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A disseminated peritoneal dialysis-related Mycobacterium abscessus infection is very rare. M. abscessus belongs to the rapidly growing mycobacteria and can be misidentified as a diphtheroid bacterium, which in our case delayed diagnosis and optimal treatment. Due to intrinsic resistance to most antimicrobials, therapeutic options in M. abscessus infections are limited. Infection often leads to catheter loss. A fatal outcome, like in our case, is not exceptional.

Background: Human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. The B27 allele is present in 90% of patients with this disease, whereas it is present in only 9% of Caucasians. Molecular detection of HLA-B27 is traditionally based on allele specific amplification of exon 2 (Olerup method) or exon 3 (Dominguez method) by PCR, followed by gel analysis., Methods: We developed a real-time TaqMan PCR based on the Dominguez method with a β-Globin PCR as internal control., Results: A total of 544 clinical samples were used to compare the real-time TaqMan PCR with the traditional Dominguez PCR, the traditional Olerup PCR and a commercial Olerup based HLA-B27 detection kit (Olerup SSPTM HLA-B27, GenoVision). While 542 samples gave concordant results, two samples showed discrepancies and were further analyzed. One sample that showed a discrepancy was negative with the traditional Olerup method and positive with the three other procedures. Sequencing analysis showed the presence of HLA-B*2712 in this sample. The other sample, positive with both Olerup based PCRs and negative with both Dominguez based methods, turned out to be positive for HLA-B*2707 by sequence analysis., Conclusions: With a correct result for 543 out of 544 samples (99.8%), we consider our real-time HLA-B27 PCR is a reliable method to detect HLA-B27 in the Dutch population, with reduced hands-on time and contamination risk compared to traditional PCR methods.

A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F., (Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Q fever is a zoonotic disease caused by Coxiella burnetii, a species of bacteria that is distributed globally. A large Q fever epidemic is currently spreading throughout the Netherlands with more than 3500 human cases notified from 2007 to 2009. Governmental measures to prevent further spread of the disease imposed in December 2009 included vaccination of all dairy goats and sheep and, in parallel, bulk tank milk testing to identify contaminated goat and sheep farms. When bulk tank milk was found to contain C. burnetii DNA, pregnant ruminants were culled. An important, but unsolved issue in this policy was whether vaccine-derived C. burnetii DNA is excreted in milk after vaccination. Using real time PCR and single nucleotide polymorphism (SNP) genotyping techniques, we show here that within hours and up to 9 days after vaccination with Coxevac(®), vaccine-derived C. burnetii DNA can be detected in the milk of dairy goats. This is the first report describing DNAlactia of vaccine-derived DNA after vaccination with a completely inactivated vaccine. This finding had implications for the Dutch policy to combat the Q fever epidemic. A 2-week interval was introduced between vaccination and bulk tank milk testing to identify infected farms., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

In 2006 a case report was published in this journal describing partial acenocoumarol- and phenprocoumon resistance in a 78-year-old man. A mutation in the VKORC1 gene was suggested to be the cause of the observed resistance. We examined the patient and found a new and hitherto unknown mutation in the VKORC1 gene which may well explain the observed resistance. The mutation concerns a polymorphism in exon 2 that predicts the substitution of tryptophan at position 59 of the VKOR protein by arginine, the p.Trp59Arg mutation. Meanwhile, we have detected the same p.Trp59Arg mutation in another patient with coumarin derivative resistance. The fact that this mutation did not occur in 100 individuals who responded well to coumarin derivatives, together with the knowledge that amino acid 59 is conserved among species, renders it likely that p.Trp59Arg was the cause of the partial acenocoumarol- and phenprocoumon resistance observed in these two patients.

The most common causes of alpha-thalassemia (thal) are deletions that remove a part, or one or both of the functional alpha-globin genes. These deletions cause diminished expression of the alpha-globin protein, which may result in relatively low hemoglobin (Hb) and/or mean corpuscular volume (MCV) values. We here report the identification of a 970 bp deletion in the alpha1-globin gene that encompasses the entire promoter region of the alpha1-globin gene and 26 bp encoding the 5' end of the mRNA. Thus, the affected alpha1-globin gene is prone to be nonfunctional. We therefore nominated the newly identified deletion allele alpha-alphaDelta970. The MCV values of four related carriers of the alpha-alphaDelta970 allele were slightly lowered, consistent with the presence of three functional alpha-globin genes.