Xinhua Weng

Analysis of microRNA expression profiling identifies miR-155 and miR-155* as potential diagnostic markers for active tuberculosis: a preliminary study

To explore biologic behaviors and disease relevance of microRNAs (miRNAs) in the development of active tuberculosis (ATB), we investigated the expression profile of Mycobacterium tuberculosis (MTB) purified protein derivative (PPD)-induced miRNAs to determine the specific miRNAs involved in the pathogenesis of ATB. The expression profile of miRNA under PPD challenge was first measured using microarray analysis in peripheral blood mononuclear cells isolated from ATB patients and healthy controls (HC). The remarkably reactive miRNAs were then validated in a larger cohort by quantitative real-time polymerase chain reaction (qRT-PCR). The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of the determined PPD-responsive miRNAs. The potential targets for those miRNAs were also predicted by computational programs. Fourteen of 866 human miRNAs exhibited at least 1.8-fold difference in the ratio of expression level before and after stimulation with PPD between the ATB and HC groups. The qRT-PCR study validated the findings from microarray-based screening, in which miR-155 exhibited a fold change of 1.4 in the HC group and 3.7 in the ATB group upon PPD stimulation (p < 0.0001); miR-155* exhibited a fold change of 1.9 in the HC and 4.6 in the ATB group (p < 0.005). In ROC plots, the area under the curve was 0.8972 for miR-155 and 0.7945 for miR-155*. The background expression of these 2 microRNAs exhibited no differences between the ATB and HC groups. miR-155 and miR-155* exhibited characteristic expression by TB-specific antigen, suggesting that they can be potential diagnostic markers under the challenge of specific MTB antigens.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Evaluation of the Diagnostic Potential of IP-10 and IL-2 as Biomarkers for the Diagnosis of Active and Latent Tuberculosis in a BCG-Vaccinated Population

Background The Mycobacterium tuberculosis (Mtb)-specific T-cell interferon gamma release assays (IGRAs) are useful in detecting Mtb infection but perform poorly at distinguishing active tuberculosis disease (ATB) and latent tuberculosis infection (LTBI). This study is aimed at evaluating additional cytokines as biomarkers besides interferon-gamma (IFN-γ) to improve the identification of ATB and LTBI. Methodology/Principal Findings Sixty-six patients with ATB, 73 household contacts (HHC) of ATB patients and 76 healthy controls (HC) were recruited to undergo QuantiFERON TB GOLD in-tube assay (QFT) and the enzyme-linked immunosorbent assay (ELISA) where the release of IFN-γ, IFN-γ inducible protein 10 (IP-10), Interleukin 2 (IL-2) and Tumor Necrosis Factor-α (TNF-α) was determined in the whole blood with or without antigen-stimulation. The positive rates of the QFT, IP-10 and IL-2 tests were 86.4%, 89.4% and 86.4% for the ATB group with no difference between them (p>0.05). However, QFT in combination with IP-10 and IL-2 significantly increased the detection rate to 95.5% in the ATB group (p = 0.03) and the indeterminate rate of all samples decreased from 2.3% (5/215) to 0.4% (1/215). The un-stimulated level of IP-10 was significantly higher in the HHC than the ATB and HC groups. The IP-10 responses were strongly associated with extended Mtb exposure time and the degree of smear-positivity of the index cases. The IL-2/IFN-γ ratio in the antigen-stimulated plasma could discriminate LTBI from ATB with a sensitivity of 77.2% and a specificity of 87.2%. Conclusion The increased Mtb-specific antigen-stimulated expression of IP-10 and IL-2 may be useful for detecting both ATB and LTBI. Combining the QFT with IP-10 and IL-2 could increase the detection accuracy of active TB over the QFT alone.

Interferon-Gamma Release Assay Performance in Pulmonary and Extrapulmonary Tuberculosis

Background The diagnosis of tuberculosis remains difficult. This study aimed to assess performance of interferon-gamma release assay (IGRA) in diagnosis of active tuberculosis (ATB) with pulmonary and extrapulmonary involvements, and to determine the diagnostic role of IGRA (T-SPOT.TB) and tuberculin skin test (TST) in BCG-vaccinated population. Methods and Findings Two hundred twenty-six ATB suspects were recruited and examined with T-SPOT.TB. Among them, fifty-two and seventy-six subjects were simultaneously tested by TST with 5TU or 1TU of purified protein derivative (PPD). The sensitivity of T-SPOT.TB was 94.7% (71/75), comparable in pulmonary and extrapulmonary disease groups (95.6% vs. 93.3%, P>0.05), while the specificity was 84.10% (90/107) but differed in two groups (69.2% vs. 88.9%, P = 0.02). Compared to T-SPOT.TB, TST with 5TU-PPD showed less sensitivity (92.3% vs. 56.4%) and specificity (84.6% vs. 61.5%) (both P<0.01); the sensitivity of TST with 1TU-PPD was 27.8%, and despite its specificity identical to T-SPOT.TB (both 82.8%) positive predictive value (PPV) was only 33.3%. By combining T-SPOT.TB with TST (1TU), the specificity rose to 95%, but the PPV stayed unchanged. Conclusions IGRA could function as a powerful immunodiagnostic test to explore pulmonary and extrapulmonary TB, while TST failed to play a reliable or auxiliary role in identifying TB disease and infection in the BCG-vaccinated population.

Evaluation of Gamma Interferon Release Assays Using Mycobacterium tuberculosis Antigens for Diagnosis of Latent and Active Tuberculosis in Mycobacterium bovis BCG-Vaccinated Populations

ABSTRACT T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.

Lactic acidosis during telbivudine treatment for HBV: A case report and literature review

All oral nucleoside analogues against hepatitis B virus, with an exception of telbivudine, have been reported causing lactic acidosis (LA). Here we report the first case of chronic hepatitis B developing severe refractory LA during telbivudine monotherapy. A 36-year-old man of Chinese origin received telbivudine antiviral treatment for chronic hepatitis B. After 11 mo of therapy, he developed anorexia, nausea, and vomiting with mild muscle weakness. The patient was found with elevated serum creatine phosphokinase up to 3683 U/L (upper limit of normal 170 U/L) and marked LA. LA did not resolve immediately following discontinuation of telbivudine. His condition began to improve after hemodialysis treatment for 16 times and usage of glucocorticosteroid. The patient fully recovered after 16 wk of treatment. This is the first documented case with severe LA caused by telbivudine monotherapy. Besides serum creatine phosphokinase, blood lactate level should also be closely monitored in patients receiving telbivudine.

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse. Methods One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS. Results For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively. Conclusions rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.

Histological improvement of long-term antiviral therapy in chronic hepatitis B patients with persistently normal alanine aminotransferase levels.

The aim of this study was to evaluate the histological outcomes of chronic hepatitis B (CHB) patients with persistently normal alanine aminotransferase (ALT) levels after long‐term antiviral therapy. Paired liver biopsies before and after lamivudine (LAM) treatment in CHB patients with normal and elevated ALT levels were compared. Histological response was defined as a 1‐point decrease according to the Scheuer scoring system, without worsening of fibrosis between pretreatment and posttreatment biopsies. Among the 48 patients who underwent paired liver biopsies, 17 had persistently normal baseline ALT level and 31 had elevated ALT level. The median age of the patients was 44 years and 72.9% of the patients were male. The median duration of antiviral treatment was 44.5 months (range 14–104). Long‐term follow‐up of liver biopsies revealed that 82.4% of patients in the normal ALT group and 61.3% in the elevated ALT group had a baseline fibrosis score of 4, which was reduced to 17.6% and 38.7% after long‐term therapy, respectively, indicating reversal of cirrhosis in a large proportion of both groups, especially in patients with normal baseline ALT levels. Long‐term antiviral treatment could achieve significant histological improvement in CHB patients with fibrosis or cirrhosis, regardless of ALT level.

Mycobacterium tuberculosis Region of Difference (RD) 2 Antigen Rv1985c and RD11 Antigen Rv3425 Have the Promising Potential To Distinguish Patients with Active Tuberculosis from M. bovis BCG-Vaccinated Individuals

ABSTRACT Antigens encoded in the region of difference (RD) of Mycobacterium tuberculosis constitute a potential source of specific immunodiagnostic antigens for distinguishing tuberculosis (TB) infection from BCG vaccination. We evaluated the diagnostic potential of specific T-cell epitopes selected from two immunodominant antigens, Rv1985c and Rv3425, from RD2 and RD11, respectively, on the basis of epitope mapping, in TB patients and BCG-vaccinated healthy individuals. Using a whole-blood gamma interferon release assay, a wide array of epitopes was recognized on both Rv1985c and Rv3425 in TB patients. Those epitopes that could specifically discriminate TB infection from BCG vaccination were carefully selected, and the most promising peptide pools from Rv1985c showed a sensitivity of 53.9% and a specificity of 95.5%. When the novel specific peptides from Rv1985c joined the diagnostic antigens in the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, the sensitivity was increased from 86.4% to 96.2%, with no drop in specificity. These results indicate that the peptide pools selected from Rv1985c and Rv3425 have the potential to diagnose TB infection by a method that may be routinely used in clinical laboratories.

Histological outcome for chronic hepatitis B patients treated with entecavir vs lamivudine-based therapy.

AIM To compare the histological outcome of chronic hepatitis B (CHB) patients treated with entecavir (ETV) or lamivudine (LAM)-based therapy. METHODS We conducted a retrospective analysis of data from 42 CHB patients with advanced fibrosis (baseline Ishak score ≥ 2) or cirrhosis who were treated with ETV or LAM-based therapy in Beilun Peoples Hospital, Ningbo between January 2005 and May 2012. The patients enrolled were more than 16 years of age and underwent a minimum of 12 mo of antiviral therapy. We collected data on the baseline characteristics of each patient and obtained paired liver biopsies pre- and post-treatment. The Knodell scoring system and Ishak fibrosis scores were used to evaluate each example. An improvement or worsening of necroinflammation was defined as ≥ 2-point change in the Knodell inflammatory score. The progression or regression of fibrosis was defined as ≥ 1-point change in the Ishak fibrosis score. The continuous variables were compared using t-test or Mann-Whitney test, and the binary variables were compared using χ(2) test or Fishers exact test. The results of paired liver biopsies were compared with a Wilcoxon signed rank test. RESULTS Nineteen patients were treated with ETV and 23 patients were treated with LAM therapy for a mean duration of 39 and 42 mo, respectively. After long-term antiviral treatment, 94.74% (18/19) of the patients in the ETV arm and 95.65% (22/23) in the LAM arm achieved an HBV DNA level less than 1000 IU/mL. The majority of the patients (94.74% in the ETV arm and 73.91% in the LAM arm) had normalized ALT levels. The median Knodell necroinflammatory score decreased from 11 to 0 in the patients receiving ETV, and the median Knodell score decreased from 9 to 3 in the patients receiving LAM (P = 0.0002 and < 0.0001, respectively). The median Ishak fibrosis score showed a 1-point reduction in ETV-treated patients and a 2-point reduction in LAM-treated patients (P = 0.0019 and 0.0205, respectively). The patients receiving ETV showed a more significant improvement in necroinflammation than the LAM-treated patients (P = 0.0003). However, there was no significant difference in fibrotic improvement between the two arms. Furthermore, two patients in each arm achieved a fibrosis score of 0 post-treatment, which indicates a full reversion of fibrosis after antiviral therapy. CONCLUSION CHB patients with advanced fibrosis or cirrhosis benefit from antiviral treatment. ETV is superior to LAM therapy in improving necroinflammatory but not fibrotic outcome.