Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy.

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of 'covert' virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Killing of gram-negative bacteria by lactoferrin and lysozyme.

Although lactoferrin has antimicrobial activity, its mechanism of action is not full defined. Recently we have shown that the protein alters the Gram-negative outer membrane. As this membrane protects Gram-negative cells from lysozyme, we have studied whether lactoferrin's membrane effect could enhance the antibacterial activity of lysozyme. We have found that while each protein alone is bacteriostatic, together they can be bactericidal for strains of V. cholerae, S. typhimurium, and E. coli. The bactericidal effect is dose dependent, blocked by iron saturation of lactoferrin, and inhibited by high calcium levels, although lactoferrin does not chelate calcium. Using differing media, the effect of lactoferrin and lysozyme can be partially or completely inhibited; the degree of inhibition correlating with media osmolarity. Transmission electron microscopy shows that E. coli cells exposed to lactoferrin and lysozyme at 40 mOsm become enlarged and hypodense, suggesting killing through osmotic damage. Dialysis chamber studies indicate that bacterial killing requires direct contact with lactoferrin, and work with purified LPS suggests that this relates to direct LPS-binding by the protein. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense.

Association of hospital contact precaution policies with emergency department admission time.

BACKGROUND: Contact precautions are a widely accepted strategy to reduce in-hospital transmission of meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). However, these practices may have unintended deleterious effects on patients. AIM: To evaluate the effect of a modification in hospital-wide contact precaution practices on emergency department (ED) admission times. METHODS: During the study period, the hospital changed its contact precaution policy from requiring contact precautions for all patients with a history of MRSA or VRE to only those who presented with clinical conditions likely to contaminate the environment with pathogens. An interrupted time series analysis of ED admission times for adults for one year preceding and one year following this change was performed at a two-campus hospital. The main outcome was admission time, defined as time from decision to admit to arrival in an inpatient bed, for patients with MRSA or VRE compared with all other patients. The in-hospital MRSA and VRE acquisition rates were evaluated over the same period and have been published previously. FINDINGS: At one campus, admission time decreased immediately by 161min for MRSA patients (P=0.008) and 135min for VRE patients (P=0.003), and both continued to decrease over the duration of the study. There was no significant change in admission time at the second campus. CONCLUSIONS: Modifying contact precaution requirements for MRSA and VRE may be associated with improved ED admission time without significantly altering in-hospital MRSA and VRE acquisition.

Pyogenic meningitis manifesting during therapy for Aeromonas hydrophila sepsis.

Pyogenic meningitis became apparent on the third day of ampicillin and gentamicin therapy for Aeromonas hydrophila sepsis in a patient with severe alcoholic hepatitis. The patient responded clinically to therapy with intravenous cefotaxime sodium and gentamicin sulfate. Antibiotic therapy that provides adequate CSF concentrations should be considered in the treatment of patients with Aeromonas sepsis.

Meningococcemia and acquired complement deficiency. Association in patients with hepatic failure.

We treated two patients with severe hepatic failure complicated by meningococcemia. Serum complement profiles performed on these patients found low total hemolytic complement assays, normal concentrations of C1q, and low or undetectable concentrations of C3 through C6, C8, C9, and factors B and I. These studies suggest that these patients developed meningococcemia in the setting of acquired complement deficiency from impaired synthesis of multiple complement system proteins.

Statistical evaluation of the role of Helicobacter pylori in stress gastritis: applications of splines and bootstrapping to the logistic model.

Stress gastritis is a serious problem in the intensive care unit population. The recent discovery of the causal nature of Helicobacter pylori (H. pylori) in the development of gastric ulcers led us to examine its relationship with stress gastritis. We investigated this relationship in 874 veterans admitted to intensive care units who were tested for the presence of H. pylori and followed for 6 weeks for the development of stress gastritis. We fit spline models to assess functional relationships and used the logistic model to determine the association between H. pylori and stress gastritis. The predictive ability of the model was assessed with receiver operating characteristic (ROC) curve analysis and validated with the bootstrapping technique. Increased anti-H. pylori immunoglobulin A concentrations were found to be an important predictor of stress gastritis independent of other known risk factors.

The effects of lactoferrin on gram-negative bacteria.

Lactoferrin is an iron-binding protein found in human mucosal secretions as well as the specific granules of polymorphonuclear leukocytes. A variety of functions have been ascribed to the protein, and it appears to contribute to antimicrobial host defense. In particular, it has been shown to have direct effects on pathogenic microorganisms including bacteriostasis and the induction of microbial iron uptake systems. Still its overall physiologic role remains to be defined. It has appeared logical that antimicrobial activity of the protein arises from sequestration of environmental iron thereby causing nutritional deprivation in susceptible organisms. This argument is buttressed by the finding that selected highly virulent pathogens have evolved techniques to subvert this effect and use the protein as an iron source. However, recent observations indicate that the protein has additional properties that contribute to host defense. Work by several groups has shown that the protein synergistically interacts with immunoglobins, complement, and neutrophil cationic proteins against Gram-negative bacteria. Further, both the whole protein and a cationic N-terminus peptide fragment directly damage the outer membrane of Gram-negative bacteria suggesting a mechanism for the supplemental effects. This review will summarize these diverse observations with a consideration of how the in vitro work relates to the physiological role of the protein.

Rapid nighttime evacuation of a veterans hospital.

Loss of essential utilities and danger of explosion forced a rapid nighttime winter evacuation of 229 patients from an acute-care Veterans Administration hospital. Although distribution of patients to recipient hospitals was not optimal, and the location of several patients could not be documented for more than 24 hours, the evacuation in subfreezing weather went smoothly. Continuity of care and careful planning permitted an orderly return to the hospital five days later. Although financial costs were high, no excess mortality or morbidity was associated with the evacuation. No changes in pharyngeal gram-negative bacterial flora of the patients were noted. Further, a critique is presented to aid in planning for similar emergencies elsewhere.

Impact of contact precautions on falls, pressure ulcers and transmission of MRSA and VRE in hospitalized patients.

BACKGROUND: Hospitals use contact precautions to prevent the spread of meticillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). There is concern that contact precautions may have adverse effects on the safety of isolated patients. In November 2010, the infection control policy at an academic medical centre was modified, and contact precautions were discontinued for patients colonized or infected with MRSA or VRE (MRSA/VRE patients). AIM: To assess the rates of falls and pressure ulcers among MRSA/VRE patients and other adult medical-surgical patients, as well as changes in MRSA and VRE transmission before and after the policy change. METHODS: A single-centre retrospective hospital-wide cohort study was performed from 1st November 2009 to 31st October 2011. FINDINGS: Rates of falls and pressure ulcers were significantly higher among MRSA/VRE patients compared with other adult medical-surgical patients before the policy change (falls: 4.57 vs 2.04 per 1000 patient-days, P < 0.0001; pressure ulcers: 4.87 vs 1.22 per 1000 patient-days, P < 0.0001) and after the policy change (falls: 4.82 vs 2.10 per 1000 patient-days, P < 0.0001; pressure ulcers: 4.17 vs 1.19 per 1000 patient-days, P < 0.0001). No significant differences in the rates of falls and pressure ulcers among MRSA/VRE patients were found after the policy change compared with before the policy change. There was no overall change in MRSA or VRE hospital-acquired transmission. CONCLUSION: MRSA/VRE patients had higher rates of falls and pressure ulcers compared with other adult medical-surgical patients. Rates were not affected by removal of contact precautions, suggesting that other factors contribute to these complications. Further research is required among this population to prevent complications.

Pulmonary secretions accelerate the metabolic rate of Escherichia coli.

A number of studies have suggested that bronchoalveolar lavage fluid (BALF) contributes to intrapulmonary antibacterial host defense, however the mechanisms underlying this interaction have not been defined. To better understand the effect of BALF on bacteria, we measured the metabolism of bacteria in the presence of human or rabbit BALF. Escherichia coli oxygen consumption significantly increases with exposure to BALF (2.9 +/- 0.2 (SEM) nmol/min) compared to incubation in a saline-glucose solution alone (1.8 +/- 0.1); the rate of 1-[14C]-glucose utilization is comparably increased. The effect on oxygen metabolism is dose dependent. The surfactant phospholipids produce a small stimulation of oxygen metabolism, but the major effect is caused by phospholipid-poor material less than 10,000 daltons in size. The activity is heat stable, pH stable, and resistant to the effects of proteases. These studies demonstrate that factor(s) within BALF increase the metabolic rate of bacteria. Further work is required to determine if this bacterial respiratory burst is a pathogenic mechanism or a reparative response to BALF induced injury.

Differentiating pyogenic arthritis from spontaneous hemarthrosis in patients with hemophilia.

Pyogenic arthritis in patients with hemophilia is predominantly monoarticular, usually involving the knee, is associated with hemophilic arthropathy and other predisposing factors for infection, is mainly due to Staphylococcus aureus and carries serious morbidity. In patients with hemophilia, it is associated significantly with fever, an increased leukocyte count, knee joint involvement and possible predisposing factors for infection. Such patients presenting with unexplained fever of more than 1 degrees C(1.8 degrees F) and acute symptoms of joint inflammation should have an arthrocentesis.

Recurrent postcoital lower-extremity streptococcal erythroderma in women. Streptococcal-sex syndrome.

Two women with underlying distortion of their lower-body lymphatic systems from neoplasia and surgery or radiation therapy had recurrent episodes of lower-extremity erythroderma temporally associated with sexual intercourse. Both women had vaginal colonization with Streptococcus agalactiae (group B); one was shown to have recurrent bacteremia with this organism at the time of the episodes. Erythroderma developed in these women possibly because of seeding of S agalactiae in the vaginal soft tissues during coitus.

Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin.

Many studies have shown that lactoferrin and transferrin have antimicrobial activity against gram-negative bacteria, but a mechanism of action has not been defined. We hypothesized that the iron-binding proteins could affect the gram-negative outer membrane in a manner similar to that of the chelator EDTA. The ability of lactoferrin and transferrin to release radiolabeled lipopolysaccharide (LPS) from a UDP-galactose epimerase-deficient Escherichia coli mutant and from wild-type Salmonella typhimurium strains was tested. Initial studies in barbital-acetate buffer showed that EDTA and lactoferrin cause significant release of LPS from all three strains. Further studies found that LPS release was blocked by iron saturation of lactoferrin, occurred between pH 6 and 7.5, was comparable for bacterial concentrations from 10(4) to 10(7) CFU/ml, and increased with increasing lactoferrin concentrations. Studies using Hanks balanced salt solution lacking calcium and magnesium showed that transferrin also could cause LPS release. Additionally, both lactoferrin and transferrin increased the antibacterial effect of a subinhibitory concentration of rifampin, a drug excluded by the bacterial outer membrane. This work demonstrates that these iron-binding proteins damage the gram-negative outer membrane and alter bacterial outer membrane permeability.

Isolation of an antibacterial peptide from human lung lavage fluid.

The contribution of extracellular secretions to the antibacterial defenses of the lungs remains poorly defined. Recent studies have demonstrated that mouse and rabbit bronchoalveolar washings contain a low-molecular-weight peptide that has antibacterial activity against Escherichia coli. In this study we investigated whether a similar peptide could be identified in human secretions. Bronchoalveolar lavage fluid was obtained from normal volunteers and patients with interstitial lung disease or pulmonary alveolar proteinosis. Cellular material and surfactant lipids were removed from the fluid by sequential centrifugations, and the supernatant was fractionated by exclusion filtration to isolate peptides with a molecular weight less than 10,000. Gel filtration chromatography separated the ultrafiltrate into several peaks, the first of which had antibacterial activity against E. coli. This material was further separated into several hydrophilic peaks by reverse-phase high-pressure liquid chromatography (RPHPLC). All samples had similar RPHPLC graphs. Material from the third RPHPLC peak produced an antibacterial effect similar to that produced by the rabbit and mouse peptide.

Effect of aerosolized Escherichia coli and Staphylococcus aureus on iron and iron-binding proteins in lung lavage fluid.

Iron-binding proteins have antibacterial activity; they have been identified in lung secretions, but their role in pulmonary antibacterial defenses is unclear. Murine lactoferrin and murine transferrin were used to generate polyclonal antiserum to lactoferrin and to transferrin, and the specificity of both antisera was shown by western blot. Mice were exposed to either aerosolized Escherichia coli or Staphylococcus aureus; they were killed 1, 4, 24, or 48 hr later; and their lungs were lavaged. We measured the levels of transferrin, lactoferrin, and albumin and did a cell count for the lavage fluid. The predominant iron-binding protein in resting animals was transferrin. Aerosolized E. coli caused a brisk PMNL response in the lungs that was associated with a major increase in the levels of lactoferrin. Challenge with S. aureus was associated with a moderate increase in the number of macrophages and a moderate decrease in the levels of transferrin and iron but no change in the levels of lactoferrin. The levels of iron-binding protein can vary according to the type of inflammatory response.

Complement levels in patients with hepatic dysfunction.

Total hemolytic complement activity and serum complement protein concentrations were compared in 17 hospitalized patients with normal hepatic function and 16 patients with liver disease due to alcohol (15 patients) or acetaminophen toxicity (one patient). In contrast to the control patients, individuals with hepatic dysfunction had decreased total CH50 levels and low concentrations of total C3, C4, C5, factor B, and the regulatory proteins factor I and beta-1H. These patients also had increased C4d/C4 ratios, indicating classical pathway activation. The level of complement deficiency appears to correlate with either prolongation of the prothrombin time or depression of serum albumin concentration. These results indicate that patients with hepatic disease have severe complement depletion that is probably multifactorial in origin. This impairment in complement function will contribute to the impaired antibacterial host defense of the patient with chronic hepatic disease.

Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni.

Evidence from developing countries and volunteer studies indicates that immunity to Campylobacter jejuni and Campylobacter coli may be acquired, but the antigenic basis for this protection is poorly defined. We have purified to homogeneity four proteins with molecular weights of 28,000 (PEB1), 29,000 (PEB2), 30,000 (PEB3), and 31,000 (PEB4) from epidemic C. jejuni strain 81-176 using acid extraction and sequential ion-exchange, hydrophobic interaction, and gel filtration chromatography. The relative amino acid compositions of these four proteins are similar. NH2-terminal sequence analysis indicates that all four proteins are different, although the first 35 amino acids of PEB2 and PEB3 are 51.4% homologous. Isoelectric focusing showed that all four are basic proteins with pI of 8.5 for PEB1 protein and greater than 9.3 for the others. Use of the purified proteins as antigens in an IgG enzyme-linked immunosorbent assay (ELISA) found that seroconversion to the PEB1 or PEB3 proteins occurred in 15 of 19 patients with sporadic C. jejuni or C. coli infection. In comparison, only two, six, and 14 of these patients seroconverted to PEB2, PEB4, or the acid extract antigen. In an ELISA with whole bacterial cells as antigens, antiserum to the acid-extracted antigens showed broad recognition of C. jejuni, C. coli, C. fetus, C. lari, and Helicobacter pylori. Antiserum to PEB1 recognized all 35 C. jejuni and all 15 C. coli strains but none of the isolates of the other three bacterial species. The PEB1 and PEB3 proteins appear to be candidate antigens for both a Campylobacter vaccine and for serological assays for the pathogen.

Bacteremia in granulocytopenic patients in a tertiary-care general hospital.

Episodes of bacteremia in granulocytopenic patients during 1985 and 1986 at a tertiary-care general hospital were reviewed to assess the adequacy of current empiric antimicrobial therapy. The major pathogens in these cases were Pseudomonas aeruginosa, Enterobacteriaceae organisms, and Staphylococcus epidermidis. This combination of pathogens differed from that found at the same facility from 1975 to 1977, when Staphylococcus aureus and streptococci predominated. When apparent, the sources of infection were predominantly venous catheters, the lower respiratory tract, and the urinary tract; most frequently there was no identifiable focus. S. epidermidis and streptococci were isolated more frequently during initial episodes of febrile bacteremia, and P. aeruginosa was isolated more often during subsequent episodes. If a narrow definition for therapeutic outcome is used, only 38% of episodes had a favorable response; response rates were no different with appropriate or inappropriate therapy. The low response rate may have been related to the use of data from the previous review to guide empiric therapy and to the subsequent inadequate treatment of infections caused by Pseudomonas and Enterobacter organisms. The overall mortality per total bacteremic episodes was 19%, and the primary factor associated with mortality was pneumonia (P less than .0001). This study emphasizes the need for ongoing surveillance of local patterns of bacteremia to direct empiric therapy.