de Bruin A; van Rotterdam B (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2012-01-30)
Q fever, caused by the bacterium Coxiella burnetii, has become an emerging public health problem in the Netherlands since 2007. Diagnosis of Q fever, both in humans and animals, is mainly based on serology. Serological techniques are less suitable for direct transmission and source-finding studies for C. burnetii infection due to the delayed detection window for serological tests. The last two decades, several PCR based diagnostic assays (conventional PCR or qPCR) have been developed for the detection of C. burnetii DNA. These assays have been applied for the detection of C. burnetii DNA in clinical samples, veterinary samples, and environmental samples. These PCR-based diagnostic tests are often "in-house" developed assays. A number of commercial PCR diagnostic tests, however, have also become available. A drawback of these commercial kits is that information on some of the components is patented. This makes a thorough assessment of assay performance in relation to other "inhouse" developed assays difficult. In the current study, we describe a comparison of various (q)PCR assays used by laboratories both nationally and internationally for the detection of C. burnetii DNA. We compare the results obtained from three ring trials, set up specifically for the detection of C. burnetii DNA in human, veterinary, or environmental matrices. In addition, we compare specific parameters, such as sensitivity, specificity, and reproducibility for each of the (q)PCR assays. In conclusion, most (q)PCR assays developed for C. burnetii include detection of the multicopy insertion element IS1111, in combination with detection of other single copy genes such as icd, com1, sod, or plasmid genes. PCR based detection assays (conventional PCR or qPCR) for C. burnetii DNA preferably target short and multiple target sequences, including an internal process control in multiplex format. Other performance characteristics have not yet been published for these qPCR assays for the detection of C. burnetii, although this would be recommended.
Janse I; Bossers A; Roest HJ; van Rotterdam B (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2011-11-29)
Coxiella burnetii is een intracellulaire bacterie die Q-koorts veroorzaakt. De genoomsequenties van een aantal isolaten die verkregen werden tijdens de Nederlandse Q-koorts uitbraak werden opgehelderd. Deze genoomsequenties dienen als basis voor verbeterde typeringsmethodes die nauwkeurigere bronopsporing en epidemiologische studies mogelijk maken. Daarnaast zijn de sequenties uiterst waardevol voor allerhande overig onderzoek, zoals naar de samenhang tussen de Nederlandse uitbraken en veranderde virulentiekenmerken. Als resultaat van dit project zijn er nu 19 C. burnetii isolaten van de Nederlandse uitbraak in kweek. Deze isolaten zijn voornamelijk afkomstig van veterinaire bronnen, maar er zijn ook enkele stammen van humane en omgevingsbronnen verkregen. De genomen van 3 isolaten zijn grotendeels opgehelderd en enkele voorlopige analyses zijn erop uitgevoerd. De ruwe sequentiedata van nog 4 isolaten komen binnenkort beschikbaar. De isolaten en genoomsequenties spelen een essentiële rol spelen in meerdere vervolgprojecten van de deelnemende onderzoeksgroepen. De ervaring die opgedaan is met het opwerken en sequencen van de C. burnetii genomen is van grote waarde voor lopend en toekomstig onderzoek naar C. burnetii en andere intracellulaire micro-organismen.
de Bruin A; van Alphen P; Janse I; van Rotterdam B (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2011-10-25)
Q fever, caused by the bacterium C. burnetii, is a zoonosis with a worldwide distribution that affects both humans and animals. From 2007 to 2010, large community outbreaks of Q fever were observed in the Netherlands. In 2008 and 2009 source finding investigations were initiated by several Municipal Health Services, primarily on commercial dairy goat farms, to pinpoint potential sources for Q fever. In that same year, the Food and Consumer Product Safety Authority initiated a project to investigate if petting zoos were a potential source for human Q fever as well. Petting zoos showed insufficient C. burnetii DNA content for molecular typing (qPCR Cq values higher than 33) and were not considered an important source for human Q fever. However, 31 samples from eight out of 57 commercial dairy farms, involved in source finding investigations in 2009, showed a relatively high C. burnetii DNA content based on qPCR data for single copy target com1 (Cq values lower than 33). These samples were selected for molecular typing using a Multi-Locus Variable number of tandem repeat Analysis (MLVA). In this study we show that samples highly positive for C. burnetii DNA can be successfully typed using a multiplex MLVA assay. Three different MLVA types were found, based on six MLVA markers. On seven out of eight locations a single MLVA type was found. On one location a mixture of two types was observed within a number of samples. Our findings show that multiple MLVA types are present in the Netherlands, which is promising for future source finding investigations to identify potential sources. However, only a few different MLVA types have been determined in human and animal samples so far, which makes identifying transmission routes and sources of C. burnetii in the Netherlands still challenging.
de Bruin A; van der Plaats R; Janse I; van Rotterdam B (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2011-10-25)
Coxiella burnetii is a bacterium that causes Q fever, a zoonosis that affects large numbers of both humans and animals. From 2007 to 2010, large outbreaks of Q fever were observed in a rural area in the Netherlands. In 2009, field studies were started to investigate if C. burnetii DNA can be detected in aerosols on and in the near vicinity of Q fever affected farms. In 2010, these studies were continued in two areas studied in 2009, in the provinces of Noord-Brabant and Zuid-Limburg, to investigate if C. burnetii DNA was still present in aerosols in these areas. In both areas, the C. burnetii DNA content in aerosols obtained in 2010 seemed to have declined in comparison to data of the same locations visited in 2009. These data are in agreement with the observed reduction in the number of reported Q fever cases in 2010 in comparison to 2009. Possible explanations for this decline could be the start of a mandatory vaccination campaign for small ruminants in 2009 and the culling of pregnant animals on Q fever affected farms that started at the end of 2009. This data will be used in future investigations, in which we will combine molecular detection and typing methods for C. burnetii in aerosols with mathematical modelling to get more insight in the transmission of C. burnetii via aerosols and track (individual) sources for C. burnetii infection.
de Bruin A; van der Plaats R; van Rotterdam B (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2011-12-19)
During the spring and summer of 2007, 2008 and 2009, large Q fever outbreaks occurred in the Netherlands affecting a rural area in the southeast of the country. Prior to and during these outbreaks Q fever related abortion waves were reported on several dairy goat farms in the same region. As a result, primarily commercial dairy goat farms were implicated as potential sources for the emerging human Q fever cases in the Netherlands. However, in 2008 and 2009 a number of (non-dairy) sheep farms were identified where C. burnetii DNA was detected in both animal (vaginal swabs) and environmental (surface area swabs) matrices. In addition, in two epidemiological studies non-dairy sheep farms were implicated as the primary source for an emerging cluster of human Q fever cases in their near vicinity. Therefore, although less important in the recent epidemic, non-dairy sheep farms cannot be ruled out as potential source for human Q fever. In the current study, we describe the presence of C. burnetii DNA in animal and environmental matrices obtained from two non-dairy sheep farms. We show that C. burnetii DNA content in surface area swabs from fences and drinking buckets and udder swabs from animals was consistently higher on farm B in comparison to farm A. This may be explained by the geographical locations of the farms, since farm B is located in a highly Q fever affected area (Noord-Brabant), while farm A is located in an area not affected by Q fever (Noord-Holland). How these results are related to shedding of C. burnetii by the non-dairy sheep on these farms is not clear. No positive relationship was found between C. burnetii content in vaginal swabs and udder swabs. Coxiella burnetii contamination of sheep udders may be a result from excrements from the same animal, direct contact with other animals (or other contaminated surfaces), or a combination of these.
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