Coinfections identified from metagenomic analysis of cervical lymph nodes from tularemia patients.

BACKGROUND: Underlying coinfections may complicate infectious disease states but commonly go unnoticed because an a priori clinical suspicion is usually required so they can be detected via targeted diagnostic tools. Shotgun metagenomics is a broad diagnostic tool that can be useful for identifying multiple microbes simultaneously especially if coupled with lymph node aspirates, a clinical matrix known to house disparate pathogens. The objective of this study was to analyze the utility of this unconventional diagnostic approach (shotgun metagenomics) using clinical samples from human tularemia cases as a test model. Tularemia, caused by the bacterium Francisella tularensis, is an emerging infectious disease in Turkey. This disease commonly manifests as swelling of the lymph nodes nearest to the entry of infection. Because swollen cervical nodes are observed from many different types of human infections we used these clinical sample types to analyze the utility of shotgun metagenomics. METHODS: We conducted an unbiased molecular survey using shotgun metagenomics sequencing of DNA extracts from fine-needle aspirates of neck lymph nodes from eight tularemia patients who displayed protracted symptoms. The resulting metagenomics data were searched for microbial sequences (bacterial and viral). RESULTS: F. tularensis sequences were detected in all samples. In addition, we detected DNA of other known pathogens in three patients. Both Hepatitis B virus (HBV) and Human Parvovirus B-19 were detected in one individual and Human Parvovirus B-19 alone was detected in two other individuals. Subsequent PCR coupled with Sanger sequencing verified the metagenomics results. The HBV status was independently confirmed via serological diagnostics, despite evading notice during the initial assessment. CONCLUSION: Our data highlight that shotgun metagenomics of fine-needle lymph node aspirates is a promising clinical diagnostic strategy to identify coinfections. Given the feasibility of the diagnostic approach demonstrated here, further steps to promote integration of this type of diagnostic capability into mainstream clinical practice are warranted.

Viability of Yersinia pestis subcultures in agar stabs.

UNLABELLED: Since its identification as the causative agent of plague in 1894, thousands of Yersinia pestis strains have been isolated and stored. Here, we report the ability of Y. pestis to survive up to 47 years in agar stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. Specifically, we found variation in the presence of plasmid and chromosomal virulence markers (genes pla, lcrV, caf1 and irp2) among multiple subcultures of Y. pestis strains in the 'Collection of Yersinia pestis' (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil. This variation, together with all of the inherent temporal, geographic and other genetic variation represented by all of the recoverable strains in this historical collection was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis. SIGNIFICANCE AND IMPACT OF THE STUDY: We report the ability of Yersinia pestis to survive up to 47 years in agar stabs, in rubber-stoppered tubes, under refrigeration (+4 to +10°C), although overall subculture recovery rates were poor and inversely related to the length of time stored. Genetic characterization of virulence gene presence among these subcultures was suggestive of significant variation in the genomic stability of Y. pestis subcultures stored under these conditions. This variation, together with all of the inherent temporal, geographic and other genetic variation represented by all of the recoverable strains in the historical 'Collection of Yersinia pestis' (Fiocruz-CYP) maintained by the SRP of FIOCRUZ-PE in Brazil was preserved in new frozen culture stocks stored at -70°C as a result of this study. These frozen culture stocks represent a valuable resource for future comparative studies of Y. pestis.

Hybridization in natural sympatric populations of Dermacentor ticks in northwestern North America.

Hybridization in ticks has been described in a handful of species and mostly as a result of laboratory experiments. We used 148 AFLP loci to describe putative hybridization events between D. andersoni and D. variabilis in sympatric populations from northwestern North America. Recently, D. variabilis has expanded its range westward into the natural range of D. andersoni. Using a sample of 235 D. andersoni and 62 D. variabilis, we identified 31 individuals as putative hybrids: four F2 individuals and 27 backcrosses to D. andersoni (as defined by NewHybrids). We found no evidence of hybrids backcrossing into D. variabilis. Furthermore, all hybrids presented 16S mtDNA signatures characteristic of D. andersoni, which indicates the directionality of the hybrid crosses: female D. andersoni × male D. variabilis. We also discovered 13 species-specific AFLP fragments for D. andersoni. These loci were found to have a decreased occurrence in the putative hybrids and were absent altogether in D. variabilis samples. AFLP profiles were also used to determine the levels of genetic population structure and gene flow among nine populations of D. andersoni and three of D. variabilis. Genetic structure exists in both species (D. andersoni, ΦST = 0.110; D. variabilis, ΦST = 0.304) as well as significant estimates of isolation by distance (D. andersoni, ρ = 0.066, P = 0.001; D. variabilis, ρ = 0.729, P = 0.001).

Identification and typing of Francisella tularensis with a highly automated genotyping assay.

A PCR assay was developed to genotypically characterize Francisella tularensis and F. novicida. An integrated and partially redundant set of markers was selected to provide positive identification of these species, identify subspecies of F. tularensis and genotype 14 variable number tandem repeat (VNTR) markers. Assay performance was evaluated with 117 Francisella samples. Sample DNA was amplified, and the masses of the PCR products were determined with electrospray ionization/time of flight mass spectrometry (ESI-MS). The base compositions of the PCR amplicons were derived from these high-accuracy mass measurements and contrasted with databased information associated with each of the 25 assay markers. Species and subspecies determinations for all samples were fully concordant with results from established typing methods, and VNTR markers provided additional discrimination among samples. Sequence variants were observed with a number of assay markers, but these did not interfere with sample characterization, and served to increase the genetic diversity detected by the assay.

Phylogeography of Francisella tularensis ssp. holarctica in France.

AIM: To investigate the phylogeography of French Francisella tularensis ssp. holarctica isolates. METHODS AND RESULTS: Canonical SNPs and MLVA were used to genotype 103 French F. tularensis ssp. holarctica isolates. We confirmed the presence of one subclade, the central and western European group (B.Br.FTNF002-00), and identified four major MLVA genotypes with no obvious geographical differentiation. CONCLUSIONS: The lack of geographical resolution among MLVA genotypes suggests rapid dispersal, convergent evolution or a combination of the two. SIGNIFICANCE AND IMPACT OF THE STUDY: This study expands knowledge of the phylogeography of one of the two dominant European F. tularensis ssp. holarctica subclades and illustrates the need for additional SNP discovery within this subclade.

An optimized, multiplexed multi-locus variable-number tandem repeat analysis system for genotyping Francisella tularensis.

We present a truncated, optimized, multiplexed multiple-locus variable-number tandem repeat analysis system for the molecular subtyping of Francisella tularensis that reduces time and cost requirements while retaining high discriminatory power.

High resolution genotyping of Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat markers.

AIMS: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. METHODS AND RESULTS: Forty-seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi-locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. CONCLUSIONS: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.

Genomic deletion marking an emerging subclone of Francisella tularensis subsp. holarctica in France and the Iberian Peninsula.

Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.

Nuclear genes resolve mesozoic-aged divergences in the insect order Lepidoptera.

Compared to the number of genes available for study of both younger and older divergences, few genes have yet been identified that can strongly resolve phylogenetic splits of Mesozoic age ( approximately 65-250 mya). Thus, reconstruction of Mesozoic-age phylogenies, exemplified by basal divergences within the major orders of holometabolous insects, is likely to be especially dependent on combining multiple lines of evidence. This study tests the potential of the 18S ribosomal RNA gene for reconstructing Mesozoic-aged divergences within the insect order Lepidoptera and its ability when combined with a second, previously analyzed nuclear gene (phosphoenolpyruvate carboxykinase, PEPCK) to strongly resolve these relationships. 18S sequences were obtained for 21 taxa, representing major clades of Lepidoptera plus outgroups from the other "panorpoid orders. A well-corroborated morphology-based "test phylogeny was used to evaluate the effects of partitioning the 18S gene according to variable versus conserved domains, paired versus unpaired sites in the secondary structure, and transition versus transversion substitutions. Likelihood and unweighted parsimony analyses of the 18S data recover the "test phylogeny" almost completely, with no improvement of agreement or support provided by any form of weighting or partitioning. No conflict in signal between 18S and PEPCK was detected by the partition homogeneity test. Combined parsimony analysis yielded strong bootstrap support for nearly all relationships, much higher than for either gene alone, thereby also providing strong evidence on several hypotheses about the early evolution of lepidopteran-plant interactions. These genes in combination may be widely useful for resolving insect divergences of comparable age.

Quality indicators: an approach to quality improvement in home healthcare.

In this article, Donna Wagner discusses using quality indicators as a means of improving quality in home healthcare. Stressing the need for an organization to have a thorough foundation in total quality management as its working context, the article demonstrates how to use quality indicators as part of a quality improvement program. Monitoring quality indicators in a home health agency will provide specific information on aspects of care that the agency has identified as measures of quality. This cost-effective monitoring can be a means of incorporating QI activities into daily work patterns.

QA in home care: the staff nurse's challenge.

The home healthcare staff nurse faces formidable challenges that can be met only by an extraordinary commitment to quality care. Functioning autonomously much of the time, the home healthcare nurse must act as the client's case manager. Coordination of multiple services and extensive client education are vital to positive outcomes, and intervention decisions must be based on a holistic view of the client's needs and environment. Reimbursement issues under Medicare often conflict with the delivery of high-quality care. To achieve the goals of the home healthcare agency and perform optimally, the home healthcare nurse needs a supportive QA program and frequent peer review procedures.

The human side of quality: service oriented employees.

Motivating employees to care about the quality of the services they provide is an essential ingredient in quality improvement. In this article the author presents eight methods that employers can use to translate expectations into actions. Becoming aware of what today's employees want is the first step; money and job security are not as important as respect, challenging work that shows results, and managers who listen. Since customer relations mirror management's attitudes toward employees, caring about staff is basic in a quality service organization. Education and training of workers are also crucial, as are communication of expectations and providing prompt feedback. Employees also need reward and recognition. Hiring the right people is another necessity for quality service. Finally, managers must be leaders who can articulate values and the organization's vision in order to enlist employees in the cause of quality service.

Quality assurance. Issues and process.

As a result of company-wide involvement in quality assurance we have been able to advance the concept of participatory management though the organization's base is centralized in Kalamazoo. Despite the great size and geographical span of UHCS, regional and office management staff across North America play an integral part in guiding our future. Our QA program has given field operations the opportunity to display to management their interest and desire to be involved in the overall process. Success in the home health care industry is due in large part to the work ethic of our employees and their desire to do their jobs in a truly professional manner. It is extremely important for a multiagency organization that all of its employees in every location, know that the company is only as good as its people. After all, an organization is perceived by the public through individual employees. Thus, expectation levels for health care employees must always be heightened. Quality improvement is an ongoing effort no matter how well we do it. We keep attempting to reduce the amount of rework or correction and to achieve higher levels of productivity. Employees of any healthcare organization, large or small, need to know that the organization cannot be satisfied with where it is today. We in the health service industry must be looking toward the care we can provide, better and more efficiently, tomorrow. Quality improvement is an ongoing effort, no matter how well we do it.