Blastocystis sp. subtype 2 detection during recurrence of gastrointestinal and urticarial symptoms

Blastocystis is a common unicellular intestinal parasite in humans. Its clinical relevance is still subject to discussion with numerous conflicting reports on its ability to cause disease. A remarkable genetic heterogeneity among isolates suggests an association between distinct subtypes (STs) and pathogenicity, although a clear correlation between symptoms and subtype is lacking. Here, we report on a clinical case which possibly links Blastocystis sp. ST2 infection with the simultaneous occurrence of gastrointestinal illness and generalized chronic urticaria. Despite repeated chemotherapy with different antimicrobial drugs, both the gastrointestinal and cutaneous disorders reoccurred after short symptom-free intervals. Eradication of the parasite and permanent resolution of the patient's medical condition was finally achieved with the combined application of metronidazole and paromomycin.     

Association between pulmonary function and peak oxygen uptake in elderly: the Generation 100 study

Chronic obstructive lung disease (COPD) is a common cause of death in industrialized countries often induced by exposure to tobacco smoke. A substantial number of patients with COPD also suffer from pulmonary hypertension that may be caused by hypoxia or other hypoxia-independent stimuli - inducing pulmonary vascular remodeling. The Ca2+ binding protein, S100A4 is known to play a role in non-COPD-driven vascular remodeling of intrapulmonary arteries. Therefore, we have investigated the potential involvement of S100A4 in COPD induced vascular remodeling. Lung tissue was obtained from explanted lungs of five COPD patients and five non-transplanted donor lungs. Additionally, mice lungs of a tobacco-smoke-induced lung emphysema model (exposure for 3 and 8 month) and controls were investigated. Real-time RT-PCR analysis of S100A4 and RAGE mRNA was performed from laser-microdissected intrapulmonary arteries. S100A4 immunohistochemistry was semi-quantitatively evaluated. Mobility shift assay and siRNA knock-down were used to prove hypoxia responsive elements (HRE) and HIF binding within the S100A4 promoter. Laser-microdissection in combination with real-time PCR analysis revealed higher expression of S100A4 mRNA in intrapulmonary arteries of COPD patients compared to donors. These findings were mirrored by semi-quantitative analysis of S100A4 immunostaining. Analogous to human lungs, in mice with tobacco-smoke-induced emphysema an up-regulation of S100A4 mRNA and protein was observed in intrapulmonary arteries. Putative HREs could be identified in the promoter region of the human S100A4 gene and their functionality was confirmed by mobility shift assay. Knock-down of HIF1/2 by siRNA attenuated hypoxia-dependent increase in S100A4 mRNA levels in human primary pulmonary artery smooth muscle cells. Interestingly, RAGE mRNA expression was enhanced in pulmonary arteries of tobacco-smoke exposed mice but not in pulmonary arteries of COPD patients. As enhanced S100A4 expression was observed in remodeled intrapulmonary arteries of COPD patients, targeting S100A4 could serve as potential therapeutic option for prevention of vascular remodeling in COPD patients.     

Azole-Resistant Invasive Aspergillosis: Relationship to Agriculture

Azole resistance in Aspergillus fumigatus has been increasingly reported particularly over the last decade. Two routes of acquisition are described: selection of resistance during long term azole therapy in the clinical setting, and primary acquisition of resistant isolates from the environment due to the considerable use of azole fungicides in agriculture and for material preservation. Three specific resistance genotypes have been found in azole naïve patients. Two of these have also been found in the environment and are characterized by a tandem repeat in the promoter region of the target gene coupled with point mutation(s) in CYP51A (TR₃₄/L98H and TR₄₆/Y121F/T289A). In the third a single target enzyme alteration (G432S) is found. These resistant “environmental” strains have been detected in many West-European countries as well as in the Asia-Pacifics. Noticeably, these two continents account for the highest fungicide use in the global perspective (37 % and 24 %, respectively). Among the 25 azole fungicides, five have been associated with the potential to select for the TR₃₄/L98H genotype; three of these are among those most frequently used. Although the number of antifungal fungicide compounds and classes available is impressive compared to the armamentarium in human medicine, azoles will remain the most important group in agriculture due to superior field performance and significant resistance in fungal pathogens to other compounds. Hence, further spread of environmental resistant Aspergillus genotypes may occur and will depend on the fitness of each resistant phenotype and the pattern of azole fungicide use.     

Genetic Diversity in the Worldwide <Emphasis Type="Italic">Botrychium lunaria</Emphasis> (Ophioglossaceae) Complex,with New Species and New Combinations

The Botrychium lunaria (Ophioglossaceae) complex worldwide includes the named species B. lunaria, B. crenulatum, B. tunux, and B. yaaxudakeit. These species have been distinguished from each other morphologically and genetically. This study further investigates the genetic diversity and geographic distribution of this complex, examining a large number of plants worldwide. Enzyme electrophoresis was used to examine allelic variation of 22 loci for 1574 plants of putative B. lunaria, B. crenulatum and B. tunux from North America, Eurasia, and New Zealand, and B. dusenii from the Falkland Islands. Variation in allelic composition assessed by genetic identity and cluster analysis using the programs PopGene and STRUCTURE as well as morphology and geography indicated that the complex is composed of six distinct entities; two of which warrant recognition as new species, B. neolunaria, endemic to North America, and B. nordicum, sister to the B. lunaria complex, from Iceland and Norway; and a new combination, B. lunaria var. melzeri , endemic to Greenland, Iceland, and Norway. The new taxa are described in this paper. Three entities within B. tunux are discussed but not proposed for recognition at this time. Botrychium lanceolatum, included in this study, is composed of three morphologically and genetically distinct entities warranting taxonomic recognition.     

Blastocystis: subtyping isolates using pyrosequencing technology

Blastocystis is a prevalent single-celled enteric parasite of unresolved clinical significance. Efforts based on molecular methodologies to establish whether pathogenicity is linked to specific isolates of the genetically diverse genus of Blastocystis have been scarce and so far yielded ambiguous results which can be difficult to interpret. To alleviate some of the problems related to unravelling the molecular epidemiology of Blastocystis infections we developed and evaluated a simple and high-throughput sequence analysis (SQA) pyrosequencing technique based on the detection of genotype-specific nucleotide polymorphisms in the 18S small subunit rRNA gene for a rapid and cost-effective post-PCR screening of Blastocystis genotypes. The method was effectively capable of genotyping 48/48 isolates positive by nested PCR in approximately one hour, and in 94% of the cases the isolate detected by PCR and pyrosequencing was also detected by one of two different PCR assays with subsequent dideoxy sequencing.     

Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population

The human gut is host to a diversity of microorganisms, including the single-celled microbial eukaryote Blastocystis. Research has shown that most carriers host a single Blastocystis subtype (ST), which is unusual given the considerable within-host species diversity observed for other microbial genera in this ecosystem. However, our limited knowledge of both the incidence and biological significance of Blastocystis diversity within hosts (i.e., so-called mixed infections) is likely due to problems with existing methodologies. Here, we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes of Blastocystis (ST1 to ST4) to a healthy human cohort (n = 50). We detected mixed infections in 22% of the cases, all of which had been identified as single-ST infections in a previous study using state-of-the-art methods. Our results show that certain STs occur predominantly as either single (ST3 and 4) or mixed (ST1) infections, which may reflect inter alia transient colonization patterns and/or cooperative or competitive interactions between different STs. Comparative analyses with other primers that have been used extensively for ST-specific analysis found them unsuitable for detection of mixed- and, in some cases, single-ST infections. Collectively, our data shed new light on the diversity of Blastocystis within and between human hosts. Moreover, the development of these PCR assays will facilitate future work on the molecular epidemiology and significance of mixed infections in groups of interest, including health and disease cohorts, and also help identify sources of Blastocystis transmission to humans, including identifying potential animal and environmental reservoirs.     

Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants

Six ssrRNA gene sequences were obtained by PCR amplification of DNA from uninucleated Entamoeba cysts isolated from fresh faeces of sheep, cows, a roe deer and a reindeer. Phylogenetic analysis using sequences of non-, uni-, quadri- and octonucleate cyst-producing Entamoeba spp. for comparison showed that all six isolates formed a separate clade nested within the clade of quadrinucleate cyst producers. The data indicate that Entamoeba bovis can be isolated from ruminant hosts other than cattle, and we suggest that organisms clustering with the sheep and cattle isolates analysed in the present study be named E. bovis.     

Detection of Blastocystis hominis in unpreserved stool specimens by using polymerase chain reaction

Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect > or =32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.