Application of GeneXpert MTB/RIF technology in rifampicin resistance gene mutation of Mycobacterium tuberculosis / 中国热带医学

@#Abstract: Objective To analyze the accuracy and feasibility of GeneXpert MTB/RIF (GeneXpert) detection in the detection of Mycobacterium tuberculosis and the characteristics of rifampicin-resistant rpoB gene mutations. Methods A total of 4 234 sputum samples from suspected tuberculosis patients diagnosed in Sanya tuberculosis designated hospitals from 2015 to 2021 were selected and subjected to sputum smear, solid culture, drug sensitivity test by solid proportion method and GeneXpert detection. Results The positive detection rates of sputum smear, solid culture and GeneXpert of 4 234 sputum samples were 29.24% (1 238/4 234), 32.17% (1 362/4 234) and 35.40% (1 499/4 234), respectively. The positive detection rate of GeneXpert was higher than that of sputum smear, and the difference was statistically significant (χ2=36.775, P<0.01). It was slightly higher than solid culture, and the difference was not statistically significant (χ2=9.908, P=0.02). Taking solid culture results as the gold standard, the sensitivity and specificity of GeneXpert for detecting MTB were 91.04% (1 240/1 362) and 90.98% (2 613/2 872), respectively. According to the proportional drug susceptibility test results as the gold standard, the sensitivity and specificity of GeneXpert in detecting rifampicin resistance were 96.96% (96/99) and 98.86% (1 128/1 141), respectively, with the consensus rate of 98.71%. The accuracy of rifampicin resistance in GeneXpert group without probe mutation was significantly lower than that in group with probe mutation. There was a statistical difference in probe mutation frequency between newly treated and retreated cases. The analysis of rpoB gene mutation frequency characteristics showed: Probe E (50.00%) > Probe A (22.12%) > Probe D (14.42%) > Probe B (6.73%) > combined probe (5.77%) > Probe C (0.96%). Conclusions GeneXpert detection can quickly and effectively diagnose rifampicin-resistant tuberculosis, which is helpful for early clinical diagnosis and treatment. In this region, the rpoB gene mutation probes of rifampicin-resistant tuberculosis mainly occurr in Probe E and Probe A, with the least mutations in Probe C.

Detection of tBid Oligomerization and Membrane Permeabilization by Graphene-Based Single-Molecule Surface-Induced Fluorescence Attenuation.

The permeabilization of organelle membranes by BCL-2 family proteins is a pivotal step during the regulation of apoptosis; the underlying mechanisms remain unclear. Based on the fluorescence attenuation by graphene oxide, we developed a single-molecule imaging method termed surface-induced fluorescence attenuation (smSIFA), which enabled us to track both vertical and lateral kinetics of singly labeled BCL-2 family protein tBid during membrane permeabilization. We found that tBid monomers lie shallowly on the lipid bilayer, where they self-assemble to form oligomers. During the initiation phase of self-assembly, the two central hydrophobic helices (α6 and α7) of tBid insert halfway into the phospholipid core, while the other helices remain on the surface. In oligomerized tBid clusters, α6 and α7 prefer to float up, and the other helices may sink to the bottom of the membrane and cause the formation of transient two-dimensional, micelle-like pore structures, which are responsible for the permeabilization of membranes and the induction of apoptosis. Our results shed light on the understanding of tBid-induced apoptosis, and this nanotechnology-based smSIFA approach could be used to dissect the kinetic interaction between membrane protein and lipid bilayer at the single-molecule level with subnanometer precision.

Nanodomain Formation of Ganglioside GM1 in Lipid Membrane: Effects of Cholera Toxin-Mediated Cross-Linking.

Cross-linking of specific lipid components by proteins mediates transmembrane signaling and material transport. In this work, we conducted coarse-grained simulation to investigate the interactions of binding units of chorela toxin (CTB) with mixed ganglioside GM1 and dipalmitoylphosphatidylcholine (DPPC) lipid bilayer membrane. We determine that the binding of CTB pentamers cross-links GM1 molecules into protein-sized nanodomains that have distinct lipid order compared with the bulk. The toxin in the nanodomain partially penetrates into the membrane. The local disordering can also transmit across the membrane via lipid coupling. Comparison simulations on CTB binding to a membrane that is composed of various lipid components demonstrate that several factors are responsible for the nanodomain formation: (a) the negatively charged headgroup of a GM1 receptor is responsible for the multivalent binding; (b) the head groups being full of hydrogen-bonding donors and receptors stabilize the GM1 cluster itself and ensure the toxin binding with high affinity; and

Theoretical insight into the relationship between the structures of antimicrobial peptides and their actions on bacterial membranes.

Antimicrobial peptides with diverse cationic charges, amphiphathicities, and secondary structures possess a variety of antimicrobial activities against bacteria, fungi, and other generalized targets. To illustrate the relationship between the structures of these peptide and their actions at microscopic level, we present systematic coarse-grained dissipative particle dynamics simulations of eight types of antimicrobial peptides with different secondary structures interacting with a lipid bilayer membrane. We find that the peptides use multiple mechanisms to exert their membrane-disruptive activities: A cationic charge is essential for the peptides to selectively target negatively charged bacterial membranes. This cationic charge is also responsible for promoting electroporation. A significant hydrophobic portion is necessary to disrupt the membrane through formation of a permeable pore or translocation. Alternatively, the secondary structure and the corresponding rigidity of the peptides determine the pore structure and the translocation pathway.

Effects of antimicrobial peptide revealed by simulations: translocation, pore formation, membrane corrugation and euler buckling.

We explore the effects of the peripheral and transmembrane antimicrobial peptides on the lipid bilayer membrane by using the coarse grained Dissipative Particle Dynamics simulations. We study peptide/lipid membrane complexes by considering peptides with various structure, hydrophobicity and peptide/lipid interaction strength. The role of lipid/water interaction is also discussed. We discuss a rich variety of membrane morphological changes induced by peptides, such as pore formation, membrane corrugation and Euler buckling.