An additional lysis procedure during arthrocentesis of the temporomandibular joint

Background Arthrocentesis of the temporomandibular joint (TMJ) is an easy, highly efficient, minimally invasive procedure for treating temporomandibular joint disorders (TMDs). However, in some cases of mouth opening limitation (MOL), routine arthrocentesis is ineffective due to severe fibrotic adhesion in the superior joint space of the TMJ. In this condition, mechanical lysis of the adhesions might be needed to resolve the MOL, as well as other symptoms, such as chronic pain. Currently, this can be achieved by arthroscopic surgery or open TMJ surgery. The objective of this study was to introduce and evaluate our trial of the adhesion lysis procedure during arthrocentesis of the TMJ using normal 18-gauge needles. Results In this study, 40 patients with MOL due to disc derangement underwent conventional arthrocentesis at first and then physical detachment was conducted using the same needle. The change in maximum mouth opening (MMO) and the pain at the TMJ were recorded before, during, and after treatment according to our protocol. The mean increase in MMO after conventional arthrocentesis was 6.6 ± 4.2mm. The mean increase in MMO after the detachment procedure with the same needle was 4.2 ± 2.0 mm. The MMO in ten patients was significantly increased after the detachment procedure than after arthrocentesis alone. In all cases, the pain intensity in the TMJ significantly decreased over time, whereas the MMO increased over time. No adverse effect was observed in all joints during our observation periods. Conclusion We confirmed that our simple lysis procedure with the same needle of the arthrocentesis of the TMJ could not only improve the MMO more than after a conventional arthrocentesis but also resolve severe adhesion of the joint space that was ineffective by conventional arthrocentesis. Although this additional lysis procedure is simple, it might reduce the number of cases of more invasive procedures such as arthroscopic surgery or open TMJ surgery.

Genomic analysis of high copy-number sequences for the targeted detection of Listeria species using a flow-through surveillance system

The bacterial foodborne pathogen Listeria monocytogenes has been implicated in fresh produce outbreaks with a significant economic impact. Given that L. monocytogenes is widespread in the environment, food production facilities constantly monitor for the presence of Listeria species. To develop a surveillance platform for food processing facilities, this study conducted a comparative genomic analysis for the identification of conserved high copy sequences in the ribosomal RNA of Listeria species. Simulated folding was performed to assess RNA accessibility in the identified genomic regions targeted for detection, and the developed singleplex assay accurately detected cell amounts lower than 5 cells, while no signals were detected for non-targeted bacteria. The singleplex assay was subsequently tested with a flow-through system, consisting of a DNA aptamer-capture step, followed by sample concentration and mechanical lysis for the detection of Listeria species. Validation experiments indicated the continuous flow-through system accurately detected Listeria species at low cell concentrations.

Evaluation of the Allplex™ GI-Helminth(I) Assay, the first marketed multiplex PCR for helminth diagnosis

Molecular biology has been gaining more importance in parasitology. Recently, a commercial multiplex PCR assay detecting helminths was marketed: the Allplex™ GI-Helminth(I) Assay. It targets Ancylostoma spp., Ascaris spp., Enterobius vermicularis, Hymenolepis spp., Necator americanus, Strongyloides spp., Taenia spp. and Trichuris trichiura, but also the two most common microsporidia genera in human health, i.e. Enterocytozoon spp. and Encephalitozoon spp. This study aimed to evaluate and compare the Allplex™ GI-Helminth(I) Assay to classical diagnostic methods, based on a cohort of 110 stool samples positive for helminths (microscopy) or for microsporidia (PCR). Samples were stored at −80 °C until analysis by the Allplex™ GI-Helminth(I) Assay. False-negatives were re-tested with bead-beating pretreatment. Without mechanical lysis, concordance and agreement between microscopy and Allplex™ GI-Helminth(I) Assay ranged from 91% to 100% and from 0.15 to 1.00, respectively depending on the target. Concordance was perfect for Taenia spp. (n = 5) and microsporidia (n = 10). False-negative results were observed in 54% (6/13), 34% (4/11) and 20% (7/35) of cases, for hookworms, E. vermicularis and Strongyloides spp. detection, respectively. For these targets, pretreatment improved the results, but only slightly. Trichuris trichiura detection was critically low without pretreatment, as only 9% (1/11) of the samples were positive, but detection reached 91% (10/11) with bead-beating pretreatment. Mechanical lysis was also needed for Ascaris spp. and Hymenolepis spp. to reduce false-negative results from 1/8 to 1/21, respectively, to none for both. Overall, with an optimized extraction process, the Allplex™ GI-Helminth(I) Assay allows the detection of numerous parasites with roughly equivalent performance to that of microscopy, except for hookworms.

Hidden Components of Microbiome Diversity Revealed by Cell Wall Lysis with Adaptive Focused Acoustics

Within the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm; while some species are quite easy to lyse, others are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly skews current microbiome results in ways that are not currently understood. To develop a cell wall thickness-agnostic lysis protocol, we used Adaptive Focused Acoustics (AFA), a tunable acoustic methodology for processing of biological samples. Using identical aliquots of mouse stool homogenate as the lysis substrate, we compared AFA with chemical/mechanical lysis methodology routinely used in microbiome studies and found that AFA-mediated lysis substantially increases both microbial DNA yield as well as alpha and beta diversity. By starting with lower AFA energy levels, sequentially removing aliquots at each step, and subjecting the remainder to progressively stronger AFA treatment, we developed a sequential lysis method that accounts for differences in cell wall thickness. This method revealed even greater levels of diversity than single-timepoint AFA treatment. 16S sequencing results from the above experiments were verified by shotgun metagenome sequencing of a subset of the AFA samples. We found that lysis-induced noise affects not just species compositions, but also functional characterization of shotgun metagenome data. AFA samples also showed a higher detection of eukaryotic and fungal DNA. We suggest that AFA-mediated lysis produces a truer representation of the native microbiota, and that this method deserves consideration as a potential addition to microbiome lysis protocols.

Hidden components of microbiome diversity revealed by cell wall lysis with Adaptive Focused Acoustics

Within the rapidly evolving field of microbiome sequencing, a primary need exists for experimentally capturing microbiota in a manner as close as possible to their in vivo composition. During microbiome profiling, the first step necessarily involves lysis of the cell wall, releasing nucleic acids for next-generation sequencing. Microbial cell wall thicknesses can vary between 5nm to 80nm; while some species are quite easy to lyse, others are particularly resistant to lysis. Despite this, current chemical/mechanical lysis protocols ignore the possibility that species with different cell wall thicknesses are lysed at differential rates. This creates noise in species compositions and possibly skews current microbiome results in ways that are not currently understood. To develop a cell wall thickness-agnostic lysis protocol, we used Adaptive Focused Acoustics (AFA), a tunable acoustic methodology for processing of biological samples. Using identical aliquots of mouse stool homogenate as the lysis substrate, we compared AFA with chemical/mechanical lysis methodology routinely used in microbiome studies and found that AFA-mediated lysis substantially increases both microbial DNA yield as well as alpha and beta diversity. By starting with lower AFA energy levels, sequentially removing aliquots at each step, and subjecting the remainder to progressively stronger AFA treatment, we developed a sequential lysis method that accounts for differences in cell wall thickness. This method revealed even greater levels of diversity than single-timepoint AFA treatment. 16S sequencing results from the above experiments were verified by shotgun metagenome sequencing of a subset of the AFA samples. We found that lysis-induced noise affects not just species compositaions, but also functional characterization of shotgun metagenome data. AFA samples also showed a higher detection of eukaryotic and fungal DNA. We suggest that AFA-mediated lysis produces a truer representation of the native microbiota, and that this method deserves consideration as a potential addition to microbiome lysis protocols.

Blood-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Carbapenemase-Producing Enterobacteriaceae Directly from Positive Blood Culture Broths

ABSTRACT A variant of the modified carbapenem inactivation method (mCIM) was developed to detect carbapenemase activity directly from positive blood culture broths. The method, termed “Blood-mCIM,” was evaluated using Bactec blood culture bottles (Becton, Dickinson and Company, Franklin Lakes, NJ) inoculated with 27 different carbapenemase-producing Enterobacteriaceae (CPE) isolates and 34 different non-CPE isolates. The assay was positive for all blood culture broths inoculated with CPE isolates and negative for all blood culture broths inoculated with non-CPE isolates, corresponding to a diagnostic sensitivity and specificity of 100%, respectively. This assay is inexpensive using “off the shelf” reagents, does not require centrifugation or mechanical lysis, and can be readily implemented in any clinical microbiology laboratory. The Blood-mCIM should facilitate expedient administration of antimicrobial therapy targeted toward CPE bloodstream infections and assist infection control and public health surveillance.

An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

Chromatin immunoprecipitation (ChIP) is a commonly used technique to investigate which parts of a genome are bound by a particular protein. The result of ChIP is often interpreted in a binary manner: bound or not bound. Due to this focus, ChIP protocols frequently lack the ability to quantitatively compare samples with each other, for example in a time series or under different growth conditions. Here, using the yeast S. cerevisiae transcription factors Cbf1, Abf1, Reb1, Mcm1 and Sum1, we optimized the five major steps of a commonly used ChIP protocol: cross-linking, quenching, cell lysis, fragmentation and immunoprecipitation. Quenching with glycine is inefficient and can lead to large degrees of variability, an issue that is resolved by using tris(hydroxymethyl)aminomethane (Tris). Another source of variability is degradation of the protein of interest during the procedure. Enzymatic cell lysis with zymolyase can lead to extensive protein degradation, which is greatly reduced by mechanical lysis through bead beating. Degradation also occurs during sonication of chromatin, affecting large proteins in particular. An optimal mix of protease inhibitors and cross-linking with a higher percentage of formaldehyde reduces the extent of this degradation. Finally we also show that the immunoprecipitation step itself can be greatly improved with magnetic beads and optimized incubation/washing steps. The study results in a highly optimized protocol, which is shorter, easier to perform and has a stronger, more reproducible signal with less background. This protocol is presented in detail. In addition, the results highlight the greatest sources of variability in many other protocols, showing which steps are important to focus on for reproducible and quantitatively comparable ChIP experiments.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.