Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B. burgdorferi (Bb) isolates derived from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bb isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bb isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ~900 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination in humans and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, have increased rates of dissemination in humans. OspC type A strains possess a unique set of strongly linked genetic elements including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. These features of OspC type A strains reflect a broader paradigm across Bb isolates, in which near-clonal genotypes are defined by strain-specific clusters of linked genetic elements, particularly those encoding surface-exposed lipoproteins. These clusters of genes are maintained by strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Actionable diagnosis of neuroleptospirosis by next-generation sequencing.

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Principles and Pitfalls: a Guide to Death Certification.

Death certificates serve the critical functions of providing documentation for legal/administrative purposes and vital statistics for epidemiologic/health policy purposes. In order to satisfy these functions, it is important that death certificates be filled out completely, accurately, and promptly. The high error rate in death certification has been documented in multiple prior studies, as has the effectiveness of educational training interventions at mitigating errors. The following guide to death certification is intended to illustrate some basic principles and common pitfalls in electronic death registration with the goal of improving death certification accuracy.

Canine blastomycosis in Wisconsin: a survey of small-animal veterinary practices.

The disease burden and impact of canine blastomycosis in Wisconsin is uncertain. We surveyed small-animal veterinary practices to obtain estimates of disease incidence, determine patient outcomes, and investigate variation in diagnostic and treatment strategies used by veterinarians. Veterinarians representing small-animal practices in Wisconsin were contacted by mail with the option to complete a paper or online questionnaire. Questionnaires were returned from 68 of 443 veterinary practices (15%) that estimated diagnosing 239 cases of canine blastomycosis annually, with an overall mortality of 36%. Annual incidence rates of canine blastomycosis were calculated for 43 individual veterinary clinics and differed significantly between clinics in endemic and nonendemic counties (P = 0.01), with the mean in endemic counties being 204/100,000/yr and nonendemic counties being 72/100,000/yr. Veterinarians reported an increase in canine blastomycosis cases from April through August. A wide variety of methods were used for diagnosis, ranging from clinical signs alone to antigen testing and "in-house" cytology. Of note, fungal culture was used rarely for diagnosis. In addition, veterinarians at these 68 clinics estimated diagnosing 36 cases of feline blastomycosis annually. The incidence of canine blastomycosis is high but quite variable among veterinary practices in Wisconsin. Diagnosis is based frequently on clinical signs exclusively due, in part, to the perceived high cost of laboratory tests. Similarly, the mortality associated with blastomycosis is likely negatively impacted because some dog owners defer therapy due to the cost of antifungal drugs.

Variation in clinical phenotype of human infection among genetic groups of Blastomyces dermatitidis.

BACKGROUND: Blastomyces dermatitidis, the etiologic agent of blastomycosis, has 2 genetic groups and shows varied clinical presentation, ranging from silent infections to fulminant respiratory disease and dissemination. The objective of this study was to determine whether clinical phenotype and outcomes vary based on the infecting organism's genetic group. METHODS: We used microsatellites to genotype 227 clinical isolates of B. dermatitidis from Wisconsin patients. For each isolate, corresponding clinical disease characteristics and patient demographic information were abstracted from electronic health records and Wisconsin Division of Health reportable disease forms and questionnaires. RESULTS: In univariate analysis, group 1 isolates were more likely to be associated with pulmonary-only infections (P < .0001) and constitutional symptoms such as fever (P < .0001). In contrast, group 2 isolates were more likely to be associated with disseminated disease (P < .0001), older patient age (P < .0001), and comorbidities (P = .0019). In multivariate analysis, disease onset to diagnosis of >1 month (P < .0001), older age at diagnosis (P < .0001), and current smoking status (P = .0001) remained predictors for group 2 infections. CONCLUSIONS: This study identified previously unknown associations between clinical phenotype of human infection and genetic groups of B. dermatitidis and provides a framework for further investigations of the genetic basis for virulence in B. dermatitidis.

Whole genome sequencing of Borrelia burgdorferi isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived B. burgdorferi sensu stricto ( Bbss ) isolates from patients in the Eastern and Midwestern US and Central Europe. We develop a WGS-based classification of Bbss isolates, confirm and extend the findings of previous single- and multi-locus typing systems, define the plasmid profiles of human-infectious Bbss isolates, annotate the core and strain-variable surface lipoproteome, and identify loci associated with disseminated infection. A core genome consisting of ∻800 open reading frames and a core set of plasmids consisting of lp17, lp25, lp36, lp28-3, lp28-4, lp54, and cp26 are found in nearly all isolates. Strain-variable (accessory) plasmids and genes correlate strongly with phylogeny. Using genetic association study methods, we identify an accessory genome signature associated with dissemination and define the individual plasmids and genes that make up this signature. Strains within the RST1/WGS A subgroup, particularly a subset marked by the OspC type A genotype, are associated with increased rates of dissemination. OspC type A strains possess a unique constellation of strongly linked genetic changes including the presence of lp56 and lp28-1 plasmids and a cluster of genes that may contribute to their enhanced virulence compared to other genotypes. The patterns of OspC type A strains typify a broader paradigm across Bbss isolates, in which genetic structure is defined by correlated groups of strain-variable genes located predominantly on plasmids, particularly for expression of surface-exposed lipoproteins. These clusters of genes are inherited in blocks through strain-specific patterns of plasmid occupancy and are associated with the probability of invasive infection.

Rapid response team-triggered procalcitonin measurement predicts infectious intensive care unit transfers*.

OBJECTIVE: Determine if procalcitonin at the time of initial rapid response team activation identifies patients who are likely to need subsequent intensive care unit transfer. DESIGN: Prospective observational cohort study. SETTING: Urban, tertiary care hospital with rapid response team activation through an electronic modified early warning score. PATIENTS: One hundred nineteen oncology and 100 consecutive non-oncology patients after initial rapid response team visit precipitated by an elevated electronic modified early warning score were recruited. Rapid response team activations by request of nursing or for other reasons were not studied. Five oncology patients seen by a rapid response team for complications of interleukin-2 therapeutic infusions were subsequently excluded. INTERVENTIONS: Residual serum from the next ordered clinical test (within 12 hrs) was retrieved, frozen, and stored for procalcitonin determination. A second sample 12-24 hrs after the initial specimen was also retrieved if available and if the patient had not yet been transferred to the intensive care unit. MEASUREMENTS AND MAIN RESULTS: Seventy-three patients (33%) were transferred to the intensive care unit. Rapid response team activations that did not result in intensive care unit transfer had significantly lower procalcitonin levels (median 0.28 ng/mL [interquartile range 0.09-1.24]) than those that resulted in intensive care unit transfer (median 0.51 ng/mL [interquartile range 0.11-1.97], p = .0001) but the area under the receiver operating curve was only 0.656. The change in procalcitonin level in patients with intensive care unit transfers was very heterogeneous but was significantly increased compared to the change in patients not transferred to the intensive care unit. Procalcitonin levels for intensive care unit transfers for probable or definite infection were 2.28 ng/mL [interquartile range 0.68-8.05], and were significantly greater than rapid response team visits that did not result in transfer (p = .0001). The difference between infectious and noninfectious intensive care unit transfers (0.95 ng/mL [interquartile range 0.26-1.89]) was also significant (p = .03). The procalcitonin levels of patients with noninfectious intensive care unit transfers were also different than the levels of patients who never transferred (p = .04). CONCLUSIONS: Preliminary results suggest procalcitonin levels in patients at the time of initial visit by a rapid response team correlate with the need for subsequent intensive care unit transfer, particularly for infectious reasons.

Legionella cardiaca sp. nov., isolated from a case of native valve endocarditis in a human heart.

A Gram-negative, rod-shaped bacterium, designated H63(T), was isolated from aortic valve tissue of a patient with native valve endocarditis. 16S rRNA gene sequencing revealed that H63(T) belongs to the genus Legionella, with its closest neighbours being the type strains of Legionella brunensis (98.8% similarity), L. londiniensis (97.0%), L. jordanis (96.8%), L. erythra (96.2%), L. dresdenensis (96.0%) and L. rubrilucens, L. feeleii, L. pneumophila and L. birminghamensis (95.7%). DNA-DNA hybridization studies yielded values of <70% relatedness between strain H63(T) and its nearest neighbours in terms of 16S rRNA gene sequence similarity, indicating that the strain represents a novel species. Phylogenetic analysis of the 16S rRNA, macrophage infectivity potentiator (mip) and RNase P (rnpB) genes confirmed that H63(T) represents a distinct species, with L. brunensis being its closest sister taxon. Fatty acid composition and biochemical traits, such as the inability to ferment glucose and reduce nitrate, supported the affiliation of H63(T) to the genus Legionella. H63(T) was distinguishable from its neighbours based on it being positive for hippurate hydrolysis. H63(T) was further differentiated by its inability to grow on BCYE agar at 17 °C, its poor growth on low-iron medium and the absence of sliding motility. Also, H63(T) did not react with antisera generated from type strains of Legionella species. H63(T) replicated within macrophages. It also grew in mouse lungs, inducing histopathological evidence of pneumonia and dissemination to the spleen. Together, these results confirm that H63(T) represents a novel, pathogenic Legionella species, for which the name Legionella cardiaca sp. nov. is proposed. The type strain is H63(T) ( = ATCC BAA-2315(T)  = DSM 25049(T)  = JCM 17854(T)).

Native valve endocarditis due to a novel strain of Legionella.

Legionellae are Gram-negative bacteria which are capable of causing disease, most commonly in the form of pneumonia. We describe a case of native valve endocarditis caused by a Legionella strain which by genotypic (16S rRNA and mip gene sequencing) and phenotypic analyses is unlike previously described strains of Legionella.

Population genetic structure of clinical and environmental isolates of Blastomyces dermatitidis, based on 27 polymorphic microsatellite markers.

Blastomyces dermatitidis, a thermally dimorphic fungus, is the etiologic agent of North American blastomycosis. Clinical presentation is varied, ranging from silent infections to fulminant respiratory disease and dissemination to skin and other sites. Exploration of the population genetic structure of B. dermatitidis would improve our knowledge regarding variation in virulence phenotypes, geographic distribution, and difference in host specificity. The objective of this study was to develop and test a panel of microsatellite markers to delineate the population genetic structure within a group of clinical and environmental isolates of B. dermatitidis. We developed 27 microsatellite markers and genotyped B. dermatitidis isolates from various hosts and environmental sources (n=112). Assembly of a neighbor-joining tree of allele-sharing distance revealed two genetically distinct groups, separated by a deep node. Bayesian admixture analysis showed that two populations were statistically supported. Principal coordinate analysis also reinforced support for two genetic groups, with the primary axis explaining 61.41% of the genetic variability. Group 1 isolates average 1.8 alleles/locus, whereas group 2 isolates are highly polymorphic, averaging 8.2 alleles/locus. In this data set, alleles at three loci are unshared between the two groups and appear diagnostic. The mating type of individual isolates was determined by PCR. Both mating type-specific genes, the HMG and α-box domains, were represented in each of the genetic groups, with slightly more isolates having the HMG allele. One interpretation of this study is that the species currently designated B. dermatitidis includes a cryptic subspecies or perhaps a separate species.

Non-rural point source blastomycosis outbreak near a yard waste collection site.

BACKGROUND: Blastomycosis is a potentially fatal infection caused by the fungus Blastomyces dermatitidis. During January 1 through March 5, 2006, twenty-one laboratory confirmed cases of blastomycosis were reported among residents of an endemic area in north-central Wisconsin; a striking increase compared with previous years. The objective of the study was to determine if an observed increase in blastomycosis among residents of an urban area in north-central Wisconsin was caused by a point-source exposure and to identify its source. METHODS: We compared epidemiologic features, and signs and symptoms of B. dermatitidis infection among 46 historic (1999-2005) and 21 possible outbreak case patients. In addition, a case-control study was conducted to compare risk factors of the outbreak case patients with those of 64 age, gender, and geographically-matched control subjects. We conducted site inspections, evaluated meteorological data, genetically compared outbreak and non-outbreak isolates, and attempted environmental detection of B. dermatitidis using polymerase chain reaction, in vitro isolation, and in vivo isolation by tail vein injection of mice. RESULTS: The unusual risk profile of this outbreak included: residence within non-rural city limits with limited time spent outdoors and an equivalent gender ratio and young median age among case patients consistent with common source rather than unrelated exposures. Thirteen of fourteen outbreak-associated clinical isolates of B. dermatitidis clustered in the same genetic group by PCR-RFLP analysis. Inspections near the cluster center suggested a yard waste collection site as the probable exposure source. B. dermatitidis nucleic acid was detected in one of 19 environmental samples. Environmental and meteorological conditions and material management practices were identified that may have facilitated growth and dispersal of B. dermatitidis conidia near this residential area. CONCLUSIONS: Results of our investigation of this large non-rural outbreak of blastomycosis suggest bioaerosol hazards may exist near yard waste collection and composting facilities, especially where pine tree litter is present, in B. dermatitidis endemic areas.

Evolution of northeastern and midwestern Borrelia burgdorferi, United States.

The per capita incidence of human Lyme disease in the northeastern United States is more than twice that in the Midwest. However, the prevalence of Borrelia burgdorferi, the bacterium that causes Lyme disease, in the tick vector is nearly identical in the 2 regions. The disparity in human Lyme disease incidence may result from a disparity in the human invasiveness of the bacteria in the Northeast and Midwest caused by fundamentally different evolutionary histories. B. burgdorferi populations in the Northeast and Midwest are geographically isolated, enabling evolutionary divergence in human invasiveness. However, we found that B. burgdorferi populations in the Northeast and Midwest shared a recent common ancestor, which suggests that substantial evolutionary divergence in human invasiveness has not occurred. We propose that differences in either animal ecology or human behavior are the root cause of the differences in human incidence between the 2 regions.

Virulence genes and genotypic associations in nasal carriage, community-associated methicillin-susceptible and methicillin-resistant USA400 Staphylococcus aureus isolates.

It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically defined clinical CA-MRSA (n = 77), clinical MSSA (n = 103), and nasal carriage MSSA (n = 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identified putative toxin genes from the USA400 lineage of CA-MRSA. Genotypically, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene profiles of the three groups showed that CA-MRSA strains harbored significantly higher percentages (≥95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.

Genetic diversity in Blastomyces dermatitidis: implications for PCR detection in clinical and environmental samples.

Blastomycosis is a serious and potentially fatal infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. Polymerase chain reaction (PCR) assays targeting the BAD-1 virulence gene promoter have been developed to aid in the detection of the pathogen in clinical and environmental samples. However, little is known regarding the genetic diversity of B. dermatitidis and how this might affect the performance characteristics of these assays. We explored the genetic relatedness of 106 clinical and environmental isolates of B. dermatitidis using a previously described rDNA PCR restriction fragment length polymorphism (RFLP) assay. In addition, we looked for polymorphisms in the promoter region upstream of BAD-1. RFLP analysis showed that all isolates fell into one of five genotypic groups, designated A through E. Genotypic groups A and B predominated, comprising 50/106 (47.2%) and 51/106 (48.1%) of isolates, respectively. Three of 106 (2.8%) isolates were genotype C. Genotypes D and E represented novel genotypes and were each associated with single clinical isolates. PCR of the BAD-1 promoter revealed significant size differences among amplification products. Fifty-one of 106 isolates (50/50 RFLP genotypic group A and 1/51 genotypic group B) had amplicons of 663-bp, nearly twice the size of the expected product. Sequence analysis of amplification products from 17 representative isolates revealed four haplotypes and showed that the size disparity was due to two large insertions. Because these insertions were present in a high percentage of isolates, the utility of the PCR assays for diagnostic purposes could be affected. However, the novel RFLP genotypes and multiple BAD-1 haplotypes may prove useful as markers in population genetic studies.

Adapterama I: universal stubs and primers for 384 unique dual-indexed or 147,456 combinatorially-indexed Illumina libraries (iTru & iNext).

Massively parallel DNA sequencing offers many benefits, but major inhibitory cost factors include: (1) start-up (i.e., purchasing initial reagents and equipment); (2) buy-in (i.e., getting the smallest possible amount of data from a run); and (3) sample preparation. Reducing sample preparation costs is commonly addressed, but start-up and buy-in costs are rarely addressed. We present dual-indexing systems to address all three of these issues. By breaking the library construction process into universal, re-usable, combinatorial components, we reduce all costs, while increasing the number of samples and the variety of library types that can be combined within runs. We accomplish this by extending the Illumina TruSeq dual-indexing approach to 768 (384 + 384) indexed primers that produce 384 unique dual-indexes or 147,456 (384 × 384) unique combinations. We maintain eight nucleotide indexes, with many that are compatible with Illumina index sequences. We synthesized these indexing primers, purifying them with only standard desalting and placing small aliquots in replicate plates. In qPCR validation tests, 206 of 208 primers tested passed (99% success). We then created hundreds of libraries in various scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter that works with indexed PCR primers to uniquely identify samples. Our approach reduces buy-in costs because: (1) relatively few oligonucleotides are needed to produce a large number of indexed libraries; and (2) the large number of possible primers allows researchers to use unique primer sets for different projects, which facilitates pooling of samples during sequencing. Our libraries make use of standard Illumina sequencing primers and index sequence length and are demultiplexed with standard Illumina software, thereby minimizing customization headaches. In subsequent Adapterama papers, we use these same primers with different adapter stubs to construct amplicon and restriction-site associated DNA libraries, but their use can be expanded to any type of library sequenced on Illumina platforms.

New classes of Gram-positive selective antibacterials: inhibitors of MRSA and surrogates of the causative agents of anthrax and tuberculosis.

An antimicrobial phenolic stilbene, (E)-3-hydroxy-5-methoxystilbene, 1 was recently isolated from the leaves of Comptonia peregrina (L.) Coulter and shown to possess inhibitory activity against several Gram-positive bacteria, including isolates of methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium bovis BCG, and avirulent Bacillusanthracis (Sterne strain), among others. These results prompted the design and synthesis of two new classes of compounds, phenoxystyrenes and phenothiostyrenes, as analogs of the natural antimicrobial stilbene. These and additional stilbenoid analogs were synthesized using new, efficient, copper-mediated coupling strategies. Minimum inhibitory concentration (MIC) antimicrobial assays were performed on all compounds prepared. These preliminary structure-activity relationship studies indicated that both new classes of synthetic analogs, as well as the stilbenes, show promising activity against Gram-positive bacteria when at least one phenolic moiety is present, but not when absent. The potencies of the phenolic phenoxystyrenes and phenothiostyrenes were found to be comparable to those of the phenolic stilbenes tested.

The detection of monkeypox in humans in the Western Hemisphere.

BACKGROUND: During May and June 2003, an outbreak of febrile illness with vesiculopustular eruptions occurred among persons in the midwestern United States who had had contact with ill pet prairie dogs obtained through a common distributor. Zoonotic transmission of a bacterial or viral pathogen was suspected. METHODS: We reviewed medical records, conducted interviews and examinations, and collected blood and tissue samples for analysis from 11 patients and one prairie dog. Histopathological and electron-microscopical examinations, microbiologic cultures, and molecular assays were performed to identify the etiologic agent. RESULTS: The initial Wisconsin cases evaluated in this outbreak occurred in five males and six females ranging in age from 3 to 43 years. All patients reported having direct contact with ill prairie dogs before experiencing a febrile illness with skin eruptions. We found immunohistochemical or ultrastructural evidence of poxvirus infection in skin-lesion tissue from four patients. Monkeypox virus was recovered in cell cultures of seven samples from patients and from the prairie dog. The virus was identified by detection of monkeypox-specific DNA sequences in tissues or isolates from six patients and the prairie dog. Epidemiologic investigation suggested that the prairie dogs had been exposed to at least one species of rodent recently imported into the United States from West Africa. CONCLUSIONS: Our investigation documents the isolation and identification of monkeypox virus from humans in the Western Hemisphere. Infection of humans was associated with direct contact with ill prairie dogs that were being kept or sold as pets.

A LightCycler TaqMan assay for detection of Borrelia burgdorferi sensu lato in clinical samples.

Lyme disease (LD) is an infection caused by an ixodid tick-borne spirochete, Borrelia burgdorferi sensu lato. LD manifests itself as a multisystem inflammatory disease that affects the skin in its early localized stage and spreads to the joints, nervous system, heart, and, to a lesser extent, other organ systems in its later disseminated stages. If diagnosed and treated early with appropriate antibiotics, LD is almost always readily cured. Developing a highly sensitive and specific real-time polymerase chain reaction assay could be very useful in improving the diagnostic accuracy and decreasing turnaround time for results. We report the development of a LightCycler TaqMan assay targeting the OspA gene for clinical detection of B. burgdorferi sensu lato in various types of biologic samples. This assay was validated by testing a variety of clinical samples including cerebrospinal fluid, synovial fluid, skin biopsies, and blood and culture isolates from skin biopsies. The TaqMan testing results were 100% concordant with previously reported results. Reference strains representing isolates from other geographic regions were also successfully amplified. The developed assay is robust, is highly sensitive and specific for B. burgdorferi sensu lato, and is suitable for clinical detection of the bacterium in biologic samples.