Alphons M. Horrevorts

RespiFinder: a New Multiparameter Test To Differentially Identify Fifteen Respiratory Viruses

ABSTRACT Broad-spectrum analysis for pathogens in patients with respiratory tract infections is becoming more relevant as the number of potential infectious agents is still increasing. Here we describe the new multiparameter RespiFinder assay, which is based on the multiplex ligation-dependent probe amplification (MLPA) technology. This assay detects 15 respiratory viruses in one reaction. The MLPA reaction is preceded by a preamplification step which ensures the detection of both RNA and DNA viruses with the same specificity and sensitivity as individual monoplex real-time reverse transcription-PCRs. The RespiFinder assay was validated with 144 clinical samples, and the results of the assay were compared to those of cell culture and a respiratory syncytial virus (RSV)-specific immunochromatography assay (ICA). Compared to the cell culture results, the RespiFinder assay showed specificities and sensitivities of 98.2% and 100%, respectively, for adenovirus; 96.4% and 100%, respectively, for human metapneumovirus; 98.2% and 100%, respectively, for influenza A virus (InfA); 99.1% and 100%, respectively, for parainfluenza virus type 1 (PIV-1); 99.1% and 80%, respectively, for PIV-3; 90.1% and 100%, respectively, for rhinovirus; and 94.6% and 100%, respectively, for RSV. Compared to the results of the RSV-specific ICA, the RespiFinder assay gave a specificity and a sensitivity of 82.4% and 80%, respectively. PIV-2, PIV-4, influenza B virus, InfA H5N1, and coronavirus 229E were not detected in the clinical specimens tested. The use of the RespiFinder assay resulted in an increase in the diagnostic yield compared to that obtained by cell culture (diagnostic yields, 60% and 35.5%, respectively). In conclusion, the RespiFinder assay provides a user-friendly and high-throughput tool for the simultaneous detection of 15 respiratory viruses with excellent overall performance statistics.

Rapid diagnosis of acute meningococcal infections by needle aspiration or biopsy of skin lesions.

OBJECTIVES--To evaluate the usefulness of Gram staining and culture of skin lesions in patients with acute meningococcal infections. DESIGN--Retrospective study. SETTING--Community hospital and intensive care unit of a teaching hospital. SUBJECTS--51 patients admitted from 1989 to 1993 with proved meningococcal infections and microbiological examination of specimens from skin lesions. INTERVENTIONS--Needle aspiration of a skin lesion before start of antibiotic treatment in 26 patients in the community hospital; punch biopsy of skin lesion after start of antibiotic treatment in 25 patients in the teaching hospital. MAIN OUTCOME MEASURES--Detection of meningococci by Gram staining of specimens from skin lesions according to category of infection (meningococcaemia, meningitis, meningitis with shock, or septic shock without meningitis). RESULTS--Bacteria were detected in the specimen from haemorrhagic skin lesions by culture or Gram staining, or both in 32 (63%) patients. The sensitivity of the Gram stain was 51% and did not differ significantly from its sensitivity in detecting bacteria in cerebrospinal fluid. In meningococcal sepsis, however, a Gram stained skin lesion was significantly more sensitive (72%) than Gram stained cerebrospinal fluid (22%). In patients with meningitis skin lesions gave positive results on staining more often if shock was present. The results for punch biopsy specimens were not affected by antibiotics as Gram staining gave positive results up to 45 hours after the start of treatment and culture gave positive results up to 13 hours. CONCLUSION--Microbiological examination of skin lesions is informative, especially in patients with sepsis and inconclusive results from cerebrospinal fluid, and may provide a diagnosis in such patients within 45 minutes. It differentiates well between meningitis with and without haemodynamic complications, and the result is not affected by previous antibiotic treatment.

DNA Microarray Format for Detection and Subtyping of Human Papillomavirus

ABSTRACT A new human papillomavirus (HPV) assay using high-density DNA microarrays is described. An HPV DNA fragment from the 3′ end of the E1 gene was amplified and digoxigenin labeled by PCR, and the resulting amplicons were hybridized onto type-specific oligonucleotides immobilized on high-density DNA microarrays. For detection, a simple immunohistochemical staining procedure was used with a substrate that has both colorimetric and fluorescent properties. This detection chemistry enables the rapid identification of reactive spots by regular light microscopy and semiquantification by laser scanning. Both single and multiple HPV infections are recognized by this assay, and the corresponding HPV types are easily identified. With this assay, 53 mucosal HPV types were detected and identified. A total of 45 HPV types were identified by a single type-specific probe, whereas the remaining 8 mucosal HPV types could be identified by a specific combination of probes. The simple assay format allows usage of this assay without expensive equipment, making it accessible to all diagnostic laboratories with PCR facilities.

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.

Clinical evaluation and reproducibility of the Pastorex aspergillus antigen latex agglutination test for diagnosing invasive aspergillosis

AIMS--The performance of the Pastorex Aspergillus antigen latex agglutination test for the detection of galactomannan in sera of patients at risk for invasive aspergillosis was evaluated, and the impact of storage on the reproducibility of the antigen titre was tested. METHODS--During a one year period, 392 serum samples were obtained from 46 patients at risk for invasive aspergillosis and tested for the presence of galactomannan using an Aspergillus latex agglutination test (Pastorex). Twenty three positive serum samples which had been stored at -20 degrees C for 2-16 months were retrospectively retested. Furthermore, two positive serum samples were stored at -20 degrees C and -70 degrees C and prospectively tested at three month intervals for a period of 15 months. RESULTS--The Pastorex Aspergillus test was positive in eight patients with microbiological, radiological, or histological evidence for invasive aspergillosis, but was negative in the initial serum sample from five of these patients. In two patients with histological evidence for invasive aspergillosis no positive reaction was found in six samples. Six of 13 (45%) serum samples which had been stored at -20 degrees C for longer than six months had lost reactivity, while one of 10 (10%) samples had lost reactivity when stored up to six months. Two serum samples which had been stored at -20 degrees C and -70 degrees C and prospectively retested at three month intervals for 15 months, maintained stable antigen titres. CONCLUSIONS--The Pastorex Aspergillus test is too insensitive to diagnose invasive aspergillosis in an early stage, but may contribute to the diagnosis when cultures remain negative and serial samples are obtained. To maintain a good reproducibility, serum samples should be stored at -70 degrees C when the period of storage exceeds six months.

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.

Cellulitis as First Clinical Presentation of Disseminated Cryptococcosis in Renal Transplant Recipients

Two renal transplant recipients with cellulitis due to Cryptococcus neoformans are described. The patients were treated empirically for a presumed bacterial erysipelas, but without response. Examination of skin biopsies revealed C. neoformans as the causative organism. In both patients the cellulitis was the presenting clinical manifestation of disseminated cryptococcosis. Therapy with antifungal agents was successful. Disseminated cryptococcal disease occurs mainly in immunocompromised patients. When left untreated, it nearly always has a fatal course. Early diagnosis and appropriate therapy are therefore essential.

A Q fever outbreak in a psychiatric care institution in The Netherlands.

In May 2008 the Nijmegen Municipal Health Service (MHS) was informed about an outbreak of atypical pneumonia in three in-patients of a long-term psychiatric institution. The patients had been hospitalized and had laboratory confirmation of acute Q fever infection. The MHS started active case finding among in-patients, employees of and visitors to the institution. In a small meadow on the institution premises a flock of sheep was present. One of the lambs in the flock had been abandoned by its mother and cuddled by the in-patients. Samples were taken of the flock. Forty-five clinical cases were identified in employees, in-patients and visitors; 28 were laboratory confirmed as Q fever. Laboratory screening of pregnant women and persons with valvular heart disease resulted in one confirmed Q fever case in a pregnant woman. Of 27 samples from animals, seven were positive and 15 suspect for Coxiella burnetii infection. This outbreak of Q fever in a unique psychiatric setting pointed to a small flock of sheep with newborn lambs as the most likely source of exposure. Care institutions that have vulnerable residents and keep flocks of sheep should be careful to take adequate hygienic measures during delivery of lambs and handling of birth products.