Potential capsule switching from serogroup Y to B: The characterization of three such Neisseria meningitidis isolates causing invasive meningococcal disease in Canada

Three group B Neisseria meningitidis isolates, recovered from meningococcal disease cases in Canada and typed as B:2c:P1.5, were characterized. Multilocus sequence typing showed that all three isolates were related because of an identical sequence type (ST) 573. Isolates typed as 2c:P1.5 are common in serogroup Y meningococci but rare in isolates from serogroups B or C. Although no serogroup Y isolates have been typed as ST-573, eight isolates showed five to six housekeeping gene alleles that were identical to that of ST-573. This suggested that the B:2c:P1.5 isolates may have originated from serogroup Y organisms, possibly by capsule switching.Key Words: Capsule switching, Neisseria meningitidis, Serogroup YNeisseria meningitidis is a significant pathogen that causes invasive meningococcal disease (IMD). The average case fatality rate of 9% to 12% remains high despite the availability of effective antibiotics and vaccines (1). Laboratory study and surveillance of N meningitidis involves the characterization of a number of surface markers of the bacterium, including its capsule and outer membrane proteins (OMPs). Most epidemiological studies of meningococcal disease rely on differentiating meningococcal isolates based on their serogroup, serotype and serosubtype. Serogrouping is determined by the demonstration of serologically distinct epitopes present on chemically and structurally different capsules. Serotyping and serosubtyping rely on the detection of distinct epitopes present on three of five different classes of OMPs of N meningitidis. Serotyping epitopes are found on the class 2 or class 3 OMP (also called PorB) of N meningitidis; these OMPs are expressed in a mutually exclusively manner (ie, a strain will only express either a class 2 or class 3 OMP but not both). Serosubtyping epitopes are present on the class 1 OMP (also called PorA). Based on this nomenclature scheme, a strain can therefore be characterized by its antigenic formula; for example, B:15:P1.7,16 refers to serogroup B, serotype 15 and serosubtype P1.7,16.One of the most important virulence factors of meningococci is the capsular polysaccharide antigen, which is also the basis for serogrouping and is the target antigen for the currently licensed vaccines against A, C, Y and W135 organisms. Of the 13 known serogroups, five (serogroups A, B, C, Y and W135) are responsible for most of the meningococcal disease worldwide (2). In North America, most endemic and epidemic strains belong to serogroups B, C, Y and W135 (3,4). Capsules of serogroups B, C, Y and W135 meningococci contain sialic acid, either as a homopolymer of sialic acids assembled by alpha-2,8 linkages (serogroup B) or alpha-2,9 linkages (serogroup C), or as a heteropolymer of sialic acids with glucose (serogroup Y) or galactose (serogroup W135). Besides demonstrating structural similarities, these four serogroups of meningococci also have very similar capsule polysaccharide synthesis (cps) gene loci (5). Because of this similarity, capsule switching has been demonstrated in vivo and in vitro by specific gene replacement within the cps loci between different serogroups. To date, a number of IMD cases have been described in the literature to be caused by organisms in which capsule switching between serogroup B and C meningococci occurred (6-8).In the present paper, the authors describe three unusual serogroup B meningococci isolated from separate IMD cases in Nanaimo, British Columbia, that presented with the OMP antigens 2c:P1.5, characteristic of serogroup Y strains found in Canada (4). This antigenic profile prompted the authors to examine the relationship of these three serogroup B strains with antigenically similar serogroup Y organisms isolated in Canada. The authors describe the characterization of these antigenically similar isolates and postulate that the B:2c:P1.5 isolates arose by capsule switching from serogroup Y organisms.     

Risk and prevention of meningococcal disease among education workers: A review

The aims of the present study were to review the risk of invasive meningococcal disease (IMD) among education workers, particularly pregnant women, and to evaluate preventive measures, in a context of endemicity, outbreak or epidemic as observed in the province of Quebec. The literature was reviewed and persons in charge of IMD surveillance in France, Quebec, the United Kingdom and the United States were interviewed. Surveys of asymptomatic carriage of Neisseria meningitidis show that transmission among students is higher than transmission between students and teachers. IMD incidence among education workers was analyzed in Cheshire (United Kingdom) in the period from 1997 to 1999, and the results indicated a risk six times higher than that in the general population. Overestimation of the magnitude of the risk is possible because the analysis focused on a cluster. None of the population-based studies of IMD mentioned a risk of secondary cases among education workers. Six IMD cases in education workers were identified in five clusters in schools in the United Kingdom, but not in the other countries. There is no epidemiological study on IMD risk among pregnant women, and this factor was not mentioned in any published review of IMD. Immunization of education workers at the beginning of their employment, using serogroup C glycoconjugate vaccine or a combined A, C, W-135, and Y conjugate vaccine (still under development), could reduce IMD risk, but the cost effectiveness of this measure should be evaluated. The societal benefit of excluding pregnant women from the work place during an outbreak seems to be very low, even if disease risk could be decreased for this specific group. When chemoprophylaxis is indicated for the control of an outbreak in an educational setting, treatment should be offered both to students and teachers in the group at risk.Key Words: Disease risk, Education, Neisseria meningitidis, Occupational health, PreventionAn epidemic of invasive meningococcal disease (IMD) caused by a virulent clone of serogroup C was observed in Quebec in the early 1990s, and resulted in a mass immunization campaign with the polysaccharide vaccine during the winter of 1992 to 1993 (1). A recrudescence was detected at the beginning of 2001 and it was decided to offer the new serogroup C conjugate meningococcal vaccine to all Quebec persons up to 20 years of age (2). In this epidemiological situation, characterized by numerous cases of IMD in day care centres and educational institutions, the question arose whether staff should be immunized and pregnant women removed from the work place. Quebec''s occupational health and safety legislation provides for the elimination of dangers to workers'' health and for protection of workers when necessary (3). In addition, there is a provision that allows for the protective reassignment of pregnant women when working conditions may be physically dangerous to them or their unborn child. The aims of the present study were to estimate the risk of IMD for workers in the education system, pregnant women in particular, and to evaluate the potential effectiveness and usefulness of various preventive measures including temporary exclusion from the work place, immunization and chemoprophylaxis.To address these questions, we did a critical review of the scientific literature using Medline and a series of key words (Neisseria meningitidis, meningococcal, school, college, nursery, university, cluster, outbreak). The personal archives of the authors covering the epidemiology of IMD during the past thirty years were also consulted. Finally, the persons in charge of IMD surveillance in the United States, France, Quebec and the United Kingdom were questioned to identify clusters involving staff in the education system.     

Legionnaires' disease - Results of a multicentre Canadian study

BACKGROUND:

There has never been a cross-Canada surveillance project to determine the rate of Legionella species as a cause of community-acquired pneumonia requiring hospitalization and to determine whether there are any regional differences in the rates of Legionnaires'' disease in Canada. Anecdotally, Legionnaires'' disease is thought to be uncommon in Western Canada.

METHODS:

From January, 1996 through to October 31, 1997, a prospective study of the etiology of community acquired pneumonia requiring admission to 15 tertiary care hospitals in eight Canadian provinces was conducted. A urine sample from each patient was tested for Legionella pneumophila serogroup 1 antigen using a commercially available ELISA assay. A culture of sputum or other respiratory specimens for Legionellaceae was carried out at the discretion of the attending physician. Two hundred thirty-four patients had acute and 6-week convalescent serum samples tested for antibodies to L pneumophila serogroups 1 through 6 using an ELISA method.

RESULTS:

28 of the 850 patients (3.2%) had Legionnaires'' disease; 18 of 823 (2.1%) were positive for L pneumophila serogroup 1 by urinary antigen testing. The rate of Legionnaires'' disease, based on urinary antigen, at the Halifax site was higher than that at the other sites (seven of 163 patients versus 11 of 660 [P=0.04]). Of the 28 cases of Legionnaires'' disease identified using all methods, 11 of 277 patients (3.9%) were enrolled from Western provinces versus 17 of 573 patients (2.9%) from Eastern provinces (P=nonsignificant).

CONCLUSIONS:

Legionnaires'' disease is just as common in Western as in Eastern Canada. L pneumophila serogroup 1 may be more common in Halifax than at the other sites studied.Key Words: Acquired pneumonia, Canada, Legionella, Legionella urine antigen, Legionnaires'' diseaseLegionnaires'' disease is an acute infectious disease of which the predominant manifestation is pneumonia (1). The most common cause is Legionella pneumophila serogroup 1, however, just under half of the over 40 recognized species in the Legionellaceae family can cause Legionnaires'' disease (2). A soluble antigen is present in the urine of patients with Legionnaires'' disease due to serogroup 1 (3-5). A commercially available enzyme immunoassay is available that detects this antigen with a specificity of 100% and a sensitivity of 94.6% (3). We used this test to determine the frequency with which L pneumophila serogroup 1 was a cause of community-acquired pneumonia (CAP) in Canada, and to determine whether there was any geographic clustering of the cases of Legionnaires'' disease. We tested a subset of the study population for Legionella species infection using a serological assay. Our objectives were to determine the rate of seroconversion among patients with a positive urinary antigen assay and to determine whether infection with serogroups 2 to 6 occurred in Canada.     

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.The Gram-negative encapsulated bacterium Neisseria meningitidis causes severe sepsis and meningococcal meningitis. Invasive meningococcal disease (IMD) is associated with 5–15% mortality; furthermore, devastating long-term sequelae such as amputations, hearing loss, and neurodevelopmental disabilities are observed in 11–19% of IMD survivors (1). Meningococcal serogroups are distinguished by the composition of their capsular polysaccharides. The five serogroups most commonly associated with invasive disease are A, B, C, W, and Y. (2). Effective mono- or polyvalent-conjugated polysaccharide vaccines against N. meningitidis serogroups A, C, W, and Y have been available since the early 1990s (3). However, serogroup B meningococcus (MenB) is responsible for the majority of endemic and epidemic meningococcal disease in developed countries (4–6). The development of an efficient capsular polysaccharide-based vaccine against MenB has been hampered by potential autoimmunity issues, namely, the structural similarity between the MenB capsular polysaccharide and the neuraminic acid present on the surface of human fetal neural tissues (7).In early 2013 the European Medicines Agency approved 4CMenB, to our knowledge the first broadly protective vaccine against MenB, for the prevention of IMD in all age groups. 4CMenB is a multicomponent vaccine formulation composed of three surface-exposed meningococcal proteins originally identified by the reverse vaccinology approach (8) plus outer membrane vesicles from the New Zealand epidemic clone. The three antigenic proteins are factor H-binding protein (fHbp), neisserial heparin-binding antigen (NHBA), and neisserial adhesin A (NadA) (9, 10).The gene encoding NadA is present in ∼30% of pathogenic meningococcal isolates and is associated mostly with strains that belong to three of the four hypervirulent serogroup B lineages (11–14). NadA expression levels can vary among isolates by more than 100-fold, and its expression is up-regulated in vivo by niche-specific signals (15). NadA induces high levels of bactericidal antibodies in humans (16–18) and is recognized by serum antibodies of children convalescent after IMD (19), suggesting that it is expressed and is immunogenic during IMD. Two main genetically distinct groups of NadA have been identified that share overall amino acid sequence identities of 45–50%. Group I includes the three most common variants (NadA1, NadA2, and NadA3, the latter being the vaccine variant), which share ∼95% sequence identity and are immunologically cross-reactive (11). Group II includes three rarer variants: NadA4, primarily associated with carriage strains (11); NadA5, found mainly in strains of clonal complex 213 (20, 21); and NadA6 (Fig. S1A); these three share ∼90% sequence identity (Fig. S1B) (22).Functionally, NadA3 expressed on the surface of Escherichia coli promotes adhesion to and invasion of Chang epithelial cells (23). This adhesive activity has been mapped, at least partially, to an N-terminal region extending to residue T132 (23, 24). Recently, interactions of NadA3 with β-1 integrin (25) and with the heat shock protein Hsp90 (26) have been reported.Structurally, NadA belongs to the class of trimeric autotransporter adhesins (TAAs) (27, 28), which are known to mediate adhesion through interaction with extracellular matrix proteins and are involved in invasion of target cells (29). TAAs are obligate homotrimers, and accordingly the recombinant NadA3 vaccine antigen, lacking the C-terminal membrane anchor region, forms soluble, stable trimers (23, 30). TAAs generally are made of a conserved C-terminal integral membrane β-barrel, which anchors the proteins to the outer membrane, and an N-terminal “passenger” domain responsible for adhesion (31). The TAA passenger domain typically is made of a central α-helical domain (stalk) that forms coiled-coil structures and a distinct N-terminal domain (head) that is mainly responsible for binding to host cellular receptors.Here we present the X-ray structure of a large ectodomain fragment of NadA5 and a structural analysis by transmission electron microscopy (TEM) of the vaccine variant NadA3. In addition, epitope mapping shows that the head of NadA3 contains immunogenic regions responsible for the generation of a protective bactericidal response.     

Epidemiology,antibiotic susceptibility,and serotype distribution of Streptococcus pneumoniae associated with invasive pneumococcal disease in British Columbia - A call to strengthen public health pneumococcal immunization programs

BACKGROUND:

This study examined the epidemiology, antibiotic susceptibility and serotype distribution of Streptococcus pneumoniae associated with invasive pneumococcal disease (IPD) in British Columbia.

METHODS:

Six hospitals and one private laboratory network participated in a prospective, sentinel laboratory based surveillance study of IPD, between October 1999 and October 2000. At each site, S pneumoniae isolates were collected and epidemiological data were gathered using a structured questionnaire, for all cases of IPD meeting the study case definition. Isolates were serotyped and tested for antimicrobial susceptibility. Bivariate associations were analyzed and multivariate logistic regression was used to identify independent risk factors associated with hospitalization or death.

RESULTS:

One hundred three reports and isolates were collected. Seventy-nine per cent of cases were community-acquired, 64% required hospitalization and 5% died. Cases with one or more assessed risk factor for IPD and of female sex were independent variables associated with hospitalization or death. One-third of isolates had reduced penicillin susceptibility and 96% of these represented serotypes contained in the 23-valent pneumococcal polysaccharide vaccine (PPV-23). Overall, 89% of serotypes identified are included in the PPV-23 vaccine and 88% of isolates from children under five years of age are found in the 7-valent pneumococcal conjugate vaccine (PCV-7). Forty-one per cent of cases qualified for publicly funded pneumococcal vaccine and 34% of eligible persons were vaccinated.

CONCLUSIONS:

Overall, pneumococcal serotypes associated with IPD in this study closely matched serotypes included in PPV-23 products currently licensed in Canada. Most serotypes associated with IPD in children under five years of age are included in a recently licenced PCV-7. One third of isolates demonstrated reduced penicillin susceptibility, most involving serotypes included in PPV-23. Effective delivery of current public health immunization programs using PPV-23 and extending protection to infants and young children using the PCV-7 will prevent many cases of IPD.Key Words: Antibiotic susceptibility, Immunization, Serotype, Streptococcus pneumoniaeStreptococcus pneumoniae (pneumococcus) is a leading cause of invasive bacterial infections, including septicemia and meningitis, as well as non-invasive infections such as community-acquired pneumonia and acute otitis media (1-3). The highest rates of invasive pneumococcal disease (IPD) are seen in children under two years of age, in whom it is currently the leading cause of invasive bacterial disease in Canada and the United States (3,4). IPD is also a leading cause of illness and death among the elderly and persons having underlying chronic medical conditions (1,2,5). Overall, IPD accounts for more deaths in Canada and the United States than any other vaccine-preventable bacterial disease (6,7).Data from the Canadian National Centre for Streptococcus indicate the proportion of invasive pneumococcal isolates with reduced penicillin susceptibility increased from 5.5% to 15.2% between 1992 and 2000 (8). The Canadian Bacterial Surveillance Network has documented a similar trend of decreasing susceptibility to penicillin and to other antibiotics among pneumococcal isolates recovered from invasive, respiratory and other sites, between 1988 and 2001 (9).The high population burden of IPD and adverse consequences of increasing antimicrobial resistance of pneumococcus causing IPD give new impetus to public health prevention programs that better exploit the potential benefits of pneumococcal immunization (1,2,10). Only within the past five years has British Columbia, along with other Canadian provinces and territories, begun to offer 23-valent pneumococcal polysaccharide vaccine (PPV-23) through public health immunization programs, according to recommendations of the National Advisory Committee on Immunization (NACI). NACI recommends immunization using PPV-23 for all persons 65 years of age and older, and for those two to 64 years of age with health conditions placing them at higher risk of IPD or its complications (2,5). Further benefits may accrue after provinces/territories introduce a 7-valent pneumococcal conjugate vaccine (PCV-7) that was licensed in Canada in June 2001, into routine universal infant immunization programs. This vaccine has demonstrated potential to decrease acute or recurrant otitis media associated with antibiotic-resistant pneumococcus; reduce carriage and spread of resistant pneumococci in community settings; and decrease antibiotic use (11).We report on a one year, prospective, sentinel laboratory-based study of IPD in British Columbia, Canada. The objectives were to characterize the epidemiology, antibiotic susceptibility and serotype distribution of S pneumoniae associated with IPD, and viewed in the context of existing and future public health prevention strategies.     

Correlation of Kidney Function,Volume and Imaging Findings,and PKHD1 Mutations in 73 Patients with Autosomal Recessive Polycystic Kidney Disease

Background and objectives: Renal function and imaging findings have not been comprehensively and prospectively characterized in a broad age range of patients with molecularly confirmed autosomal recessive polycystic kidney disease (ARPKD).Design, setting, participants, & measurements: Ninety potential ARPKD patients were examined at the National Institutes of Health Clinical Center. Seventy-three fulfilled clinical diagnostic criteria, had at least one PKHD1 mutation, and were prospectively evaluated using magnetic resonance imaging (MRI), high-resolution ultrasonography (HR-USG), and measures of glomerular and tubular function.Results: Among 31 perinatally symptomatic patients, 25% required renal replacement therapy by age 11 years; among 42 patients who became symptomatic beyond 1 month (nonperinatal), 25% required kidney transplantation by age 32 years. Creatinine clearance (CrCl) for nonperinatal patients (103 ± 54 ml/min/1.73 m²) was greater than for perinatal patients (62 ± 33) (P = 0.002). Corticomedullary involvement on HR-USG was associated with a significantly worse mean CrCl (61 ± 32) in comparison with medullary involvement only (131 ± 46) (P < 0.0001). Among children with enlarged kidneys, volume correlated inversely with function, although with wide variability. Severity of PKHD1 mutations did not determine kidney size or function. In 35% of patients with medullary-only abnormalities, standard ultrasound was normal and the pathology was detectable with HR-USG.Conclusions: In ARPKD, perinatal presentation and corticomedullary involvement are associated with faster progression of kidney disease. Mild ARPKD is best detected by HR-USG. Considerable variability occurs that is not explained by the type of PKHD1 mutation.Autosomal recessive polycystic kidney disease (ARPKD) occurs in 1 in 20,000 births and is the most common hepatorenal fibrocystic disease of childhood (1–7). It is caused by mutations in PKHD1, which encodes fibrocystin/polyductin (8,9), a protein localized to the primary cilium, an organelle functioning as the cell''s “sensory antenna” (10). Proteins defective in other diseases having fibrocystic pathology, such as autosomal dominant polycystic kidney disease, nephronophthisis, Bardet–Biedl, Meckel, and Joubert syndromes, also localize to the primary cilium; these disorders, along with ARPKD, comprise the “ciliopathies” (10–12).Individuals with ARPKD have nonobstructive fusiform dilations of the renal collecting ducts, leading to progressive renal insufficiency. All ARPKD patients manifest some degree of congenital hepatic fibrosis (CHF) caused by ductal plate malformation of the developing portobiliary system; some patients also have macroscopic dilations of the intrahepatic bile ducts, a combination termed Caroli''s syndrome (7,13,14). Portal hypertension complicates CHF and often results in esophageal varices and hypersplenism (15–18). Early-onset severe hypertension, often requiring multiagent therapy, occurs in most ARPKD patients (5).Most ARPKD patients present perinatally with oligohydramnios and massively enlarged, diffusely microcystic kidneys. Many such newborns subsequently succumb to pulmonary hypoplasia. Characterization of the clinical phenotype of ARPKD has been based primarily upon this subtype (i.e., perinatally symptomatic patients) (1,4,5). Documentation of the kidney disease in patients presenting late in childhood or adulthood has been more limited (3,19,20). In this paper, we detail the clinical, biochemical, imaging, and molecular characteristics of 73 children and adults with PKHD1 mutations and a spectrum of clinical presentations. Our data document the extent of renal glomerular and tubular dysfunction; correlate molecular, functional, and imaging findings; and provide prognostic information.     

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.Diphtheria is a contagious respiratory disease that was a major cause of death among children around the world until mass vaccination was introduced in the 1920s. Although diphtheria has now been virtually eliminated in the industrialized world, rare outbreaks still occur worldwide (1, 2). Corynebacterium diphtheriae was shown to be the causative agent of diphtheria by Loeffler in 1885 (3), and Roux and Yersin showed that an extracellular toxin, diphtheria toxin (DT) secreted by C. diphtheriae is responsible for toxicity (4). The formaldehyde-treated detoxified form of DT, diphtheria toxoid, has been successfully used for mass vaccination and is still widely used as a component of combination vaccines (5, 6).A major contribution to the understanding of the mode of action of DT was the discovery of mutated forms in the early 1970s (7). Several phages encoding mutants of DT, named cross-reactive materials (CRMs), were isolated following nitrosoguanidine-based mutagenesis of the phage containing the gene encoding DT. Being naturally nontoxic, CRMs were immediately recognized as having great potential for vaccine development. The most important CRM identified was CRM197, an enzymatically inactive and nontoxic form of DT that contains a single amino acid substitution from Glycine to Glutamate in position 52 (8). Subsequently CRM197 was found to be an ideal carrier for conjugate vaccines against encapsulated bacteria. Here, the carrier protein is covalently linked to poorly immunogenic and T-cell-independent capsular polysaccharides, thus creating T-cell-dependent conjugate antigens that are highly immunogenic in infants (9–11).Vaccines containing CRM197 as a carrier protein have been successfully used to immunize hundreds of millions of children. Such vaccines currently include Menveo®, a recently approved tetravalent conjugate vaccine against serogroups A-C-W135-Y of Neisseria meningitidis, Menjugate® and Meningitec® (against serotype C of N. meningitidis), Vaxem-Hib® and HibTITER® (against Haemophilus influenzae type B, Hib), and the multivalent pneumococcal conjugate Prevnar™ (12).The widespread use of diphtheria toxoid and CRM197 has prompted many investigations of DT and related proteins. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a proenzyme of 535 residues and is processed by trypsin-like proteases with release of two fragments (A and B). Fragment A uses NAD as a substrate, catalyzing the cleavage of the N-glycosidic bond between the nicotinamide ring and the N-ribose and mediating the covalent transfer of the ADP-ribose (ADPRT activity) to the modified Histidine 715 (diphthamide) of the elongation factor EF-2. This posttranslational diphthamide modification inactivates EF-2, halting protein synthesis and resulting in cell death. Extensive structural studies elucidated the molecular architecture of DT (13–19). The A fragment of DT (also named C domain) carries the catalytic active site and is the only fragment of the toxin required for the final step of intoxication, while the B fragment carries the R and T domains, which mediate binding to receptors on the host cell surface and promote the pH-dependent transfer of fragment A to the cytoplasm, respectively. An Arginine-rich disulfide-linked loop connects fragment A to fragment B (or domain C to domains TR), and this interchain disulfide bond is the only covalent link between the two fragments after proteolytic cleavage of the chain at position 186.While much progress has been made on the molecular characterization of DT over the last two decades, an understanding of the molecular basis for the lack of toxicity of CRM197 has so far been elusive. Here we present the crystal structures of full-length nucleotide-free (NF)-CRM197, and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA). In addition, the differences between CRM197 and DT were elucidated using surface plasmon resonance (SPR), a NAD-glycohydrolase (NADase) activity assay, molecular dynamics (MD) simulations, and differential scanning fluorimetry (DSF).     

Coxiella burnetii seroprevalence of shepherds and their flocks in the lower Saint-Lawrence River region of Quebec,Canada

OBJECTIVE:

To determine the seroprevalence of Coxiella burnetii among the shepherds and their sheep in the lower Saint-Lawrence River region (LSLRR) of Quebec, Canada.

DESIGN:

A prospective human-animal comparative study was conducted with 81 shepherds from 46 farms and a control group matched for sex and age. All participants answered a standardized questionnaire to evaluate their risk factors for Q fever, including a specific section on the work practices of the shepherds. All human subjects had a blood sample taken for serology to phase I and phase II antigens of C burnetii performed by indirect immunofluorescence assay. At each participating farm, seven to nine sheep had blood samples taken for C burnetii serology to be assessed by the complement fixation test.

RESULTS:

The seroprevalence to C burnetii was higher in the group of shepherds (28.4%) than the control group (1.2%) (P<0.005). Among the group of shepherds, spending more than 5 h/week in the sheep barn (P=0.06) and buying and/or trading sheep within the past six months (P=0.004) were associated with positive C burnetii serology. A total of 137 of 334 sheep (41%) were seropositive for C burnetii. These positive sheep were distributed in 41 of the 46 flocks (89%). No correlation could be demonstrated between a serology for C burnetii in the herds and the shepherds.

CONCLUSION:

Q fever is highly prevalent in the LSLRR of Quebec, affecting 89% of the flocks and 28% of the shepherds. Shepherds in this region are at increased risk for C burnetii infection in comparison to the general population.Key Words: Coxiella burnetii, Flock, Q fever, Quebec, Serology, Seroprevalence, Sheep, ShepherdQ fever is a worldwide zoonosis caused by an intracellular rickettsial agent, Coxiella burnetii. This bacteria shows a characteristic phase conversion: the virulent phase I is directly isolated from infected animals, and the avirulent phase II is obtained after successive passages on cellular cultures of embryonated eggs (1,2). Q fever is predominantly transmitted to humans by inhalation of contaminated aerosols from infected animal litters (3-5). The parturition products and the feces and urine of sheep, cats, goats and dogs are the more common sources of transmission of the bacteria (6-9). Although these animals rarely show any symptoms of the infection (10,11), abortions in sheep, goats and cattle can be seen in some cases (5,6,12-14).Human Q fever is often asymptomatic or manifests as a mild illness. Atypical pneumonia and hepatitis are the principal clinical manifestations of the acute symptomatic infection, while endocarditis is the most frequent presentation of chronic Q fever (1,3,15,16). Very few studies in North America have addressed the issues of prevention of Q fever. In Australia, a highly effective vaccine (Q-Vax, Commonwealth Serum Laboratories, USA) is available for workers with occupational exposition to C burnetii (17,18).Seroprevalence studies conducted in Canada have shown that 5% to 36% of blood donors (19,20), 20% of slaughterhouse workers of the Mauricie region of Quebec (12), 25% of personnel of the animal pathology laboratory in Rimouski in the lower Saint-Lawrence River region (LSLRR) (M Rochette, unpublished data) and 49% of veterinarians in Nova Scotia (21) were positive for C burnetii.Epizootiological studies have also been done on animal populations in Canada. A seroprevalence study in the Mauricie region of Quebec showed that C burnetii had infected 26.7% of the cats, 11.5% of the cattle and 7.7% of the sheep (22). Cats were reported as important vectors of transmission of C burnetii to humans in the Mauricie region of Quebec and in Nova Scotia (23,24). In Ontario, a seroprevalence study demonstrated that 21.3% of the sheep were positive for antibodies to C burnetii (25).Q fever is a notifiable disease in Quebec. From January 1990 to December 1998, 43 (36.1%) of 119 reported cases were from the LSLRR, which had the highest incidence rate of the province, although it represents only 5% of the population. A review of the cases of Q fever from the LSLRR between 1991 and 1999 showed that 67% of the Q fever cases were related to exposure to sheep (P Jutras, unpublished data). It is noteworthy that an important rise in the number of sheep was observed in LSLRR, with an increase from 11,000 animals distributed in 110 flocks in 1983, to more than 33,000 in 180 flocks in 1999 (Union des producteurs agricoles of Quebec, personal communication).To evaluate the association between Q fever and exposure to ovine in the LSLRR, a prospective human-animal seroprevalence study was undertaken to compare the seroprevalence of C burnetii between the shepherds, their flocks and the general population. The work practices of the shepherds were also evaluated.     

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.     

Genome-wide association analysis reveals a SOD1 mutation in canine degenerative myelopathy that resembles amyotrophic lateral sclerosis

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.Amyotrophic lateral sclerosis (ALS) refers to a heterogeneous group of adult onset human diseases, in which progressive neurodegeneration affecting both the upper and lower motor neuron systems causes advancing weakness and muscle atrophy, and culminates in paralysis and death. Approximately 5 to 10% of ALS cases are familial; the rest appear to be sporadic (1–3). Mutations in SOD1 account for ≈20% of the familial ALS cases and 1 to 5% of the cases of sporadic ALS (1–4); >120 different SOD1 mutations have been identified in ALS patients (http://alsod.iop.kcl.ac.uk/Als/index.aspx). Elucidation of mechanisms underlying ALS has been hampered by a paucity of biological material from affected individuals in early stages of the disease (5). To our knowledge, there are no previous reports of spontaneously occurring animal models of ALS. Thus, ALS research has relied heavily on transgenic rodents expressing mutant human SOD1 (hSOD1^m) to produce a motor neuron disease, which recapitulates many features of ALS (5–7). In contrast, nullizygous SOD1 knockout mice develop normally (8), suggesting that the neurodegeneration in hSOD1^m mice and in ALS patients results from a toxic gain of function (1, 5–8). Although the nature of the toxin is unclear, several experiments suggest that the neurodegeneration occurs because conformational changes in the mutant superoxide dismutase 1 protein (SOD1) alter the biological activity and/or promote the formation of intracellular SOD1 aggregates (1, 4, 9, 10).Canine degenerative myelopathy (DM) has been recognized for >35 years as a spontaneously occurring, adult-onset spinal cord disorder of dogs (11). When pelvic limb hyporeflexia and nerve root involvement were observed, the disease was termed chronic degenerative radiculomyelopathy (12). Initially thought to be specific to German Shepherds, it has also been called German Shepherd dog myelopathy (13). Since these early reports, DM has been diagnosed in several other breeds. The disease is common in certain breeds including the Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, and Chesapeake Bay retriever (14).With DM, there is no sex predilection. Most dogs are at least 8 years old before the onset of clinical signs (11–18). The initial clinical sign is a spastic and general proprioceptive ataxia in the pelvic limbs. At this stage of the disease, the presence of spinal reflexes indicates an upper motor neuron paresis (11). The asymmetric weakness frequently reported at disease onset progresses to paraplegia (11, 12, 14, 16, 18). Hyporeflexia of the myotatic and withdrawal reflexes occur in the latter disease stage (11, 12, 14, 16, 18). The disease duration can exceed 3 years; however, dog owners usually elect euthanasia within a year of diagnosis when their dogs become paraplegic. If the disease is allowed to progress, clinical signs will ascend to affect the thoracic limbs (11, 14, 16). Because various common acquired compressive spinal cord diseases can mimic DM by compromising the upper motor neuron and general proprioceptive pathways, a definitive diagnosis of DM can only be accomplished postmortem by the histopathologic observation of axonal and myelin degeneration, which can occur at all levels of the spinal cord (16–18) and in all spinal cord funiculi, but are consistently most severe in the dorsal portion of the lateral funiculus within the middle to caudal thoracic region (11, 13–18).     

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.     

The management of infection and colonization due to methicillin-resistant Staphylococcus aureus: A CIDS/CAMM position paper

Methicillin-resistant Staphylococcus aureus (MRSA) is being seen with greater frequency in most hospitals and other health care facilities across Canada. The organism may cause life-threatening infections and has been associated with institutional outbreaks. Several studies have confirmed that MRSA infection is associated with increased morbidity and mortality compared with infections caused by susceptible strains, even when the presence of comorbidities is accounted for. Treatment of MRSA infection is complicated by the fact that these organisms are resistant to multiple antimicrobial agents, so treatment options are limited. The effectiveness of decolonization therapy (attempting to eradicate MRSA carriage) is also uncertain. This paper reviews the medical management of MRSA infections, discusses the potential role of decolonization and provides an overview of evidence to support recommended infection control practices.Key Words: Methicillin resistance, MRSA, Staphylococcus aureusThe past few decades have witnessed the emergence of methicillin-resistant Staphylococcus aureus (MRSA) as a major hospital-acquired pathogen worldwide (1-4). Although MRSA was first reported in Canada in 1981 (5), MRSA rates in Canadian hospitals have only increased substantially in the last few years. The Canadian Nosocomial Infection Surveillance Program (CNISP) reported that the incidence of MRSA in sentinel hospitals across the country increased from a mean of 0.9 per 100 S aureus isolates in 1995 to 8.2 per 100 isolates in 2001, and from 0.5 cases per 1000 admissions in 1995 to 4.4 per 1000 admissions in 2001 (6,7). Part of this increase may have been related to more frequent screening for MRSA colonization in high risk patients (8). However, a fourfold increase in MRSA infection rates was also observed (from 0.3 infections per 1000 admissions in 1995 to 1.2 infections per 1000 admissions in 2001) (6,7)Although there have been recent reports describing community-onset MRSA in the United States, CNISP data would suggest that MRSA remains predominantly a hospital-acquired pathogen in Canada (6). Nevertheless, it would seem reasonable to expect that an increase in MRSA rates in hospitals will eventually lead to spread of the organism in long term care facilities and the community. In Canada, community-acquired MRSA has been reported most frequently in western Canada, especially among native Aboriginals and intravenous drug users (9,10). Recognized risk factors for MRSA acquisition have included previous hospitalization, admission to an intensive care unit, prolonged hospital stay, proximity to another patient with MRSA, older age, invasive procedures, presence of wounds or skin lesions, and prior antimicrobial therapy (11-15).If MRSA only colonized patients, there would be little reason for concern. However, 20% to 60% of patients identified as being colonized with MRSA in hospital subsequently develop an MRSA infection (12). Using standard criteria for identification of infections, CNISP data indicated that approximately 31% of patients with MRSA in Canadian hospitals were infected (7). In certain high risk populations, staphylococcal infections including bacteremia occur more frequently following colonization with MRSA than after colonization with susceptible strains of S aureus (16). Moreover, MRSA does not merely replace susceptible strains of S aureus as a hospital-acquired pathogen, but rather, it appears to add substantially to the total burden of nosocomial infections (17,18). Although the results are somewhat controversial, several studies have also indicated increased mortality and prolonged hospitalization associated with MRSA infections (19-21). After adjustment for comorbidities, methicillin resistance has been found to be a significant independent risk factor for mortality in bacteremic patients (21-23).Several studies have also documented the economic impact of MRSA in hospitalized patients, demonstrating increased costs associated with managing infections and with the implementation of control measures (19,24,25). The average attributable cost of managing an MRSA infection in a Canadian hospital was estimated to be approximately $14,360, whereas costs associated with managing a patient with MRSA colonization were approximately $1,363 per hospital admission (25).The first strain of S aureus, an MRSA, with reduced susceptibility to vancomycin was reported from Japan in 1996 (26,27). Since then, such strains with vancomycin minimum inhibitory concentrations (MIC) of 8 μg/mL to 16 μg/mL (vancomycin-intermediate S aureus [VISA]) have been reported from several countries in southeast Asia, South America, Europe and the United States (28-30). Of even greater concern has been the recent identification of two infections caused by MRSA with high level resistance to vancomycin (MIC greater than 128 μg/mL; vancomycin-resistant S aureus [VRSA]), mediated by the vanA gene determinant found in vancomycin-resistant enterococci (31,32). These developments have emphasized the need for appropriate use of glycopeptides and other antimicrobial agents in the management of patients with MRSA. This paper reviews options for the treatment of patients with MRSA infection or colonization. The treatment options should be considered appropriate for hospitalized patients as well as for out-patients, and for those residing in long term care facilities.     

A Phase III,Randomized, Double-Blind,Placebo-Controlled Study of Tenecteplase for Improvement of Hemodialysis Catheter Function: TROPICS 3

Background and objectives: Despite widespread use of tunneled hemodialysis (HD) catheters, their utility is limited by the development of thrombotic complications. To address this problem, this study investigated whether the thrombolytic agent tenecteplase can restore blood flow rates (BFRs) in dysfunctional HD catheters.Design, setting, participants, & measurements: In this randomized, double-blind study, patients with dysfunctional tunneled HD catheters, defined as a BFR <300 ml/min at −250 mmHg pressure in the arterial line, received 1-hour intracatheter dwell with tenecteplase (2 mg) or placebo. The primary endpoint was the percentage of patients with BFR ≥300 ml/min and an increase of ≥25 ml/min above baseline 30 minutes before and at the end of HD. Safety endpoints included the incidence of hemorrhagic, thrombotic, and infectious complications.Results: Eligible patients (n = 149) were treated with tenecteplase (n = 74) or placebo (n = 75). Mean baseline BFR was similar for the tenecteplase and placebo groups at 151 and 137 ml/min, respectively. After a 1-hour dwell, 22% of patients in the tenecteplase group had functional catheters compared with 5% among placebo controls (P = 0.004). At the end of dialysis, mean change in BFR was 47 ml/min in the tenecteplase group versus 12 ml/min in the placebo group (P = 0.008). Four catheter-related bloodstream infections (one tenecteplase, three placebo) and one thrombosis (tenecteplase) were observed. There were no reports of intracranial hemorrhage, major bleeding, embolic events, or catheter-related complications.Conclusions: Tenecteplase improved HD catheter function and had a favorable safety profile compared with placebo.Effective hemodialysis (HD) requires reliable vascular access. Arteriovenous fistulas and grafts are preferred over catheters for their higher patency rates, prolonged survival, and lower complication rates (1–3). However, HD catheters are used by most dialysis patients to provide temporary access or to allow maturation of surgically placed fistulas (4). Tunneled catheters may also be used because of comorbidities or exhaustion of all graft and fistula sites (5,6).HD catheters must be carefully managed to mitigate a high complication rate. For example, catheter thrombosis is estimated to occur at a frequency of 0.5 to 3.0 events/1000 catheter-days (7–9). Among patients who experience access loss, catheter thrombosis is the precipitating event in 30% to 40% (1). Thrombotic obstruction of the catheter lumen reduces blood flow rate (BFR), frequently impeding the delivery of adequate HD. Treatment of partial occlusions of catheter lumens is often postponed by catheter line reversal, contributing to recirculation without addressing the underlying thrombus. The National Kidney Foundation''s Kidney Disease Outcomes Quality Initiative guidelines for HD vascular access advise against regular use of line reversal to manage low BFR (1).Maintenance of catheter patency is critical because many patients rely on catheter access for HD, and catheter insertion sites are limited. Administration of a thrombolytic directly into a dysfunctional HD catheter lumen may provide a way to salvage catheters with suboptimal BFRs while minimizing the risk of adverse events (AEs) associated with systemic delivery of these agents and avoid the need for catheter replacement. Previous studies evaluating the efficacy of alteplase or reteplase for clearance of HD catheters have yielded conflicting results (10–25). Limitations of prior studies include differences in trial design, sample size, thrombolytic dose, and definitions of treatment success. Until now there have been no large-scale, randomized, double-blind controlled trials using well defined efficacy and safety endpoints. As a result, many questions remain regarding the efficacy and safety of thrombolytics in the treatment of dysfunctional HD catheters.Tenecteplase is a recombinant serine protease that binds to fibrin and converts thrombus-bound plasminogen to plasmin, thereby stimulating local fibrinolysis. Tenecteplase has three engineered amino acid changes, resulting in greater fibrin specificity and an increased resistance to plasminogen activator inhibitor-1 compared with alteplase (26,27). When injected systemically, tenecteplase has a plasma half-life of approximately 22 minutes (28) and is primarily cleared by the liver (29). However, when tenecteplase is administered intraluminally for HD catheter dysfunction and subsequently withdrawn, circulating tenecteplase levels are not expected to reach detectable concentrations (30). Tenecteplase maintained clot lysis ability for 72 hours in a catheter in in vitro studies (Genentech data on file). In this study, we compared the efficacy of tenecteplase with placebo in improving BFR in dysfunctional HD catheters.     

Chest wall hydatidosis as the unique location of the disease: Case report and review of the literature

The chest wall is a rare location of secondary hydatidosis, but secondary hydatidosis may occur from the rupture of a lung cyst, from a liver cyst invading the diaphragm into the pleural cavity, following previous thoracic surgery for hydatidosis, or by hematogenous spread. This report describes a case of chest wall hydatidosis, which was the primary disease site in the patient, who had no previous history or current disease (hydatidosis) at other sites. The cyst invaded and partially destroyed the 9th and 10th ribs and the 10th thoracic vertebra, and protruded outside the pleural cavity through the 9th intercostal space. Preoperative albendazole administration for 10 days, surgical resection of the disease through a posterolateral thoracotomy incision, and postoperative albendazole treatment resulted in a cure with no evidence of local recurrence or disease at other sites in four years of follow-up.Key Words: Chest wall echinococcosis, Echinococcosis, Hydatidosis, Pseudotumours of the chest wallHuman echinococcosis (hydatidosis) is a parasitic disease that was first identified by Hippocrates. The disease is endemic in many parts of the world (1,2). The majority of cases - approximately 90% - are caused by the parasite Echinococcus granulosus (1,3). Less common species of Echinococcus that cause human disease are: Echinococcus multilocularis or Echinococcus sibericensis caused by the foxes and rodents of Siberia, Alaska and Central Europe, which cause alveolar hydatid disease of the liver, Echinococcus vogeli, in South America and Echinococcus oligarthrus in South and Central America (1-5). In Greece, the disease is endemic with an annual incidence of hydatidosis, caused exclusively by E granulosus, of 10 cases per 100,000 inhabitants (unpublished data). In 85% of the cases E granulosus affects either the liver, lungs, or both, although any organ can be affected (5). Hydatidosis of the chest wall as the primary manifestation of the disease is rare (1,3,6). We report a case of chest wall hydatidosis and discuss the differential diagnosis of chest wall tumors and treatment.     

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.     

CRP Polymorphisms and Progression of Chronic Kidney Disease in African Americans

Background and objectives: Chronic inflammation may play a role in chronic kidney disease (CKD) progression. CRP gene polymorphisms are associated with serum C-reactive protein (CRP) concentrations. It is unknown if CRP polymorphisms are associated with CKD progression or modify the effectiveness of anti-hypertensive therapy in delaying CKD progression.Design, setting, participants, & measurements: We genotyped 642 participants with CKD from the African American Study of Kidney Disease and Hypertension (AASK), selecting five tag polymorphisms: rs2808630, rs1205, rs3093066, rs1417938, and rs3093058. We compared the minor allele frequencies (MAF) of single nucleotide polymorphisms (SNPs) in AASK to MAFs of African Americans from NHANES III. Among AASK participants, we evaluated the association of SNPs with CRP levels and prospectively with a composite: halving the GFR, ESRD, or death.Results: The MAF was higher for the rs2808630_G allele (P = 0.03) and lower for the rs1205_A allele (P = 0.03) in the AASK compared with NHANES III. Among AASK participants, the rs3093058_T allele predicted higher CRP concentrations (P < 0.0001) but not CKD progression. The rs2808630_GG genotype was associated with higher risk of the composite endpoint compared with the AA genotype (P = 0.002). Participants with the rs2808630_GG genotype on angiotensin converting enzyme inhibitors (ACEIs) versus β blockers had increased risk of progression (P = 0.03).Conclusion: CRP SNPs that were associated with higher levels of CRP did not predict CKD progression. The rs2808630_GG genotype was associated with higher risk of CKD progression, and in patients with this genotype, ACEIs did not slow progression.Familial clustering of chronic kidney disease (CKD) and ESRD has been reported in populations throughout the world for most types of nephropathy (1–6). This genetic predisposition to ESRD seems to be strongly associated with race (7,8). Compared with people with no family history of kidney disease, African Americans with a first-degree relative with ESRD have a nine-fold increase in the risk of ESRD compared with a three- to five-fold increase in whites (8). Recently, the candidate gene MYH9 has been identified as associated with nondiabetic ERSD in African Americans, and this association explains some of the disparity in incidence of ESRD observed between whites and African Americans (7,9). However, it is possible that additional genetic variants, such as those related to inflammatory pathways, may also be associated with ESRD.Biomarkers of inflammation, including C-reactive protein (CRP), are increased even in early stages of CKD and have been linked to the risk of CKD progression (10–15). These observations have led to studies examining the genetic basis of inflammation and identification of several candidate genes for ESRD susceptibility (16–19). Recently, several large population-based studies showed that plasma CRP levels are under genetic influence (20–25). Some of these polymorphisms have been consistently associated with CRP levels (higher levels associated with rs3093058_T and lower levels associated with rs1205_A and rs2808630_G) and the risk of cardiovascular events (rs3093058_T) in African Americans (23).CRP gene polymorphisms that affect CRP concentrations may reflect lifetime exposure to CRP more accurately than single time point measurements of serum CRP concentrations. The primary goal of this study was to characterize CRP gene polymorphisms and evaluate their association with CKD progression. We hypothesized that polymorphisms associated with higher levels of CRP would be associated with higher risk of CKD progression. Additionally, we examined whether these polymorphisms modify the renoprotective effects of angiotensin converting enzyme inhibitors (ACEIs), a drug class known to have anti-inflammatory effects (26–28). We hypothesized that patients with polymorphisms associated with higher levels of CRP would benefit most from ACEIs.     

The HALT Polycystic Kidney Disease Trials: Design and Implementation

Background and objectives: Two HALT PKD trials will investigate interventions that potentially slow kidney disease progression in hypertensive autosomal dominant polycystic kidney disease (ADPKD) patients. Studies were designed in early and later stages of ADPKD to assess the impact of intensive blockade of the renin-angiotensin-aldosterone system and level of BP control on progressive renal disease.Design, settings, participants, and measurements: PKD-HALT trials are multicenter, randomized, double-blind, placebo-controlled trials studying 1018 hypertensive ADPKD patients enrolled over 3 yr with 4 to 8 yr of follow-up. In study A, 548 participants, estimated GFR (eGFR) of >60 ml/min per 1.73 m² were randomized to one of four arms in a 2-by-2 design: combination angiotensin converting enzyme inhibitor (ACEi) and angiotensin receptor blocker (ARB) therapy versus ACEi monotherapy at two levels of BP control. In study B, 470 participants, eGFR of 25 to 60 ml/min per 1.73 m² compared ACEi/ARB therapy versus ACEi monotherapy, with BP control of 120 to 130/70 to 80 mmHg. Primary outcomes of studies A and B are MR-based percent change kidney volume and a composite endpoint of time to 50% reduction of baseline estimated eGFR, ESRD, or death, respectively.Results: This report describes design issues related to (1) novel endpoints such as kidney volume, (2) home versus office BP measures, and (3) the impact of RAAS inhibition on kidney and patient outcomes, safety, and quality of life.Conclusions: HALT PKD will evaluate potential benefits of rigorous BP control and inhibition of the renin-angiotensin-aldosterone system on kidney disease progression in ADPKD.Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease occurring in 1/400 to 1/1000 live births and accounts for ∼4.6% of the prevalent kidney replacement population in the United States (1,2). ADPKD is a systemic disorder characterized by early onset hypertension before loss of kidney function. Hypertension relates to progressive kidney enlargement and is a significant independent risk factor for progression to ESRD (3).As kidney cysts enlarge, kidney architecture and vasculature are compressed, resulting in interstitial fibrosis and tubular atrophy. Despite cyst growth and kidney enlargement, kidney function remains intact for decades. However, once GFR begins to decrease, a progressive decline in kidney function occurs, with 50% of patients requiring renal replacement therapy by age 53 yr (4,5).MR imaging can accurately and reproducibly measure kidney volume and small changes in total kidney volume over short periods of time in ADPKD (6–8). Imaging studies in early ADPKD indicate that >90% show significant kidney enlargement (4 to 5%/yr), while renal function remains intact (6,8). In The Consortium of Radiologic Imaging Study of ADPKD (CRISP), hypertensive ADPKD patients showed a greater increase in kidney volume compared with normotensives with normal renal function (6.4 versus 4.3%/yr) (9). However, a causal role for hypertension in accelerated kidney growth in ADPKD cannot be proven from this observational cohort. The Polycystic Kidney Disease Treatment Network (HALT PKD) will directly test whether BP has a causal role in increased kidney volume in ADPKD.The renin-angiotensin-aldosterone system (RAAS) plays a role in the pathophysiology of hypertension and is activated in ADPKD patients (10–14). Some (12,13), but not all (14), have found higher plasma renin and aldosterone levels and a more pronounced decrease in renal vascular resistance after administration of angiotensin converting enzyme inhibitor (ACEi) in ADPKD compared with essential hypertensives. Angiotensin II is an important growth factor for kidney epithelial and interstitial fibroblasts, indicating that the RAAS may play also a role in cyst growth and expansion and kidney fibrosis. With increasing cyst size, activation of the RAAS occurs, BP increases, and a vicious cycle ensues with enhanced cyst growth, hypertension, and more cyst growth, ultimately leading to ESRD.There are multiple randomized controlled trials in kidney disease addressing the impact of inhibition of RAAS on disease progression using ACEi that include ADPKD subjects (4,15–22). To date, no benefit of inhibition of the RAAS has shown benefit on progression to ESRD or rate of GFR decline (7). Importantly, a meta-analysis of 142 ADPKD subjects from eight trials in nondiabetic kidney disease reported a 25% nonsignificant relative risk reduction in the composite endpoint of ESRD or doubling of serum creatinine in individuals on ACEi compared with other anti-hypertensive agents (19). The meta-analysis also noted that most enrolled ADPKD subjects had late-stage disease, with a mean age of 48 yr and a mean baseline serum creatinine of 3.0 mg/dl. Overall, past studies have been limited by small numbers of patients who have been studied at relatively late stages of disease.Renal chymase, which locally activates angiotensin II through non-ACE pathways, is elevated in ADPKD kidneys (23). Systemic angiotensin II levels do not suppress with chronic ACEi therapy in ADPKD, suggesting that non–ACEi dependent activation of the RAAS exists in ADPKD. Systemic and renal hemodynamic responses to exogenous angiotensin I and II persist in the presence of ACEi therapy in ADPKD (24,25). Additionally, although angiotensin receptor blocker (ARB) therapy prevents the action of angiotensin II in systemic and renal circulations by binding with the angiotensin type 1 II receptor, angiotensin II levels increase with chronic ARB therapy and exogenous angiotensin II responses are also not totally suppressed (24,25). Therefore, if angiotensin II levels are important in regulating BP and renal plasma flow as well as promoting cyst growth in ADPKD, combination therapy with ACEi and ARB may be warranted.On this background, the HALT-PKD trials, constituting two concurrent multicenter randomized placebo controlled trials have been initiated to compare the impact of rigorous versus standard BP control as well as combined ACEi + ARB therapy versus ACEi monotherapy on progression in both early and later stage ADPKD. This report will present the study design and rationale for these trials.     

Gemella species endocarditis in a child

Organisms of the genus Gemella can, on occasion, cause serious systemic illness. The present paper reports a successfully treated case of endocarditis in a 12-year-old girl with congenital heart disease caused by species of Gemella. The child presented with cough, fatigue and decreased appetite without fever. Echocardiogram demonstrated marked mitral insufficiency with flail posterior mitral valve leaflet, mitral valve vegetations, and an enlarged left atrium and ventricle. While being treated with vancomycin, the child initially had persistent bacteremia, which resolved after the addition of gentamycin; the course of therapy was completed with penicillin G and gentamycin once antimicrobial susceptibilities were available. Attempts to identify the species of Gemella were unsuccessful in the local laboratory, and at reference laboratories in Canada and the United States. The isolate is undergoing further evaluation to determine its taxonomic status.Key Words: Endocarditis, Gemella species, PaediatricsThe genus Gemella has five known species: Gemella haemolysans, Gemella morbillorum, Gemella bergeri, Gemella sanguinis and Gemella palaticanis (1-4). All but the last are opportunistic human pathogens that may cause severe infections; G palaticanis has been identified only in dogs. These organisms have been implicated in serious systemic disease, including meningitis (5) and septic shock (6). In addition, several cases of endocarditis have been reported in the past 20 years, primarily in the adult population (4,7-9). The present article describes a case of endocarditis in a child caused by a species of Gemella.