ABSTRACT
BACKGROUND: Plague is an acute infectious disease caused by the Yersinia pestis. Historically, it has been a major pandemic with high mortality rates, known as the "Black Death" in the 14th century, which resulted in millions of deaths in Europe. With increasing economic prosperity, more and more people are traveling to Xizang. However, this trend also hides significant safety hazards. Currently, there are few recent reports on plague, especially those with imaging manifestations available. In this study, we report the detailed clinical and radiological data of the patient with pneumonic plague in Xizang, China, in 2023. CASE PRESENTATION: We report a case of pneumonic plague in Xizang, which occurred in a herdsman living in an area where dead marmots were found. The patient presented with symptoms such as fever, hemoptysis, dyspnea and coma. Chest computed tomography (CT) scans showed multiple nodules distributed in the central regions of lung lobes, consolidation distributed in secondary pulmonary lobules, and had a gravity-dependent distribution pattern. These imaging findings were consistent with pulmonary hemorrhage and diffuse alveolar damage. Despite emergency treatment, the patient died within 48 h of admission. Through retrospective medical history investigation, laboratory examination and autopsy, the final diagnosis was confirmed as pneumonic plague. CONCLUSION: Pneumonic plague is the most deadly infectious disease, and its pathological features mainly include damage to the alveoli, pulmonary hemorrhage, and pulmonary edema. Corresponding to CT, it manifests as acute and rapidly progressing pneumonia, alveolar damage, and pulmonary hemorrhage. The value of this article lies in the completeness and typicality of the imaging data, vivid hand-drawn illustrations of transmission pathways, and comprehensive literature review, all of which serve to enhance public understanding of plague and play an important warning role.
Subject(s)
Plague , Tomography, X-Ray Computed , Humans , Plague/diagnosis , China , Male , Fatal Outcome , Lung/diagnostic imaging , Lung/pathology , Yersinia pestis/isolation & purification , Animals , MarmotaABSTRACT
Yersinia pestis has been infecting humans since the Late Neolithic (LN). Whether those early infections were isolated zoonoses or initiators of a pandemic remains unclear. We report Y. pestis infections in two individuals (of 133) from the LN necropolis at Warburg (Germany, 5300-4900 cal BP). Our analyses show that the two genomes belong to distinct strains and reflect independent infection events. All LN genomes known today (n = 4) are basal in the phylogeny and represent separate lineages that probably originated in different animal hosts. In the LN, an opening of the landscape resulted in the introduction of new rodent species, which may have acted as Y. pestis reservoirs. Coincidentally, the number of dogs increased, possibly leading to Y. pestis infections in canines. Indeed, we detect Y. pestis in an LN dog. Collectively, our data suggest that Y. pestis frequently entered human settlements at the time without causing significant outbreaks.
Subject(s)
Dog Diseases , Phylogeny , Plague , Yersinia pestis , Animals , Yersinia pestis/genetics , Yersinia pestis/isolation & purification , Dogs/microbiology , Plague/microbiology , Plague/epidemiology , Plague/history , Plague/transmission , Humans , Dog Diseases/microbiology , Germany/epidemiology , Genome, Bacterial , History, AncientABSTRACT
In the period between 5,300 and 4,900 calibrated years before present (cal. BP), populations across large parts of Europe underwent a period of demographic decline1,2. However, the cause of this so-called Neolithic decline is still debated. Some argue for an agricultural crisis resulting in the decline3, others for the spread of an early form of plague4. Here we use population-scale ancient genomics to infer ancestry, social structure and pathogen infection in 108 Scandinavian Neolithic individuals from eight megalithic graves and a stone cist. We find that the Neolithic plague was widespread, detected in at least 17% of the sampled population and across large geographical distances. We demonstrate that the disease spread within the Neolithic community in three distinct infection events within a period of around 120 years. Variant graph-based pan-genomics shows that the Neolithic plague genomes retained ancestral genomic variation present in Yersinia pseudotuberculosis, including virulence factors associated with disease outcomes. In addition, we reconstruct four multigeneration pedigrees, the largest of which consists of 38 individuals spanning six generations, showing a patrilineal social organization. Lastly, we document direct genomic evidence for Neolithic female exogamy in a woman buried in a different megalithic tomb than her brothers. Taken together, our findings provide a detailed reconstruction of plague spread within a large patrilineal kinship group and identify multiple plague infections in a population dated to the beginning of the Neolithic decline.
Subject(s)
Farmers , Genomics , Pedigree , Plague , Population Dynamics , Yersinia pestis , Female , Humans , Male , Cemeteries/history , Farmers/history , Genome, Bacterial/genetics , History, Ancient , Phylogeny , Plague/epidemiology , Plague/history , Plague/microbiology , Plague/mortality , Scandinavian and Nordic Countries/epidemiology , Time Factors , Virulence Factors/genetics , Yersinia pestis/genetics , Yersinia pestis/isolation & purificationABSTRACT
BACKGROUND: Plague, a zoonotic disease caused by Yersinia pestis, was responsible for 3 historical human pandemics that killed millions of people. It remains endemic in rodent populations in Africa, Asia, North America, and South America but human plague is rare in most of these locations. However, human plague is still highly prevalent in Madagascar, which typically records a significant part of all annual global cases. This has afforded an opportunity to study contemporary human plague in detail using various typing methods for Y. pestis. AIM: This review aims to summarize the methods that have been used to type Y. pestis in Madagascar along with the major discoveries that have been made using these approaches. METHODS: Pubmed and Google Scholar were used to search for the keywords: "typing Yersinia pestis Madagascar," "evolution Yersinia pestis Madagascar," and "diversity Yersinia pestis Madagascar." Eleven publications were relevant to our topic and further information was retrieved from references cited in those publications. RESULTS: The history of Y. pestis typing in Madagascar can be divided in 2 periods: the pre-genomics and genomics eras. During the pre-genomics era, ribotyping, direct observation of plasmid content and plasmid restriction fragment length polymorphisms (RFLP) were employed but only revealed a limited amount of diversity among Malagasy Y. pestis strains. Extensive diversity only started to be revealed in the genomics era with the use of clustered regularly interspaced palindromic repeats (CRISPR), multiple-locus variable number tandem repeats (VNTR) analysis (MLVA), and single-nucleotide polymorphisms (SNPs) discovered from whole genome sequences. These higher-resolution genotyping methods have made it possible to highlight the distribution and persistence of genotypes in the different plague foci of Madagascar (Mahajanga and the Central and Northern Highlands) by genotyping strains from the same locations across years, to detect transfers between foci, to date the emergence of genotypes, and even to document the transmission of antimicrobial resistant (AMR) strains during a pneumonic plague outbreak. Despite these discoveries, there still remain topics that deserve to be explored, such as the contribution of horizontal gene transfer to the evolution of Malagasy Y. pestis strains and the evolutionary history of Y. pestis in Madagascar. CONCLUSIONS: Genotyping of Y. pestis has yielded important insights on plague in Madagascar, particularly since the advent of whole-genome sequencing (WGS). These include a better understanding of plague persistence in the environment, antimicrobial AMR and multi-drug resistance in Y. pestis, and the person-to-person spread of pneumonic plague. Considering that human plague is still a significant public health threat in Madagascar, these insights can be useful for controlling and preventing human plague in Madagascar and elsewhere, and also are relevant for understanding the historical pandemics and the possible use of Y. pestis as a biological weapon.
Subject(s)
Plague , Yersinia pestis , Yersinia pestis/genetics , Yersinia pestis/classification , Yersinia pestis/isolation & purification , Madagascar/epidemiology , Plague/microbiology , Plague/epidemiology , Humans , Animals , Genotype , Genotyping Techniques/methodsABSTRACT
BACKGROUND: Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. METHODS: Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). RESULTS: The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. CONCLUSION: The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.
Subject(s)
Plague , Sensitivity and Specificity , Soil Microbiology , Yersinia pestis , Yersinia pestis/genetics , Yersinia pestis/isolation & purification , Animals , Plague/diagnosis , Plague/microbiology , Mice , Polymerase Chain Reaction/methods , DNA, Bacterial/genetics , Reproducibility of Results , Bacterial Proteins/genetics , Liver/microbiology , Limit of Detection , HumansABSTRACT
Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.
Subject(s)
Tandem Mass Spectrometry , Yersinia pestis , Yersinia pestis/isolation & purification , Tandem Mass Spectrometry/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/instrumentation , Bacteria/isolation & purificationABSTRACT
Background: The control and prevention of rodent-borne diseases are mainly based on our knowledge of ecology and the infectious status of their reservoir hosts. This study aimed to evaluate the prevalence of Francisella tularensis, Yersinia pestis, and arenavirus infections in small mammals and to assess the potential of disease occurrence in East Azerbaijan, northwest of Iran, in 2017 and 2018. Methods: Spleen and lung samples were obtained from all trapped small mammals. The real-time quantitative PCR (qPCR) method was used to detect nucleic acid sequences of F. tularensis, Y. pestis, and arenaviruses. Serum samples were tested for antibodies indicating the host response to F. tularensis and Y. pestis infections using the standard tube agglutination test and enzyme-linked immunosorbent assay (ELISA), respectively. Results: A total of 205 rodents, four Eulipotyphla, and one carnivore were captured. The most common rodent species captured (123 of 205 rodents, 60%) belonged to the genus Meriones (mainly Persian jird, Meriones persicus). In total, 317 fleas were removed from trapped animals. Flea species belonged to Xenopsylla buxtoni, Xenopsylla nuttalli, Stenoponia tripectinata, Paraceras melis, Ctenophthalmus rettigi smiti, Rhadinopsylla bivirgis, Paradoxopsyllus grenieri, and Nosopsyllus iranus. Using the qPCR tests, five spleen samples from M. persicus were positive for F. tularensis. The qPCR tests were negative for the detection of Y. pestis and arenaviruses. Finally, all serum samples tested were negative for antibodies against Y. pestis and F. tularensis. Conclusions: F. tularensis was the only zoonotic agent detected in rodents captured in East Azerbaijan. However, the diversity of trapped rodents and fleas provides the potential for the spread of various rodent-borne viral and bacterial diseases in the studied areas.
Subject(s)
Arenaviridae Infections , Francisella tularensis , Plague , Rodentia , Tularemia , Yersinia pestis , Animals , Iran/epidemiology , Yersinia pestis/isolation & purification , Yersinia pestis/genetics , Tularemia/epidemiology , Tularemia/veterinary , Plague/epidemiology , Plague/veterinary , Francisella tularensis/isolation & purification , Francisella tularensis/genetics , Arenaviridae Infections/epidemiology , Arenaviridae Infections/veterinary , Rodent Diseases/epidemiology , Rodent Diseases/virology , Rodent Diseases/microbiologyABSTRACT
Graves in Kyrgyzstan hold early victims of plague that swept medieval Europe.
Subject(s)
Epidemics , Plague , Yersinia pestis , Burial/history , DNA, Ancient , Epidemics/history , History, Medieval , Humans , Kyrgyzstan/epidemiology , Plague/epidemiology , Plague/history , Yersinia pestis/isolation & purificationABSTRACT
The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.
Subject(s)
Genome, Bacterial , Plague , Yersinia pestis , Animal Husbandry/history , Animals , DNA, Ancient , Genetic Variation , History, Ancient , Human Migration/history , Humans , Phylogeny , Plague/epidemiology , Plague/history , Plague/microbiology , Yersinia pestis/classification , Yersinia pestis/genetics , Yersinia pestis/isolation & purificationABSTRACT
On 12 November 2019, one couple from the Sonid Left Qi (County) in the Inner Mongolia Autonomous Region was diagnosed with pneumonic plague in Beijing. The wife acquired the infection from her husband. Thereafter, two bubonic plague cases were identified in Inner Mongolia on November 16th and 24th. In this study, genome-wide single nucleotide polymorphism (SNP) analysis was used to identify the phylogenetic relationship of Yersinia pestis strains isolated in Inner Mongolia. Strains isolated from reservoirs in 2018 and 2019 in Inner Mongolia, together with the strain isolated from Patient C, were further clustered into 2.MED3m, and two novel lineages (2.MED3q, 2.MED3r) in the 2.MED3 population. According to the analysis of PCR-based molecular subtyping methods, such as the MLVA 14 scheme and seven SNP allele sequencing, Patients A/B and D were classified as 2.MED3m. In addition, strains from rodents living near the patients' residences were clustered into the same lineage as patients. Such observations indicated that human plague cases originated from local reservoirs. Corresponding phylogenetic analysis also indicated that rodent plague strains in different areas in Inner Mongolia belong to different epizootics rather than being caused by spreading from the same epizootic in Meriones unguiculatus in 2019.
Subject(s)
Plague/epidemiology , Yersinia pestis/genetics , Yersinia pestis/pathogenicity , Adult , Animals , Beijing/epidemiology , China/epidemiology , Fatal Outcome , Female , Humans , Male , Middle Aged , Phylogeny , Plague/etiology , Rodentia/microbiology , Yersinia pestis/isolation & purificationABSTRACT
Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category 'A' agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5' 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.
Subject(s)
Plague/microbiology , Polymerase Chain Reaction/methods , Yersinia pestis/genetics , Yersinia pestis/isolation & purification , Animals , Base Sequence , DNA Primers/genetics , Humans , Yersinia pestis/physiologyABSTRACT
Plague, a fleaborne rodent-associated zoonosis, is a neglected disease with most recent cases reported from east and central Africa and Madagascar. Because of its low incidence and sporadic occurrence, most of our knowledge of plague ecology, prevention, and control derives from investigations conducted in response to human cases. Long-term studies (which are uncommon) are required to generate data to support plague surveillance, prevention, and control recommendations. Here we describe a 15-year, multidisciplinary commitment to plague in the West Nile region of Uganda that led to significant advances in our understanding of where and when persons are at risk for plague infection and how to reduce morbidity and mortality. These findings provide data-driven support for several existing recommendations on plague surveillance and prevention and may be generalizable to other plague foci.
Subject(s)
Ecology , Epidemiological Monitoring , Plague/epidemiology , Plague/prevention & control , Primary Prevention/organization & administration , Primary Prevention/statistics & numerical data , Yersinia pestis/isolation & purification , Humans , Incidence , Longitudinal Studies , Risk Factors , Uganda/epidemiologyABSTRACT
Streptomycin is considered to be one of the effective antibiotics for the treatment of plague. In order to investigate the streptomycin resistance of Y. pestis in China, we evaluated streptomycin susceptibility of 536 Y. pestis strains in China in vitro using the minimal inhibitory concentration (MIC) and screened streptomycin resistance-associated genes (strA and strB) by PCR method. A clinical Y. pestis isolate (S19960127) exhibited high-level resistance to streptomycin (the MIC was 4,096 mg/L). The strain (biovar antiqua) was isolated from a pneumonic plague outbreak in 1996 in Tibet Autonomous Region, China, belonging to the Marmota himalayana Qinghai-Tibet Plateau plague focus. In contrast to previously reported streptomycin resistance mediated by conjugative plasmids, the genome sequencing and allelic replacement experiments demonstrated that an rpsL gene (ribosomal protein S12) mutation with substitution of amino-acid 43 (K43R) was responsible for the high-level resistance to streptomycin in strain S19960127, which is consistent with the mutation reported in some streptomycin-resistant Mycobacterium tuberculosis strains. Streptomycin is used as the first-line treatment against plague in many countries. The emergence of streptomycin resistance in Y. pestis represents a critical public health problem. So streptomycin susceptibility monitoring of Y. pestis isolates should not only include plasmid-mediated resistance but also include the ribosomal protein S12 gene (rpsL) mutation, especially when treatment failure is suspected due to antibiotic resistance.
Subject(s)
Anti-Bacterial Agents/pharmacology , Plasmids/genetics , Yersinia pestis/drug effects , Yersinia pestis/genetics , Animals , DNA, Bacterial/genetics , Marmota , Microbial Sensitivity Tests , Mutation/drug effects , Plague/microbiology , Streptomycin/pharmacology , Tibet , Yersinia pestis/isolation & purificationABSTRACT
Yersinia pestis, causative agent of plague, occurs throughout the western United States in rodent populations and periodically causes epizootics in susceptible species, including black-tailed prairie dogs (Cynomys ludovicianus). How Y. pestis persists long-term in the environment between these epizootics is poorly understood but multiple mechanisms have been proposed, including, among others, a separate enzootic transmission cycle that maintains Y. pestis without involvement of epizootic hosts and persistence of Y. pestis within epizootic host populations without causing high mortality within those populations. We live-trapped and collected fleas from black-tailed prairie dogs and other mammal species from sites with and without black-tailed prairie dogs in 2004 and 2005 and tested all fleas for presence of Y. pestis. Y. pestis was not detected in 2126 fleas collected in 2004 but was detected in 294 fleas collected from multiple sites in 2005, before and during a widespread epizootic that drastically reduced black-tailed prairie dog populations in the affected colonies. Temporal and spatial patterns of Y. pestis occurrence in fleas and genotyping of Y. pestis present in some infected fleas suggest Y. pestis was introduced multiple times from sources outside the study area and once introduced, was dispersed between several sites. We conclude Y. pestis likely was not present in these black-tailed prairie dog colonies prior to epizootic activity in these colonies. Although we did not identify likely enzootic hosts, we found evidence that deer mice (Peromyscus maniculatus) may serve as bridging hosts for Y. pestis between unknown enzootic hosts and black-tailed prairie dogs.
Subject(s)
Flea Infestations/veterinary , Plague/veterinary , Sciuridae/microbiology , Siphonaptera/microbiology , Yersinia pestis/isolation & purification , Animals , Colorado/epidemiology , Flea Infestations/epidemiology , Flea Infestations/microbiology , Plague/epidemiology , Serologic Tests/veterinaryABSTRACT
BACKGROUND: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. METHODS/PRINCIPLE FINDINGS: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. CONCLUSIONS/SIGNIFICANCE: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.
Subject(s)
Pathology, Molecular/methods , Plague/diagnosis , Polymerase Chain Reaction/methods , Yersinia pestis/genetics , Yersinia pestis/isolation & purification , DNA Primers , Equipment Design , Genes, Bacterial/genetics , Nucleic Acid Amplification Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Array Sequence Analysis , Pathology, Molecular/instrumentation , Polymerase Chain Reaction/instrumentation , Sensitivity and SpecificityABSTRACT
AIM: Rapid detection of biological agents in biodefense is critical for operational, tactical and strategic levels as well as for medical countermeasures. Yersinia pestis, Francisella tularensis, and Bacillus anthracis are high priority agents of biological warfare or bioterrorism and many response forces use lateral flow assays (LFAs) for their detection. Several companies produce these assays, which offer results in short time and are easy to use. Despite their importance, only few publications on the limits of detection (LOD) for LFAs are available. Most of these studies used inactivated bacteria or risk group-2 strains. As the inactivation process in previous studies might have affected the tests' performances, it was our aim in this study to determine and compare the LOD of several commercially available LFAs using viable risk group-3 strains. METHODS AND RESULTS: Lateral flow assays from four different companies for the detection of following bacteria were evaluated: Y. pestis, F. tularensis and B. anthracis spores. Two independent quantification methods for each target organism were applied, in order to ensure high quantification accuracy. LODs varied greatly between tests and organisms and ranged between 104 for Y. pestis-tests and as high as >109 for one B. anthracis-test. CONCLUSION: This work precisely determined the LODs of LFAs from four commercial suppliers. The herein determined LODs differed from results of previous studies. This illustrates the need for using accurately quantified viable risk group 3-strains for determining such LODs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work bridges an important knowledge gap with regard to LFA LOD. The LODs determined in this study will facilitate better assessment of LFA-results. They illustrate that a negative LFA result is not suited to exclude the presence of the respective agent in the analyzed sample.
Subject(s)
Bacillus anthracis/isolation & purification , Bacteriological Techniques/methods , Francisella tularensis/isolation & purification , Immunoassay/methods , Yersinia pestis/isolation & purification , Bacterial Infections/diagnosis , Bacterial Infections/microbiology , Humans , Limit of Detection , Microbial Viability , Spores, Bacterial/isolation & purificationABSTRACT
The Gram-negative bacterium Yersinia pestis is responsible for deadly plague, a zoonotic disease established in stable foci in the Americas, Africa, and Eurasia. Its persistence in the environment relies on the subtle balance between Y. pestis-contaminated soils, burrowing and nonburrowing mammals exhibiting variable degrees of plague susceptibility, and their associated fleas. Transmission from one host to another relies mainly on infected flea bites, inducing typical painful, enlarged lymph nodes referred to as buboes, followed by septicemic dissemination of the pathogen. In contrast, droplet inhalation after close contact with infected mammals induces primary pneumonic plague. Finally, the rarely reported consumption of contaminated raw meat causes pharyngeal and gastrointestinal plague. Point-of-care diagnosis, early antibiotic treatment, and confinement measures contribute to outbreak control despite residual mortality. Mandatory primary prevention relies on the active surveillance of established plague foci and ectoparasite control. Plague is acknowledged to have infected human populations for at least 5,000 years in Eurasia. Y. pestis genomes recovered from affected archaeological sites have suggested clonal evolution from a common ancestor shared with the closely related enteric pathogen Yersinia pseudotuberculosis and have indicated that ymt gene acquisition during the Bronze Age conferred Y. pestis with ectoparasite transmissibility while maintaining its enteric transmissibility. Three historic pandemics, starting in 541 AD and continuing until today, have been described. At present, the third pandemic has become largely quiescent, with hundreds of human cases being reported mainly in a few impoverished African countries, where zoonotic plague is mostly transmitted to people by rodent-associated flea bites.
Subject(s)
Plague/epidemiology , Plague/transmission , Rodentia/microbiology , Yersinia pestis/classification , Animals , Archaeology , Clonal Evolution , Humans , Insect Vectors/microbiology , Phylogeny , Population Surveillance , Siphonaptera/microbiology , Soil Microbiology , Yersinia pestis/genetics , Yersinia pestis/isolation & purificationABSTRACT
Yersinia pestis, the causative agent of plague mainly infects rodents, while humans are the accidental host. The conventional diagnostic methods available for Y. pestis exhibit cross-reactivity with other enteropathogenic bacteria which makes its detection difficult. Rapid and reliable point-of-care detection of Y. pestis is essential for timely initiation of medical treatment. In the present study, a pair of loop mediated isothermal amplification (LAMP) assays has been developed for rapid detection of Y. pestis. Two sets of LAMP primers, each containing 6 primers were specifically designed targeting caf1 and 3a genes located on pFra plasmid and chromosome of Y. pestis, respectively. Isothermal amplification was accomplished at 65 °C for 40 min for caf1 target, and at 63 °C for 50 min for 3a choromosomal target. The analytical sensitivity of the assay for the caf1 and 3a targets was found to be 500 fg and 100 fg genomic DNA of Y. pestis, respectively. The caf1 and 3a LAMP assays detected as few as 100 copies of caf1 and 10 copies of 3a gene targets harboured in the respective recombinant plasmids. The amplified products were detected visually under visible and UV light using SYBR Green 1 dye. The assay pair was found to be highly specific as it did not cross-react with closely related and other bacterial species.
Subject(s)
Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Plague/microbiology , Yersinia pestis/isolation & purification , Benzothiazoles/metabolism , Diamines/metabolism , Humans , Limit of Detection , Plague/blood , Quinolines/metabolism , Sensitivity and SpecificityABSTRACT
Yersinia pestis, the causative agent of plague, is a highly lethal pathogen transmitted by the bite of infected fleas. Once ingested by a flea, Y. pestis establish a replicative niche in the gut and produce a biofilm that promotes foregut colonization and transmission. The rat flea Xenopsylla cheopis is an important vector to several zoonotic bacterial pathogens including Y. pestis. Some fleas naturally clear themselves of infection; however, the physiological and immunological mechanisms by which this occurs are largely uncharacterized. To address this, RNA was extracted, sequenced, and distinct transcript profiles were assembled de novo from X. cheopis digestive tracts isolated from fleas that were either: 1) not fed for 5 days; 2) fed sterile blood; or 3) fed blood containing ~5x108 CFU/ml Y. pestis KIM6+. Analysis and comparison of the transcript profiles resulted in identification of 23 annotated (and 11 unknown or uncharacterized) digestive tract transcripts that comprise the early transcriptional response of the rat flea gut to infection with Y. pestis. The data indicate that production of antimicrobial peptides regulated by the immune-deficiency pathway (IMD) is the primary flea immune response to infection with Y. pestis. The remaining infection-responsive transcripts, not obviously associated with the immune response, were involved in at least one of 3 physiological themes: 1) alterations to chemosensation and gut peristalsis; 2) modification of digestion and metabolism; and 3) production of chitin-binding proteins (peritrophins). Despite producing several peritrophin transcripts shortly after feeding, including a subset that were infection-responsive, no thick peritrophic membrane was detectable by histochemistry or electron microscopy of rat flea guts for the first 24 hours following blood-feeding. Here we discuss the physiological implications of rat flea infection-responsive transcripts, the function of X. cheopis peritrophins, and the mechanisms by which Y. pestis may be cleared from the flea gut.
Subject(s)
Gastrointestinal Tract/microbiology , Transcriptome , Xenopsylla/microbiology , Yersinia pestis/genetics , Yersinia pestis/metabolism , Animals , Biofilms , Epithelium/microbiology , Epithelium/pathology , Female , Gastrointestinal Tract/pathology , Gene Expression Profiling , Insect Vectors/microbiology , Plague/microbiology , Plague/veterinary , Rats , Sequence Analysis, RNA , Yersinia pestis/growth & development , Yersinia pestis/isolation & purificationABSTRACT
Ancient genomic studies have identified Yersinia pestis (Y. pestis) as the causative agent of the second plague pandemic (fourteenth-eighteenth century) that started with the Black Death (1,347-1,353). Most of the Y. pestis strains investigated from this pandemic have been isolated from western Europe, and not much is known about the diversity and microevolution of this bacterium in eastern European countries. In this study, we investigated human remains excavated from two cemeteries in Riga (Latvia). Historical evidence suggests that the burials were a consequence of plague outbreaks during the seventeenth century. DNA was extracted from teeth of 16 individuals and subjected to shotgun sequencing. Analysis of the metagenomic data revealed the presence of Y. pestis sequences in four remains, confirming that the buried individuals were victims of plague. In two samples, Y. pestis DNA coverage was sufficient for genome reconstruction. Subsequent phylogenetic analysis showed that the Riga strains fell within the diversity of the already known post-Black Death genomes. Interestingly, the two Latvian isolates did not cluster together. Moreover, we detected a drop in coverage of the pPCP1 plasmid region containing the pla gene. Further analysis indicated the presence of two pPCP1 plasmids, one with and one without the pla gene region, and only one bacterial chromosome, indicating that the same bacterium carried two distinct pPCP1 plasmids. In addition, we found the same pattern in the majority of previously published post-Black Death strains, but not in the Black Death strains. The pla gene is an important virulence factor for the infection of and transmission in humans. Thus, the spread of pla-depleted strains may, among other causes, have contributed to the disappearance of the second plague pandemic in eighteenth century Europe.