The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: a genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achievement of rapid, universal drug susceptibility testing for Mycobacterium tuberculosis complex (MTBC). We aimed to generate a WHO-endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: In this systematic analysis, we used a candidate gene approach to identify mutations associated with resistance or consistent with susceptibility for 13 WHO-endorsed antituberculosis drugs. We collected existing worldwide MTBC whole-genome sequencing data and phenotypic data from academic groups and consortia, reference laboratories, public health organisations, and published literature. We categorised phenotypes as follows: methods and critical concentrations currently endorsed by WHO (category 1); critical concentrations previously endorsed by WHO for those methods (category 2); methods or critical concentrations not currently endorsed by WHO (category 3). For each mutation, we used a contingency table of binary phenotypes and presence or absence of the mutation to compute positive predictive value, and we used Fisher's exact tests to generate odds ratios and Benjamini-Hochberg corrected p values. Mutations were graded as associated with resistance if present in at least five isolates, if the odds ratio was more than 1 with a statistically significant corrected p value, and if the lower bound of the 95% CI on the positive predictive value for phenotypic resistance was greater than 25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: We analysed 41 137 MTBC isolates with phenotypic and whole-genome sequencing data from 45 countries. 38 215 MTBC isolates passed quality control steps and were included in the final analysis. 15 667 associations were computed for 13 211 unique mutations linked to one or more drugs. 1149 (7·3%) of 15 667 mutations were classified as associated with phenotypic resistance and 107 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was more than 80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were identified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: We present the first WHO-endorsed catalogue of molecular targets for MTBC drug susceptibility testing, which is intended to provide a global standard for resistance interpretation. The existence of this catalogue should encourage the implementation of molecular diagnostics by national tuberculosis programmes. Funding: Unitaid, Wellcome Trust, UK Medical Research Council, and Bill and Melinda Gates Foundation

IDENTIFICATION OF DEVELOPMENTALLY REGULATED GENES OF DICTYOSTELIUM DISCOIDEUM USING A NOVEL TECHNIQUE: INSERTIONAL INACTIVATION.

IDENTIFICATION OF DEVELOPMENTALLY REGULATED GENES OF DICTYOSTELIUM DISCOIDEUM USING A NOVEL TECHNIQUE: INSERTIONAL INACTIVATION

Prosthetic mesh contamination during NOTES(®) transgastric hernia repair: A randomized controlled trial with swine explants

PURPOSE: Natural Orifice Translumenal Endoscopic Surgery (NOTES(®)) is a developing field in minimally invasive surgery that has been applied across a wide range of procedures; however, infectious concerns remain. Most of the applications have been for extraction, rather than reconstructive procedures. Prosthetic hernia repair, is a constructive procedure, has the unique challenge of avoiding contamination and infection of a permanent implant. Utilizing a novel device, we hypothesize that we can significantly reduce or eliminate prosthetic contamination during a transgastric approach for delivery of a clinically relevant, permanent, synthetic prosthetic. METHODS: 20 swine explants of stomach with attached esophagus were prepared by placing an ultraviolet (UV) light sensitive gel within the lumen of the stomach. Each stomach then underwent endoscopic gastrotomy utilizing a needle, wire guide, and 18-mm balloon dilator. A 10 × 15 cm polypropylene prosthetic was rolled and tied with a 2-0 silk suture, and delivered with one of two methods. Group A (control) utilized a snare to grasp the prosthetic adjacent to the endoscope, which was used to drag it through the gastrotomy. Group B (device) utilized a modified esophageal stent delivery system to deliver the prosthetic through the gastrotomy. Each prosthetic was then digitally photographed with UV illumination, with the contaminated areas illuminating brightly. Software analysis was performed on the photographs to quantify areas of contamination for each group. Statistical analysis was performed using a two-tailed t test with unequal variance. RESULTS: Group A demonstrated a mean of 57 % of the surface area of the prosthetic contaminated with UV light sensitive gel. Group B (experimental group) showed a mean of 0.01 % of the surface area contaminated (p \u3c 0.0001). 95 % confidence intervals indicated that the unprotected delivery technique exposes approximately 6,000 times more of the surface area to contamination than the delivery device. CONCLUSION: Use of this modified stent delivery system can nearly eliminate prosthetic contamination when placed via a transgastric approach in a swine explants model. Theoretically, the reduced inoculum size would reduce or eliminate clinical infection. Since the inoculum size required for clinical prosthetic infection for intraperitoneal mesh is unknown, further study is warranted to test the ability to eliminate clinical infection related to prosthetic delivery with this technique

wtPDV infection and binding is increased in CHO-proHB-EGF cells.

<p>CHO-empty and CHO-proHB-EGF cells were (A) examined for pro-HB-EGF expression by staining with goat anti-HB-EGF antibody or control goat serum followed by fixation and staining with rabbit anti-goat FITC. (B) Inoculated with wtPDV/USA2006, wtPDV/NL88n, wtCDV or wtMV (MOI 0.1) for 2 days. Cells were viewed by phase contrast microscopy (1st panel) or fixed before staining with SSPE serum and rabbit anti-human FITC (all other panels). Images were taken using a Nikon Eclipse TE2000-U UV microscope (X400). (C) Monolayers were inoculated with wtPDV/USA2006, wtCDV or wtMV (MOI 10) at 4°C for 2 hr. After washing, Sybr green qRT-PCR was carried out and the copy number of virus RNA determined from a standard curve.</p

Anti-β1 integrin treatment of Vero cells increases infection of wtPDV and Edmonston MV.

<p>(A) Vero cells were examined for β1 integrin expression by staining cells with anti-β1 integrin antibody or mouse isotype control followed by fixation and staining with rabbit anti-mouse FITC. Nuclei were stained with DAPI. Immunofluorescent images were taken using a Nikon Eclipse TE2000-U UV microscope (x100). (B) and (C) Vero cells were incubated with anti-β1 integrin (blocking) antibody or with control mouse isotype, prior to infection (MOI 0.1) for 2 days (Onderstepoort CDV and Edmonston MV) or 5 days (wtPDV/USA2006). (B) Cells were fixed before incubating with SSPE serum followed by staining with rabbit anti-human FITC and analysed by flow cytometry. (C) Virus was harvested and the titre determined by TCID₅₀/ml in VDS cells. The results are representative of two independent experiments.</p

wtPDV infects Vero cells.

<p>Vero and VDS cells were infected at an MOI of 0.1. (A) CPE observed in Vero and VDS cultures infected with wtPDV/NL88n, wt PDV/USA2006, wtCDV and wtMV at 2 dpi by phase contrast microscopy (Magnification X100). Foci of rounded cells are indicated by arrows. (B) Cells were infected with wtPDV/NL88n, wtCDV and wtMV, fixed, permeabilised and stained with SSPE serum and rabbit anti-human FITC; nuclei were stained with propidium iodide. Images were taken using a Nikon Eclipse TE2000-U UV microscope (x400). (C) Vero cells and VDS cells were infected with wtPDV/NL88n, wtPDVUSA2006, wtMV and wtCDV for up to 5 days. Titres were determined by TCID₅₀ in VDS cells. The results are representative of two independent experiments.</p

Sodium chlorate and heparinase treatment decreases cell fusion.

<p>Vero cells were untreated or treated with 30 mM or 60 mM sodium chlorate for 2 days prior to infection (MOI 0.1) with Schwarz-GFP MV or wtPDV/USA2006. (A) CPE and GFP expression by Schwarz-GFP MV was examined at 2 days by dual phase contrast-UV microscopy (top panel). Cultures of wtPDV were fixed and permeablised at 5 dpi before staining with SSPE antibody and rabbit anti-human FITC (bottom panel). Nuclei were stained with DAPI. (B) Vero cells were treated with 10 U/ml of heparinase for 90 min and infected at an MOI of 0.1 with Schwarz GFP MV or wtPDV/NL88n. CPE was examined at 2 days by dual phase contrast-UV microscopy (top panel) and wtPDV CPE by phase contrast microscopy (bottom panel). Images were taken using a Nikon Eclipse TE2000-U UV microscope (X100).</p

Virus Origin and Isolation.

<p>Virus Origin and Isolation.</p

Increased titres of wtPDV are obtained in CHO cells expressing either SLAM or PVRL4.

<p>Cells were infected (MOI of 0.1) with wtPDV/USA2006 (all CHO cell lines) and wtCDV (CHO, CHO-DSLAM and CHO-PVRL4). Virus was harvested at 1 to 5 dpi from wtPDV and wtCDV infected cultures and the titre determined by TCID50 in VDS cells. The results are representative of two independent experiments.</p