Quantitative Analysis of Nucleic Acid Extraction Methods for Vibrio cholerae Using Real-time PCR and Conventional PCR.

The devastating diarrheal disease cholera is caused by the bacterium Vibrio cholerae. Nucleic acid extraction is the primary step for several molecular detection approaches. In order to identify the sources of cholera illness, an efficient, fast and easy DNA extraction method for toxigenic Vibrio cholerae is desired which can be applied on diverse type of samples. This methodology developmental setup study was performed in the Environmental Microbiology Lab, Department of Microbiology, University of Dhaka, Bangladesh from March 2015 to May 2015 attempted to compare three DNA extraction methods for efficient detection and quantification of Vibrio cholerae. Three nucleic acid extraction methods: (Boiled template, Phenol: Chloroform: Isoamyl alcohol, QiaAmp® mini kit), were assessed for four, routinely tested, templates: crude culture, suspension in water, phosphate buffer saline (PBS), and food by conventional and real time PCR targeting the toxin-coding ctxA gene. Finally, the results were compared in context of processing time and overall cost. Cycle threshold (Ct) values were within acceptable parameters by real time PCR (5 to 35 cycles). All the three extraction methods produced sufficient yield of DNA and copy number for detection by real time and conventional PCR. The boiled template method for water samples yielded low amount of DNA in comparison to the other methods, and is therefore sensitive to detect by non-quantitative, conventional PCR only. Despite an overall low detectability from water samples, our comparison reveals that the boiled template method is the most suitable method for high quality and quantity pathogenic DNA particularly in light of limited access to expensive kits and reagents, time constraints, and high sample load.

Morphological and molecular analyses of larval taeniid species in small mammals from contrasting habitats in Denmark.

Taeniid infections in intermediate hosts manifest themselves as extraintestinal larval stages which, in early development, lack species-specific characteristics. The inability to distinguish infections of zoonotic importance such as Echinococcus multilocularis from other taeniid infections that have mainly veterinary significance stimulated the development of species-specific molecular diagnostics. In this study, the prevalence of taeniid infections in potential intermediate hosts was evaluated using both morphological diagnosis and a newly described multiplex polymerase chain reaction (PCR) for species determination. Small mammals (N= 719) were trapped in three different types of habitats in north-east Zealand, Denmark. The sensitivity of the multiplex PCR (90.5%) exceeded that of morphological examination (57.9%) for identifying 95 taeniid infections. The use of the multiplex PCR resulted in higher prevalence rates due to improved detection of immature liver infections with Hydatigera taeniaeformis and Versteria mustelae, but did not affect the observed prevalence rates of peritoneal metacestodes of Taenia polyacantha. The prevalence of taeniid infections showed a significant difference according to habitat type, potentially identifying a 'sylvatic' transmission and an 'urban' transmission, with marked variation among different taeniid species. Versteria mustelae and T. polyacantha were more prevalent in rural forests, while infections with H. taeniaeformis were dominant in urban parks/forests and in residential and farm gardens. The multiplex PCR facilitated a better utilization of wildlife samples by yielding a higher number of definitive diagnoses of ambiguous taeniid infections in liver lesions, allowing for more accurate epidemiological data and, hence, a more accurate risk assessment.

How health systems in sub-Saharan Africa can benefit from tuberculosis and other infectious disease programmes.

Weak and dysfunctional health systems in low-income countries, particularly in sub-Saharan Africa, are recognised as major obstacles to attaining the health-related Millennium Development Goals by 2015. Some progress is being made towards achieving the targets of Millennium Development Goal 6 for tuberculosis (TB), HIV/AIDS and malaria, with the achievements largely resulting from clearly defined strategies and intervention delivery systems combined with large amounts of external funding. This article is divided into four main sections. The first highlights the crucial elements that are needed in low-income countries in sub-Saharan Africa to deliver good quality health care through general health systems. The second discusses the main characteristics of infectious disease and TB control programmes. The third illustrates how TB control and other infectious disease programmes can help to strengthen these components, particularly in human resources; infrastructure; procurement and distribution; monitoring, evaluation and supervision; leadership and stewardship. The fourth and final section looks at progress made to date at the international level in terms of policy and guidelines, with some specific suggestions about this might be moved forward at the national level. For TB and other infectious disease programmes to drive broad improvements in health care systems and patient care, the lessons that have been learnt must be consciously applied to the broader health system, and sufficient financial input and the engagement of all players are essential.

Occurrence of multiple infections with different Borrelia burgdorferi genospecies in Danish Ixodes ricinus nymphs.

The pathogen Borrelia burgdorferi causes Lyme Borreliosis in human and animals world-wide. In Europe the pathogen is transmitted to the host by the vector Ixodes ricinus. The nymph is the primary instar for transmission to humans. We here study the infection rate of five Borrelia genospecies: B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae in nymphs, by IFA and PCR. 600 nymphs were collected in North Zealand of Denmark. Each nymph was first analysed by IFA. If positive for spirochaetal infection, the genospecies was determined by PCR. The infection rate of B. burgdorferi sensu lato was 15.5%, with the primary genospecies being B. afzelii (64.3%), B. garinii (57.1%), and B. lusitaniae (26.8%). It is the first time B. lusitaniae is documented in Denmark. Even though, the highest infection rate was discovered for B. afzelii and B. garinii, mixed infections are more common than single infections. Fifty-one percent (29/56) of these were infected with two genospecies, 7.1% (4/56) with three, and 5.3% (3/56) with four. We try to explain the high infection rate and the peculiar number of multiple infections, with a discussion of changes host abundance and occurrence of different transmission patterns.

Ixodes ricinus: the potential of two-dimensional gel electrophoresis as a tool for studying host-vector-pathogen interactions.

Ixodes ricinus is a three-host tick, with three active instars. For moulting to occur the tick has to find a host where it can take a blood meal. Throughout feeding I. ricinus can be infected or infect the host with different pathogens, e.g., Tick-Borne Encephalitis virus or Borrelia burgdorferi. The host-vector-pathogen interaction is very complex, making a detailed study difficult. Here we analyse the potential of two-dimensional gel electrophoresis (2DE) to study the host-vector-pathogen interaction. We examined 20 nymphs, which as larvae parasitised either mouse or hen. After moulting, they were kept alive for up to 30 weeks, to analyse whether tick ageing influenced host determination, and for comparison of the 2D-gels. Even though the number of proteins in the gel decreased during ageing, some proteins of the host determination persisted for all 30 weeks. We also discovered persisting proteins in relation to nymphs. These findings showed that 2DE is suitable as a tool for studying host-vector-pathogen interactions.

Five decades of tick-man interaction in Denmark--an analysis.

The tick Ixodes ricinus (L.) is a known vector of several zoonotic diseases such as Tick Borne Encephalitis (TBE), Lyme borreliosis and ehrlichiosis. The interaction between humans and ticks are at the very core of our understanding of the epidemiology of tick borne zoonoses, but only few experimental studies have been performed. Hence our understanding of the epidemiology of tick-borne disease has to rest on the assumption that the potential tick activity, which is reflected in tick samples from the vegetation or samples from animals, corresponds with tick-human interaction. Observations which may reflect the long term tick-human contact are available in Denmark. These observations are records of requests for information on I. ricinus forwarded to the Danish Pest Infestation Laboratory (DPIL). This article was based on the assumption that the request could be seen as a measure of tick-human interaction reflecting the tick densities in relative terms. The perceived tick densities given by the number of requests on I. ricinus divided by the total number of requests to DPIL was fairly stable from 1965 to 1985, while it doubled during the late 1980's reaching a higher level in the beginning of the 1990's. The perceived density was well explained by the variation in temperature, solar radiation (monthly measurement) and deer abundance (yearly assessments). The measure of deviation, i.e. the ratio between observed requests + 1 and modelled requests + 1, for the individual years varied between 0.91 and 1.20, with considerable within-year variation. A gradual change in periodicity of the residual might suggest gradually changing tick population dynamics. In conclusion, the perceived tick densities appear to be consistent with the current knowledge of tick ecology and tick-transmitted diseases.

A comparative study of skills in virtual laparoscopy and endoscopy.

BACKGROUND: The present study was designed to investigate whether there is a correlation between manual skills in laparoscopic procedures and manual skills in flexible endoscopy. METHODS: In a prospective study using laparoscopy and endoscopy simulators (MIST-VR, and GI-Mentor II), 24 consecutive subjects (gastrointestinal surgeons, novice and experienced gastroenterologists, and untrained subjects) were asked to perform laparoscopic and endoscopic tasks. Their performance was assessed by the simulators' software and by observers blinded to the levels of subjects' experience. Performance in experienced vs inexperienced subjects was compared. Score pairs of three parameters--time, errors, and economy of movement--were also compared. RESULTS: Experienced subjects performed significantly better than inexperienced subjects on both tasks in terms of time, errors, and economy of movement (p < 0.05). All three performance parameters in laparoscopy and endoscopy correlated significantly (p < 0.02). CONCLUSION: Both simulators can distinguish between experienced and inexperienced subjects. Observed skills in simulated laparoscopy correlate with skills in simulated flexible endoscopy. This finding may have an impact on the design of training programs involving both procedures.

Seasonal and diel activity of Ixodes ricinus (Acari: Ixodidae) subpopulations in Denmark. Aspects of size, physiological age, and malate dehydrogenase genotype in a forest site without any undergrowth.

UNLABELLED: The underlying population dynamics and the behavioural patterns of the vectors are key issues in understanding the transmission of vector borne pathogens. For the tick Ixodes ricinus both seasonal and diel activity have been described as bimodal patterns, which in seasonal aspect has been interpreted as representing two cohorts. However, recent studies have shown that this interpretation may be incorrect. The aim of this study was to obtain more detailed information on nymph host seeking by studying subpopulations of ticks during the day and season. The study was designed to allow for comparisons of the diel variation and seasonal variation in their dependency in a number of tick characteristics. The study took place in a forest with planted beech trees without any undergrowth. Ticks were collected by flagging the dead leaves on the forest floor. For each nymph, a number of visual observations were made. The size and physiological age was observed and the nymphs were genotyped in the malate dehydrogenase locus (MDH, E.C. 1.1.1.37). Briefly the main results can be given as: (i) There were significant differences in the composition of size classes during the season, but only limited trends in time. (ii) The proportion of the small nymphs was highly variable, with a variation from 3% to 24% in October and September, respectively. (iii) The diel variation in MDH genotypes was significant in May and August. (iv) Nymph size classes and physiological age appeared to interact. The non-random interaction was caused by a relatively even distribution of small nymphs in all four age classes, while large nymphs tended to fall into age class 2 and 3. The length by age interaction for the individual months was noted to be significant in May, July, August and September, but not in June. Similarly the interaction was significant in the morning and afternoon, but not at midday. The overall results describe the seasonal and diel activity patterns as changing systematically for several characteristics under the influence of weather condition and population dynamics. IN CONCLUSION: The observations are best interpreted as being produced of a single cohort of ticks, but the revealed complexity of the host seeking activity suggest that measures of activity x abundance should be interpreted very cautiously in relation to population dynamics.

[Gastroesophageal reflux surgery in Denmark 1997-1999]. / Operation for gastroøsofageal refluks i Danmark 1997-1999.

INTRODUCTION: The incidence and length of postoperative stay in hospital in Denmark after oesophageal reflux surgery has not been described previously. MATERIAL AND METHODS: We examined the national patient hospital register and discharge information from the hospitals for the number of reflux operations carried out, the length of postoperative hospital stay, readmissions, and postoperative complications occurring within 30 days in the period, 1997-1999. RESULTS: Twenty-four departments performed 4.9 operations/100,000 inhabitants per year, corresponding to 788 operations. The postoperative 30-day mortality was 0.4% and 6.6% of the operations were followed by serious surgical complications. The combined primary + readmission hospital stay was 3.7 days in the first postoperative month. DISCUSSION: The small number of oesophageal reflux operations performed in many departments in Denmark is not in agreement with the Danish National Board of Health recommendations from 1996, which say that the operations should be done in only five departments. The spreading of surgery means that only few surgeons can reach sufficient training of surgery and that statistical comparison between the departments of the early results of treatment is not meaningful. Overall, national results are comparable to international results. The incidence of oesophageal reflux surgery is about three times lower than in other Scandinavian countries. On these results, it is recommended that the operation is performed in fewer departments and by fewer surgeons.

[Complications of cholecystectomy before and after introduction of laparoscopic surgery]. / Komplikationer til kolecystektomi før og efter indførelse af laparoskopisk kirurgi.

OBJECTIVES: In the University Department of Surgical Gastroenterology, Arhus University Hospital, the frequency of complications to acute and elective cholecystectomy was investigated before and after introduction of laparoscopic cholecystectomy. MATERIAL AND METHODS: A retrospective survey turned up 1768 patients in the period of conventional open surgery (1974-1985) and 1432 patients in the laparoscopic period (1991-1998). All patients with exploration of the common bile duct were excluded. RESULTS: In the open period 12 patients (0.68%) had lesions of the common bile duct requiring reconstruction. In patients operated on with laparoscopic cholecystectomy, lesions of the common bile duct were found in three patients (0.21%) (p = 0.06). Lesions of the cystic duct or an aberrant bile duct occurred in 14 patients (0.79%) during the open period and in 24 patients (1.7%) (p < 0.05) operated on with the laparoscopic technique. Infectious and cardiopulmonary complications were seen in significantly more patients operated on with the open technique. DISCUSSION: It is concluded that laparoscopic cholecystectomy offers a safe procedure, although the technique can still be improved, especially with respect to decreasing leakage from the cystic duct.

Temporal risk assessment for Lyme borreliosis in Denmark.

The prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus nymphs in Denmark was found to be approximately 5%. The mean abundance of infected nymphs varied from 0.3 to 4.4 per 100 m2 according to site. The seasonal occurrence of infected nymphs in a beech forest coincided with seasonal distribution of neuroborreliosis cases. In order to establish a working hypothesis, it was assumed that the availability of habitats and human habitat preferences is one of the factors leading to low number of neuroborreliosis cases in the spring. In addition, this paper gives a description of the neuroborreliosis cases in Denmark in the period 1985-97 and offers a possible explanation for the variation in cases. The explanation is based on an assessment of tick density, which by comparison with the number of neuroborreliosis cases provides information on the infectivity of ticks. The model suggests that high temperatures and low precipitation in the autumn is essential for the transmission of B. burgdorferi sensu lato to reservoir hosts or development of B. burgdorferi sensu lato within ticks, which secures high tick infectivity in the following season.

Spatial risk assessment for Lyme borreliosis in Denmark.

A study of nymphal and adult Ixodes ricinus density was performed in well-defined spruce and beech forest habitats with different levels of roe deer abundance and soil water capacity. In 35 habitats, a total of 489 larvae, 1,611 nymphs and 193 adult I. ricinus ticks were collected. Tick density was found to be influenced by roe deer abundance and soil water capacity. Based on this evaluation, a model predicting increasing number of ticks with increasing roe deer abundance and soil water capacity was suggested. A total of 1,045 nymphs and 106 adult ticks were tested for infection with Borrelia burgdorferi sensu lato Of these, 53 nymphs and 6 adults were found to be infected, leading to an general infection rate of 5% and 6% for nymphs and adults, respectively. Prevalences of Borrelia burgdorferi sensu lato in nymphal I. ricinus were found to be independent of roe deer abundance and soil water capacity. The correlation between human neuroborreliosis incidence and the estimated number of I. ricinus based on roe deer abundance and soil water capacity was examined. Differences in human neuroborreliosis incidence were found to correspond with the expected spatial differences in tick density in 12 counties in Denmark.

Peculiarities of behaviour of taiga (Ixodes persulcatus) and sheep (Ixodes ricinus) ticks (Acarina: Ixodidae) determined by different methods.

A comparison of the behavioural peculiarities of Ixodes persulcatus Schulze, 1930 (north-western population, Russia) and Ixodes ricinus (L., 1758) from western Russia and Denmark was determined by using two methods. Method 1 involved a sojourn of ticks on vertical plastic sticks and showed that the questing behaviour of I. ricinus nymphs was dependent on temperature and relative humidity (RH). A significantly greater number of nymphs quested at 22 degrees C and 100% RH than at 18 degrees C. When the humidity was reduced to 30% all of the nymphs departed. In the second method, the activity of ticks on an inclined "ticksdrome" was estimated. The activity of I. ricinus adults from the Danish population was 1.2 times greater than that of ticks from Russia. Females of the species studied and specimens from all study areas were more active than all other stages of development. The locomotor activity of both adult and immature I. ricinus that were infected with Borrelia burgdorferi sensu lato was suppressed when compared with uninfected specimens. The locomotor activity of I. persulcatus females infected by borreliae with exoskeleton anomalies was 1.3 times greater (P<0.05) than that of infected ticks without anomalies. Our data showed that infected females with exoskeleton anomalies could crawl faster on a human and reach uncovered parts of the body that are vulnerable for attachment and feeding. A study of locomotor activity and questing behaviour may be useful for comparing the risk for different tick species and populations to transmit tick-borne pathogens.

Host seeking activity of ixodes ricinus ticks based on daily consecutive flagging samples.

The seasonality of I. ricinus was studied by three daily consecutive flaggings in four sites situated in an ecotone between a spruce forest and a peaty meadow, a spruce forest, a beech forest and a dense spruce forest. First day flagging samples represent the most commonly used method for description of tick seasonality. Since two consecutive flaggings remove ticks in the vegetation, third day flagging samples represent the number of ticks ascending into the vegetation, which was interpreted as being closely related to the size of the free living tick population. First day samples of nymphs were reasonably consistent with the typical bimodal seasonal pattern of activity (Gray, 1991). Third day samples showed a more stable seasonal development with no or only limited bimodality. The host seeking period or retention time in the vegetation, as defined by the ratio between first and third day samples differed within the season. The host seeking periods were found to be long in the spring, short in midsummer and intermediate in autumn. By analysing the host seeking period for dependency of weather parameters, it was found that host seeking periods primarily depend on relative humidity and solar radiation. No significant difference in tick abundance in third day samples could be related to weather conditions.

Diurnal activity of Ixodes ricinus in Denmark: aspects of physiological age and genotypic variation.

Four different alleles in the nymphal Ixodes ricinus were found for the Malate Dehydrogenase enzyme (MDH). The allele frequency of the MDH genotypes in nymphal I. ricinus was observed to change during the day. Some genotypes were present in the morning, but not in the afternoon and evening. The physiological age of the nymphs was also determined. No significant correlation could be found between physiological age and genotypes. Based on the results it must be anticipated that the comparability of tick samples not only differs in quantity but also in quality for various hours, but not necessarily for various months. Therefore isoenzyme analysis of nymphs at least as regards the MDH alleles appears to be a convenient biological tool to analyse differences in host seeking behaviour.

Criteria, performance and diagnostic problems in diagnosing acute otitis media.

OBJECTIVE: We aimed to assess criteria when diagnosing acute otitis media and related performance in general practice in Denmark. Furthermore, we aimed to identify the scale of and the reasons for diagnostic uncertainty. METHODS: We conducted: (i) a survey among GPs assessing criteria; and (ii) prospective registration of acute otitis-media-related consultations performed by GPs assessing performance. The survey was sent to all 790 GPs in Funen, North Jutland and Ringkøbing counties, Denmark. A total of 568 (72%) of all GPs in the three counties responded. A total of 368 children with acute otitis media or previous acute otitis media visiting 151 GPs were studied. The main outcome measures were: (i) criteria for symptoms and findings suggesting the diagnosis acute otitis media, criteria for use of equipment and reasons for diagnostic uncertainty; and (ii) prevalence of symptoms and findings in diagnosed cases, equipment used and multivariate analysis of factors predicting diagnostic certainty. RESULTS: The symptoms of earache, fever, reduced hearing, findings of bulging eardrum, red eardrum and purulent otorrhea were important criteria used during both diagnosis of acute otitis media by the GPs and assessment of performance. In the prospective study, diagnostic certainty of acute otitis media was 67% (95% CI 58-76) in children under 2 years and 75% (95% CI 69-81) in older children. Diagnostic certainty was statistically related (P < 0.05) to a good view of the eardrum and the findings of purulent otorrhea or a bulging eardrum. Logistic regression revealed that the two most important factors predicting diagnostic certainty were a satisfactory view of the eardrum, with an odds ratio (OR) 11.0 (95% CI 4.1-29.5), and purulent otorrhea OR 10.1 (95% CI 3.1-32.9). Main reasons for diagnostic uncertainty given by GPs were differential diagnostic doubts, insufficient view of the eardrum and lack of knowledge. CONCLUSION: Danish GPs' criteria for the diagnosis of acute otitis media were stricter than criteria used internationally. The discrepancy between diagnostic criteria and performance was small. Diagnostic accuracy and certainty could be substantially improved by cleaning the ear canal when needed and by widespread use of pneumatic otoscopy.

[National surveillance of Helicobacter pylori eradication in Denmark. Results based on registration of 34.582 drug prescriptions]. / National overvågning af Helicobacter pylori-eradikationsterapi i Danmark. Resultater fra registreringen af 34.582 receptudskrivninger.

We wanted to characterize the use of H. pylori eradication therapy in Denmark (inhabitants 5,227,862). All H. pylori eradication treatments from a nation wide database covering all drug prescriptions in the period January 1994-June 1996 were identified. We found 28,784 out-patients having a prescription with drugs for H. pylori eradication, accounting for 34,582 prescriptions in total. The incidence of new consumers was 220 per 10(5) inhabitants per year, with a maximum at 70-79 years of age. Eighty-six percent of the patients had only one treatment course. Forty-five percent had an anti-ulcer drug prescribed 1-12 months after the H. pylori eradication therapy. Consumption of antibiotics used for H. pylori eradication accounted for 1.4% of the total consumption of antibiotics. In conclusion, the incidence of H. pylori eradication therapy was fairly stable but with changes in the pattern of drug regimens used. Anti-ulcer drugs were often given after H. pylori eradication therapy, suggesting an inappropriate use of eradication treatment.

Antibiotic treatment of acute otitis media. Criteria and performance in Danish general practice.

OBJECTIVE: To assess implicit criteria (i.e. what the general practitioner (GP) considers good clinical practice) for and performance (i.e. what the GP actually does) with regard to antibiotic treatment of acute otitis media in Danish general practice. DESIGN: a) Criteria assessed by survey among general practitioners. b) Performance assessed by prospective registration of consultations with general practitioners related to otitis media. SETTING: General practices in three Danish counties. SUBJECTS: a) All the GPs in the three countries (n = 790). b) 368 children with acute otitis media. MAIN OUTCOME MEASURES: a) Criteria for timing of treatment and first drug of choice for acute otitis media; b) prescribed antibiotics and multivariate analysis of factors predicting antibiotic prescription. RESULTS: a) The response rate was 72%. Only 51% (95% CI 47-55) of GPs would give antibiotics to all children with acute otitis media, and 79% (95% CI 76-82) of GPs would use penicillin-V as first drug of choice. b) Seventy-four per cent (95% CI 68-81) of children with acute otitis media were given antibiotics. Factors predicting the GPs' decision to prescribe antibiotics were the general condition of the child and the factors that are normally used in diagnosing the condition. CONCLUSION: Danish general practitioners' criteria for antibiotic treatment of acute otitis media are restrictive, with non-antibiotic treatment in cases of short duration and penicillin-V as first drug of choice. Performance suggests a less restrictive pattern.