abscessus and Mycobacterium fortuitum. Methods. We performed susceptibility tests on 37 clinical NTM isolates to 30 antibiotics with the microdilution method recommended by the Clinical and Laboratory Standards Institute. RESULTS: Both M. abscessus and M. fortuitum were highly resistant to antitubercular drugs such as isoniazid, rifampin, ethambutol, clofazimine, ethionamide, and rifabutin. M. abscessus showed the lowest resistant rates to cefoxitin (10%), azithromycin (10%), amikacin (10%), and clarithromycin (20%) and very high resistant to sulfamethoxazole, vancomycin, oxacillin, clindamycin, and all fluoroquinolones. M. fortuitum showed low resistance to tigecycline (0%), tetracycline (0%), cefmetazole (12%), imipenem (12%), linezolid (18%), and the aminoglycosides amikacin (0%), tobramycin (0%), neomycin (0%), and gentamycin (24%). CONCLUSION: Amikacin, cefoxitin, and azithromycin have the highest in vitro activity against M. abscessus. Isolates of M. fortuitum need to be individually evaluated for drug susceptibility before choosing an effective antimicrobial regimen for treatment of infections.

Evaluation of the dynamic pattern of viral evolution in patients with virological breakthrough during treatment with nucleoside/nucleotide analogs by ultra­deep pyrosequencing.

Virological breakthrough is a clinical manifestation in patients infected with chronic hepatitis B (CHB), who undergo treatment with nucleoside/nucleotide analogs (NUCs). The current understanding of the underlying mechanism of virological breakthrough is limited. Ultra­deep pyrosequencing (UDPS) is a novel and powerful tool used to investigate minor viral variants and viral evolution. The present study used UDPS to investigate the viral evolution pattern during virological breakthrough in patients with CHB treated with NUCs. A total of 12 patients who experienced virological breakthrough were recruited in the present study. During the treatment with lamivudine, adefovir was added as a rescue therapy when virological breakthrough emerged, and the therapy was continued until week 96. Serum samples from each patient were collected at different time points for UDPS analysis. Treatment with lamivudine resulted in an increased rate of the viral mutations, rtM204V/I, rtL180M and rtL80I. Virological breakthrough was accompanied by significant rtM204I/V substitutions in eight of the patients. A total of three types of rt204 mutation, associated with virological breakthrough, were observed, including YIDD variant­dominated, YVDD variant­dominated and YMDD wild­type­dominated virological breakthrough. YVDD variants reverted to the wild­type following the adefovir add­on rescue therapy, although the YIDD variants remained dominant following the combination therapy. The mechanism underlying virological breakthrough was revealed to be complex and associated with the rapid replication of mutated variants. UDPS analysis, therefore, provided a useful tool to investigate the dynamic evolution pattern of hepatitis B virus.

Fluctuating Behavior and Influential Factors in the Performance of the QuantiFERON-TB Gold In-Tube Assay in the Diagnosis of Tuberculosis.

BACKGROUND: The QuantiFERON-TB Gold In-Tube (QFT-GIT) is a newly developed but widely used interferon-γ release assay for diagnosing tuberculosis (TB). However, research has not determined whether age or the use of an immune suppressive or anti-TB treatment influences this assay's ability to detect TB. We assessed the QFT-GIT diagnostic performance for active tuberculosis (ATB) in children and adults in an endemic country and explored the effects of glucocorticoids and anti-TB therapy on the diagnostic value of the QFT-GIT. METHODS: A total of 60 children and 212 adults with suspected ATB were evaluated with the QFT-GIT. The association between the QFT-GIT diagnostic value and pretreatment factors was qualitatively and quantitatively assessed. RESULTS: The sensitivity of the QFT-GIT was 83.9% (95% CI 66.3%-94.6%) in children, and 73.7% (95% CI 57.8%-85.2%) in adults. Glucocorticoids affected the mitogen-stimulated response in both children and adults. In subjects undergoing glucocorticoid pretreatment, 25.0% of the children presented with false-negative QFT-GIT results, 28.6% of adults presented with indeterminate results. For subjects pre-treated with anti-TB drugs, 44.4% presented with false-negative QFT-GIT results. CONCLUSIONS: The QFT-GIT has higher sensitivity and specificity in children than adults. Glucocorticoid treatment negatively impacts the diagnostic value of the QFT-GIT in all age groups. Anti-TB treatment decreases the sensitivity of the QFT-GIT. Therefore, we recommend that the QFT-GIT assay be performed before TB-specific treatment is initiated and the test should not be used on people undergoing immunosuppression treatment, regardless of their age. A quantitative analysis of the QFT-GIT could be useful for assessing and monitoring TB-specific and non-specific immunity during conversion of the disease.

Evolution and transmission patterns of extensively drug-resistant tuberculosis in China.

The emergence and transmission of extensively drug-resistant tuberculosis (XDR-TB) pose an increasing threat to global TB control. This study aimed to identify the patterns of evolution and transmission dynamics of XDR-TB in populations in a region of China where TB is highly endemic. We analyzed a total of 95 XDR-TB isolates collected from 2003 to 2009 in Chongqing, China. Eight drug resistance genes covering 7 drugs that define XDR-TB were amplified by PCR followed by DNA sequencing. Variable-number tandem repeat 16-locus (VNTR-16) genotyping and genotypic drug resistance profiles were used to determine the evolution or transmission patterns of XDR-TB strains. Our results indicated that the Beijing genotype was predominant (85/95 [89.5%]) in XDR-TB strains, and as many as 40.0% (38/95) of the isolates were distributed into 6 clusters based on VNTR-16 genotyping and drug resistance mutation profiles. All isolates of each cluster harbored as many as six identical resistance mutations in the drug resistance genes rpoB, katG, inhA promoter, embB, rpsL, and gidB. Among the nine cases with continuous isolates from multidrug-resistant (MDR) to XDR-TB, 4 cases represented acquired drug resistance, 4 cases were caused by transmission, and 1 case was due to exogenous superinfection. The XDR-TB epidemic in China is mainly caused by a high degree of clonal transmission, but evolution from MDR to XDR and even superinfection with a new XDR strain can also occur.

Detection and identification of viral pathogens in patients with hand, foot, and mouth disease by multilocus PCR, reverse-transcription PCR and electrospray ionization mass spectrometry.

BACKGROUND: Rapid detection and identification of viruses are important for early diagnosis and effective surveillance of hand, foot, and mouth disease (HFMD). We described a novel assay using multilocus PCR and reverse transcription-PCR coupled with electrospray ionization mass spectrometry (RT-PCR/ESI-MS) to simultaneously detect and identify human enterovirus A-D, adenovirus A-F, human herpesvirus 1-8, parvovirus B19 and polyomavirus. OBJECTIVES: To evaluate the accuracy and efficacy of the RT-PCR/ESI-MS method, to detect and type enterovirus from specimens of clinical diagnosed HFMD patients. STUDY DESIGN: In this study, 152 specimens of clinically diagnosed HFMD patients were studied by the assay using RT-PCR/ESI-MS method. The detection and typing of enterovirus by RT-PCR/ESI-MS were compared with results from reference molecular methods. RESULTS: The assay detected enteroviruses in 97 (63.8%) specimens, resulting in a sensitivity of 93.8% (95% CI: 91.8-95.7%) and a specificity of 87.5% (95% CI: 84.8-90.2%) compared with a reference clinical diagnostic test. Most enterovirus genotypes (65/84; 77%) determined by the platform were consistent with 5' UTR sequence analysis, and most misidentifications resulted from the virus library, which could be further improved by updating the enterovirus database. In addition to enteroviruses, herpesviruses, polyomaviruses, adenoviruses and human rhinoviruses were detected and identified in 55 (36%) HFMD specimens by RT-PCR/ESI-MS. CONCLUSION: With the capability of high throughput and detection and typing of multiple clinically relevant viruses simultaneously, RT-PCR/ESI-MS can be a rapid and robust laboratory tool for identifying viral pathogens.

Sputum microbiota associated with new, recurrent and treatment failure tuberculosis.

Microbiota have recently been shown to be associated with many disease conditions. However, the microbiota associated with tuberculosis (TB) infection, recurrence and treatment outcome have not been systematically characterized. Here, we used high throughput 16S RNA sequencing to analyze the sputum microbiota associated with Mycobacterium tuberculosis infection and also to identify the microorganisms associated with different outcomes of TB treatment. We recruited 25 new TB patients, 30 recurrent TB patients and 20 TB patients with treatment failure, as well as 20 healthy controls. Streptococcus, Gramulicatella and Pseudomonas were more abundant in TB patients while Prevotella, Leptotrichia, Treponema, Catonella and Coprococcus were less abundant in TB patients than in the healthy controls. We found reduced frequency and abundance of some genera such as Bulleidia and Atopobium in recurrent TB patients compared with those in new TB patients. In addition, the ratio of Pseudomonas / Mycobacterium in recurrent TB was higher than that in new TB while the ratio of Treponema / Mycobacterium in recurrent TB was lower than that in new TB, indicating that disruption of these bacteria may be a risk factor of TB recurrence. Furthermore, Pseudomonas was more abundant and more frequently present in treatment failure patients than in cured new patients, and the ratio of Pseudomonas / Mycobacterium in treatment failure was higher than that in new TB. Our data suggest that the presence of certain bacteria and the disorder of lung microbiota may be associated with not only onset of TB but also its recurrence and treatment failure. These findings indicate that lung microbiota may play a role in pathogenesis and treatment outcome of TB and may need to be taken into consideration for improved treatment and control of TB in the future.

Underestimation of the resistance of Mycobacterium tuberculosis to second-line drugs by the new GenoType MTBDRsl test.

The GenoType MTBDRsl is a new-generation PCR-based line-probe assay for the detection of extensively drug-resistant tuberculosis (XDR-TB). This study evaluated the performance of MTBDRsl in detecting genotypic resistance to ethambutol, kanamycin, and ofloxacin in Mycobacterium tuberculosis (MTB) strains. The drug resistance of 262 unique clinical MTB isolates from China was analyzed with MTBDRsl, traditional TB drug susceptibility testing (DST), and sequencing. Sensitivity of MTBDRsl was 62.4% (93/149; 95% CI = 54.1 to 70.2) for detection of ethambutol resistance, 57.9% (55/95; 95% CI = 47.3 to 68) for kanamycin resistance, and 81% (111/137; 95% CI = 73.4 to 87.2) for ofloxacin resistance; specificity was 76.8% (86/112; 95% CI = 67.9 to 84.2), 98.8% (164/166; 95% CI = 95.7 to 99.9), and 91.1% (113/124; 95% CI = 84.7 to 95.5), respectively. Sequencing suggested that 36.9% (55/149) of ethambutol-resistant strains had no embB306 mutation and that 26.8% (40/149) had embB497 mutation not covered by MTBDRsl. Furthermore, MTBDRsl indicated ethambutol resistance in 23.2% (26/112) of ethambutol-susceptible strains, of which 92.3% (24/26) were confirmed resistant by sequencing. This study demonstrated that genotypic resistance to ethambutol, kanamycin, and ofloxacin in MTB can be quickly determined with the MTBDRsl. As a rapid and convenient genetic method, this assay could function as a supplement to traditional DST. More relevant genetic markers are needed to improve sensitivity.

Improved diagnostic power by combined interferon-gamma release assay and nested-PCR in tuberculous pleurisy in high tuberculosis prevalence area.

The conventional acid fast bacilli (AFB) smear and Mycobacterium tuberculosis (M.tb) culture of pleural effusion and tuberculin skin test (TST) in tuberculous pleurisy are unable to meet clinical needs because of their low sensitivities and specificities. To evaluate the diagnostic accuracies of QuantiFERON TB Gold In-Tube test (QFT-GIT) and nested-PCR in tuberculous pleurisy, we conducted a cross-sectional study in regions of China with a high tuberculosis (TB) epidemic. Seventy-eight participants were enrolled: 58 TB patients with diagnosis of confirmed or probable tuberculous pleurisy and 20 non-TB patients with a diagnosis of other non-TB diseases. The positive rates of AFB smear and M.tb culture in the pleural effusion were 5.8% (2/42) and 10.6% (5/47), respectively. The sensitivity and specificity of QFT-GIT were 93.1% (54/58) and 90.0% (18/20), whereas those of TST were 68.5% (37/54) and 86.7% (13/15), respectively; the sensitivity of QFT-GIT was significantly higher than TST (P = 0.013). The sensitivity and specificity of M.tb-specific nested-PCR in pleural effusion were 94.8% (55/58) and 90.0% (18/20), respectively, with a turnaround time of 7 h. Furthermore, combined QFT-GIT and nested-PCR detection improves the specificity to 100% with a sensitivity of up to 90.0%. This combination of immunoassay and molecular detection holds promise for the clinical diagnosis of tuberculous pleurisy.