Next-generation ion torrent sequencing of drug resistance mutations in Mycobacterium tuberculosis strains.

A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes-rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)-were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

Performance of five FDA-approved rapid antigen tests in the detection of 2009 H1N1 influenza A virus.

Rapid antigen tests are commonly used by clinicians for rapid, simple, point-of-care testing. Five rapid antigen tests were shown to have low sensitivity (40.3-58.8%) when compared to real-time RT-PCR using nasal wash specimens from patients with influenza-like-illness (N = 167) that were collected previously and confirmed as 2009 pandemic influenza A (H1N1)-positive by PCR. Rapid antigen test sensitivity correlated with virus levels in nasal secretions when comparisons were made to cycle threshold (C(T)) values obtained from real-time RT-PCR. When C(T) values are <25 (equating to viral concentrations of >10(4) TCID(50)/ml) sensitivity for all five rapid antigen kits was high (range: 83-94% positive); however, when C(T) values are >30 (10(2) TCID(50)/ml), sensitivities of only 16-18% were observed for four of five rapid antigen kits. The Directigen EZ Flu A + B test detected more positive samples (35%) at lower viral concentrations with C(T) values >30 when compared with other commercial kits (P = 0.05). Rapid antigen test results must be interpreted with caution, and negative specimens may need confirmation by sensitive molecular assays.

Failure of pentoxifylline or cilostazol to improve blood and plasma viscosity, fibrinogen, and erythrocyte deformability in claudication.

Peripheral artery disease is associated with altered blood rheologic properties, including increased viscosity and decreased red blood cell (RBC) deformability. Pentoxifylline and cilostazol are available therapies for intermittent claudication. Improvement of blood viscosity and erythrocyte deformability have been cited as potential mechanisms of action for pentoxifylline. Cilostazol is a new drug with antiplatelet and vasodilating activity, but the mechanism by which it promotes an improvement in walking is not known. This study was performed to evaluate and compare the hemorheologic effects of pentoxifylline and cilostazol on viscosity, fibrinogen levels, and erythrocyte deformability when administered to adults with moderate to severe claudication. A double-blind, controlled study was conducted and included 59 patients (46 male, 13 female; mean age 65 yr) randomized to pentoxifylline 400 mg orally thrice daily (n=20), cilostazol 100 mg orally twice daily (n=19), or placebo (n=20); all subjects were observed for 24 weeks. Walking ability was assessed before, during, and at the conclusion of treatment by standard constant speed, variable grade treadmill testing. Erythrocyte deformability was measured by passage of washed RBCs, 10% hematocrit in phosphate buffered saline (PBS), through a polycarbonate membrane with 4.7 to 5.0 microm pores. Whole blood and plasma viscosity were measured using a cone/plate viscometer at variable shear rates (from 4.5 to 450 sec(-1)). Erythrocyte sedimentation rate was measured by a modified Westergren technique. Fibrinogen was assayed by a commercial reference laboratory. Plasma viscosities did not change significantly in any treatment group. Within-group comparisons demonstrated a significant (p<0.01) drop in whole blood viscosity (week 24 compared with week 0) for cilostazol-treated subjects (at shear rates of 45, 90, 225, and 450 sec(-1)), but these changes were not significantly different from those in the placebo group. There were no significant changes in whole blood viscosity for subjects treated with pentoxifylline or placebo. There were no significant changes in erythrocyte deformability, fibrinogen, or erythrocyte sedimentation rate. A trend toward improved walking distances was noted for both pentoxifylline and cilostazol in comparison with placebo. This trend was not correlated with changes in any underlying rheologic parameter. Ex vivo rheologic characteristics of blood from patients with intermittent claudication are not significantly affected by long-term administration of pentoxifylline or cilostazol. Pentoxifylline did not modulate viscosity or red cell deformability, a finding at variance with its putative mechanism of action. Pentoxifylline cannot be differentiated from cilostazol based on specific hemorheologic effects evaluated in this study. Different mechanisms of action for these medications should be considered.