Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics.

BACKGROUND: Ixodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba. METHODS: A cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data. RESULTS: The study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs. CONCLUSIONS: Overall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management.

First molecular characterization of Dirofilaria Immitis in Cuba.

BACKGROUND: Dirofilarioses are widespread diseases caused by mosquito-borne nematodes of the family Onchocercidae, genus Dirofilaria. The major etiologic agent of canine dirofilariosis in the American continent is the zoonotic parasite Dirofilaria immitis. Existing reports of filarioid nematodes in Cuba are based solely on morphological and immunological analysis which do not allow unambiguous identification and/or direct detection of causal agents. RESULTS: Here we present the molecular characterization of filarioid nematodes found in a dog in Cuba. Based on the molecular and phylogenetic analysis of the 5.8S-ITS2-28S region and cox1 gene fragments, the worms were unambiguously classified as D. immitis. Sequence analysis showed high identity of the gene fragments in this study with others previously obtained from D. immitis found in dogs, wolfs and jackals but also from mosquito vectors of D. immitis. CONCLUSIONS: Further studies are guarantee to better understand the epidemiological impact of canine dirofilariosis in Cuba as well as the competence of different species of culicid mosquitoes as vectors of Dirofilaria in the country.

Rickettsia helvetica infection is associated with microbiome modulation in Ixodes ricinus collected from humans in Serbia.

Rickettsia helvetica is an emerging pathogen of the Spotted Fever Group Rickettsia (SFGR) causing spotted fever diseases in various European countries. This tick-borne pathogen replicates in tick tissues such as the midgut and salivary gland, but its potential interactions with the vector microbiota is poorly characterized. The vector microbiome plays a pivotal role in tick-pathogen interactions, and some microbiota members facilitate or impede tick-borne pathogen infection. Manipulations of the tick microbiome have led to reduction in pathogen colonization in the tick vector. However, translating these findings into disease control applications requires a thorough characterization of vector microbiota response to different pathogens. In this study, we analyzed and compared the microbiota of Ixodes ricinus ticks attached on humans and collected in Serbia. Ticks were either infected with R. helvetica, or uninfected with major tick-borne pathogens (referred hereafter as 'pathogen-free'). We used microbial co-occurrence network analysis to determine keystone taxa of each set of samples, and to study the interaction patterns of the microbial communities in response to pathogen infection. The inferred functional profiles of the tick microbiome in R. helvetica-positive and pathogen-free samples were also compared. Our results show that R. helvetica infection reduces significantly the diversity of the microbiota and the connectivity of the co-occurrence network. In addition, using co-occurrence network we identified bacterial taxa (i.e., Enterobacteriaceae, Comamonadaceae, and Bacillus) that were negatively associated with 'Rickettsia' in R. helvetica-infected ticks, suggesting competition between R. helvetica and some members of the tick microbiota. The reconstruction of microbial metabolic pathways shows that the presence of R. helvetica might have a major impact on the metabolic functions of the tick microbiome. These results can inform novel interventions for the prevention of R. helvetica, or other SFGR infections in humans.

First molecular evidence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' infections and its association with epidemiological factors in dogs from Cuba.

Haemotrophic mycoplasmas (haemoplasmas) are unculturable, epicellular, cell wall-less gram-negative bacteria distributed worldwide, which infect several mammalian species. In dogs, Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' have been reported as causative agents of infectious anaemia, especially in splenectomised or immunocompromised animals. The present cross-sectional study aims to assess the prevalence, risk factors, and molecular characterization of canine haemoplasmas in Cuba. A total of 391 dog blood samples and 247 tick samples were tested for the presence of canine haemoplasmas by species-specific quantitative TaqMan® real-time PCR assays. Overall, 17.9% (70/391; 95% CI: 14.1-21.7) blood samples were PCR-positive for at least one canine haemoplasmas species, where 15.1% (59/391; 95% CI: 11.5-18.7) for Mycoplasma haemocanis, 4.4% (17/391; 95% CI: 2.3-6.4) for 'Candidatus Mycoplasma haematoparvum', and 1.5% (6/391; 95% CI: 0.3-2.8) were co-infected. All collected ticks were identified morphologically as Rhipicephalus sanguineus sensu lato, and none of the tested tick samples was found PCR-positive for the presence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum'. Risk factors for canine haemoplasmas species infection included the presence of tick infestation, crossbreeding and living in kennels, while no association was found with the occurrence of anaemia. Phylogenetic analyses based on the 16S rRNA gene sequences of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' revealed >99% identity to other isolates distributed worldwide, indicating low genetic variability amongst these canine haemoplasmas species. To the best of the authors´ knowledge, this is the first molecular evidence of Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum' infections in dogs from Cuba.

Clinical Aspects and Detection of Emerging Rickettsial Pathogens: A "One Health" Approach Study in Serbia, 2020.

Ticks carry numerous pathogens that, if transmitted, can cause disease in susceptible humans and animals. The present study describes our approach on how to investigate clinical presentations following tick bites in humans. To this aim, the occurrence of major tick-borne pathogens (TBPs) in human blood samples (n = 85) and the ticks collected (n = 93) from the same individuals were tested using an unbiased high-throughput pathogen detection microfluidic system. The clinical symptoms were characterized in enrolled patients. In patients with suspected TBP infection, serological assays were conducted to test for the presence of antibodies against specific TBPs. A field study based on One Health tenets was further designed to identify components of a potential chain of infection resulting in Rickettsia felis infection in one of the patients. Ticks species infesting humans were identified as Ixodes ricinus, Rhipicephalus sanguineus sensu lato (s.l.), Dermacentor reticulatus, and Haemaphysalis punctata. Five patients developed local skin lesions at the site of the tick bite including erythema migrans, local non-specific reactions, and cutaneous hypersensitivity reaction. Although Borrelia burgdorferi s.l., Babesia microti, Anaplasma phagocytophilum, and Candidatus Cryptoplasma sp. DNAs were detected in tick samples, different Rickettsia species were the most common TBPs identified in the ticks. The presence of TBPs such as Rickettsia helvetica, Rickettsia monacensis, Borrelia lusitaniae, Borrelia burgdorferi, Borrelia afzelii, A. phagocytophilum, and B. microti in ticks was further confirmed by DNA sequencing. Two of the patients with local skin lesions had IgG reactive against spotted fever group rickettsiae, while IgM specific to B. afzelii, Borrelia garinii, and Borrelia spielmanii were detected in the patient with erythema migrans. Although R. felis infection was detected in one human blood sample, none of the components of the potential chain of infection considered in this study tested positive to this pathogen either using direct pathogen detection in domestic dogs or xenodiagnosis in ticks collected from domestic cats. The combination of high-throughput screening of TBPs and One Health approaches might help characterize chains of infection leading to human infection by TBPs, as well as prevalence of emerging rickettsial pathogens in the Balkan region.

Unraveling the Global Phylodynamic and Phylogeographic Expansion of Mycoplasma gallisepticum: Understanding the Origin and Expansion of This Pathogen in Ecuador.

Mycoplasma gallisepticum (MG) is among the most significant problems in the poultry industry worldwide, representing a serious threat to international trade. Despite the fact that the mgc2 gene has been widely used for diagnostic and molecular characterization purposes, there is a lack of evidence supporting the reliability of this gene as a marker for molecular epidemiology approaches. Therefore, the current study aimed to assess the accuracy of the mgc2 gene for phylogenetic, phylodynamic, and phylogeographic evaluations. Furthermore, the global phylodynamic expansion of MG is described, and the origin and extension of the outbreak caused by MG in Ecuador were tracked and characterized. The results obtained strongly supported the use of the mgc2 gene as a reliable phylogenetic marker and accurate estimator for the temporal and phylogeographic structure reconstruction of MG. The phylodynamic analysis denoted the failures in the current policies to control MG and highlighted the imperative need to implement more sensitive methodologies of diagnosis and more efficient vaccines. Framed in Ecuador, the present study provides the first piece of evidence of the circulation of virulent field MG strains in Ecuadorian commercial poultry. The findings derived from the current study provide novel and significant insights into the origin, diversification, and evolutionary process of MG globally.

Ticks and Tick-Borne Diseases in Cuba, Half a Century of Scientific Research.

Ticks and the vast array of pathogens they transmit, including bacteria, viruses, protozoa, and helminths, constitute a growing burden for human and animal health worldwide. In Cuba, the major tropical island in the Caribbean, ticks are an important cause of vector-borne diseases affecting livestock production, pet animal health and, to a lesser extent, human health. The higher number of tick species in the country belong to the Argasidae family and, probably less known, is the presence of an autochthonous tick species in the island, Ixodes capromydis. Herein, we provide a comprehensive review of the ticks and tick-borne pathogens (TBPs) affecting animal and human health in Cuba. The review covers research results including ecophysiology of ticks, the epidemiology of TBPs, and the diagnostic tools used currently in the country for the surveillance of TBPs. We also introduce the programs implemented in the country for tick control and the biotechnology research applied to the development of anti-tick vaccines.

Portable abdomen radiography: moving to thickness-based protocols.

BACKGROUND: Default pediatric protocols on many digital radiography systems are configured based on patient age. However, age does not adequately characterize patient size, which is the principal determinant of proper imaging technique. Use of default pediatric protocols by inexperienced technologists can result in patient overexposure, inadequate image quality, or repeated examinations. OBJECTIVE: To ensure diagnostic image quality at a well-managed patient radiation exposure by transitioning to thickness-based protocols for pediatric portable abdomen radiography. MATERIALS AND METHODS: We aggregated patient thickness data, milliamperes (mAs), kilovoltage peak (kVp), exposure index (EI), source-to-detector distance, and grid use for all portable abdomen radiographs performed in our pediatric hospital in a database with a combination of automated and manual data collection techniques. We then analyzed the database and used it as the basis to construct thickness-based protocols with consistent image quality across varying patient thicknesses, as determined by the EI. RESULTS: Retrospective analysis of pediatric portable exams performed at our adult-focused hospitals demonstrated substantial variability in EI relative to our pediatric hospital. Data collection at our pediatric hospital over 4 months accumulated roughly 800 portable abdomen exams, which we used to develop a thickness-based technique chart. CONCLUSION: Through automated retrieval of data in our systems' digital radiography exposure logs and recording of patient abdomen thickness, we successfully developed thickness-based techniques for portable abdomen radiography.

Investigating simulation-based metrics for characterizing linear iterative reconstruction in digital breast tomosynthesis.

PURPOSE: Simulation-based image quality metrics are adapted and investigated for characterizing the parameter dependences of linear iterative image reconstruction for DBT. METHODS: Three metrics based on a 2D DBT simulation are investigated: (1) a root-mean-square-error (RMSE) between the test phantom and reconstructed image, (2) a gradient RMSE where the comparison is made after taking a spatial gradient of both image and phantom, and (3) a region-of-interest (ROI) Hotelling observer (HO) for signal-known-exactly/background-known-exactly (SKE/BKE) and signal-known-exactly/background-known-statistically (SKE/BKS) detection tasks. Two simulation studies are performed using the aforementioned metrics, varying voxel aspect ratio, and regularization strength for two types of Tikhonov-regularized least-squares optimization. The RMSE metrics are applied to a 2D test phantom with resolution bar patterns at varying angles, and the ROI-HO metric is applied to two tasks relevant to DBT: lesion detection, modeled by use of a large, low-contrast signal, and microcalcification detection, modeled by use of a small, high-contrast signal. The RMSE metric trends are compared with visual assessment of the reconstructed bar-pattern phantom. The ROI-HO metric trends are compared with 3D reconstructed images from ACR phantom data acquired with a Hologic Selenia Dimensions DBT system. RESULTS: Sensitivity of the image RMSE to mean pixel value is found to limit its applicability to the assessment of DBT image reconstruction. The image gradient RMSE is insensitive to mean pixel value and appears to track better with subjective visualization of the reconstructed bar-pattern phantom. The ROI-HO metric shows an increasing trend with regularization strength for both forms of Tikhonov-regularized least-squares; however, this metric saturates at intermediate regularization strength indicating a point of diminishing returns for signal detection. Visualization with the reconstructed ACR phantom images appear to show a similar dependence with regularization strength. CONCLUSIONS: From the limited studies presented it appears that image gradient RMSE trends correspond with visual assessment better than image RMSE for DBT image reconstruction. The ROI-HO metric for both detection tasks also appears to reflect visual trends in the ACR phantom reconstructions as a function of regularization strength. We point out, however, that the true utility of these metrics can only be assessed after amassing more data.

Assessment of vectorial total variation penalties on realistic dual-energy CT data.

Vectorial extensions of total variation have recently been developed for regularizing the reconstruction and denoising of multi-channel images, such as those arising in spectral computed tomography. Early studies have focused mainly on simulated, piecewise-constant images whose structure may favor total-variation penalties. In the current manuscript, we apply vectorial total variation to real dual-energy CT data of a whole turkey in order to determine if the same benefits can be observed in more complex images with anatomically realistic textures. We consider the total nuclear variation ([Formula: see text]) as well as another vectorial total variation based on the Frobenius norm ([Formula: see text]) and standard channel-by-channel total variation ([Formula: see text]). We performed a series of 3D TV denoising experiments comparing the three TV variants across a wide range of smoothness parameter settings, optimizing each regularizer according to a very-high-dose 'ground truth' image. Consistent with the simulation studies, we find that both vectorial TV variants achieve a lower error than the channel-by-channel TV and are better able to suppress noise while preserving actual image features. In this real data study, the advantages are subtler than in the previous simulation study, although the [Formula: see text] penalty is found to have clear advantages over either [Formula: see text] or [Formula: see text] when comparing material images formed from linear combinations of the denoised energy images.

Unified Database for Rejected Image Analysis Across Multiple Vendors in Radiography.

Reject rate analysis has been part of radiography departments' quality control since the days of screen-film radiography. In the era of digital radiography, one might expect that reject rate analysis is easily facilitated because of readily available information produced by the modality during the examination procedure. Unfortunately, this is not always the case. The lack of an industry standard and the wide variety of system log entries and formats have made it difficult to implement a robust multivendor reject analysis program, and logs do not always include all relevant information. The increased use of digital detectors exacerbates this problem because of higher reject rates associated with digital radiography compared with computed radiography. In this article, the authors report on the development of a unified database for vendor-neutral reject analysis across multiple sites within an academic institution and share their experience from a team-based approach to reduce reject rates.

Use of the Hotelling observer to optimize image reconstruction in digital breast tomosynthesis.

We propose an implementation of the Hotelling observer that can be applied to the optimization of linear image reconstruction algorithms in digital breast tomosynthesis. The method is based on considering information within a specific region of interest, and it is applied to the optimization of algorithms for detectability of microcalcifications. Several linear algorithms are considered: simple back-projection, filtered back-projection, back-projection filtration, and [Formula: see text]-tomography. The optimized algorithms are then evaluated through the reconstruction of phantom data. The method appears robust across algorithms and parameters and leads to the generation of algorithm implementations which subjectively appear optimized for the task of interest.

Estimation of noise properties for TV-regularized image reconstruction in computed tomography.

A method for predicting the image covariance resulting from total-variation-penalized iterative image reconstruction (TV-penalized IIR) is presented and demonstrated in a variety of contexts. The method is validated against the sample covariance from statistical noise realizations for a small image using a variety of comparison metrics. Potential applications for the covariance approximation include investigation of image properties such as object- and signal-dependence of noise, and noise stationarity. These applications are demonstrated, along with the construction of image pixel variance maps for two-dimensional 128 × 128 pixel images. Methods for extending the proposed covariance approximation to larger images and improving computational efficiency are discussed. Future work will apply the developed methodology to the construction of task-based image quality metrics such as the Hotelling observer detectability for TV-based IIR.

Task-based optimization of dedicated breast CT via Hotelling observer metrics.

PURPOSE: The purpose of this work is to develop and demonstrate a set of practical metrics for CT systems optimization. These metrics, based on the Hotelling observer (HO) figure of merit, are task-based. The authors therefore take the specific example of optimizing a dedicated breast CT system, including the reconstruction algorithm, for two relevant tasks, signal detection and Rayleigh discrimination. METHODS: A dedicated breast CT system is simulated using specifications in the literature from an existing prototype. The authors optimize configuration and image reconstruction algorithm parameters for two tasks: the detection of simulated microcalcifications and the discrimination of two adjacent, high-contrast signals, known as the Rayleigh discrimination task. The effects on task performance of breast diameter, signal location, image grid size, projection view number, and reconstruction filter were all investigated. Two HO metrics were evaluated: the percentage of correct decisions in a two-alternative forced choice experiment (equivalent to area under the ROC curve or AUC), and the HO efficiency, defined as the squared ratio of HO signal-to-noise ratio (SNR) in the reconstructed image to HO SNR in the projection data. RESULTS: The ease and efficiency of the HO metric computation allows a rapid high-resolution survey of many system parameters. Optimization of a range of system parameters using the HO results in images that subjectively appear optimal for the tasks investigated. Further, the results of assessment through the HO reproduce closely many existing results in the literature regarding the impact of parameter selection on image quality. CONCLUSIONS: This study demonstrates the utility of a task-based approach to system design, evaluation, and optimization. The methodology presented is equally applicable to determining the impact of a wide range of factors, including patient parameters, system and acquisition design, and the reconstruction algorithm. The results demonstrate the versatility of the proposed HO formalism by not only generating a set of parameters that are optimal for a given task but also by qualitatively reproducing many existing results from the breast CT literature. Meanwhile, the implementation of the proposed methodology is straightforward and entirely simulation-based. This is an attractive feature for many system optimization problems, where the goal is to analyze the individual system components such as the image reconstruction algorithm. Final assessment of the system as a whole should be based also on real data studies.

Region of interest based Hotelling observer for computed tomography with comparison to alternative methods.

We compare several approaches to estimation of Hotelling observer (HO) performance in x-ray computed tomography (CT). We consider the case where the signal of interest is small so that the reconstructed image can be restricted to a small region of interest (ROI) surrounding the signal. This reduces the dimensionality of the image covariance matrix so that direct computation of HO metrics within the ROI is feasible. We propose that this approach is directly applicable to systems optimization in CT; however, many alternative approaches exist, which make computation of HO performance tractable through a range of approximations, assumptions, or estimation strategies. Here, we compare several of these methods, including the use of Laguerre-Gauss channels, discrete Fourier domain computation of the HO (which assumes noise stationarity), and two approaches to HO estimation through samples of noisy images. Since our method computes HO performance exactly within an ROI, this allows us to investigate the validity of the assumptions inherent in various common approaches to HO estimation, such as the stationarity assumption in the case of the discrete Fourier transform domain method.

Comparison of human and Hotelling observer performance for a fan-beam CT signal detection task.

PURPOSE: A human observer study was performed for a signal detection task for the case of fan-beam x-ray computed tomography. Hotelling observer (HO) performance was calculated for the same detection task without the use of efficient channels. By considering the full image covariance produced by the filtered backprojection (FBP) algorithm and avoiding the use of channels in the computation of HO performance, the authors establish an absolute upper bound on signal detectability. Therefore, this study serves as a baseline for relating human and ideal observer performance in the case of fan-beam CT. METHODS: Eight human observers participated in a two-alternative forced choice experiment where the signal of interest was a small simulated ellipsoid in the presence of independent, identically distributed Gaussian detector noise. Theoretical performance of the HO, which is equivalent to the ideal observer in this case (see Sec. 13.2.12 in Barrett and Myers [Foundations of Image Science (Wiley, Hoboken, NJ, 2004)], was also computed and compared to the performance of the human observers. In addition to a reference FBP implementation, two FBP implementations with inherent loss of HO signal detectability (e.g., by apodizing the ramp filter) were also investigated. Each of these latter two implementations takes the form of a discrete-to-discrete linear operator (i.e., a matrix), which has a nontrivial null-space resulting in the loss of detectability. RESULTS: Estimated observer detectability index (d(A)) values for the human observers and SNR values for the HO were obtained. While Hanning filtering in the FBP implementation with a cutoff frequency of 1/4 of the Nyquist frequency reduces HO SNR (due to the reconstruction matrix's nontrivial null-space), this filtering was shown to consistently improve human observer performance. By contrast, increasing the image pixel size was seen to have a comparable effect on both the HO and the human observers, degrading performance. CONCLUSIONS: These results, which characterize HO and human observer performance for a signal detection task in fan-beam FBP noise, form a basis for applying model observer metrics to fan-beam CT when knowledge of the full image-domain noise statistics is important. Further, by calculating HO performance without relying on channels, these results are particularly relevant when an information theoretic approach is considered, e.g., in optimization of the image reconstruction algorithm with respect to preservation of signal detectability. Finally, the HO (which is here equivalent to the ideal observer) provides an absolute upper bound on detection performance, and our results therefore provide insight into the performance of human observers relative to the optimum for two different cases wherein ideal observer performance is compromised through degradation of the data quality. In one case (regularization), human performance is improved to practically ideal performance, and in the other (larger pixel size), ideal and human observer performance are approximately degraded equivalently.