Assessments of Different Methods for Testing Heteroresistance to Rifampicin in Tubercle Bacillus.

This study was undertaken to compare the ability of four clinically used methods to detect the heteroresistance of the tubercle bacillus to Rifampicin (Rif). A total of 94 sputum smear-positive samples of re-treated tuberculosis (TB) patients were employed. Rif-resistant tubercle bacillus tests were carried out by four methods: Lowenstein-Jensen cultures (L-J), reverse transcription-polymerase chain reaction (RT-PCR), microscopic observation drug susceptibility (MODS), and high-resolution melting (HRM). The rates of detection of Rif-resistant bacteria by of L-J, RT-PCR, MODS, and HRM were 35.1% (33), 36.4% (35), 36.2% (34), and 16.0% (15), respectively. The rate of detection by RT-PCR was slightly higher than that of L-J and MODS (P > 0.05) and significantly higher than that of HRM (P < 0.01). The median detection time of L-J, RT-PCR, MODS, and HRM was 60 d, 1 d, 9 d, and 1 d, respectively. Smear-positive TB can be directly and rapidly detected using RT-PCR and MODS technologies. The positive rates of RT-PCR and MODS were not only highly consistent with that of L-J and remarkably higher than that of HRM, but also were obtained in a markedly shortened time. The combination of MODS and RT-PCR has a synergistic effect on the speed of detection of heteroresistance, which is of great value for the timely and accurate diagnosis, guiding the clinicians in formulating the optimal treatment of smear-positive pulmonary tuberculosis.

The morphological changes of monocytes in peripheral blood as a potential indicator for predicting active pulmonary tuberculosis.

BACKGROUND: Monocytes play a crucial role in immune response against Mycobacterium tuberculosis infection. The purpose of this current study was to investigate the morphology present on monocytes in peripheral blood from patients with active pulmonary tuberculosis (APTB) and the laboratory performance of the changes for discriminating cases from normal healthy subjects (NHS). METHOD: A total of 71 peripheral blood samples from patients with APTB, and 65 samples from NHS were analyzed. The mean monocyte volume with its distribution width and mean monocyte conductivity as well as monocyte light scatter were detected by VCS technology used on the LH750 hematology analyzer. Correlations of these changes with the serum cytokine level in the immune alterations were further evaluated. The Receiver operating characteristic curve (ROC) analysis was used to highlight the clinical implication. RESULTS: In APTB patients, the mean monocyte volume showed significant difference associated with an evident elevation in the mean monocyte volume distribution width compared to those in NHS. Furthermore, the mean monocyte volume had positive relationship with the serum level of interleukine-1ß response to M. tuberculosis infection. Simultaneous measurement of the mean monocyte volume and its distribution width was able to distinguish active infection with an excellent sensitivity of 84.5% and specificity of 90.5% comparable to those obtained from pro-inflammatory cytokine interleukine-6 identifying APTB with great accuracy. CONCLUSION: The morphological changes of monocytes particular increased mean volume may be a potential indicator to predict active tuberculosis infection.

Construction of a recombinant pIRES2-EGFP-ARTS plasmid and its effect on LX-2 cells.

The inhibition of the activation of hepatic stellate cells (HSCs) and the induction of their apoptosis have been investigated as potential strategies to counteract the development and progression of liver fibrosis. Previous research has suggested that apoptosis­related protein in the transforming growth factor­ß signaling pathway (ARTS) may serve a significant role in numerous cell types; however, little is known regarding its roles in HSCs. Total RNA was extracted from LX­2 cells, and the human full­length ARTS gene was obtained by reverse transcription­polymerase chain reaction and inserted into the pIRES2­EGFP cloning vector. Subsequently, the recombinant pIRES2­EGFP­ARTS plasmid was transfected into LX­2 cells by FuGENE 6 transfection reagent, and the expression of ARTS was detected by western blotting and fluorescent microscopy. In addition, the effects of pIRES2­EGFP­ARTS on the activation, apoptosis, viability and migration of LX­2 cells were assessed by western blot analysis, TUNEL staining, an MTT assay, and scratch and Transwell assays, respectively. The present results demonstrated that the pIRES2­EGFP­ARTS vector expressing human ARTS was successfully constructed, and the overexpression of ARTS contributed to enhance the apoptosis and inhibit the activation of human LX­2 HSCs. The present findings suggested that ARTS overexpression may have potential as a novel therapeutic strategy to reverse hepatic fibrosis.

The VCS parameters: Potential hematological indicators for predicting antituberculosis drug-induced neutropenia.

BACKGROUND: The morphological changes in activated neutrophils associated with antituberculosis drugs can be measured by volume, conductivity, and scatter (VCS) technology on the Coulter LH750 hematology analyzer. We conducted the current study to further validate the clinical usefulness of the neutrophil VCS parameters in predicting drug-induced neutropenia. METHODS: Peripheral blood samples were collected from 52 patients with drug-induced neutropenia, 309 patients without any abnormal CBC, and 237 healthy controls. The mean neutrophil volume (MNV) with its distribution width (NDW) and the mean neutrophil scatter (MNS) were studied. RESULTS: We observed a significant increase in the MNV and NDW as well as a significant decrease in the MNS in neutropenia patients approximately one week prior to development of neutropenia compared to healthy controls as well as to case controls. In addition, the delta MNV and delta MNS were respectively correlated well with delta absolute neutrophil counts when neutropenia occurred. The ROC curve analyses showed that the MNV、NDW and MNS had larger areas under curves compared to conventional parameters. With a cutoff of 150.15 for the MNV, a sensitivity of 84.4% and specificity of 75.7% were achieved prior to neutropenia. CONCLUSION: The neutrophil VCS parameters may be clinically useful as potential hematological indicators for predicting antituberculosis drug-induced neutropenia.

Macrophage-Microglia Networks Drive M1 Microglia Polarization After Mycobacterium Infection.

Central nervous system tuberculosis (CNS-TB) is caused by infection with Mycobacterium tuberculosis (Mtb). The inflammatory response following CNS-TB involves the activation of resident microglia and the infiltration of macrophages. However, it has not been clarified whether microglia can be polarized into the classically activated proinflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype after Mtb infection. In this study, we found that BV2 treated with conditioned media from cultures of macrophages infected with Mycobacterium marinum (Mm) induced the expression of M1 phenotypic genes including iNOS, TNF-α, IL-1ß, IL-6, CCL2, and CXCL10 but reduced that of M2 phenotypic genes such as Arginase 1, Ym1, and CD163. These results suggest that polarization of microglia is partly mediated through macrophage-microglia interactions as a priming signal. Overall, these results provide new insights into the modulatory mechanisms of microglial polarization, thereby possibly facilitating the development of new therapies for CNS-TB infection via the regulation of microglial polarization through signalling derived from macrophages infected with mycobacteria.

Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study.

BACKGROUND: Mycobacterial culture and identification provide a definitive diagnosis of TB. Culture on Löwenstein-Jensen (L-J) medium is invariably delayed because of the slow growth of M. tuberculosis on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients. METHODS AND FINDINGS: The biphasic medium consisted of 7 ml units of L-J slant medium, 3 ml units of liquid culture medium, growth indicator and a mixture of antimicrobial agents. The decontamination sediments of sputum specimens were incubated in the biphasic culture medium at 37°C. Mycobacterial growth was determined based on the appearance of red granule sediments and the examination using acid-fast bacilli (AFB). The clinical sputum specimens were cultured in the biphasic medium, on L-J slants and in the Bactec MGIT 960 culture system. Among smear-positive specimens, the mycobacteria recovery rate of the biphasic medium was higher than that of the L-J slants (P<0.001) and similar to that of MGIT 960 (P>0.05). Among smear-negative specimens, the mycobacterial recovery rate of the biphasic medium was higher than that of L-J slants (P<0.001) and lower than that of MGIT 960 (P<0.05). The median times to detection of mycobacteria were 14 days, 20 days and 30 days for cultures grown in MGIT, in biphasic medium, on L-J slants for smear negative specimens, respectively (P<0.001). CONCLUSIONS: The biphasic culture medium developed in this study is low-cost and suitable for mycobacterial recovery. It does not require any expensive detection instrumentation, decreases the time required for detection of M. tuberculosis complex, and increases the detection rate of M. tuberculosis complex.