Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens.
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ABSTRACT:
SUBMITTER: Jain K
PROVIDER: S-EPMC8196166 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Assay sensitivity can be a limiting factor in the use of PCR as a tool for the detection of tick-borne pathogens in blood. We evaluated the performance of Tick-borne disease Capture Sequencing Assay (TBDCapSeq), a capture sequencing assay targeting tick-borne agents, to test 158 whole blood specimens obtained from the Lyme Disease Biobank. These included samples from 98 individuals with signs and symptoms of acute Lyme disease, 25 healthy individuals residing in Lyme disease endemic areas, and 35 samples collected from patients admitted to the Massachusetts General Hospital or referred to the infectious disease clinic. Compared to PCR, TBDCapSeq had better sensitivity and could identify infections with a wider range of tick-borne agents. TBDCapSeq identified a higher rate of samples positive for Borrelia burgdorferi (8 vs. 1 by PCR) and Babesia microti (26 vs. 15 by PCR). TBDCapSeq also identified previously unknown infections with Borrelia miyamotoi, Ehrlichia, and Rickettsia species. Overall, TBDCapSeq identified a pathogen in 43 samples vs. 23 using PCR, with four co-infections detected versus zero by PCR. We conclude that capture sequencing enables superior detection of tick-borne agents relative to PCR.
| S-EPMC8940530 | biostudies-literature
Project description:Infectious disease diagnostics often depend on costly serological testing with poor sensitivity, low specificity, and long turnaround time. Here, we demonstrate proof of the principle for simultaneous detection of two tick-borne pathogens from a single test sample using barcoded magnetic bead technology on the BioCode 2500 system. Specific primer sets complementary to the conserved genes of Anaplasma phagocytophilum and Borrelia burgdorferi were used in PCR amplification of the target, followed by the hybridization of the resulting biotinylated PCR products with specific probes tethered to the barcoded magnetic beads for simultaneous detection, using a fluorophore with high quantum yield. The assay has an extremely high signal to background ratio, with a limit of detection (LOD) of 2.81 50% tissue culture infection dose (TCID50)/mL and 1 CFU/mL for A. phagocytophilum and B. burgdorferi, respectively. The observed LOD for gene blocks was 1.8 copies/reaction for both the pathogens. The assay demonstrated 100% positive and negative agreement on performance evaluation using patient specimens and blood samples spiked with 1 × LOD of pathogen stock. No cross-reactivity was observed with other related tick-borne pathogens and genomic DNA of human, cattle, and canine origin. The assay can be upgraded to a sensitive and cost-effective multiplex diagnostic approach that can simultaneously detect multiple clinically important tick-borne pathogens in a single sample with a short turnaround time. IMPORTANCE The low pathogen load in the tick-borne disease test samples and the lack of highly sensitive multiplex diagnostic approaches have impacted diagnosis during clinical testing and limited surveillance studies to gauge prior insight about the prevalence of tick-borne infections in a geographical area. This article demonstrates proof of the principle for simultaneous detection of two important tick-borne pathogens from a single test sample using digital barcoded magnetic bead technology. Using a fluorophore of high quantum yield, the diagnostic approach showed high sensitivity and specificity. The LOD was 1.8 genome copies per reaction for both A. phagocytophilum and B. burgdorferi. The assay can be upgraded for the detection of all clinically important tick-borne pathogens from a single patient sample with high sensitivity and specificity. The assay can provide a diagnostic answer to the clinician in a short turnaround time to facilitate speedy therapeutic intervention to infected patients and implement public health measures to prevent community spread.
| S-EPMC10269837 | biostudies-literature
Project description:Tick-borne diseases (TBDs) cause significant losses among livestock and impact the livelihoods of resource-poor farming communities worldwide. In Ethiopia, detailed studies on the epidemiology of tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to determine the prevalence and species composition of bovine TBPs of veterinary significance in local cattle populations. A comprehensive cross-sectional epidemiological study was conducted in cattle populations of Illubabor zone in Southwestern Ethiopia from June to August 2013. For this purpose, blood samples were collected from 392 cattle. A combination of polymerase chain reaction (PCR) and a Reverse Line Blot (RLB) hybridization assay was employed for the detection of TBPs in these samples. The PCR/RLB results of the 392 blood samples indicated a high overall prevalence of 96.9% for TBPs, including Theileria mutans (66.1%), Theileria orientalis (51.8%), Anaplasma sp. Omatjenne (25.5%), Anaplasma marginale (14.5%), Babesia bigemina (14.0%) and Theileria velifera (13.0%) and minor occurrences of Ehrlichia ruminantium (0.5%) and Ehrlichia minasensis (0.26%). Moreover, three novel Anaplasma genotypes were detected in bovine blood samples. A phylogenetic analysis revealed that they most likely represent three, but at least two, new species. The prevalence of the three novel Anaplasma species, preliminary designated as Anaplasma sp. Hadesa, Anaplasma sp. Saso and Anaplasma sp. Dedessa, was 12.5%, 14.3% and 5.6%, respectively. Overall, a total of 227 cattle (57.9%) were found to be co-infected with two or more TBPs simultaneously and 86 different species combinations were observed. The findings show a very high burden of infection of cattle with TBPs in Ethiopia. The high frequency of co-infections suggests that clinical manifestations might be complex. Further research is required to determine the pathogenicity, host cell types and vector of the three novel Anaplasma species identified in this study.
| S-EPMC5695778 | biostudies-literature
Project description:BackgroundThe populations of wild felids in Africa, of especially lions (Panthera leo) and cheetahs (Acinonyx jubatus), are declining and the species are classified as vulnerable to extinction by the International Union for Conservation of Nature. As infections with tick-borne pathogens (TBP) can become more of a problem in wild felids, there are relatively few studies on TBP in wild felids in Africa and on how these infections might influence population numbers.MethodsTo gain further knowledge on TBP in captive wild felids in Southern Africa, we collected whole blood from captive lions, Southern African wildcats, cheetahs and servals in Zimbabwe for PCRs against the 18S rRNA gene of the piroplasmids (Babesia, Theileria, Cytauxzoon) and Hepatozoon spp., and the 16S rRNA gene of Ehrlichia and Anaplasma spp.ResultsOverall, 78% of the lions (67/86) and all the Southern African wildcats (6/6), cheetahs (4/4) and servals (2/2) had evidence of infection with at least one organism. The organisms most commonly detected in the lions were B. leo (59%; 51/86), B. vogeli (12%; 10/86) and H. felis (11%; 9/86) while all the Southern African wildcats and servals were positive for B. vogeli and all the cheetahs were positive for B. leo. Mixed infections were found in 22% (15/67) of the PCR positive lions, most commonly B. leo and H. felis (27%; 4/15), and in 1 (50%) of the servals (B. vogeli and A. phagocytophilum). Two lions were infected with three TBP, mainly B. leo, H. canis and T. parva, and B. leo, A. phagocytophilum and T. sinensis. Mixed infections with B. vogeli and A. phagocytophilum were seen in a serval and a Southern African wildcat. Other TBP were detected at a low prevalence (≤2%) in lions, mainly H. canis, T. sinensis, T. parva, C. manul, E. canis, and E. canis-like and B. odocoilei-like organisms.ConclusionsInfections with tick-borne agents are common in captive wild felids in Zimbabwe.
| S-EPMC4243927 | biostudies-literature
Project description:BackgroundDisease maps are used increasingly in the health sciences, with applications ranging from the diagnosis of individual cases to regional and global assessments of public health. However, data on the distributions of emerging infectious diseases are often available from only a limited number of samples. We compared several spatial modelling approaches for predicting the geographic distributions of two tick-borne pathogens: Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, and Anaplasma phagocytophilum, the causative agent of human granulocytotropic anaplasmosis. These approaches extended environmental modelling based on logistic regression by incorporating both spatial autocorrelation (the tendency for pathogen distributions to be clustered in space) and spatial heterogeneity (the potential for environmental relationships to vary spatially).ResultsIncorporating either spatial autocorrelation or spatial heterogeneity resulted in substantial improvements over the standard logistic regression model. For E. chaffeensis, which was common within the boundaries of its geographic range and had a highly clustered distribution, the model based only on spatial autocorrelation was most accurate. For A. phagocytophilum, which has a more complex zoonotic cycle and a comparatively weak spatial pattern, the model that incorporated both spatial autocorrelation and spatially heterogeneous relationships with environmental variables was most accurate.ConclusionSpatial autocorrelation can improve the accuracy of predictive disease risk models by incorporating spatial patterns as a proxy for unmeasured environmental variables and spatial processes. Spatial heterogeneity can also improve prediction accuracy by accounting for unique ecological conditions in different regions that affect the relative importance of environmental drivers on disease risk.
| S-EPMC2373776 | biostudies-literature
Project description:BACKGROUND:Species of Canidae in Russia can be infested with up to 24 different tick species; however, the frequency of different tick species infesting domestic dogs across Russia is not known. In addition, tick-borne disease risks for domestic dogs in Russia are not well quantified. The goal of this study was to conduct a nationwide survey of ticks collected from infested dogs admitted to veterinary clinics in Russian cities and to identify pathogens found in these ticks. METHODS:Ticks feeding on dogs admitted to 32 veterinary clinics in 27 major cities across Russia were preserved in ethanol and submitted to a central facility for examination. After identification, each tick was evaluated for infection with known tick-borne pathogens using PCR. RESULTS:There were 990 individual ticks collected from 636 dogs. All collected ticks belonged to the Ixodidae (hard ticks) and represented 11 species of four genera, Dermacentor, Ixodes, Rhipicephalus and Haemaphysalis. Four most common tick species were D. reticulatus, followed by I. persulcatus, I. ricinus and R. sanguineus. Ixodes persulcatus ticks were found to be infected with 10 different pathogens, and ticks of this species were more frequently infected than either D. reticulatus or I. ricinus. Ixodes persulcatus females were also more frequently co-infected with two or more pathogens than any other tick. Pathogenic species of five genera were detected in ticks: Anaplasma centrale, A. phagocytophilum and A. marginale; Babesia canis, B. microti, B. venatorum, B. divergens, B. crassa and B. vogeli; Borrelia miyamotoi, B. afzelii and B. garinii; Ehrlichia muris, E. canis and E. ruminantum; and Theileria cervi. Anaplasma marginale, E. canis, B. crassa, B. vogeli and T. cervi were detected in I. persulcatus, and Babesia canis in D. marginatum, for the first time in Russia. CONCLUSIONS:Multiple ticks from four genera and 11 species of the family Ixodidae were collected from domestic dogs across Russia. These ticks commonly carry pathogens and act as disease vectors. Ixodes persulcatus ticks present the greatest risk for transmission of multiple arthropod-borne pathogens.
| S-EPMC6234536 | biostudies-literature
Project description:The prevalence of tick-borne infections has been steadily increasing in both number and geographic distribution in the United States and abroad. This increase, in conjunction with the continued recognition of novel pathogens transmitted by ticks, has made accurate diagnosis of these infections challenging. Mainstay serologic tests are insensitive during the acute phase of infection and are often cross-reactive with similar pathogenic and nonpathogenic organisms. Further, they are unable to reliably differentiate active versus past infection which can lead to misdiagnosis and incorrect understanding of the epidemiology and incidence of specific tick-borne pathogens. We evaluated a novel multiplexed high-definition PCR (HDPCR) Tickborne Panel (TBP) assay (ChromaCode, Carlsbad, CA) for the detection of nine tick-borne pathogens or groups associated with human illness. The HDPCR technology enables multiplex identification of multiple targets in a single fluorometric channel based on fluorescent signal modulation using a limiting probe design. A collection of 530 whole-blood specimens collected from patients being evaluated for tick-borne infections, in addition to a panel of 93 simulated specimens, were used to challenge the HDPCR TBP. The results were compared to a clinically validated traditional multiplexed PCR test with additional sequence analysis and clinical history collected to aid in resolving discrepancies. Among clinical specimens the TBP demonstrated 100% sensitivity for the identification of Anaplasma phagocytophilum, Borrelia miyamotoi, Borrelia mayonii, and Rickettsia rickettsii The sensitivity for identification of B. burgdorferi was 44.4% compared to a composite gold standard. Among simulated specimens containing single or multiple targets present at 103 to 105 copies/PCR, the sensitivity of TBP was 100% for all targets, with a combined specificity of 99.5%. Of note, an increased rate of false-positive results was observed among simulated specimens that contained multiple targets. Based on these data, we find the HDPCR TBP to be a useful adjunct for the diagnosis of tick-borne infections in patients with suspected tick-borne illness.
| S-EPMC6812998 | biostudies-literature
Project description:Tick-borne diseases, due to a diversity of bacterial pathogens, represent a significant and increasing public health threat throughout the Northern Hemisphere. A high-throughput 16S V1-V2 rRNA gene-based metagenomics assay was developed and evaluated using >13,000 residual samples from patients suspected of having tick-borne illness and >1,000 controls. Taxonomic predictions for tick-borne bacteria were exceptionally accurate, as independently validated by secondary testing. Overall, 881 specimens were positive for bacterial tick-borne agents. Twelve tick-borne bacterial species were detected, including two novel pathogens, representing a 100% increase in the number of tick-borne bacteria identified compared to what was possible by initial PCR testing. In three blood specimens, two tick-borne bacteria were simultaneously detected. Seven bacteria, not known to be tick transmitted, were also confirmed to be unique to samples from persons suspected of having tick-borne illness. These results indicate that 16S V1-V2 metagenomics can greatly simplify diagnosis and accelerate the discovery of bacterial tick-borne pathogens.
| S-EPMC7587092 | biostudies-literature
Project description:In this study, blood samples obtained from 162 dogs in Jiangxi, China, were employed in molecular screening of canine tick-borne pathogens by PCR and sequencing. Babesia spp. gene fragment was detected in 12 (7.41%) dogs. All samples were negative for Hepatozoon spp., Ehrlichia canis, Coxiella spp., Borrelia spp., Rickettsia spp. and Anaplasma platys. Species-specific PCR analysis further confirmed that 8 (4.94%) and 4 (2.47%) dogs were infected by Babesia canis vogeli and Babesia gibsoni, respectively. Based on our analyses, Babesia spp. infection in Jiangxi appeared not related to age, gender, breed, usage, activity and health status or tick infestation history of the dogs. This is the first molecular report of Babesia canis vogeli and Babesia gibsoni in dogs from Jiangxi, China.
| S-EPMC5326926 | biostudies-literature
Project description:Deer represent a major vertebrate host for all feeding stages of the hard tick Ixodes ricinus in the United Kingdom (UK), and could play a role in the persistence of tick-borne pathogens. However, there have been few studies reporting the presence of Babesia spp. and Anaplasma phagocytophilum in deer in the UK, and those that detected Babesia were unable to confirm the species. To address this, we have investigated blood samples from red deer (Cervus elaphus) for the presence of tick-borne pathogens. Total DNA was extracted from haemolysed blood that was removed from clotted blood sampled from culled, captive red deer. Babesia spp. were detected with a pan-piroplasm PCR that amplifies a fragment of the 18S rRNA gene. Species were identified based on identity with published sequences. Anaplasma phagocytophilum was detected with a probe-based PCR targeting the msp2 gene. In addition, residual serum samples from a subset of animals were tested for the presence of anti-flavivirus antibodies. Of 105 red deer samples tested from three locations in the United Kingdom, 5 were positive for piroplasm and 5 were positive for A. phagocytophilum. Co-infection with both pathogens was detected in two samples from one location. No evidence for antibodies against West Nile virus were detected. However, 12% of sera tested were positive for tick-borne encephalitis virus antibodies.
| S-EPMC8224737 | biostudies-literature