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| 5. |
- Forsgren, Eva, et al.
(författare)
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Sample preservation, transport and processing strategies for honeybee RNA extraction: Influence on RNA yield, quality, target quantification and data normalization
- 2017
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 246, s. 81-89
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Tidskriftsartikel (refereegranskat)abstract
- Viral infections in managed honey bees are numerous, and most of them are caused by viruses with an RNA genome. Since RNA degrades rapidly, appropriate sample management and RNA extraction methods are imperative to get high quality RNA for downstream assays. This study evaluated the effect of various sampling transport scenarios (combinations of temperature, RNA stabilizers, and duration) of transport on six RNA quality parameters; yield, purity, integrity, cDNA synthesis efficiency, target detection and quantification. The use of water and extraction buffer were also compared for a primary bee tissue homogenate prior to RNA extraction. The strategy least affected by time was preservation of samples at -80 degrees C. All other regimens turned out to be poor alternatives unless the samples were frozen or processed within 24 h. Chemical stabilizers have the greatest impact on RNA quality and adding an extra homogenization step (a QIAshredder (TM) homogenizer) to the extraction protocol significantly improves the RNA yield and chemical purity. This study confirms that RIN values (RNA Integrity Number), should be used cautiously with bee RNA. Using water for the primary homogenate has no negative effect on RNA quality as long as this step is no longer than 15 min.
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| 6. |
- Forsgren, Eva
(författare)
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Trueness and precision of the real-time RT-PCR method for quantifying the chronic bee paralysis virus genome in bee homogenates evaluated by a comparative inter-laboratory study
- 2017
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 248, s. 217-225
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Tidskriftsartikel (refereegranskat)abstract
- The Chronic bee paralysis virus (CBPV) is the aetiological agent of chronic bee paralysis, a contagious disease associated with nervous disorders in adult honeybees leading to massive mortalities in front of the hives. Some of the clinical signs frequently reported, such as trembling, may be confused with intoxication syndromes. Therefore, laboratory diagnosis using real-time PCR to quantify CBPV loads is used to confirm disease. Clinical signs of chronic paralysis are usually associated with viral loads higher than 108 copies of CBPV genome copies per bee (8 log(10) CBPV/bee). This threshold is used by the European Union Reference Laboratory for Bee Health to diagnose the disease. In 2015, the accuracy of measurements of three CBPV loads (5, 8 and 9 log(10) CBPV/bee) was assessed through an inter-laboratory study. Twenty-one participants, including 16 European National Reference Laboratories, received 13 homogenates of CBPV-infected bees adjusted to the three loads. Participants were requested to use the method usually employed for routine diagnosis. The quantitative results (n = 270) were analysed according to international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). The standard deviations of measurement reproducibility (S-R) were 0.83, 1.06 and 1.16 at viral loads 5, 8 and 9 log(10) CBPV/bee, respectively. The inter-laboratory confidence of viral quantification (+/- 1.96 S-R) at the diagnostic threshold (8 log(10) CBPV/bee) was +/- 2.08 log(10) CBPV/bee. These results highlight the need to take into account the confidence of measurements in epidemiological studies using results from different laboratories. Considering this confidence, viral loads over 6 log(10) CBPV/bee may be considered to indicate probable cases of chronic paralysis.
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| 8. |
- Lillsunde Larsson, Gabriella, 1971-, et al.
(författare)
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HPV Genotyping from the high risk mRNA Aptima assay : a direct approach using DNA from Aptima sample tubes
- 2016
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Ingår i: Journal of Virological Methods. - Amsterdam, Netherlands : Elsevier. - 0166-0934 .- 1879-0984. ; 235, s. 80-84
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Tidskriftsartikel (refereegranskat)abstract
- The underlying cause of cervical cancer is infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.
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| 9. |
- Lindahl, Johanna, et al.
(författare)
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A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya
- 2019
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Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 269, s. 70-76
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Tidskriftsartikel (refereegranskat)abstract
- Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocapsid Np, glycoprotein Gn, and non-structural protein NSs. Sheep and cattle sera from a region in Kenya with previous outbreaks were tested by FMIA and two commercially available competitive ELISAs (BDSL and IDvet). Our results revealed strong detection of RVFV antibodies against the Np, Gn and NSs antigen targets. Additionally, testing of samples with FMIA Np and Gn had 100% agreement with the IDvet ELISA. The targets developed in the FMIA assay provided a basis for a larger ruminant disease panel that can simultaneously screen several abortive and zoonotic pathogens.
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| 12. |
- Semberg, Emilia, et al.
(författare)
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Diagnostic protocols for the detection of Acheta domesticus densovirus (AdDV) in cricket frass
- 2019
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 264, s. 61-64
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Tidskriftsartikel (refereegranskat)abstract
- The European house cricket (Acheta domesticus) is a species of interest for the emerging inseot-as-food industry. Acheta domesticus densovirus (AdDV) is a member of the Parvoviridae virus family which infects A. domesticus, causing widespread mortality and even extinction of local cricket populations. Despite the well-known detrimental effects of AdDV in commercial rearing of A. domesticus there are no optimized protocols to accurately and non-destructively detect and quantify the virus. This study establishes a new protocol for the detection of AdDV in faecal material from A.domesticus. The protocol includes methodological improvements, such as upgrading from conventional PCR to quantitative real-time PCR and is much more sensitive than previously published protocols. Moreover, this study shows that cricket faeces are a suitable, non-destructive sample substrate to infer reliably if a cricket population is infected with AdDV or not. Early detection of lethal or economic threats, such as disease-causing viruses, is an essential part of commercial cricket management as well as for monitoring the risk of spread to wild cricket populations or to (human) consumers.
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| 13. |
- Thulin Hedberg, Sara, 1980-, et al.
(författare)
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Droplet digital PCR for absolute quantification of proviral load of human T-cell lymphotropic virus (HTLV) types 1 and 2
- 2018
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Ingår i: Journal of Virological Methods. - : Elsevier. - 0166-0934 .- 1879-0984. ; 260, s. 70-74
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Human T-lymphotrophic virus (HTLV) types 1 and 2 cause lifelong infection whereby most infected individuals are asymptomatic whilst a minority develop infection-related disease. These latter patients invariably have been found to have high proviral load (PVL). Therefore, infected patients are monitored by determining the proportion of lymphocytes that are infected with HTLV-1/2. An increase in PVL has been shown to represent an increasing risk of developing HTLV-associated diseases. Monitoring of PVL requires a reliable and sensitive method. In this study assays based on droplet digital PCR (ddPCR) were established and evaluated for detection and quantification of HTLV-1/2.OBJECTIVES: To develop two parallel assays to detect the tax genes and determine the PVL of HTLV-1 and -2.STUDY DESIGN: Sixty-seven clinical samples from patients infected with HTLV-1 or HTLV-2 were analysed. The samples had previously been analysed with a qPCR and a comparison between ddPCR and qPCR was performed. The specificity of the assays were determined by analyzing samples from 20 healthy blood donors.RESULTS: The ddPCR was a stable and sensitive method for detection and quantification of HTLV-1 and -2. When comparing the qPCR and ddPCR the correlation was high (Pearsons correlation coefficient 0.96). The variability of the ddPCR was very low with intra-assay coefficient of variation (CV) of 0.97-3.3% (HTLV-1) and 1.7-8.2% (HTLV-2) and inter-assay CV of 1.8-6.1% (HTLV-1) and 1.2-12.9% (HTLV-2).CONCLUSIONS: The ddPCR reliably quantified HTLV DNA in clinical samples and could be a useful tool for monitoring of PVLs in HTLV-infected individuals.
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| 14. |
- Wille, Michelle, et al.
(författare)
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RNAlater (R) is a viable storage option for avian influenza sampling in logistically challenging conditions
- 2018
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Ingår i: Journal of Virological Methods. - : ELSEVIER SCIENCE BV. - 0166-0934 .- 1879-0984. ; 252, s. 32-36
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Tidskriftsartikel (refereegranskat)abstract
- Surveillance of wild birds is critical in monitoring for highly pathogenic avian influenza A viruses (ATVs). However, a successful surveillance regime requires proper treatment of samples in the field - rapid placement of samples in -80 degrees C and subsequent maintenance of cold-chain. Given the logistical difficulties of this, many avian taxa and/or geographic locations are not sampled, or, when sampled may result in false negatives due to poor sample treatment in the field. Here, we assessed the utility of RNAlater (R) as a stabilization agent for AIV sampling. We found no difference in real time PCR performance between virus transport media at optimal conditions and RNAlater (R) at -80 degrees C, -20 degrees C, 4 degrees C or room temperature up to two weeks, at either low or high virus load. Not only was RNAlater (R) useful in comparison of spiked samples or those from duck experiments, it was employed successfully in a field study of backyard birds in China. We detected AIV in cloacal and oropharyngeal samples from chickens and a sample with a low Cq was successfully subtyped as H9, although sample storage conditions were suboptimal. Thus, despite limitations in downstream characterization such virus isolation and typing, RNAlater (R) is a viable option for AIV sampling under logistically challenging circumstances.
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| 15. |
- Borgfeldt, Christer, et al.
(författare)
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Increased HPV detection by the use of a pre-heating step on vaginal self-samples analysed by Aptima HPV assay
- 2019
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 270, s. 18-20
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Tidskriftsartikel (refereegranskat)abstract
- Background: We recently reported a sensitivity of 85.5% to detect high-grade squamous intraepithelial lesions (HSIL)/adenocarcinoma in situ (AIS)/cancer by the use of self-collected vaginal samples analysed by the Aptima mRNA HPV assay (AHPV). Objectives: To increase detection of HPV among self-samples. Study design: We used a pre-heating step at 90 °C for 1 h on our previously AHPV-negative self-samples (N = 20) among women with AHPV-positive cervical samples. We also analysed AHPV results before and after the heating among a series of self-samples from women who had not attended cervical screening for > 7 years (N = 173). Results: After heating, 55% (11/20) of the self-samples became AHPV-positive. By updating our original series 93.1% (121/130, 95% CI: 87.3–96.8) of the self-samples were AHPV-positive among women with AHPV-positive cervical samples, and among women with histologically confirmed cervical intraepithelial neoplasia or worse (CIN2+) now 95.3% (61/64, 95% CI: 86.9–99.0) of the self-samples were AHPV-positive. Among the 11 AHPV-positive self-samples we detected high-risk HPV types in 10 of the samples (HPV16 3 cases, HPV18 1, HPV31 1, HPV33 1, HPV 45 1, HPV51 2, HPV 56 and 58 1, HPV42 and 90 1 [low risk]) by multiplex PCR and Luminex assay. Among the self-samples from the non-attenders 16% (27/170) and 5.3% (8/152) were AHPV-positive after and before the heating step, respectively (P = 0.0022). Concerning validity of AHPV-results, 99% (170/172) were valid after the heating step compared to 88% (152/172) before the heating step (P < 0.0001). Conclusions: A pre-heating step on vaginal self-samples increased HPV detection by the AHPV assay.
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| 16. |
- Lindman, Jacob, et al.
(författare)
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Performance of Bio-Rad HIV-1/2 Confirmatory Assay in HIV-1, HIV-2 and HIV-1/2 dually reactive patients - comparison with INNO-LIA and immunocomb discriminatory assays
- 2019
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 268, s. 42-47
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Tidskriftsartikel (refereegranskat)abstract
- Background: Being able to discriminate between HIV-1, HIV-2 and HIV-1/2 dual infection is imperative for the appropriate selection of antiretroviral therapy (ART) in regions with high HIV-2 endemicity. Objectives: To evaluate Bio-Rad Geenius HIV-1/2 Confirmatory Assay against INNO-LIA HIV 1/2 Score and ImmunoComb HIV 1/2 BiSpot with an emphasis towards ability to discriminate between HIV-1, HIV-2 and HIV-1/2 dual infection. Material and Methods: 131 samples from ART naïve HIV infected patients in Guinea-Bissau were selected retrospectively and tested with Geenius, INNO-LIA and Immunocomb. HIV-1/2 RNA were measured in all samples and HIV-1/2 DNA in 59 samples. Results: The Geenius reader typed 62 samples as HIV-1 reactive, 37 samples as HIV-2 reactive and 32 samples as HIV-1/2 dually reactive. Geenius manual reading classified 10% more samples as HIV-1/2 dually reactive (n = 35). INNO-LIA typed 63 samples as HIV-1 reactive, 36 samples as HIV-2 reactive and 32 samples as HIV-1/2 dually reactive while Immunocomb classified a large proportion of samples as HIV-1/2 dually reactive (n = 45). The measurement of agreement of the Geenius reader compared with INNO-LIA and Immunocomb was 92.4% and 84.0% respectively while the measurement of agreement of Geenius manual reading compared with INNO-LIA and Immuncomb was 93.1% and 89.3% respectively. Conclusions: Geenius has similar performance characteristics as INNO-LIA, and performs considerably better than Immunocomb, for differentiating between HIV types. This is especially true when using the Geenius reader while manual reading of the Geenius assay seemed to overestimate the numbers of HIV-1/2 dually reactive samples.
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| 17. |
- Asker, Noomi, 1968, et al.
(författare)
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Biomarker responses in eelpouts from four coastal areas in Sweden, Denmark and Germany
- 2016
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Ingår i: Marine Environmental Research. - : Elsevier BV. - 0141-1136 .- 1879-0291. ; 120, s. 32-43
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Tidskriftsartikel (refereegranskat)abstract
- To increase our understanding of possible chemical impacts on coastal fish populations in the Baltic Sea, Kattegat and Skagerrak, the viviparous eelpout (Zoarces viviparus) was used as sentinel species in two major sampling campaigns (spring and autumn) in 16 different coastal sites. Condition factor (CF), liver somatic index (LSI), gonad somatic index (GSI) were measured and the activity of the hepatic enzymes ethoxyresorufin-O-deethylase (EROD), glutathione reductase GR), glutathione S-transferase (GST), catalase (CAT) and muscular activity of acetylcholinesterase (AChE) were assessed. PAH metabolites in bile were also analyzed. The most notable finding in the data set was the low EROD activity in eelpouts collected at the relatively polluted region in Germany compared to the other regions, which could be due to an inhibition of the CYP1A-system or to adaptation to chronic exposure of pollutants in this area. Additionally, low AChE activity was noted in the German region in the autumn campaign and low AChE activity detected in the Danish region in the spring campaign. These differences suggest possible season-specific differences in the use and release of AChE-inhibiting chemicals in the Danish and German regions. Clustering of biomarkers on site level indicated a relationship between CF and GSI and suggested that sites with a high CF contained eelpout that put a larger effort into their larvae development. Clustering of the oxidative stress markers GR, GST and CAT on the individual level reflected a possible coordinated regulation of these enzymes. Overall, the results support the importance of taking into account general regional differences and seasonal variation in biomarker activity when monitoring and assessing the effects of pollution. Despite the expected seasonal variation for most of the measured endpoint, several markers (GSI, EROD and CF) vary similarly between all selected sites in both spring and autumn. This suggests that the differences between sites for these endpoints are independent of season.
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