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PubMed2000

Outbreak of Bacillus cereus infections in a neonatal intensive care unit traced to balloons used in manual ventilation.

In 1998, an outbreak of systemic infections caused by Bacillus cereus occurred in the Neonatal Intensive Care Unit of the University Hospital Vrije Universiteit, Amsterdam, The Netherlands. Three neonates developed sepsis with positive blood cultures. One neonate died, and the other two neonates recovered. An environmental survey, a prospective surveillance study of neonates, and a case control study were performed, in combination with molecular typing, in order to identify potential sources and transmission routes of infection. Genotypic fingerprinting by amplified-fragment length polymorphism (AFLP) showed that the three infections were caused by a single clonal type of B. cereus. The same strain was found in trachea aspirate specimens of 35 other neonates. The case control study showed mechanical ventilation with a Sensormedics ventilation machine to be a risk factor for colonization and/or infection (odds ratio, 9.8; 95% confidence interval, 1.1 to 88.2). Prospective surveillance showed that colonization with B. cereus occurred exclusively in the respiratory tract of mechanically ventilated neonates. The epidemic strain of B. cereus was found on the hands of nursing staff and in balloons used for manual ventilation. Sterilization of these balloons ended the outbreak. We conclude that B. cereus can cause outbreaks of severe opportunistic infection in neonates. Typing by AFLP proved very useful in the identification of the outbreak and in the analysis of strains recovered from the environment to trace the cause of the epidemic.

Bacillaceae Infections↗

PubMed2000

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea.

Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive C. difficile were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of C. difficile that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C. difficile. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C. difficile-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.

Adolescent↗

PubMed2001

Molecular analysis of a hospital cafeteria-associated salmonellosis outbreak using modified repetitive element PCR fingerprinting.

A hospital cafeteria-associated outbreak of gastroenteritis due to Salmonella enterica serotype Infantis was retrospectively evaluated using modified repetitive element PCR (rep-PCR) fingerprinting with the ERIC2 and BOXA1R primers and computer-assisted gel analysis and dendrogram construction. Rep-PCR yielded objective between-cycler, same-strain similarity values of from 92% (composite fingerprints) to 96% (ERIC2 fingerprints). The 70 Salmonella isolates (which included 19 serotype Infantis isolates from the hospital outbreak, 10 other serotype Infantis isolates, and 41 isolates representing 14 other serotypes) were resolved well to the serotype level with each of the three fingerprint types (ERIC2, BOXA1R, and composite). Rep-PCR typing uncovered several historical serotyping errors and provided presumptive serotype assignments for other isolates with incomplete or undetermined serotypes. Analysis of replicate fingerprints for each isolate, as generated on two different thermal cyclers, indicated that most of the seeming subserotype discrimination noted in single-cycler dendrograms actually represented assay variability, since it was not reproducible in combined-cycler dendrograms. Rep-PCR typing, which would have been able to identify the presence of the hospital-associated serotype Infantis outbreak after the second outbreak isolate, could be used as a simple surrogate for serotyping by clinical microbiology laboratories that are equipped for diagnostic PCR.

Bacterial Typing Techniques↗

PubMed2001

Analysis of Clostridium difficile isolates from nosocomial outbreaks at three hospitals in diverse areas of Japan.

Clostridium difficile isolates recovered from patients with C. difficile-associated diarrhea (CDAD) at three hospitals located in diverse areas of Japan were analyzed by three typing systems, PCR ribotyping, pulsed-field gel electrophoresis (PFGE), and Western immunoblotting. At the three hospitals examined, a single PCR ribotype strain (type smz) was predominant and accounted for 22 (65%) of 34, 18 (64%) of 28, and 11 (44%) of 25 isolates, respectively. All of the 51 isolates that represented PCR ribotype smz were nontypeable by PFGE because of DNA degradation. Since the type smz strain did not react with any of the antisera against 10 different serogroups (A, B, C, D, F, G, H, I, K, and X), we prepared a new antiserum against a type smz isolate. All 51 type smz isolates presented identical banding patterns, reacting with the newly prepared antiserum (designated subserogroup JP-0 of serogroup JP). These results were compared with those of a strain from a hospital outbreak that occurred in New York, which has been identified as type J9 by restriction enzyme analysis and type 01/A by arbitrarily primed PCR but was nontypeable by PFGE because of DNA degradation. This strain was reported to be epidemic at multiple hospitals in the United States. The J9 strain represented a PCR ribotype pattern different from that of a type smz strain and was typed as subserogroup G-1 of serogroup G by immunoblot analysis. A single outbreak type causing nosocomial CDAD in Japan was found to be different from the strain causing multiple outbreaks in the United States, even though the outbreak strains from the two countries were nontypeable by PFGE because of DNA degradation.

Bacterial Typing Techniques↗

PubMed2001

Outbreak of vancomycin-resistant Enterococcus faecium of the phenotype VanB in a hospital in Warsaw, Poland: probable transmission of the resistance determinants into an endemic vancomycin-susceptible strain.

The first outbreak caused by vancomycin-resistant enterococci of the VanB phenotype in Poland was analyzed. It occurred in a single ward of a Warsaw hospital which is a specialized center for the treatment of hematological disorders. Between July 1999 and February 2000, 11 patients in the ward were found to be infected and/or colonized by Enterococcus faecium that was resistant in vitro to vancomycin and susceptible to teicoplanin. PCR analysis confirmed that the vancomycin-resistant E. faecium (VREM) isolates carried the vanB gene, which is responsible for the VanB phenotype. Pulsed-field gel electrophoresis (PFGE) typing revealed that the isolates belonged to four distinct PFGE types and that one of these was clearly predominant, including isolates collected from seven different patients. The isolates contained one or more copies of the vanB gene cluster of the identical, unique DraI/PagI (BspHI) restriction fragment length polymorphism type, which resided in either the same or different plasmid molecules or chromosomal regions. All this data suggested that the outbreak was due to both clonal spread of a single strain and horizontal transfer of resistance genes among nonrelated strains, which could be mediated by plasmids and/or by vanB gene cluster-containing transposons. The comparative analysis of vancomycin-susceptible E. faecium (VSEM) isolates collected from infections in the same ward at the time of the VREM outbreak has led to identification of a widespread VSEM strain that was possibly related to the major VREM clone. It is very likely that this endemic VSEM strain has acquired vancomycin-resistance determinants and that the acquisition occurred more than once during the outbreak.

Anti-Bacterial Agents↗

PubMed2002

Molecular epidemiological analysis of the changing nature of a meningococcal outbreak following a vaccination campaign.

A serogroup C meningococcal outbreak that occurred in an Israeli Arab village led to a massive vaccination campaign. During the subsequent 18 months, new cases of type B Neisseria meningitidis infection were revealed. To investigate the influence of vaccination on bacteriological epidemiology, bacteria were isolated from individuals at the outbreak location, patients with several additional other sporadic cases, and patients involved in another outbreak. Haploid bacterial genomic DNA was mixed with a consensus PCR product to form a heteroduplex state that enabled multilocus sequence typing (MLST) to be combined with denaturing high-performance liquid chromatography (DHPLC) for a novel high-throughput molecular typing method called MLST-DHPLC. A 100% correlation was found to exist between the sequencing by MLST alone and the MLST-DHPLC method. Independent molecular typing by repetitive extragenic palindromic PCR discriminated the neisserial clones as well as the MLST-DHPLC method did. The occurrence of type B N. meningitidis in the postvaccination period might be attributed to the selection pressure applied to the bacteria by vaccination, suggesting a possible unwarranted outcome of vaccination with the quadrivalent vaccine for control of a serogroup C meningococcal outbreak. This is the first time that DHPLC has been applied to the genotyping of bacteria, and it proved to be more efficient than MLST alone.

Adolescent↗

PubMed2002

Molecular subtyping methods for detection of Salmonella enterica serovar Oranienburg outbreaks.

This study involved 82 Salmonella enterica serovar Oranienburg isolates from patients with gastroenteritis and/or focal infections, healthy carriers, and cuttlefish chips which were epidemiologically linked to a major outbreak that had affected 1,505 people in Japan between 1998 and 1999. We concurrently investigated four different molecular subtyping methods using human salmonellosis-associated Salmonella serovars and their applicability in detection of serovar Oranienburg in an outbreak. Pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic sequence PCR (ERIC2-PCR), or 16S/23S rRNA ribotyping provided a high degree of interserovar discrimination for most of the serovars, with PFGE being the most discriminatory. For intraserovar typing of serovar Oranienburg, ERIC2-PCR was found to be the most sensitive. Native plasmid profiling, however, revealed nine different subgroups among epidemiologically and genetically related outbreak strains. Using these methods, a link was confirmed between food (cuttlefish chips) and patients in the serovar Oranienburg outbreak. This study underscores the limitations of chromosome-based and plasmid-based typing methods.

Animals↗

PubMed2002

Issues associated with and recommendations for using PCR to detect outbreaks of pertussis.

Two outbreaks of respiratory tract illness associated with prolonged cough occurring in 1998 and 1999 in New York State were investigated. A PCR test for Bordetella pertussis was primarily used by a private laboratory to confirm 680 pertussis cases. Several clinical specimens had positive culture results for B. pertussis during both outbreaks, which confirmed that B. pertussis was circulating during the outbreaks. However, testing by the New York State Department of Health reference laboratory suggested that some of the PCR results may have been falsely positive. In addition, features of the outbreak that suggested that B. pertussis may not have been the primary agent of infection included a low attack rate among incompletely vaccinated children and a significant amount of illness among patients testing PCR negative for B. pertussis. These investigations highlight the importance of appropriate clinical laboratory quality assurance programs, of the limitations of the PCR test, and of interpreting laboratory results in context of clinical disease.

Bordetella pertussis↗

PubMed2002

Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers.

A recent outbreak of hand, foot, and mouth disease in Singapore in 2000 affected several thousand children and resulted in four deaths. The aim of this study was to determine the applicability of reverse transcription-PCR (RT-PCR) with universal pan-enterovirus primers and enterovirus 71 (EV71) type-specific primers for the direct detection of enteroviruses in clinical specimens derived from this outbreak. With the universal primers, EV71 RNA sequences were successfully detected by RT-PCR and direct sequencing in 71% of positive specimens. Three pairs of EV71 type-specific primers were evaluated for rapid detection of EV71 directly from clinical specimens and cell culture isolates. By using a seminested RT-PCR strategy, specific identification of EV71 sequences directly in clinical specimens was achieved, with a detection rate of 53%. In contrast, cell culture could isolate EV71 in only 20% of positive specimens. EV71 was detected directly from brain, heart, and lung specimens of two deceased siblings. Although more than one type of enterovirus was identified in clinical specimens from this outbreak, 90% of the enteroviruses were confirmed as EV71. The data demonstrate the clinical applicability of pan-enterovirus and seminested RT-PCR for the detection of EV71 RNA directly from clinical specimens in an outbreak situation.

Child↗

PubMed2002

Molecular epidemiology of caliciviruses causing outbreaks and sporadic cases of acute gastroenteritis in Spain.

The molecular epidemiology of human caliciviruses (HuCVs) causing sporadic cases and outbreaks of acute gastroenteritis around eastern Spain (Catalonia and the Valencian Community) was studied by reverse transcription-PCR (RT-PCR) and by sequencing part of the RNA polymerase gene in open reading frame 1. HuCVs were detected in 44 of 310 stool specimens (14.19%) negative for other enteric pathogens obtained from children with acute gastroenteritis. Norwalk-like viruses (NLVs) were the most common cause of the gastroenteritis outbreaks investigated here. They were detected in 14 out of 25 (56%) outbreaks with an identified pathogen. Genotypes producing both sporadic cases and outbreaks were diverse, with a predominance of GGII strains related to genotypes Melksham and Lordsdale. Five strains clustered with a "new variant" designated GGIIb, which was detected circulating throughout quite a few European countries in the years 2000 and 2001. The emergence mechanism of these strains might be the occurrence of intertypic recombinations between different viruses. The nucleotide sequence of part of the capsid gene (ORF2) from three of these strains demonstrated their relationship with Mexico virus.

Acute Disease↗

PubMed2003

Detection, isolation, and molecular subtyping of Escherichia coli O157:H7 and Campylobacter jejuni associated with a large waterborne outbreak.

The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx(1) and stx(2) Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx(1) and stx(2) was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.

Campylobacter Infections↗

PubMed2003

Use of multienzyme multiplex PCR amplified fragment length polymorphism typing in analysis of outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit.

We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal results with regard to band resolution and discriminatory power were selected and combined. The computer-assisted analysis of fingerprint patterns was performed with Pearson's product-moment correlation values of densitometric curves, without assigning bands to peaks. Thus, the analysis is not subject to human interpretation errors. With this method, we investigated two outbreaks of multiresistant Klebsiella pneumoniae in an intensive care unit and various sporadic isolates of K. pneumoniae and Klebsiella oxytoca. Cluster analysis of isolates analyzed in different experiments and on different gels showed that fingerprint patterns clustered correctly according to subspecies or to the outbreaks. Multienzyme multiplex PCR AFLP revealed that the first outbreak was caused by two different types of strains. Outbreak two was caused by yet another strain of K. pneumoniae. In conclusion, the typing method used here is easy to perform and highly reproducible, and due to generation of complex banding patterns, it has a higher discriminatory power. Furthermore, the multienzyme multiplex PCR fingerprints are easy to analyze, and a reliable database can be stored in the computer to facilitate comparison of future isolates of Klebsiella spp. The method can be performed in every clinical microbiology laboratory.

Cross Infection↗

PubMed2003

Evaluation and utilization as a public health tool of a national molecular epidemiological tuberculosis outbreak database within the United Kingdom from 1997 to 2001.

The aim of this study was to develop a national model and analyze the value of a molecular epidemiological Mycobacterium tuberculosis DNA fingerprint-outbreak database. Incidents were investigated by the United Kingdom PHLS Mycobacterium Reference Unit (MRU) from June 1997 to December 2001, inclusive. A total of 124 incidents involving 972 tuberculosis cases, including 520 patient cultures from referred incidents and 452 patient cultures related to two population studies, were examined by using restriction fragment length polymorphism IS6110 fingerprinting and rapid epidemiological typing. Investigations were divided into the following three categories, reflecting different operational strategies: retrospective passive analysis, retrospective active analysis, and retrospective prospective analysis. The majority of incidents were in the retrospective passive analysis category, i.e., the individual submitting isolates has a suspicion they may be linked. Outbreaks were examined in schools, hospitals, farms, prisons, and public houses, and laboratory cross-contamination events and unusual clinical presentations were investigated. Retrospective active analysis involved a major outbreak centered on a high school. Contact tracing of a teenager with smear-positive pulmonary tuberculosis matched 14 individuals, including members of his class, and another 60 cases were identified in schools clinically and radiologically and by skin testing. Retrospective prospective analysis involved an outbreak of 94 isoniazid-resistant tuberculosis cases in London, United Kingdom, that began after cases were identified at one hospital in January 2000. Contact tracing and comparison with MRU databases indicated that the earliest matched case had occurred in 1995. Subsequently, the MRU changed to an active prospective analysis targeting linked isoniazid-monoresistant isolates for follow up. The patients were multiethnic, born mainly in the United Kingdom, and included professionals, individuals from the music industry, intravenous drug abusers, and prisoners.

Antitubercular Agents↗

PubMed2005

Multiprefectural spread of gastroenteritis outbreaks attributable to a single genogroup II norovirus strain from a tourist restaurant in Nagasaki, Japan.

A series of gastroenteritis outbreaks caused by noroviruses (NVs) among tourist groups from several prefectures was associated with eating a lunch prepared by a restaurant in Nagasaki City, Japan, on 18 and 19 November 2003. A retrospective cohort study was performed to estimate the magnitude of the outbreak and identify the source of infection. Epidemiological information was obtained through the local public health centers in the areas where the illness occurred. Stool and vomit specimens and food and environmental samples were analyzed by reverse transcription-PCR with genogroup-specific primers. Positive samples were sequenced and analyzed phylogenetically. Of 1,492 tourists who ate a lunch prepared by the restaurant during the 2-day period, 660 (44.2%) developed illness, with an average incubation time of 31.2 h. Whereas NVs were not detected in any food samples, identical sequences most closely related to the Mexico genotype of genogroup II NV were found in specimens from case patients, restaurant staff, and the kitchen table. Food handlers were concluded to be the source of the outbreak as a result of the contamination of several meals. The series of outbreaks described here exemplifies the role of tourism as a contemporary way to distribute a single infectious agent to multiple and geographically remote areas.

Adolescent↗

PubMed2005

Use of the oxford multilocus sequence typing protocol and sequencing of the flagellin short variable region to characterize isolates from a large outbreak of waterborne Campylobacter sp. strains in Walkerton, Ontario, Canada.

The Walkerton (Ontario, Canada) outbreak of waterborne Escherichia coli O157:H7 and Campylobacter jejuni was quite limited in both space and time, making it a good model for exploring the utility of different typing and subtyping methods for the characterization of relationships among isolates of these organisms. We have extended previous work with these organisms through analysis by the Oxford multilocus sequence typing (MLST) and the flagellin short variable region (fla-SVR) sequencing methods. Additional isolates not epidemiologically related to the Walkerton outbreak have also been included. Both sequencing methods identified and differentiated between Walkerton outbreak strains 1 and 2. When these strains were compared with isolates that were not part of the outbreak, the information produced by the fla-SVR method more often correlated with epidemiological findings than that produced by MLST, though both methods were required for optimal discrimination. The MLST data were more relevant in terms of the overall population structure of the organisms. Both mutation and recombination appeared to be responsible for generating diversity among the isolates tested.

Animals↗

PubMed2005

Outbreak of dysentery associated with ceftriaxone-resistant Shigella sonnei: First report of plasmid-mediated CMY-2-type AmpC beta-lactamase resistance in S. sonnei.

We document the first report of plasmid-encoded CMY-2-type AmpC beta-lactamase identified among Shigella sonnei isolates resistant to ceftriaxone and obtained after an outbreak of bacillary dysentery in Taiwan. One hundred eighty-two children in two elementary schools in Yu-Li, Taiwan, where an outbreak occurred after a typhoon hit this area in 2001, were enrolled in this study. Clinical and epidemiologic data on the infected children were collected. Pulsed-field gel electrophoresis (PFGE) was performed on the isolates to determine the genetic relatedness of outbreak strains. Plasmid analysis and PCR were performed to identify beta-lactamase genes responsible for ceftriaxone resistance. Forty-seven children from the two elementary schools were culture positive for S. sonnei in this outbreak. Twenty-three children were asymptomatic. Of the total isolates 55.3% were resistant to ampicillin. One hundred percent of the isolates obtained from children in school A were initially susceptible to both ampicillin and ceftriaxone. Of isolates obtained from school B 96.2% were nonsusceptible to ceftriaxone. However, two isolates from school A developed resistance to ampicillin during the course of treatment. All 18 available isolates showed closely related PFGE patterns (4, 4a, 4b, and 4c). CMY-2-type AmpC beta-lactamase was responsible for ceftriaxone resistance in ceftriaxone-nonsusceptible isolates; Southern blot hybridization confirmed that such a resistance gene was located on the plasmid. This is the first report of plasmid-mediated CMY-2-type AmpC beta-lactamase in S. sonnei. Ampicillin-resistant isolates can develop during the course of antibiotic treatment.

Adolescent↗

PubMed2001

Low incidence of reactive arthritis in children following a salmonella outbreak.

OBJECTIVES: To assess the incidence of reactive arthritis (ReA) in an outbreak of salmonella infection in a large cohort of children in Germany. METHODS: A few days after the salmonella outbreak all parents of affected children and all paediatricians and general practitioners in the region were provided with detailed information about the possibility of ReA. Six weeks after the outbreak a telephone call was made to all general practitioners and paediatricians to identify patients with ReA. Ten weeks after the outbreak a questionnaire assessing symptoms of ReA was mailed to all parents, and after a period of 4 months paediatricians and general practitioners were contacted again to search for additional unreported cases of ReA. RESULTS: Of the 286 children (age range 11 months to 9 years) with diarrhoea and stool cultures positive for Salmonella enteritidis lysotype 8/7, not a single case of arthritis was reported over the 4 month period. However, six children (2%) had arthralgia of various duration (1 day to 6 weeks) with a single recurrence in one child. The joint pattern was oligoarticular and lower limb joints (knee/ankle) were affected exclusively. CONCLUSION: The incidence of ReA after salmonella infection in children appears to be very low which may be related to differences in the immune response between children and adults.

Arthritis, Reactive↗

PubMed1979

Hospital outbreak of trimethoprim resistance in pathogenic coliform bacteria.

In an investigation of outbreak of infection caused by coliform bacilli with plasmid-mediated trimethoprim (TMP) resistance many patients were found to be asymptomatic carriers of TMP-resistant coliform bacilli. Analysis of factors predisposing to rectal carriage of these organisms showed that the most important was previous treatment with co-trimoxazole, a sulphonamide, or ampicillin. The outbreak was controlled by a policy restricting the antibiotics given. Geriatric units are an important source of hospital infection. When an outbreak occurs the logical sequence of steps to be taken is to monitor cases, identify the outbreaks, analyse the causative factors, plan corrective action jointly with laboratory staff, and monitor the outcome.

Aged↗