Three cases of Q fever osteomyelitis in children and a review of the literature

Q fever is a common zoonosis worldwide. Awareness of the disease and newer diagnostic modalities have resulted in increasing recognition of unusual manifestations. We report 3 cases of Q fever osteomyelitis in children and review the literature on 11 other reported cases. The cases demonstrate that Coxiella burnetii can cause granulomatous osteomyelitis that presents without systemic symptoms and frequently results in a chronic, relapsing, multifocal clinical course. Optimal selection and duration of antimicrobial therapy and methods of monitoring therapy are currently uncertain.

Antibiotic therapy for Klebsiella pneumoniae bacteremia: Implications of production of extended-spectrum beta-lactamases

The prevalence of extended-spectrum beta-lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of 455 consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL-producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem ( primarily imipenem) was associated with a significantly lower 14-day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

Providing treatment for hepatitis C in an Australian district centre

Hepatitis C virus (HCV) poses a major public health problem world wide. The introduction of combined therapy (interferon and ribavirin) and the recent development of pegylated interferon have offered the opportunity to alter the natural history of HCV, potentially reducing morbidity and mortality. Until recently, treatment has been confined to larger Australian cities. This paper describes the establishment of a clinic for the treatment of HCV in a regional Australian city. The facilities of the sexual health clinic were utilised. Factors contributing to the success of the clinic include the specialist nurse, a multidisciplinary approach, and the service model of shared care with general practitioners. The patient population and the outcomes of managing HCV in a regional centre are described. The sustained viral response rate is comparable to the published data from specialist centres.

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.

Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype of West Nile virus involves blockage of STAT1 and STAT2 activation by nonstructural proteins

The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-alpha/beta) and gamma IFN (IFN-gamma) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-alpha. IFN-alpha signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins. responsible for this inhibition.

Continuous infusion of ticarcillin-clavulanate for home treatment of serious infections: clinical efficacy, safety, pharmacokinetics and pharmacodynamics

Continuous infusion (CI) ticarcillin-clavulanate is a potential therapeutic improvement over conventional intermittent dosing because the major pharmacodynamic (PD) predictor of efficacy of beta-lactams is the time that free drug levels exceed the MIC. This study incorporated a 6-year retrospective arm evaluating efficacy and safety of CI ticarcillin-clavulanate in the home treatment of serious infections and a prospective arm additionally evaluating pharmacokinetics (PK) and PD. In the prospective arm, steady-state serum ticarcillin and clavulanate levels and MIC testing of significant pathogens were performed. One hundred and twelve patients (median age, 56 years) were treated with a CI dose of 9.3-12.4 g/day and mean CI duration of 18.0 days. Infections treated included osteomyelitis (50 patients), septic arthritis (6), cellulitis (17), pulmonary infections (12), febrile neutropenia (7), vascular infections (7), intra-abdominal infections (2), and Gram-negative endocarditis (2); 91/112 (81%) of patients were cured, 14 (13%) had partial response and 7 (6%) failed therapy. Nine patients had PICC line complications and five patients had drug adverse events. Eighteen patients had prospective PK/PD assessment although only four patients had sufficient data for a full PK/PD evaluation (both serum steady-state drug levels and ticarcillin and clavulanate MICs from a bacteriological isolate), as this was difficult to obtain in home-based patients, particularly as serum clavulanate levels were found to deteriorate rapidly on storage. Three of four patients with matched PK/PD assessment had free drug levels exceeding the MIC of the pathogen. Home Cl of ticarcillin-clavulanate is a safe, effective, convenient and practical therapy and is a therapeutic advance over traditional intermittent dosing when used in the home setting. (c) 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Sequence variation in primer targets affects the accuracy of viral quantitative PCR

This study was designed to assess the effect of sequence mismatches between primers and their targets on viral quantitative PCR. Numerous primers were constructed incorporating various mismatches with a target sequence on the BKV T antigen gene. When using these primers in standard Taqman two-step cycling conditions, as few as two mismatches in a single primer increased cycle threshold values and significantly influenced the calculation of viral load. (C) 2005 Elsevier B.V. All rights reserved.

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Encapsulation of lipopeptides within liposomes: Effect of number of lipid chains, chain length and method of liposome preparation

The purpose of this study was to systematically investigate the effect of lipid chain length and number of lipid chains present on lipopeptides on their ability to be incorporated within liposomes. The peptide KAVYNFATM was synthesized and conjugated to lipoamino acids having acyl chain lengths of C-8, C-12 and C-16. The C-12 construct was also prepared in the monomeric, dimeric and trimeric form. Liposomes were prepared by two techniques: hydration of dried lipid films (Bangham method) and hydration of freeze-dried monophase systems. Encapsulation of lipopeptide within liposomes prepared by hydration of dried lipid films was incomplete in all cases ranging from an entrapment efficiency of 70% for monomeric lipoamino acids at a 5% (w/w) loading to less than 20% for di- and trimeric forms at loadings of 20% (w/w). The incomplete entrapment of lipopeptides within liposomes appeared to be a result of the different solubilities of the lipopeptide and the phospholipids in the solvent used for the preparation of the lipid film. In contrast, encapsulation of lipopeptide within liposomes prepared by hydration of freeze-dried monophase systems was high, even up to a loading of 20% (w/w) and was much less affected by the acyl chain length and number than when liposomes were prepared by hydration of dried lipid films. Freeze drying of monophase systems is better at maintaining a molecular dispersion of the lipopeptide within the solid phospholipid matrix compared to preparation of lipid film by evaporation, particularly if the solubility of the lipopeptide in solvents is markedly different from that of the polar lipids used for liposome preparation. Consequently, upon hydration, the lipopeptide is more efficiently intercalated within the phospholipid bilayers. (C) 2005 Elsevier B.V. All rights reserved.

Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity

Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC - NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus ( MCMV) - infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Development time and survival of Verrallina funerea (Theobald) (Diptera : Culicidae) immatures and other brackish water mosquito species in southeast Queensland, Australia

Verrallina funerea (Theobald) is a brackish water mosquito that is recognised as an important pest and vector in southeast Queensland, Australia. Immature development time and survival of Ve. funerea was defined in the laboratory in response to a range of temperatures (17-34 degrees C) and salinities (0-35 parts per thousand (p.p.t)). The expression of autogeny in this species was also assessed. Salinity only had a slight effect on mean development time from hatching to adult emergence (7.0-7.4 d at salinities of 0, 17.5 and 31.5 p.p.t) and survival was uniformly high (97.5-99.0%). Mean development times were shorter at 26, 29 and 32 degrees C (7.0, 6.8 and 6.8 d, respectively) and longest at 17 degrees C (12.2 d). The threshold temperature (t) was 5.8 degrees C and the thermal constant (K) was 142.9 degree-days above t. Survival to adulthood decreased from > 95% (at 17-29 degrees C) to 78% (at 32 degrees C) and 0% (at 34 degrees C). No expression of autogeny was observed. Immature development times of Ve. funerea, Ochlerotatus vigilax (Skuse) and Oc. procax (Skuse) were then determined under field conditions at Maroochy Shire. Following tide and rain inundation, cohorts of newly hatched larvae were monitored daily by dipping, and time until pupation was noted. Tidal inundation triggered hatching of Ve. funerea and Oc. vigilax larvae whereas Oc. procax larvae were found only after rain inundation. Estimates of Ve. funerea and Oc. vigilax field development times were similar (8-9 d) while Oc. procax development time was slightly longer (9-10 d). Based on these survey results, control activities targeting Ve. funerea must be initiated 4 d (if using Bacillus thuringiensis var. israelensis de Barjac) or 5 d (if using s-methoprene) after inundation. However, Casuarina glauca Sieber canopy and branchlets covering breeding habitats may present a problem for the penetration of such treatments.

Semliki Forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity

RNA replicons offer a number of qualities which make them attractive as vaccination vectors. Both alphavirus and flavivirus replicon vaccines have been investigated in preclinical models yet there has been little direct comparison of the two vector systems. To determine whether differences in the biology of the two vectors influence immunogenicity, we compared two prototypic replicon vectors based on Semliki Forest virus (SFV) (alphavirus) and Kunjin virus (KUN) (flavivirus). Both vectors when delivered as naked RNAs elicited comparable CD8+ T cell responses but the SFV vectors elicited greater humoral responses to an encoded cytoplasmic antigen beta-galactosidase. Studies in MHC class II-deficient mice revealed that neither vector could overcome the dependence of CD4+ T cell help in the development of humoral and cellular responses following immunization. These studies indicate that the distinct biology of the two replicon systems may differentially impact the adaptive immune response and this may need to be considered when designing vaccination strategies. (c) 2005 Elsevier Ltd. All rights reserved.

Investigation of ansB and sspA derived promoters for multi- and single-copy antigen expression in attenuated Salmonella enterica var. typhimurium

Five candidate promoters were examined to determine their utility in directing immunogenic levels of expression of the C fragment from tetanus toxin in attenuated S. enterica used as an oral vaccine in mice. Promoters derived from the genes encoding the stringent starvation protein (sspA) from E. coli and S. enterica, but not ansB derived promoters, expressed immunogenic levels of C fragment from multi-copy plasmids in attenuated S. enterica in vivo and, following oral immunization, induced high titre specific anti-tetanus toxoid serum antibodies. We also demonstrate that not only the choice of promoter, replicon and growth conditions but also how expression constructs are assembled in the chosen plasmid is critical for the successful development of plasmid-based antigen delivery systems using attenuated S. enterica. In addition, the S. enterica sspA promoter is able to elicit anti-tetanus toxoid antibodies in mice when the psspA-tetC expression cassette is integrated in single copy on the S. enterica chromosome.