Generic publications

2018 Article - "New rapid one-step PCR diagnostic assay for Plasmodium falciparum infective mosquitoes" (pdf, 1MB)

Summary: An essential component of malaria vector control programmes is the detection of Plasmodium falciparum within its mosquito vectors, particularly in the salivary glands where the infective sporozoites reside. Several protocols have been developed for this purpose; however they require dissection of mosquito specimens prior to analysis. Here, a novel one-step RT-qPCR TaqMan diagnostic assay was developed for mosquitoes with infective Plasmodium falciparum sporozoites in the salivary glands. It is based on detection of the sporozoite-specific Pfslarp and Pfplp1 gene transcripts. These transcripts were chosen based on bioinformatics analysis, and experimentally verified to be overexpressed in the salivary gland sporozoite stage of the parasite compared to other mosquito parasite stages. The proof of principle and the performance of the assay were demonstrated using RNAlater preserved mosquito samples. Tests of analytical sensitivity showed the novel TaqMan assay to be 100% accurate, although its performance in the field needs to be further demonstrated. This method has no requirement for dissection and post-PCR processing and thus is simple and rapid to perform in individual mosquitoes or mosquito pools. It can be used in single or multiplex formats also targeting additional markers expressed in different tissues, such as detoxification enzymes associated with insecticide resistance.

Kit used: Fast Track mastermix

2017 Article - "Detection of West Nile Virus – Lineage 2 in Culex pipiens mosquitoes, associated with disease outbreak in Greece, 2017" (limited access)

Summary: During July-October 2017 a WNV outbreak took place in the Peloponnese, Southern Greece with five confirmed deaths. During routine monitoring survey in the Peloponnese, supported by the local Prefecture, we have confirmed the presence of all three Culex pipiens biotypes in the region, with a high percentage of Culex pipiens/molestus hybrids (37.0%) which are considered a highly competent vector of WNV. Kdr mutations related to pyrethroid resistance were found at relatively low levels (14.3% homozygosity) while no mosquitoes harboring the recently identified chitin synthase diflubenzuron-resistance mutations were detected in the region. As an immediate action, following the disease outbreak (within days), we collected a large number of mosquitoes using CO2 CDC traps from the villages in the Argolis area of the Peloponnese, where high incidence of WNV human infections were reported. WNV lineage 2 was detected in 3 out of 47 Cx. pipiens mosquito pools (detection rate=6.38%). The virus was not detected in any other mosquito species, such as Aedes albopictus, sampled from the region at the time of the disease outbreak. Our results show that detection of WNV lineage 2 in Cx. pipiens pools is spatially and chronologically associated with human clinical cases, thus implicating Cx. pipiens mosquitoes as the most likely WNV vector. The absence of diflubenzuron resistance mutations and the low frequency of pyrethroid (kdr) resistance mutations indicates the suitability of these insecticides for Cx. pipiens control, in the format of larvicides and/or residual spraying applications respectively, which was indeed the main (evidence based) response, following the disease outbreak.

Kit used: Fast Track mastermix
Website link

2016 Poster - "Comparison of two exctraction methods: NucleoSpin® Blood and NucleoSpin® DNA/RNA Virus Kit (Macherey-Nagel) and NucliSENS® easyMag® (bioMérieux)" (pdf, 648KB)

Summary:The aim of this study was to compare two extraction methods, one semi-automated platform NucliSENS® easyMag® (bioMérieux) and one handprep extraction NucleoSpin® Blood and NucleoSpin® DNA/RNA Virus Kit (Macherey-Nagel). To confirm the compatibility of the performance of NucleoSpin® Blood and NucleoSpin® DNA/RNA Virus Kit (Macherey-Nagel) with FTD products we investigated nucleic acid extraction from 40 clinical specimens (human plasma, whole blood, swabs, faeces, culture and plasmids) and dilution series of certain samples. NucleoSpin® Blood was only tested with human plasma and whole blood, while NucleoSpin® DNA/RNA Virus Kit was validated with swab, faeces, culture, plasma and plasmids. For analysis of the extracted material commercial real-time multiplex assays from FTD (FTD Viral gastroenteritis, FTD Bacterial gastroenteritis, FTD Stool parasites, FTD ACE, FTD Respiratory pathogens 21 plus, FTD Viral meningitis, FTD Bacterial meningitis, FTD Hepatitis B DNA and FTD Urethritis basic) were performed on Applied BiosystemsTM 7500 (ThermoFisher Scientific) with extracted material from NucliSENS® easyMag® and with extracted material from NucleoSpin® Blood and DNA/RNA Virus Kit.

Both were tested with Fast-track mastermix (FTD)

2015 Poster - "Lyophilised real-time PCR kits from Fast-track diagnostics: faster and even easier" (pdf, 445KB)

Summary: The easy to use application of lyophilised Fast-track diagnostics real-time multiplexes is a time saving and more flexible alternative compared to our existing liquid Fast-track diagnostics real-time multiplexes. The simple addition of extracted material to the lyophilised reagent allows people to save time and improve flexibility by testing anything between 1 and 62 patients without discarding any reagent afterwards.

Kits used: FTD Bacterial gastroenteritis, FTD ACE and FTD Respiratory pathogens 21

2012 Poster - "Comparison of two extraction methods for detection of viruses, bacteria" (pdf, 873KB)

Summary: The study obtained just small differences (less or equal 1 log Ct range) in the recovery of nucleic acid for all of the tested pathogens. For Gram-pos bacteria (Spneu and Saur) they received the same range of linearity with both tested methods. Also for the Gram-neg bacteria (Kpneu and Morax) the linear range was equal for both extraction methods. Except for Cpneu where the last dilution step with the RTP® Pathogens kit was missed. In general the outcome of the RTP® Pathogens kit was equal or slightly more efficient than the nucleic acid isolation with the easyMAG. It is clear that, as long as one selects an extraction kit which is designed for the required pathogens, that there is little to choose between many kits on the market (two examples are compared here). However, for an integrated infection laboratory, it is crucial that extraction kits are chosen that are equally efficient for all types of pathogens. The major decision now facing laboratories is not extraction, but sensitivity of the downstream nucleic acid detection system.

Kits used: FTD Viral gastroenteritis, FTD EPA, FTD Stool parasites, FTD Bacterial gastroenteritis, FTD Respiratory pathogens 21 and FTD Respiratory pathogens 33

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