N-CDAD in Canada: Results of the Canadian Nosocomial Infection Surveillance Program 1997 N-CDAD Prevalence Surveillance Project

BACKGROUND:

A 1996 preproject survey among Canadian Hospital Epidemiology Committee (CHEC) sites revealed variations in the prevention, detection, management and surveillance of Clostridium difficile-associated diarrhea (CDAD). Facilities wanted to establish national rates of nosocomially acquired CDAD (N-CDAD) to understand the impact of control or prevention measures, and the burden of N-CDAD on health care resources. The CHEC, in collaboration with the Laboratory Centre for Disease Control (Health Canada) and under the Canadian Nosocomial Infection Surveillance Program, undertook a prevalence surveillance project among selected hospitals throughout Canada.

OBJECTIVE:

To establish national prevalence rates of N-CDAD.

METHODS:

For six weeks in 1997, selected CHEC sites tested all diarrheal stools from inpatients for either C difficile toxin or C difficile bacteria with evidence of toxin production. Questionnaires were completed for patients with positive stool assays who met the case definitions.

RESULTS:

Nineteen health care facilities in eight provinces participated in the project. The overall prevalence of N-CDAD was 13.0% (95% CI 9.5% to 16.5%). The mean number of N-CDAD cases were 66.3 cases/100,000 patient days (95% CI 37.5 to 95.1) and 5.9 cases/1000 patient admissions (95% CI 3.4 to 8.4). N-CDAD was found most frequently in older patients and those who had been hospitalized for longer than two weeks in medical or surgical wards.

CONCLUSIONS:

This national prevalence surveillance project, which established N-CDAD rates, is useful as ''benchmark'' data for Canadian health care facilities, and in understanding the patterns and impact of N-CDAD.Key Words: Canada, CDAD, Clostridium difficile-associated diarrhea, Hospital, Nosocomial diarrhea, PrevalenceNosocomial acquisition and transmission of Clostridium difficile are well known (1-4). Despite efforts to control and prevent infections in health care facilities, nosocomially acquired C difficile-associated diarrhea (N-CDAD) persists; some have reported that the number of N-CDAD infections are increasing (5-10). Although the majority of patients remain asymptomatic following acquisition of C difficile (5), it is still the most commonly identified cause of nosocomial diarrhea (5,11,12). While specific antibiotic therapy for C difficile has reduced morbidity and mortality among people with CDAD (13-15), evidence exists that C difficile infection contributes to patient morbidity (7,10) and significantly impacts hospital costs (15-17).Published literature related to the prevalence of CDAD primarily describes periodic outbreaks or endemic situations in health care facilities (7,10,16-19). Because elderly people and those exposed to large amounts of antibiotics have a higher risk of acquiring CDAD, they are commonly surveyed (15,20,21). Specific wards (eg, medical and surgical) where the rates of CDAD are higher are also more frequently studied (2,6,22,23). Multicentre and national surveillance of CDAD in North America and Europe is rare (24-28). In Canada, individual health centres have data on the prevalence and demographics of CDAD cases (6,9,21); however, no national data exist.Many CDAD surveillance studies include community cases (16,17,24-28); however, it is useful to examine specifically N-CDAD cases, because they represent illness that may be prevented by hospital infection prevention and control practices. The primary reservoirs of C difficile in the hospital are humans and the environment (29). Consequently, the nosocomial acquisition of this organism may represent inadequate infection control practices (30). This underscores the importance of instigating measures to monitor the prevalence of N-CDAD, and implementing and assessing the efficacy of any prevention or control practices.There is no Canadian literature that examines the hospital costs of C difficile infections. Worldwide, there are limited data regarding the hospital costs associated with CDAD (16,17). One British study specifically examined the costs of N-CDAD (15). However, all studies suggest that these costs are substantial, which include the expenses of caring for and treating patients with CDAD, combined with the costs associated with C difficile outbreaks (15-17).An N-CDAD prevalence project was undertaken by the Canadian Nosocomial Infection Surveillance Program (CNISP) through participating Canadian health care facilities. CNISP is a collaborative national surveillance program among the Laboratory Centre for Disease Control, Health Canada and the Canadian Hospital Epidemiology Committee (CHEC), a subcommittee of the Canadian Infectious Disease Society. CHEC members participated voluntarily in the CNISP project. The intent of this project was to establish health care facility N-CDAD prevalence rates that could be used as ''benchmark'' data for other Canadian health care facilities, and to assist with the development and evaluation of guidelines that may decrease the incidence and cost of N-CDAD within Canadian health care facilities. The project used standardized case definitions for CDAD and N-CDAD. Non-nominal data were collected and submitted to the Laboratory Centre for Disease Control for compilation, analysis and interpretation. To estimate the burden of N-CDAD on the Canadian health care system, it was necessary to first determine national N-CDAD prevalence rates through a multicentre, geographically diverse surveillance project.     

The Origin of Multiple Molecular Forms in Urine of HNL/NGAL

Background and objectives: Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report.Design, setting, participants, & measurements: The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions.Results: The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL.Conclusions: The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.Human neutrophil lipocalin(HNL) (1), also named neutrophil gelatinase-associated lipocalin (NGAL) (2), is a ubiquitous glycoprotein originally isolated from human neutrophils and localized in their specific granules. HNL/NGAL exists as a 25-kD monomer, or as a 45-kD disulfide-linked homodimer, and it is covalently conjugated with gelatinase (matrix metalloproteinase 9) via an intermolecular disulfide bridge as a 135-kD heterodimeric form (2).Although HNL/NGAL was originally identified in and purified from human neutrophils, it is also expressed in kidney, liver, and epithelial cells under certain conditions (3,4). Pathologic or stressful conditions such as inflammation, infection, cancer, intoxication, ischemia, kidney injury, and cardiac surgery can induce the upregulation of the synthesis of HNL/NGAL (5–13). In addition, several studies have shown that upregulation of HNL/NGAL in human cell lines (A459 (14), MCF-7 (15), and HepG2 (11)) is induced by oxidative stress, cytokines, or other stimuli.HNL/NGAL has recently been highlighted as a novel and early biomarker of acute kidney injury (AKI) (12,13,16–19). Thus, the levels of HNL/NGAL were significantly increased in serum/plasma and urine after cardiac surgery and paralleled reduction in renal function (12,16,19). Several immunoassays have been developed for the measurement of HNL/NGAL. The assays are based on different formats and include RIA (20), Western blotting (21), ELISA (22,23), Triage device (24), and the Architect platform (16). Several research groups used one of these assays to determine the levels of HNL/NGAL in urine and drew the conclusion that HNL/NGAL is a biomarker of AKI (12,13,16–18). Our previous results indicated that the antibody configuration had an effect on the clinical performance of the assay (19,25). We also reported, for the first time, the existence of several molecular forms of HNL/NGAL in urine obtained from patients after cardiac surgery and that the presence of dimeric and monomeric forms and their ratios changed after operation (19). The source of the different molecular forms of HNL/NGAL and what they might reflect has not yet been elucidated. The aim of this report was therefore to study the possible cellular source of these different molecular forms and to investigate the possible effect of these different forms on the assay performances of HNL/NGAL assays using several different monoclonal and polyclonal antibodies with different epitope specificities.     

Clonality of mouse and human cardiomyogenesis in vivo

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1–5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2–4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.Fate mapping protocols establish a lineage relationship between ancestors carrying the reporter gene and their descendents (1, 2), but do not provide information on the self-renewing property and clonogenicity of progenitor cells or clonal origin of daughter cells in vivo (3). Because of these limitations, viral gene-tagging remains the most accurate strategy for the analysis of stem cell growth (3–8). The semi-random insertion of retroviral and lentiviral vectors represents an effective tool for genetic marking, enabling the identification of the progeny generated by stem cell differentiation. Retroviruses and lentiviruses integrate permanently in the genome of the host cells; the insertion site of the viral genome is inherited by the population derived from the parental cell (6) and can be amplified by PCR. Thus, the detection of the sites of integration constitutes a unique approach for the documentation of self-renewal, clonogenicity, and multipotentiality of stem cells in vivo. So far, this methodology has been applied to the bone marrow (4–6) and the brain (3, 7, 8) and has not been used to characterize the mechanisms regulating cardiac homeostasis and pathology.The implementation of this technique in the adult heart is relevant for the incontrovertible demonstration of resident cardiac stem cells and the ability of the myocardium to undergo spontaneous regeneration. Moreover, the notion that cardiomyocytes have a long lifespan and their turnover is slow and age-dependent (2, 9) remains controversial (10–12). This view is not consistent with a series of reports documenting the critical role that apoptosis has in the removal of damaged myocytes and the necessary replacement of dying cells with new cardiomyocytes in animals and humans (13). The recent reconsideration of the immortal strand hypothesis of stem cell division (14), together with the need to reinterpret the long-term label retaining assay for the evaluation of stem cell growth kinetics, has required the use of complementary strategies for the measurement of cardiac cell turnover. In the current study, we have combined viral tagging with BrdU pulse-chasing in vivo to characterize the biology of c-kit-positive cardiac progenitor cells (CPCs) of the mouse and human heart (11, 15, 16).     

Urinary Biomarkers in the Clinical Prognosis and Early Detection of Acute Kidney Injury

Background and objectives: Several novel urinary biomarkers have shown promise in the early detection and diagnostic evaluation of acute kidney injury (AKI). Clinicians have limited tools to determine which patients will progress to more severe forms of AKI at the time of serum creatinine increase. The diagnostic and prognostic utility of novel and traditional AKI biomarkers was evaluated during a prospective study of 123 adults undergoing cardiac surgery.Design, setting, participants, & measurements: Urinary neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CyC), kidney injury molecule-1 (KIM-1), hepatocyte growth factor (HGF), π-glutathione-S-transferase (π-GST), α-GST, and fractional excretions of sodium and urea were all measured at preoperative baseline, postoperatively, and at the time of the initial clinical diagnosis of AKI. Receiver operator characteristic curves were generated and the areas under the curve (AUCs) were compared.Results: Forty-six (37.4%) subjects developed AKI Network stage 1 AKI; 9 (7.3%) of whom progressed to stage 3. Preoperative KIM-1 and α-GST were able to predict the future development of stage 1 and stage 3 AKI. Urine CyC at intensive care unit (ICU) arrival best detected early stage 1 AKI (AUC = 0.70, P < 0.001); the 6-hour ICU NGAL (AUC = 0.88; P < 0.001) best detected early stage 3 AKI. π-GST best predicted the progression to stage 3 AKI at the time of creatinine increase (AUC = 0.86; P = 0.002).Conclusion: Urinary biomarkers may improve the ability to detect early AKI and determine the clinical prognosis of AKI at the time of diagnosis.Acute kidney injury (AKI) is a common and serious complication of cardiothoracic surgery (1); depending on the definition of AKI used, it may occur in over 40% of adults, with 1% to 5% requiring renal replacement therapy (RRT) (2–9). Recently, standardized clinical definitions of AKI have been implemented through the use of the RIFLE (Risk, Injury, Failure, Loss, and ESRD) and AKIN (Acute Kidney Injury Network) criteria (10,11). However, these criteria are still very much dependent on delayed serum creatinine elevations, the current gold standard for the diagnosis of AKI. Furthermore, as a functional marker of glomerular filtration, serum creatinine is not ideally suited to diagnose AKI caused by renal tubular injury, rather than reversible prerenal azotemia (10).In recent years, several novel human biomarkers have been demonstrated to detect acute tubular injury and have shown promise in their ability to precede and/or complement serum creatinine in the diagnosis of AKI (12–15). Cardiac surgery has long been used to study AKI because of the ability to prospectively follow patients before and after a well timed renal insult; for this reason, several urinary proteins have been shown to serve as biomarkers of AKI after cardiac surgery, including neutrophil gelatinase-associated lipocalin (NGAL) (16–20), cystatin C (CyC) (19,21), kidney injury molecule-1 (KIM-1) (18,21), interleukin-18 (IL-18) (22), and α-glutathione-S-transferase (α-GST) (23,24). Limited data are available comparing the ability of these markers to predict renal outcomes at the time of AKI diagnosis. In fact, nephrologists have limited tools in their arsenal to assess the presence and severity of renal tubular injury at the time of AKI diagnosis. Although urinalysis with microscopy has been shown to be of some utility in the differential diagnosis of AKI in a generalized hospital-based cohort (25), data supporting its use in the specific setting of cardiac surgery are lacking (24). Similarly, diagnostic mainstays of AKI evaluation such as the fractional excretion of sodium (FENa) have long been shown to be suboptimal tools in the complex setting of cardiac surgery AKI (24), in which volume status, fluid responsiveness, and diuretic use confound inferences regarding the relationship between tubular function and injury (26,27). Additionally, although recent data support the utility of the fractional excretion of urea (FEUrea) as a diagnostic tool in AKI (28), not all data support its use (29). Furthermore, very little is known about the utility of FENa or FEUrea compared with the novel urinary biomarkers discussed above for the differential diagnosis and prognostic evaluation of AKI.In this study, we assessed the diagnostic utility of urinary NGAL, CyC, KIM-1, hepatocyte growth factor (HGF), α-GST (a proximal tubular damage marker), π-GST (a marker specific to distal tubule damage), FENa, and FEUrea as biomarkers for the detection of early and severe AKI after adult cardiac surgery. These novel biomarkers can be thought of as falling into two categories: constitutive markers (proteins/enzymes that are normally present in renal tubular cells and not normally found in the urine in significant concentration but are released into the urine in direct response to cellular injury), and inducible biomarkers (proteins that are not normally found in high concentrations in renal tubular cells or urine until their production is directly upregulated in response to cellular injury). CyC, α-GST, and π-GST are constitutive proteins that are extruded into urine in the presence of site-specific renal tubular injury (CyC and α-GST are proximal and π-GST is distal); intracellular GSTs are released into urine by damaged tubular cells, whereas injured proximal tubules fail to reabsorb filtered CyC. In contrast, KIM-1 and NGAL are inducible biomarkers, gene products that are increased in direct response to nephron damage (30,31). We also evaluated the ability of these markers to predict the severity/stage of AKI at the time of clinical diagnosis by serum creatinine increase. We performed all of the above analyses for those subjects who developed AKI as defined by the AKIN (11). Recent data demonstrate that urine NGAL after cardiac surgery varies with baseline renal function (32); as such, a secondary analysis of baseline GFR was conducted for the aforementioned panel of biomarkers (32). Finally, we interpreted the data for all novel biomarker concentrations adjusted and unadjusted for dilution by indexing to urinary creatinine, but for brevity''s sake, we only report the indexed values unless otherwise noted.     

Training of Surgeons in Peritoneal Dialysis Catheter Placement in the United States: A National Survey

Background and objectives: Peritoneal dialysis (PD) depends on timely and skilled placement of a PD catheter (PDC). Most PDCs are placed surgically, but little is known about the residency training of surgeons in this procedure. Inadequate residency training could limit surgical expertise in PDCs, resulting in high complication rates that discourage PD use. This study assessed surgical PDC training in the United States to explore this issue.Design, setting, participants, & measurements: A survey was sent to program directors of 248 U.S. surgery residency programs regarding the amount of PDC training, attitudes toward PDCs, and barriers to PDC training. Results were compared between academic and private centers.Results: Ninety-three surgery programs (38%) responded: 82% provided training in PDC and 69% were academic centers. Most surgeons placed 2 to ≤5 catheters during residency. Forty-eight percent of program directors felt that PDC training was important, 61% felt PDC training affected outcomes and increased the likelihood surgeons would place PDCs in practice, and 62% of programs expressed willingness to provide more PDC training. Lack of referrals from nephrology was the most frequently cited barrier to PDC training.Conclusions: Although many U.S. surgery residency programs provide PDC training, this training appears inadequate. Low PD use and lack of referrals limits surgical training at most centers. Nephrologists need to develop initiatives with surgeons to improve PDC training and outcomes.The use of peritoneal dialysis (PD) in the United States is declining. Despite comparable efficacy, improving outcomes, and cost savings compared with hemodialysis (HD), only 6% of incident and 7.2% of prevalent dialysis patients are treated with PD (1–4). Although many factors determine success on PD, a well functioning PD catheter (PDC) is absolutely necessary. Placement of a PDC by an experienced operator is strongly recommended to reduce complications (5–9). Little attention has been given to the potential effect of surgical PDC training on PD use and outcomes (1–2,10). Conversely, considerable focus has been placed on improving surgical training and outcomes for HD access (11–15).Problems with PDC placement and malfunction can disrupt efforts to grow and develop a PD program (5,9,16–18). PDC problems frustrate patients, nurses, and nephrologists alike, leading to dissatisfaction with PD and an early switch to HD (18). PDC malfunction is second only to infection as the cause of technique failure in PD (19,20). Surgeons insert most PD catheters in the United States because most nephrologists are not trained in PDC placement (5,21–23). Unfortunately, there is a shortage of surgeons interested and skilled in performing this procedure (5).Surgical outcomes correlate strongly with training during residency (24). Reluctance by surgeons to place PDCs and suboptimal PDC outcomes might stem from inadequate residency training. Unfortunately, little is known about the training surgeons undergo in this outwardly simple, yet critical procedure. We sought to investigate PDC training in U.S. surgery residency programs and explore surgical program directors'' attitudes toward this procedure.     

Prevalence of Atrial Fibrillation and Its Predictors in Nondialysis Patients with Chronic Kidney Disease

Background and objectives: Chronic kidney disease (CKD) increases systemic inflammation, which is implicated in development and maintenance of atrial fibrillation (AF); therefore, we hypothesized that the prevalence of AF would be increased among nondialysis patients with CKD. This study also reports independent predictors of the presence of AF in this population.Design, setting, participants, & measurements: A retrospective, cross-sectional analysis of 1010 consecutive nondialysis patients with CKD from two community-based hospitals was conducted. Estimated GFRs (eGFRs) were calculated using the Modification of Diet in Renal Disease (MDRD) equation. Multivariate logistic regression was used to determine independent predictors.Results: Of 1010 nondialysis patients with CKD, 214 (21.2%) had AF. Patients with AF were older than patients without AF (76 ± 11 versus 63 ± 15 yr). The prevalence of AF among white patients (42.7%) was higher than among black patients (12.7%) or other races (5.7%). In multivariate analyses, age, white race, increasing left atrial diameter, lower systolic BP, and congestive heart failure were identified as independent predictors of the presence of AF. Although serum high-sensitivity C-reactive protein levels were elevated in our population (5.2 ± 7.4 mg/L), levels did not correlate with the presence of AF or with eGFR. Finally, eGFR did not correlate with the presence of AF in our population.Conclusions: The prevalence of AF was increased in our population, and independent predictors were age, white race, increasing left atrial diameter, lower systolic BP, and congestive heart failure.Atrial fibrillation (AF) is the most common arrhythmia in clinical practice (1). Cardiac comorbidities that are associated with AF include hypertension, coronary artery disease (CAD), valvular heart disease (VHD), congestive heart failure (CHF), cardiomyopathy, pericarditis, congenital heart disease (CHD), and cardiac surgery (2–9). Noncardiac comorbidities that are associated with AF include acute pulmonary embolism, chronic obstructive pulmonary disease (COPD), obstructive sleep apnea, hyperthyroidism, and obesity (10–14).Evidence suggests that inflammation is involved in the pathogenesis of AF (15–20). For example, AF after cardiac surgery is associated with proinflammatory cytokine and complement activation (16,19). Moreover, patients with refractory lone AF have inflammatory infiltrates, myocyte necrosis, and fibrosis on biopsy (18). Several studies also reported elevated serum high-sensitivity C-reactive protein (hsCRP) levels in patients with AF (15–17,20).Evidence suggests that inflammation is associated with renal dysfunction (21–24). Proposed mechanisms include decreased proinflammatory cytokine clearance, endotoxemia, oxidative stress, and reduced antioxidant levels (23,24). Moreover, hsCRP levels are higher among elderly patients with renal insufficiency (24). In hemodialysis (HD) patients with ESRD, hsCRP, IL-6, and fibrinogen levels are elevated (21,22).HD patients with ESRD have an increased prevalence of AF; however, prevalence among nondialysis patients with CKD has not been investigated (25–30). Because CKD promotes inflammation, which promotes AF, we hypothesized the prevalence of AF would be increased among nondialysis patients with CKD. This study reports the prevalence and independent predictors of the presence of AF in a nondialysis population with CKD.     

Renal Involvement in Primary Sj?gren's Syndrome: A Clinicopathologic Study

Background & objectives: Renal pathology and clinical outcomes in patients with primary Sjögren''s syndrome (pSS) who underwent kidney biopsy (KB) because of renal impairment are reported.Design, setting, participants, & measurements: Twenty-four of 7276 patients with pSS underwent KB over 40 years. Patient cases were reviewed by a renal pathologist, nephrologist, and rheumatologist. Presentation, laboratory findings, renal pathology, initial treatment, and therapeutic response were noted.Results: Seventeen patients (17 of 24; 71%) had acute or chronic tubulointerstitial nephritis (TIN) as the primary lesion, with chronic TIN (11 of 17; 65%) the most common presentation. Two had cryoglobulinemic GN. Two had focal segmental glomerulosclerosis. Twenty patients (83%) were initially treated with corticosteroids. In addition, three received rituximab during follow-up. Sixteen were followed after biopsy for more than 12 mo (median 76 mo; range 17 to 192), and 14 of 16 maintained or improved renal function through follow-up. Of the seven patients presenting in stage IV chronic kidney disease, none progressed to stage V with treatment.Conclusions: This case series supports chronic TIN as the predominant KB finding in patients with renal involvement from pSS and illustrates diverse glomerular lesions. KB should be considered in the clinical evaluation of kidney dysfunction in pSS. Treatment with glucocorticoids or other immunosuppressive agents appears to slow progression of renal disease. Screening for renal involvement in pSS should include urinalysis, serum creatinine, and KB where indicated. KB with characteristic findings (TIN) should be considered as an additional supportive criterion to the classification criteria for pSS because it may affect management and renal outcome.Primary Sjögren''s syndrome (pSS) is a progressive autoimmune disorder involving the exocrine glands (1), typically presenting with keratoconjunctivitis and xerostomia (2). It is characterized pathologically by a predominant lymphocytic infiltrate around epithelial ducts of exocrine glands on salivary gland biopsy (3). Extraglandular manifestations of pSS, once thought to be uncommon, occur in up to 25% of patients. Patients can be afflicted by severe interstitial lung disease (4), cutaneous vasculitis (5), peripheral neuropathy (6), and hematologic complications such as lymphoma (7). They are also at increased risk for celiac sprue (8) and complications from Helicobacter pylori infection (9) such as mucosa-associated lymphatic tissue (MALT)-type lymphoma.Much of our understanding of the clinical presentation of renal involvement in pSS is based on case reports (10–26) and small retrospective cohorts (27–29). Tubulointerstitial nephritis (TIN) remains the most common presentation of renal involvement in pSS and CD4/CD8 T cell subsets are reported to predominate (27,30). This is often characterized by a distal (type I) renal tubular acidosis (RTA) and less commonly proximal (type II) RTA (Fanconi syndrome) (11,31–33). GN is thought to be a rare occurrence, with only case reports available in the literature (10,12–23), and tends to be a late development (34) in the course of the disease.We examined the renal pathologic findings and clinical trends of all patients with pSS who underwent kidney biopsy (KB) at Mayo Clinic since 1967 and assembled a case series of patients with pSS with renal pathologic disease evaluated by renal biopsy at a single center in the United States. This case series aimed to describe the common clinical presentations of renal disease in pSS, the array of pathologic findings of renal involvement in pSS, and trends during follow-up and treatment.     

Outcomes Associated with Serum Calcium Level in Men with Non-Dialysis-Dependent Chronic Kidney Disease

Background and objectives: Elevated serum calcium has been associated with increased mortality in dialysis patients, but it is unclear whether the same is true in non-dialysis-dependent (NDD) chronic kidney disease (CKD). Outcomes associated with low serum calcium are also not well-characterized.Design, setting, participants, & measurements: We examined associations of baseline, time-varying, and time-averaged serum calcium with all-cause mortality in a historic prospective cohort of 1243 men with moderate and advanced NDD CKD by using Cox models.Results: The association of serum calcium with mortality varied according to the applied statistical models. Higher baseline calcium and time-averaged calcium were associated with higher mortality (multivariable adjusted hazard ratio (95% confidence interval): 1.31 (1.13, 1.53); P < 0.001 for a baseline calcium 1 mg/dl higher). However, in time-varying analyses, lower calcium levels were associated with increased mortality.Conclusions: Higher serum calcium is associated with increased long-term mortality (as reflected by the baseline and time-averaged models), and lower serum calcium is associated with increased short-term mortality (as reflected by the time-varying models) in patients with NDD CKD. Clinical trials are warranted to determine whether maintaining normal serum calcium can improve outcomes in these patients.Mineral and bone disorders in chronic kidney disease (CKD) (1) have emerged as novel mortality risk factors in dialysis patients (2–8). Some of these abnormalities (such as serum phosphorus and parathyroid hormone (PTH) levels) have also been implicated in similar ways in patients with non-dialysis-dependent (NDD) CKD (9–12). Serum calcium''s effect on outcomes has been the focus of attention mainly in dialysis patients, where calcium metabolism is significantly distorted (13–19). The use of calcium-containing phosphate binders further complicates the picture because these medications could be involved in the etiology of vascular calcification (20,21), and their roles as therapeutic agents have been intensely debated (22). Supporting the potential role for calcium in cardiovascular disease were epidemiologic studies showing an association between higher calcium and increased mortality (2–8). Some of the same studies have also suggested that extremely low calcium levels may themselves be deleterious (2,3), which has ultimately resulted in recommendations to attain a low-normal serum calcium level in dialysis patients (23). Studies examining the role of calcium in NDD CKD patients are fewer and failed to unequivocally show an association between abnormal calcium levels and vascular calcification (24–27). No study has yet examined the association of calcium levels with mortality in NDD CKD.We examined the association of serum calcium levels with all-cause mortality in a large number of male US veterans with moderate and advanced NDD CKD at a single medical institution.     

Reduced susceptibility to penicillin among pneumococci causing invasive infection in children - Canada, 1991 to 1998

OBJECTIVE:

To determine, over time, the rate and serotypes of pneumococci with reduced penicillin susceptibility obtained from children with invasive infection.

DESIGN:

Active, hospital-based, multicentre surveillance spanning from 1991 to 1998.

SETTING:

Eleven Canadian tertiary care paediatric facilities located from coast to coast.

POPULATION STUDIED:

1847 children with invasive pneumococcal infection whose isolates (from a normally sterile site) were available for serotyping and standardized testing for penicillin susceptibility at the National Centre for Streptococcus.

MAIN RESULTS:

The prevalence of reduced penicillin susceptibility increased from 2.5% of 197 cases in 1991 to 13.0% of 276 cases in 1998. In the latter year, 8.7% of isolates had intermediate level resistance, and 4.3% had high level resistance. Since they were first detected in 1992, strains with high level resistance have been encountered only sporadically at most centres, but by 1998, all centres but two had encountered examples. Of 40 isolates with high level resistance and 101 isolates with intermediate level resistance, serotypes matched those included in new seven-valent conjugate vaccines for children in 97.5% and 79.2% of cases, respectively.

CONCLUSIONS:

Pneumococci with reduced susceptibility to penicillin are increasing in frequency across Canada among children with invasive infection. The Immunization Monitoring Program, Active data indicate that new conjugate vaccines could help to curb infections due to pneumococci with reduced susceptibility to penicillin but are unlikely to control completely the problem of antibiotic resistance.Key Words: Children, Penicillin resistance, Pneumococcus, Steptococcus pneumoniaeInfections with Streptococcus pneumoniae (pneumococcus) are a leading cause of infection-related morbidity and mortality in children (1,2). Manifestations include middle ear and upper respiratory infections, pneumonia and invasive infections, including bacteremia and meningitis. The latter most often affect young children (1,2) and those with various chronic conditions (2,3). The recent emergence of resistance to penicillin and other antibiotics (4) adds to the challenge of treating pneumococcal infections.In Canada, antibiotic resistance has emerged more slowly than it did in the United States, but the gap is narrowing with time. National surveillance in the United States indicated rates of reduced susceptibility to penicillin (minimal inhibitory concentration [MIC] 0.1 μg/mL or greater) averaging 5.0% during 1979 to 1987 (5), but by 1993 to 1994, the rate had increased to 14.1% (6). During 1997 to 1998, the rate had increased further to 29.5%, with 12.1% having high level penicillin resistance (MIC 2.0 μg/mL or greater) (7). Another national survey (8) in 1997, which was limited to invasive isolates, reported that 25.0% had reduced susceptibility to penicillin (referring to both intermediate and high level resistance), and 13.6% were highly resistant. In both recent studies (7,8), the proportion of isolates with reduced susceptibility to penicillin varied significantly by region of the country, from 12.8% to 64.6%, and among hospitals within a region.Canadian reports from the 1970s indicated a low rate of intermediate level penicillin resistance (MIC 0.1 to 1.0 μg/mL) among collected strains (9) and cases in children (10). No increase was evident in surveys from Ontario (11) and Quebec (12) reported in 1989. By 1994, the rate of reduced penicillin susceptibility had increased in the Toronto area to 7.3%, and high level resistance was evident in 2.2% of isolates (13). Canada-wide surveys subsequently showed further increases, with at least one reporting a statistically significant increase in high level resistance between 1992 and 1995 (14). In serial surveys of respiratory isolates from 50 hospitals across the country, rates increased between 1994 and 1996 from 8.5% to 13.3% for reduced susceptibility, and from 2.1% to 4.4% for high level resistance (15). Allowing for sample size effects, resistance rates were similar across the country. Another survey in 1997 (16) involving lower respiratory tract isolates from seven large Canadian hospitals showed that 30.2% of isolates had reduced penicillin susceptibility, and 8.4% were highly resistant. All of the hospitals encountered isolates with intermediate level resistance (rates ranging from 15.0% to 33.3%) but differed markedly in experiences with highly resistant isolates (rates ranging from 0% to 17%). Another survey (17) in 1997 involving eight Canadian tertiary care hospitals focused on bloodstream isolates, and 30.5% of isolates were reported to have reduced penicillin susceptibility, while American centres in the same survey reported a rate of 41%.Limited information is available about invasive infections in children. A study of such infections carried out in 1995 in the Toronto area (18) reported reduced penicillin susceptibility in 11% of isolates. The Canadian Paediatric Society/Laboratory Centre for Disease Control Immunization Monitoring Program, Active (IMPACT) network of 10 children''s hospitals across Canada (19) reported that 3.4% of invasive isolates encountered in 1994 had reduced penicillin susceptibility, with high level resistance in 1.3%, but almost all of the latter isolates were from hospitals in Montreal. Surveillance has continued at IMPACT centres, and results through 1998 are presented in this report. Of particular interest were the extent of increase in rates of intermediate and high level resistance, geographic distribution of nonsusceptible isolates, and their association with particular clinical syndromes and serotypes.     

Phosphoinositides and SNARE chaperones synergistically assemble and remodel SNARE complexes for membrane fusion

Yeast vacuole fusion requires 4 SNAREs, 2 SNARE chaperone systems (Sec17p/Sec18p/ATP and the HOPS complex), and 2 phosphoinositides, phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. By reconstituting proteoliposomal fusion with purified components, we now show that phosphoinositides have 4 distinct roles: PI(3)P is recognized by the PX domain of the SNARE Vam7p; PI(3)P enhances the capacity of membrane-bound SNAREs to drive fusion in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ can activate SNARE chaperones for the recruitment of Vam7p into fusion-competent SNARE complexes; and either PI(3)P or PI(4,5)P₂ strikingly promotes synergistic SNARE complex remodeling by Sec17p/Sec18p/ATP and HOPS. This ternary synergy of phosphoinositides and 2 SNARE chaperone systems is required for rapid fusion.Intracellular membrane fusion is a conserved reaction, vital for vesicle trafficking, hormone secretion, and neurotransmission. Fusion is regulated by NSF (N-ethylmaleimide-sensitive factor)/Sec18p, αSNAP (soluble NSF attachment protein)/Sec17p, SNAREs (SNAP receptors), Sec1p/Munc18–1p family (SM) proteins, Rab GTPases, and Rab:GTP-binding proteins, termed “Rab effectors” (1–3). Lipids, including phosphoinositides, sterols, diacylglycerol (DAG), and phosphatidic acid (PA), have specific roles in fusion (4–14). Proteins and lipids cooperate for their enrichment in membrane fusion microdomains (6, 8, 15, 16).SNARE proteins are integral or peripheral membrane proteins required for membrane fusion. SNAREs have either a Q or R residue at the center of their SNARE domain and associate in 4-helical QabcR complexes in cis (anchored to one membrane) or in trans (anchored to apposed membranes), where a, b, and c are families of related Q-SNAREs (2, 17, 18). Reconstituted proteoliposomes (RPLs) bearing Q-SNAREs fuse with RPLs bearing an R-SNARE through trans-SNARE-complex assembly (19, 20). This fusion has slow kinetics, requires nonphysiologically high SNARE densities, and causes substantial leakage of luminal contents of the RPLs (21–24).We study membrane fusion with yeast vacuoles (lysosomes). Vacuole fusion (25) requires 3 Q-SNAREs (Vam3p, Vti1p, and Vam7p) and 1R-SNARE (Nyv1p) (26, 27), two SNARE chaperone systems, Sec17p/Sec18p/ATP (28), and the HOPS (homotypic fusion and vacuole protein sorting)/Vps Class C complex (29, 30), the Rab-GTPase Ypt7p (31), and chemically minor but functionally vital “regulatory lipids”: ergosterol (ERG), DAG, PI(3)P, and PI(4,5)P₂ (8). Inactive 4SNARE cis-complexes on isolated organelles are disassembled by Sec17p/Sec18p/ATP (27). The heterohexameric HOPS complex, containing the SM protein Vps33p as a subunit, promotes and proofreads SNARE-complex assembly (32–34). HOPS can physically interact with the Q-SNAREs [Vam7p (35) and Vam3p (36, 37)], 4SNARE cis-complexes (32), GTP-bound Ypt7p (29), and phosphoinositides (35). PI(3)P supports the membrane association of the Qc-SNARE Vam7p, which has no transmembrane domain, through binding its PX domain (38). SNAREs, HOPS, Ypt7p, and regulatory lipids assemble in an interdependent fashion to form a fusion-competent membrane microdomain, the “vertex ring” (8, 16, 39). Trans-SNARE complexes are essential for fusion (26), yet fusion can be accelerated by SNARE-associating factors such as HOPS (14, 35) and by cycles of SNARE complex disassembly and reassembly, termed “remodeling” (40).Membrane fusion has been reconstituted with all purified yeast vacuolar components, including 4SNAREs, vacuolar lipids, 2 SNARE chaperone systems, and phosphoinositides (14). We now show distinct functions of phosphoinositides in RPL fusion: the PX-domain of the SNARE Vam7p recognizes PI(3)P, as reported (38); PI(3)P activates the 3Q-SNAREs to be more fusogenic in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ accelerates fusion by promoting the synergy between Sec17p/Sec18p and HOPS, although this synergy is not a function of the membrane recruitments of these SNARE chaperones. This ternary synergy between phosphoinositides and SNARE chaperones is essential for the assembly and remodeling of SNARE complexes.     

Evaluation of Fluoroquinolones for the Prevention of BK Viremia after Renal Transplantation

Background and objectives: Nearly 30% of renal transplant recipients develops BK viremia, a prerequisite for BK nephropathy. Case reports have evaluated treatment options for BK virus, but no controlled studies have assessed prophylactic therapies. Fluoroquinolone antibiotics were studied for prevention of BK viremia after renal transplantation.Design, setting, participants, & measurements: This retrospective analysis evaluated adult renal transplant recipients with at least one BK viral load (blood) between 90 and 400 days after transplantation. Six to 12 months of co-trimoxazole was used for Pneumocystis prophylaxis. In sulfa-allergic/-intolerant patients, 6 to 12 months of atovaquone with 1 month of a fluoroquinolone was used. Fluoroquinolones can inhibit BK DNA topoisomerase. The two groups studied were those that received 30 days of levofloxacin or ciprofloxacin after transplantation and those that did not. The primary endpoint was BK viremia rates at 1 year. Of note, of the 160 patients not receiving fluoroquinolone prophylaxis, 40 received a fluoroquinolone for treatment of a bacterial infection within 3 months after transplantation. Subgroup analysis evaluating these 40 patients against the 120 who had no exposure to fluoroquinolones was completed.Results: A 1-month fluoroquinolone course after transplantation was associated with significantly lower rates of BK viremia at 1 year compared with those with no fluoroquinolone. In the subgroup analysis, exposure to fluoroquinolone for treatment of bacterial infections within 3 months after transplantation was associated with significantly lower 1-year rates of BK viremia.Conclusions: This analysis demonstrates that fluoroquinolones are effective at preventing BK viremia after renal transplantation.One complication of increasing significance in renal transplant recipients (RTR) is BK polyomavirus. Polyomaviruses belong to a family of small, circularized, double-stranded DNA viruses called Papovaviridae (1–3). Twelve members of the polyomavirus family have been described in a variety of species including mice, monkeys, and humans, with SV40 being the most studied (4). Many studies related to viral replication, assembly, structure, gene expression, and DNA replication have been performed with SV40 and its large T antigen (4). In humans, there are two known pathologic polyoma strains, BK and JC. The majority of healthy individuals are seropositive for antibodies against both viruses. Seroconversion normally occurs in childhood, possibly associated with mild upper respiratory tract symptoms. These viruses rarely are associated with disease in immunocompetent individuals. During periods of immunosuppression the virus is reactivated and can be associated with significant morbidity (1–3).In RTR, the major diseases caused by BK virus (BKV) are tubulointerstitial nephritis and ureteral stenosis, which occur after BKV reactivates from its latent state with the onset of immunosuppression (3,5,6). BKV causes disease of the genitourinary tract, due in part to its tropism for genitourinary epithelium. BKV-induced nephropathy (BKVN) presents with evidence of allograft dysfunction, resulting in either an asymptomatic acute or a slowly progressive rise in serum creatinine concentrations (1–3,5,6). Some studies have reported the incidence of BK viremia to be as high as 29% (7). BK viremia is believed to be a precursor to BKVN with BK viremia preceding nephropathy by 1 to 12 weeks (5,6,8–10). One analysis showed a peak in BK viremia occurring at 3 months after transplantation (11). The onset of BKVN occurs at a mean period of 9 to 12 months after transplantation; however, some cases have been reported as early as 7 days after transplantation (1–3,5,6). It is estimated that BKVN affects up to 10% of RTR, frequently resulting in permanent renal dysfunction or allograft loss (5,6). The temporal relationship between the introduction of more potent immunosuppressive agents and BKV has led to the proposal that intensity of immunosuppression is a risk factor for BKVN. Other possible risk factors include increased age, male gender, Caucasian race, diabetes mellitus, and acute rejection (12–14). Some donor-related risk factors are the presence of active BKV or cytomegalovirus (CMV) infection and deceased donor versus living donor transplant (12–14).Currently, treatment options for BKV are limited and management recommendations are formulated on the basis of individual case reports and small case series. Pharmacologic options with activity against BKV are limited; therefore, a reduction in the degree of immunosuppression in patients with BK viremia is often thought to be the first-line option to prevent BKVN (1–3). This strategy focuses on routine patient monitoring for development of BK viremia and then on reducing immunosuppression upon diagnosis. However, in immunosuppressive regimens utilizing drug-minimization or -withdrawal strategies, it may be inconceivable to reduce immunosuppression in some patients without increasing the risk of acute rejection. Therefore, the search for a pharmacologic option for the prevention and management of BK infection remains a priority.Despite the lack of a directed antiviral intervention, there are several agents that have anti-BK activity, including intravenous immune globulin (IVIG), cidofovir, leflunomide, and the fluoroquinolone antibiotics (6,15,16). All of these agents have been reported to provide some benefit in managing BKV in anecdotal cases. It should be noted that these cases have been complicated by the fact that administration of the antiviral agent was also done simultaneously with immunosuppression reductions, making it difficult to comment on the true effectiveness of the pharmacologic intervention. Beyond the paucity of data and the poor quality of the existing evidence, there may be several reasons why the use of IVIG, cidofovir, and leflunomide are not ideal in RTR.The use of IVIG is limited by its expense, frequent nationwide shortages, potential for nephrotoxicity from sucrose-containing formulations, and lengthy administration times that may necessitate hospital admission (16).The active metabolite of leflunomide, A771726, has wide interpatient variability and also has a long half-life (mean = 15 days). Therapeutic drug monitoring is suggested when using leflunomide, but the limited availability of A771726 levels may preclude its routine use at some institutions. This agent is also associated with significant hematologic and hepatic toxicities (17).Cidofovir is highly nephrotoxic and, on the basis of in vitro data, appears to have minimal antiviral activity against BKV (18,19).Clinically, the fluoroquinolone antibiotics may represent a class of medications that could manage BKV infections with a low incidence of adverse events at a reasonable expense. Fluoroquinolones display anti-BK properties through inhibition of DNA topoisomerase and polyomavirus associated large T-antigen helicase (2,16). An in vitro analysis using older fluoroquinolone compounds, nalidixic acid and oxolinic acid, demonstrated that these agents are capable of inhibiting BKV DNA replication (20). In another analysis using contemporary fluoroquinolones (i.e., levofloxacin, trovafloxacin, ciprofloxacin, ofloxacin, and gatifloxacin), it was shown that these antibiotics have the ability to inhibit viral DNA replication and T-antigen helicase activity of SV40 (21). These agents demonstrate the ability to inhibit viral replication and block the cytopathic effect of SV40 virus in permissive monkey cells. Given the homology among SV40, BK, and JC T-antigens, these data could be applied to management of BKV. In a clinical analysis of RTR, 7 of 10 patients with active BKV replication had a reduction in viremia or urinary decoy cells 2 months after receiving a 10-day course of gatifloxacin without any reduction in immunosuppression (6,22).Given the literature suggesting the activity of fluoroquinolones against BKV, we hypothesize that patients receiving a 1-month postoperative course of fluoroquinolones would have a lower frequency of BK viremia than those patients not receiving this therapy. To test this hypothesis, we compared the rates of BK viremia in patients who were exposed with those who were not exposed to fluoroquinolones for posttransplant prophylaxis.     

Treatment with IFN-α, -β, or -γ Is Associated with Collapsing Focal Segmental Glomerulosclerosis

Background and objectives: Treatment with IFN is rarely associated with nephrotic syndrome and renal biopsy findings of minimal-change disease or FSGS.Design, setting, participants, & measurements: We report 11 cases of collapsing FSGS that developed during treatment with IFN and improved after discontinuation of therapy.Results: The cohort consists of seven women and four men with a mean age of 48.2 yr. Ten of the 11 patients were black. Six patients were receiving IFN-α for hepatitis C virus infection (n = 5) or malignant melanoma (n = 1), three were receiving IFN-β for multiple sclerosis, and two were treated with IFN-γ for idiopathic pulmonary fibrosis. After a mean and median duration of therapy of 4.0 and 12.6 months, respectively, patients presented with acute renal failure (mean creatinine 3.5 mg/dl) and nephrotic-range proteinuria (mean 24-hour urine protein 9.7 g). Renal biopsy revealed collapsing FSGS with extensive foot process effacement and many endothelial tubuloreticular inclusions. Follow-up was available for 10 patients, all of whom discontinued IFN. At a mean of 23.6 months, nine of 10 patients had improvement in renal function, including one with complete remission and two with partial remission. Among the seven patients with available data, mean proteinuria declined from 9.9 to 3.0 g/d. Four of the seven patients were treated with immunosuppression, and there was no detectable benefit.Conclusions: Collapsing FSGS may occur after treatment with IFN-α, -β, or -γ and is typically accompanied by the ultrastructural finding of endothelial tubuloreticular inclusions. Optimal therapy includes discontinuation of IFN.FSGS is the most common cause of idiopathic nephrotic syndrome in black patients and may be the most frequent cause of nephrotic syndrome in the general population (1–6). The spectrum of FSGS includes primary forms mediated by a putative circulating or permeability factor and a number of secondary forms caused by such diverse insults as hereditable mutations in podocyte genes, drugs, viral infections, and adaptive responses to reduced renal mass or other hemodynamic stress (1). A variety of histologic variants of FSGS have been identified and can be applied to both primary and secondary forms (7–9). Many secondary forms tend to manifest as particular morphologic subtypes (1).The collapsing variant of FSGS is defined by implosive wrinkling and “collapse” of the glomerular basement membrane associated with hypertrophy and hyperplasia of overlying podocytes (10–12). Collapsing FSGS was mainly described in patients with HIV-associated nephropathy (HIVAN) (13) but also was recognized as a variant of idiopathic FSGS (11,12). Both idiopathic collapsing FSGS and HIVAN are most commonly seen in young black patients (8–12,14). Compared with the usual, most common form of FSGS with discrete segmental scars (FSGS not otherwise specified [FSGS NOS]), collapsing FSGS is distinguished by more severe nephrotic syndrome and renal insufficiency at presentation and a more rapid course to renal failure (8–12,14). Central to the morphogenesis of the collapsing variant is podocyte injury that leads to podocyte dedifferentiation, apoptosis, and proliferation, in part through dysregulation of cell cycle–related proteins (15–19). Podocyte precursor cells from the parietal cell layer may contribute to the glomerular epithelial cell proliferation (20).HIVAN is not the only established secondary cause of collapsing FSGS. Collapsing FSGS has been reported in the setting of Parvovirus B19 infection (21) and in patients with hemophagocytic syndrome (with or without underlying lymphoma) (22). Collapsing FSGS also may follow treatment with pamidronate (23), with 15 cases reported in the medical literature (23,24). In contrast, FSGS NOS has been reported to result from treatment with lithium (25), sirolimus (26), and more recently anabolic steroids (27). Although rare cases of collapsing FSGS also have been reported after treatment with IFN-α (28–30), this therapeutic agent is more commonly associated with minimal-change disease (MCD) (31–38) and FSGS NOS (39–47). We report 11 additional cases of collapsing FSGS that developed during treatment with IFN, including six IFN-α (for hepatitis C virus [HCV] infection or melanoma), three IFN-β (for multiple sclerosis [MS]), and two IFN-γ (for idiopathic pulmonary fibrosis).     

Potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding

This study describes the isolation and characterization of a neutralizing monoclonal antibody (mAb) against anthrax edema factor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic AMP (cAMP) responses in cells and protected mice from both ET-induced footpad edema and systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. The mAb was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. EF-mAb binding affinity (0.05–0.12 nM) was 50- to 130-fold higher than that reported for EF-CaM. This anti-EF neutralizing mAb could potentially be used alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection.Infection by inhalational anthrax is often fatal if treatment is delayed. Anthrax bacteria can be killed by vigorous treatment with antibiotics, but patients may still die because the lethality of anthrax is largely because of the action of toxins (1). Anti-toxin neutralizing monoclonal antibodies (mAbs) are the only viable choice for immediate neutralization of toxin and they could augment the effectiveness of antibiotics.Anthrax bacteria produce 3 toxin components: Protective antigen (PA), lethal factor (LF), and edema factor (EF) (2, 3). PA binds to cellular receptors and acts as a vehicle to deliver LF or EF into the cytosol where they exert their enzymatic activities (4–8). LF is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinases and causes lysis of macrophages (9, 10). EF is a calcium-calmodulin (CaM)-dependent adenylate cyclase and causes local inflammation and edema (11). The combination of PA with LF results in lethal toxin (LT). LT can replicate symptoms of anthrax disease when injected into animals (12). PA together with EF forms edema toxin (ET) and ET can produce a range of toxic effects in the host (11, 13).PA has been regarded as the most important target for prophylaxis and therapy of anthrax, because PA is common to both LTs and ETs, initiates the toxic process via receptor binding, and is highly immunogenic. In fact, PA is the major component in the current anthrax vaccine and the target for most of the available human or human-like neutralizing mAbs that have been shown to be very effective in protection against anthrax toxin or spore challenge (14–19). However, there is evidence that LF and EF may play important roles in providing protective immunity (20–22). Furthermore, concerns that PA could potentially be manipulated, such that it would no longer be neutralized by current anti-PA neutralizing mAbs have led to interest in therapeutics against the other 2 toxin components. A mixture of mAbs that recognize distinct epitopes on multiple toxin components (PA, LF, or EF) would not only enhance the protective efficacy but also broaden the spectrum of protection. Thus, in recent years, several anti-LF mAbs have also been reported (23–27). However, no anti-EF neutralizing mAbs have been reported to date. A previous report had indicated that immunization with the PA-binding N-terminal domain of EF (amino acids 1–254) resulted in polyclonal sera containing both EF and LF neutralizing activities (28).The purpose of this study was to determine (i) if anti-EF neutralizing mAbs could be isolated; (ii) the effectiveness of such antibodies against anthrax ET effects; and (iii) the neutralization mechanism of these antibodies. We have made a Fab combinatorial phage display library from chimpanzees that were immunized with anthrax toxins (17). From the library, 4 EF-specific Fabs were recovered, and 1 of them had potent neutralizing activity independent of the homologous PA-binding N-terminal (1–254) domain of LF. In this report, we describe the detailed characterization of these anti-EF clones.     

Pharmacokinetics and Pharmacodynamics of Intensified versus Standard Dosing of Mycophenolate Sodium in Renal Transplant Patients

Background and objectives: Adequate early mycophenolic acid (MPA) exposure is an important determinant for effective rejection prophylaxis. This pharmacokinetic study investigated whether an intensified dosing regimen of enteric-coated mycophenolate sodium (EC-MPS) could achieve higher mycophenolic acid (MPA) exposure early after transplantation versus a standard dosing regimen.Design, setting, participants, & measurements: De novo kidney transplant recipients (n = 75) who were treated with basiliximab induction and cyclosporine were randomly assigned to receive EC-MPS as either standard dosing (1440 mg/d; n = 37) or intensified dosing (days 0 through 14: 2880 mg/d; days 15 through 42: 2160 mg/d; followed by 1440 mg/d; n = 38). Full 12-hour pharmacokinetic and pharmacodynamic profiles were taken at six time points during the first 3 months. Exploratory analysis of inosine monophosphate dehydrogenase (IMPDH) activity was also performed for better understanding of the pharmacokinetic–pharmacodynamic relationship between MPA exposure and IMPDH activity in the early posttransplantation period. Preliminary efficacy parameters, safety, and tolerability were assessed.Results: Exposure to MPA was significantly higher on days 3 and 10 after transplantation in the intensified versus standard EC-MPS group, with 52.9 versus 22.2% (P < 0.05) of patients reaching MPA exposure >40 mg/h per L in the first week. The intensified regimen resulted in significantly lower IMPDH activity on day 3 after transplantation, and the overall safety was comparable for both groups.Conclusions: These pharmacokinetic and safety data support further research on the hypothesis that early adequate MPA exposure could improve clinical outcome.The combination of mycophenolic acid (MPA), given as mycophenolate mofetil (MMF) or enteric-coated mycophenolate sodium (EC-MPS), with steroids and calcineurin inhibitors (either cyclosporine A [CsA] or tacrolimus) has become standard immunosuppressive therapy worldwide. MMF and EC-MPS have a similar efficacy and safety profile (1,2) but differ in their pharmacokinetic characteristics (3).A large number of retrospective and prospective studies support the hypothesis that adequate early MPA exposure is an important determinant for effective rejection prophylaxis (4–13). Whereas the majority of tacrolimus-treated patients achieve adequate MPA exposure early after transplantation (13,14), studies have demonstrated that approximately 50% of patients who are treated with CsA and standard MPA dosages are underexposed (4,7,12,13). Larger initial MMF dosages (up to 4 g/d) have been suggested early after transplantation for achievement of sufficient MPA exposure in combination with CsA (13,15,16).There are only limited data on the pharmacokinetics, safety, and efficacy of higher (>3 g/d) MMF dosages (4,5,17), and data on higher EC-MPS dosages are lacking. The aim of this pilot study was to investigate the feasibility and safety of achieving target MPA exposure levels (≥40 mg/h per L), measured as area under time-concentration curve (AUC), using an intensified EC-MPS dosing regimen, compared with a standard dosing regimen, in de novo CsA-treated renal transplant patients. In addition, an exploratory analysis of inosine-monophosphate dehydrogenase (IMPDH) activity was performed for better understanding of the pharmacokinetic–pharmacodynamic relationship between MPA exposure and IMPDH activity early after transplantation.     

Reactogenicity to a live attenuated varicella vaccine in Canadian children

OBJECTIVE:

To assess the reactogenicity and safety of a thermostable, high titre, varicella vaccine in healthy infants and children.

DESIGN:

Open study of 505 children monitored for 42 days after vaccination.

SETTING:

Three urban Canadian centres (Halifax, Ottawa and Vancouver).

PARTICIPANTS:

505 healthy children one to 12 years of age were enrolled and 504 completed the study. All were susceptible to varicella by history.

INTERVENTIONS:

All participants received one dose of live attenuated varicella vaccine (1x10^4.5 plaque forming units/dose) subcutaneously.

MAIN OUTCOME MEASURES:

The children were monitored from the day of vaccine administration (day 0) until day 42. All local and general symptoms and signs were recorded on diary cards by the patients'' parents, who were encouraged to fill in the cards on days 2 to 3 and 18 to 24 via telephone calls from study personnel.

RESULTS:

Most of the symptoms noted after vaccine administration were mild and transient, and all resolved within the respective follow-up periods. Injection site symptoms included pain (17.5%, 13.9% and 30.4% in centres 1, 2 and 3 respectively), redness (21.1%, 32.1% and 48.8%) and swelling (7%, 10.3% and 29.2%). The general symptoms reported were fever 37.5°C or higher (3.5%, 4.8% and 3.0%) and varicella-like rashes (6.4%, 2.4% and 0%). Two subjects had severe symptoms (one with cervical lymphadenopathy, and one with a fever higher than 39°C) probably related to vaccine administration. No serious adverse events were reported during the entire study.

CONCLUSION:

The vaccine was well tolerated.Key Words: Chickenpox, Reactogenicity, Varicella vaccineInfection by varicella zoster virus (VZV) usually results in benign disease in children. However, complications such as pneumonia, encephalitis and bacterial superinfection of the skin lesions (1,2) occur in some patients, mainly in adolescents and adults, and in immunocompromised children (1,2). In addition, children born to nonimmune mothers who contract varicella during pregnancy can develop congenital varicella syndrome (1,3), with limb hypoplasia and central nervous system damage.A live varicella vaccine was developed in Japan in 1974 (4) using the OKA strain of the virus. The original wild type virus was isolated from a boy with natural varicella, and then attenuated by passages through human and guinea pig embryonic cells, and two human diploid cell lines, WI-38 and MRC-5 (4,5). The vaccine strain obtained after this treatment has different thermosensitivity and host range spectrum than the wild type virus (6). Additionally, it can be easily differentiated from wild type strains currently circulating in North America by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) testing (6,7).All OKA varicella vaccine production lots are derived directly from a working seed lot (8), following the World Health Organization guidelines (9). Many clinical trials have demonstrated that this strain is safe and immunogenic (2,5,10,11). The vaccine produced by SmithKline Beecham Biologicals (Rixensart, Belgium), VARILRIX, was first licensed in Europe in 1984 (10,11). An OKA-strain varicella vaccine, Oka/Biken (Biken, Osaka, Japan), has been licensed in Japan since 1987 (5). Vaccines using the same strain, were licensed in the United States in 1995 (Oka/Merck or VARIVAX, Merck and Company Inc, Whitehouse Station, New Jersey [12]) and in France in 1997 (Pasteur Merieux, Lyon, France).SmithKline Beecham Biologicals introduced recently a reformulation of the vaccine to increase its stability at 2 to 8°C and, therefore, facilitate its use for general vaccination (10). The vaccine will retain a titre of 1x10^3.3 plaque forming units (PFU) or more/dose after two years at 2 to 8°C. The safety and immunogenicity of this vaccine have been extensively tested (10,11).We conducted a multicentre study to assess the reactogenicity and safety of two lots of the reformulated varicella zoster vaccine produced by SmithKline Beecham Biologicals in children from one to 12 years of age. This vaccine has a higher titre (approximately 1x10^4.5 PFU per dose) of virus at release and is more thermostable than the vaccine presently licensed in Canada. The purpose of the study was to obtain daily information on local and general reactogenicity to the vaccine. Such information is required by licensing agencies before a vaccine is made available to the public.     

A state-mutating genetic algorithm to design ion-channel models

Realistic computational models of single neurons require component ion channels that reproduce experimental findings. Here, a topology-mutating genetic algorithm that searches for the best state diagram and transition-rate parameters to model macroscopic ion-channel behavior is described. Important features of the algorithm include a topology-altering strategy, automatic satisfaction of equilibrium constraints (microscopic reversibility), and multiple-protocol fitting using sequential goal programming rather than explicit weighting. Application of this genetic algorithm to design a sodium-channel model exhibiting both fast and prolonged inactivation yields a six-state model that produces realistic activity-dependent attenuation of action-potential backpropagation in current-clamp simulations of a CA1 pyramidal neuron.The importance of modeling ion channels has been understood since Hodgkin and Huxley''s seminal work with the squid giant axon (1). Subsequently, the development of the patch-clamp method (2) enabled the characterization of the properties of a wide range of channels, and intensive efforts followed to produce quantitative models that could predict and explain specific ion-channel behavior (3–7). Such efforts have led to two broad classes of models: those describing single-channel and gating currents, and those describing macroscopic currents.Models of single-channel and gating currents can be used to analyze properties such as open-channel probabilities, dwell times, and activation kinetics; they therefore facilitate an improved understanding of channel biophysics (3–5, 7–9). By contrast, models of macroscopic currents are usually intended as empirical tools as part of larger compartmental models of neurons. Such a macroscopic model may not necessarily describe the actual molecular state changes of the channel; the goal is rather for it to function as a “black-box” that reproduces the mean behavior of a population of channels. Hodgkin and Huxley''s (1) original formulation of sodium- and potassium-channel models is a prime example of this case, and its basic formalism continues to be widely used to generate empirical ion-channel models.An alternative to the Hodgkin–Huxley formalism is the state-dependent modeling approach (3). In reality, state-dependent models subsume Hodgkin–Huxley models because the latter can be recast as the former (3). State-dependent models are more general, however, because they can describe certain behaviors more easily than Hodgkin–Huxley type models, such as having widely different transition rates into and out of a given state (10). In general, the gold standard for the use of state-dependent models is single-channel recording (3, 5, 11), but the state-dependent formalism is also often employed in models of macroscopic currents (12–15) because of the generality and flexibility it affords.Methods to make empirical state-dependent models conform to data have been studied extensively and have involved a multitude of techniques such as hand fitting (12–14, 16), principal-axis fitting (17), maximum-likelihood estimation (5, 7, 17, 18), and genetic algorithms (15), among others. Here, we present a new fitting technique based on a topology-mutating genetic algorithm. Genetic algorithms have a number of useful characteristics: First, they have been shown to explore a large area of parameter space with relatively quick convergence, especially for problems with many parameters (19). Second, they are easily parallelizable. Third, they have been successfully applied to neuronal modeling, both for Hodgkin–Huxley-type ion-channel parameters and for compartmental neuronal models with voltage-activated conductances (15). Here we show that if the ion-channel model is formulated properly, genetic algorithms provide a natural way to incorporate changes in model topology as mutations.The algorithm presented here has several key features. Most notably, whereas other published optimization algorithms fix the model topology and optimize the rate constants, our algorithm searches over the space of model topologies and the space of rate parameters simultaneously. In order to design such an algorithm appropriate for state-dependent ion-channel models, we formulated an automated, computationally efficient method to satisfy the principle of microscopic reversibility, an equilibrium condition that imposes constraints on topologies with loops (20, 21). Finally, our algorithm uses a sequential approach, also known as goal programming (22), to optimize multiple protocols without the need to assign weights to each of the objective functions. The ability of this genetic algorithm to select and examine topologies not previously explored demonstrates its flexibility in developing working empirical models.We applied this genetic algorithm to devise a sodium-channel model that exhibits both fast and slow inactivation. Fast inactivation refers to a nonconducting channel state that follows quickly after depolarization and activation (within milliseconds) and from which channels recover quickly when the voltage is restored to resting levels (1). In response to either sustained depolarization (23, 24) or a train of depolarizing pulses (16, 25), however, the fraction of sodium channels available for activation also decreases rapidly, but in this case recovery occurs much more slowly, on the order of seconds rather than milliseconds. This form of inactivation has therefore been called “prolonged” or “slow” (16, 25). The presence of such widely disparate time scales makes the creation of state-dependent models of these channels a challenge. At the same time, the effect of prolonged inactivation on processes such as action-potential backpropagation (26, 27), transitions from bursting to spiking (28), and dendritic spiking (29, 30) makes the development of accurate models of prolonged inactivation important for computational simulations of neuronal function.     

Hypophosphatemic Effect of Niacin in Patients without Renal Failure: A Randomized Trial

Background and objectives: Niacin administration lowers the marked hyperphosphatemia that is characteristic of renal failure. We examined whether niacin administration also reduces serum phosphorus concentrations in patients who have dyslipidemia and are free of advanced renal disease.Design, setting, participants, & measurements: We performed a post hoc data analysis of serum phosphorus concentrations that had been determined serially (at baseline and weeks 4, 8, 12, 18, and 24) among 1547 patients who had dyslipidemia and were randomly assigned in a 3:2:1 ratio to treatment with extended release niacin (ERN; 1 g/d for 4 weeks and dose advanced to 2 g/d for 20 weeks) combined with the selective prostaglandin D2 receptor subtype 1 inhibitor laropiprant (L; n = 761), ERN alone (n = 518), or placebo (n = 268).Results: Repeated measures analysis revealed that ERN-L treatment resulted in a net mean (95% confidence interval) serum phosphorus change comparing ERN-L with placebo treatment of −0.13 mmol/L (−0.15 to −0.13 mmol/L; −0.41 mg/dl [−0.46 to −0.37 mg/dl]). These results were consistent across the subgroups defined by estimated GFR of <60 or ≥60 ml/min per 1.73 m², a serum phosphorus of >1.13 mmol/L (3.5 mg/dl) versus ≤1.13 mmol/L (3.5 mg/dl), the presence of clinical diabetes, or concomitant statin use.Conclusions: We have provided definitive evidence that once-daily ERN-L treatment causes a sustained 0.13-mmol/L (0.4-mg/dl) reduction in serum phosphorus concentrations, approximately 10% from baseline, which is unaffected by estimated GFR ranging from 30 to ≥90 ml/min per 1.73 m² (i.e., stages 1 through 3 chronic kidney disease).Abnormalities in calcium-phosphorus homeostasis, including significant elevations in serum phosphorus concentrations, are thought to contribute to arterial stiffening, hypertension, and cardiovascular disease (CVD) risk in patients with advanced chronic kidney disease and ESRD that requires maintenance dialysis (1–6). Observational data from population-based studies suggested that even serum phosphorus concentrations within the normative range are linearly associated with measures of subclinical arteriosclerosis and the development of incident CVD outcomes (7–12). Two cross-sectional studies from patients who underwent cardiac catheterization have further indicated that serum phosphorus concentrations, primarily within the normative range, were directly associated with both the presence and the severity of angiographic coronary artery disease (13,14). Moreover, a graded, independent association between serum phosphorus concentrations (again, within the normative range) and recurrent CVD events was reported among a large clinical trial cohort of patients with a previous myocardial infarction (15).Supplementation of calcium salts, despite their efficacy and tolerability as a phosphorus-lowering treatment in ESRD, may enhance coronary artery and aortic valve calcification (16,17). This observation highlights the need for hyperphosphatemia treatment protocols to balance potential benefits and adverse effects (18–22). Phosphorus-lowering drugs that target other cardiovascular risk factors in chronic kidney disease (CKD), simultaneously, including, for example, dyslipidemia (23), might have additive or synergistic benefits. These findings may also be relevant to populations with less advanced CKD or normal renal function.Preliminary studies suggested that niacin administration (as niacinamide, niceritrol, or nicotinic acid) could be a useful primary or adjunctive treatment for the marked hyperphosphatemia that is characteristic of ESRD (24–30). Several reports from clinical trials of extended-release niacin (ERN) that was given to patients who had dyslipidemia and were free of clinical renal disease and hyperphosphatemia have contained limited additional data noting up to 10% reductions in the serum phosphorus concentrations of actively treated patients (31–34). These repeated clinical observations (24–34) are most plausibly explained by the direct inhibitory effect of niacin compounds on active transport-mediated phosphorus absorption in the mammalian small intestine (35–39).Published studies of patient populations who had dyslipidemia and were receiving ERN that included phosphorus data may have failed to provide information on baseline phosphorus values (33,34), and none (31–34) performed repeated measures analyses to examine the potential effects of niacin treatment on serum phosphorus and calcium concentrations, as well as the calcium-phosphorus products.Focused reexamination of the large, placebo-controlled clinical trial data set assembled by Maccubbin et al. (34) afforded us a unique opportunity to elucidate these and other unresolved issues regarding the impact of niacin given as the fixed-dose combination of ERN and laropiprant (ERN-L), a selective prostaglandin D2 receptor subtype 1 inhibitor that reduces niacin-induced flushing (34) or ERN alone on serum phosphorus and calcium concentrations and calcium-phosphorus products. We further evaluated whether there was evidence for significant effect modification by estimated GFR (eGFR), baseline serum phosphorus concentration, the presence of diabetes, or concurrent hepatic hydroxymethyl glutaryl–CoA reductase inhibitor (statin) use when assessing the potential impact of niacin on these routine clinical measures of calcium-phosphorus homeostasis.     

Phase 1 Study of Anti-CTGF Monoclonal Antibody in Patients with Diabetes and Microalbuminuria

Background and objectives: This report summarizes the first phase 1 trial treating patients with microalbuminuric diabetic kidney disease (DKD) using FG-3019, a human monoclonal antibody to connective tissue growth factor (CTGF). CTGF is critically involved in processes of progressive fibrosis, including DKD. This phase 1, open-label, dose-escalation trial evaluated safety, pharmacokinetics, and possible therapeutic effects of FG-3019 on albuminuria, proteinuria, and tubular proteins.Design, setting, participants, and measurements: Microalbuminuric subjects (n = 24) with type 2 (79%) or type 1 (21%) diabetes received 3 or 10 mg/kg FG-3019 dosed intravenously every 14 days for four doses. Albuminuria and safety follow-up were to days 62 and 365, respectively.Results: No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg than at 3 mg/kg, suggesting a saturable elimination pathway. Although this study was not designed for efficacy testing, it was notable that urinary albumin/creatinine ratio (ACR) decreased significantly from mean pretreatment ACR of 48 mg/g to mean post-treatment (day 56) ACR of 20 mg/g (P = 0.027) without evidence for a dose-response relationship.Conclusions: Treatment of microalbuminuric DKD subjects using FG-3019 was well tolerated and associated with a decrease in albuminuria. The data demonstrate a saturable pathway for drug elimination, minimal infusion adverse events, and no significant drug-attributable adverse effects over the year of follow-up. Changes in albuminuria were promising but require validation in a prospective, randomized, blinded study.Patients with diabetic kidney disease (DKD) are at increased risk for cardiovascular complications and early mortality. Those who survive long enough tend to progress to ESRD requiring dialysis or transplantation. Although advances in therapy with angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor type II blockers (ARBs) have attenuated the incident rate of ESRD (1), disease progression remains common (2–4) and diabetes continues to be the leading cause for initiation of dialysis in the United States (1).Connective tissue growth factor (CTGF) is a 349-amino-acid secreted pleiotropic protein belonging to the cysteine-rich CCN (CTGF/Cyr61/Cef10/NOVH) family. Numerous glomerular, tubulointerstitial, and vascular cells types can produce CTGF, and many factors associated with the diabetic condition can stimulate CTGF expression, including hypertension, hyperglycemia, and hyperlipidemia (5–24).CTGF is a critical mediator of extracellular matrix accumulation and coordinates a final common pathway of fibrosis (5,25,26). CTGF has been shown to amplify the fibrogenic activity of TGFβ (27) and IGF-1 (17) and to inhibit the action of antifibrotic and regenerative factors bone morphogenic protein-7 (27,28) and vascular endothelial growth factor (29,30).In type 1 diabetes, plasma and urine CTGF levels correlate with the level of albuminuria and the stage of progressive renal insufficiency (31–34), and the plasma CTGF level is an independent predictor of vascular disease as assessed by intimal medial thickness (35) and of mortality and progression to ESRD (36). In renal biopsy specimens from patients with diabetes, elevated levels of CTGF mRNA are associated with chronic tubulointerstitial damage, albuminuria, and progression of renal insufficiency (37–39).FG-3019 is a recombinant human anti-CTGF monoclonal IgG₁ antibody that has shown activity in rodent models of kidney dysfunction associated with type 1 and 2 diabetes (40–42). Here, we report results of an open-label dose-escalation trial of FG-3019 infusions administered biweekly over 56 days in patients with DKD, the first study designed to evaluate safety and potential therapeutic effect of FG-3019 in this patient population.