Development and application of MLVA methods as a tool for inter-laboratory surveillance.

Multiple-locus variable-number of tandem-repeats analysis (MLVA) has emerged as a valuable method for subtyping bacterial pathogens and has been adopted in many countries as a critical component of their laboratory-based surveillance. Lack of harmonisation and standardisation of the method, however, has made comparison of results generated in different laboratories difficult, if not impossible, and has therefore hampered its use in international surveillance. This paper proposes an international consensus on the development, validation, nomenclature and quality control for MLVA used for molecular surveillance and outbreak detection based on a review of the current state of knowledge.

Proof-of-concept study for successful inter-laboratory comparison of MLVA results.

Multiple-locus variable-number of tandem repeats analysis (MLVA) is widely used for typing of pathogens. Methods such as MLVA based on determining DNA fragment size by the use of capillary electrophoresis have an inherent problem as a considerable offset between measured and real (sequenced) lengths is commonly observed. This discrepancy arises from variation within the laboratory set-up used for fragment analysis. To obtain comparable results between laboratories using different set-ups, some form of calibration is a necessity. A simple approach is to use a set of calibration strains with known allele sizes and determine what compensation factors need to be applied under the chosen set-up conditions in order to obtain the correct allele sizes. We present here a proof-of-concept study showing that using such a set of calibration strains makes inter-laboratory comparison possible. In this study, 20 international laboratories analysed 15 test strains using a five-locus Salmonella enterica serovar Typhimurium MLVA scheme. When using compensation factors derived from a calibration set of 33 isolates, 99.4% (1,461/1,470) of the MLVA alleles of the test strains were assigned correctly, compared with 64.8% (952/1,470) without any compensation. After final analysis, 97.3% (286/294) of the test strains were assigned correct MLVA profiles. We therefore recommend this concept for obtaining comparable MLVA results.

Substantial increase in listeriosis, Denmark 2009.

In 2009, 97 cases of listeriosis were reported in Denmark (1.8 per 100,000), a significant rise over the previous year. The increase was seen both in cases of bacteraemia and meningitis and affected mainly people aged 70 years and older. A foodborne outbreak of eight cases was identified by pulsed-field gel electrophoresis typing. No explanation has so far been found for the marked increase in incidence. An increasing trend has been observed since 2003 and possible explanations are discussed.

Systematic serotyping and riboprinting of Campylobacter spp. improves surveillance: experiences from two Danish counties.

In order to monitor the distribution of subtypes of Campylobacter and to identify clusters, 975 isolates of Campylobacter spp., obtained from human infections occurring in two Danish counties, were studied during a 1-year period. The isolates were characterised by Penner serotyping and automated ribotyping. Pulsed-field gel electrophoresis (PFGE) profiling was used to confirm clustering of identical serotypes and ribotypes. The 975 isolates were divided into 48 serotypes, 210 ribotypes and 277 serotype-ribotype combinations. The overall distribution of serotypes and ribotypes was similar between the two counties. After taking into account the rare or common occurrence of subtypes, a model identified 43 clusters of subtypes during the study period. Clustered isolates represented 28% (273/975) of the study population, with clusters containing between three and 20 isolates. PFGE confirmed the validity of selected clusters identified by serotyping and ribotyping. The observed clustering of Campylobacter isolates, with identical types in time and place, indicates that common-source outbreaks of campylobacteriosis are more common than is usually thought.

An outbreak of Campylobacter jejuni associated with consumption of chicken, Copenhagen, 2005.

In May/June 2005 an outbreak of diarrhoeal illness occurred among company employees in Copenhagen. Cases were reported from seven of eight companies that received food from the same catering kitchen. Stool specimens from three patients from two companies were positive for Campylobacter jejuni. We performed a retrospective cohort study among employees exposed to canteen food in the three largest companies to identify the source of the outbreak and to prevent further spread. Using self-administered questionnaires we collected information on disease, days of canteen food eaten and food items consumed. The catering kitchen was inspected and food samples were taken. Questionnaires were returned by 295/348 (85%) employees. Of 247 employees who ate canteen food, 79 were cases, and the attack rate (AR) was 32%. Consuming canteen food on 25 May was associated with illness (AR 75/204, RR=3.2, 95%CI 1.3-8.2). Consumption of chicken salad on this day, but not other types of food, was associated with illness (AR=43/97, RR=2.3, 95%CI 1.3-4.1). Interviews with kitchen staff indicated the likelihood of cross-contamination from raw chicken to the chicken salad during storage. This is the first recognised major Campylobacter outbreak associated with contaminated chicken documented in Denmark. It is plausible that food handling practices contributed to transmission, and awareness of safe food handling and storage has since been raised among kitchen staff. The low number of positive specimens accrued in this outbreak suggests a general underascertainment of adult cases in the laboratory reporting system by a factor of 20.

Evidence of transmission of verocytotoxin-producing Escherichia coli O111 from a cattle stable to a child.

Infections with verocytotoxigenic Escherichia coli (VTEC) other than O157 have been assumed to have the same epidemiology as those with VTEC O157, but the source of infection is rarely defined for sporadic cases. This report describes a child with VTEC O111:H- infection who was probably infected by playing in a cattle stable and/or by drinking raw milk from the cows in this stable. E. coli O111 isolates colonising the cattle were indistinguishable from the patient isolate by the use of serotyping, pulsed-field gel electrophoresis, and virulence profiling.

The Escherichia coli K-12 gntP gene allows E. coli F-18 to occupy a distinct nutritional niche in the streptomycin-treated mouse large intestine.

Escherichia coli F-18 is a human fecal isolate that makes type 1 fimbriae, encoded by the fim gene cluster, and is an excellent colonizer of the streptomycin-treated mouse intestine. E. coli F-18 fimA::tet, lacking type 1 fimbriae, was constructed by bacteriophage P1 transduction of the fim region of the E. coli K-12 strain ORN151, containing the tetracycline resistance gene from Tn10 inserted in the fimA gene, into E. coli F-18. E. coli F-18 fimA::tet was found to occupy a distinct niche in the streptomycin-treated mouse intestine when fed in small numbers (10(4) CFU) to mice, along with large numbers (10(10) CFU) of E. coli F-18, as defined by the ability of the E. coli F-18 fimA::tet strain to grow and colonize only 1 order of magnitude below E. coli F-18. The same effect was observed when mice already colonized with E. coli F-18 were fed small numbers of E. coli F-18 fimA::tet. Experiments which show that the E. coli K-12 gene responsible for this effect is not fim::tet but gntP, which maps immediately downstream of the fim gene cluster, are presented. gntP encodes a high-affinity gluconate permease, suggesting that the distinct niche in the mouse large intestine is defined by the presence of gluconate. The data presented here support the idea that small numbers of an ingested microorganism can colonize the intestine as long as it can utilize an available nutrient better than any of the other resident species can.

A double blind comparative multicentre study of remoxipride and haloperidol in schizophrenia.

Seventy-two patients fulfilling the DSM-III criteria for schizophrenia and schizophreniform psychosis were admitted to a multicentre, double-blind controlled study to evaluate the efficacy and safety of remoxipride in comparison to haloperidol. The mean daily dose of remoxipride at the end of treatment was 353 mg and of haloperidol, 11 mg. Patients were assessed each week on the Brief Psychiatric Rating Scale (BPRS), the Clinical Global Impression (CGI) and the symptoms checklist. No significant differences in efficacy were found between the two treatments. The median total BPRS score in the remoxipride group was 25 at start of active treatment and 17 at the last valid rating (n = 31). For the haloperidol group the corresponding figures were 24 and 15 (n = 29). According to the CGI, 40% of remoxipride patients and 50% of haloperidol patients were much or very much improved. Treatment-emergent extrapyramidal symptoms, such as akathisia and rigidity, occurred significantly more frequently, and were more severe during treatment with haloperidol than with remoxipride (p = 0.012 and 0.024, respectively). Haloperidol-treated patients reported significantly more drowsiness and increased sleep during treatment (p = 0.026 and 0.012, respectively). No statistically significant differences were seen in endocrine or autonomic symptoms. Remoxipride seemed to be as effective as haloperidol, had a lower frequency of side effects, and was used safely in the dose range 150-600 mg/day.

Paroxetine: pharmacokinetic and antidepressant effect in the elderly.

To evaluate the pharmacokinetic properties, efficacy, and tolerability of paroxetine in elderly depressed patients, a clinical study was set up--initially at Aalborg Psychiatric Hospital in Denmark, and subsequently at the University Hospital in Linköping, Sweden. A total of 21 patients with a median age of 72 years were included in the study. After a single dose of 20 or 30 mg of paroxetine followed by two drug-free days, treatment continued with 20 or 30 mg daily for seven weeks. The majority of patients showed a continuous reduction in their HAMD scores, starting in the second week of treatment. Paroxetine was well tolerated at the doses given, and side-effects were mostly mild and transient. Steady-state, pre-dose plasma levels of paroxetine showed considerable variability, and the median steady-state concentration was higher in elderly patients compared with data from a previous study in young volunteers. Elimination half-lives also showed variability between these elderly patients, but tended to be longer after cessation of multiple dosing than after a single dose. They also tended to be longer than in the young volunteers. The results of this study do not advocate reduced doses of paroxetine in the elderly, but further studies are warranted.