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PubMed2006

Molecular epidemiology of metallo-beta-lactamase-producing Pseudomonas aeruginosa in the Calgary Health Region: emergence of VIM-2-producing isolates.

A study was designed to describe the molecular epidemiology of carbapenem-resistant (CR) Pseudomonas aeruginosa in a large well-defined geographical region with a centralized laboratory system serving one pediatric and three large adult hospitals (acute care centers I, II, and III). Molecular characterization was done using PCR with sequencing of the integron-associated gene cassettes. Pulsed-field gel electrophoresis (PFGE) using a modified combined Stenotrophomas maltophilia and Streptococcus pneumoniae protocol with SpeI was performed on CR P. aeruginosa strains isolated in the Calgary Health Region during 2002-2006. The majority (96%) of metallo-beta-lactamase (MBL)-producing isolates produced VIM-2 with gene cassettes aacC1 and aacA4, while 4% produced IMP-7 with gene cassettes aacC4 and aacC1. Eighty-six percent of VIM-2 producers belonged to a cluster (MBLV) that was responsible for nosocomial outbreaks during 2003 (intensive care unit) and 2004 (bone marrow transplant unit) at acute care center I. Environmental isolates from these units also belonged to MBLV. The majority of strains from cluster MBLVR (related to MBLV) were present in acute care center III. Isolates producing IMP-7 belonged to a different cluster (MBLI) and were related to strains described during the 1990 s. PFGE of the MBL-negative CR strains showed that 37% belonged to a closely related cluster, NMBL, whose members were predominantly isolated from acute care center II. Our findings suggest that CR and dissemination of MBL clusters among P. aeruginosa populations in large geographic healthcare regions are dynamic processes that require continuous molecular surveillance.

Alberta↗

PubMed1984

Bacterial growth and endotoxin production in lipid emulsion.

Klebsiella pneumoniae serotypes 21 and 24 and Enterobacter cloacae were responsible for an outbreak of polymicrobial bacteremia associated with the receipt of lipid emulsion. Since it is recommended that lipid emulsion be kept refrigerated between uses, we undertook a study to determine the growth characteristics of these organisms in lipid emulsion at 5 and 25 degrees C and to examine the use of alternative measurements (pH and endotoxin) to determine contamination by viable and nonviable microorganisms. The bacteria survived but did not proliferate at 5 degrees C; no endotoxin was detected, and the pH remained unchanged. In contrast, after a 2-h lag phase, all three organisms proliferated rapidly when incubated at 25 degrees C and reached concentrations of greater than or equal to 10(7) CFU/ml at 24 h. A decrease in pH was detected after proliferation to 10(7) CFU/ml. Endotoxin was detected after proliferation reached 10(2) CFU/ml. The amount of endotoxin elaborated by the three organisms differed and ranged from 0.013 ng per 8 X 10(2) CFU/ml to 1.3 ng per 2 X 10(3) CFU/ml at 8 h. Our findings show that these microorganisms do not proliferate at refrigerator temperature in lipid emulsion, but can reach significant levels at room temperature. It is, therefore, important to keep lipid emulsion refrigerated between uses. Furthermore, when lipid emulsion contamination is suspected, endotoxin and pH determinations should be considered as possible adjunctive tests while results of bacterial cultures are pending. The results of the present study are applicable to only selected gram-negative bacteria and may not apply to gram-positive bacteria and fungi. However, these data demonstrate that measurement of pH and detection of endotoxin is quite useful when lipid emulsion contamination occurs with selected gram-negative bacteria.

Drug Contamination↗

PubMed1997

Use of a PCR method based on IS6110 polymorphism for typing Mycobacterium tuberculosis strains from BACTEC cultures.

Two PCR typing methods, based on polymorphism of the insertion sequence IS6110, were compared with Mycobacterium tuberculosis strains by using a single primer complementary to the inverted repeats of IS6110. Total M. tuberculosis DNA either was amplified directly (IS6110-PCR) or was amplified following digestion and ligation (IS6110-inverse-PCR). Both PCR techniques showed a similar degree of discrimination. Because of its simplicity, IS6110-PCR was chosen to confirm that a single M. tuberculosis strain was responsible for an outbreak of tuberculosis in a secondary school. IS6110-PCR was used to study the degree of differentiation in 85 clinical M. tuberculosis isolates from BACTEC 12B broth cultures. Results were consistent with those of the standardized IS6110 restriction fragment length polymorphism (RFLP) analysis method, showing identical PCR types for identical RFLPs, although the degree of discrimination was greater by RFLP analysis. The study concludes that due to its simplicity, IS6110-PCR is a good screening method when quick differentiation between M. tuberculosis strains is needed because BACTEC cultures may be used directly.

Bacterial Typing Techniques↗

PubMed2003

Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone.

To study the epidemiology of Pseudomonas aeruginosa colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental P. aeruginosa isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with P. aeruginosa, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal P. aeruginosa colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD(r)). The MDR strain was found at a higher frequency in sputum samples than the SSD(r) strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (oprI, oprL, and oprD) and the pyoverdine type revealed that all predominant strains except the SSD(r) strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.

Anti-Infective Agents, Local↗

PubMed2003

Molecular epidemiology of Shigella in a Taiwan township during 1996 to 2000.

A previously identified Shigella flexneri serotype 2a strain was responsible for an outbreak of shigellosis in a Taiwan township in August 1996. In order to find the relationship between this outbreak strain and subsequent Shigella infections in the area, 59, 47, 35, and 20 Shigella isolates recovered in 1997, 1998, 1999, and 2000, respectively, were collected and typed by serological and pulsed-field gel electrophoresis (PFGE) techniques. Of these 161 isolates, 139 isolates were S. flexneri serotype 2a, and one-third of them (47 isolates) exhibited the outbreak pattern. The remaining 92 S. flexneri serotype 2a isolates displayed 49 different NotI-PFGE patterns. Forty-five patterns were closely related to the outbreak pattern, with deletions of three specific NotI fragments occurring with high frequency. While the outbreak strain remained the main cause of shigellosis after the outbreak, the continuous emergence of closely related though poorly transmissible strains from the outbreak strain contributed to the observed annual decrease of shigellosis in the area.

Deoxyribonucleases, Type II Site-Specific↗

PubMed2004

Noninfectious recombinant antigen for detection of St. Louis encephalitis virus-specific antibodies in serum by enzyme-linked immunosorbent assay.

Proper surveillance of virus activity and a timely response to viral outbreaks depend upon the rapid diagnosis of viral infections. The immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a fast, sensitive test routinely used for the diagnosis of the medically important West Nile and St. Louis encephalitis flaviviruses. However, the suckling mouse brain-derived (SMB) antigen used in this assay is tedious to prepare and has a risk of exposing personnel to live virus and hazardous chemicals. We report the development of a St. Louis encephalitis virus (SLEV) noninfectious recombinant antigen that is a safe and easily produced alternative antigen for use in diagnostic assays. The expression plasmid pCB8SJ2, containing the premembrane and envelope structural protein-encoding regions of SLEV, was constructed to express secreted extracellular virus-like particles (VLPs) from CHO cells. Blind-coded human serum panels were assembled from patients having recent SLEV, West Nile virus (WNV), Powassan virus, or La Crosse encephalitis virus infections to assess the sensitivity and specificity of assays with SLEV VLP or SMB antigen. MAC-ELISAs with either antigen had comparable sensitivity for the detection of IgM antibodies against SLEV. Importantly, when these two antigens were tested against a human serum panel from patients having recent WNV or Powassan virus infections, the SLEV VLPs were less likely than SMB antigen to detect flavivirus cross-reactive IgM antibodies. An optimized IgG antibody capture ELISA (GAC-ELISA) with both WNV and SLEV VLPs was developed to circumvent the frequently observed higher background in the antigen-capture IgG-ELISA (ACG-ELISA). For the detection of IgG antibodies against WNV, the GAC-ELISA resulted in a statistically significant higher performance accuracy (P = 0.003) than the ACG-ELISA when the WNV VLP antigen was used in both assays. However, no statistical difference was observed in the assay performance of the GAC-ELISA with SLEV VLP or the ACG-ELISA with SLEV SMB antigen.

Animals↗

PubMed2005

Evaluation of ribotyping as a tool for molecular typing of Yersinia pseudotuberculosis strains of worldwide origin.

Yersinia pseudotuberculosis is a gram-negative bacterium that infects a wide range of animals, including humans, and is transmitted by the fecal-oral route. This species is found globally and is responsible for human outbreaks, mainly in cold countries. The aim of this study was to evaluate the potential of ribotyping for the molecular typing of worldwide isolates. For this purpose, 80 strains of Y. pseudotuberculosis belonging to the six classical serotypes and nine subserotypes and isolated from various countries and different hosts were analyzed. Combination of the EcoRI and EcoRV ribopatterns allowed the delineation of 27 ribotypes. In most instances, ribotypes were associated with specific subserotypes and allowed their subdivision. No association between the ribotype and the geographical origin of the strains was observed, arguing for a global spread of this organism. Similarly, no marked association between the ribotype and the type of host was noted, confirming the circulation of this pathogen in the environment, different animal species, and human hosts. Y. pseudotuberculosis exhibited ribopatterns very close to those of Y. pestis, although not completely identical. Altogether, the present study demonstrates that ribotyping may be a useful tool for molecular typing of global isolates of Y. pseudotuberculosis but that it has some limitations due to the small number of hybridizing bands that generate the diversity of the profiles.

Animals↗

PubMed2002

C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans.

Ebola virus is a highly lethal pathogen responsible for several outbreaks of hemorrhagic fever. Here we show that the primate lentiviral binding C-type lectins DC-SIGN and L-SIGN act as cofactors for cellular entry by Ebola virus. Furthermore, DC-SIGN on the surface of dendritic cells is able to function as a trans receptor, binding Ebola virus-pseudotyped lentiviral particles and transmitting infection to susceptible cells. Our data underscore a role for DC-SIGN and L-SIGN in the infective process and pathogenicity of Ebola virus infection.

Cell Adhesion Molecules↗

PubMed2002

Structural requirements for the assembly of Norwalk virus-like particles.

Norwalk virus (NV) is the prototype strain of a group of human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant NV virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. The X-ray structure of the rNV VLPs has revealed that the capsid protein folds into two principal domains: a shell (S) domain and a protruding (P) domain (B. V. V. Prasad, M. E. Hardy, T. Dokland, J. Bella, M. G. Rossmann, and M. K. Estes, Science 286:287-290, 1999). To investigate the structural requirements for the assembly of rNV VLPs, we performed mutational analyses of the capsid protein. We examined the ability of 10 deletion mutants of the capsid protein to assemble into VLPs in insect cell cultures. Deletion of the N-terminal 20 residues, suggested by the X-ray structure to be involved in a switching mechanism during assembly, did not affect the ability of the mutant capsid protein to self-assemble into 38-nm VLPs with a T=3 icosahedral symmetry. Further deletions in the N-terminal region affected particle assembly. Deletions in the C-terminal regions of the P domain, involved in the interactions between the P and S domains, did not block the assembly process, but they affected the size and stability of the particles. Mutants carrying three internal deletion mutations in the P domain, involved in maintaining dimeric interactions, produced significantly larger 45-nm particles, albeit in low yields. The complete removal of the protruding domain resulted in the formation of smooth particles with a diameter that is slightly smaller than the 30-nm diameter expected from the rNV structure. These studies indicate that the shell domain of the NV capsid protein contains everything required to initiate the assembly of the capsid, whereas the entire protruding domain contributes to the increased stability of the capsid by adding intermolecular contacts between the dimeric subunits and may control the size of the capsid.

Animals↗

PubMed2003

The 3' end of Norwalk virus mRNA contains determinants that regulate the expression and stability of the viral capsid protein VP1: a novel function for the VP2 protein.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3' untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3' terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3' elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3'UTR (ORF2+3+3'UTR). In contrast, expression of VP1 from constructs that lacked the 3'UTR (ORF2+3), ORF3 (ORF2+3'UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3'UTR revealed that the 3'UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.

3' Untranslated Regions↗

PubMed2001

Solar disinfection of drinking water protects against cholera in children under 6 years of age.

BACKGROUND AND AIMS: We have previously reported a reduction in risk of diarrhoeal disease in children who used solar disinfected drinking water. A cholera epidemic, occurring in an area of Kenya in which a controlled trial of solar disinfection and diarrhoeal disease in children aged under 6 had recently finished, offered an opportunity to examine the protection offered by solar disinfection against cholera. METHODS: In the original trial, all children aged under 6 in a Maasai community were randomised by household: in the solar disinfection arm, children drank water disinfected by leaving it on the roof in a clear plastic bottle, while controls drank water kept indoors. We revisited all households which had participated in the original trial. RESULTS: There were 131 households in the trial area, of which 67 had been randomised to solar disinfection (a further 19 households had migrated as a result of severe drought). There was no significant difference in the risk of cholera in adults or in older children in households randomised to solar disinfection; however, there were only three cases of cholera in the 155 children aged under 6 years drinking solar disinfected water compared with 20 of 144 controls. CONCLUSIONS: Results confirm the usefulness of solar disinfection in reducing risk of water borne disease in children. Point of consumption solar disinfection can be done with minimal resources, which are readily available, and may be an important first line response to cholera outbreaks. Its potential in chorine resistant cholera merits further investigation.

Child, Preschool↗

PubMed2003

Late clinical and magnetic resonance imaging follow up of Nipah virus infection.

The Nipah virus is a newly identified paramyxovirus responsible for an outbreak of fatal encephalitis in Malaysia and Singapore. This paper reports the follow up clinical and magnetic resonance imaging findings in 22 affected subjects. Of 13 patients with encephalitis, one died, one was lost to follow up, and seven recovered. Among the four remaining patients, one had residual sixth nerve palsy, another suffered from severe clinical depression, and a third patient had evidence of retinal artery occlusion. One patient with delayed onset Horner syndrome had a single lesion in the cervical spinal cord. The brain magnetic resonance findings were stable or improved in nine patients over 18 months of follow up. Among a second group of nine asymptomatic seropositive abattoir workers, magnetic resonance examination in seven subjects revealed discrete small lesions in the brain; similar to those detected in encephalitis patients. These findings suggest that in addition to encephalitis, the newly discovered Nipah virus affects the spinal cord and the retina. Late clinical and radiological findings can occur in Nipah virus infections as with other paramyxoviruses.

Abducens Nerve Diseases↗

PubMed2003

Characterization of Neisseria meningitidis strains isolated from invasive meningococcal disease cases in Canada in 2001.

With the recent introduction of polysaccharide-protein conjugated vaccines for the control of serogroup C meningococcal disease and the emergence of different variants of serogroup C meningococci, it is likely the epidemiology of meningococcal disease in many countries may be affected. We have therefore analysed and reported the characteristics of Neisseria meningitidis strains collected in 2001 from the Canadian surveillance program on invasive meningococcal disease. Only strains collected from normally sterile clinical sites of patients were studied. Of the 289 isolates obtained from individual patients, 173 (59.9%) were serogroup C, 76 (26.3%) were serogroup B, 30 (10.4%) were serogroup Y, and 10 (3.5%) were serogroup W135. Ninety-six percent of the serogroup C isolates belonged to the ET-15 clone, with an additional 2.3% belonging to other electrophoretic types within the ET-37 clonal complex. Different antigenic variants of the endemic serogroup C ET-15 clone were responsible for localized outbreaks in different parts of the country. One novel variant with the antigenic composition of C:2a:P1.1,7 was reported in two provinces, Quebec and Ontario. Eighteen percent of the meningococci isolated from patients in Ontario belonged to serogroup Y, compared with only 8% in the rest of Canada. The current data highlight the importance of strain characterization by serogroup, serotype, and serosubtype antigens in providing useful information for the surveillance of meningococcal disease in Canada.

Bacterial Typing Techniques↗

PubMed1986

Epidemiology of human rotaviruses in Argentina as determined by RNA genome electrophoresis.

Rotavirus gastroenteritis was studied by enzyme-linked immunosorbent assay (ELISA) and by PAGE from April 1983 to April 1985 at the Buenos Aires Ricardo Gutierrez Children's Hospital and at the San Justo Children's Hospital. Based on examination of 576 cases, rotavirus was identified in 109 (18.9%) cases by ELISA and in 99 (17.2%) cases by PAGE. As a diagnostic tool PAGE presented a sensitivity of 90.8% compared with ELISA. Compared with the control SA-11 genome, 84 samples (84.9%) presented a long electropherotype and 15 isolates (15.1%) presented a short electropherotype. We detected 35 different long electropherotypes and report a short electropherotype that did not show genome variation and was responsible for an outbreak in the San Justo Children's Hospital population from September 1983 to July 1984.

Argentina↗

PubMed1997

Alexithymia, immunity and cervical intraepithelial neoplasia: replication.

BACKGROUND: In a previous study [Psychother Psychosom 1994;61:199-204] we investigated the relationship between alexithymia, carcinogenesis and immunity in a group of women who were unconscious sufferers from precancerous lesions of the cervix (CIN). The results of this study showed a high level of association between alexithymia and CIN and, an even more interesting fact, between alexithymia and reduced levels of immunity. METHODS: The aim of the present study is to check the results of the previous one by testing a larger group (43 women affected by cervical dysplasia and 67 healthy women) and by the use of a self-administered test for detection of alexithymia, the well-validated Twenty-Item Toronto Alexithymia Scale (TAS-20). RESULTS: The results confirm that women suffering from CIN have higher average TAS-20 ratings (55) than normal women (47.32) and that the level of alexithymia detected in the group of women suffering from dysplasia (42.5%) is higher than that of normal women (12.85%). Moreover, the present study confirms that alexithymic women have lower rates of a number of lymphocyte subsets than non-alexithymic women. CONCLUSIONS: This study fully confirms the results of our previous work and those of a number of other studies: (1) personality might be one of the factors jointly responsible for the outbreak of cancer; (2) the immune system appears to play an important part as a mediator between personality and cancer.

Adult↗

PubMed1996

Myeloencephalitis associated with a viridans group Streptococcus in a colony of Japanese macaques (Macaca fuscata).

Five weaned immature Japanese macaques (Macaca fuscata), bred in captivity, showed nervous signs over a 12-month period. Hemorrhagic cerebral infarcts with vasculitis were detected in four necropsied animals. The distribution and nature of the lesions were consistent with bacterial embolism, and a Streptococcus isolate, biochemically similar to S. salivarius, was recovered from the cerebral lesions from three of the four necropsied macaques. Treatment with antibacterial agents (enrofloxacin) improved the clinical condition of the surviving affected animal. These observations strongly suggest that this Streptococcus spp., member of the viridans group, is responsible for this outbreak. Dental pulpitis, present in two of the four macaques, probably served as the entry for this bacterium.

Animals↗

PubMedDate not supplied

Eradicating polio: today's challenges and tomorrow's legacy.

Since its launch in 1988, the Global Polio Eradication Initiative has grown into one of the largest international health efforts in history, operating in every country and area in the world. The burden of polio disease has been reduced by over 99%, and the number of countries with indigenous virus has fallen from more than 125 to just four. As importantly, a strong surveillance and laboratory infrastructure has been established for vaccine-preventable diseases (including measles, tetanus, yellow fever, rubella and Japanese encephalitis), and a massive investment has been made in the physical infrastructure and human resources needed to deliver routine immunizations and other health services in developing countries. Between 2000 and 2003, new challenges to polio eradication emerged, threatening the interruption of the transmission of wild poliovirus globally and the eventual elimination of any residual polio disease as the result of the continued use of oral polio vaccines. By the end of 2005, a range of solutions had been developed to address these late challenges, including two new monovalent oral polio vaccines, new and robust international standards for the response to polio outbreaks, and renewed political commitment across the countries that remain infected. As importantly, a comprehensive strategy had been established for managing the long-term risks of paralytic polio, centred, ironically, on the eventual elimination from routine immunizations of the vaccine that is still central to the success of the global eradication effort.

Disease Outbreaks↗

PubMed2006

Genotyping of Newcastle disease viruses isolated from 2002 to 2004 in China.

The main function region of the fusion (F) protein gene of 124 strains of Newcastle disease virus isolated from 2002 to 2004 in China was amplified and sequenced for further phylogenetic and residue substitutive analysis. Most of the isolates were classified into genotype VIIc, VIId, VIf, and VIb, while others into genotype IX, III, or II. The genotype IX, a unique genotype which includes strain F48, the first Chinese virulent NDV strain isolated in 1948, were still found inducing sporadic infections in certain areas. Subgenotype VIIc, VIId, and VIIe viruses, which were distributed in clusters in the phylogenetic tree distinct from members of subgenotypes VIIa and VIIb, were responsible for most outbreaks in China and circulated predominantly in China in recent years. Strain NDV03-026, an isolate of the genotype II which was normally lentogenic, was found carrying (112)RRQKRF(117) motif at the cleavage site of F protein as the virulent strain.

Amino Acid Sequence↗