Effects of amoxicillin treatment on the salivary microbiota in children with acute otitis media.

Amoxicillin is a first-line antibiotic treatment for acute otitis media in children and one of the most commonly used antibiotics for human bacterial infections. We investigated changes in salivary bacterial communities among children treated with amoxicillin for acute otitis media (n = 18), using a culture-independent approach based on pyrosequencing of the V3 region of the bacterial 16S rRNA gene. The control group consisted of children with acute otitis media who were not given antibiotics (n = 15). One species-level phylotype assigned to the genus Streptococcus was identified across all (n = 99) saliva samples. Two additional species-level phylotypes from the genera Gemella and Granulicatella were shared by all (n = 45) samples of control subjects. Amoxicillin treatment resulted in reduced species richness and diversity, and a significant shift in the relative abundance of 35 taxa at different ranks from phylum to species-level phylotype. At the phylum level, prevalence of TM7 and Actinobacteria decreased at the end of treatment, whereas Proteobacteria had a higher relative abundance post-treatment. Multivariate analysis showed that samples from the same control subject taken over time intervals tended to cluster together. Among antibiotic-treated subjects, samples taken before and at the end of amoxicillin treatment formed two relatively well-separated clusters both of which greatly overlapped with samples taken about 3 weeks post-treatment. Our results point to a substantial but incomplete recovery of the salivary bacterial community from the antibiotic about 3 weeks after the end of treatment.

Survival effect of lung transplantation among patients with cystic fibrosis.

CONTEXT: Patients with cystic fibrosis (CF) are the second largest group of lung transplant recipients in the United States. The survival effect of transplantation on a general CF population has not previously been measured. OBJECTIVE: To determine the impact of bilateral lung transplantation on survival in patients with CF. DESIGN, SETTING, AND PATIENTS: Retrospective observational cohort study of 11 630 CF patients who did not undergo lung transplantation (controls) and 468 transplant recipients with CF from 115 CF centers in the United States, 1992-1998. Patients were stratified into 5 groups based on a 5-year survival prediction model (survival group 1: <30%; survival group 2: 30 to <50%; survival groups 3-5: 50 to <100%.) MAIN OUTCOME MEASURE: Five-year survival from date of transplantation in 1992-1997 in the transplant group and from January 1, 1993, in the control group. RESULTS: Lung transplantation increased 5-year survival of CF patients in survival group 1. Survival group 2 had equivocal survival effects, and groups 3-5 had negative survival effects from transplantation. From 1994-1997, there was a mean annual prevalence of 238 patients in survival group 1 and mean annual incidence of 154 patients entering the group, approximately 1.5 times the number of lung transplantations performed each year in CF patients (mean, 104). Use of the criterion of forced expiratory volume in 1 second of less than 30% resulted in an equivocal survival benefit and identified 1458 potential candidates for transplantation in 1993. CONCLUSIONS: Cystic fibrosis patients in group 1 have improved 5-year survival after lung transplantation. The majority of patients with CF have equivocal or negative survival effects from the procedure. Selection of patients with CF for transplantation based on group 1 survival predictions maximizes survival benefits to individuals and may reduce the demand for scarce donor organs.

Recurrent Listeria monocytogenes infection: relapse or reinfection with a unique strain confirmed by molecular subtyping.

We report a case of recurrent listeriosis for which molecular subtyping by automated ribotyping and pulsed-field gel electrophoresis confirmed either relapse of infection or reinfection due to a common source almost 9 months after initial infection due to a unique Listeria monocytogenes strain in a patient with colorectal cancer. This case report illustrates the potential use of molecular subtyping to further understand the pathogenesis and epidemiology of listeriosis and the potential for relapse of Listeria infections in humans.

Predictive 5-year survivorship model of cystic fibrosis.

The objective of this study was to create a 5-year survivorship model to identify key clinical features of cystic fibrosis. Such a model could help researchers and clinicians to evaluate therapies, improve the design of prospective studies, monitor practice patterns, counsel individual patients, and determine the best candidates for lung transplantation. The authors used information from the Cystic Fibrosis Foundation Patient Registry (CFFPR), which has collected longitudinal data on approximately 90% of cystic fibrosis patients diagnosed in the United States since 1986. They developed multivariate logistic regression models by using data on 5,820 patients randomly selected from 11,630 in the CFFPR in 1993. Models were tested for goodness of fit and were validated for the remaining 5,810 patients for 1993. The validated 5-year survivorship model included age, forced expiratory volume in 1 second as a percentage of predicted normal, gender, weight-for-age z score, pancreatic sufficiency, diabetes mellitus, Staphylococcus aureus infection, Burkerholderia cepacia infection, and annual number of acute pulmonary exacerbations. The model provides insights into the complex nature of cystic fibrosis and supplies a rigorous tool for clinical practice and research.

Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes.

A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro. The PERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL). We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes. Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL. Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested). Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase. Primers targeting the pol gene of PERV were used for PCR. Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene. Levels of PERV sequences were estimated by serial dilution. All positive samples were tested for infectivity in HEK293 cells. Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively. Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA). PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes. The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA. In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera). These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.

Effective use of polymerase chain reaction for diagnosis of central nervous system infections.

Polymerase chain reaction (PCR)-based testing of cerebrospinal fluid (CSF) specimens has become standard for confirmatory diagnosis of central nervous system (CNS) infections; however, these tests increase health care costs. We reviewed 3-year data from 974 consecutive CSF specimens submitted for detection of seven pathogens by PCR. In 1997, 237 of 367 specimens (64.6%) were submitted for multiple tests, compared with 203 of 522 (38.9%) in 1996 and 18 of 85 (21.2%) in 1995. In each year the arrival of new house officers coincided with a peak in multiple testing. Among 732 specimens submitted for herpesvirus detection, results were positive for 24 (4.6%) of 523 specimens with increased leukocyte counts or protein levels. None of 209 specimens with normal leukocyte and protein levels were positive for herpesviruses. None of 471 CSF specimens submitted for Borrelia burgdorferi detection were PCR-positive. Use of protein and leukocytes to screen CSF specimens before employing PCR for herpesvirus detection would save almost one-third of costs without reducing sensitivity.

Transfer of porcine endogenous retrovirus across hollow fiber membranes: significance to a bioartificial liver.

BACKGROUND: A porcine endogenous retrovirus (PERV) capable of infecting human cells has been identified. This study was designed to determine whether hollow fiber membranes, such as those used in a bioartificial liver, block the transfer of PERV. METHODS: Three hollow fiber cartridges (HFCs) were studied in duplicate: cellulose fibers with 70 kD nominal molecular weight cut-off (MWCO), polysulfone fibers with 400 kD MWCO, and mixed cellulose fibers with 200 nm porosity. PK15 cells (porcine kidney cell line), known to produce PERV, were grown in the intraluminal compartment of HFCs fiber cartridges. Samples of medium were collected from both intraluminal and extraluminal compartments of the HFCs fiber cartridge during 14 days of culture. Samples were screened for PERV using reverse transcription polymerase chain reaction. All positive samples were tested for PERV infectivity in human 293 cells. RESULTS: PERV was detected in all samples from the intraluminal space and all intraluminal samples seemed to infect 293 cells. All extraluminal samples from the fibers of 200 nm porosity tested positive for PERV. Detection of PERV in the extraluminal space was delayed by fibers of 400 kD MWCO and 70 kD MWCO until at least day 3 and day 7, respectively, after inoculation of PK15 cells. Positive extraluminal samples from fibers of 400 kD MWCO and 70 kD MWCO did not infect 293 cells. CONCLUSION: Pore size, membrane composition, and duration of exposure influenced the transfer of PERV across HFCs. Some HFCs decrease the risk of viral exposure to patients during bioartificial liver therapy.

Diagnosis and outcome of 100 consecutive patients with extreme granulocytic leukocytosis.

PURPOSE: To determine the clinical features, causes, and prognostic significance of extreme leukocytosis in adults. PATIENTS AND METHODS: Medical records of 100 consecutive patients who presented at the Minneapolis Veterans Affairs Medical Center between March 1993 and January 1994 with more than 25,000 leukocytes/microL blood and with more than 50% granulocytes were reviewed. Demographic, clinical, and outcome information was recorded, and a cause of extreme leukocytosis was sought in each case. RESULTS: Extreme leukocytosis was attributed to infection in 48 cases, advanced malignancy in 13 cases, hemorrhage in 9 cases, glucocorticoids in 8 cases, and other causes in 22 cases. Four patients had previously diagnosed conditions resulting in chronic leukocytosis. Higher leukocyte counts were associated with malignancy (chi2 for trend=12.5, P <0.002). Fever was more common in patients with infection (weighted rate ratio=3.7, 95% Confidence interval [CI]=2.2 to 6.2). Mortality was high overall (31%), and was greater in patients with noninfectious diagnoses compared with infected patients, an association which persisted after stratification by leukocyte count (weighted rate ratio=2.5, 95% CI=1.2 to 4.9). CONCLUSION: Clinicians should be aware that extreme leukocytosis with a predominance of granulocytes is associated with infection in only 48% of cases. The presence of fever increases the likelihood that infection is the cause. Mortality is high, particularly in patients without infection.

Aspergillus airway colonization and invasive disease after lung transplantation.

BACKGROUND: Invasive Aspergillus is an important cause of morbidity and mortality among lung transplant recipients. The diagnosis can be difficult and treatment is often unsuccessful so many centers preemptively treat all Aspergillus airway isolates to prevent invasive disease. This approach is untested as little is known about the relationship between Aspergillus airway colonization and invasive disease. This study was undertaken to evaluate the incidence of Aspergillus airway colonization after lung transplantation and the risk of invasive disease after colonization. DESIGN: All cultures and histologic specimens obtained from a consecutive series of 151 lung transplant cases were reviewed for the presence of Aspergillus and compared with clinical data. RESULTS: Aspergillus was isolated from the airway in 69 (46%) of 151 transplant recipients. Invasive disease occurred in five cases and was uniformly fatal, accounting for 13% of all posttransplant deaths. Results of cytologic examination of BAL fluid were normal in all cases of invasive disease and cultures were positive in only one of five patients prior to invasion. Invasive disease occurred exclusively in patients who died or were colonized with Aspergillus fumigatus within the first 6 months posttransplant. Patients growing A. fumigatus from the airway during the first 6 months were 11 times more likely to develop invasive disease relative to those not colonized. CONCLUSION: Aspergillus airway colonization after lung transplantation is common and in most cases, transient. In contrast, invasive Aspergillus disease is less common, but fatal. Bronchoscopy with cytologic examination and fungal culture are not sensitive or timely predictors of invasive disease. Invasive Aspergillus occurred only in patients initially colonized with A. fumigatus within the first 6 months posttransplant. A trial of empiric anti-Aspergillus therapy limited to the first 6 months posttransplant may be warranted.

Prevalence of human immunodeficiency virus infection, mortality rate, and serogroup distribution among patients with pneumococcal bacteremia at Denver General Hospital, 1984-1994.

Pandemics of human immunodeficiency virus (HIV) type 1 infection and penicillin resistance highlight the urgency of preventing invasive pneumococcal disease with vaccination. We characterized pneumococcal serogroup distribution and the mortality rate among 460 patients with pneumococcal bacteremia from 1984 through 1994 at Denver General Hospital and the prevalence of HIV infection in patients for whom pneumococcal bacteremia was diagnosed from 1989 to 1994. Vaccine-related serogroups accounted for 426 isolates (92.6%), including 48 (92.3%) of 52 isolates from HIV-infected patients. Mortality among patients 15 years of age or older was higher during 1984-1988 (18[12.9%] of 140) than during 1989-1994 (10 [5.2%] of 191: rate ratio, 2.5; 95% confidence interval, 1.2-5.2). Of patients 15-59 years of age from 1989 to 1994, 44 (39.6%) of 111 men and three (7.3%) of 41 women were HIV-infected. Four (8.5%) of 47 HIV-infected patients and four (3.8%) of 105 other patients in this group died (age-weighted rate ratio, 1.8; 95% confidence interval, 0.5-6.2). We recommend routine screening of young adults with pneumococcal bacteremia for HIV infection and immunization of HIV-infected patients with pneumococcal vaccine (which includes most serogroups of infecting strains).

Nitric oxide production during adjuvant-induced and collagen-induced arthritis.

OBJECTIVE: To investigate the role of nitric oxide (NO) production and NO synthase (NOS) induction during adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in Dark Agouti rats. METHODS: Urinary nitrate excretion and immune NOS (INOS) messenger RNA (mRNA) expression were measured in the joint, lymph node, spleen, and liver tissues following the induction of either AIA or CIA. RESULTS: Urinary nitrate excretion and iNOS mRNA expression increased substantially during joint inflammation in both models of arthritis. However, the increases in urinary nitrate excretion and iNOS mRNA expression observed in the joint, liver, and spleen tissues during AIA were greater than those observed during CIA, although iNOS induction in the lymph nodes was similar for both models. A prior injection with Mycobacterium bovis heat-shock protein resulted in suppression of arthritis and NO production in AIA, but not in CIA. CONCLUSION: Differences in NO production during AIA versus CIA are a reflection of the fundamental pathophysiologic differences between these 2 models of arthritis. Thus, NO production in these 2 models could not be merely a nonspecific reaction to the adjuvant injection, nor simply a byproduct of local inflammation in the joint.

Nitric oxide production in murine leishmaniasis: correlation of progressive infection with increasing systemic synthesis of nitric oxide.

Previous studies have shown that nitric oxide (NO) production is a major effector mechanism in the control of Leishmania major infection in the BALB/c and C3H murine models. The susceptibility of mice correlates with intrinsic NO production after infection. We have previously shown that blocking of systemic NO production with oral N-Omega-monomethyl-L-arginine results in decreased NO and exacerbated infection. The C3H mice also synthesize markedly more NO than BALB/c mice shortly after initial infection. We now show that late in infection, as the lesion size is increasing, the BALB/c NO production actually exceeds that seen during the curative stages of the C3H infection. In addition, treatment with meglumine antimoniate, which ameliorates but does not cure the BALB/c mouse, results in decreased systemic parasite load with a concomitant decrease in NO production. These results imply that in vivo, systemically measured levels of NO may in some circumstances reflect ongoing parasitic load, and that NO production is not always correlated with a curative response.

Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor.

IL-2 therapy is a potent inductive stimulus for nitric oxide (NO.) synthesis in mice and humans. It is not yet clear whether NO. can contribute to IL-2-induced therapeutic responses. The murine skin cancer Meth A is relatively resistant to lymphokine-activated killer (LAK) cell killing, allowing evaluation of the role of IL-2-induced NO. synthesis in vivo, without contribution by LAK cells. Subcutaneous IL-2 treatment of mice bearing i.p. Meth A tumor increased nitrite production by cells derived from ascites (63 +/- 14 microM vs 3.2 +/- 1.5 microM in untreated controls). N omega-monomethyl-L-arginine (MLA), NO. synthase inhibitor, prevented this increase. NO. production correlated in an inverse fashion with tumor cell proliferation in vitro. Evidence for IL-2-induced heme nitrosylation was demonstrated in tumor cells by electron paramagnetic resonance spectroscopy. By immunomagnetic depletion experiments, macrophages were implicated as a major source of NO. synthesis. Cytologic and flow-cytometric evaluation revealed that IL-2 treatment resulted in enhanced lymphocyte and macrophage recruitment into malignant ascites, and decreases in tumor cell recovery. MLA administration further increased host cell recovery. Subcutaneous IL-2 therapy increased urinary nitrate excretion up to eightfold in mice, and appeared to produce a significant survival advantage that was prevented by MLA administration.

Nitric oxide production during murine Lyme disease: lack of involvement in host resistance or pathology.

The murine model of Lyme disease was used to determine the role of inflammatory induced nitric oxide (NO) during infection by the spirochete Borrelia burgdorferi. The outer surface lipoproteins of B. burgdorferi are potent stimulators of inflammatory cytokines and NO production by cultured macrophages in vitro. The addition of NO to cultures of B. burgdorferi prevents growth, suggesting a protective role of NO for the infected host. NO is also a crucial effector in some models of arthritis. Therefore, the involvement of NO in controlling B. burgdorferi infection and its participation in pathological development of arthritis were investigated. Both mildly arthritic (BALB/c) and severely arthritic (C3H/HeJ) strains of mice systemically produced high levels of NO 1 week after infection with B. burgdorferi, as determined by urinary nitrate. NO production remained high throughout the infection in BALB/c mice, while in C3H/HeJ mice NO production returned rapidly to uninfected levels. The in vivo inhibitor of the NO synthase enzyme NG-L-monomethyl arginine (LMMA) was given to mice to investigate whether decreasing NO production would alter the course of disease. LMMA effectively blocked NO production in infected mice; however, there was no significant difference in arthritis development, spirochete infection of tissues, or production of specific antibody in LMMA-treated mice. These results indicate that B. burgdorferi is able to persist in the host even in the presence of high levels of NO. Furthermore, NO is not involved in the control of spirochete infection of tissues, nor is it involved in the development of arthritis. The potent activity of NO against intracellular pathogens and the in vivo resistance of B. burgdorferi to NO suggest that this organism is not located in an intracellular compartment during an essential portion of its infection of the mammalian host.

Effectiveness and toxicity of protracted nitric oxide synthesis inhibition during IL-2 treatment of mice.

The current study was designed to characterize nitric oxide (NO.) synthesis during interleukin-2 (IL-2) treatment of mice, and to determine whether NO. mediated IL-2-induced "vascular leak." We developed a technique for chronic subcutaneous infusion of the NO. synthase inhibitor N omega monomethyl-L-arginine (MLA) via osmotic minipump to aid in further study of these processes. After IL-2 administration to C3H/HeN mice (180,000 IU i.p. b.i.d. for 5 days), NO. synthesis increased two-to-three fold, peaking on days 5-8. Administration of MLA reduced NO. synthesis in both IL-2-treated mice (from 2.7 to 1 microM/mouse/day), and normal mice (from 1 to 0.5 microM/mouse/day). This agent decreased IL-2-induced radiolabeled albumin accumulation in the liver after i.p. IL-2 administration (p < 0.02). MLA infusions resulted in minimal systemic toxicity in mice, as reflected by complete blood counts or serum chemistries. MLA also did not impair lymphokine-activated killer cell induction in vitro or in vivo, or alter IL-2-induced tumor responses in a 3-day pulmonary metastasis model. These experiments demonstrated that NO. is a mediator involved in the genesis of vascular permeability induced by IL-2 treatment. Studies designed to further evaluate the toxicity and usefulness of MLA infusions to modify this IL-2 induced toxicity appear to be warranted.

Ampicillin/sulbactam versus ampicillin alone for cesarean section prophylaxis: a randomized double-blind trial.

To study the effectiveness of anaerobic coverage in prevention of postpartum endometritis in women undergoing nonelective cesarean sections, we conducted a randomized prospective double-blind study of women undergoing cesarean sections and requiring antibiotic prophylaxis from April 1, 1989, through December 31, 1990. Ninety-four patients were enrolled in the study. Forty-five patients received ampicillin alone and 46 received ampicillin in conjunction with sulbactam. All patients were evaluated prior to surgery and in the postoperative period. Ninety-one patients completed the study and their records were analyzed. Patients were divided into two groups depending on the presence or absence of ruptured membranes. Seventy-five percent of patients had ruptured membranes. Failure of prophylaxis and subsequent endometritis was documented in 8.8% of patients who received ampicillin and sulbactam and 35.3% of patients who received ampicillin alone. This difference was statistically significant (p < 0.02). In conclusion, single-dose ampicillin and sulbactam provides better prophylaxis than single-dose ampicillin in women undergoing cesarean section with rupture of membranes.

Intracellular bacteria in blood smears in patients with central venous catheters.

The presence of intracellular bacteria in blood smears is usually associated with overwhelming sepsis and an ominous prognosis. Recently, the hematology laboratory at our institution documented this finding in a group of mostly asymptomatic patients. We studied seven adult patients from a tertiary care university hospital in whom intracellular bacteria were found incidentally on routine manual differential cell counts of 100 white blood cells during a 12-month period. A retrospective review of the clinical and laboratory data was performed. All seven patients were immunosuppressed and had central venous catheters in place. The blood samples positive for intracellular bacteria were all catheter derived. Six patients were asymptomatic at the time of bacteria detection, but they had blood cultures that were positive for coagulase-negative Staphylococcus; five of these patients became symptomatic 1 to 14 days after bacteria detection. Bacteremia persisted in five of these six patients until the eventual removal of the catheters. The one symptomatic patient had Pseudomonas aeruginosa bacteremia and died shortly after admission. The finding of intracellular bacteria in routine differential blood cell counts from a central venous catheter blood specimen most likely indicates active infection. We recommend that central venous catheters be removed in such patients, even if the patient is asymptomatic.