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PubMed2006

Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet.

Standardized rapid pulsed-field gel electrophoresis (PFGE) protocols for the subtyping of Escherichia coli O157:H7, Salmonella serotypes, and Shigella species are described. These protocols are used by laboratories in PulseNet, a network of state and local health departments, and other public health laboratories that perform real-time PFGE subtyping of these bacterial foodborne pathogens for surveillance and outbreak investigations. Development and standardization of these protocols consisted of a thorough optimization of reagents and reaction conditions to ensure that the protocols yielded consistent results and high-quality PFGE pattern data in all the PulseNet participating laboratories. These rapid PFGE protocols are based on the original 3-4-day standardized procedure developed at Centers for Disease Control and Prevention that was validated in 1996 and 1997 by eight independent laboratories. By using these rapid standardized PFGE protocols, PulseNet laboratories are able to subtype foodborne pathogens in approximately 24 h, allowing for the early detection of foodborne disease case clusters and often aiding in the identification of the source responsible for the infections.

Cluster Analysis↗

PubMed2002

Dendritic cells transport conidia and hyphae of Aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate Th responses to the fungus.

Aspergilli are respiratory pathogens and pulmonary infections are usually acquired through the inhalation of conidia, able to reach small airways and the alveolar space where the impaired host defense mechanisms allow hyphal germination and subsequent tissue invasion. The invasive pulmonary aspergillosis is the most common manifestation of Aspergillus fumigatus infection in immunocompromised patients and is characterized by hyphal invasion and destruction of pulmonary tissue. A Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development and unfavorable outcome of invasive pulmonary aspergillosis. Dendritic cells (DC) have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them. In the present study, we assessed the functional activity of pulmonary DC in response to A. fumigatus conidia and hyphae, both in vitro and in vivo. We analyzed mechanisms and receptors for phagocytosis by DC as well as DC migration, maturation, and Th priming in vivo upon exposure to either form of the fungus. We found a remarkable functional plasticity of DC in response to the different forms of the fungus, as pulmonary DC were able to: 1) internalize conidia and hyphae of A. fumigatus through distinct phagocytic mechanisms and recognition receptors; 2) discriminate between the different forms in terms of cytokine production; 3) undergo functional maturation upon migration to the draining lymph nodes and spleens; and 4) instruct local and peripheral Th cell reactivity to the fungus.

Animals↗

PubMed2000

Incidence of foodborne illnesses reported by the foodborne diseases active surveillance network (FoodNet)-1997. FoodNet Working Group.

In 1997, the Foodborne Diseases Active Surveillance Program (FoodNet) conducted active surveillance for culture-confirmed cases of Campylobacter, Escherichia coli O157, Listeria, Salmonella, Shigella, Vibrio, Yersinia, Cyclospora, and Cryptosporidium in five Emerging Infections Program sites. FoodNet is a collaborative effort of the Centers for Disease Control and Prevention's National Center for Infectious Diseases, the United States Department of Agriculture's Food Safety and Inspection Service, the Food and Drug Administration's Center for Food Safety and Applied Nutrition, and state health departments in California, Connecticut, Georgia, Minnesota, and Oregon. The population under active surveillance for foodborne infections was approximately 16.1 million persons or roughly 6% of the United States Population. Through weekly or monthly contact with all clinical laboratories in these sites, 8,576 total isolations were recorded: 2,205 cases of salmonellosis, 1,273 cases of shigellosis, 468 cases of cryptosporidiosis, 340 of E. coli O157:H7 infections, 139 of yersiniosis, 77 of listeriosis, 51 of Vibrio infections, and 49 of cyclosporiasis. Results from 1997 demonstrate that while there are regional and seasonal differences in reported incidence rates of certain bacterial and parasitic diseases, and that some pathogens showed a change in incidence from 1996, the overall incidence of illness caused by pathogens under surveillance was stable. More data over more years are needed to assess if observed variations in incidence reflect yearly fluctuations or true changes in the burden of foodborne illness.

Animals↗

PubMed2001

[Introduction of ongoing research projects on echinococcosis at Asahikawa Medical College and some comments on the surveillance, prevention and control of alveolar echinococcosis in Japan].

All researchers working at Department of Parasitology, Asahikawa Medical College (AMC) have been involved in either basic or applied research on echinococcosis (alveolar echinococcosis; AE, cystic echinococcosis; CE) and neurocysticercosis (NCC). As these parasitic diseases are 1) most serious helminthic infections threatening human life, 2) caused by the larval stage of very closely related taeniid cestodes and 3) recognized as global emerging parasitic diseases, we have to establish research reference center for differentiation of these diseases, since the recommendations for the treatment of these three diseases differ critically. We at AMC have already established highly reliable differential serodiagnosis for these all using native antigens and recombinant antigens for AE and NCC. Based on such scientific contribution, we have been involved in many international collaboration projects on AE, CE and NCC in Asia, Europe, Africa and America. Basic research from mitochondrial DNA analysis of Echinococcus multilocularis (causative agent of AE) and Taenia solium (NCC) has revealed and/or implied the origin of the polymorphism of these parasites in the world and diversity of pathogenicity. Epidemiological surveillance of AE in China has strongly suggested that the risk factor is dog carer but not dog feces, dog owned, wild herbs or fox hunting. As the ongoing serology in Hokkaido carried out by the Hokkaido Institute of Public Health is for the primary screening of AE, it is impossible to identify AE patients. It is, therefore, recommended that serology for identification of AE without surgical confirmation is the key to be introduced for differentiation of AE patients, either asymptomatic or symptomatic, in order to recognize the incidence of AE in Hokkaido. Without the true incidence of AE, it is impossible to establish any reliable systems for the surveillance, prevention and control of AE in Hokkaido, Japan.

Echinococcosis↗

PubMed2025

Prospective characterisation of drug-resistant bloodstream infections in Africa and Asia (ACORN2): a surveillance network assessment.

BACKGROUND: Antimicrobial resistance (AMR) is a major global health threat, but there is scarcity of laboratory surveillance data linked to clinical information to determine burden and inform interventions, especially from low-income and middle-income countries. The ACORN2 study sought to address this through prospective case-based surveillance in 19 hospitals across Africa and Asia to characterise drug-resistant infections by origin, clinical syndrome, patient age, outcome, and geographical location. METHODS: Patients were enrolled on selected wards and clinical data were collected daily for community-acquired infections (CAIs). Point prevalence surveys for hospital-acquired infections (HAIs) were conducted weekly. Mortality was assessed at discharge and after 28 days. Linked microbiology data were extracted from local laboratory databases. Primary descriptive analyses focused on WHO Global Antimicrobial Resistance and Use Surveillance System pathogen (target organism) bloodstream infections (BSIs). Comparisons were adjusted for clustering by site using random effects models. FINDINGS: Over 31 months, 41 907 infections were characterised from 41 032 admissions. Two-thirds were children (19 351; 47·2%) or neonates (6649; 16·2%). There were marked differences in pathogen incidence and antibiotic resistance when clinical infections were stratified by patient age category and infection origin (CAI/HAI). The highest rates of target organism AMR BSI were third-generation cephalosporin-resistant (3GC-R) Escherichia coli (718·56/100 000 blood cultured infection episodes), meticillin-resistant Staphylococcus aureus (586·89/100 000 blood cultured infection episodes), and 3GC-R Klebsiella pneumoniae (364·92/100 000 blood cultured infection episodes). In-hospital mortality was 13·1% (166/1265) in patients with target organism BSI versus 6·2% (1357/21 845) in those with negative blood cultures, p<0·0001. INTERPRETATION: ACORN2 has shown practical implementation of collecting linked clinical-laboratory AMR data in low-income and middle-income countries and identified a significant burden of WHO GLASS BSI. Adoption of the ACORN2 approach at scale might enhance use of diagnostic microbiology and improve the volume of clinical data included in national and global AMR surveillance datasets. FUNDING: Wellcome.

Humans↗

PubMed2006

A reappraisal of humoral immunity based on mechanisms of antibody-mediated protection against intracellular pathogens.

Sometime in the mid to late twentieth century the study of antibody-mediated immunity (AMI) entered the doldrums, as many immunologists believed that the function of AMI was well understood, and was no longer deserving of intensive investigation. However, beginning in the 1990s studies using monoclonal antibodies (mAbs) revealed new functions for antibodies, including direct antimicrobial effects and their ability to modify host inflammatory and cellular responses. Furthermore, the demonstration that mAbs to several intracellular bacterial and fungal pathogens were protective issued a serious challenge to the paradigm that host defense against such microbes was strictly governed by cell-mediated immunity (CMI). Hence, a new view of AMI is emerging. This view is based on the concept that a major function of antibody (Ab) is to amplify or subdue the inflammatory response to a microbe. In this regard, the "damage-response framework" of microbial pathogenesis provides a new conceptual viewpoint for understanding mechanisms of AMI. According to this view, the ability of an Ab to affect the outcome of a host-microbe interaction is a function of its capacity to modify the damage ensuing from such an interaction. In fact, it is increasingly apparent that the efficacy of an Ab cannot be defined either by immunoglobulin or epitope characteristics alone, but rather by a complex function of Ab variables, such as specificity, isotype, and amount, host variables, such as genetic background and immune status, and microbial variables, such as inoculum, mechanisms of avoiding host immune surveillance and pathogenic strategy. Consequently, far from being understood, recent findings in AMI imply a system with unfathomable complexity and the field is poised for a long overdue renaissance.

Animals↗

PubMed2002

Long-term antibiotic resistance surveillance of gram-negative pathogens suggests that temporal trends can be used as a resistance warning system.

Antibiotic resistance among Gram-negative bacteria and antibiotic consumption were investigated at the Karolinska Hospital, Stockholm, Sweden over a 12-y period. The investigation showed an increase in ciprofloxacin resistance of Escherichia coli from 0% in 1991 to 7% in 1997 and to 11% in 1999. Resistance among Pseudomonas aeruginosa isolates to ciprofloxacin increased from 2.5% in 1991 to 9.0% in 1997 and to 13% in 1999. Resistance levels for norfloxacin showed the same high statistical significance in terms of the temporal trend. A more detailed analysis showed higher resistance against norfloxacin in specific wards. Relationships between antibiotic use and antibiotic susceptibility showed different patterns. The increased ciprofloxacin resistance of E. coli and P. aeruginosa during the study period was paralleled by an increased consumption of quinolones. During the 12-y study period the total use of cephalosporins increased 2.5-fold, while the levels of E. coli resistance to cefuroxime and cefotaxime remained stable. A third pattern was seen with trimethoprim-sulfamethoxazole, namely increasing resistance of E. coli as the use of trimethoprim-sulfamethoxazole declined. The analysis of resistance levels and antibiotic consumption in the present study suggests different mechanisms for the increased resistance. The significant trend of increased resistance to antibiotics over time constitutes an important warning system.

Anti-Bacterial Agents↗

PubMed2000

In vitro activities of LY333328 and comparative agents against nosocomial gram-positive pathogens collected in a 1997 global surveillance study.

The in vitro activity of LY333328 was evaluated for 1,479 nosocomial gram-positive pathogens isolated in 12 countries during 1997. LY333328 MICs at which 90% of the isolates tested were inhibited for Enterococcus faecalis (n = 351), Enterococcus faecium (n = 100), Staphylococcus aureus (n = 593), coagulase-negative Staphylococcus species (n = 325), and Streptococcus pneumoniae (n = 110) were 1, 1, 2, 2, and 0.015 microg/ml, respectively. LY333328 demonstrated potent activity against isolates of vancomycin-resistant enterococci, oxacillin-resistant staphylococci, and penicillin-resistant pneumococci.

Anti-Bacterial Agents↗

PubMed2000

Human afferent lymph from normal skin contains an increased number of mainly memory / effector CD4(+) T cells expressing activation, adhesion and co-stimulatory molecules.

We investigated the T cell population in the afferent lymph and the peripheral blood with regard to expression of activation, adhesion and co-stimulatory molecules and cytokine profile by immunohistochemistry and flow cytometry. The majority of the lymphoid cell population in the afferent lymph were CD4(+), CD45RO(+) T cells expressing the alpha beta TCR. An increased percentage of the T cells expressed activation molecules like HLA-DR, CD25, CD26, CD69 as well as adhesion and co-stimulatory molecules like CD54, CD154 / 40 ligand. Furthermore, T cells in the afferent lymph predominantly expressed the type 1 cytokine IFN-gamma, whereas type 2 cytokines such as IL-4, IL-5, IL-10 were not or barely detectable. Interestingly, dendritic cells expressing IL-12 were also found in close association with some lymphocytes, indicating that these contacts may be important in promoting Th 1 cells. In conclusion, an increased number of mainly CD4(+) memory / effector T cells, expressing activation, adhesion and co-stimulatory molecules migrate through the afferent skin-derived lymph in humans. Furthermore, our data demonstrate the dominance of a type 1 cytokine profile in these T cells and suggest that they have an important function in the immune surveillance against pathogens or other antigens in the skin and its associated lymphoid tissue.

Antigens, CD↗

PubMed2002

Update on Listeria monocytogenes infection.

Listeria monocytogenes is a gram-positive bacillus that causes meningitis, encephalitis, bacteremia, and febrile gastroenteritis. Most disease occurs in immunosuppressed individuals. Recent seroepidemiologic studies show that the infection is foodborne. Due to the increasing number of immunosuppressed individuals at risk for listeriosis, as well as the persistence of substantial foodborne outbreaks, L. monocytogenes has gained worldwide attention as an important pathogen. Heightened surveillance and quality control by the food industry have been instituted, leading to a reduction in the number of cases and deaths from this infection in the past decade. However, due to the ubiquity of the organism in the environment, outbreaks and sporadic disease continue to occur. The standard therapy for listeriosis is a combination of ampicillin and gentamicin or, for patients who are intolerant of b-lactam agents, trimethoprim-sulfamethazole. Despite the availability of therapy, the mortality rate remains high in those with T-cell immunodeficiencies.

Adult↗

PubMed1981

Infection in the renal transplant recipient.

The incidence of infection in the renal transplant patient is directly related to the net immunosuppressive effect achieved and the duration of time over which this therapy is administered. A second major factor in the causation of infections in this population is the nosocomial hazards to which these patients are exposed, ranging from invasive instrumentation to environmental contamination with Aspergillus species, Legionella pneumophila, Pseudomonas aeruginosa and other microbial pathogens. Careful surveillance is necessary to identify and eliminate such nosocomial sources of infection. The major types of infection observed can be categorized according to the time period post-transplant in which they occur: postsurgical bacterial infection in the first month after transplantation; opportunistic infection, with cytomegalovirus playing a major role, and transplant pyelonephritis in the period one to four months post-transplant; and a mixture of conventional and opportunistic infections in the last post-transplant period. Conventional infection in this late period occurs primarily in patients with good renal function who are receiving minimal immunosuppressive therapy; opportunistic infection occurs primarily in patients with poor renal function who are receiving higher levels of immunosuppression.

Central Nervous System Diseases↗

PubMed2026

A species-discriminatory aerA TaqMan qPCR assay for rapid quantification of Aeromonas veronii in fish tissues and aquaculture water.

Aeromonas veronii is a major bacterial pathogen in freshwater aquaculture, yet rapid species-level quantification remains challenging within the genetically complex genus Aeromonas. We developed a singleplex hydrolysis-probe (TaqMan) quantitative PCR (qPCR) assay targeting an A. veronii-discriminatory region of the aerolysin gene (aerA) and validated it according to MIQE recommendations. Plasmid standards gave a linear range of 2 to 2 × 106 copies/reaction (R2 = 0.9962) with 100.5% amplification efficiency. The endpoint limit of detection was 2 copies per reaction, and 20 copies per reaction was set as the practical reporting limit based on reproducible detection and low intra- and inter-assay variation. Analytical specificity was evaluated with genomic DNA from an 18-strain panel, with reproducible amplification observed only for A. veronii. The assay was further tested in 55 fish-tissue and 11 aquaculture-water DNA extracts. NH8B-1D2 sample-process monitoring was used for matrix-level recovery correction, and tissue and water extraction blanks were undetermined. The aerA target was detected in all tested gill, stomach/intestine, spleen, kidney/head kidney, pond-water filter and Xiamen seawater filter extracts, and in 10/11 liver extracts. Median NH8B-corrected loads were highest in gill among tissues and higher in pond-water filters than in Xiamen seawater filters. A separate Vibrio harveyi inhibition-check assay indicated no obvious amplification-stage inhibition. This assay supports rapid quantification of aerA-positive A. veronii in fish and aquaculture-water matrices.

Animals↗

PubMed2003

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Gram's stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.

Base Sequence↗

PubMed2002

Sharps injury recordkeeping activities and safety product use in California health care facilities: pilot study results from the sharps injury control program.

BACKGROUND: In 1999, licensed health care facilities in California (N = 2532) were invited to participate in a survey about occupational bloodborne pathogens exposure surveillance activities, recordkeeping methods, and use of safety-enhanced sharps devices. RESULTS: A total of 1274 facilities responded to the survey from January 1999 through August 1999 (response rate = 50%). Sharps-related injuries were recorded for multiple departments on various forms in diverse manners. Only 66% of hospitals, 37% of home health agencies, and 33% of skilled nursing facilities reported sharps injuries on a mandated sharps injury log. More than 70% of facilities stated that they used some type of safety device or needleless system, but this figure varied by type of device and facility type. Eighty-four percent of general acute care hospitals, 28% of home health agencies and skilled nursing facilities each had evaluated at least 1 safety-enhanced device. Almost 90% of all facilities expressed a need for educational materials on topics such as device selection and evaluation. CONCLUSIONS: Standardization of surveillance and recordkeeping activities does not exist across facility types. Standards and regulations demand complex recordkeeping activities. Increased funding for distribution of educational materials and on-site training should accompany changes in mandated reporting activities when appropriate. Increased testing and evaluation of devices across facility types are necessary to ensure that safety-enhanced devices are protective of health care workers and patients.

California↗

PubMed2000

Resistance to ciprofloxacin and the new fluoroquinolones.

Resistance to ciprofloxacin has generally remained low in the UK. A low rate of resistance development in vitro has been suggested for the newer fluoroquinolones such as grepafloxacin and trovafloxacin. Levofloxacin is likely to have a pattern of development of resistance similar to that of ofloxacin. Resistance problems with ofloxacin are being reported in the USA, but it is unclear whether similar problems will arise with levofloxacin. Restrained quinolone use, surveillance of pathogen susceptibility and control of cross-infection with resistant clones remain important strategies for managing fluoroquinolone resistance.

Anti-Infective Agents↗

PubMed2025

Reference-Free Variant Calling with Local Graph Construction with ska lo (SKA).

The study of genomic variants is increasingly important for public health surveillance of pathogens. Traditional variant-calling methods from whole-genome sequencing data rely on reference-based alignment, which can introduce biases and require significant computational resources. Alignment- and reference-free approaches offer an alternative by leveraging k-mer-based methods, but existing implementations often suffer from sensitivity limitations, particularly in high mutation density genomic regions. Here, we present ska lo, a graph-based algorithm that aims to identify within-strain variants in pathogen whole-genome sequencing data by traversing a colored De Bruijn graph and building variant groups (i.e. sets of variant combinations). Through in silico benchmarking and real-world dataset analyses, we demonstrate that ska lo achieves high sensitivity in single-nucleotide polymorphism (SNP) calls while also enabling the detection of insertions and deletions, as well as SNP positioning on a reference genome for recombination analyses. These findings highlight ska lo as a simple, fast, and effective tool for pathogen genomic epidemiology, extending the range of reference-free variant-calling approaches. ska lo is freely available as part of the SKA program (https://github.com/bacpop/ska.rust).

Polymorphism, Single Nucleotide↗

PubMed1999

Processing of bacterial antigens for peptide presentation on MHC class I molecules.

Professional antigen-presenting cells (pAPC) can process and present exogenous antigens on major histocompatibility complex class I (MHC-I) molecules. This unusual pathway for antigen presentation may represent a physiologically important step in the course of priming and tolerance induction of CD8+ T cells. In addition, it may play an important role in immunological surveillance for pathogens that survive in vacuolar compartments in APC. The goal of the present review is to discuss recent studies on the processing of bacterial-derived antigens for presentation on MHC-I molecules. The antigen presentation emphasized will include bacteria that remain confined in vacuolar compartments. This is in contrast to antigens derived from bacteria that have intrinsic properties allowing translocation across membranes and access into the classical MHC-I presentation pathway. In particular, presentation of bacterial antigens by dendritic cells (DC) will be emphasized, and MHC-I presentation of antigens derived from apoptotic cells, particularly cells induced to undergo apoptosis by microbial infection, will be presented. Finally, some special aspects of the interaction between bacteria and DC will be discussed as it relates to DC maturation, antigen presentation and T-cell stimulation.

Animals↗

PubMed2001

Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.

DNA Primers↗