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Leptospirosis is a zoonosis caused by the spirochete Leptospira spp. It is often not clear why certain areas appear to be hotspots for human leptospirosis. Therefore, a predictive risk map for the Netherlands was developed and assessed, based on a random forest model for human leptospirosis incidence levels with various environmental factors and rat density as variables. Next, it was tested whether misclassifications of the risk map could be explained by the prevalence of Leptospira spp. in brown rats. Three recreational areas were chosen, and rats (≥25/location) were tested for Leptospira spp. Concurrently, it was investigated whether Leptospira spp. prevalence in brown rats was associated with Leptospira DNA concentration in surface water, to explore the usability of this parameter in future studies. Approximately 1 L of surface water sample was collected from 10 sites and was tested for Leptospira spp. Although the model predicted the locations of patients relatively well, this study showed that the prevalence of Leptospira spp. infection in rats may be an explaining variable that could improve the predictive model performance. Surface water samples were all negative, even if they had been taken at sites with a high Leptospira spp. prevalence in rats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

A 6-month-old girl with an isolated bald spot was seen at the general practice. We diagnosed aplasia cutis congenita, a rare disorder with a wide variation in clinical symptoms. Most lesions can be managed conservatively. Larger defects, however, require surgery.

Parasites often have complex developmental cycles that account for their presence in a variety of difficult-to-analyze matrices, including feces, water, soil, and food. Detection of parasites in these matrices still involves laborious methods. Untargeted sequencing of nucleic acids extracted from those matrices in metagenomic projects may represent an attractive alternative method for unbiased detection of these pathogens. Here, we show how publicly available metagenomic datasets can be mined to detect parasite specific sequences, and generate data useful for environmental surveillance. We use the protozoan parasite Cryptosporidium parvum as a test organism, and show that detection is influenced by the reference sequence chosen. Indeed, the use of the whole genome yields high sensitivity but low specificity, whereas specificity is improved through the use of signature sequences. In conclusion, querying metagenomic datasets for parasites is feasible and relevant, but requires optimization and validation. Nevertheless, this approach provides access to the large, and rapidly increasing, number of datasets from metagenomic and meta-transcriptomic studies, allowing unlocking hitherto idle signals of parasites in our environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Franssen, Janse, Janssen, Caccio, Vatta, van der Giessen and van Passel.)

A 14-year-old girl presented with brownish grey macules on and around the lips. Genetic testing revealed a mutation in the LKB1 tumor suppressor gene. The diagnosis made was Peutz-Jeghers syndrome, a rare inherited disease that is characterized by gastrointestinal polyps and an increased risk of cancer.

Genome sequences provide information on the genetic elements present in an organism, and currently there are databases containing hundreds of thousands of bacterial genome sequences. These repositories allow for mining patterns concerning antibiotic resistance gene occurrence in both pathogenic and non-pathogenic bacteria in e.g. natural or animal environments, and link these to relevant metadata such as bacterial host species, country and year of isolation, and co-occurrence with other resistance genes. In addition, the advances in the prediction of mobile genetic elements, and discerning chromosomal from plasmid DNA, broadens our view on the mechanism mediating dissemination. In this study we utilize the vast amount of data in the public database PATRIC to investigate the dissemination of carbapenemase-encoding genes (CEGs), the emergence and spread of which is considered a grave public health concern. Based on publicly available genome sequences from PATRIC and manually curated CEG sequences from the beta lactam database, we found 7,964 bacterial genomes, belonging to at least 70 distinct species, that carry in total 9,892 CEGs, amongst which bla NDM , bla OXA , bla . We were able to distinguish between chromosomally located resistance genes (4,137; 42%) and plasmid-located resistance genes (5,753; 58%). We found that a large proportion of the identified CEGs were identical, i.e. displayed 100% nucleotide similarity in multiple bacterial species (8,361 out of 9,892 genes; 85%). For example, the New Delhi metallo-beta-lactamase NDM-1 was found in 42 distinct bacterial species, and present in seven different environments. Our data show the extent of carbapenem-resistance far beyond the canonical species VIM , bla IMP and bla KPC . We were able to distinguish between chromosomally located resistance genes (4,137; 42%) and plasmid-located resistance genes (5,753; 58%). We found that a large proportion of the identified CEGs were identical, i.e. displayed 100% nucleotide similarity in multiple bacterial species (8,361 out of 9,892 genes; 85%). For example, the New Delhi metallo-beta-lactamase NDM-1 was found in 42 distinct bacterial species, and present in seven different environments. Our data show the extent of carbapenem-resistance far beyond the canonical species Acetinobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa . These types of data complement previous systematic reviews, in which carbapenem-resistant Enterobacteriaceae were found in wildlife, livestock and companion animals. Considering the widespread distribution of CEGs, we see a need for comprehensive surveillance and transmission studies covering more host species and environments, akin to previous extensive surveys that focused on extended spectrum beta-lactamases. This may help to fully appreciate the spread of CEGs and improve the understanding of mechanisms underlying transmission, which could lead to interventions minimizing transmission to humans., Competing Interests: The authors declare there are no competing interests., (©2021 Janse et al.)

Unsafe drinking water is a recognized health threat in Ethiopia, and climate change, rapid population growth, urbanization and agricultural practices put intense pressure on availability and quality of water. Climate change-related health problems due to floods and waterborne diseases are increasing. With increasing insight into impacts of climate change and urbanization on water availability and quality and of required adaptations, a shift towards climate-resilient water safety planning was introduced into an Ethiopian strategy and guidance document to guarantee safe drinking water. Climate-resilient water safety planning was implemented in the urban water supplies of Addis Ababa and Adama, providing drinking water to 5 million and 500,000 people, respectively. Based on the risks identified with climate-resilient water safety planning, water quality monitoring can be optimized by prioritizing parameters and events which pose a higher risk for contaminating the drinking water. Water quality monitoring was improved at both drinking water utilities and at the Public Health Institute to provide relevant data used as input for climate-resilient water safety planning. By continuously linking water quality monitoring and climate-resilient water safety planning, utilization of information was optimized, and both approaches benefit from linking these activities.

Background: Hidradenitis suppurativa (HS) is a chronic debilitating skin disease, frequently located in the groin and anogenital area, leading to a substantial impact on quality of life and sexual health in patients with HS. Skin-tissue-sparing excision with electrosurgical peeling (STEEP) is a procedure with known low recurrence rates and high patient satisfaction in retrospective series. However, a prospective study to investigate the impact of any major surgery on specific aspects of the quality of life has not yet been performed., Objective: To assess surgical outcomes and the effect of major surgery on the general quality of life, sexual health and activity impairment in patients with HS., Materials and Methods: A single centre prospective survey study was conducted among 40 patients undergoing major surgery. Surveys were completed prior to the surgery and 2, 6, 12 and 26 weeks after surgery. Besides the objective parameters (time to wound closure and surface of the wound), patient-reported outcomes were reported., Results: Thirty-nine patients with a total of 171 survey responses were included for analysis. Patients with Hurley stage I or II had a shorter time to wound closure (TTWC) compared to patients with Hurley stage III (P = 0.005). TTWC was significantly prolonged in patients treated with biologics (P < 0.001). Smoking did not significantly influence TTWC. For patient-reported outcomes, DLQI and ASEX scores did not significantly improve during the study period of 6 months. However, activity and overall work impairment showed considerable improvement after surgery., Conclusion: Time to wound closure is significantly prolonged by higher Hurley stage and treatment with biologics, contrastingly not by smoking. Major surgery improved the overall work and daily activity impairment., (© 2019 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Microbial air pollution from livestock farms has raised concerns regarding public health. Little is known about airborne livestock-related microbial levels in residential areas. We aimed to increase insights into this issue. Air measurements were performed in 2014 and 2015 at 61 residential sites in The Netherlands. Quantitative-PCR was used to assess DNA concentrations of selected bacteria (commensals: Escherichia coli and Staphylococcus spp.; a zoonotic pathogen: Campylobacter jejuni) and antimicrobial resistance (AMR) genes ( tetW, mecA) in airborne dust. Mixed models were used to explore spatial associations (temporal adjusted) with livestock-related characteristics of the surroundings. DNA from commensals and AMR genes was detectable even at sites furthest away from farms (1200 m), albeit at lower levels. Concentrations, distinctly different between sites, were strongly associated with the density of farms in the surroundings especially with poultry and pigs. C. jejuni DNA was less prevalent (42% of samples positive). Presence of C. jejuni was solely associated with poultry (OR: 4.7 (95% CI: 1.7-14), high versus low poultry density). Residential exposure to livestock-related bacteria and AMR genes was demonstrated. Identified associations suggest contribution of livestock farms to microbial air pollution in general and attribution differences between farm types. This supports the plausibility of recent studies showing health effects in relation to residential proximity to farms.

Background: Hidradenitis suppurativa (HS) is a chronic, debilitating, heterogeneous disease requiring different treatment approaches. Recently, we refined the classic Hurley classification into a seven-stage classification in order to guide these treatment choices. This new classification subdivides Hurley stage I and II into three substages, namely mild (A), moderate (B) and severe (C) HS disease. Hurley stage III is not subcategorized and is always severe., Objectives: To investigate the correlation between the given severity grades of Hurley I and Hurley II in the refined Hurley classification, and the patient-reported quality of life and physician-assessed objective severity score., Materials and Methods: In this cross-sectional study, patients with HS participating in the observational cohorts of two Dutch tertiary referral centres were included before June 2017. The patient-reported Dermatology Life Quality Index (DLQI) and physician-assessed International HS Severity Score System (IHS4) scores were compared between the refined Hurley stages., Results: In total, 433 patients were analysed. DLQI and IHS4 scores increased within Hurley stage I and II from A through C. There was a significant positive correlation of DLQI and IHS4 with increasing refined Hurley substages [refined Hurley stage I (A, B and C) to DLQI: r s = 0·259, P < 0·001 and refined Hurley stage II (A, B and C) to DLQI: r s = 0·185, P = 0·010; refined Hurley stage I (A, B and C) to IHS4: r s = 0·603, P < 0·001 and refined Hurley stage II (A, B and C) to IHS4: r s = 0·532, P < 0·001]., Conclusions: The refined Hurley classification accurately correlates with HS severity assessed by both patients and clinicians. Therefore, the refined Hurley classification is a useful tool for the quick assessment of severity in HS., (© 2018 British Association of Dermatologists.)

From 2007 through 2010, the Netherlands experienced the largest Q fever epidemic ever reported. This study integrates the outcomes of a multidisciplinary research programme on spatial airborne transmission of Coxiella burnetii and reflects these outcomes in relation to other scientific Q fever studies worldwide. We have identified lessons learned and remaining knowledge gaps. This synthesis was structured according to the four steps of quantitative microbial risk assessment (QMRA): (a) Rapid source identification was improved by newly developed techniques using mathematical disease modelling; (b) source characterization efforts improved knowledge but did not provide accurate C. burnetii emission patterns; (c) ambient air sampling, dispersion and spatial modelling promoted exposure assessment; and (d) risk characterization was enabled by applying refined dose-response analyses. The results may support proper and timely risk assessment and risk management during future outbreaks, provided that accurate and structured data are available and exchanged readily between responsible actors., (© 2018 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.)

Tularemia is an emerging zoonosis caused by the Gram-negative bacterium Francisella tularensis , which is able to infect a range of animal species and humans. Human infections occur through contact with animals, ingestion of food, insect bites or exposure to aerosols or water, and may lead to serious disease. F. tularensis may persist in aquatic reservoirs. In the Netherland, no human tularemia cases were notified for over 60 years until in 2011 an endemic patient was diagnosed, followed by 17 cases in the 6 years since. The re-emergence of tularemia could be caused by changes in reservoirs or transmission routes. We performed environmental surveillance of F. tularensis in surface waters in the Netherlands by using two approaches. Firstly, 339 samples were obtained from routine monitoring -not related to tularemia- at 127 locations that were visited between 1 and 8 times in 2015 and 2016. Secondly, sampling efforts were performed after reported tularemia cases (n = 8) among hares or humans in the period 2013-2017. F. tularensis DNA was detected at 17% of randomly selected surface water locations from different parts of the country. At most of these positive locations, DNA was not detected at each time point and levels were very low, but at two locations contamination was clearly higher. From 7 out of the 8 investigated tularemia cases, F. tularensis DNA was detected in at least one surface water sample collected after the case. By using a protocol tailored for amplification of low amounts of environmental DNA, 10 gene targets were sequenced. Presence of F. tularensis subspecies holarctica was confirmed in 4 samples, and in 2 of these, clades B.12 and B.6 were identified. This study shows that for tularemia, information regarding the spatial and temporal distribution of its causative agent could be derived from environmental surveillance of surface waters. Tracking a particular strain in the environment as source of infection is feasible and could be substantiated by genotyping, which was achieved in water samples with only low levels of F. tularemia present. These techniques allow the establishment of a link between tularemia cases and environmental samples without the need for cultivation.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife., (This article is copyright of The Authors, 2017.)

Background: Hidradenitis suppurativa (HS) has a major impact on patients' quality of life (QoL). Although it has commonly been assumed that HS impairs sexual health, only a single case-control study has been performed on sexual functioning in a small group of patients with HS., Objectives: To investigate the QoL with a particular focus on sexual health in a substantial population of patients with HS., Methods: In total 916 patients with HS received an invitation to participate in this multicentre cross-sectional survey., Results: Three hundred patients completed the questionnaires. This study showed a diminished QoL and sexual health in patients with HS (Female Sexual Function Index: 21·6 ± 9·6, International Index of Erectile Function: 49·7 ± 20·7, Arizona Sexual Experience Scale: 16·7 ± 5·3, Dermatology Life Quality Index: 12·5 ± 7·5). Sexual health was associated with QoL in women but not in men. Female sex and late onset of HS were associated with poor sexual function. Impairment of QoL was associated with anogenital involvement, early onset of HS, disease severity and disease activity., Conclusions: HS is associated with impaired sexual health and QoL. Physicians should not hesitate to ask patients with HS about their sexual function and, when needed, offer them psychological support., (© 2016 British Association of Dermatologists.)

The largest global Q fever outbreak occurred in The Netherlands during 2007 to 2010. Goats and sheep were identified as the major sources of disease. Here, we report the first complete genome sequence of ITALIC! Coxiella burnetiigoat outbreak strain NL3262 and that of an epidemiologically linked chronic human strain, both having the outbreak-related ITALIC! CbNL01multilocus variable-number tandem-repeat analysis (MLVA) genotype., (Copyright © 2016 Kuley et al.)

One of the largest Q fever outbreaks ever occurred in the Netherlands from 2007-2010, with 25 fatalities among 4,026 notified cases. Airborne dispersion of Coxiella burnetii was suspected but not studied extensively at the time. We investigated temporal and spatial variation of Coxiella burnetii in ambient air at residential locations in the most affected area in the Netherlands (the South-East), in the year immediately following the outbreak. One-week average ambient particulate matter < 10 μm samples were collected at eight locations from March till September 2011. Presence of Coxiella burnetii DNA was determined by quantitative polymerase chain reaction. Associations with various spatial and temporal characteristics were analyzed by mixed logistic regression. Coxiella burnetii DNA was detected in 56 out of 202 samples (28%). Airborne Coxiella burnetii presence showed a clear seasonal pattern coinciding with goat kidding. The spatial variation was significantly associated with number of goats on the nearest goat farm weighted by the distance to the farm (OR per IQR: 1.89, CI: 1.31-2.76). We conclude that in the year after a large Q fever outbreak, temporal variation of airborne Coxiella burnetii is suggestive to be associated with goat kidding, and spatial variation with distance to and size of goat farms. Aerosol measurements show to have potential for source identification and attribution of an airborne pathogen, which may also be applicable in early stages of an outbreak.

Hidradenitis suppurativa is difficult to treat owing to its complex pathomechanism; beside the extensive inflammation with abscesses and inflammatory nodules, there is also an architectural loss with sinus tract formation and in severe cases with extensive scarring. Therefore, surgery is mandatory in moderate and severe HS., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping, assay accessibility and inter-laboratory comparisons.

Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S-layer, and prophage-lambda. Following a review of the literature, an in silico analysis of all signature sequences reported for identification of B. anthracis was conducted. Published primer and probe sequences were compared for specificity against 134 available Bacillus spp. genomes. Although many of the chromosomal targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European laboratories was then performed to evaluate six assays, including the WHO recommended procedures, using a collection of 90 Bacillus strains. Three assays performed adequately, yielding no false positive or negative results. All three assays target chromosomal markers located within the lambdaBa03 prophage region (PL3, BA5345, and BA5357). Detection limit was further assessed for one of these highly specific assays.

Many pathogens that can cause major public health, economic, and social damage are relatively easily accessible and could be used as biological weapons. Wildlife is a natural reservoir for many potential bioterrorism agents, and, as history has shown, eliminating a pathogen that has dispersed among wild fauna can be extremely challenging. Since a number of wild rodent species live close to humans, rodents constitute a vector for pathogens to circulate among wildlife, domestic animals, and humans. This article reviews the possible consequences of a deliberate spread of rodentborne pathogens. It is relatively easy to infect wild rodents with certain pathogens or to release infected rodents, and the action would be difficult to trace. Rodents can also function as reservoirs for diseases that have been spread during a bioterrorism attack and cause recurring disease outbreaks. As rats and mice are common in both urban and rural settlements, deliberately released rodentborne infections have the capacity to spread very rapidly. The majority of pathogens that are listed as potential agents of bioterrorism by the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases exploit rodents as vectors or reservoirs. In addition to zoonotic diseases, deliberately released rodentborne epizootics can have serious economic consequences for society, for example, in the area of international trade restrictions. The ability to rapidly detect introduced diseases and effectively communicate with the public in crisis situations enables a quick response and is essential for successful and cost-effective disease control.

Aim: To investigate the Coxiella burnetii DNA content in environmental samples that may contribute to the transmission of C. burnetii., Methods and Results: During a large Q fever outbreak in the Netherlands, surface swabs and aerosol samples were collected inside stables and around six Q fever-affected ruminant farms, which are located in municipalities varying in Q fever incidence. After the outbreak in 2010, aerosol samples were collected in the same geographical areas. The use of an optimized multiplex qPCR for the detection of C. burnetii DNA revealed that all samples obtained inside stables were positive. In addition, the C. burnetii DNA content in aerosol samples collected in stables is significantly higher than in aerosol samples collected around the farms. Finally, the C. burnetii DNA content in aerosol samples collected in the same geographical locations was lower in 2010 in comparison with 2009., Conclusions: The reduction in C. burnetii DNA content in aerosol samples between 2009 and 2010 is in agreement with the reduction in Q fever incidence in the same geographical areas., Significance and Impact of the Study: The presence of C. burnetii DNA in environmental samples collected on and around ruminant farms supports the hypothesis that C. burnetii can be disseminated from ruminant farms to the surrounding areas., (© 2013 National Institute for Public Health © 2013 The Society for Applied Microbiology.)

Background: Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance., Methods: Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated., Results: A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction., Conclusions: The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

Background: The bacterium Coxiella burnetii has caused unprecedented outbreaks of Q fever in the Netherlands between 2007 and 2010. Since 2007, over 4000 human cases have been reported, with 2354 cases in 2009 alone. Dairy goat farms were identified as most probable sources for emerging clusters of human Q fever cases in their vicinity. However, identifying individual farms as primary source for specific clusters of human cases remains a challenge, partly due to limited knowledge of the different C. burnetii strains circulating in livestock, the environment and humans., Results: We used a multiplex multi-locus variable number of tandem repeats analysis (MLVA) assay to investigate the genotypic diversity of C. burnetii in different types of samples that were collected nationwide during the Dutch Q fever outbreaks between 2007 and 2010. Typing was performed on C. burnetii positive samples obtained from several independent studies investigating C. burnetii presence in animals and the environment. Six different genotypes were identified on 45 farm locations, based on sequence-confirmed estimates of repeat numbers of six MLVA markers. MLVA genotype A was observed on 38 of the 45 selected farm locations in animals and in environmental samples., Conclusions: Sequence confirmation of the numbers of tandem repeats within each locus and consensus about repeat identification is essential for accurate MLVA typing of C. burnetii. MLVA genotype A is the most common genotype in animal samples obtained from goat, sheep, and rats, as well as in environmental samples such as (aerosolized) dust, which is considered to be the major transmission route from animals via the environment to humans. The finding of a single dominant MLVA genotype in patients, the environment, and livestock complicates accurate source-finding. Pinpointing individual sources in the Netherlands requires discrimination of genotypes at a higher resolution than attained by using MLVA, as it is likely that the dominant C. burnetii MLVA type will be detected on several farms and in different patients in a particular area of interest.

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.

Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targets icd, com1, and IS1111, respectively; and no cross-reactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10- or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.

Coxiella burnetii is the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of the C. burnetii strain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination of C. burnetii strains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 different C. burnetii genotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 different C. burnetii genotypes are involved in the Dutch outbreak.

Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples., Results: Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains., Conclusions: The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.

The high incidence of coral disease in shallow coastal marine environments suggests seawater depth and coastal pollution have an impact on the microbial communities inhabiting healthy coral tissues. A study was undertaken to determine how bacterial communities inhabiting tissues of the coral Montastraea annularis change at 5 m, 10 m and 20 m water depth in varying proximity to the urban centre and seaport of Willemstad, Curaçao, Netherlands Antilles. Analyses of terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA gene sequences show significant differences in bacterial communities of polluted and control localities only at the shallowest seawater depth. Furthermore, distinct differences in bacterial communities were found with increasing water depth. Comparisons of TRFLP peaks with sequenced clone libraries indicate the black band disease cyanobacterium clone CD1C11 is common and most abundant on healthy corals in less than 10 m water depth. Similarly, sequences belonging to a previously unrecognized group of likely phototrophic bacteria, herein referred to as CAB-I, were also more common in shallow water. To assess the influence of environmental and physiologic factors on bacterial community structure, canonical correspondence analysis was performed using explanatory variables associated with: (i) light availability; (ii) seawater pollution; (iii) coral mucus composition; (iv) the community structure of symbiotic algae; and (v) the photosynthetic activity of symbiotic algae. Eleven per cent of the variation in bacterial communities was accounted for by covariation with these variables; the most important being photosynthetically active radiation (sunlight) and the coral uptake of sewage-derived compounds as recorded by the delta(15)N of coral tissue.

The cyanobacterium Microcystis can produce microcystins, a family of toxins that are of major concern in water management. In several lakes, the average microcystin content per cell gradually declines from high levels at the onset of Microcystis blooms to low levels at the height of the bloom. Such seasonal dynamics might result from a succession of toxic to nontoxic strains. To investigate this hypothesis, we ran competition experiments with two toxic and two nontoxic Microcystis strains using light-limited chemostats. The population dynamics of these closely related strains were monitored by means of characteristic changes in light absorbance spectra and by PCR amplification of the rRNA internal transcribed spacer region in combination with denaturing gradient gel electrophoresis, which allowed identification and semiquantification of the competing strains. In all experiments, the toxic strains lost competition for light from nontoxic strains. As a consequence, the total microcystin concentrations in the competition experiments gradually declined. We did not find evidence for allelopathic interactions, as nontoxic strains became dominant even when toxic strains were given a major initial advantage. These findings show that, in our experiments, nontoxic strains of Microcystis were better competitors for light than toxic strains. The generality of this finding deserves further investigation with other Microcystis strains. The competitive replacement of toxic by nontoxic strains offers a plausible explanation for the gradual decrease in average toxicity per cell during the development of dense Microcystis blooms.

Microcystin concentrations in two Dutch lakes with an important Planktothrix component were related to the dynamics of cyanobacterial genotypes and biovolumes. Genotype composition was analysed by using denaturing gradient gel electrophoresis (DGGE) profiling of the intergenic transcribed spacer region of the rrn operon (rRNA-ITS), and biovolumes were measured by using microscopy. In Lake Tjeukemeer, microcystins were present throughout summer (maximum concentration 30 microg l(-1)) while cyanobacterial diversity was low and very constant. The dominant phototroph was Planktothrix agardhii. In contrast, Lake Klinckenberg showed a high microcystin peak (up to 140 microg l(-1)) of short duration. In this lake, cyanobacterial diversity was higher and very dynamic with apparent genotype successions. Several genotypes derived from DGGE field profiles matched with genotypes from cultures isolated from field samples. The microcystin peak measured in Lake Klinckenberg could be confidently linked to a bloom of Planktothrix rubescens, as microscopic and genotypic analysis showed identity of bloom samples and a toxin-producing P. rubescens culture. Toxin-producing genotypes were detected in the microbial community before they reached densities at which they were detected by using microscopy. Cyanobacterial biovolumes provided additional insights in bloom dynamics. In both lakes, the microcystin content per cell was highest at the onset of the blooms. Our results suggest that while genotypic characterization of a lake can be valuable for detection of toxic organisms, for some lakes a monitoring of algal biomass has sufficient predictive value for an assessment of toxin production.

Assessing and predicting bloom dynamics and toxin production by Microcystis requires analysis of toxic and nontoxic Microcystis genotypes in natural communities. We show that genetic differentiation of Microcystis colonies based on rRNA internal transcribed spacer (ITS) sequences provides an adequate basis for recognition of microcystin producers. Consequently, ecological studies of toxic and nontoxic cyanobacteria are now possible through studies of rRNA ITS genotypic diversity in isolated cultures or colonies and in natural communities. A total of 107 Microcystis colonies were isolated from 15 lakes in Europe and Morocco, the presence of microcystins in each colony was examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and they were grouped by rRNA ITS denaturing gradient gel electrophoresis (DGGE) typing. Based on DGGE analysis of amplified ITSa and ITSc fragments, yielding supplementary resolution (I. Janse et al., Appl. Environ. Microbiol. 69:6634-6643, 2003), the colonies could be differentiated into 59 classes. Microcystin-producing and non-microcystin-producing colonies ended up in different classes. Sequences from the rRNA ITS of representative strains were congruent with the classification based on DGGE and confirmed the recognition of microcystin producers on the basis of rRNA ITS. The rRNA ITS sequences also confirmed inconsistencies reported for Microcystis identification based on morphology. There was no indication for geographical restriction of strains, since identical sequences originated from geographically distant lakes. About 28% of the analyzed colonies gave rise to multiple bands in DGGE profiles, indicating either aggregation of different colonies, or the occurrence of sequence differences between multiple operons. Cyanobacterial community profiles from two Dutch lakes from which colonies had been isolated showed different relative abundances of genotypes between bloom stages and between the water column and surface scum. Although not all bands in the community profiles could be matched with isolated colonies, the profiles suggest a dominance of nontoxic colonies, mainly later in the season and in scums.

Denaturant gradient gel electrophoresis (DGGE) is a widely used method for mutation analysis and for studies of microbial diversity. Particular combinations of target gene fragments and primers may give rise to erroneous DGGE profiles. We report on a very straightforward means to eliminate the artifactual 'double bands' that can be encountered in several applications.

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis: Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.

HEp-2 cells, human epithelial cells derived from a larynx carcinoma, were found to be highly susceptible to infection with HIV-1 stain IIIb and MN, but not to infection with the monotropic strain IIIBa-L or the clinical isolate HIV-1AT. HEp-2 cells infected with HIV-1 IIIb continuously secreted high levels of p24 antigen, while no cytopathic effects were observed. Although no CD4 antigen could be detected on the cells by flow cytometric analysis, CD4 mRNA was detected by reverse transcriptase PCR. Furthermore, infection could be blocked by anti-CD4 monoclonal antibody OKT4a indicating a CD4 mediated viral entry in HEp-2 cells. HEp-2 cells are commonly used in clinical virology for the culture of different viruses from clinical specimens. HEp-2 cells should therefore be handled with caution as they may potentially become infected with HIV.

Low levels of anti-viral antibodies may facilitate virus infection of Fc-receptor bearing cells. For human immunodeficiency virus (HIV) it has been reported that antibodies can enhance infection of phagocytic cells. We show that HIV-1 can infect an Epstein-Barr virus transformed B cell line and that low levels of anti-HIV antibodies enhance infection. The enhanced infection was characterized by an increase in viral DNA and increased HIV p24 protein production. Detection of cell surface antigen expression of CD4, the receptor for HIV, Fc-receptor type II for IgG, but not of type I and III could be demonstrated by immunofluorescence cytometry. The enhancement was abrogated when infection was performed in presence of a monoclonal antibody directed against CD4. Based on these results we conclude that antibody mediated enhancement of HIV-1 infection can also occur in non-phagocytic cells in a CD4 dependent manner and that IgG Fc-receptors other than types I or III are involved in this process.