What Is Resistance? Impact of Phenotypic versus Molecular Drug Resistance Testing on Therapy for Multi- and Extensively Drug-Resistant Tuberculosis.

Rapid and accurate drug susceptibility testing (DST) is essential for the treatment of multi- and extensively drug-resistant tuberculosis (M/XDR-TB). We compared the utility of genotypic DST assays with phenotypic DST (pDST) using Bactec 960 MGIT or Löwenstein-Jensen to construct M/XDR-TB treatment regimens for a cohort of 25 consecutive M/XDR-TB patients and 15 possible anti-TB drugs. Genotypic DST results from Cepheid GeneXpert MTB/RIF (Xpert) and line probe assays (LPAs; Hain GenoType MTBDRplus 2.0 and MTBDRsl 2.0) and whole-genome sequencing (WGS) were translated into individual algorithm-derived treatment regimens for each patient. We further analyzed if discrepancies between the various methods were due to flaws in the genotypic or phenotypic test using MIC results. Compared with pDST, the average agreement in the number of drugs prescribed in genotypic regimens ranged from just 49% (95% confidence interval [CI], 39 to 59%) for Xpert and 63% (95% CI, 56 to 70%) for LPAs to 93% (95% CI, 88 to 98%) for WGS. Only the WGS regimens did not contain any drugs to which pDST showed resistance. Importantly, MIC testing revealed that pDST likely underestimated the true rate of resistance for key drugs (rifampin, levofloxacin, moxifloxacin, and kanamycin) because critical concentrations (CCs) were too high. WGS can be used to rule in resistance even in M/XDR strains with complex resistance patterns, but pDST for some drugs is still needed to confirm susceptibility and construct the final regimens. Some CCs for pDST need to be reexamined to avoid systematic false-susceptible results in low-level resistant isolates.

Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDRsl Assays.

In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

Procalcitonin Impairs Endothelial Cell Function and Viability.

BACKGROUND: Procalcitonin is used as a diagnostic tool for the identification and risk stratification of septic patients. Procalcitonin plasma concentrations tightly correlate with the severity of the ongoing inflammatory reaction and can rise up to 10,000-fold. Impairment of endothelial cell function plays an important role in the pathogenesis of hypotension and disturbed organ perfusion during sepsis. We investigated the possible effects of procalcitonin itself on endothelial cell function and viability. METHODS: Human endothelial cells were exposed to 0.01 to 100 ng/mL procalcitonin and investigated for endothelial permeability using transwells, migration in a scratch wound assay and new capillary formation on extracellular matrix in vitro. Tumor necrosis factor-α and vascular endothelial growth factor served as positive controls. Procalcitonin's impact on the response of endothelial cells toward ischemia was investigated in vivo in the murine model of unilateral femoral artery ligation. Procalcitonin-exposed endothelial cells were subjected to immunoblot for the investigation of vascular endothelial-cadherin expression and angiogenic signaling pathways. Flow cytometry was used for the detection of inflammatory activation and viability, and genomic analysis was performed. Data are presented as difference in means and 95% confidence intervals; statistical analyses were performed using analysis of variance/Bonferroni, and P values are reported as adjusted for multiple comparisons (Padjust). RESULTS: Tumor necrosis factor-α and 0.1 ng/mL procalcitonin induced endothelial barrier disruption after incubation of endothelial monolayers for 6 hours (-2.53 [-4.16 to -0.89], P = .0008 and -2.09 [-3.73 to -0.45], Padjust = .0064 compared with vehicle-treated control, respectively). Procalcitonin beginning at concentrations of 0.02 ng/mL reduced endothelial cell migration (0.26 [0.06 to 0.47], Padjust = .0069) and new capillary formation in vitro (0.47 [0.28 to 0.66], Padjust < .0001) contrasting the proangiogenic action of vascular endothelial growth factor. Left ventricular injection of procalcitonin in mice on postoperative day 1, 3, and 5 after induction of ischemia impaired new capillary formation and recovery of hindlimb perfusion in vivo (number of capillaries/mm in the ischemic leg of vehicle-treated versus procalcitonin-treated mice, 852.6 [383.4-1322], Padjust = .0002). Twenty-four-hour incubation with procalcitonin reduced the expression of vascular endothelial-cadherin at 100 ng/mL (0.39 [0.06-0.71], Padjust = .0167) and induced endothelial cell death (apoptosis, -5.4 [-10.67 to -0.13], Padjust = .0431). No alteration in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or extracellular signal-regulated kinase 1/2, and AKT signaling pathways was observed. Genomic analysis revealed regulation of a variety of genes involved in inflammation, angiogenesis, and cell growth. CONCLUSIONS: This study found that procalcitonin itself impaired several aspects of endothelial cell function. Procalcitonin-induced loss of endothelial barrier function may contribute to capillary leakage and therapy-refractory hypotension during sepsis. Anti-angiogenic properties of procalcitonin at low concentrations could also identify procalcitonin as a mediator of vascular disease associated with the metabolic syndrome. Future studies are needed to further test procalcitonin as a potential therapeutic target for preserving vascular dysfunction during acute and chronic inflammatory disorders.

Influence of different infracardial positions of central venous catheters in hemodynamic monitoring using the transpulmonal thermodilution method.

Hemodynamic measurements are often conducted by the transpulmonary thermodilution (TPTD)-based PiCCO(®)-system. This requires a central-venous (CVC) and a thermistor-tipped arterial catheter, usually placed in the femoral artery. In certain clinical situations, CVC devices have to be placed in the inferior vena cava. However, little is known about the influence of different CVC positions (i.e. ipsi- vs. contra-lateral to the arterial catheter) on the accuracy of the TPTD measurement results. In this prospective observational study surgical intensive care unit patients who had been inserted with CVCs either into the superior (CVCVCS) or the inferior vena cava (CVCinf) in addition to an arterial PiCCO(®)-catheter, were enrolled. Patients were then divided into two groups: Group I was provided with a CVC in the contralateral (CVCcontra) and Group II in the ipsilateral (CVCipsi) inferior vena cava. Thermodilution via injection of ice-cold saline was then performed via CVCsup and CVCinf. Bland-Altman analysis for cardiac index (CI), extra-vascular lung water index (EVLWI) and global end-diastolic volume index (GEDVI) were employed. Additional correction formulas for femorally assed parameters were determined. In a total of 28 patients, bias (limits of agreement) for measurements of CI in CVCcontra was found to be +0.2 (-0.4; +0.9) and +0.3 (-0.4; +1.0) L/min/m(2) in CVCipsi. GEDVI showed a bias of +274.8 (-47.3; +596.9) mL/m(2) in CVCcontra and +274.7 (-100.7; +650.1) mL/m(2) in CVCipsi. The mean EVLWI were 9.4 ± 4.3 mL/kg for EVLWIVCS and 10.7 ± 5.2 mL/kg for EVLWIinf. The LoA yielded at -3.4 and +6.1 mL/kg with a bias of +1.3 mL/kg. Percentage errors revealed clinically acceptable limits for CI and GEDVI, but not for EVLWI. Using TPTD via an infracardial central vein, measurements of CI showed high accuracy and precision while GEDVI measurements were precise with a lower accuracy, irrespective of the position of the infracardial CVC.

PhyResSE: a Web Tool Delineating Mycobacterium tuberculosis Antibiotic Resistance and Lineage from Whole-Genome Sequencing Data.

Antibiotic-resistant tuberculosis poses a global threat, causing the deaths of hundreds of thousands of people annually. While whole-genome sequencing (WGS), with its unprecedented level of detail, promises to play an increasingly important role in diagnosis, data analysis is a daunting challenge. Here, we present a simple-to-use web service (free for academic use at http://phyresse.org). Delineating both lineage and resistance, it provides state-of-the-art methodology to life scientists and physicians untrained in bioinformatics. It combines elaborate data processing and quality control, as befits human diagnostics, with a treasure trove of validated resistance data collected from well-characterized samples in-house and worldwide.

MTBseq: a comprehensive pipeline for whole genome sequence analysis of Mycobacterium tuberculosis complex isolates.

Analyzing whole-genome sequencing data of Mycobacterium tuberculosis complex (MTBC) isolates in a standardized workflow enables both comprehensive antibiotic resistance profiling and outbreak surveillance with highest resolution up to the identification of recent transmission chains. Here, we present MTBseq, a bioinformatics pipeline for next-generation genome sequence data analysis of MTBC isolates. Employing a reference mapping based workflow, MTBseq reports detected variant positions annotated with known association to antibiotic resistance and performs a lineage classification based on phylogenetic single nucleotide polymorphisms (SNPs). When comparing multiple datasets, MTBseq provides a joint list of variants and a FASTA alignment of SNP positions for use in phylogenomic analysis, and identifies groups of related isolates. The pipeline is customizable, expandable and can be used on a desktop computer or laptop without any internet connection, ensuring mobile usage and data security. MTBseq and accompanying documentation is available from https://github.com/ngs-fzb/MTBseq_source.

Mycobacterium tuberculosis resistance prediction and lineage classification from genome sequencing: comparison of automated analysis tools.

Whole-genome sequencing (WGS) has the potential to accelerate drug-susceptibility testing (DST) to design appropriate regimens for drug-resistant tuberculosis (TB). Several recently developed automated software tools promise to standardize the analysis and interpretation of WGS data. We assessed five tools (CASTB, KvarQ, Mykrobe Predictor TB, PhyResSE, and TBProfiler) with regards to DST and phylogenetic lineage classification, which we compared with phenotypic DST, Sanger sequencing, and traditional typing results for a collection of 91 strains. The lineage classifications by the tools generally only differed in the resolution of the results. However, some strains could not be classified at all and one strain was misclassified. The sensitivities and specificities for isoniazid and rifampicin resistance of the tools were high, whereas the results for ethambutol, pyrazinamide, and streptomycin resistance were more variable. False-susceptible DST results were mainly due to missing mutations in the resistance catalogues that the respective tools employed for data interpretation. Notably, we also found cases of false-resistance because of the misclassification of polymorphisms as resistance mutations. In conclusion, the performance of current WGS analysis tools for DST is highly variable. Sustainable business models and a shared, high-quality catalogue of resistance mutations are needed to ensure the clinical utility of these tools.