Discovery Of New Pathogens Research Articles

A One Health Evaluation of the Surveillance Systems on Tick-Borne Diseases in the Netherlands, Spain and Italy.

Simple SummaryIxodid ticks and tick-borne diseases are expanding their geographical range, but surveillance activities vary among countries. We analysed the surveillance systems in place in the Netherlands, Spain and Italy, to identify ideal elements to monitor tick-borne diseases, by using a One Health evaluation protocol. We identified differences among the three surveillance systems, with the Dutch initiative showing a high level of transdisciplinary collaboration, good identification of the actors and engagement of the public in research and education. Measurable outcomes have been generated, such as the reduction in tick bites and the discovery of new pathogens and tick species. In Italy and Spain, surveillance systems are based on compulsory notification to health authorities; legislation seems relevant but law enforcement alongside the availability of economic resources is rather fragmented and limited to the most severe diseases. The non-scientific community is marginally considered and collaborations are limited to local initiatives. Research activities in both countries have mostly contributed to gaining knowledge on the distribution of tick species and the discovery of new pathogens. Although all TBD surveillance plans comply with the EU regulations, the initiatives characterised by trans-disciplinary collaboration may be more effective for the surveillance and prevention of tick-transmitted diseases.To identify ideal elements for the monitoring and prevention of tick-borne diseases (TBD), we analysed the surveillance systems in place in the Netherlands, Spain and Italy. We applied a semi-quantitative evaluation to identify outcomes and assess the degree of One Health implementation. Differences emerged in the surveillance initiatives, as well as the One Health scores. The Dutch surveillance is dominated by a high level of transdisciplinary and trans-sectoral collaboration, enabling communication and data sharing among actors. Different project-based monitoring, research and educational activities are centrally coordinated and the non-scientific community is actively involved. All this yielded measurable health outcomes. In Italy and Spain, TBD surveillance and reporting systems are based on compulsory notification. Law enforcement, alongside dedicated time and availability of economic resources, is fragmented and limited to the most severe health issues. Veterinary and human medicine are the most involved disciplines, with the first prevailing in some contexts. Stakeholders are marginally considered and collaborations limited to local initiatives. Research activities have mostly contributed to gaining knowledge on the distribution of tick vectors and discovery of new pathogens. Although all TBD surveillance plans comply with EU regulations, initiatives characterised by transdisciplinary collaboration may be more effective for the surveillance and prevention of TBD.

Th e Clues of Th e Discovery of New Pathogens Eventuated by the Horizontal Gene Transfer Among the Bacillus cereus Group Members

Th e Clues of Th e Discovery of New Pathogens Eventuated by the Horizontal Gene Transfer Among the Bacillus cereus Group Members

Identification of emerging viral genomes in transcriptomic datasets of alfalfa (Medicago sativa L.)

BackgroundPublicly available transcriptomic datasets have become a valuable tool for the discovery of new pathogens, particularly viruses. In this study, several coding-complete viral genomes previously not found or experimentally confirmed in alfalfa were identified in the plant datasets retrieved from the NCBI Sequence Read Archive.MethodsPublicly available Medicago spp. transcriptomic datasets were retrieved from the NCBI SRA database. The raw reads were first mapped to the reference genomes of Medicago sativa and Medigago truncatula followed by the alignment of the unmapped reads to the NCBI viral genome database and de novo assembly using the SPAdes tool. When possible, assemblies were experimentally confirmed using 5′/3′ RACE and RT-PCRs.ResultsTwenty three different viruses were identified in the analyzed datasets, of which several represented emerging viruses not reported in alfalfa prior to this study. Among them were two strains of cnidium vein yellowing virus, lychnis mottle virus and Cactus virus X, for which coding-complete genomic sequences were obtained by a de novo assembly.ConclusionsThe results improve our knowledge of the diversity and host range of viruses infecting alfalfa, provide essential tools for their diagnostics and characterization and demonstrate the utility of transcriptomic datasets for the discovery of new pathogens.

Diagnostic accuracy of two DNA-based molecular assays for detection of porcine circovirus 3 in swine population using Bayesian latent class analysis.

Molecular-based tools sometimes are the only laboratory techniques available to detect a recently discovered agent, and their validation without the existence of previously described 'gold standard' methods poses a challenge for the diagnosticians. A good example within this scenario is the recently described porcine circovirus 3 (PCV-3) in the swine population worldwide, from which only few PCR methods have been described. Therefore, the primary objective of this study was to estimate the diagnostic accuracy of a direct PCR (dPCR) and a real-time qPCR (qPCR) for detection of PCV-3 in Italian swine population. Bayesian latent class analysis approach was used to rigorously assess their features and applicability in routine diagnostic activity. Data on dPCR and qPCR were available from 116 domestic pigs, which were randomly selected from 55 farms located at different regions in Northern Italy. The sensitivity (Se) estimates of dPCR (94%; posterior credible interval (PCI%) 84-100) and qPCR (96%; PCI% 90-100) were high and similar. The estimated specificity (Sp) of both dPCR and qPCR assays was around 97%. dPCR and qPCR assays showed a high and comparable Se and Sp estimates for the detection of PCV-3 in Italian swine population. SIGNIFICANCE AND IMPACT OF THE STUDY: The continuous discovery of new pathogens poses a challenge in the development and evaluation of adequate diagnostic tools. In fact, since molecular-based tools sometimes are the only available laboratory techniques, it is typically difficult to evaluate their diagnostic performances in the absence of a gold standard. The present study assesses this issue, demonstrating the excellent performances of two PCR-based assays for porcine circovirus 3 (PCV-3) detection using a Bayesian latent class analysis approach. Therefore, the molecular tests evaluated under this study constitute reliable tools for the routine diagnosis and surveillance programs of PCV-3 circulating in swine populations.

Infections due to Rickettsia and Related Organisms

Infections caused by organisms of the genus Rickettsia, Orientia, Ehrlichia, Anaplasma, and Coxiella occur throughout the world and are important, yet often overlooked, causes of febrile illness. They are transmitted by ticks, lice, mites, fleas, and, in the case of Coxiella, infected aerosols. Some are considered emerging and reemerging infectious diseases, as exemplified by the emergence of Rocky Mountain spotted fever in the American Southwest and Mexico; the reemergence of murine typhus in parts of Texas; and the discovery of new pathogens, such as Ehrlichia muris–like agent. Manifestations are usually of an acute undifferentiated febrile illness, with associated headache, malaise, myalgias, and varying frequency of rash. Since confirmation of diagnosis is often retrospective, requiring the dynamic change in antibody titers from acute and convalescent phase sera, clinical recognition for empirical treatment is imperative. Indeed, timely treatment is effective at abating symptoms and preventing complications. This review discusses important aspects of the epidemiology, clinical manifestations, diagnostic methods, and treatment of infections caused by Rickettsia and related organisms. This review contains 5 figures, 9 tables, and 50 references. Key words: anaplasmosis, ehrlichiosis, Q fever, Rocky Mountain spotted fever, scrub typhus, spotted fever group rickettsioses, typhus group rickettsioses

Innovative Approaches to Improve Anti-Infective Vaccine Efficacy.

Safe and efficacious vaccines are arguably the most successful medical interventions of all time. Yet the ongoing discovery of new pathogens, along with emergence of antibiotic-resistant pathogens and a burgeoning population at risk of such infections, imposes unprecedented public health challenges. To meet these challenges, innovative strategies to discover and develop new or improved anti-infective vaccines are necessary. These approaches must intersect the most meaningful insights into protective immunity and advanced technologies with capabilities to deliver immunogens for optimal immune protection. This goal is considered through several recent advances in host-pathogen relationships, conceptual strides in vaccinology, and emerging technologies. Given a clear and growing risk of pandemic disease should the threat of infection go unmet, developing vaccines that optimize protective immunity against high-priority and antibiotic-resistant pathogens represents an urgent and unifying imperative.

The role and challenges of new diagnostic technology in plant biosecurity

Pest and pathogens pose a major threat to food security and the natural environment, and these threats are moving around the globe. Trade, travel and transport have major roles to play in this and thus to improve plant biosecurity, we need enhanced phytosanitary inspection systems. In order to achieve this, there is a role for the more effective use of diagnostic technology, such as field-based testing or the use of next generation sequencing technology. In this review we examine the opportunities and challenges posed by using new technology within a plant biosecurity context, but in contrast to previous reviews, here we focus on practical challenges associated with deployment and routine use, rather than specific technical issues. These key challenges include the need to accelerate the development and deployment of new technologies into the field, the accelerated discovery of new pathogens and the need for new risk assessment approaches, and improvements to our understanding of how best to deploy and use new diagnostic tools for maximum impact. Throughout we focus on how interdisciplinary approaches are important to help us improve our understanding and achieve our goals.

Preferential Amplification of Pathogenic Sequences.

The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called “Preferential Amplification of Pathogenic Sequences (PATHseq)” that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called “non-human primers” that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.

:Emerging Infections 8

DICTIONARIES DEFINE THAT FOR SOMETHING TO “EMERGE” IT has to rise from an obscure or inferior position. One could say that humans, by vanquishing the threats of infectious diseases, have emerged as one of the dominant species on the planet. But an ominous foreshadowing of the history of infectious diseases is contained in another dictionary definition of “emerge,” in which emergence refers to coming into being through evolution. While the human species has developed an armamentarium to ward off the known infectious threats to health, the microbial world has not been idle. One only has to peruse newspaper headlines to realize that reports of emerging infections are increasing at an alarming rate. Humans around the world are continually battling viruses, bacteria, prions, and parasites. Sorting through the plethora of new infectious threats to human health and biosecurity can be daunting, even for infectious disease specialists. The rapid-fire pace with which new discoveries of unknown pathogens are announced makes most textbooks irrelevant when it comes to this topic, and one is left to use Internet search engines when colleagues or patients inquire about some exotic new infection creating havoc on the other side of the globe. A recent review on this topic begins with a fitting 19th-century quote from Rudolf Virchow: “Not a single year passes without [which] . . . we can tell the world: here is a new disease!” Emerging Infections 8, edited by a renowned team of clinicians, should be on the bookshelf of anyone desiring to keep abreast of these developments. It is the product of various symposia from the Intersciences Conference on Antimicrobial Agents and Chemotherapy. Spanning diseases caused not only by exotic or newly described pathogens, the book features chapters on microbes as disparate as Clostridium difficile and arenaviruses. Each chapter provides summaries that are both comprehensive and concise. Chapters that bring rarely discussed concepts to light include an insightful piece on norovirus infection susceptibility and the relationship to blood group antigens, an encouraging overview of the Japanese encephalitis virus vaccine pipeline, as well as a discussion of Streptococcus suis infection. I found the discussion of bats as disease reservoirs, addressed in the chapter on Nipah and Hendra viruses, thoroughly illuminating and informative. Integrating the facts that bats can cover large distances via their ability to fly, are long-lived, roost in numbers, and have unique immune features, the authors of that chapter explain what makes these organisms attractive hosts for viruses such as the severe acute respiratory syndrome virus, coronavirus, Marburg, and possibly the elusive Ebola. Chapters likewise contain information that would be useful to a clinician. For example, treatment suggestions for adenovirus infections in immunocompromised hosts are presented, providing clinicians with guidance for what is often an exceedingly complex decision. Other topics of high clinical relevance include comparisons of C difficile–associated diarrhea with the newly described entity of Klebsiella oxytoca hemorrhagic colitis, a detailed look at human immunodeficiency virus–associated immune reconstitution inflammatory syndromes, and pertussis. The closing chapter is, fittingly, devoted to the topic of new diagnostic techniques that have aided the discovery of new pathogens. The major strength of this book is that it is premised that a disease does not have to be new or flashy to be subsumed in the definition of “emerging.” Finding deficiencies was a hard task; however, in the chapter on Lyssaviruses, one notable deficiency was the detailed discussion of a rabies case successfully treated using a novel regimen. In an era when the power of medicine has dramatically shifted to unveiling the cryptic causes of new diseases, a book like this one will become as necessary and ubiquitous as the Sanford Guide to Antimicrobial Therapy. It is in this vein that I recommend Emerging Infections 8 to provide a foundation for what will likely be an exciting and frightening era of infectious disease.

Microbial combinatorics: a simplified approach for isolating insecticidal bacteria

Bacteria can control pest insects that damage food crops, vector diseases and defoliate trees. Conventionally, isolation of these bacteria has been from soil and sporadically from dead insects. A simplified approach for isolating insecticidal bacteria from soil using the target insect as the selective agent was employed in this study. Instead of isolating single strains of bacteria from soil and testing each individual strain for insect toxicity, mixtures of bacteria present in each soil sample were tested together directly for toxicity using Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) as a model insect. Thirty-five soil suspensions or bacterial suspensions of the 40 suspensions tested killed at least one M. sexta larva. All but one bacterial culture isolated from dead larvae and retested for toxicity, killed at least one M. sexta larva. Nineteen bacterial strains isolated from larvae killed in the first test, were identical to the bacteria fed to the retested larvae. Of the 19 strains isolated, 14 were identified by 16S rDNA sequencing as belonging to the Bacillus cereus group including three strains that formed crystals that were identified as B. thuringiensis. Of the three other spore-forming strains, two were identified as psychrotrophic B. weihenstephanensis and the third as Lysinibacillus fusiformis. Two others were identified as Enterococcus faecalis. This approach, microbial combinatorics, reduces the number of insects necessary for toxicity screening and associated time and resources compared to conventional methods that first isolate bacteria and then individually test for toxicity as well as a means of discovery of new pathogens using the insect as the selective agent.

A Recently Identified Rhinovirus Genotype Is Associated with Severe Respiratory‐Tract Infection in Children in Germany

Acute respiratory infection is a significant cause of morbidity and mortality in children worldwide. Accurate identification of causative agents is critical to case management and to prioritization in vaccine development. Sensitive multiplex diagnostics provide us with an opportunity to investigate the relative contributions of individual agents andmayalso facilitate the discovery of new pathogens. Recently, application of MassTag polymerase chain reaction (PCR) to undiagnosed infuenza-like illness in New York State led to the discovery of a novel rhinovirus genotype. Here we report the investigation, by MassTag PCR, of pediatric respiratory-tract infections in Germany, studying 97 cases for which no pathogen was identified through routine laboratory evaluation. Respiratory viruses were identified in 49 cases (51%); of the 55 identified viruses, 41 (75%) were rhinoviruses. The novel genotype represented 73% of rhinoviruses and 55% of all identified viruses. Infections with the novel genotype were associated with upper-respiratory-tract symptoms but, more frequently, with bronchitis, bronchiolitis, and pneumonia.

Detection and Identification of Microorganisms by Gene Amplification and Sequencing

Gene amplification and sequencing have led to the discovery of new pathogens as agents of disease and have enabled us to better classify microorganisms from culture. Sequence-based identification of bacteria and fungi using culture is more objective and accurate than conventional methods, especially for classifying unusual microorganisms that are emerging pathogens in immunocompromised hosts. Although a powerful tool, the interpretation of sequence-based classification can be challenging as microbial taxonomy grows more complex, without known clinical correlatives. Additionally, broad-range gene polymerase chain reaction and sequencing have emerged as alternative, culture-independent methods for detecting pathogens from clinical material. The promise of this technique has remained strong, limited mainly by contamination and inadequate sensitivity issues. This review explains sequence-based microbial classification, with emphasis on relating the complex world of microbial taxonomy to a clinical context. Additionally, this review discusses a rational approach to broad-range bacterial polymerase chain reaction and gene sequencing when applied directly to clinical samples.

Parasitic protozoa transmission by drinking water

The pathogenic protozoa Cryptosporidium sp. and Giardia sp. showed their infectivity and negative effects on the health of thousands of people in both developed and developing countries. Protozoa have ambient resistant stages that permit survived to physical and chemical treatment of drinking water. This risk is mainly related to the consumption of water whose microbiological quality indicators typically fulfill with current standards (fecal coliforms and Escherichia coli). Likewise, the discovery of new pathogens is calling for the development of new microbiological quality indicators that allow the production of really safe goods and services such as water for human consumption. Herein the authors present a literature review showing the impact of this microbiological risk in different populations.

Microbial considerations in genetically engineered mouse research.

Microbial infections have long been of concern to scientists using laboratory rodents because of their potential to confound and invalidate research. With the explosion of genetically engineered mice (GEM), new concerns over the impact of microbial agents have emerged because these rodents in many cases are more susceptible to disease than their inbred or outbred counterparts. Moreover, interaction between microbe and host and the resulting manifestation of disease conceivably differ between GEM and their inbred and outbred counterparts. As a result, infections may alter the GEM phenotype and confound interpretation of results and conclusions about mutated gene function. In addition, because GEM are expensive to produce and maintain, contamination by pathogens or opportunists has severe economic consequences. This review addresses how microbial infections may influence phenotype, how immunomodulation of the host as the result of induced mutations may modify host susceptibility to microbial infections, how novel host:microbe interactions have led to the development of new animal models for disease, how phenotype changes have led to the discovery of new pathogens, and new challenges associated with prevention and control of microbial infections in GEM. Although the focus is on naturally occurring infections, extensive literature on the use of GEM in studies of microbial pathogenesis also exists, and the reader is referred to this literature if microbial infection is a suspected culprit in phenotype alteration.

Les maladies infectieuses émergentes : importance en santé publique, aspects épidémiologiques, déterminants et prévention

At the end of the 70s, it was said that eradicating infectious diseases was possible. With the occurrence of AIDS and the discovery of new pathogens the come-back of infectious diseases was noted, 15 to 20 years later, and the concept of emerging infections was defined. An emerging infection is the appearance of a new infection or the increase of its incidence if not new. It also includes infections for which the incidence could increase because of favorable conditions. Several scenarios can be proposed: 1) the appearance of a new infection (AIDS or SARS); 2) the appearance of a known infection where it did not exist previously (West Nile virus infection in the USA); 3) an infection that existed but that was not diagnosed or could not be linked precisely to a particular infectious agent (hepatitis C before the discovery of the hepatitis C virus; 4) a known infection that increases its usual incidence (outbreaks…); 5) infectious diseases for which current conditions may facilitate transmission (decrease of immunization coverage...). The deliberate release of a biological agent is one of the possible scenarios of emergence. Qualitative changes of infectious agents such as the resistance to anti-infectious agents may also contribute, through a selection mechanism, to the emergence of new infectious threat. Emerging infections result from the interaction between the agent, the host, and environment evolving according to human activity. Surveillance, research, laboratory capacity, and an effective public health system are key factors for their control.

Identificación bacteriana mediante secuenciación del ARNr 16S: fundamento, metodología y aplicaciones en microbiología clínica

La comparación de las secuencias de los ARNr 16S (o de los genes que los codifican) permite establecer las relaciones filogenéticas existentes entre los organismos procariotas. Este hecho ha tenido una enorme repercusión en taxonomía bacteriana, dando lugar al sistema de clasificación vigente y permitiendo la identificación rápida y precisa de las bacterias. En microbiología clínica la identificación molecular basada en el ADNr 16S se utiliza fundamentalmente para bacterias cuya identificación mediante otro tipo de técnicas resulta imposible, difícil o requiere mucho tiempo. La amplificación del gen, para su posterior secuenciación, parte preferentemente de ADN extraído de un cultivo puro de la bacteria, pero también puede conseguirse directamente de una muestra clínica. Esto último ha conducido al descubrimiento de nuevos agentes patógenos. Teniendo en cuenta su potencialidad, a medida que los recursos técnicos aumenten y el precio se haga más competitivo, la identificación bacteriana basada en el ADNr 16S encontrará probablemente una aplicación más amplia en el laboratorio de microbiología clínica. Phylogenetic relationships among prokaryotes can be inferred from comparisons of their 16S rRNA (or 16S rDNA) sequences. This has had an enormous repercussion on bacterial taxonomy, leading to the currently applied system of classification, and allowing a rapid and precise identification of bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques turns out impossible, difficult, or requires a lot of time. Amplification of the gene to be sequenced uses preferably DNA extracted from a bacterial pure culture, but can be achieved also directly from a clinical sample. The latter has led to the discovery of new pathogens. Bearing in mind its potential, as the technical resources improve and the prize becomes more competitive, the identification based on 16S rDNA sequencing will certainly find a wider application in the clinical microbiology laboratory.

Iridovirus in the root weevil Diaprepes abbreviatus

Invertebrate iridescent virus 6 (IIV6) was evaluated for mode of transmission and ability to cause infection in the root weevil, Diaprepes abbreviatus (L.). This is the first evidence of IIV6 infection in D. abbreviatus, which caused both patent and sub-lethal covert infections in both larvae and adults. Adults and larvae were successfully infected with IIV6 by puncture, injection and per os. Transmission of IIV6 was demonstrated between infected and healthy individuals regardless of gender. Virus was detected in egg masses produced by virus-infected females suggesting IIV6 is transmitted transovarially. Virus particles were observed in the cytoplasm of weevil cells, and were shown to infect fat bodies, muscle, and nerve tissues, as visualized using transmission electron microscopy. Patent infections resulted in death of individuals within 3 to 4 days post infection. Individuals with covert infections tested positive for virus infection on day 7 by polymerase chain reaction analysis. Sequencing of PCR amplicons confirmed virus infection. Discovery of new pathogens against root weevils may provide new management tools for development of control strategies based on induced epizootics. This is the first report of a virus infecting D. abbreviatus.

Iridovirus in the root weevil Diaprepes abbreviatus.

Invertebrate iridescent virus 6 (IIV6) was evaluated for mode of transmission and ability to cause infection in the root weevil, Diaprepes abbreviatus (L.). This is the first evidence of IIV6 infection in D. abbreviatus, which caused both patent and sub-lethal covert infections in both larvae and adults. Adults and larvae were successfully infected with IIV6 by puncture, injection and per os. Transmission of IIV6 was demonstrated between infected and healthy individuals regardless of gender. Virus was detected in egg masses produced by virus-infected females suggesting IIV6 is transmitted transovarially. Virus particles were observed in the cytoplasm of weevil cells, and were shown to infect fat bodies, muscle, and nerve tissues, as visualized using transmission electron microscopy. Patent infections resulted in death of individuals within 3 to 4 days post infection. Individuals with covert infections tested positive for virus infection on day 7 by polymerase chain reaction analysis. Sequencing of PCR amplicons confirmed virus infection. Discovery of new pathogens against root weevils may provide new management tools for development of control strategies based on induced epizootics. This is the first report of a virus infecting D. abbreviatus.

Community-acquired pneumonia: an update.

Despite the discovery of new pathogens and the evolving problem of antibiotic resistance, the basic trends in community-acquired pneumonia remain remarkably constant. This article reviews the common pathogens, new pathogens, their clinical presentations, the diagnostic workup, the decision to hospitalize, antibiotic resistance, and antibiotic choices.

Sulphonamides and streptomycin in bacillary dysentery; a controlled trial.

<h3>SUMMARY</h3> Despite the discovery of new pathogens and the evolving problem of antibiotic resistance, the basic trends in community-acquired pneumonia remain remarkably constant. This article reviews the common pathogens, new pathogens, their clinical presentations, the diagnostic workup, the decision to hospitalize, antibiotic resistance, and antibiotic choices.