A Rare Tick Tale: A Novel Case of the Australian Paralysis Tick Causing Multiple Cranial Neuropathies.

The Australian paralysis tick (Ixodes holocyclus) is found along the east coast of Australia. Tick bites may result in paralysis ranging from muscular weakness to ascending paralysis requiring respiratory support. Ocular complications and facial nerve involvement are rare. We present a rare occurrence of tick-bite-associated visual loss, proptosis, and multiple cranial neuropathies not previously reported in the literature. The tick was removed, and the patient's symptoms improved following treatment with steroids and oral doxycycline. The vision and sensory changes are not explained by the Ixodes toxin; thus, we hypothesize this is related to orbital apex inflammation.

Spatiotemporal dynamics of Ixodes ricinus abundance in northern Spain.

Ixodes ricinus is the most medically relevant tick species in Europe because it transmits the pathogens that cause Lyme borreliosis and tick-borne encephalitis. Northern Spain represents the southernmost margin of its main European range and has the highest rate of Lyme borreliosis hospitalisations in the country. Currently, the environmental determinants of the spatiotemporal patterns of I. ricinus abundance remain unknown in this region and these may differ from drivers in highly favourable areas for the species in Europe. Therefore, our study aimed to understand the main factors modulating questing I. ricinus population dynamics to map abundance patterns in northern Spain. From 2012 to 2014, monthly/fortnightly samplings were conducted at 13 sites in two regions of northern Spain to estimate spatiotemporal variation in I. ricinus questing abundance. Local abundance of I. ricinus was modelled in relation to variation in local biotic and abiotic environmental conditions by constructing generalised linear mixed models with a zero-inflated negative binomial distribution for overdispersed data. The different developmental stages of I. ricinus were most active at different times of the year. Adults and nymphs showed a peak of abundance in spring, while questing larvae were more frequent in summer. The main determinants affecting the spatiotemporal abundance of the different stages were related to humidity and temperature. For adults and larvae, summer seemed to be the most influential period for their abundance, while for nymphs, winter conditions and those of the preceding months seemed to be determining factors. The highest abundances of nymphs and adults were predicted for the regions of northern Spain with the highest rate of Lyme borreliosis hospitalisations. Our models could be the basis on which to build more accurate predictive models to identify the spatiotemporal windows of greatest potential interaction between animals/humans and I. ricinus that may lead to the transmission of I. ricinus-borne pathogens.

Assessing the value and knowledge gains from an online tick identification and tick-borne disease management course for the Southeastern United States.

BACKGROUND: Tick-borne diseases are a growing public health threat in the United States. Despite the prevalence and rising burden of tick-borne diseases, there are major gaps in baseline knowledge and surveillance efforts for tick vectors, even among vector control districts and public health agencies. To address this issue, an online tick training course (OTTC) was developed through the Southeastern Center of Excellence in Vector-Borne Diseases (SECOEVBD) to provide a comprehensive knowledge base on ticks, tick-borne diseases, and their management. METHODS: The OTTC consisted of training modules covering topics including tick biology, tick identification, tick-borne diseases, and public health, personal tick safety, and tick surveillance. The course was largely promoted to vector control specialists and public health employees throughout the Southeastern US. We collected assessment and survey data on participants to gauge learning outcomes, perceptions of the utility of knowledge gained, and barriers and facilitators to applying the knowledge in the field. RESULTS: The OTTC was successful in increasing participants' baseline knowledge across all course subject areas, with the average score on assessment increasing from 62.6% (pre-course) to 86.7% (post-course). More than half of participants (63.6%) indicated that they would definitely use information from the course in their work. Barriers to using information identified in the delayed assessment included lack of opportunities to apply skills (18.5%) and the need for additional specialized training beyond what the OTTC currently offers (18.5%), while the main facilitator (70.4%) for applying knowledge was having opportunities at work, such as an existing tick surveillance program. CONCLUSIONS: Overall, this OTTC demonstrated capacity to improve knowledge in a necessary and underserved public health field, and more than half of participants use or plan to use the information in their work. The geographic reach of this online resource was much larger than simply for the Southeastern region for which it was designed, suggesting a much broader need for this resource. Understanding the utility and penetrance of training programs such as these is important for refining materials and assessing optimal targets for training.

Potential of mRNA-based vaccines for the control of tick-borne pathogens in one health perspective.

The One Health approach, which integrates the health of humans, animals, plants, and ecosystems at various levels, is crucial for addressing interconnected health threats. This is complemented by the advent of mRNA vaccines, which have revolutionized disease prevention. They offer broad-spectrum effectiveness and can be rapidly customized to target specific pathogens. Their utility extends beyond human medicine, showing potential in veterinary practices to control diseases and reduce the risk of zoonotic transmissions. This review place mRNA vaccines and One Health in the context of tick-borne diseases. The potential of these vaccines to confer cross-species immunity is significant, potentially disrupting zoonotic disease transmission cycles and protecting the health of both humans and animals, while reducing tick populations, infestations and circulation of pathogens. The development and application of mRNA vaccines for tick and tick-borne pathogens represent a comprehensive strategy in global health, fostering a healthier ecosystem for all species in our interconnected world.

NMR-based metabolomic investigation of dogs with acute flaccid paralysis due to tick paralysis.

BACKGROUND: Acute flaccid paralysis (AFP) is a complex clinical syndrome with various aetiologies. If untreated, AFP may lead to death due to failure of respiratory muscles. Tick paralysis, which is a noninfectious neurologic syndrome of AFP, occurs following tick attachment, engorgement, and injection of tick saliva toxins. There is no specific diagnostic test for tick paralysis, and mortality increases as definitive diagnosis is delayed. Although metabolomic investigation of tick saliva was conducted, there is a lack of research on metabolomic evaluation of hosts affected by tick paralysis. OBJECTIVES: Thus, the aim of this study is to investigate metabolomic changes in serum samples of dogs with tick paralysis due to Rhipicephalus sanguineus using NMR-based metabolomics and to identify potential diagnostic/prognostic markers. MATERIALS AND METHODS: Forty dogs infested with R. sanguineus, with clinical findings compatible with AFP and with a confirmed tick paralysis diagnosis ex juvantibus, constituted the Paralysis Group. Ten healthy dogs, which were admitted either for vaccination and/or check-up purposes, constituted the Control Group. After the confirmation tick paralysis, medical history, vaccination and nutritional status, body surface area and estimated tick numbers of all the dogs were noted. Physical examination included body temperature, heart and respiratory rate, capillary refill time evaluation and Modified Glasgow Coma Scale calculation. Serum samples were extracted from venous blood samples of all the dogs and were prepared for NMR analysis, and NMR-based metabolomics identification and quantification were performed. RESULTS: NMR-based serum metabolomics of the present study revealed distinct up/down-regulated expressions, presenting a promising avenue. Moreover, it was observed that energy metabolism and especially liver functions were impaired in dogs with tick paralysis, and not only the respiratory system but also the kidneys were affected. CONCLUSION: It was concluded that the present approach may help to better understand the pathological mechanisms developing in cases of AFP due to tick paralysis.

Changes in cerebrospinal fluid proteome of patients with tick-borne encephalitis.

Tick-borne encephalitis (TBE) is one of the main diseases transmitted by ticks, the incidence of which is increasing. Moreover, its diagnosis and therapy are often long and difficult according to nonspecific symptoms and complex etiology. This study aimed to observe changes in the proteome of cerebrospinal fluid from TBE patients. Cerebrospinal fluid (CSF) of TBE patients (n = 20) and healthy individuals (n = 10) was analyzed using a proteomic approach (QExactiveHF-Orbitrap mass spectrometer) and zymography. Obtained results show that in CSF of TBE patients, the top-upregulated proteins are involved in pro-inflammatory reaction (interleukins), as well as antioxidant/protective response (peroxiredoxins, heat shock proteins). Moreover, changes in the proteome of CSF are not only the result of this disease development, but they can also be an indicator of its course. This mainly applies to proteins involved in proteolysis including serpins and metalloproteinases, whose activity is proportional to the length of patients' convalescence. The obtained proteomic data strongly direct attention to the changes caused by the development of TBE to antioxidant, pro-inflammatory, and proteolytic proteins, knowledge about which can significantly contribute to faster and more accurate diagnosis of various clinical forms of TBE.

A retrospective study of Crimean-Congo hemorrhagic fever in Iraq.

Background: CCHF is transmitted via ticks biting and directly by contact with tissue or blood of infected patients or viremic animals. This study intends to determine the occurrence of CCHF in Iraq between 2015 and 2019. Methods: This study was designed as a retrospective and descriptive cross-sectional study. It was approved the occurrence of CCHF in Iraq with relation to some epidemiological and demographic data reported in the Iraqi Communicable Diseases Control Center (CDC)/zoonotic diseases section between 2015-2019. Results: Out of 206 suspected cases, only 17 were diagnosed as CCHF with a total fatality ratio of 52%, 25%, and 80% in 2015 and 2018 respectively. However, no mortality was reported during 2016, 2017, and 2019. The mean age of the patients was 33 years± 18 SD, in males mainly (76%). Moreover, the risk groups were 29 %, 23 %, 18 % 12 %, and 6 % for butchers, animal dealers, gainers, both housewives and students and children respectively. Conclusion: Strict precautions and precise surveillance should be implemented to control the disease and protect the community by improving the diagnosis and treatment of CCHF. The authors recommend another future study to detect the genotyping and sequencing of CCHFV in Iraq.

Haemaphysalis longicornis (Acari: Ixodidae) does not transmit Babesia bovis, a causative agent of cattle fever.

The Asian longhorned tick (Haemaphysalis longicornis) was first reported in the United States in 2017 and has since been detected in at least 17 states. This tick infests cattle and can produce large populations quickly due to its parthenogenetic nature, leading to significant livestock mortalities and economic losses. While H. longicornis has not been detected in Texas, species distribution models have identified southern Texas as a possible hospitable region for this tick. Southern Texas is currently home to the southern cattle tick (Rhipicephalus microplus), which can transmit the causative agent of cattle fever (Babesia bovis). With the potential for H. longicornis and B. bovis to overlap in southern Texas and their potential to negatively impact the national and global livestock industry, it is imperative to identify the role H. longicornis may play in the cattle fever disease system. A controlled acquisition and transmission experiment tested whether H. longicornis is a vector for B. bovis, with the R. microplus-B. bovis system used as a positive control. Transstadial (nymphs to adults) and transovarial (adults to larvae) transmission and subsequent transstadial maintenance (nymphs and adults) routes were tested in this study. Acquisition-fed, splenectomized animals were used to increase the probability of tick infection. Acquisition nymphs were macerated whole and acquisition adults were dissected to remove midguts and ovaries at five time points (4, 6, 8, 10, and 12 days post-repletion), with 40 ticks processed per time point and life stage. The greatest percentage of nymphs with detectable B. bovis DNA occurred six days post-repletion (20.0 %). For adults, the percentage of positive midguts and ovaries increased as days post-repletion progressed, with day 12 having the highest percentage of positive samples (67.5 % and 60.0 %, respectively). When egg batches were tested in triplicate, all H. longicornis egg batches were negative for B. bovis, while all R. microplus egg batches were positive for B. bovis. During the transmission phase, the subsequent life stages for transstadial (adults) and transovarial transmission/transstadial maintenance (larvae, nymphs, and adults) were fed on naïve, splenectomized calves. All life stages of H. longicornis ticks tested during transmission were negative for B. bovis. Furthermore, the transmission fed animals were also negative for B. bovis and did not show signs of bovine babesiosis during the 45-day post tick transmission period. Given the lack of successful transstadial or transovarial transmission, it is unlikely that H. longicornis is a vector for B. bovis.

Tick salivary proteome and lipidome with low glycan content correlate with allergic type reactions in the zebrafish model.

Ticks, as hematophagous ectoparasites, can manipulate host immune and metabolic processes, causing tick-borne allergies such as α-Gal syndrome (AGS). Glycolipids with bound galactose-alpha-1-3-galactose (α-Gal) are potential allergenic molecules associated with AGS. Nevertheless, proteins and lipids lacking α-Gal modifications may contribute to tick salivary allergies and be linked to AGS. In this study, we characterized the effect of deglycosylated tick salivary proteins without lipids on treated zebrafish fed with dog food formulated with mammalian (beef, lamb, pork) meat by quantitative proteomics analysis of intestinal samples. The characterization and functional annotations of tick salivary lipids with low representation of glycolipids was conducted using a lipidomics approach. Results showed a significant effect of treatment with saliva and saliva deglycosylated protein fraction on zebrafish abnormal or no feeding (p < 0.005). Treatment with this fraction affected multiple metabolic pathways, defense responses to pathogens and protein metabolism, which correlated with abnormal or no feeding. Lipidomics analysis identified 23 lipid classes with low representation of glycolipids (0.70% of identified lipids). The lipid class with highest representation was phosphatidylcholine (PC; 26.66%) and for glycolipids it corresponded to diacylglycerol (DG; 0.48%). Qualitative analysis of PC antibodies revealed that individuals bitten by ticks were more likely to produce PC-IgG antibodies (p < 0.001). DG levels were significantly higher in tick salivary glands (p < 0.05) compared with tick saliva and salivary fractions. The α-Gal content was higher in tick saliva than in deglycosylated saliva and lipid fractions. These results support a possible role for tick salivary proteins and lipids without α-Gal modifications in AGS.

Update on tick-borne pathogens detection methods within ticks.

Ticks are known vectors of various pathogens, including bacteria, parasites and viruses, that impact both animal and human health. Improving knowledge of the distribution of tick-borne pathogens, combined with their early detection in ticks, are essential steps to fight against tick-borne diseases and mitigate their impacts. Here we give an overview of what are the common methods of pathogen detection in tick samples, including recent developments concerning how to handle tick samples, get access to tick-borne pathogens by chemical or physical disruption of the ticks, and methods used for the RNA/DNA extraction steps. Furthermore, we discuss promising tools that are developed for other sample types such as serum or blood to detect tick-borne pathogens, and those that could be used in the future for tick samples.

Molecular analysis of human tick-bitten skin yields signatures associated with distinct spatial and temporal trajectories - A proof-of-concept study.

Tick-associated diseases present challenges due to tridirectional interactions among host-specific responses, tick toxins and salivary proteins as well as microbes. We aimed to uncover molecular mechanisms in tick-bitten skin samples (cases) and contralateral skin samples (controls) collected simultaneously from the same participants, using spatial transcriptomics. Cases and controls analysed using NanoString GeoMx Digital Spatial Profiler identified 274 upregulated and 840 downregulated differentially expressed genes (DEGs), revealing perturbations in keratinization and immune system regulation. Samples of skin biopsies taken within 72 h post tick-bite DEGs had changes in protein metabolism and viral infection pathways as compared to samples taken 3 months post tick-bite, which instead displayed significant perturbations in several epigenetic regulatory pathways, highlighting the temporal nature of the host response following tick bites. Within-individual signatures distinguished tick-bitten samples from controls and identified between-individual signatures, offering promise for future biomarker discovery to guide prognosis and therapy.

Vaccine approaches and treatment aspects against Crimean Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever [CCHF] is a severe infectious viral disease caused by a tick borne virus which can lead to fatal hemorrhagic disease in humans. It has been reported from some continents including Africa, Asia and Europe. Virus is transmitted to human mainly through tick bite, whose acquire infection from reservoirs wild and domesticated mammalians and ostriches. Currently no approved vaccine or drug is available for CCHF and prevention is mainly based on biosecurity measures. Ribavirin is the only approved drug that has been used in some countries to treat human disease, however some new studies did not prove the Ribavirin efficacy. Different strategies to design effective vaccines, have been conducted through years, from inactivated virus to nucleotide-based ones including DNA and mRNA vaccines. In this study we review of pioneering vaccine candidate platforms.

Robust CXCL10/IP-10 and CCL5/RANTES Production Induced by Tick-Borne Encephalitis Virus in Human Brain Pericytes Despite Weak Infection.

Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.

A novel rapid visual nucleic acid detection technique for tick-borne encephalitis virus by combining RT-recombinase-aided amplification and CRISPR/Cas13a coupled with a lateral flow dipstick.

Tick-borne encephalitis virus (TBEV), a zoonotic pathogen, can cause severe neurological complications and fatal outcomes in humans. Early diagnosis of TBEV infection is crucial for clinical practice. Although serological assays are frequently employed for detection, the lack of antibodies in the early stages of infection and the cross-reactivity of antibodies limit their efficacy. Conventional molecular diagnostic methods such as RT-qPCR can achieve early and accurate identification but require specialized instrumentation and professionals, hindering their application in resource-limited areas. Our study developed a rapid and visual TBEV molecular detection method by combining RT-recombinase-aided amplification, the CRISPR/Cas13a system, and lateral flow dipsticks. The diagnostic sensitivity of this method is 50 CFU/ml, with no cross-reactivity with a variety of viruses. The detection can be carried out within 1 h at a temperature between 37 and 42 °C, and the results can be visually determined without the need for complex instruments and professionals. Subsequently, this assay was used to analyze clinical samples from 15 patients suspected of TBEV infection and 10 healthy volunteers, and its sensitivity and specificity reached 100 %, which was consistent with the results of RT-qPCR. These results indicate that this new method can be a promising point-of-care test for the diagnosis of tick-borne encephalitis.

Molecular detection of Coxiella burnetii in ticks collected from animals and the environment in Uganda.

AIMS: Coxiella burnetii is a highly infectious organism that is easily spread through aerosols causing Q fever in humans. Ticks can harbour and transmit C. burnetii to animals, contributing to disease maintenance. Our aim was to examine the presence of C. burnetii in ticks in Uganda. METHODS AND RESULTS: In this study, ticks were collected from five Ugandan districts and tested by real-time PCR for C. burnetii (Coxiella outer membrane protein 1 gene). A total of 859 tick pools (9602 individual ticks) were tested, and pool positivity for C. burnetii was 5.5% (n = 47). Pooled prevalence differed by district; the highest was Luwero (7.3%), then Gulu (6.6%), and Kasese had the lowest (1.3%). However, district variation was not statistically significant (Fisher's exact = 0.07). Ticks collected from dogs and cats had the highest positivity rates [23/47, (48.9%)] followed by livestock (cattle, goats, sheep, and pigs) [18/47, (38.3%)] and vegetation [6/47, (12.8%)]. Haemaphysalis elliptica had the highest infection rates, followed by Rhipicephalus appendiculatus, Amblyomma variegatum and Rhipicephalus decoloratus had similar prevalence. CONCLUSIONS: Although ticks are not the primary transmitters of C. burnetii to humans, pathogen detection in ticks can be an indirect indicator of risk among animal hosts. Vulnerable populations, including occupations with close animal contact such as farming, butchery, and veterinary practice, have an increased risk of C. burnetii exposure. Veterinarians and clinicians should be aware that C. burnetii may cause human and animal illness in these regions.

Co-infection dynamics of B. afzelii and TBEV in C3H mice: insights and implications for future research.

Ticks are important vectors of disease, particularly in the context of One Health, where tick-borne diseases (TBDs) are increasingly prevalent worldwide. TBDs often involve co-infections, where multiple pathogens co-exist in a single host. Patients with chronic Lyme disease often have co-infections with other bacteria or parasites. This study aimed to create a co-infection model with Borrelia afzelii and tick-borne encephalitis virus (TBEV) in C3H mice and to evaluate symptoms, mortality, and pathogen level compared to single infections. Successful co-infection of C3H mice with B. afzelii and TBEV was achieved. Outcomes varied, depending on the timing of infection. When TBEV infection followed B. afzelii infection by 9 days, TBEV symptoms worsened and virus levels increased. Conversely, mice infected 21 days apart with TBEV showed milder symptoms and lower mortality. Simultaneous infection resulted in mild symptoms and no deaths. However, our model did not effectively infect ticks with TBEV, possibly due to suboptimal dosing, highlighting the challenges of replicating natural conditions. Understanding the consequences of co-infection is crucial, given the increasing prevalence of TBD. Co-infected individuals may experience exacerbated symptoms, highlighting the need for a comprehensive understanding through refined animal models. This study advances knowledge of TBD and highlights the importance of exploring co-infection dynamics in host-pathogen interactions.

Development of microsatellite markers for the soft tick Ornithodoros phacochoerus.

BACKGROUND: Soft ticks of the genus Ornithodoros are responsible for the maintenance and transmission of the African swine fever (ASF) virus in the sylvatic and domestic viral cycles in Southern Africa. They are also the main vectors of the Borrelia species causing relapsing fevers. Currently, no genetic markers are available for Afrotropical Ornithodoros ticks. As ASF spreads globally, such markers are needed to assess the role of ticks in the emergence of new outbreaks. The aim of this study is to design microsatellite markers that could be used for ticks of the Ornithodoros moubata complex, particularly Ornithodoros phacochoerus, to assess population structure and tick movements in ASF endemic areas. METHODS: A total of 151 markers were designed using the O. moubata and O. porcinus genomes after elimination of repeated sequences in the genomes. All designed markers were tested on O. phacochoerus and O. porcinus DNA to select the best markers. RESULTS: A total of 24 microsatellite markers were genotyped on two populations of O. phacochoerus and on individuals from four other Ornithodoros species. Nineteen markers were selected to be as robust as possible for population genetic studies on O. phacochoerus. CONCLUSIONS: The microsatellite markers developed here represent the first genetic tool to study nidicolous populations of O. phacochoerus.

Discovery of a novel inhibitor of macropinocytosis with antiviral activity.

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using high-throughput microscopy, where we identified chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as mpox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present the discovery of a molecule that inhibits macropinocytosis, thereby limiting the infectivity of viruses that use this entry route such as SARS-CoV2.

A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan.

Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.

Sample-to-answer direct real-time PCR detection of Anaplasma phagocytophilum, Ehrlichia spp., and Babesia spp. infections in whole-blood specimens.

Emerging tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, are caused by obligate intracellular pathogens that have clinically comparable presentations. Diagnostics used in laboratories today are serologic assays and blood smear analyses, which have known diagnostic limits. This study evaluated the performance of a sample-to-answer direct real-time PCR laboratory-developed test for the multiplex qualitative detection of Anaplasma, Babesia, and Ehrlichia DNA in whole-blood specimens. Compared to two standard-of-care (SOC) methods, the DiaSorin tick-borne laboratory-developed test for Anaplasma detection demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 100% (95% CI, 0.80 to 1.0) and 89% (95% CI, 0.74 to 0.97), respectively with a discordant rate of 9.3% against microscopy. After discordant resolution, the NPA increased to 100%. For Babesia, the test demonstrated a PPA of 100% (95% CI, 0.90 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Compared to a SOC PCR method Anaplasma samples showed a PPA of 100% (95% CI, 0.66 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Ehrlichia results showed a PPA of 100% (95% CI, 0.69 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). The total percent agreement was 98% (95% CI, 0.95 to 0.99) with a κ statistic of 0.95 (95% CI, 0.90 to 0.99) or almost perfect agreement compared to SOC methods. This laboratory-developed test for detecting Anaplasma, Babesia, and Ehrlichia DNA provides rapid and reliable detection of tick-borne infections without nucleic acid extraction. IMPORTANCE: This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance.