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In 2005 human bocavirus (HBoV) was discovered in respiratory tract samples of children. The role of HBoV as the single causative agent for respiratory tract infections remains unclear. Detection of HBoV in children with respiratory disease is frequently in combination with other viruses or bacteria. We set up an algorithm to study whether HBoV alone can cause severe acute respiratory tract infection (SARI) in children. The algorithm was developed to exclude cases with no other likely cause than HBoV for the need for admission to the paediatric intensive care unit (PICU) with SARI. We searched for other viruses by next-generation sequencing (NGS) in these cases and studied their HBoV viral loads. To benchmark our algorithm, the same was applied to respiratory syncytial virus (RSV)-positive patients. From our total group of 990 patients who tested positive for a respiratory virus by means of RT-PCR, HBoV and RSV were detected in 178 and 366 children admitted to our hospital. Forty-nine HBoV-positive patients and 72 RSV-positive patients were admitted to the PICU. We found seven single HBoV-infected cases with SARI admitted to PICU (7/49, 14%). They had no other detectable virus by NGS. They had much higher HBoV loads than other patients positive for HBoV. We identified 14 RSV-infected SARI patients with a single RSV infection (14/72, 19%). We conclude that our study provides strong support that HBoV can cause SARI in children in the absence of viral and bacterial co-infections., (Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Almost five decades after their first application in diagnostics, dried blood spot (DBS) cards remain to be of key interest in many research areas and clinical applications. The advantages of sample stability during transport and storage, can now be combined with the high sensitivity of novel diagnostic techniques for the measurement and analysis of nucleic acids, proteins and small molecules which may overcome the limitations of the small samples sizes in DBS cards. Here we present a survey of the literature on the use of DBS cards for diagnosis, monitoring and epidemiological studies of virus infections other than HIV, including CMV, HBV, HCV, HAV, HEV, HTLV, EBV, HSV, measles-, rubella- and dengue-virus. The minimal invasiveness of sampling and the relative ease of handling and storing DBS cards is expected to offer additional opportunities to measure and analyze biomarkers of viral disease in resource poor settings or when limited amount of blood can be obtained. Large retrospective studies of virus infections in newborns using stored DBS cards have already been undertaken for screening of congenital infections. In addition, DBS cards have been used prospectively for prevalence studies, outbreak surveillance, mass screening for viral infections, follow-up of chronic infection and its treatment in resource-limited areas. We do not expect that current wet sampling techniques of plasma or serum will be replaced by DBS sampling but it allows extension of sampling in persons and settings that are currently difficult to access or that lack suitable storage facilities. In conclusion, DBS card sampling and storage will aid adequate outbreak management of existing and emerging viral diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nucleoside reverse transcriptase inhibitors (NRTIs) are a key class of drugs for the treatment of HIV infection. NRTIs are intracellularly phosphorylated to their active triphosphate metabolites and compete with endogenous deoxynucleotides (dNTP) for substrate binding. It is therefore important to analyze the intracellular concentrations of these compounds to understand drug efficacy and toxicity. To that purpose an analytical platform was developed that is capable of analyzing 8 NRTIs, 12 phosphorylated NRTIs and 4 dNTPs in small numbers of peripheral blood mononuclear cells, i.e. 1 × 10(6) cells. The platform consists of two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: a reversed-phase method for NRTIs using positive electrospray ionization (ESI) and an ion-pair LC-MS/MS method for the phosphorylated compounds using negative ESI. The methods use the same LC-MS system and column and changing from one method to the other only includes changing the mobile phase. The methods were partially validated, focussing on sensitivity, accuracy and precision. Successful transfer of the methods to ultra performance liquid chromatography (UPLC) led to a significant improvement of speed for the analysis of NRTIs and sensitivity for both NRTIs and phosphorylated NRTIs. The latter was demonstrated by the improved separation by UHPLC of dGTP vs. AZT-TP and ATP which made direct analysis of dGTP possible using the optimal MS/MS transition thereby significantly improving the detection limit of dGTP. Typically LLOQs observed for both the NRTIs and phosphorylated NRTIs were 1 nM, while the mean accuracy varied between 82 and 120% and inter- and intra-assay precision was generally <20%., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mass spectrometry is a powerful tool for studying the intracellular pharmacokinetics of antiretroviral drugs. However, the biohazard of HIV-1 calls for a safety protocol for such analyses. To this end, we extracted HIV-1 producing cells with methanol or ethanol at 4 degrees C. After extraction, no viral infectivity was detected, as shown by a reduction in infectious titers of more than 6log. In addition, this protocol is compatible with the quantitative analysis of antiretroviral drugs in cell extracts using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Thus, using this protocol, infectious HIV-1 is inactivated and antiretroviral drugs are extracted from cells in a single step.

We have developed a new method based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) for analysis of zidovudine-triphosphate and (deoxy)nucleotide-triphosphates, which ultimately can be used for nucleoside reverse transcriptase inhibitor (NRTI) treatment monitoring in HIV-1 infected children and adults. Four different matrices were compared for sensitivity and reproducibility of zidovudine-triphosphate detection and anthranilic acid mixed with nicotinic acid (AA/NA) was selected as most suitable matrix. Solutions of zidovudine-triphosphate, ATP, and dGTP were detected up to 0.5fmol per sample. Furthermore, intracellular zidovudine-triphosphate, ATP, and dGTP were detected in peripheral blood mononuclear cells (PBMCs). Zidovudine-triphosphate, ATP, and dGTP yield identical mass spectra, however MALDI-TOF post-source decay analysis can be used for discrimination between these compounds. We conclude that this method based on MALDI-TOF MS can be used for analysis of intracellular zidovudine-triphosphate and (deoxy)nucleotide-triphosphates in PBMCs.