Infectious Agents Research Articles

A Transmission Assay of 'Candidatus Liberibacter asiaticus' Using Citrus Phloem Sap and Topical Feeding to Its Insect Vector, Diaphorina citri.

'Candidatus Liberibacter asiaticus', the putative causal agent of citrus greening disease, is transmitted by the Asian citrus psyllid, Diaphorina citri, in a propagative, circulative, and persistent manner. Unfortunately, 'Ca. L. asiaticus' is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (wt/vol) sucrose and 0.1% ascorbic acid (wt/vol) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (10 nymphs per seedling) for an inoculation period of 2 weeks. A transmission rate of up to 80% was recorded at 6 months postinoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of 'Ca. L. asiaticus' becomes available.

Novel report of Bipolaris heliconiae causing frog-eye-like leaf spot on Dypsis lutescens in Indian Sub-Continent

Dypsis lutescens, commonly known as areca palm, is a highly valued ornamental species due to its aesthetic value. However, the foliage is vulnerable to various pathogens, particularly those responsible for fungal leaf spot diseases. In October 2023, a severe incidence (93 %) of destructive leaf spots was recorded on Dypsis lutescens at the University of Agricultural Sciences, GKVK, Bangalore, and surrounding areas. The leaf spot symptoms manifested as frog-eye-like lesions, leading to complete leaf desiccation and significantly reducing the palms ornamental value. The pathogen exhibited the highest radial growth (90.00 mm) and prominent sporulation on oat meal agar, whereas Richard's synthetic agar resulted in the lowest radial growth (38.00 mm) with no sporulation. Morphological and multilocus phylogenetic analyses confirmed the pathogen as Bipolaris heliconiae. Pathogenicity tests fulfilled Koch's postulates, confirming that Bipolaris heliconiae is the causative agent of leaf spot disease in Dypsis lutescens in India. This novel finding underscores the emergence of a new disease and highlights the urgent need for effective management strategies.

Antiparasitary potential of Sarcomphalus joazeiro (Mart.) Hauenschild (Rhamnaceae)

Leishmaniasis and American trypanosomiasis are part of a set of diseases characterized by the World Health Organization as neglected diseases caused by infectious and parasitic agents in populations in situations of poverty and poor sanitation. Pharmaceutical industries neglect such diseases, which have limited treatments, mainly due to few drug options and significant toxicity. Therefore, it is essential to search for new substances that have the potential to replace these medications. In this context, this study aimed to evaluate the leishmanicidal and trypanocidal activity of the ethanolic extract of the leaves and bark of Sarcomphalus joazeiro (Mart.) Hauenschild. These extracts were prepared by maceration with cold extraction. When carrying out the susceptibility tests, the extracts were tested in four different concentrations against the epimastigote forms of the CL-B5 parasite strains of Trypanosoma cruzi and promastigote forms of Leishmania braziliensis and Leishmania infantum, both tests were carried out "in vitro" in plates 96-well microdilution system, with nifurtimox and metronidazole as reference medicine. The cytotoxicity assay was performed with mammalian fibroblast lines from the NCTC 929 clone. The results indicate that the ethanolic extract of the bark of the stem of S. joazeiro (EBSJ) presented a more significant antiparasitic effect when compared to the ethanolic extract of the leaves of Sarcomphalus joazeiro (ELSJ) particularly against L. braziliensis and L. infantum. Regarding cytotoxicity, EBSJ and ELSJ presented moderate and low degrees, respectively. Based on the data obtained, further investigations into the properties of the bark extract are necessary, mainly due to its leishmanicidal potential.

Metagenomic analysis of atheroma plaques for identification of microorganisms indicates presence of Toxoplasma gondii as a possible etiological agent

BackgroundCardiovascular diseases (CVDs) are the leading cause of death worldwide. Vital organs like the heart are affected by the occlusion of blood vessels due to atherosclerotic plaque formation. However, the role of infectious agents has always been an essential subject of investigation. This study investigated the presence of microorganisms, including nanobacteria, in atherosclerotic plaques removed from human carotid arteries by microbiological and metagenomic examination. MethodsAtheroma plaque samples were obtained from 20 patients with carotid artery stenosis who had atherectomy by surgery or percutaneous intervention. Nanobacteria were grown by culturing homogenates of the atheroma plaques. Whole genome sequencing was done for samples. Because of the high percentage of Toxoplasma gondii (T. gondii) DNA, PCR investigation was applied to detect T. gondii DNA in the samples. ResultsA molecular analysis of nanobacteria revealed them to be made of human proteins, supporting the theory that they are not living organisms. According to sequencing results, samples showed that more than 50 % of the metagenomic sequences belonged to Toxoplasma gondii. PCR investigation indicated that T. gondii DNA was positive in 8 (40 %) of 20 plaques. ConclusionsFurther evidence regarding the role of T. gondii in the etiology of plaque formation may help determine the strategy for prevention and treatment of infections in preventing atheroma plaque formation in the future.

The unique Legionella longbeachae capsule favors intracellular replication and immune evasion.

Legionella longbeachae and Legionella pneumophila are the most common causative agents of Legionnaires' disease. While the clinical manifestations caused by both species are similar, species-specific differences exist in environmental niches, disease epidemiology, and genomic content. One such difference is the presence of a genomic locus predicted to encode a capsule. Here, we show that L. longbeachae indeed expresses a capsule in post-exponential growth phase as evidenced by electron microscopy analyses, and that capsule expression is abrogated when deleting a capsule transporter gene. Capsule purification and its analysis via HLPC revealed the presence of a highly anionic polysaccharide that is absent in the capsule mutant. The capsule is important for replication and virulence in vivo in a mouse model of infection and in the natural host Acanthamoeba castellanii. It has anti-phagocytic function when encountering innate immune cells such as human macrophages and it is involved in the low cytokine responses in mice and in human monocyte derived macrophages, thus dampening the innate immune response. Thus, the here characterized L. longbeachae capsule is a novel virulence factor, unique among the known Legionella species, which may aid L. longbeachae to survive in its specific niches and which partly confers L. longbeachae its unique infection characteristics.

Discovery of Two Novel Phthalide Phytotoxins from the Wheat Tan Spot Fungal Pathogen Pyrenophora tritici-repentis.

The Dothideomycete fungal pathogen Pyrenophora tritici-repentis (Ptr) is the causal agent of the tan spot disease of wheat. The proteinaceous necrotrophic effectors ToxA and ToxB are well characterized. A nonproteinaceous effector called ToxC has also been partially characterized. Ptr produces a number of other small molecular weight compounds, but these remain poorly characterized. In this study, two novel compounds, designated ToxE1 and ToxE2, capable of inducing chlorotic symptoms on wheat leaves in a cultivar-specific manner, were purified from Ptr liquid cultures. There is no evidence that these compounds correspond to ToxC. Most isolates produced ToxE1, ToxE2, or both, and both compounds were detected in infected wheat leaves. The structures of both analogues were elucidated by NMR spectroscopy and comprise a phthalide core structure with an amide moiety. We postulate that these compounds have a general phytotoxic effect and may have an ancillary role in disease development.

Cancer and One Health: tumor-bearing individuals can act as super spreaders of symbionts in communities

Recent theoretical advances in the One Health approach have suggested that cancer pathologies should be given greater consideration, as cancers often render their hosts more vulnerable to infectious agents, which could turn them into super spreaders within ecosystems. Although biologically plausible, this hypothesis has not yet been validated experimentally. Using a community of cnidarians of the Hydra genus (Hydra oligactis, Hydra viridissima, Hydra vulgaris) and a commensal ciliate species (Kerona pediculus) that colonizes them, we tested whether tumoral polyps of H. oligactis, compared to healthy ones, played an amplifying role in the number of ciliates, potentially resulting in a higher likelihood of infection for other community members through spillovers. Our results indicate that K. pediculus has a higher proliferation rate on tumoral polyps of H. oligactis than on healthy ones, which results in the infestation of other hydras. However, the magnitude of the spillover differed between recipient species. This study provides to our knowledge the first elements of proof of concept that tumoral individuals in communities could act as super spreaders of symbionts within and between species, and thus affect biotic interactions and dynamics in ecosystems.

Assessment of the antimicrobial and antibiofilm activity of the combination of N-acetyl cysteine and carvacrol against Staphylococcus aureus, the most common orthopedic infectious agent

BackgroundThe increasing prevalence of antibiotic-resistant bacterial infections has led to the search for new approaches. ObjectiveThis study aimed to evaluate the effects of carvacrol and N-acetyl cysteine, both individually and in combination, on the planktonic cells and biofilm formations of Staphylococcus aureus, including methicillin-resistant and methicillin-sensitive strains. Additionally, the study sought to perform cytotoxicity tests and chemical characterization to further understand the properties and potential applications of these substances. MethodsA total of 19 S. aureus strains were included in the study. Minimum inhibitory concentration and minimum bactericidal concentration were determined by assays. Synergy analysis tests were carried out. Cytotoxicity tests were conducted on the fibroblast cell line. Characterization test was performed. ResultsWhile Minimum inhibitory concentration and minimum bactericidal concentration values for carvacrol varied between 250 and 500 μg/ml, these values were in the range of 32–64 mg/ml for N-acetyl cysteine. Biofilm formation activities were identified. A total of eight strains, including six clinical and two standard strains with the highest biofilm-forming ability, were selected for combination studies. The combination of Carvacrol and N-acetyl cysteine exhibited synergistic and partially synergistic effects on the tested planktonic and biofilm strains, and these effects were dose-dependent. Carvacrol was found to be the most active drug at the end of 24, 48, and 72 h. Regarding the synergistic effect of N-acetyl cysteine + carvacrol, it was revealed to exhibit higher activity than N-acetyl cysteine and lower activity than carvacrol. ConclusionThe combination of carvacrol and N-acetyl cysteine demonstrated synergistic and partially synergistic effects against both planktonic and biofilm forms of Staphylococcus aureus. These results suggest potential for novel approaches in managing orthopedic infections, warranting further research to explore their therapeutic applications.

Macroalgal Diseases: Exploring Biology, Pathogenesis, and Management Strategies

The global seaweed market is expected to reach USD 17.8 billion by 2032, fuelled by growing demand for sustainable and healthy food solutions and expanding applications in agriculture and aquaculture. However, this rapid growth poses significant challenges, particularly in managing diseases that often establish themselves in intensive macroalgal culture facilities. Red rot disease, Olpidiopsis, and green spot disease often affect marine macroalgae species of high commercial interest, as seen in Pyropia/Porphyra as has already happened for “ice-ice” malaise on Kappaphycus, causing huge economic losses. These diseases are caused by infectious agents that find their place in extreme environmental conditions, such as those characterized by sudden changes in temperature and pollution. Despite technological advances aimed at monitoring the well-being of cultivated seaweed, discrepancies between regions’ technological capabilities and species vulnerability exacerbate management difficulties. This review provides an overview of diseases prevalent among marine algae, their impact on aquaculture, and the effectiveness of currently adopted treatments. This study highlights the need to improve disease management strategies and highlights the importance of understanding host–pathogen interactions in order to mitigate future epidemics.

An Enteric Bacterial Infection Triggers Neuroinflammation and Neurobehavioral Impairment in 3xTg-AD Transgenic Mice.

Klebsiella pneumoniae is infamous for hospital-acquired infections and sepsis, which have also been linked to Alzheimer disease (AD)-related neuroinflammatory and neurodegenerative impairment. However, its causative and mechanistic role in AD pathology remains unstudied. A preclinical model of K. pneumoniae enteric infection and colonization is developed in an AD model (3xTg-AD mice) to investigate whether and how K. pneumoniae pathogenesis exacerbates neuropathogenesis via the gut-blood-brain axis. K. pneumoniae, particularly under antibiotic-induced dysbiosis, was able to translocate from the gut to the bloodstream by penetrating the gut epithelial barrier. Subsequently, K. pneumoniae infiltrated the brain by breaching the blood-brain barrier. Significant neuroinflammatory phenotype was observed in mice with K. pneumoniae brain infection. K. pneumoniae-infected mice also exhibited impaired neurobehavioral function and elevated total tau levels in the brain. Metagenomic analyses revealed an inverse correlation of K. pneumoniae with gut biome diversity and commensal bacteria, highlighting how antibiotic-induced dysbiosis triggers an enteroseptic "pathobiome" signature implicated in gut-brain perturbations. The findings demonstrate how infectious agents following hospital-acquired infections and consequent antibiotic regimen may induce gut dysbiosis and pathobiome and increase the risk of sepsis, thereby increasing the predisposition to neuroinflammatory and neurobehavioral impairments via breaching the gut-blood-brain barrier.

Understanding the transmission of bacterial agents of sapronotic diseases using an ecosystem-based approach: A first spatially realistic metacommunity model.

Pathogens such as bacteria, fungi and viruses are important components of soil and aquatic communities, where they can benefit from decaying and living organic matter, and may opportunistically infect human and animal hosts. One-third of human infectious diseases is constituted by sapronotic disease agents that are natural inhabitants of soil or aquatic ecosystems. They are capable of existing and reproducing in the environment outside of the host for extended periods of time. However, as ecological research on sapronosis is infrequent and epidemiological models are even rarer, very little information is currently available. Their importance is overlooked in medical and veterinary research, as well as the relationships between free environmental forms and those that are pathogenic. Here, using dynamical models in realistic aquatic metacommunity systems, we analyze sapronosis transmission, using the human pathogen Mycobacterium ulcerans that is responsible for Buruli ulcer. We show that the persistence of bacilli in aquatic ecosystems is driven by a seasonal upstream supply, and that the attachment and development of cells to aquatic living forms is essential for such pathogen persistence and population dynamics. Our work constitutes the first set of metacommunity models of sapronotic disease transmission, and is highly flexible for adaptation to other types of sapronosis. The importance of sapronotic agents on animal and human disease burden needs better understanding and new models of sapronosis disease ecology to guide the management and prevention of this important group of pathogens.

Etiological stratification and prognostic assessment of haemophagocytic lymphohistiocytosis by machine learning on onco-mNGS data and clinical data

IntroductionHemophagocytic lymphohistiocytosis (HLH) is a rare, complicated and life threatening hyperinflammatory syndrome that maybe triggered by various infectious agents, malignancies and rheumatologic disorders. Early diagnosis and identification of the cause is essential to initiate appropriate treatment and improve the quality of life and survival of patients. The recently developed Onco-mNGS technology can be successfully used for simultaneous detection of infections and tumors.MethodsIn the present study, 92 patients with clinically confirmed HLH were etiologically subtyped for infection, tumor and autoimmunity based on CNV and microbial data generated by Onco-mNGS technology, and a predictive model was developed and validated for the differential diagnosis of the underlying disease leading to secondary HLH. Furthermore, the treatment outcomes of patients with HLH triggered by EBV infection and non-EBV infection were evaluated, respectively.ResultsThe current study demonstrated that the novel Onco-mNGS can identify the infection and malignancy- related triggers among patients with secondary HLH. A random forest classification model based on CNV profile, infectious pathogen spectrum and blood microbial community was developed to better identify the different HLH subtypes and determine the underlying triggers. The prognosis for treatment of HLH patients is not only associated with CNV, but also with the presence of pathogens and non- pathogens in peripheral blood. Higher CNV burden along with frequent deletions on chromosome 19, higher pathogen burden and lower non-pathogenic microbes were prognosis factors that significantly related with unfavorable treatment outcomes.DiscussionOur study provided comprehensive knowledge in the triggers and prognostic predictors of patients with secondary HLH, which may help early diagnosis and appropriate targeted therapy, thus improving the survival and prognosis of the patients.

The most common dermatoses of athletes

Introduction and purpose The skin and subcutaneous tissue constitute the body's protective barrier. It also fulfills important physiological functions. It protects internal organs against the harmful effects of environmental factors and maintains the balance between the human body and the environment. Athlete's dermatoses are a wide group of skin diseases that may occur as a result of specific conditions to which people who practise sports are exposed. Some skin diseases are characteristic of specific sports, while others may occur regardless of the type of physical activity performed. Mechanical damage to the epidermis, excessive humidity, contact with pathogens and exposure to various environmental factors play a key role in the development of these diseases. Regular dermatological check-ups are crucial for identifying and treating skin lesions and preventing mass infections caused by infectious agents. The study discusses the most common dermatoses in athletes, including fungal, bacterial and viral infections, contact dermatitis and dermatoses caused by mechanical and environmental factors. Materials and methods The literature included in the PubMed, BioMed Central and Polish Medical Platform databases is searched by means of the words such as dermatosis, sport, skin, fungal, bacterial, viral infection, contact dermatitis. Quoted sources in selected works were also used. ConclusionsMany factors can have a harmful effect on the skin, negatively affect its function as an integral barrier of the body and, consequently, cause the development of dermatoses, which are quite common among athletes due to frequent contact with sweat, clothing, sports equipment and the environments in which they train. Prevention is an important element in minimizing the risk of skin problems and this is key to maintaining health and continuing physical activity.

Native valve endocarditis caused by Corynebacterium striatum without underlying structural heart disease or indwelling cardiovascular medical devices: a case report

BackgroundCorynebacterium striatum (C. striatum) is a gram-positive, anaerobic bacillus found both environmentally and in human skin and nasal mucosa flora. It is reportedly the etiologic agent of community-acquired and nosocomial diseases and is significantly associated with bacteremia and medical endovascular devices. This is the rare case of mitral valve native valve endocarditis (NVE) caused by C. striatum occurring in a young adult without underlying structural heart disease or indwelling cardiovascular medical devices successfully treated with multidisciplinary therapy.Case presentationThe patient was a 28-year-old female with no medical history. She was transferred our hospital due to sudden onset of vertigo and vomit. A computed tomography on day 2 revealed the hydrocephalus due to the cerebellar infarction, and she underwent posterior fossa decompression for cerebellar infarction. An angiography on day 8 revealed a left vertebral artery dissection, which was suspected be the etiology. Afterwards, a sudden fever of 39 degrees developed on day 38. She was diagnosed with aspiration pneumonia and treated with ampicillin/sulbactam but was still febrile at the time of transfer for rehabilitation. Treatment continued with levofloxacin, the patient had no fever decline, and she was readmitted to our hospital. Readmission blood cultures (3/3 sets) revealed C. striatum, and an echocardiogram revealed an 11 mm long mitral valve vegetation, leading to NVE diagnosis. On the sixth illness day, cardiac failure symptoms manifested. Echocardiography revealed mitral valve rupture. She was transferred again on the 11th day of illness, during which time her mitral valve was replaced. C. striatum was detected in the vegetation. Following surgery, she returned to our hospital, and vancomycin administration continued. The patient was discharged after 31 total days of postoperative antimicrobial therapy. The patient experienced no exacerbations thereafter.ConclusionsWe report the rare case of C. striatum mitral valve NVE in a young adult without structural heart disease or indwelling cardiovascular devices.Clinical trial numberNot applicable.

Inorganic Polyphosphate Is in the Surface of Trypanosoma cruzi but Is Not Significantly Secreted

Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-β), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.

Two Polyketide Synthase Genes, VpPKS10 and VpPKS33, Regulated by VpLaeA Are Essential to the Virulence of Valsa pyri.

Valsa pyri, the causal agent of pear canker disease, typically induces cankers on the bark of infected trees and even leads to tree mortality. Secondary metabolites produced by pathogenic fungi play a crucial role in the pathogenic process. In this study, secondary metabolic regulator VpLaeA was identified in V. pyri. VpLaeA was found to strongly affect the pathogenicity, fruiting body formation, and toxicity of secondary metabolites of V. pyri. Additionally, VpLaeA was found to be required for the response of V. pyri to some abiotic stresses. Transcriptome data analysis revealed that many of differentially expressed genes were involved in the secondary metabolite biosynthesis. Among them, about one third of secondary metabolite biosynthesis core genes were regulated by VpLaeA at different periods. Seven differentially expressed secondary metabolite biosynthesis core genes (VpPKS9, VpPKS10, VpPKS33, VpNRPS6, VpNRPS7, VpNRPS16, and VpNRPS17) were selected for knockout. Two modular polyketide synthase genes (VpPKS10 and VpPKS33) that were closely related to the virulence of V. pyri from the above seven genes were identified. Notably, VpPKS10 and VpPKS33 also affected the production of fruiting body of V. pyri but did not participate in the resistance of V. pyri to abiotic stresses. Overall, this study demonstrates the multifaceted biological functions of VpLaeA in V. pyri and identifies two toxicity-associated polyketide synthase genes in Valsa species fungi for the first time.

Plant-Driven Assembly of Disease-Suppressive Soil Microbiomes

Plants have coevolved together with the microbes that surround them and this assemblage of host and microbes functions as a discrete ecological unit called a holobiont. This review outlines plant-driven assembly of disease-suppressive microbiomes. Plants are colonized by microbes from seed, soil, and air but selectively shape the microbiome with root exudates, creating microenvironment hot spots where microbes thrive. Using plant immunity for gatekeeping and surveillance, host-plant genetic properties govern microbiome assembly and can confer adaptive advantages to the holobiont. These advantages manifest in disease-suppressive soils, where buildup of specific microbes inhibits the causal agent of disease, that typically develop after an initial disease outbreak. Based on disease-suppressive soils such as take-all decline, we developed a conceptual model of how plants in response to pathogen attack cry for help and recruit plant-protective microbes that confer increased resistance. Thereby, plants create a soilborne legacy that protects subsequent generations and forms disease-suppressive soils.

Prevention of contaminated aerosol and the transmission during nebulized therapy in hospital settings: a systematic review

Inhalation nebulization therapy is important for administering medications to patients in aerosolized form. However, there are persistent apprehensions in healthcare settings regarding aerosol contamination because of the significant infection risk. Despite rigorous adherence to established hospital protocols, concerns about potential contamination and transmission persist, raising considerable apprehension about nosocomial pneumonia. This condition shows the urgent need for implementing highly effective strategies to ensure patient safety during nebulization therapy. Therefore, this study aimed to review current investigations, focusing on interventions to mitigate aerosol contamination and minimize the transmission of contaminated aerosols.Adhering to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, this systematic review included an exhaustive analysis of randomized and non-randomized clinical trials as well as, simulated experimental and in vitro studies published in English in the past decade. A meticulous search was conducted across four major databases, namely ScienceDirect, Cumulative Index to Nursing & Allied Health (CINAHL), PubMed, and Scopus. A total of 37 pertinent studies were identified and subjected to rigorous analysis.The preventive measures include a range of strategies, such as the use of masks by therapists, thorough disinfection of nebulizers, integration of filters, and regular environmental cleaning in the vicinity of the patient.In conclusion, these multifaceted interventions are significant in preventing the administration of contaminated aerosols and curbing the proliferation of infectious agents in the hospital environment.

Evaluating metagenomics and targeted approaches for diagnosis and surveillance of viruses

BackgroundMetagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity.MethodsWe evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers.ResultsCapture with the Twist CVRP increased sensitivity by at least 10–100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600–6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT.ConclusionsMetagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.

A Draft Genome Resource for ‘Candidatus Phytoplasma prunorum’, the Agent Associated with European Stone Fruit Yellows Disease

‘Candidatus Phytoplasma prunorum’ is associated with European stone fruit yellows disease, affecting wild and cultivated species of Prunus at different degrees of susceptibility, so far being mainly restricted to Europe. Here, we report draft genome sequences for ‘Ca. Phytoplasma prunorum’ strains ESFY1 and LNS1, which represent the first available for this species. Strain ESFY1 is the causal agent of the European stone fruit yellows disease of Prunus persica in Germany and LNS1, leptonecrosis disease of Prunus salicina in Italy. Their draft genomes consist of 480,123 and 490,930 bases, assembled into 28 and 16 contigs, respectively, with an average GC content of 21.1% and 20.7%, respectively. Analysis of average nucleotide identity values between genomes further supported the delineation of ‘Ca. Phytoplasma prunorum’ as a species distinct from ‘Ca. Phytoplasma mali’, in agreement with previous phylogenetic data.