Virulent hypermucoviscous Klebsiella pneumoniae strains associated with the magA and rmpA genes have mainly emerged in Asia. We analysed the frequency and the clinical and molecular epidemiology of K. pneumoniae bacteraemia isolates obtained over a 7-year period (2007-2013). Fifty-three of 878 K. pneumoniae invasive isolates (5.4%) showed a hypermucoviscous phenotype (by the string test). Of these, 16 (30.2%) were magA(+)/rmpA(+), 12 (22.6%) were magA(-)/rmpA(+), and the remaining 25 (47.2%) were magA(-)/rmpA(-). After multilocus sequence typing and wzi sequencing, all magA(+)/rmpA(+) isolates were serotype K1 and sequence type (ST)23. Of the 12 magA(-)/rmpA(+) isolates, nine were K2 (ST380, ST86, ST65, ST25 and ST493), and three magA(-)/rmpA(+) isolates had the new wzi allele 122, an unknown serotype, and the new ST1013. The remaining isolates, which were magA(-)/rmpA(-), showed different serotypes and STs. Patients with magA(+)/rmpA(+) or magA(-)/rmpA(+)K. pneumoniae bacteraemia more frequently had pyogenic liver abscesses (PLAs) and pneumonia than patients with magA(-)/rmpA(-)K. pneumoniae bacteraemia (respectively: 21.4% vs. 8%, p 0.26; and 17.9% vs. 0%, p 0.05). In fact, magA(-)/rmpA(-) isolates were similar to the those termed 'classic' K. pneumoniae isolates causing bacteraemia, the urinary and biliary tracts being the main foci of infection. In conclusion, hypervirulent clones (CC23K1, CC86K2, CC65K2, and CC380K2) were infrequent among K. pneumoniae isolates causing bacteraemia in our geographical area. A hypermucoviscous phenotype as determined with the string test is not enough to recognize these clones; additional molecular studies are needed. Patients with magA(+) and/or rmpA(+)K. pneumoniae bacteraemia more frequently had PLAs and pneumonia than patients without hypermucoviscosity genes.

There are seven confirmed hepatitis C virus (HCV) genotypes, with whole-genome nucleotide sequences differing by Ͼ30%, and each can be further subdivided into related subtypes (67 confirmed), with nucleotide sequence divergence of between 15% and 30% (1).Genotype identification has long been used in clinical practice, because major genotypes have different response rates and require different doses and durations of pegylated interferon and ribavirin (PR) treatment. In contrast, until recently, subtype identification was mainly used in epidemiological studies. However, both in vitro studies and clinical trials with different classes of direct-acting antiviral (DAA) agents (NS3 protease, NS5A-, and nucleos[t]ide and nonnucleos[t]ide NS5B-polymerase inhibitors), given with PR or in interferon-free combinations, have shown lower response rates for HCV genotype 1a than for HCV genotype 1b (2-8). Moreover, at least for HCV genotype 1, both the frequency and the pattern of resistance to different DAA classes are subtype specific (9). A striking example is the NS3-Q80K polymorphism, naturally found in Ͼ30% of naive subtype 1a patients but in Ͻ1% of subtype 1b patients (10), which conveys 30%-to-40%-lower sustained-virologic-response (SVR) rates to the macrocyclic protease inhibitor simeprevir (2). Similarly, all subtype 1g sequences identified naturally carry a mutation conferring resistance to linear NS3 protease inhibitors (11).Subtype-specific differences in the genetic barrier to resistance appear to correlate to the RNA-dependent RNA polymerase mu-

No abstract

An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3-protease inhibitors.

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GS-FLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%.Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity.

BackgroundSelection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region.Methodology/Principal FindingsSerum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples.ConclusionsUDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants.

BackgroundPaclitaxel, a widely-used antineoplastic drug, produces a painful peripheral neuropathy that in rodents is associated with peripheral-nerve mitochondrial alterations. The sigma-1 receptor (σ1R) is a ligand-regulated molecular chaperone involved in mitochondrial calcium homeostasis and pain hypersensitivity. This receptor plays a key role in paclitaxel-induced neuropathic pain, but it is not known whether it also modulates mitochondrial abnormalities.In this study, we used a mouse model of paclitaxel-induced neuropathic pain to test the involvement of the σ1R in the mitochondrial abnormalities associated with paclitaxel, by using genetic (σ1R knockout mice) and pharmacological (σ1R antagonist) approaches.ResultsPaclitaxel administration to wild-type (WT) mice produced cold- and mechanical-allodynia, and an increase in the frequency of swollen and vacuolated mitochondria in myelinated A-fibers, but not in C-fibers, of the saphenous nerve. Behavioral and mitochondrial alterations were marked at 10 days after paclitaxel-administration and had resolved at day 28. In contrast, paclitaxel treatment did not induce allodynia or mitochondrial abnormalities in σ1R knockout mice. Moreover, the prophylactic treatment of WT mice with BD-1063 also prevented the neuropathic pain and mitochondrial abnormalities induced by paclitaxel.ConclusionsThese results suggest that activation of the σ1R is necessary for development of the sensory nerve mitochondrial damage and neuropathic pain produced by paclitaxel. Therefore, σ1R antagonists might have therapeutic value for the prevention of paclitaxel-induced neuropathy.

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.