An online method for the analysis of volatile organic compounds in electronic cigarette aerosol based on proton transfer reaction mass spectrometry.

RATIONALE: Due to the recent rapid increase in electronic cigarette (e-cigarette) use worldwide, there is a strong scientific but also practical interest in analyzing e-cigarette aerosols. Most studies to date have used standardized but time-consuming offline technologies. Here a proof-of-concept for a fast online quantification setup based on proton transfer reaction mass spectrometry (PTR-MS) is presented. METHODS: The combination of a novel sampling interface with a time-of-flight PTR-MS instrument specially designed for three scenarios is introduced: (i) mainstream aerosol analysis (aerosol that the user inhales prior to exhalation), and analysis of exhaled breath following (ii) mouth-hold (no inhalation) and (iii) inhalation of e-cigarette aerosols. A double-stage dilution setup allows the various concentration ranges in these scenarios to be accessed. RESULTS: First, the instrument is calibrated for the three principal constituents of the e-cigarettes' liquids, namely propylene glycol, vegetable glycerol and nicotine. With the double-stage dilution the instrument's dynamic range was easily adapted to cover the concentration ranges obtained in the three scenarios: 20-1100 ppmv for the mainstream aerosol characterisation; 4-300 ppmv for the mouth-hold; and 2 ppbv to 20 ppmv for the inhalation experiment. CONCLUSIONS: It is demonstrated that the novel setup enables fast, high time resolution e-cigarette studies with online quantification. This enables the analysis and understanding of any puff-by-puff variations in e-cigarette aerosols. Large-scale studies involving a high number of volunteers will benefit from considerably higher sample throughput and shorter data processing times.

A strategy for correlative microscopy of large skin samples: towards a holistic view of axillary skin complexity.

Knowledge about the structural elements of skin and its appendices is an essential prerequisite for understanding their complex functions and interactions. The hence necessary morphological description across several orders of scale not only requires the investigation at the light microscopic level but also ultrastructural investigation, ideally on the identical sample. For a correlative and multimodal observation one unique preparation protocol is mandatory. As a compromise between sample sizes of >500 microm in diameter on the one hand and optimal preservation of antigenicity and morphology on the other, we developed a new preparation protocol that allows (i) 3D reconstruction of the resin-embedded sample by confocal light microscopy prior to (ii) direct immunolocalization of target proteins within selected sample planes by light and fluorescence microscopy or transmission electron microscopy. Alternatively, (iii) serial cryosections of the frozen sample can be taken for characterizing the sample in toto. With this unique approach we were able to fully demonstrate the structural complexity of axillary skin samples, increasing the structural resolution from 3D reconstruction of the whole gland up to ultrastructural investigations at the subcellular level. We could demonstrate that axillary sweat glands are not separately distributed, as has been assumed to date; instead, they seem to be intricately twisted into one another. This promotes the concept of a complex axillary sweat gland organ instead of single sweat gland entities.

Noninvasive measurement of cell volume changes by negative staining.

To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.

Quality control assessment for the serological diagnosis of dengue virus infections.

BACKGROUND: A major drawback of modern society's rapidly increasing mobility is the ease with which dangerous infections can be imported into Europe. Often these infections are not diagnosed because physicians are not familiar with the symptoms and laboratory tests are not always available in local diagnostic centres. Improving diagnostics is the most important step in detecting and dealing with these pathogens and quality control measures are, therefore, essential tools. OBJECTIVES: To assess the diagnosis of imported dengue virus infections in Europe by (1) running a pre-evaluation panel (four serum samples, sent out in 1999) and optimising sample preparation and shipping procedures and (2) initiating an External Quality Assurance (EQA) program (20 serum samples, sent out in 2002). STUDY DESIGN: All serum samples sent out were to be tested for the presence of dengue virus-specific IgM and IgG. For the pre-evaluation panel, four samples were distributed (one sample IgM+/IgG+, one sample IgM-/IgG+, two samples IgM-/IgG-) and for the EQA 20 samples (12 samples IgM+/IgG+, five samples IgM-/lgG+, one sample lgM+/IgG- two samples IgM-/IgG-). 13 laboratories took part in the pre-evaluation panel and 18 laboratories participated in the first EQA run. RESULTS: For the pre-evaluation panel, the participants reported concurrent and correct results for 88% of the IgG-positive samples and for 100% of the IgG-negative samples. The results for the IgM-positive sample were correct in 91% of the reported tests and in 97% of the IgM-negative samples. For the EQA, the participants reported concurrent and correct results for 71% of the IgG-positive samples and 89% of the IgG-negative samples. 58% concurrent and correct results were reported for the IgM-positive samples and 97% for the IgM-negative samples. CONCLUSIONS: The results presented here demonstrate the importance of quality measures for imported viral pathogens like dengue viruses and clearly indicate the need for improving the existing test systems.

Quality control measures for the serological diagnosis of hantavirus infections.

BACKGROUND: With society's rapidly increasing mobility, patients infected with severe viral infections can become seriously ill at any place in Europe and elsewhere. Improving the diagnostics of these infections is the most important step in detecting the pathogens and dealing with them, and for this purpose, quality control measures are essential tools. OBJECTIVES: To assess the diagnostic reality for rare hantavirus infections in Europe by (1) running a pre-evaluation panel (four samples, sent out in 1999) to optimise sample preparation and shipping procedure and afterwards (2) starting an External Quality Assurance (EQA) program (20 samples, sent out in 2001). STUDY DESIGN: All samples sent out had to be tested for the presence of specific IgG and IgM antibodies against hantavirus. For the pre-evaluation panel, four samples were distributed (two samples IgG+/IgM-, one sample IgG-borderline/IgM-, one sample IgG-/IgM-), for the EQA 20 samples (six samples IgG+/IgM+, eight samples IgG+/IgM-, one sample IgG-borderline/IgM-, five samples IgG-/IgM-). Thirteen laboratories took part in the pre-evaluation panel, 18 laboratories participated in the first EQA run. RESULTS: For the pre-evaluation panel, the participants reported correct results for 64% of the IgG-positive samples (85% excluding borderline-positive sample), and 92% for the IgG-negative sample. IgM testing was correctly negative in all laboratories. For the EQA, the participants reported correct results for 76% of the IgG-positive samples, and 97% correct results for the IgG-negative samples. For the IgM-positive samples, 53% correct results were reported, and 98% correct results for the IgM-negative samples. CONCLUSIONS: The results presented here prove the importance of quality measures also for viruses only rarely suspected, like hantavirus, and they clearly demonstrate the need for improvement of the existing test systems.

Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.

Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).

For debate: is disinfection of specimens, which may contain unknown or bio-terrorist organisms, essential before electron microscopic examination?

Severe and unexpected infections may be due to bioterrorism (BT), or the natural emergence of novel micro-organisms. Whatever the cause, there will be an urgent need to identify it for several reasons: defusing public anxiety, providing logical management and countermeasures, etc. For viruses and some bacteria, electron microscopy provides the fastest answers and identifying the cause quickly may eliminate BT. In the interests of safety, many would recommend that the specimen be disinfected at some stage before examination, but with unknown agents, however, reliable disinfection cannot be guaranteed and attempting to do so may also impair structure sufficiently to make recognition difficult or even impossible. As a basis for debate, this paper discusses the pros and cons of disinfecting such specimens.

Detection of human polyomaviruses in urine from bone marrow transplant patients: comparison of electron microscopy with PCR.

BACKGROUND: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. METHODS: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. RESULTS: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 10(6) genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <10(3) GE/mL and 100% for urine samples containing 10(9) GE/mL. CONCLUSIONS: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.