Health care resource utilization and costs associated with atrial fibrillation and rural-urban disparities.

BACKGROUND: Atrial fibrillation (AF) imposes substantial health care and economic burden on health care systems and patients. Previous studies failed to examine health care resource utilization (HCRU) and costs among patients with incident AF and potential disparity with regard to geographic location. OBJECTIVES: To examine HCRU and costs among patients with incident AF compared with patients without AF and examine whether a geographic disparity exists. METHODS: This was a retrospective cohort study. We selected patients with AF and patients without AF from IBM/Watson MarketScan Research Databases 2014-2019. HCRU and costs were collected 12 months following an AF index date. We used 2-part models with bootstrapping to obtain the marginal estimates and CIs. Rural status was identified based on Metropolitan Statistical Area. We adjusted for age, sex, plan type, US region, and comorbidities. RESULTS: Among 156,732 patients with AF and 3,398,490 patients without AF, patients with AF had 9.04 (95% CI = 8.96-9.12) more outpatient visits, 0.82 (95% CI = 0.81-0.83) more emergency department (ED) visits, 0.33 (95% CI = 0.33-0.34) more inpatient admission, and $15,095 (95% CI = 14,871-15,324) higher total costs, compared with patients without AF. Among patients with AF, rural patients had 1.99 fewer (95% CI = -2.26 to -1.71) outpatient visits and 0.05 (95% CI = 0.02-0.08) more ED visits than urban patients. Overall, rural patients with AF had decreased total costs compared with urban patients (mean = $751; 95% CI = -1,227 to -228). CONCLUSIONS: Incident AF was associated with substantial burden of health care resources and an economic burden, and the burden was not equally distributed across patients in urban vs rural settings. DISCLOSURES: Dr Hansen reports grants from the National Science Foundation during the conduct of the study.

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.

Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction.

OBJECTIVES: Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. METHODS: FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. RESULTS: FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. CONCLUSIONS: While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.

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.

Proposal to reclassify Ehrlichia muris as Ehrlichia muris subsp. muris subsp. nov. and description of Ehrlichia muris subsp. eauclairensis subsp. nov., a newly recognized tick-borne pathogen of humans.

We have previously described a novel taxon of the genus Ehrlichia (type strain WisconsinT), closely related to Ehrlichia muris, that causes human ehrlichiosis among patients with exposures to ticks in the upper midwestern USA. DNA from this bacterium was also detected in Ixodes scapularis and Peromyscus leucopus collected in Minnesota and Wisconsin. To determine the relationship between the E. muris-like agent (EMLA) and other species of the genus Ehrlichia phenotypic, genotypic and epidemiologic comparisons were undertaken, including sequence analysis of eight gene loci (3906 nucleotides) for 39 EMLA DNA samples and the type strain of E. muris AS145T. Three loci were also sequenced from DNA of nine strains of E. muris from mouse spleens from Japan. All sequences from E. muris were distinct from homologous EMLA sequences, but differences between them were less than those observed among other species of the genus Ehrlichia. Phenotypic comparison of EMLA and E. muris revealed similar culture and electron microscopic characteristics, but important differences were noted in their geographic distribution, ecological associations and behavior in mouse models of infection. Based on these comparisons, we propose that type strain WisconsinT represents a novel subspecies, Ehrlichia murissubsp. eauclairensis,subsp. nov. This strain is available through the Centers for Disease Control and Prevention Rickettsial Isolate Reference Collection (CRIRC EMU002T) and through the Collection de Souches de l'Unité des Rickettsies (CSURP2883 T). The subspecies Ehrlichia murissubsp. muris subsp. nov. is automatically created and the type strain AS145T is also available through the same collections (CRIRC EMU001T, CSUR E2T). Included is an emended description of E. muris.

Possible Transfusion-Transmitted Babesia divergens-like/MO-1 Infection in an Arkansas Patient.

A patient with asplenia and multiple red blood cell transfusions acquired babesiosis infection with Babesia divergens-like/MO-1 organisms and not Babesia microti, the common United States species. He had no known tick exposure. This is believed to be the first transfusion-transmitted case and the fifth documented case of B. divergens-like/MO-1 infection.

Thermal Contrast Amplification Reader Yielding 8-Fold Analytical Improvement for Disease Detection with Lateral Flow Assays.

There is an increasing need for highly sensitive and quantitative diagnostics at the point-of-care. The lateral flow immunoassay (LFA) is one of the most widely used point-of-care diagnostic tests; however, LFAs generally suffer from low sensitivity and lack of quantification. To overcome these limitations, thermal contrast amplification (TCA) is a new method that is based on the laser excitation of gold nanoparticles (GNPs), the most commonly used visual signature, to evoke a thermal signature. To facilitate the clinical translation of the TCA technology, we present the development of a TCA reader, a platform technology that significantly improves the limit of detection and provides quantification of disease antigens in LFAs. This TCA reader provides enhanced sensitivity over visual detection by the human eye or by a colorimetric reader (e.g., BD Veritor System Reader). More specifically, the TCA reader demonstrated up to an 8-fold enhanced analytical sensitivity and quantification among LFAs for influenza, malaria, and Clostridium difficile. Systematic characterization of the laser, infrared camera, and other components of the reader and their integration into a working reader instrument are described. The development of the TCA reader enables simple, highly sensitive quantification of LFAs at the point-of-care.

A global map of genetic diversity in Babesia microti reveals strong population structure and identifies variants associated with clinical relapse.

Human babesiosis caused by Babesia microti is an emerging tick-borne zoonosis of increasing importance due to its rising incidence and expanding geographic range(1). Infection with this organism, an intraerythrocytic parasite of the phylum Apicomplexa, causes a febrile syndrome similar to malaria(2). Relapsing disease is common among immunocompromised and asplenic individuals(3,4) and drug resistance has recently been reported(5). To investigate the origin and genetic diversity of this parasite, we sequenced the complete genomes of 42 B. microti samples from around the world, including deep coverage of clinical infections at endemic sites in the continental USA. Samples from the continental USA segregate into a Northeast lineage and a Midwest lineage, with subsequent divergence of subpopulations along geographic lines. We identify parasite variants that associate with relapsing disease, including amino acid substitutions in the atovaquone-binding regions of cytochrome b (cytb) and the azithromycin-binding region of ribosomal protein subunit L4 (rpl4). Our results shed light on the origin, diversity and evolution of B. microti, suggest possible mechanisms for clinical relapse, and create the foundation for further research on this emerging pathogen.

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).

Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study.

BACKGROUND: Lyme borreliosis is the most common tick-borne disease in the northern hemisphere. It is a multisystem disease caused by Borrelia burgdorferi sensu lato genospecies and characterised by tissue localisation and low spirochaetaemia. In this study we aimed to describe a novel Borrelia species causing Lyme borreliosis in the USA. METHODS: At the Mayo clinic, from 2003 to 2014, we tested routine clinical diagnostic specimens from patients in the USA with PCR targeting the oppA1 gene of B burgdorferi sensu lato. We identified positive specimens with an atypical PCR result (melting temperature outside of the expected range) by sequencing, microscopy, or culture. We collected Ixodes scapularis ticks from regions of suspected patient tick exposure and tested them by oppA1 PCR. FINDINGS: 100 545 specimens were submitted by physicians for routine PCR from Jan 1, 2003 to Sept 30, 2014. From these samples, six clinical specimens (five blood, one synovial fluid) yielded an atypical oppA1 PCR product, but no atypical results were detected before 2012. Five of the six patients with atypical PCR results had presented with fever, four had diffuse or focal rash, three had symptoms suggestive of neurological inclusion, and two were admitted to hospital. The sixth patient presented with knee pain and swelling. Motile spirochaetes were seen in blood samples from one patient and cultured from blood samples from two patients. Among the five blood specimens, the median oppA1 copy number was 180 times higher than that in 13 specimens that tested positive for B burgdorferi sensu stricto during the same time period. Multigene sequencing identified the spirochaete as a novel B burgdorferi sensu lato genospecies. This same genospecies was detected in ticks collected at a probable patient exposure site. INTERPRETATION: We describe a new pathogenic Borrelia burgdorferi sensu lato genospecies (candidatus Borrelia mayonii) in the upper midwestern USA, which causes Lyme borreliosis with unusually high spirochaetaemia. Clinicians should be aware of this new B burgdorferi sensu lato genospecies, its distinct clinical features, and the usefulness of oppA1 PCR for diagnosis. FUNDING: US Centers for Disease Control and Prevention Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) Cooperative Agreement and Mayo Clinic Small Grant programme.

A multi-laboratory comparison of two molecular methods for the detection of toxigenic Clostridium difficile.

INTRODUCTION: Diarrheal disease due to toxigenic Clostridium difficile (CD) accounts for an increased number of hospitalizations and deaths each year. Published guidelines recommend reflex testing of CD antigen-positive samples to molecular testing or testing samples directly by a molecular assay. This multicenter study was designed to compare the accuracy of two different molecular methods targeting different CD genes: Xpert C. difficile Epi RUO RT-PCR assay (XPCR) which targets toxin B (Cepheid, Sunnyvale, CA) and a laboratory-developed PCR (LDPCR) which targets mutations in the tcdC regulatory gene. METHODOLOGY: Two molecular methods for toxigenic CD detection, the Xpert C. difficile Epi RUO RT-PCR assay (XPCR) [Cepheid, Sunnyvale, CA] and a laboratory-developed PCR assay (LDPCR) were compared to a consensus gold standard (CGS) or toxigenic culture (TC) as the reference method. A subset of specimens was subjected to additional molecular characterization of toxigenic CD. RESULTS: Both molecular methods were >90% sensitive for CD detection. Discordant results were noted when molecular test results were compared to non-molecular methods. Supplemental molecular characterization illustrated inherent difficulties in comparisons using different molecular methods for CD. CONCLUSION: Laboratories may consider using multiple CD detection methods or combinations of methods, including molecular detection for rapid and accurate diagnosis of CD, as driven by best practices for the respective healthcare environment. Laboratories must be aware of intrinsic differences when comparing performance characteristics of different molecular assays.

A real-time PCR assay for detection of the Ehrlichia muris-like agent, a newly recognized pathogen of humans in the upper Midwestern United States.

The Ehrlichia muris-like agent (EMLA) is an emerging, tick-transmitted human pathogen that occurs in the upper Midwestern United States. Here, we describe the development and validation of a p13-based quantitative real-time PCR TaqMan assay to detect EMLA in blood or tissues of ticks, humans, and rodents. The primer and probe specificities of the assay were ascertained using a large panel of various Ehrlichia species and other members of Rickettsiales. In addition to control DNA, both non-infected and EMLA-infected human blood, Mus musculus blood, and M. musculus tissue extracts were evaluated, as were non-infected and EMLA-infected Ixodes scapularis and uninfected Dermacentor variabilis DNA lysates. The specificity of the probe was determined via real-time PCR. An EMLA p13 control plasmid was constructed, and serial dilutions were used to determine the analytical sensitivity, which was found to be 1 copy per 4µl of template DNA. The sensitivity and specificity of this assay provides a powerful tool for ecological studies involving arthropod vectors and their mammalian hosts.

Human Infection with Ehrlichia muris-like Pathogen, United States, 2007-2013(1).

An Ehrlichia muris-like (EML) pathogen was detected among 4 patients in Minnesota and Wisconsin during 2009. We characterized additional cases clinically and epidemiologically. During 2004-2013, blood samples from 75,077 patients from all 50 United States were tested by PCR from the groEL gene for Ehrlichia spp. and Anaplasma phagocytophilum. During 2007-2013, samples from 69 (0.1%) patients were positive for the EML pathogen; patients were from 5 states: Indiana (1), Michigan (1), Minnesota (33), North Dakota (3), and Wisconsin (31). Most (64%) patients were male; median age was 63 (range 15-94) years; and all 69 patients reported likely tick exposure in Minnesota or Wisconsin. Fever, malaise, thrombocytopenia, and lymphopenia were the most common symptoms. Sixteen (23%) patients were hospitalized (median 4 days); all recovered, and 96% received doxycycline. Infection with the EML pathogen should be considered for persons reporting tick exposure in Minnesota or Wisconsin.

Detection of human pathogenic Ehrlichia muris-like agent in Peromyscus leucopus.

An Ehrlichia muris-like (EML) bacterium was recently detected in humans and Ixodes scapularis ticks in Minnesota and Wisconsin. The reservoir for this agent is unknown. To investigate the occurrence of the EML agent, groEL PCR testing and sequencing was performed on blood from small mammals and white-tailed deer that were collected in areas where human and tick infections were previously demonstrated. DNA of the EML agent was detected in two Peromyscus leucopus of 146 small mammals (1.4%); while 181 O. virginianus tested negative. This report provides the first evidence that DNA from the EML agent is found in P. leucopus, the same animal that is a reservoir for Anaplasma phagocytophilum in this region. The role of white-tailed deer remains inconclusive. Further sampling is warranted to understand the spatial and temporal distribution, transmission and maintenance of this pathogen.

Comparison of phenology and pathogen prevalence, including infection with the Ehrlichia muris-like (EML) agent, of Ixodes scapularis removed from soldiers in the midwestern and the northeastern United States over a 15 year period (1997-2012).

BACKGROUND: Since 1997, human-biting ticks submitted to the Department of Defense Human Tick Test Kit Program (HTTKP) of the US Army Public Health Command have been tested for pathogens by PCR. We noted differences in the phenology and infection prevalence among Ixodes scapularis ticks submitted from military installations in different geographic regions. The aim of this study was to characterize these observed differences, comparing the phenology and pathogen infection rates of I. scapularis submitted from soldiers at two sites in the upper Midwest (Camp Ripley, MN, and Ft. McCoy, WI) and one site in the northeastern US (Ft. Indiantown Gap, PA). METHODS: From 1997 through 2012, the HTTKP received 1,981 I. scapularis from the three installations and tested them for Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi and the Ehrlichia muris-like (EML) agent using PCR; pathogen presence was confirmed via sequencing or amplification of a second gene target. Pathogen and co-infection prevalence, tick engorgement status, and phenology were compared among installations. RESULTS: Greater rates of A. phagocytophilum and Ba. microti infections were detected in ticks submitted from installations in Minnesota than in Wisconsin or Pennsylvania, and the EML agent was only detected in ticks from Minnesota and Wisconsin. Midwestern ticks were also more likely to be co-infected than those from Pennsylvania. Both adult and nymphal ticks showed evidence of feeding on people, although nymphs were more often submitted engorged. Adult I. scapularis were received more frequently in June from Minnesota than from either of the other sites. Minnesota adult and nymphal peaks overlapped in June, and submissions of adults exceeded nymphs in that month. CONCLUSIONS: There were clear differences in I. scapularis phenology, pathogen prevalence and rates of co-infection among the three military installations. Seasonal and temperature differences between the three sites and length of time a population had been established in each region may contribute to the observed differences. The synchrony of adults and nymphs observed in the upper Midwest has implications for pathogen infection prevalence. The EML agent was only detected in Minnesota and Wisconsin, supporting the previous assertion that this pathogen is currently limited to the upper Midwest.

First reported case of Ehrlichia ewingii involving human bone marrow.

A 65-year-old female with a history of multiple tick bites presented with fever and pancytopenia. Intracytoplasmic rickettsial morulae were detected on peripheral smear and bone marrow biopsy specimens, and PCR amplified Ehrlichia ewingii DNA from both specimens. To our knowledge, this is the first report of E. ewingii infection of human bone marrow.

Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens.

The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples (n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 µl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 µl raw stool; however, 100 µl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

Cutaneous manifestations of a zoonotic Onchocerca species in an adult male, acquired in Nova Scotia, Canada.

A 65-year-old male with known hypertension and hypercholesterolemia sought medical attention because of a 3-month history of skin swelling on his upper back. Histopathology and molecular techniques were employed and identified an organism in the Onchocerca genus. This represents a very uncommon example of cutaneous infection by a zoonotic Onchocerca species.

Real-time PCR detection of Mycoplasma pneumoniae in respiratory specimens.

A real-time PCR assay targeting the phosphotransferase system I gene (ptsI) of Mycoplasma pneumoniae was compared to 2 commercially available PCR assays targeting the P1 cytadhesion gene (the LightMix®Kit Mycoplasma pneumoniae [TIB MOLBIOL, Adelphia, NJ, USA] and M. pneumoniae Analyte Specific Reagent [Focus Diagnostics, Cypress, CA, USA] assays) and to a PCR assay targeting the M. pneumoniae repetitive element, RepMP1. Thirty previously positive specimens including 15 throat swab, 10 bronchoalveolar lavage, and 5 sputum specimens, all tested positive with the ptsI and M. pneumoniae ASR assays. Among the previously positive specimens, 14/15 throat swab, 9/10 bronchoalveolar lavage, and 4/5 sputum specimens tested positive with the LightMix®Kit Mycoplasma pneumoniae assay and 13/15 throat swab, 10/10 bronchoalveolar lavage, and 4/5 sputum specimens tested positive with the RepMP1 assay. Forty previously negative clinical specimens tested negative using the ptsI assay. A PCR assay targeting M. pneumoniae ptsI performs equivalently to assays targeting the P1 cytadhesion gene or RepMP1.