Marrigje H. Nabuurs-Franssen

Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii. One of the largest reported outbreaks of Q fever in humans occurred in the Netherlands starting in 2007; epidemiologic investigations identified small ruminants as the source. To determine the genetic background of C. burnetii in domestic ruminants responsible for the human Q fever outbreak, we genotyped 126 C. burnetii–positive samples from ruminants by using a 10-loci multilocus variable-number tandem-repeat analyses panel and compared them with internationally known genotypes. One unique genotype predominated in dairy goat herds and 1 sheep herd in the human Q fever outbreak area in the south of the Netherlands. On the basis of 4 loci, this genotype is similar to a human genotype from the Netherlands. This finding strengthens the probability that this genotype of C. burnetii is responsible for the human Q fever epidemic in the Netherlands.

Chronic Q fever: review of the literature and a proposal of new diagnostic criteria.

A review was performed to determine clinical aspects and diagnostic tools for chronic Q fever. We present a Dutch guideline based on literature and clinical experience with chronic Q fever patients in The Netherlands so far. In this guideline diagnosis is categorized as proven, possible or probable chronic infection based on serology, PCR, clinical symptoms, risk factors and diagnostic imaging.

Genotypic Diversity of Coxiella burnetii in the 2007-2010 Q Fever Outbreak Episodes in The Netherlands

ABSTRACT The genotypic diversity of Coxiella burnetii in clinical samples obtained from the Dutch Q fever outbreak episodes of 2007-2010 was determined by using a 6-locus variable-number tandem repeat analysis panel. The results are consistent with the introduction of one founder genotype that is gradually diversifying over time while spreading throughout The Netherlands.

Epidemic Genotype of Coxiella burnetii among Goats, Sheep, and Humans in the Netherlands

To the Editor: The 2007–2010 Q fever epidemic among humans in the Netherlands was among the largest reported in magnitude and duration (1). The increase in human Q fever cases coincided with an increase in spontaneous abortions among dairy goats in the southeastern part of the Netherlands, an area that is densely populated with goat farms (1). Genotypic analyses of the involved isolates could confirm the possible link between the human and animal Q fever cases. In previous studies, genotypic investigations of human and animal samples in the Netherlands were performed by using a 3-locus multilocus variable-number tandem repeats analysis (MLVA) panel and single-nucleotide polymorphism genotyping, respectively (2,3). The first study, performed on relatively few samples from a minor part of the affected area, showed that farm animals and humans in the Netherlands were infected by different but apparently closely related genotypes. More recently, genotyping by using a 10-locus MLVA panel provided additional information about the genotypic diversity of Coxiella burnetii among ruminants in the Netherlands: 1 dominant MLVA genotype was identified among goats and sheep throughout the entire affected Q fever area (4). A different panel of MLVA markers was applied to human samples (5). Four markers that are shared by both panels showed identical alleles in human and animal samples, again implicating goats and sheep as possible sources of the outbreak. MLVA, which is based on relatively unstable repetitive DNA elements, is sometimes criticized for producing results that are too discriminatory or difficult to reproduce in different settings (6). Because of their instability, use of tandem repeats as genotyping targets can lead to problems with data interpretation and to overestimation of genotypic diversity by showing small variations in MLVA genotypes in isolates of otherwise identical background. We used a more stable, sequence-based typing method, multispacer sequence typing (MST), on samples from humans and a group of ruminant animals (goats, sheep, and cattle) to establish a firmer correlation between Q fever cases in humans and animals (7). We identified MST genotypes using a Web-based MST database (http://ifr48.timone.univ-mrs.fr/MST_Coxiella/mst) containing genotypes from several countries in Europe. Ultimately, this study could answer the question of whether the current outbreak situation could have been caused by a specific C. burnetii strain in the ruminant population in the Netherlands. Real-time PCR-positive specimens from 10 humans and 9 Q fever–positive specimens from goats and sheep collected from various locations throughout the affected area were used (8). We also included Q fever-positive specimens from cattle to rule out cattle as a possible source of Q fever infection. Five samples of cow’s milk and 1 bovine vaginal swab sample were analyzed (Table A1). MST33 was identified in 9 of 10 tested human samples and in the remaining 8 of 9 clinical samples from goats and sheep (Table A1). MST33 has been isolated incidentally in nonoutbreak situations in human clinical samples obtained in France during 1996, 1998, and 1999 and from a placenta of an asymptomatic ewe in Germany during 1992. All samples from cattle in the Netherlands, 1 goat, and cow’s milk contained genotype MST20. Genotype MST20 has also been identified in human clinical samples from France, in a cow’s placenta from Germany isolated in 1992 and in rodents from the United States isolated in 1958. In 1 human bronchoalveolar lavage sample, a novel (partial) MST genotype was found. This may be an incidental Q fever case unrelated to the outbreak situation. Because no historical genotyping data for the period before the outbreak of Q fever in the Netherlands are available, this explanation needs further research. MST genotyping shows the presence of genotype MST33 in clinical samples from humans, goats and sheep. These results confirm that goats and sheep are the source of human Q fever in the Netherlands. Few worldwide genotyping studies have been conducted, and therefore information about a possible global persistence of this genotype is lacking. This study also indicates that the outbreak among humans is not linked to C. burnetii in cattle, although the infection is widespread among dairy herds in the Netherlands (10), exemplifying that most outbreaks are related to goats and sheep rather than to cattle. In conclusion, the increase in the number of Q fever cases in the Netherlands among humans most likely results from MST33 in the goat population in the Netherlands and could have been facilitated by intensive goat farming in the affected area and its proximity to the human population.

Chronic Q Fever in the Netherlands 5 Years after the Start of the Q Fever Epidemic: Results from the Dutch Chronic Q Fever Database

ABSTRACT Coxiella burnetii causes Q fever, a zoonosis, which has acute and chronic manifestations. From 2007 to 2010, the Netherlands experienced a large Q fever outbreak, which has offered a unique opportunity to analyze chronic Q fever cases. In an observational cohort study, baseline characteristics and clinical characteristics, as well as mortality, of patients with proven, probable, or possible chronic Q fever in the Netherlands, were analyzed. In total, 284 chronic Q fever patients were identified, of which 151 (53.7%) had proven, 64 (22.5%) probable, and 69 (24.3%) possible chronic Q fever. Among proven and probable chronic Q fever patients, vascular infection focus (56.7%) was more prevalent than endocarditis (34.9%). An acute Q fever episode was recalled by 27.0% of the patients. The all-cause mortality rate was 19.1%, while the chronic Q fever-related mortality rate was 13.0%, with mortality rates of 9.3% among endocarditis patients and 18% among patients with a vascular focus of infection. Increasing age (P = 0.004 and 0.010), proven chronic Q fever (P = 0.020 and 0.002), vascular chronic Q fever (P = 0.024 and 0.005), acute presentation with chronic Q fever (P = 0.002 and P < 0.001), and surgical treatment of chronic Q fever (P = 0.025 and P < 0.001) were significantly associated with all-cause mortality and chronic Q fever-related mortality, respectively.

Interlaboratory Evaluation of Different Extraction and Real-Time PCR Methods for Detection of Coxiella burnetii DNA in Serum

ABSTRACT In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever.

Genotyping Reveals the Presence of a Predominant Genotype of Coxiella burnetii in Consumer Milk Products

ABSTRACT Real-time PCR shows the widespread presence of Coxiella burnetii DNA in a broad range of commercially available milk and milk products. MLVA genotyping shows that this is the result of the presence of a predominant C. burnetii genotype in the dairy cattle population.

Genotypic diversity of clinical Coxiella burnetii isolates from Portugal based on MST and MLVA typing

The temporal and spatial diversity of Coxiella burnetii genotypes associated with human and animal disease in Portugal was analysed using a 6-locus multiple-locus variable-number tandem repeat analysis (MLVA) and a 10-locus multi-spacer sequence typing (MST) panel. Fifteen cultured C. burnetii isolates from 13 Q fever patients and a stillborn goat and 6 additional PCR-positive ruminant tissue samples obtained during 2006-2011 were included in this study. Seven MLVA genotypes (types S-Y) were obtained, including 4 new MLVA types (U, V, W, and X), all corresponding to 3 MST profiles (types 4, 8, and 13) previously reported from France and Spain. MLVA types U-Y, all belonging to MST type 4, were found in acute Q fever patients from the districts of Évora, Faro, Lisbon, and Setúbal. Different MLVA types were associated with goats from Castelo Branco district (S) and chronic Q fever patients from both Castelo Branco and Lisboa districts (S and T), matching with MST types 13 and 8, respectively. In conclusion, a genotypic diversity of C. burnetii consistent with a non-outbreak situation was identified. The involvement of different genotypes in acute and chronic Q fever was found, linking one of the chronic genotypes to goats from the eastern region of the country.

Genotyping of Coxiella burnetii from domestic ruminants in northern Spain

BackgroundInformation on the genotypic diversity of Coxiella burnetii isolates from infected domestic ruminants in Spain is limited. The aim of this study was to identify the C. burnetii genotypes infecting livestock in Northern Spain and compare them to other European genotypes. A commercial real-time PCR targeting the IS1111a insertion element was used to detect the presence of C. burnetii DNA in domestic ruminants from Spain. Genotypes were determined by a 6-loci Multiple Locus Variable number tandem repeat analysis (MLVA) panel and Multispacer Sequence Typing (MST).ResultsA total of 45 samples from 4 goat herds (placentas, N = 4), 12 dairy cattle herds (vaginal mucus, individual milk, bulk tank milk, aerosols, N = 20) and 5 sheep flocks (placenta, vaginal swabs, faeces, air samples, dust, N = 21) were included in the study. Samples from goats and sheep were obtained from herds which had suffered abortions suspected to be caused by C. burnetii, whereas cattle samples were obtained from animals with reproductive problems compatible with C. burnetii infection, or consisted of bulk tank milk (BTM) samples from a Q fever surveillance programme. C. burnetii genotypes identified in ruminants from Spain were compared to those detected in other countries. Three MLVA genotypes were found in 4 goat farms, 7 MLVA genotypes were identified in 12 cattle herds and 4 MLVA genotypes were identified in 5 sheep flocks. Clustering of the MLVA genotypes using the minimum spanning tree method showed a high degree of genetic similarity between most MLVA genotypes. Overall 11 different MLVA genotypes were obtained corresponding to 4 different MST genotypes: MST genotype 13, identified in goat, sheep and cattle from Spain; MST genotype 18, only identified in goats; and, MST genotypes 8 and 20, identified in small ruminants and cattle, respectively. All these genotypes had been previously identified in animal and human clinical samples from several European countries, but some of the MLVA genotypes are described here for the first time.ConclusionsGenotyping revealed a substantial genetic diversity among domestic ruminants from Northern Spain.

Microbiological Challenges in the Diagnosis of Chronic Q Fever

ABSTRACT Diagnosis of chronic Q fever is difficult. PCR and culture lack sensitivity; hence, diagnosis relies mainly on serologic tests using an immunofluorescence assay (IFA). Optimal phase I IgG cutoff titers are debated but are estimated to be between 1:800 and 1:1,600. In patients with proven, probable, or possible chronic Q fever, we studied phase I IgG antibody titers at the time of positive blood PCR, at diagnosis, and at peak levels during chronic Q fever. We evaluated 200 patients, of whom 93 (46.5%) had proven, 51 (25.5%) had probable, and 56 (28.0%) had possible chronic Q fever. Sixty-five percent of proven cases had positive Coxiella burnetii PCR results for blood, which was associated with high phase I IgG. Median phase I IgG titers at diagnosis and peak titers in patients with proven chronic Q fever were significantly higher than those for patients with probable and possible chronic Q fever. The positive predictive values for proven chronic Q fever, compared to possible chronic Q fever, at titers 1:1,024, 1:2,048, 1:4,096, and ≥1:8,192 were 62.2%, 66.7%, 76.5%, and ≥86.2%, respectively. However, sensitivity dropped to <60% when cutoff titers of ≥1:8,192 were used. Although our study demonstrated a strong association between high phase I IgG titers and proven chronic Q fever, increasing the current diagnostic phase I IgG cutoff to >1:1,024 is not recommended due to increased false-negative findings (sensitivity < 60%) and the high morbidity and mortality of untreated chronic Q fever. Our study emphasizes that serologic results are not diagnostic on their own but should always be interpreted in combination with clinical parameters.