Kurt D. Reed

Multilocus Sequence Typing of Borrelia burgdorferi Suggests Existence of Lineages with Differential Pathogenic Properties in Humans

The clinical manifestations of Lyme disease, caused by Borrelia burgdorferi, vary considerably in different patients, possibly due to infection by strains with varying pathogenicity. Both rRNA intergenic spacer and ospC typing methods have proven to be useful tools for categorizing B. burgdorferi strains that vary in their tendency to disseminate in humans. Neither method, however, is suitable for inferring intraspecific relationships among strains that are important for understanding the evolution of pathogenicity and the geographic spread of disease. In this study, multilocus sequence typing (MLST) was employed to investigate the population structure of B. burgdorferi recovered from human Lyme disease patients. A total of 146 clinical isolates from patients in New York and Wisconsin were divided into 53 sequence types (STs). A goeBURST analysis, that also included previously published STs from the northeastern and upper Midwestern US and adjoining areas of Canada, identified 11 major and 3 minor clonal complexes, as well as 14 singletons. The data revealed that patients from New York and Wisconsin were infected with two distinct, but genetically and phylogenetically closely related, populations of B. burgdorferi. Importantly, the data suggest the existence of B. burgdorferi lineages with differential capabilities for dissemination in humans. Interestingly, the data also indicate that MLST is better able to predict the outcome of localized or disseminated infection than is ospC typing.

Adapterama I: Universal stubs and primers for thousands of dual-indexed Illumina libraries (iTru & iNext)

Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing the time and costs associated with: 1) start-up (i.e., doing NGS for the first time), 2) buy-in (i.e., getting any data from a run), and 3) sample preparation. Although many researchers have focused on reducing sample preparation costs, few have addressed the first two problems. Here, we present iTru and iNext, dual-indexing systems for Illumina libraries that help address all three of these issues. By breaking the library construction process into re-usable, combinatorial components, we achieve low start-up, buy-in, and per-sample costs, while simultaneously increasing the number of samples that can be combined within a single run. We accomplish this by extending the Illumina TruSeq dual-indexing approach from 20 (8+12) indexed adapters that produce 96 (8x12) unique combinations to 579 (192+387) indexed primers that produce 74,304 (192x387) unique combinations. We synthesized 208 of these indexed primers for validation, and 206 of them passed our validation criteria (99% success). We also used the indexed primers to create hundreds of libraries in a variety of scenarios. Our approach reduces start-up and per-sample costs by requiring only one universal adapter which 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. Although the methods we present are highly customizable, resulting libraries can be used with the standard Illumina sequencing primers and demultiplexed with the standard Illumina software packages, thereby minimizing instrument and software customization headaches. In subsequent Adapterama papers, we use these same iTru primers with different adapter stubs to construct double- to quadruple-indexed amplicon libraries and double-digest restriction-site associated DNA (RAD) libraries. For additional details and updates, please see http://baddna.org.Next-generation DNA sequencing (NGS) offers many benefits, but major factors limiting NGS include reducing costs of: 1) start-up (i.e., doing NGS for the first time); 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.

Macrolide and Clindamycin Resistance in Group A Streptococci Isolated from Children With Pharyngitis.

Group A streptococcus (GAS) is responsible for 15%–30% of cases of acute pharyngitis in children. Macrolides such as azithromycin have become popular for treating GAS pharyngitis. We report macrolide resistance rates in a primary care setting in our geographic area over the past 5 years and discuss the implications of resistance in making treatment decisions. Throat swabs were collected from children with pharyngitis from May 2011 to May 2015 in a primary care setting in Madison, Wisconsin. Susceptibility testing was performed for erythromycin and clindamycin using the Kirby–Bauer disk diffusion method. GAS was identified on 143 throat cultures. Overall, 15% of GAS isolates demonstrated nonsusceptibility for both clindamycin and erythromycin. Inducible resistance (positive D-test) was detected in 17 isolates (12%). The rate of detection of nonsusceptibility in each year of the study did not change over time. Azithromycin should only be used for patients with pharyngitis and substantial manifestations of penicillin hypersensitivity and when used, susceptibility testing should always be performed.

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.

Limb Embolism in a 52-Year-Old Woman

Diagnosis: Arterial thrombus caused by Histoplasma capsulatum deriving from native valve endocarditis caused by the same organism. Microscopic examination of the surgically extracted thrombus revealed numerous narrow budding, round to oval yeast and hyaline, septate hyphae branching at 90 degrees with occasional pseudohyphael forms (Figure 1). Based on this analysis, the patient was placed on liposomal amphotericin B, voriconazole, and micafungin to provide coverage for dimorphic fungi as well as Candida and Aspergillus species. Transesophageal echocardiography demonstrated a 6 × 9-mm vegetation on the ventricular surface of the noncoronary cusp of the aortic valve. Computed tomography of the chest was negative for any parenchymal infiltrates, and fungal cultures of blood and the resected thrombus remained negative. Serum galactomannan, serum and urinary Blastomyces antigen, and human immunodeficiency virus antibody tests were all negative. While the urinary Histoplasma antigen was negative, the serum (1,3)-βD-glucan (163 pg/mL) and Histoplasma antigen (Miravista Diagnostic, Indianapolis, Indiana) (1.22 ng/mL) tests were positive. Broad-range (18S ribosomal RNA and internal transcribed spacer) polymerase chain reaction (PCR) of the paraffin-embedded thrombus confirmed infection caused by H. capsulatum. Figure 1. A, Most of the thrombus contains fungal elements. The yeast and hyphae are both intermixed and in discrete areas. B, Budding yeast (white arrow) and hyaline, septated hyphae (black arrow) are present. Hyphae branch at right angles (dashed arrow).

Respiratory Tract Infections: A Clinical Approach

Respiratory tract infections are among the most common and important problems in clinical medicine. The approach to these infections begins with a basic understanding of the pathophysiology of the respiratory tract and how immune defences interact with pathogens. A number of distinct clinical syndromes can be defined which help physicians and clinical microbiologists develop a differential diagnosis of potential infecting pathogens. The type and severity of infection is often modulated by the medical characteristics of the patient, circumstances surrounding infection, geography and environmental exposures. Multiple laboratory test methodologies are available to aid clinical diagnosis and close communication between clinicians and laboratory personnel can optimise the efficiency and cost-effectiveness of diagnosis. Molecular testing for respiratory viruses has become widely used in the clinical setting and will expand to include other pathogens of the respiratory tract.n n Respiratory tract infections are among the most common and important problems in clinical medicine. The approach to these infections begins with a basic understanding of the pathophysiology of the respiratory tract and how immune defences interact with pathogens. A number of distinct clinical syndromes can be defined which help physicians and clinical microbiologists develop a differential diagnosis of potential infecting pathogens. The type and severity of infection is often modulated by the medical characteristics of the patient, circumstances surrounding infection, geography and environmental exposures. Multiple laboratory test methodologies are available to aid clinical diagnosis and close communication between clinicians and laboratory personnel can optimise the efficiency and cost-effectiveness of diagnosis. Molecular testing for respiratory viruses has become widely used in the clinical setting and will expand to include other pathogens of the respiratory tract.n n