Safety and pharmacokinetics of aciclovir in women following release from a silicone elastomer vaginal ring.

OBJECTIVES: Systemic aciclovir and its prodrug valaciclovir are effective in treating and reducing recurrences of genital herpes simplex virus (HSV) and reducing transmission. Local aciclovir delivery, if it can achieve and maintain comparable intracellular genital tract levels, may be equally effective in the treatment and suppression of genital HSV. Intravaginal ring (IVR) delivery of aciclovir may provide pre-exposure prophylaxis against HSV acquisition. METHODS: Tolerability and pharmacokinetics were evaluated in six HIV-negative women with recurrent genital HSV who switched their daily oral valaciclovir suppression to an aciclovir IVR for 7 days (n = 3) or 14 days (n = 3). Blood and cervicovaginal lavage (CVL) were collected after oral and IVR dosing to measure aciclovir concentrations and genital swabs were obtained to quantify HSV shedding by PCR. RESULTS: The rings were well tolerated. Median plasma aciclovir concentrations were 110.2 ng/mL (IQR, 85.9-233.5) 12-18 h after oral valaciclovir. Little or no drug was detected in plasma following IVR dosing. Median (IQR) CVL aciclovir levels were 127.3 ng/mL (21-660.8) 2 h after oral valaciclovir, 154.4 ng/mL (60.7-327.5) 12-18 h after oral valaciclovir and 438 ng/mL (178.5-618.5) after 7 days and 393 ng/mL (31.6-1615) after 14 days of aciclovir ring use. Median CVL aciclovir levels 2 h after oral dosing were similar to levels observed 7 (P = 0.99) and 14 (P = 0.75) days after ring use. HSV DNA was not detected in genital swabs and there was no significant change in inflammatory mediators. CONCLUSIONS: This first-in-human study demonstrated that an IVR could safely deliver mucosal levels of aciclovir similar to oral valaciclovir without systemic absorption. More intensive site-specific pharmacokinetic studies are needed to determine whether higher local concentrations are needed to achieve optimal drug distribution within the genital tract.

Amp C beta-lactamase-producing Escherichia coli in neonatal meningitis: diagnostic and therapeutic challenge.

Antibiotic resistance is a global health priority. Major defenses for Gram-negative bacteria are beta-lactamase enzymes, which have co-evolved with the development and increasing utilization of new antibiotics. Bacteria harboring the plasmid-mediated AmpC enzymes are increasingly prevalent among adult patients, but have not previously been reported in neonates. Early-onset neonatal meningitis caused by an AmpC beta-lactamase-producing Escherichia coli is described for the first time; the plasmid was identified as a transferable CMY-2 family beta-lactamase. Limited experience with newer antibiotics and pharmacokinetics in neonates presents a therapeutic challenge. Currently, there are no Clinical Laboratory Standards Institute (CLSI) recommendations for detecting AmpC nor is the optimal treatment for AmpC-producing organisms known. Thus, it is imperative that clinicians have a high index of suspicion when antimicrobial susceptibility patterns are inconsistent. Development of better microbiology screening tests to rapidly detect resistance is essential. Additionally, pharmacokinetic studies with newer antibiotics in neonates are warranted.

Cryoprecipitates in Kawasaki syndrome: association with coronary artery aneurysms.

Cryoprecipitates are postulated to play a role in the pathogenesis of several vasculitis illnesses and infectious diseases. To investigate the presence of cryoprecipitates in Kawasaki syndrome, we studied sera from 25 children with acute Kawasaki syndrome. None of the subjects was treated with intravenous gamma-globulin. Cryoprecipitates were detectable in sera of 11 of 25 (44%) children studied. The mean (+/- SE protein concentration of the cryoprecipitates was 88.0 (+/- 20.2) micrograms/ml serum. Cryoprecipitates consisted primarily of IgG and IgM; no complement components were detected but highly sensitive methods were not used. The presence of cryoprecipitates in the serum of children with acute Kawasaki syndrome was associated with the subsequent development of coronary artery aneurysms detected by echocardiogram (P less than 0.05). There was no association between detectable cryoprecipitates and either peak platelet count or erythrocyte sedimentation rate. In one patient, measurement of cryoprecipitates in serial samples showed a reduction in concentrations that paralleled subsidence of disease activity. We speculate that cryoprecipitates may be a marker for increased risk of coronary aneurysm formation and may play a role in the pathogenesis of the cardiac disease in Kawasaki syndrome.

Heparan sulfate glycosaminoglycans as primary cell surface receptors for herpes simplex virus.

Our current incomplete picture of the earliest events in HSV infection may be summarized as follows. The initial interaction of virus with cells is the binding of virion gC to heparan sulfate moieties of cell surface proteoglycans. Stable binding of virus to cells may require the interaction of other virion glycoproteins with other cell surface receptors as well (including the interaction of gB with heparan sulfate). Penetration of virus into the cell is mediated by fusion of the virion envelope with the cell plasma membrane. Events leading up to this fusion require the action of at least three viral glycoproteins (gB, gD and gH), one or more of which may interact with specific cell surface components. It seems likely that binding of gB to cell surface heparan sulfate may occur and may be important in the activation of some event required for virus penetration. Heparan sulfate is present not only as a constituent of cell surface proteoglycans but also as a component of the extracellular matrix and basement membranes in organized tissues. In addition, body fluids contain both heparin and heparin-binding proteins, either of which can prevent the binding of HSV to cells (WuDunn and Spear, 1989). As a consequence, the spread of HSV infection is probably influenced, not only by immune responses to the virus, but also by the probability that virus will be entrapped or inhibited from binding to cells by extracellular forms of heparin or heparan sulfate.

A compartmental pharmacokinetic evaluation of long-acting rilpivirine in HIV-negative volunteers for pre-exposure prophylaxis.

Rilpivirine long-acting (RPV-LA) is a parenteral formulation enabling prolonged plasma exposure. We explored its multiple-compartment pharmacokinetics (PK) after a single dose, for pre-exposure prophylaxis. Sixty-six HIV-negative volunteers were enrolled: women received an intramuscular dose of 300, 600, or 1,200 mg, with plasma and genital levels measured to 84 days postdose; men receiving 600 mg had similar PK determined in plasma and rectum. Ex vivo antiviral activity of cervicovaginal lavage (CVL) was also assessed. After a single dose, RPV concentrations peaked at days 6-8 and were present in plasma and genital-tract fluid to day 84. Vaginal and male rectal tissue levels matched those in plasma. At the 1,200 mg dose, CVL showed greater antiviral activity, above baseline, at days 28 and 56. All doses were well tolerated. All doses gave prolonged plasma and genital-tract rilpivirine exposure. PK and viral inhibition of repeated doses will be important in further dose selection.

Neomycin inhibits glycoprotein C (gC)-dependent binding of herpes simplex virus type 1 to cells and also inhibits postbinding events in entry.

Previous studies have identified requirements for the binding of herpes simplex virus type 1 (HSV-1) to cells, including the presence of particular glycoproteins in the virion envelope (gC or gB) and the presence of particular glycosaminoglycan chains (principally heparan sulfate) on cell surface proteoglycans. We show here that neomycin, a known inhibitor of HSV infection, blocked early events in HSV infection by two mechanisms: partial inhibition of the gC-dependent binding of virions, but not the gB-dependent binding, and inhibition of events that occurred after the binding of virus to cells. Near-maximal (but incomplete) inhibition of virus binding occurred at low concentrations of neomycin (1 mM) for wild-type and gB-negative virions only. Neomycin also inhibited the binding of isolated gC to cells at a similar concentration. Concentrations of neomycin as high as 50 mM had little or no effect on the binding of gC-negative virions to cells. Nevertheless, neomycin significantly inhibited infection by both wild-type and gC-negative virions, at concentrations greater than 10 mM, indicating that the inhibition at higher doses was not due to effects on virus binding. The effects of neomycin on virus binding suggest that gC (but not gB) and neomycin compete for binding to similar structural features of cell surface heparan sulfate.

Characterization of a BHK(TK-) cell clone resistant to postattachment entry by herpes simplex virus types 1 and 2.

BHK(TK-) cells selected for resistance to polyethylene glycol-mediated fusion give rise to clones that are resistant to herpes simplex virus (HSV) infection. We have characterized one such clone, designated 95-19, and found that it is resistant to entry of HSV type 1 (HSV-1), HSV-2, and the related alphaherpesvirus pseudorabies virus (PRV). Single-step growth experiments show no detectable replication of multiple strains of HSV-1 and HSV-2 on 95-19 cells. Three lines of evidence suggest that these cells are resistant to postattachment entry. (i) Measurements of binding of radiolabeled virus show that heparin-sensitive binding of HSV-1 and HSV-2 to 95-19 cells is identical to binding to BHK(TK-) cells, suggesting that the block to replication occurs after attachment to heparan sulfate proteoglycan. (ii) 95-19 cells exposed to HSV-1 or HSV-2 at high multiplicity show no detectable immediate-early (IE) mRNA expression. (iii) Exposure of attached virus and cells to polyethylene glycol results in partial recovery of both IE gene expression and virus yield in single-step growth. The degrees of recovery of single-step yield and IE gene expression are similar, suggesting that the only block to single-step replication is at the point of virus entry and that these cells are deficient in some cellular factor required for efficient postattachment entry of free virus. 95-19 cells are also highly resistant to entry by cell-to-cell spread, suggesting that the same cellular factor participates in both types of entry.

Differences in the role of glycoprotein C of HSV-1 and HSV-2 in viral binding may contribute to serotype differences in cell tropism.

Heparan sulfate serves as a receptor for several herpesviruses. For herpes simplex virus 1 (HSV-1), pseudorabies virus, and bovine herpesvirus 1, glycoprotein C homologues have been shown to mediate the binding to cell-surface heparan sulfate. It has been assumed that glycoprotein C of HSV-2 (gC-2) plays a similar role in HSV-2 entry, but this has not been established experimentally. We first determined, using heparin-affinity chromatography, that gC-2 is a heparin-binding glycoprotein. To examine the role of gC-2 in HSV-2 infection, we constructed a gC-2 deletion mutant, HSV-2(G)gC-. In contrast to results obtained for the other alpha herpesviruses, we found that the HSV-2(G)gC- virus showed no loss in specific binding activity (particles bound/cell) or specific infectivity (PFU/particle) compared to the parental wild-type virus. Moreover, while gC-1 mutants show a marked lag in the rate of viral penetration, the gC-2-deletion virus did not. We did find that gC-2, like gC-1, protects virus from complement-mediated neutralization. These results suggest that, in contrast to HSV-1, gC-2 does not play the key role in viral binding. The major role of gC-2 may be to protect virus from complement-mediated neutralization. We speculate that serotype differences in the contribution of gC to viral binding may contribute to serotype differences in cell tropism.

Development of topical microbicides for prevention of human immunodeficiency virus and herpes simplex virus.

Topical microbicides, designed for vaginal or rectal administration, are needed to prevent human immunodeficiency virus (HIV) and other sexually transmitted infections (STI). Presently marketed topical microbicides are cytotoxic and damage the vaginal epithelium with frequent use. Rational development of new candidate compounds should build on knowledge of the pathways of microbial invasion. The establishment of assays and models that predict efficacy and safety is critical. Comprehensive pre-clinical evaluation of promising microbicides should include rigorous assessment of the effects of repeated application of topical agents on mucosal inflammatory cells, cytokines, and the genital tract virus population. These studies will lay the groundwork for future clinical trials.

Peptostreptococcus asaccharolyticus renal abscess: a rare cause of fever of unknown origin.

Renal abscess is uncommon in pediatrics and is rarely a cause of fever of unknown origin. We recently cared for a patient who presented with a 3-week history of fever. An indium scan ultimately led to the diagnosis of a renal abscess. Aspiration yielded Peptostreptococcus asaccharolyticus. This unusual case prompted a review of the clinical and microbiologic features of renal abscess in pediatric patients at our hospital over the past 10 years. Seven additional patients with a discharge diagnosis of renal abscess were identified. Only 2 of the patients had identifiable risk factors (diabetes mellitus and polycystic kidneys). Staphylococcus aureus or Enterobacteriaceae were responsible for most infections, consistent with hematogenous and urinary tract sources, respectively. No other cases of anaerobic abscess were identified. This case highlights the importance of considering a renal abscess in the differential diagnosis of fever of unknown origin and of processing specimens for both aerobic and anaerobic organisms.

Herpes simplex virus entry is associated with tyrosine phosphorylation of cellular proteins.

The initial step in herpes simplex virus (HSV) entry is binding of virion glycoprotein (g)C and/or gB to cell surface heparan sulfate. After this initial attachment, gD interacts with cell surface receptor or receptors, and the virion envelope fuses with the cell membrane. Fusion requires viral glycoproteins gB, gD, gL, and gH, but the cellular factors that participate in or the pathways activated by viral entry have not been defined. To determine whether signal transduction pathways are triggered by viral-cell fusion, we examined the association of viral entry with tyrosine phosphorylation of cellular proteins. Using immunoprecipitation and Western blotting, we found that at least three cytoplasmic host cell proteins, designated p80, p104, and p140, become tyrosine phosphorylated within 5-10 min after exposure to HSV-1 or HSV-2. However, no phosphorylation is detected when cells are exposed to a mutant virus deleted in gL that binds but fails to penetrate. Phosphorylation is restored when the gL-deletion virus is grown on a complementing cell line. Viral entry and the phosphorylation of p80, p104, and p140 are inhibited when cells are infected with virus in the presence of protein tyrosine kinase inhibitors. Taken together, these studies suggest that tyrosine phosphorylation of host cellular proteins is triggered by viral entry.

Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity.

The purpose of this study was to identify the herpes simplex virus glycoprotein(s) that mediates the adsorption of virions to cells. Because heparan sulfate moieties of cell surface proteoglycans serve as the receptors for herpes simplex virus adsorption, we tested whether any of the viral glycoproteins could bind to heparin-Sepharose in affinity chromatography experiments. Two glycoproteins, gB and gC, bound to heparin-Sepharose and could be eluted with soluble heparin. In order to determine whether virions devoid of gC or gB were impaired for adsorption, we quantitated the binding of wild-type and mutant virions to cells. We found that at equivalent input concentrations of purified virions, significantly fewer gC-negative virions bound to cells than did wild-type or gB-negative virions. In addition, the gC-negative virions that bound to cells showed a significant delay in penetration compared with wild-type virus. The impairments in adsorption and penetration of the gC-negative virions can account for their reduced PFU/particle ratios, which were found to be about 5 to 10% that of wild-type virions, depending on the host cell. Although gC is dispensable for replication of herpes simplex virus in cell culture, it clearly facilitates virion adsorption and enhances infectivity by about a factor of 10.

Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B.

Previous studies have shown that the initial interaction of herpes simplex virus (HSV) with cells is binding to heparan sulphate and that HSV-1 glycoprotein C (gC) is principally responsible for this binding. Although gC-negative viral mutants are impaired for binding and entry, they retain significant infectivity. The purpose of the studies reported here was to explore the requirements for infectivity of gC-negative HSV-1 mutants. We found that absence or alteration of cell surface heparan sulphate significantly reduced the binding of gC-negative mutant virus and rendered cells resistant to infection, as shown previously for the wild-type virus. We isolated a recombinant double-mutated HSV strain that produces virions devoid of both of the known heparin-binding glycoproteins, gB and gC. The drastically impaired binding of these mutant virions to cells, relative to gC-negative and wild-type virions, indicates that gB mediates the binding of gC-negative virions to cells. Thus at least two HSV glycoproteins can independently mediate the binding of HSV to cell surface heparan sulphate to start the process of viral entry into cells.