Antimicrobial Susceptibility of Francisella tularensis Isolates in the United States, 2009-2018.

Francisella tularensis is the causative agent of tularemia. We tested the susceptibility of 278 F. tularensis isolates from the United States received during 2009-2018 to 8 antimicrobial drugs (ciprofloxacin, levofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, chloramphenicol, and erythromycin). All isolates were susceptible to all tested drugs.

Francisella tularensis Bone and Joint Infections: United States, 2004-2023.

Tularemia is caused by the highly infectious bacterium Francisella tularensis, which is recognized as a Tier 1 bioterrorism agent. Tularemia has a range of recognized clinical manifestations, but fewer than 20 bone or joint infections from 6 countries have been reported in the literature to date. This series includes 13 cases of F. tularensis septic arthritis or osteomyelitis in the United States during 2004-2023 and describes exposures, clinical presentation, diagnosis, and outcomes for this rare but severe form of tularemia. Clinicians should consider F. tularensis in patients with compatible exposures or a history of joint replacement or immunosuppression.

Anaplasma bovis-Like Infections in Humans, United States, 2015-2017.

We detected the DNA of an Anaplasma bovis-like bacterium in blood specimens from 4 patients from the United States with suspected tickborne illnesses. Initial molecular characterization of this novel agent reveals identity to A. bovis-like bacteria detected in Dermacentor variabilis ticks collected from multiple US states.

Soft Tick Relapsing Fever - United States, 2012-2021.

Soft tick relapsing fever (STRF) (also known as tickborne relapsing fever) is a rare infection caused by certain Borrelia spirochetes and transmitted to humans by soft-bodied Ornithodoros ticks. In the United States, acquisition of STRF is commonly associated with exposure to rustic cabins, camping, and caves. Antibiotic treatment is highly effective for STRF, but without timely treatment, STRF can result in severe complications, including death. No nationally standardized case definition for STRF exists; however, the disease is reportable in 12 states. This report summarizes demographic and clinical information for STRF cases reported during 2012-2021 from states where STRF is reportable. During this period, 251 cases were identified in 11 states. The median annual case count was 24. Most patients with STRF (55%) were hospitalized; no fatalities were reported. The geographic distribution and seasonal pattern of STRF have remained relatively constant since the 1990s. Persons should avoid rodent-infested structures and rodent habitats, such as caves, in areas where STRF is endemic. STRF surveillance, prevention, and control efforts would benefit from a standardized case definition and increased awareness of the disease among the public and clinicians.

Simultaneous Detection and Differentiation of Clinically Relevant Relapsing Fever Borrelia with Semimultiplex Real-Time PCR.

Bacterial vector-borne diseases, including Borrelia species, present a significant diagnostic, clinical, and public health challenge due to their overlapping symptoms and the breadth of causative agents and arthropod vectors. The relapsing fever (RF) borreliae encompass both established and emerging pathogens and are transmitted to humans by soft ticks, hard ticks, or lice. We developed a real-time semimultiplex PCR assay that detects multiple RF borreliae causing human illness and classifies them into one of three groups. The groups are based on genetic similarity and include agents of soft-tick relapsing fever (Borrelia hermsii and others), the emerging hard-tick-transmitted pathogen B. miyamotoi, and the agent of louse-borne relapsing fever (B. recurrentis). The real-time PCR assay uses a single primer pair designed to amplify all known pathogenic RF borreliae and multiple TaqMan probes to allow the detection of and differentiation among the three groups. The assay detects all RF borreliae tested, with an analytical limit of detection below 15 genome equivalents per reaction. Thirty isolates of RF borreliae encompassing six species were accurately identified. Thirty-nine of 41 residual specimens (EDTA whole blood, serum, or plasma) from patients with RF were detected and correctly classified. None of 42 clinical samples from patients with other infections and 46 culture specimens from non-RF bacteria were detected. The development of a single-assay real-time PCR approach will help to improve the diagnosis of RF by simplifying the selection of tests to aid in the clinical management of acutely ill RF patients.

Detection of Tick-Borne Bacteria from Whole Blood Using 16S Ribosomal RNA Gene PCR Followed by Next-Generation Sequencing.

Reported cases of tick-borne diseases have steadily increased for more than a decade. In the United States, a majority of tick-borne infections are caused by bacteria. Clinical diagnosis may be challenging, as tick-borne diseases can present with similar symptoms. Laboratory diagnosis has historically relied on serologic methods, which have limited utility during the acute phase of disease. Pathogen-specific molecular methods have improved early diagnosis, but can be expensive when bundled together and may miss unexpected or novel pathogens. To address these shortcomings, we developed a 16S rRNA gene PCR with a next-generation sequencing (NGS) approach to detect tick-borne bacteria in whole blood. A workflow was optimized by comparing combinations of two extraction platforms and two primer sets, ultimately pursuing DNA extraction from blood with the MagNA Pure 96 and PCR amplification using dual-priming oligonucleotide primers specific to the V1-V3 region of the 16S rRNA gene. The amplified product underwent modified Illumina 16S metagenomics sequencing library preparation and sequencing on a MiSeq V2 Nano flow cell, with data analysis using Pathogenomix RipSeq NGS software. Results with the developed method were compared to those from a V1-V2 16S rRNA gene primer set described by the Centers for Disease Control and Prevention (CDC). The V1-V3 assay demonstrated equivalent performance to the CDC assay, with each method showing concordance with targeted PCR results in 31 of 32 samples, and detecting 22 of 23 expected organisms. These data demonstrate the potential for using a broad-range bacterial detection approach for diagnosis of tick-borne bacterial infection from blood.

Targeted Metagenomics for Clinical Detection and Discovery of Bacterial Tick-Borne Pathogens.

Tick-borne diseases, due to a diversity of bacterial pathogens, represent a significant and increasing public health threat throughout the Northern Hemisphere. A high-throughput 16S V1-V2 rRNA gene-based metagenomics assay was developed and evaluated using >13,000 residual samples from patients suspected of having tick-borne illness and >1,000 controls. Taxonomic predictions for tick-borne bacteria were exceptionally accurate, as independently validated by secondary testing. Overall, 881 specimens were positive for bacterial tick-borne agents. Twelve tick-borne bacterial species were detected, including two novel pathogens, representing a 100% increase in the number of tick-borne bacteria identified compared to what was possible by initial PCR testing. In three blood specimens, two tick-borne bacteria were simultaneously detected. Seven bacteria, not known to be tick transmitted, were also confirmed to be unique to samples from persons suspected of having tick-borne illness. These results indicate that 16S V1-V2 metagenomics can greatly simplify diagnosis and accelerate the discovery of bacterial tick-borne pathogens.

Intervention To Stop Transmission of Imported Pneumonic Plague - Uganda, 2019.

Plague, an acute zoonosis caused by Yersinia pestis, is endemic in the West Nile region of northwestern Uganda and neighboring northeastern Democratic Republic of the Congo (DRC) (1-4). The illness manifests in multiple clinical forms, including bubonic and pneumonic plague. Pneumonic plague is rare, rapidly fatal, and transmissible from person to person via respiratory droplets. On March 4, 2019, a patient with suspected pneumonic plague was hospitalized in West Nile, Uganda, 4 days after caring for her sister, who had come to Uganda from DRC and died shortly thereafter, and 2 days after area officials received a message from a clinic in DRC warning of possible plague. The West Nile-based Uganda Virus Research Institute (UVRI) plague program, together with local health officials, commenced a multipronged response to suspected person-to-person transmission of pneumonic plague, including contact tracing, prophylaxis, and education. Plague was laboratory-confirmed, and no additional transmission occurred in Uganda. This event transpired in the context of heightened awareness of cross-border disease spread caused by ongoing Ebola virus disease transmission in DRC, approximately 400 km to the south. Building expertise in areas of plague endemicity can provide the rapid detection and effective response needed to mitigate epidemic spread and minimize mortality. Cross-border agreements can improve ability to respond effectively.

Francisella opportunistica sp. nov., isolated from human blood and cerebrospinal fluid.

Two isolates of a Gram-negative, non-spore-forming coccobacillus cultured from the blood and cerebrospinal fluid of immunocompromised patients in the United States were described previously. Biochemical and phylogenetic analyses revealed that they belong to a novel species within the Francisella genus. Here we describe a third isolate of this species, recovered from blood of a febrile patient with renal failure, and formally name the Francisella species. Whole genome comparisons indicated the three isolates display greater than 99.9 % average nucleotide identity (ANI) to each other and are most closely related to the tick endosymbiont F. persica, with only 88.6-88.8 % ANI to the type strain of F. persica. Based on biochemical, metabolic and genomic comparisons, we propose that these three isolates should be recognized as Francisella opportunistica sp. nov, with the type strain of the species, PA05-1188T, available through the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 107100) and the American Type Culture Collection (ATCC BAA-2974).

Francisella tularensis Transmission by Solid Organ Transplantation, 20171.

In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor's residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation.

Updated CDC Recommendation for Serologic Diagnosis of Lyme Disease.

Lyme disease is a tickborne zoonosis for which serologic testing is the principal means of laboratory diagnosis. In 1994, the Association of State and Territorial Public Health Laboratory Directors, CDC, the Food and Drug Administration (FDA), the National Institutes of Health (NIH), the Council of State and Territorial Epidemiologists, and the National Committee for Clinical Laboratory Standards convened the Second National Conference on Serologic Diagnosis of Lyme Disease (1).

Surveillance for and Discovery of Borrelia Species in US Patients Suspected of Tickborne Illness.

Background: Tick-transmitted Borrelia fall into 2 heterogeneous bacterial complexes comprised of multiple species, the relapsing fever (RF) group and the Borrelia burgdorferi sensu lato group, which are the causative agents of Lyme borreliosis (LB), the most common tickborne disease in the Northern Hemisphere. Geographic expansion of LB in the United States and discovery of emerging Borrelia pathogens underscores the importance of surveillance for disease-causing Borrelia. Methods: De-identified clinical specimens, submitted by providers throughout the United States, for patients suspected of LB, anaplasmosis, ehrlichiosis, or babesiosis were screened using a Borrelia genus-level TaqMan polymerase chain reaction (PCR). Borrelia species and sequence types (STs) were characterized by multilocus sequence typing (MLST) utilizing next-generation sequencing. Results: Among 7292 specimens tested, 5 Borrelia species were identified: 2 causing LB, B. burgdorferi (n = 25) and B. mayonii (n = 9), and 3 RF borreliae, B. hermsii (n = 1), B. miyamotoi (n = 8), and Candidatus B. johnsonii (n = 1), a species previously detected only in the bat tick, Carios kelleyi. ST diversity was greatest for B. burgdorferi-positive specimens, with new STs identified primarily among synovial fluids. Conclusions: These results demonstrate that broad PCR screening followed by MLST is a powerful surveillance tool for uncovering the spectrum of disease-causing Borrelia species, understanding their geographic distribution, and investigating the correlation between B. burgdorferi STs and joint involvement. Detection of Candidatus B. johnsonii in a patient with suspected tickborne disease suggests this species may be a previously undetected cause of illness in humans exposed to bat ticks.

A Bead-Based Flow Cytometric Assay for Monitoring Yersinia pestis Exposure in Wildlife.

Yersinia pestis is the causative agent of plague and is considered a category A priority pathogen due to its potential for high transmissibility and the significant morbidity and mortality it causes in humans. Y. pestis is endemic to the western United States and much of the world, necessitating programs to monitor for this pathogen on the landscape. Elevated human risk of plague infection has been spatially correlated with spikes in seropositive wildlife numbers, particularly rodent-eating carnivores, which are frequently in contact with the enzootic hosts and the associated arthropod vectors of Y. pestis In this study, we describe a semiautomated bead-based flow cytometric assay developed for plague monitoring in wildlife called the F1 Luminex plague assay (F1-LPA). Based upon Luminex/Bio-Plex technology, the F1-LPA targets serological responses to the F1 capsular antigen of Y. pestis and was optimized to analyze antibodies eluted from wildlife blood samples preserved on Nobuto filter paper strips. In comparative evaluations with passive hemagglutination, the gold standard tool for wildlife plague serodiagnosis, the F1-LPA demonstrated as much as 64× improvement in analytical sensitivity for F1-specific IgG detection and allowed for unambiguous classification of IgG status. The functionality of the F1-LPA was demonstrated for coyotes and other canids, which are the primary sentinels in wildlife plague monitoring, as well as felids and raccoons. Additionally, assay formats that do not require species-specific immunological reagents, which are not routinely available for several wildlife species used in plague monitoring, were determined to be functional in the F1-LPA.

Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection.

Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features-for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE: DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

Investigation of and Response to 2 Plague Cases, Yosemite National Park, California, USA, 2015.

In August 2015, plague was diagnosed for 2 persons who had visited Yosemite National Park in California, USA. One case was septicemic and the other bubonic. Subsequent environmental investigation identified probable locations of exposure for each patient and evidence of epizootic plague in other areas of the park. Transmission of Yersinia pestis was detected by testing rodent serum, fleas, and rodent carcasses. The environmental investigation and whole-genome multilocus sequence typing of Y. pestis isolates from the patients and environmental samples indicated that the patients had been exposed in different locations and that at least 2 distinct strains of Y. pestis were circulating among vector-host populations in the area. Public education efforts and insecticide applications in select areas to control rodent fleas probably reduced the risk for plague transmission to park visitors and staff.

Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus.

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus.

Borrelia mayonii sp. nov., a member of the Borrelia burgdorferi sensu lato complex, detected in patients and ticks in the upper midwestern United States.

Lyme borreliosis (LB) is a multisystem disease caused by spirochetes in the Borrelia burgdorferisensu lato (Bbsl) genospecies complex. We previously described a novel Bbsl genospecies (type strain MN14-1420T) that causes LB among patients with exposures to ticks in the upper midwestern USA. Patients infected with the novel Bbsl genospecies demonstrated higher levels of spirochetemia and somewhat differing clinical symptoms as compared with those infected with other Bbsl genospecies. The organism was detected from human specimens using PCR, microscopy, serology and culture. The taxonomic status was determined using an eight-housekeeping-gene (uvrA, rplB, recG, pyrG, pepX, clpX, clpA and nifS) multi-locus sequence analysis (MLSA) and comparison of 16S rRNA gene, flaB, rrf-rrl, ospC and oppA2 nucleotide sequences. Using a system threshold of 98.3 % similarity for delineation of Bbsl genospecies by MLSA, we demonstrated that the novel species is a member of the Bbsl genospecies complex, most closely related to B. burgdorferisensu stricto (94.7-94.9 % similarity). This same species was identified in Ixodes scapularis ticks collected in Minnesota and Wisconsin. This novel species, Borrelia mayonii sp. nov, is formally described here. The type strain, MN14-1420, is available through the Deutsche Sammlung von Mikroorganismen und Zelkulturen GmbH (DSM 102811) and the American Type Culture Collection (ATCC BAA-2743).

Notes from the Field: Increase in Human Cases of Tularemia--Colorado, Nebraska, South Dakota, and Wyoming, January-September 2015.

Tularemia is a rare, often serious disease caused by a gram-negative coccobacillus, Francisella tularensis, which infects humans and animals in the Northern Hemisphere. Approximately 125 cases have been reported annually in the United States during the last two decades. As of September 30, a total of 100 tularemia cases were reported in 2015 among residents of Colorado (n = 43), Nebraska (n = 21), South Dakota (n = 20), and Wyoming (n = 16) (Figure). This represents a substantial increase in the annual mean number of four (975% increase), seven (200%), seven (186%) and two (70%) cases, respectively, reported in each state during 2004-2014.

Outbreak of Human Pneumonic Plague with Dog-to-Human and Possible Human-to-Human Transmission--Colorado, June-July 2014.

On July 8, 2014, the Colorado Department of Public Health and Environment (CDPHE) laboratory identified Yersinia pestis, the bacterium that causes plague, in a blood specimen collected from a man (patient A) hospitalized with pneumonia. The organism had been previously misidentified as Pseudomonas luteola by an automated system in the hospital laboratory. An investigation led by Tri-County Health Department (TCHD) revealed that patient A's dog had died recently with hemoptysis. Three other persons who had contact with the dog, one of whom also had contact with patient A, were ill with fever and respiratory symptoms, including two with radiographic evidence of pneumonia. Specimens from the dog and all three human contacts yielded evidence of acute Y. pestis infection. One of the pneumonia cases might have resulted through human-to-human transmission from patient A, which would be the first such event reported in the United States since 1924. This outbreak highlights 1) the need to consider plague in the differential diagnosis of ill domestic animals, including dogs, in areas where plague is endemic; 2) the limitations of automated diagnostic systems for identifying rare bacteria such as Y. pestis; and 3) the potential for milder plague illness in patients taking antimicrobial agents. Hospital laboratorians should be aware of the limitations of automated identification systems, and clinicians should suspect plague in patients with clinically compatible symptoms from whom P. luteola is isolated.

Human Plague - United States, 2015.

Since April 1, 2015, a total of 11 cases of human plague have been reported in residents of six states: Arizona (two), California (one), Colorado (four), Georgia (one), New Mexico (two), and Oregon (one). The two cases in Georgia and California residents have been linked to exposures at or near Yosemite National Park in the southern Sierra Nevada Mountains of California. Nine of the 11 patients were male; median age was 52 years (range = 14-79 years). Three patients aged 16, 52, and 79 years died.