Parallel artificial liquid membrane extraction of psychoactive analytes: a novel approach in therapeutic drug monitoring.

AIM: Liquid-liquid extraction is widely used in therapeutic drug monitoring of antipsychotics, but difficulties in automation of the technique can result in long operational time. In this paper, parallel artificial liquid membrane extraction was used for extraction of serotonin- and serotonin-norepinephrine reuptake inhibitors from human plasma, and an approach to automate the technique was investigated. RESULTS: Eight model analytes were extracted from 125 µl human plasma with recoveries in the range 72-111% (relative standard deviation [RSD] ≤12.8%). A semiautomated pipettor was successfully utilized in the procedure, reducing the manual handling time. Real patient samples were analyzed with satisfying accuracy. CONCLUSION: A semiautomated extraction of serotonin-and serotonin-norepinephrine reuptake inhibitors by parallel artificial liquid membrane extraction extraction was successfully performed.

CRISPR Diversity and Microevolution in Clostridium difficile.

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.

Standardised surveillance of Clostridium difficile infection in European acute care hospitals: a pilot study, 2013.

Clostridium difficile infection (CDI) remains poorly controlled in many European countries, of which several have not yet implemented national CDI surveillance. In 2013, experts from the European CDI Surveillance Network project and from the European Centre for Disease Prevention and Control developed a protocol with three options of CDI surveillance for acute care hospitals: a 'minimal' option (aggregated hospital data), a 'light' option (including patient data for CDI cases) and an 'enhanced' option (including microbiological data on the first 10 CDI episodes per hospital). A total of 37 hospitals in 14 European countries tested these options for a three-month period (between 13 May and 1 November 2013). All 37 hospitals successfully completed the minimal surveillance option (for 1,152 patients). Clinical data were submitted for 94% (1,078/1,152) of the patients in the light option; information on CDI origin and outcome was complete for 94% (1,016/1,078) and 98% (294/300) of the patients in the light and enhanced options, respectively. The workload of the options was 1.1, 2.0 and 3.0 person-days per 10,000 hospital discharges, respectively. Enhanced surveillance was tested and was successful in 32 of the hospitals, showing that C. difficile PCR ribotype 027 was predominant (30% (79/267)). This study showed that standardised multicountry surveillance, with the option of integrating clinical and molecular data, is a feasible strategy for monitoring CDI in Europe.

Enteroaggregative Escherichia coli in Daycare-A 1-Year Dynamic Cohort Study.

Enteroaggregative Escherichia coli (EAEC) has been associated with persistent diarrhea, reduced growth acceleration, and failure to thrive in children living in developing countries and with childhood diarrhea in general in industrialized countries. The clinical implications of an EAEC carrier-status in children in industrialized countries warrants clarification. To investigate the pathological significance of an EAEC carrier-state in the industrialized countries, we designed a 1-year dynamic cohort study and performed follow-up every second month, where the study participants submitted a stool sample and answered a questionnaire regarding gastrointestinal symptoms and exposures. Exposures included foreign travel, consumption of antibiotics, and contact with a diseased animal. In the capital area of Denmark, a total of 179 children aged 0-6 years were followed in a cohort study, in the period between 2009 and 2013. This is the first investigation of the incidence and pathological significance of EAEC in Danish children attending daycare facilities. Conventional microbiological detection of enteric pathogens was performed at Statens Serum Institute, Copenhagen, Denmark, and at Hvidovre Hospital, Copenhagen, Denmark. Parents completed questionnaires regarding gastrointestinal symptoms. The EAEC strains were further characterized by serotyping, phylogenetic analysis, and susceptibility testing. EAEC was detected in 25 (14%) of the children during the observational period of 1 year. One or more gastrointestinal symptoms were reported from 56% of the EAEC-positive children. Diarrhea was reported in six (24%) of the EAEC positive children, but no cases of weight loss, and general failure to thrive were observed. The EAEC strains detected comprised a large number of different serotypes, confirming the genetic heterogeneity of this pathotype. EAEC was highly prevalent (n = 25, 14%) in Danish children in daycare centers and was accompanied by gastrointestinal symptoms in 56% of the infected children. No serotype or phylogenetic group was specifically linked to children with disease.

Childhood diarrhoea in Danish day care centres could be associated with infant colic, low birthweight and antibiotics.

AIM: Diarrhoea is very common in children attending day care centres. The aim of this study was to examine certain predisposing risk factors for an association with diarrhoea, including foreign travel, treatment with antibiotics, having household pets, infant colic, bottle feeding, using a pacifier and low birthweight. METHODS: A dynamic one-year follow-up cohort study comprising 179 children from 36 day care centres was conducted from September 2009 to July 2013 in Copenhagen, Denmark. Questionnaires were sent to the children's parents or legal guardians every two months for a year, requesting information on gastrointestinal symptoms and exposure. A logistic regression was performed to identify the odds ratios of different risk factors for diarrhoea. RESULTS: The odds ratios for diarrhoea were 1.97 (0.93-4.20) for children with a history of infant colic, 1.91 (0.90-4.04) for low birthweight children and 1.45 (0.74-2.82) for children who had used antibiotics. Having a pet in the household had a possible protective effect towards diarrhoeal events, with an odds ratio of 0.47 (0.20-1.09). CONCLUSION: A history of infant colic, low birthweight, and to a lesser extent antibiotic use, possibly increased the risk of diarrhoea in Danish children in day care centres.

Treatment of Clostridium difficile infection in mice with vancomycin alone is as effective as treatment with vancomycin and metronidazole in combination.

OBJECTIVE: Clostridium difficile is a major cause of nosocomial infectious diarrhoea. Treatment of C. difficile infection (CDI) depends on disease severity. A combination of vancomycin and metronidazole is often recommended in severe cases. The aim of this study was to examine, in a murine model of CDI, if mice treated with a combination of vancomycin and metronidazole had a better clinical outcome than mice treated with vancomycin or metronidazole alone. DESIGN: C57BL/6J mice pretreated with an antimicrobial mixture were challenged with C. difficile VPI 10463 or phosphate-buffered saline by oral gavage. After the challenge, the mice were treated with placebo, vancomycin, metronidazole or a combination of vancomycin and metronidazole for 10 days. The mice were monitored for 20 days with weight and a clinical score. Stool samples were examined for C. difficile spore load and presence of C. difficile toxins. RESULTS: None of the mice in the vancomycin-treated group died during the treatment phase compared to a mortality of 17%, 33% and 55% in the combination, metronidazole and infected control group, respectively. Mice treated with vancomycin alone or in combination with metronidazole recovered from CDI faster than mice treated with metronidazole alone. However, after discontinuation of treatment, vancomycin-treated and combination-treated mice succumbed to clinical and bacteriological relapse. CONCLUSIONS: Mice treated with vancomycin alone had a better clinical outcome in the treatment phase of CDI than mice treated with metronidazole alone. A combination of vancomycin and metronidazole did not improve the clinical outcome when compared to treatment with vancomycin alone. TRIAL REGISTRATION NUMBER: The trial registration number from the Danish Experimental Animal Inspectorate is J number 2012-15-2934-00422.

Novel multiplex format of an extended multilocus variable-number-tandem-repeat analysis of Clostridium difficile correlates with tandem repeat sequence typing.

Subtyping of Clostridium difficile is crucial for outbreak investigations. An extended multilocus variable-number tandem-repeat analysis (eMLVA) of 14 variable number tandem repeat (VNTR) loci was validated in multiplex format compatible with a routine typing laboratory and showed excellent concordance with tandem repeat sequence typing (TRST) and high discriminatory power.

Identification of clinical aeromonas species by rpoB and gyrB sequencing and development of a multiplex PCR method for detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media.

Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.

Epidemiology and clinical manifestations of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) represents a heterogeneous group of E. coli strains. The pathogenicity and clinical relevance of these bacteria are still controversial. In this review, we describe the clinical significance of EAEC regarding patterns of infection in humans, transmission, reservoirs, and symptoms. Manifestations associated with EAEC infection include watery diarrhea, mucoid diarrhea, low-grade fever, nausea, tenesmus, and borborygmi. In early studies, EAEC was considered to be an opportunistic pathogen associated with diarrhea in HIV patients and in malnourished children in developing countries. In recent studies, associations with traveler's diarrhea, the occurrence of diarrhea cases in industrialized countries, and outbreaks of diarrhea in Europe and Asia have been reported. In the spring of 2011, a large outbreak of hemolytic-uremic syndrome (HUS) and hemorrhagic colitis occurred in Germany due to an EAEC O104:H4 strain, causing 54 deaths and 855 cases of HUS. This strain produces the potent Shiga toxin along with the aggregative fimbriae. An outbreak of urinary tract infection associated with EAEC in Copenhagen, Denmark, occurred in 1991; this involved extensive production of biofilm, an important characteristic of the pathogenicity of EAEC. However, the heterogeneity of EAEC continues to complicate diagnostics and also our understanding of pathogenicity.

Metronidazole therapy for treating dientamoebiasis in children is not associated with better clinical outcomes: a randomized, double-blinded and placebo-controlled clinical trial.

BACKGROUND: There is a paucity of evidence documenting the pathogenicity of Dientamoeba fragilis, an intestinal protozoan common in children. As case reports on successful treatment are numerous, many authors advocate treatment, despite no placebo-controlled trials being available. Metronidazole is often used for treatment, though eradication rates are relatively low (60%-80%). In the present study we determined the clinical and microbiological efficacy of metronidazole in Danish children. METHODS: In this parallel placebo-controlled double-blinded trial, children aged 3-12 years with >4 weeks of gastrointestinal symptoms were allocated using block randomization in a 1:1 ratio to a 10-day course of oral metronidazole or placebo. Primary outcome was change in level of gastrointestinal symptoms, measured on a visual-analog-scale (VAS), and secondary outcome was eradication of D. fragilis infection. Participants, caregivers, investigators, and sponsor were all blinded to group assignment. The trial was registered with clinicaltrials.gov (NCT01314976) prior to start. RESULTS: Of 96 participants, 48 were allocated to the metronidazole and placebo group each. Mean VAS change from pre- to post-treatment did not differ significantly (P = .8) between the metronidazole (-1.8 CI, [-2.5, -1.1]) and the placebo group (-1.6 CI, [-2.3, -.9]). Eradication of D. fragilis was significantly greater in the metronidazole group, although it declined rapidly from 62.5% 2 weeks after end of treatment to 24.9% 8 weeks after end of treatment. CONCLUSIONS: These findings do not provide evidence to support routine metronidazole treatment of D. fragilis positive children with chronic gastrointestinal symptoms. Study funded by Statens Serum Institut. CLINICAL TRIALS REGISTRATION: Trial was registered with clinicaltrials.gov (NCT01314976).

Biased hand use in captive emperor tamarins (Saguinus imperator).

Comparing forelimb use across primates can improve the understanding of the mechanisms and functions of brain asymmetry. We broadened the comparative framework by investigating hand use biases across spontaneous behaviors in 11 captive emperor tamarins of 2 family groups. We found a right preference across 58% of pooled unimanual bouts and 60% of all tasks. The maximum biases for a single task were 88% right-hand use and 78% left-hand use. On an individual basis, for most tasks, 6 tamarins preferred their right hand, 2 tamarins the left, and 2 animals were ambidextrous. Only a single tamarin did not switch between left and right hand bias according to task, but displayed a consistent right-hand bias. Our accompanying review of the 2 dozen previous callitrichid hand use studies confirms this picture. Across the board, these report hand preferences for single tasks. Task specialization is found in less than half of the studies, hand specialization in less than a third, whereas population level handedness is almost never reported. Importantly, right-hand use is preponderant, but very rarely consistent across tasks or subjects. This might be attributable to the fact that often-employed simple (food-reaching) tasks may not be coupled to a specialized hemisphere and therefore not constrain forelimb use. A better understanding of what causes directional forelimb use in callitrichids and other animals is currently hampered by a lack of standardized methods including measurement criteria, task difficulty, and social setting as well as a dearth of taxonomic diversity and field studies.

Poultry as a possible source of non-typhoidal Salmonella enterica serovars in humans in Bangladesh.

We investigated Salmonella enterica isolates from human clinical cases of gastroenteritis to determine the distribution of non-typhoidal Salmonella serovars in the human population, and compared them to isolates originating from poultry by serotyping, phage typing, plasmid profiling, pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) to evaluate the potential role of poultry in human non-typhoidal salmonellosis in Bangladesh. Nine different serovars were identified among the human isolates of which Salmonella Paratyphi B var Java (S. Java), S. Kentucky, S. Enteritidis, S. Virchow and S. Weltevreden also were commonly isolated from poultry. The poultry isolates belonging to S. Java, S. Kentucky and S. Enteritidis were indistinguishable from human isolates or genetically closely related, based on PFGE profiles and MLST. S. Kentucky clone ST198 and S. Java clone ST43 both well-known cause of human infections were also isolated from poultry.

Molecular characterization of motile serovars of Salmonella enterica from breeder and commercial broiler poultry farms in Bangladesh.

Contaminated poultry and poultry products are a major source of motile Salmonellae for human salmonellosis worldwide. Local circulation of any motile Salmonella serovar in poultry has a wider public health impact beyond its source of origin for being dispersed elsewhere through poultry trades or human travels. To investigate the status of motile Salmonella serovars in breeder farms in Bangladesh, multiple flocks of two breeder farms were observed for a period of six months. In addition, a cross-sectional survey was carried out to determine the prevalence and serovar distribution of motile Salmonella by randomly selecting 100 commercial broiler poultry farms. Five pooled faecal samples representing an entire housed flock of breeders or broilers were screened for presence of motile Salmonella following conventional bacteriological procedures. The Salmonella isolates obtained were subsequently serotyped, and characterized by plasmid profiling and pulsed-field gel electrophoresis (PFGE). The results revealed that both the breeder farms were positive with three Salmonella serovars: S. Virchow, S. Paratyphi B var Java (S. Java) and S. Enteritidis. Eleven of the 100 broiler farms investigated were positive for motile Salmonella, giving a farm-level prevalence of 11% (95% confidence interval 5-17%). S. Virchow and S. Kentucky were the two predominant serovars isolated from the broiler farms. The PFGE genotyping demonstrated that the isolates belonging to the same serovars were closely related due to variation in only 1-4 bands. All the S. Virchow and S. Java isolates, irrespective of breeder or broiler farm origin, were plasmid-free, except for one S. Virchow isolate from a broiler farm that harboured a 9.7 kb-sized plasmid. The S. Kentucky isolates belonged to three plasmid profiles having plasmids of four different sizes, ranging from 2.7 to 109 kb. This is the first report of any motile Salmonella serovars from breeder and commercial broiler poultry farms in Bangladesh.

Real-time TaqMan polymerase chain reaction-based genus-identification and pyrosequencing-based species identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus directly on stool samples.

A new method was developed for Campylobacter identification and applied directly on 599 stool samples from diarrhoeagenic patients. Here, the gyrase B gene of Campylobacter was targeted in a 2-step process: first, TaqMan polymerase chain reaction (PCR)-based identification of C. jejuni, C. coli, C. upsaliensis, C. lari, and C. fetus at the genus level, and, second, pyrosequencing-based identification at the species level. The TaqMan PCR method was compared to culturing and identified 87 Campylobacter-positive samples of which 64 were culture positive. Among the discrepant 23 samples, 18 were confirmed positive by conventional PCR, underlining a significant increase in diagnostic yield by use of this molecular and culture-independent method. For species identification, the pyrosequencing method was compared to conventional PCR and among the 87 TaqMan PCR-positive samples, 74 Campylobacter species were identified by both methods, 10 samples gave discrepant results, and 3 samples were negative by both methods.

Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh.

Salmonella is a globally widespread food-borne pathogen having major impact on public health. All motile serovars of Salmonella enterica of poultry origin are zoonotic, and contaminated meat and raw eggs are an important source to human infections. Information on the prevalence of Salmonella at farm/holding level, and the zoonotic serovars circulating in layer poultry in the South and South-East Asian countries including Bangladesh, where small-scale commercial farms are predominant, is limited. To investigate the prevalence of Salmonella at layer farm level, and to identify the prevalent serovars we conducted a cross-sectional survey by randomly selecting 500 commercial layer poultry farms in Bangladesh. Faecal samples from the selected farms were collected following standard procedure, and examined for the presence of Salmonella using conventional bacteriological procedures. Thirty isolates were randomly selected, from the ninety obtained from the survey, for serotyping and characterized further by plasmid profiling and pulsed-field gel electrophoresis (PFGE). Results of the survey showed that the prevalence of motile Salmonella at layer farm level was 18% (95% confidence interval 15-21%), and Salmonella Kentucky was identified to be the only serovar circulating in the study population. Plasmid analysis of the S. Kentucky and non-serotyped isolates revealed two distinct profiles with a variation of two different sizes (2.7 and 4.8 kb). PFGE of the 30 S. Kentucky and 30 non-serotyped isolates showed that all of them were clonally related because only one genotype and three subtypes were determined based on the variation in two or three bands. This is also the first report on the presence of any specific serovar of Salmonella enterica in poultry in Bangladesh.

[Intracerebral abscess after infection with non-toxigenic Vibrio cholerae]. / Cerebral absces med Vibrio cholerae non-01 efter badning i dansk havvand.

We present the first case of intracerebral abscess after blood-borne infection with non-toxigenic Vibrio cholerae (non-01). The patient was a 66 year-old woman who was infected after swimming in Danish seawater during an unusually hot summer. She had predisposing haemochromatosis and a skin lesion on the ankle. We treated the patient with meropenem and ciprofloxacin for six weeks followed by ciprofloxacin for four weeks, and she recovered with hemiparesis and speech impairment. Marine Vibrio species may produce intracranial infection in predisposed individuals, even in temperate climate zones.

Multiplex PCR method for detection of Clostridium difficile tcdA, tcdB, cdtA, and cdtB and internal in-frame deletion of tcdC.

A multiplex PCR method was developed for the detection of Clostridium difficile toxin genes tcdA, tcdB, ctdA, and cdtB and the major in-frame deletion types (18, 39, and 54 bp) of tcdC. The method has high specificity for PCR ribotype 027 and may identify other C. difficile strains of clinical and epidemiological importance.

Binary toxin and death after Clostridium difficile infection.

We compared 30-day case-fatality rates for patients infected with Clostridium difficile possessing genes for toxins A and B without binary toxin (n = 212) with rates for patients infected with C. difficile possessing genes for A, B, and binary toxin. The latter group comprised patients infected with strains of PCR ribotype 027 (CD027, n = 193) or non-027 (CD non-027, n = 72). Patients with binary toxin had higher case-fatality rates than patients without binary toxin, in univariate analysis (relative risk [RR] 1.8, 95% confidence interval [CI] 1.2-2.7) and multivariate analysis after adjustment for age, sex, and geographic region (RR 1.6, 95% CI 1.0-2.4). Similar case-fatality rates (27.8%, 28.0%) were observed for patients infected with CD027 or CD non-027. Binary toxin either is a marker for more virulent C. difficile strains or contributes directly to strain virulence. Efforts to control C. difficile infection should target all virulent strains irrespective of PCR ribotype.

Blastocystis sp. subtype 4 is common in Danish Blastocystis-positive patients presenting with acute diarrhea.

Fecal samples from 444 Danish patients presenting with acute diarrhea were tested for Blastocystis and positive samples were subtyped to investigate the prevalence and subtype distribution of Blastocystis in this patient group. A total of 25 patients (5.6%) were positive, and 19 of these patients (76.0%) were positive for Blastocystis sp. ST4. Because the relative prevalence of ST4 in other patients presenting with other types of diarrhea (persistent, travel-related, and human immunodeficiency virus-related) in Denmark is low, the role of Blastocystis sp. ST4 in the etiology of acute diarrhea should be investigated further.

Five commercial DNA extraction systems tested and compared on a stool sample collection.

In this study, 5 different commercial DNA extraction systems were tested on a stool sample collection containing 81 clinical stool specimens that were culture-positive for diarrheagenic Escherichia coli, Campylobacter jejuni, Salmonella enterica, or Clostridium difficile. The purified DNAs were analyzed by polymerase chain reaction (PCR) directed toward the relevant organisms. The results showed that conventional PCR combined with the extraction systems BioRobot EZ1 (Qiagen, Hilden, Germany), Bugs'n Beads (Genpoint, Oslo, Norway), ChargeSwitch (Invitrogen, Paisley, UK), QIAamp Stool Mini Kit (Qiagen), and 2 protocols (generic and Specific A) for EasyMag (BioMérieux, Marcy I'Etoile, France) were able to identify 89%, 62%, 85%, 88%, 85%, and 91%, respectively, of the pathogens originally identified by conventional culture-based methods. When TaqMan PCR was combined with the EasyMag Specific A protocol, 99% of the samples were correctly identified. The results demonstrate that the extraction efficiencies can vary significantly among different extraction systems, careful optimization may have a significant positive effect, and the use of sensitive and specific detection methods like TaqMan PCR is an ideal choice for this type of analysis.