Martin E. Schriefer

Serodiagnosis of Lyme Disease by Kinetic Enzyme-Linked Immunosorbent Assay Using Recombinant VlsE1 or Peptide Antigens of Borrelia burgdorferi Compared with 2-Tiered Testing Using Whole-Cell Lysates

Abstract In a study of US patients with Lyme disease, immunoglobulin (Ig) G and IgM antibody responses to recombinant Borrelia burgdorferi antigen VlsE1 (rVlsE1), IgG responses to a synthetic peptide homologous to a conserved internal sequence of VlsE (C6), and IgM responses to a synthetic peptide comprising the C-terminal 10 amino acid residues of a B. burgdorferi outer-surface protein C (pepC10) were evaluated by kinetic enzyme-linked immunoassay. At 99% specificity, the overall sensitivities for detecting IgG antibody to rVlsE1 or C6 in samples from patients with diverse manifestations of Lyme disease were equivalent to that of 2-tiered testing. When data were considered in parallel, 2 combinations (IgG responses to either rVlsE1 or C6 in parallel with IgM responses to pepC10) maintained high specificity (98%) and were significantly more sensitive than 2-tiered analysis in detecting antibodies to B. burgdorferi in patients with acute erythema migrans. In later stages of Lyme disease, the sensitivities of the in parallel tests and 2-tiered testing were high and statistically equivalent

Francisella tularensis: an arthropod-borne pathogen.

Arthropod transmission of tularemia occurs throughout the northern hemisphere. Few pathogens show the adaptability of Francisella tularensis to such a wide array of arthropod vectors. Nonetheless, arthropod transmission of F. tularensis was last actively investigated in the first half of the 20th century. This review will focus on arthropod transmission to humans with respect to vector species, modes of transmission, geographic differences and F. tularensis subspecies and clades.

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.

Clinical and Epidemiological Features of Early Lyme Disease and Human Granulocytic Ehrlichiosis in Wisconsin

To compare clinical features and assess risk factors for human granulocytic ehrlichiosis (HGE) and early Lyme disease, we enrolled patients in a case-control study during the 1996 and 1997 tick seasons. Clinical and demographic characteristics were assessed for patients with laboratory-confirmed cases of HGE or Lyme disease, and risk factors were compared with those of matched control subjects. We identified 83 persons with Lyme disease, 27 with HGE, and 11 with apparent coinfection. Unsuspected Ehrlichia infection was identified in 8 (13%) of 60 patients with Lyme disease. Patients with HGE were older and more likely to have fever, chills, or dyspnea than were those with Lyme disease only. Most patients with apparent coinfection did not have hematologic abnormalities. In the risk factor analysis, tickborne illness was independently associated with rural residence and camping. The clinical spectrum of HGE overlaps that of Lyme disease, and physicians in areas of endemicity should consider both diseases in treating patients with a compatible rash or febrile illness.

Single-tier testing with the C6 peptide ELISA kit compared with two-tier testing for Lyme disease

For the diagnosis of Lyme disease, the 2-tier serologic testing protocol for Lyme disease has a number of shortcomings including low sensitivity in early disease; increased cost, time, and labor; and subjectivity in the interpretation of immunoblots. In this study, the diagnostic accuracy of a single-tier commercial C6 ELISA kit was compared with 2-tier testing. The results showed that the C6 ELISA was significantly more sensitive than 2-tier testing with sensitivities of 66.5% (95% confidence interval [CI] 61.7-71.1) and 35.2% (95% CI 30.6-40.1), respectively (P < 0.001) in 403 sera from patients with erythema migrans. The C6 ELISA had sensitivity statistically comparable to 2-tier testing in sera from Lyme disease patients with early neurologic manifestations (88.6% versus 77.3%, P = 0.13) or arthritis (98.3% versus 95.6%, P = 0.38). The specificities of C6 ELISA and 2-tier testing in over 2200 blood donors, patients with other conditions, and Lyme disease vaccine recipients were found to be 98.9% and 99.5%, respectively (P < 0.05, 95% CI surrounding the 0.6 percentage point difference of 0.04 to 1.15). In conclusion, using a reference standard of 2-tier testing, the C6 ELISA as a single-step serodiagnostic test provided increased sensitivity in early Lyme disease with comparable sensitivity in later manifestations of Lyme disease. The C6 ELISA had slightly decreased specificity. Future studies should evaluate the performance of the C6 ELISA compared with 2-tier testing in routine clinical practice.

Detection of Rickettsia, Borrelia, and Bartonella in Carios kelleyi (Acari: Argasidae)

Abstract Carios kelleyi (Colley & Kohls 1941), a tick associated with bats and bat habitats, has been reported to feed on humans, but there is little published data regarding the presence of vector-borne pathogens in these ticks. C. kelleyi nymphs and adults were collected from residential and community buildings in Jackson County, Iowa, and tested by polymerase chain reaction for Rickettsia, Borrelia, Bartonella, Coxiella, and Anaplasma. Rickettsia DNA was detected in 28 of 31 live ticks. Sequences of the 17-kDa and rOmpA genes suggest that this agent is a novel spotted fever group Rickettsia. Transstadial and transovarial transmission of this Rickettsia were demonstrated. The flagellin gene of a Borrelia, closely related to B. turicatae, was detected in one of 31 live ticks. The 16S-23S intergenic spacer region of Bartonella henselae also was detected in one of 31 live ticks. Coxiella or A. phagocytophilum DNA were not detected in these ticks.

Persistence of Yersinia pestis in Soil Under Natural Conditions

As part of a fatal human plague case investigation, we showed that the plague bacterium, Yersinia pestis, can survive for at least 24 days in contaminated soil under natural conditions. These results have implications for defining plague foci, persistence, transmission, and bioremediation after a natural or intentional exposure to Y. pestis.

Wide Distribution of a High-Virulence Borrelia burgdorferi Clone in Europe and North America

We found substantial population differentiation and recent trans-Atlantic dispersal of a high-virulence B. burgdorferi clone.

Seroepidemiology of Emerging Tickborne Infectious Diseases in a Northern California Community

A seroprevalence and risk factor study of emerging tickborne infectious diseases (Lyme disease, ehrlichiosis, and babesiosis) was conducted among 230 residents of a semirural community in Sonoma County, California. Over 50% of residents reported finding a tick on themselves in the preceding 12 months. Samples from 51(23%) residents were seroreactive to antigens from one or more tickborne disease agents: 1.4% to Borrelia burgdorferi, 0.4% to Ehrlichia equi, 4.6% to Ehrlichia chaffeensis, and 17.8% to the Babesia-like piroplasm WA1. Only 14 (27%) of these seroreactive residents reported one or more symptoms compatible with these diseases. Seroreactivity was significantly associated with younger age (<16 years), longer residence in the community (11-20 years), and having had a physicians diagnosis of Lyme disease. In northern California, the risk of infection with these emerging tickborne diseases, particularly in children, may be greater than previously recognized.

Whole genome single nucleotide polymorphism based phylogeny of Francisella tularensis and its application to the development of a strain typing assay

BackgroundA low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.ResultsWe have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).ConclusionWhole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.