Insights to Genetic Characterization Tools for Epidemiological Tracking of Francisella tularensis in Sweden

Wahab, Tara; Birdsell, Dawn N.; Hjertqvist, Marika; Mitchell, C. Madeline; Wagner, David M.; Keim, Paul; Hedenström, Ingela; Löfdahl, Sven

doi:10.1371/journal.pone.0112167

Cited by 8 publications

(7 citation statements)

References 20 publications

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“…We selected six VNTR markers (Ft-M03, Ft-M06, Ft-M22, Ft-M24, Ft-M20, and Ft-M21) with the highest variability on a subspecies level ( Johansson et al 2004, Wahab et al 2014 and performed MLVA directly from real-time PCR-positive clinical material. The VNTR markers, Ft-M03, Ft-M06, Ft-M22, Ft-M24 (Vogler et al 2009b), Ft-M20, and Ft-M21 ( Johansson et al 2004), were amplified as follows: 1 • PCR supermix (Invitrogen), 2.5 lL of template DNA, at different concentrations of forward and reverse primers (Table 1), in a total volume of 25 lL.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis

Biškup

Kogoj

Korva

et al. 2021

Vector-Borne and Zoonotic Diseases

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Francisella tularensis is the etiologic agent of tularemia, a bacterial zoonotic disease. The genome of F. tularensis shows a recent evolutionary change, especially in reservoirs. Variable number of tandem repeats (VNTR) is described as a high-speed molecular clock and can thus be used as a high-resolution typing system. The main objective of our study was to investigate the molecular diversity of F. tularensis strains and reveal possible sources of infection. Using real-time PCR targeting the ISFtu2 region, we successfully amplified targeted DNA in 13/31 Slovenian patients with a clinical diagnosis of tularemia, and with PCR targeting the fopA gene, we obtained 11/13 PCR products. Sequencing revealed that all samples were identified as F. tularensis subsp. holarctica. We successfully obtained one F. tularensis isolate from a lymph node aspirate by culture on chocolate agar. Our isolate was clustered into major clade B12 (subclade B43). We optimized VNTR typing to be used directly on clinical samples. Multiple-locus VNTR analysis (MLVA) revealed five unique MLVA types; 45.5% samples had the same MLVA type, another 27.3% shared a different MLVA type, and each of the remaining had a unique MLVA type. Most samples differed at only two VNTR markers (Ft-M03 and Ft-M06). Additionally, we investigated samples from small mammals (n = 532) and Ixodes ricinus ticks (n = 232) captured in the same geographical area in which patients with tularemia were found. No F. tularensis DNA was detected in samples of small mammals or I. ricinus ticks. The diversity of MLVA types in Slovenia was high, despite the small region, but most of the samples from the same region shared the same MLVA type. Our results suggest that MLVA is a useful tool for quick molecular characterization of F. tularensis directly from patient samples, especially when investigating geographically localized outbreaks.

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Section: Methodsmentioning

confidence: 99%

“…VNTR is described as a high-speed molecular clock (van Belkum 1999) and can thus be used as a high-resolution typing system ( Johansson et al 2004, Wahab et al 2014). The genome of F. tularensis has shown a recent evolutionary change, especially in reservoirs (Larsson et al 2009).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis

Biškup

Kogoj

Korva

et al. 2021

Vector-Borne and Zoonotic Diseases

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“…F. tularensis is the causative agent of the zoonotic disease tularemia and it can infect humans by several routes, including the ingestion of contaminated water or food. Exposure to F. tularensis -contaminated water [ 13 , 14 ], blood-feeding vectors [ 15 ], or, on rare occasion, aerosolized particulates [ 16 ] each lead to distinct clinical forms of tularemia: orophyryngeal, ulcergrandular, and respiratory tularemia, respectively. In the rapidly developing nation of Turkey, tularemia has been on the rise since 2009 and oropharyngeal disease is the most common form [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

Coinfections identified from metagenomic analysis of cervical lymph nodes from tularemia patients

et al. 2018

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BackgroundUnderlying coinfections may complicate infectious disease states but commonly go unnoticed because an a priori clinical suspicion is usually required so they can be detected via targeted diagnostic tools. Shotgun metagenomics is a broad diagnostic tool that can be useful for identifying multiple microbes simultaneously especially if coupled with lymph node aspirates, a clinical matrix known to house disparate pathogens. The objective of this study was to analyze the utility of this unconventional diagnostic approach (shotgun metagenomics) using clinical samples from human tularemia cases as a test model. Tularemia, caused by the bacterium Francisella tularensis, is an emerging infectious disease in Turkey. This disease commonly manifests as swelling of the lymph nodes nearest to the entry of infection. Because swollen cervical nodes are observed from many different types of human infections we used these clinical sample types to analyze the utility of shotgun metagenomics.MethodsWe conducted an unbiased molecular survey using shotgun metagenomics sequencing of DNA extracts from fine-needle aspirates of neck lymph nodes from eight tularemia patients who displayed protracted symptoms. The resulting metagenomics data were searched for microbial sequences (bacterial and viral).ResultsF. tularensis sequences were detected in all samples. In addition, we detected DNA of other known pathogens in three patients. Both Hepatitis B virus (HBV) and Human Parvovirus B-19 were detected in one individual and Human Parvovirus B-19 alone was detected in two other individuals. Subsequent PCR coupled with Sanger sequencing verified the metagenomics results. The HBV status was independently confirmed via serological diagnostics, despite evading notice during the initial assessment.ConclusionOur data highlight that shotgun metagenomics of fine-needle lymph node aspirates is a promising clinical diagnostic strategy to identify coinfections. Given the feasibility of the diagnostic approach demonstrated here, further steps to promote integration of this type of diagnostic capability into mainstream clinical practice are warranted.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3218-2) contains supplementary material, which is available to authorized users.

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“…Single nucleotide polymorphisms (SNPs) are highly valuable genetic markers that have advanced our knowledge of diverse biological fields such as human health [ 1 , 2 ], infectious disease epidemiology [ 3 – 5 ], agriculture [ 6 ], and ecology [ 7 ], among others. In the study of infectious diseases SNPs can be informative of bacterial phenotype, such as antibiotic susceptibility [ 8 , 9 ], and also can be used to classify unknown strains.…”

Section: Introductionmentioning

confidence: 99%

“…These stable signatures can be used to classify unknown strains into known phylogenetic groups according to SNP profiles [ 3 , 11 , 12 ]. Within the context of epidemiological investigations, these SNP profiles can link isolates from active outbreak sites to possible sources and help track disease transmission patterns [ 5 , 13 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

Low cost, low tech SNP genotyping tools for resource-limited areas: Plague in Madagascar as a model

et al. 2017

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BackgroundGenetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs.Methodology/Principal findingsTo demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar), we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM). We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort.ConclusionsThe success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable financial cost for resource constraint laboratories. This is a practical formula that reduces resource-driven limitations to genetic research and promises to advance global collective knowledge of infectious diseases emanating from resource limited regions of the world.

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Insights to Genetic Characterization Tools for Epidemiological Tracking of Francisella tularensis in Sweden

Cited by 8 publications

References 20 publications

Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis

Characterization of Tularemia Cases in Slovenia with Multiple-Locus Variable-Number Tandem Repeat Analysis

Coinfections identified from metagenomic analysis of cervical lymph nodes from tularemia patients

Low cost, low tech SNP genotyping tools for resource-limited areas: Plague in Madagascar as a model

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