| 1. |
- Lager, Malin, 1975-
(author)
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Molecular and serological tools for clinical diagnostics of Lyme borreliosis - can the laboratory analysis be improved?
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Lyme borreliosis (LB) is caused by spirochetes within the Borrelia burgdorferi sensu lato complex and is the most common tick-transmitted disease in the northern hemisphere. The transmission of the spirochetes to humans in Europe is done by the Ixodes ricinus ticks, which can also transmit the relapsing fever species Borrelia miyamotoi. LB may cause clinical manifestations in the skin, in the central nervous system, in joints, and in the heart. Diagnosis of LB is mainly based on the patient´s medical history, self-described symptoms, and clinical signs in combination with the detection of Borrelia-specific antibodies (serological methods). In some cases/issues, detection of Borrelia-specific deoxyribonucleic acid (molecular methods) may be used as a complement to serology. All diagnosed LB infections are treated with antibiotics to prevent disease progression, and most patients fully recover without further sequelae. The overall aims of this thesis were to evaluate molecular and serological tools for laboratory diagnosis of LB, with a special focus on Lyme neuroborreliosis (LNB), and to identify potential improvements.The results presented in this thesis showed that the immunoglobulin (Ig) G assays, currently in use in northern Europe for detection of antibodies in serum, had high diagnostic sensitivity (88 %) together with comparable results both between and within assays. For the IgM assays, the diagnostic sensitivity was lower (59 %) with more heterogeneous results. Small variations in diagnostic performance for IgM and IgG were mainly presented for samples within the borderline zone. These results support the theory that separate testing of IgM antibodies in serum has low diagnostic value. However, simultaneous detection in serum and cerebrospinal fluid (CSF) for both IgM and IgG antibodies was essential for the diagnosis of LNB, at least for certain assays.So far (to our knowledge), no systematic evaluation and optimisation of the pre-analytical handling of CSF samples before molecular testing has been performed. By use of the precipitate concentrated by moderate centrifugation, extraction of total nucleic acid followed by reversetranscription to complementary deoxyribonucleic acid, in combination with the absence of polymerase chain reaction (PCR) inhibitors, detection of Borrelia garinii, Borrelia afzelii, Borrelia burgdorferi sensu stricto, and B. miyamotoi was possible. These four species are all known to be pathogenic to humans. The results revealed a high analytical sensitivity and specificity for the optimised pre-analytical conditions. The thesis also presents results showing that the real-time PCR protocols currently used in Scandinavia have high analytical sensitivity, specificity, and concordance. This indicates that the low diagnostic sensitivity for detection of Borrelia in CSF was not a result of poorly designed and evaluated PCR protocols, but was possibly due to the low number of spirochetes in the samples. However, to further evaluate the diagnostic performance for detection of Borrelia in CSF by PCR, clinical samples need to be evaluated based on our new recommendations for the pre-analytical handling of CSF samples.In conclusion, this thesis presents results revealing that both molecular and serological tools for detection of Borrelia have, in general high sensitivity and specificity with results comparable between different protocols and different laboratories. It also presents recommendations for pre-analytical handling of CSF samples before PCR-analysis, and shows the benefits in diagnostic performance by simultaneous detection of IgM and IgG antibodies in serum and CSF for accurate diagnosis of LNB. Even though the techniques mentioned above have high analytical performance, the ability to discriminate an active infection from a previous one is limited and further studies need to be carried out. These studies need to focus on finding diagnostic tools that can help physicians to determine ongoing infection to ensure adequate treatment. It is also desirable to improve the standardisation of the diagnostic tools and to find methods that can discriminate between different Borrelia species.
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| 2. |
- Hoffman, Tove, et al.
(author)
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Co-occurrence of Francisella and spotted fever group Rickettsia in avian-associated Hyalomma rufipes
- 2022
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In: Microorganisms. - : MDPI. - 2076-2607. ; 10:7
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Journal article (peer-reviewed)abstract
- Introduction: The migratory behaviour of wild birds aids in the geographical spread of ticks and their microorganisms. Ticks are known to harbor both pathogenic and symbiotic bacteria - such as species of the genera Francisella, Rickettsia,and Midichloria - and multiple bacterial species may occur within them. Francisella occurs in different tick taxa andconsists of closely related pathogenic and non-pathogenic species. Spotted fever group Rickettsia are transmitted to humans by different tick genera and are emerging human pathogens in Europe. The aims of this study were to investigate dispersal of Francisella as well as co-occurrence of Francisella and spotted fever group Rickettsia in ticks infesting northward migrating birds in the African-Western Palaearctic region.Materials and methods: Birds were trapped using mist nets at bird observatories in Spain, Italy, Greece, and Israel during their spring migration of 2014 and 2015. Ticks were screened for the genus Francisella, the species Francisella tularensis, and spotted fever group Rickettsia by microfluidic qPCR. Ticks with putative positive results for F. tularensiswere subjected to confirmation analyses, metagenomics analysis, enrichment, and whole genome sequencing.Results: There was a high prevalence of Francisella species (76.7%) and co-occurrence of Francisella species and spotted fever group Rickettsia (50.6%) in the tick species Hyalomma rufipes. Two H. rufipes yielded putative positive test results for the human pathogen F. tularensis during initial screening. Metagenomics analysis revealed presence of Francisella sp., Rickettsia sp., and Midichloria sp. DNA in the two H. rufipes ticks. The levels of Rickettsia and Midichloria DNA were relatively high while the level of Francisella DNA was low and required enrichment for the construction of metagenome-assembled genomes. Phylogenetic inference and calculations of the average nucleotide identity (ANI) indicated that: i) the Francisella genomes belonged to the Francisella-like endosymbiont (FLE) group in Clade 1 of Francisella and had highest sequence identity to an FLE found in Ornithodoros moubata (ANI: 96.7/97.0%), ii) the Rickettsia genomes had highest resemblance to Rickettsia aeschlimannii (ANI: 98.8 - 99.9%), and iii) the Midichloria genomes resembled Midichloria mitochondrii (ANI: 91.5 - 92.3%).Conclusions: The results of this study suggest ticks containing Francisella species, FLEs, and spotted fever groupRickettsia are dispersed by northbound migratory birds in the African-Western Palaearctic and suggest H. rufipes may not be involved in the transmission of F. tularensis in the study region. Future studies should aim at confirming the prevalence of Francisella spp. and spotted fever group Rickettsia in H. rufipes, in addition to focusing on the influence of FLEs on H. rufipes and their interaction with pathogenic and symbiotic bacteria of the genera Rickettsia and Midichloria.
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| 3. |
- Wilhelmsson, Peter, 1981-, et al.
(author)
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Candidatus Rickettsia Vini DNA in Ticks Collected from Nest Burrows of the European Sand Martin (Riparia riparia) in Sweden
- 2023
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In: Vector Borne and Zoonotic Diseases. - : Mary Ann Liebert. - 1530-3667 .- 1557-7759. ; 23:7, s. 378-383
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Journal article (peer-reviewed)abstract
- Background: Birds can cross geographical and environmental barriers and thereby facilitate dispersal of tick-borne pathogens both as carriers of infected ticks and as reservoirs of pathogenic microorganisms. Ixodes lividus (Ixodida: Ixodidae) is an endophilic tick in the Palearctic region that is highly specialized on its host, the European sand martin Riparia riparia. The purpose of this study was to determine whether I. lividus ticks sampled from sand martin nests in Sweden carry vector-borne pathogens.Materials and Methods: Fed ticks were collected in the autumns of 2017 and 2019 from the nests of a European sand martin colony in southern Sweden. Ticks were identified morphologically to developmental stage and species and were tested for tick-borne pathogens using PCR-based methods.Results: None of the 41 ticks tested positive for five tick-borne pathogens including Borrelia spp., tick-borne encephalitis virus (TBEV), Neoehrlichia mikurensis, Anaplasma phagocytophilum, and Babesia spp. Thirty-seven (13 females, 23 nymphs and 1 larva) of the 41 ticks tested positive for the gltA gene of Rickettsia spp. The sequences of the 17 kDa and gltA genes were most closely related to Candidatus Rickettsia vini.Conclusion: Our study confirms other reports that I. lividus ticks associated with the European sand martin have high infection prevalence of Ca. R. vini.
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