Significance of Pretreatment Analysis of Hepatitis C Virus Genotype 1b Hypervariable Region 1 Sequences To Predict Antiviral Outcome

The heterogeneity of hypervariable region 1 (HVR1), located at the amino terminus of the E2 envelope, may be involved in resistance to alpha interferon (IFN-α) treatment. We investigated whether peculiar HVR1 domain profiles before treatment were associated with the maintenance of sensitivity or the appearance of resistance to treatment. Fifteen patients infected with hepatitis C virus genotype 1b and treated with IFN with or without ribavirin were selected. Ten responded to treatment (groups R1 and R2) and five did not (group NR). The amino acid sequences of 150 naturally occurring HVR1 variants present in the serum before therapy were compared in relation to treatment outcome. HVR1 variants from the NR group contained a constant nonantigenic amino acid segment that was not found in HVR1 variants from the R groups

Comparative microbial analysis of paired amniotic fluid and cord blood from pregnancies complicated by preterm birth and early-onset neonatal sepsis.

BACKGROUND: 16S rRNA-based genomic analyses have revolutionized our understanding of infectious diseases. Many cases which were recognized as "idiopathic" are now known to have an infectious etiology. Here, we present a proof-of-concept study to examine the microbial link between intra-amniotic infection (IAI) and early-onset neonatal sepsis (EONS). RESULTS: Using culture independent methods, we analyzed paired amniotic fluid (AF) and cord blood (CB) samples from 36 singleton pregnancies complicated by preterm birth (PTB), IAI, and/or EONS. PTB cases were grouped as 1) Group 1- neonatal blood culture-positive EONS (n=6). 2) Group 2- neonatal blood culture-negative presumed EONS with positive IAI (n=16). 3) Group 3- neonatal blood culture-negative presumed EONS with no IAI (n=7); 4) Group 4- no EONS or IAI (n=7). In addition, samples from term healthy deliveries (n=8) served as technical controls. A total of 31 species (15 non-redundant) were identified in AF, of which only 1/3 were cultivated. Significantly fewer microorganisms were detected in CB, with a total of 18 species (7 non-redundant) identified, of which only 2 (Escherichia coli, Streptococcus agalactiae) were cultivated. Of those, Bergeyella, Fusobacterium nucleatum, and Sneathia sanguinegens had not been detected in EONS before. The novel species identified in AF by PCR include Peptoniphilus harei and Lachnospiraceae sp. The majority (72%) of CB species were also detected in the matching AF, with E. coli and F. nucleatum as the most prevalent. The 16S rRNA sequences of paired AF and CB were 99.9-100% identical, while no identical sequences were found between different pregnancies. CONCLUSIONS: Previously unrecognized, uncultivated or difficult-to-cultivate species are implicated in EONS. Microbial species in paired AF and CB likely share the same infectious origin. Given its prevalence in EONS, F. nucleatum should be placed on the same importance scale as E. coli

A Listeria monocytogenes Strain Is Still Virulent despite Nonfunctional Major Virulence Genes

International audienceThe low-virulence Listeria monocytogenes strains have been previously assigned to 4 phenotypic groups. This study aimed to characterize the A23 strain, which exhibits a pulsed-field gel electrophoresis profile specific to low-virulence strains. This strain has the same causal mutations as the group III strains and a supplementary mutation in the mpl gene, leading to the absence of internalin A expression and the presence of inactive internalin B, phosphatidyl-inositol phospholipase C, and phosphatidylcholine phospholipase C. Despite these mutations in major virulence genes, the A23 strain formed plaques in cell monolayers and contaminated 100% of inoculated mice, suggesting that it evolved from group III strains by acquiring new virulence gene

A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity

International audienceThe sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDΔprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix αH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA boxes. PrfAK220T did not form a PrfA–DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T–RNA polymerase–DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix–turn–helix (HTH) motif

Summary of total number of species detected in amniotic fluid and cord blood by culture-dependent and independent methods.

*<p>Data presented as n (%).</p>†<p>Data presented as n.</p

Bacterial species identified in matching amniotic fluid, cord and neonatal blood samples by culture and/or culture-independent methods.^*.

*<p>Results are shown for Groups 1 and 2. No bacterial DNA was detected in any samples in Groups 3 and 4. The species identified in more than one compartment are shown in <b>bold</b>.</p

Similarity of 16S rRNA gene sequences of the 13 common species shared between AF and CB from the same pregnancy.

*<p>Expressed in percent identity with number of identical bases/total bases in alignment shown in parentheses.</p

Accession numbers of 16S rRNA genes detected in matching amniotic fluid and cord blood samples by PCR and clone analysis.

*<p>Two subspecies of Fusobacterium nucleatum were identified. JN546083, F. nucleatum sbsp animalis; JN584646, F. nucleatum sbsp polymorphum.</p

Results of clinical laboratory tests and placental pathology for preterm birth cases.

*<p>Data presented as median [interquartile range] and analyzed by Kruskal-Wallis ANOVA.</p>†<p>Data presented as n (%) and analyzed by Chi square.</p><p><u>Abbreviations:</u> EONS, early-onset neonatal sepsis; IAI, intra-amniotic infection; LDH, lactate dehydrogenase; WBC, white blood cell; HCA, histological chorioamnionitis; ANC, absolute neutrophil count; ABC, absolute band count; I:T ratio, ratio of immature-to-total neutrophils.</p

Alphabetical list of bacterial species identified by culture and culture-independent methods.

*<p>identified only by culture.</p>**<p>identified only by culture-independent methods.</p>***<p>Kappa coefficient is calculated for species identified in the paired AF-CB samples.</p