Carol A. Spiegel

Bacterial vaginosis associated with increased risk of female-to-male HIV-1 transmission: a prospective cohort analysis among African couples

In a prospective study, Craig Cohen and colleagues investigate the association between bacterial vaginosis and the risk of female-to-male HIV-1 transmission.

Identification of Plasmid-Mediated AmpC β-Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results

ABSTRACT The goal of this study was to determine if the interpretations of extended-spectrum and advanced-spectrum cephalosporins (ESCs and ASCs, respectively) for isolates of Enterobacteriaceae would be impacted by the results of aminophenylboronic acid (APBA) testing. Fifty-three isolates of Escherichia coli, 21 Klebsiella species, and 6 Proteus species that were resistant to at least one ESC were tested by disk diffusion with ceftazidime and cefotetan disks with and without APBA. Ceftazidime disks with and without clavulanic acid (CLAV) were also tested to confirm extended-spectrum β-lactamase (ESBL) carriage. Twenty-nine (36.3%) isolates were only APBA test positive, 27 were only CLAV test positive, 2 were positive with both substrates, and 22 were negative with both substrates. Thirteen (41.9%) of the 31 APBA-test-positive isolates (all E. coli) tested susceptible to cefotaxime, ceftriaxone, or ceftazidime. Since clinical data suggest that AmpC-producing isolates should be reported as resistant to all ESCs, APBA testing can be helpful in identifying such organisms. Screening for AmpC-producing organisms using nonsusceptibility to cefoxitin and amoxicillin-clavulanate was less specific than APBA testing; it identified ESBL as well as AmpC-producing organisms. Only 18 of 31 APBA-positive isolates were positive by PCR for an AmpC β-lactamase gene. Thus, testing with APBA could improve the accuracy of reporting ESCs, especially for E. coli. However, results of APBA and CLAV testing did not correlate well for isolates containing both AmpC β-lactamases and ESBLs. Thus, additional data are needed before formal recommendations can be made on changing the reporting of ASC test results.

An outbreak of the 2009 influenza a (H1N1) virus in a children's hospital.

Please cite this paper as: Bearden et al. (2012) An outbreak of the 2009 influenza a (H1N1) virus in a children’s hospital. Influenza and Other Respiratory Viruses 6(5), 374–379.

Bacteremic sepsis disturbs alveolar perfusion distribution in the lungs of rats

Objective: Sepsis often leads to lung injury, although the mechanisms that initiate this are unclear. One preinjury phenomenon that has not been explored previously is the effect of bacterial (nonlipopolysaccharide) sepsis on the distribution of alveolar perfusion. The goals of our studies were to measure this. Design: Randomized, controlled, prospective animal study. Setting: University animal laboratory. Subjects: Male Sprague-Dawley rats (450–550 g). Interventions: We induced sepsis by placing gelatin capsules containing Escherichia coli and Bacteroides fragilis into the abdomens of rats (n = 9). Empty capsules (n = 6) were placed into the abdomens of controls. After 24 hrs, 4-&mgr;m-diameter fluorescent latex particles (2 × 108) were infused into the pulmonary circulation. Sepsis was induced in additional rats and controls to assess lung injury, as follows: Lung histology was performed on eight septic rats and on seven controls; lung lavage was performed on three septic rats and three controls after their plasma albumin had been labeled with Evans blue dye. Measurements and Main Results: Confocal microscopy was used to prepare digital maps of latex particle trapping patterns (eight per lung). Analysis of these patterns revealed statistically more clustering (perfusion inhomogeneity) down to tissue volumes less than that of ten alveoli in septic lungs compared with controls (p ≤ .05). Bacterial counts and neutrophil counts were significantly higher in the circulation of septic rats (p ≤ .05). Blood pressures and arterial Po2s were unchanged. Cell counts in histological images were three-fold higher in septic lungs than in controls (p ≤ .05). Lung lavage revealed 0.41 ± 0.03 mL of plasma in the lungs of septic rats, and 0.06 ± 0.05 mL in the lungs of controls (p ≤ .05). Conclusions: Bacterial sepsis caused significant maldistribution of interalveolar perfusion in the lungs of rats in the absence of significant lung injury.

Efficacy of bilateral bronchoalveolar lavage for diagnosis of ventilator-associated pneumonia.

ABSTRACT Ventilator-associated pneumonia (VAP) is a common nosocomial infection causing significant morbidity and mortality. The goal of this study was to determine the efficacy of bilateral versus unilateral bronchoalveolar lavage (BAL) for the detection of the causative bacterial agents of VAP. We retrospectively studied the quantitative bacterial cultures of 399 BAL sample pairs collected from 287 mechanically ventilated patients over a 5-year period at a U.S. tertiary-care teaching hospital. Trauma was the underlying illness in 69% of patients. No evidence of bacterial infection was found in 226 BAL pairs (56.6%). Among 173 positive BAL sample pairs, significant bacterial counts were detected exclusively in 6.4% of left-lung and 12.1% of right-lung samples. In contrast, 81.5% of positive sample pairs had significant bacterial counts in both lungs. All bacteria recovered at significant concentrations from bilateral samples would have been detected in a unilateral right-lung sample in 89% of positive sample pairs. Unilateral sampling would have failed to recover one or more significant isolates in 11% of positive pairs had only the right lung been sampled and in 16.7% had only the left lung been sampled. Our study shows that preferential sampling of the right lung improves the diagnostic efficacy of unilateral BAL for the detection of the etiologic agents of VAP. If bilateral sampling is performed, our results also indicate that pooling left- and right-lung samples for a single quantitative culture is comparable to processing samples individually.

Ribonucleoside 3'-phosphates as pro-moieties for an orally administered drug.

Oral administration of chemotherapeutic agents is the mainstay for the treatment of disease. Sustained release formulations have been crucial for the safe and effective dosing of orally administered drugs.[1] Such formulations allow for the prolonged maintenance of therapeutic drug concentrations, reducing the required dosages per day and thereby enhancing patient compliance. Sustained release formulations also provide tighter control over the pharmacokinetics of a drug, thereby minimizing side effects.[1] Aqueous solubility is likewise a critical attribute for an orally available drug.[2] Robust absorption across the intestinal epithelium relies upon a high concentration of the drug to drive diffusion into enterocytes and, eventually, into the circulatory system. On average, 35–40% of lead compounds have aqueous solubilities of <5 mg/mL, which is defined by the U.S. Pharmacopeia as being slightly soluble or worse.[3] Accordingly, the bioavailability and consequent efficacy of many compounds relies on enhancing their aqueous solubility.[2c] The formation of a phosphomonoester can improve the oral bioavailability of poorly water-soluble chemotherapeutic agents.[2b,2c,4] Endogenous phosphatases near the surface of enterocytes can catalyze the hydrolysis of the phosphoryl group, releasing the lipophilic drug and allowing for its efficient absorption into the body. Several prodrugs approved by the U.S. Food and Drug Administration rely on this strategy, including estramustine, fosamprenavir, and prednisolone phosphate.[4] Recently, we reported on the potential utility of a phosphodiester as the pro-moiety for a drug administered intravenously.[5] Specifically, we found that the coupling of 4-hydroxytamoxifen to uridine 3′-phosphate enabled its timed-release in serum by human pancreatic ribonuclease (RNase 1[6]; EC 3.1.27.5). This modification also increased the aqueous solubility of 4-hydroxytamoxifen. RNase 1 is an ideal endogenous enzyme to elicit pro-moiety release. A major excreted enzyme, RNase 1 has a concentration of 6.4 mg/mL in human pancreatic juice and 0.2 mg/mL in saliva, according to a radioimmunoassay.[7] Moreover, like its renowned homologue bovine pancreatic ribonuclease (RNase A[8]), RNase 1 catalyzes the cleavage of RNA by a transphosphorylation reaction[9] with little specificity for its leaving group.[10] Herein, we report on the utility of several ribonucleoside 3′-phosphates as pro-moieties for a model orally available drug, metronidazole. Metronidazole is a commonly used antibiotic for a variety of protozoa and anaerobic bacterial infections, including Bacteroides fragilis, Helicobacter pylori, Clostridium difficile, Trichomonas vaginalis, and Entamoeba histolytica.[11] In 1997, Flagyl ER, an extended release formulation of metronidazole, was approved by the FDA as a superior treatment for bacterial vaginosis. Still, metronidazole has several common side effects, such as nausea, diarrhea, and metallic taste. Moreover, metronidazole therapy can occasionally cause more severe side effects, such as pancreatitis, neutropenia, neuropathies, or CNS toxicities.[12] These adverse effects could be attenuated with better control over the pharmacokinetics of metronidazole.[13] Hence, in this proof-of-concept study, we elected to attach metronidazole to ribonucleoside 3′-phosphates to assess the attributes of this promoiety for orally available drugs (Figure 1). Figure 1 Scheme showing catalysis of the cleavage of a ribonucleoside 3′-(metronidazole phosphate) (NpMet) by RNase 1 to yield a nucleoside 2′,3′-cyclic phosphate (N>p) and Met. Each ribonucleoside 3′-(metronidazole phosphate) (NpMet) was synthesized in four steps from commercially available metronidazole (Met) and a ribonucleoside phosphoramidite (Scheme 1). Briefly, Met was coupled to the phosphoramidite by using N-methylbenzimidazolium triflate (MBIT) as a catalyst.[14] The coupled product was oxidized with iodine and deprotected stepwise. The final products were purified by chromatography on silica gel. This route was used to synthesize three different NpMets: cytidine 3′-(metronidazole phosphate) (CpMet, 18% non-optimized yield), uridine 3′-(metronidazole phosphate) (UpMet, 80%), and adenosine 3′-(metronidazole phosphate) (ApMet, 64%). Scheme 1 Route for the synthesis of NpMets. We expected the ribonucleoside 3′-phosphate moiety of an NpMet to endow the prodrug with greater hydrophilicity than the parent drug, which could improve its oral bioavailability. To investigate this issue, we calculated the partition (log P) and distribution (log D) coefficients of Met, CpMet, UpMet, and ApMet.[15] The calculated log P and log D values for the NpMets were indeed significantly lower than those of the parent drug (Table 1), indicative of increased hydrophilicity and decreased tendency to aggregate. Table 1 Calculated Partition and Distribution Coefficients of Met and NpMets[15] To be the basis for an effective timed-release prodrug strategy, the pro-moiety needs to be released by the activating enzyme over time. Hence, we used 1H NMR spectroscopy to assess the rate at which RNase 1 catalyzed the release of Met from the prodrugs (Figure S1). We assumed that pancreatic juice is diluted in the intestine, which led us to use RNase 1 at concentrations of 0.1 mg/mL and 0.01 mg/mL in these assays. Because inorganic phosphate inhibits RNase A with a Ki value of 2.3 mM,[16] we initially investigated the effect of phosphate in simulated intestinal fluid (SIF) on the rates of UpMet unmasking (Figure 2A). Compared to a buffer with no inorganic phosphate (19.5 mM Tris–HCl, pH 7.4, 2.5% v/v D2O), the rate of Met release in SIF was only marginally slower. RNase 1 cleaves after pyrimidine residues more readily than after purine residues.[6,7] Accordingly, we predicted that RNase 1 would unmask CpMet and UpMet faster than ApMet. For both concentrations of RNase 1, we did indeed observe that the cytidine and uridine prodrugs were unmasked faster than the adenosine prodrug (Figure 2B). Moreover, unlike the uridine 3′-phosphate–4-hydroxytamoxifen conjugate that cleaved spontaneously in aqueous solutions lacking ribonucleases, the NpMet conjugates were stable in SIF, which has pH 7.5, and in simulated gastric fluid (SGF), which has pH 1.1. The absence of appreciable degradation (<5%) in either medium (Figure S2) is attributable to the alkoxyl group of metronidazole being a much worse leaving group than the aryloxyl group of 4-hydroxytamoxifen. Figure 2 Progress curves for the release of Met from NpMets under various conditions as determined by 1H NMR spectroscopy. (A) Comparison of UpMet cleavage rates in Tris–HCl buffer, pH 7.4, and simulated intestinal fluid (SIF). (B) Comparison of CpMet, ... Finally, we sought to assess the antimicrobial activity of our NpMet prodrugs on B. fragilis. This penicillin-resistant Gram-negative bacillus is common in anaerobic infections, like those that originate from the gastrointestinal tract. We determined the minimum inhibitory concentration (MIC) of UpMet and ApMet, as well as Met (Figure S3). We found that both UpMet and ApMet had considerably higher MIC values than did Met (Table 2), demonstrating that the prodrugs were relatively inactive. Incubating UpMet with 0.1 mg/mL RNase 1 overnight resulted in an MIC similar to that of Met. Finally, adding UpMet to a culture medium containing RNase 1 gave an intermediate MIC, demonstrating the in situ release of the drug. Table 2 MIC Values of NpMets for Bacteroides fragilis

Bacteremia Does Not Affect Cellular Uptake of Ultrafine Particles in the Lungs of Rats

To assess the effects of intra‐abdominal bacteremia on lung cellular function in vivo, we used electron microscopy to quantify the uptake of 6 nm diameter, albumin‐coated colloidal gold particles (overall diam. 20.8 nm) by cells in the lungs of rats made septic by the introduction of live bacteria (E.coli and B. fragilis) into their abdomens. Gold particles were instilled into the trachea 24 hr after bacteremia induction, and lungs were harvested and prepared for electron microscopy 24 hr later. Because bacteremia produces an increase in metabolism, we hypothesized that this might be associated with increased cellular uptake of these particles and also with increased permeability of the alveolar epithelial barrier to them, as bacteremia is also associated with lung injury. We quantified particle uptake by counting particle densities (particles/μm2) within type I and type II epithelial cells, capillary endothelial cells, erythrocytes and neutrophils in the lungs of five septic rats and five sham‐sepsis controls. We also counted particle densities within organelles of these cells (nuclei, mitochondria, type II cell lamellar bodies) and within the alveolar interstitium. We found particles to be present within all of these compartments, although we found no differences in particle densities between bacteremic rats and sham‐sepsis controls. Our results suggest that these 6 nm particles were able to freely cross cell and organelle membranes, and further suggest that this ability was not altered by bacteremia. Anat Rec, 2011.