Prevalence and Antimicrobial Agent Susceptibility of Methicillin-resistant Staphylococcus aureus in Healthy Pediatric Outpatients in Las Vegas

Colonization and infection by community-associated resistant strains of Staphylococcus aureus are being reported in epidemic proportions. The purpose of this study was to determine the local prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization in children and to characterize the MRSA isolates in the laboratory with regard to antimicrobial agent susceptibility patterns, and the presence of the mecA and the Panton-Valentine leukocidin (PVL) genes. Nasal swabs were collected at two pediatric clinics from a total of 505 children during health maintenance visits. A brief questionnaire was administered to collect demographic data and pertinent medical, family, and social history. Samples were cultured onto 2 selective media for S. aureus and MRSA. Potential MRSA isolates were further evaluated by real-time polymerase chain reaction (PCR), and for susceptibility to eight antibiotics by disk diffusion. Culture results showed that MRSA was present in 15 of the 505 specimens (3.0%). Six different antimicrobial susceptibility profiles were observed among the MRSA isolates. PCR amplification results showed that all 15 MRSA isolates were positive for the presence of the mecA gene, and 10 MRSA isolates contained the PVL gene. Understanding local prevalence rates and the role of colonization in infection are needed to develop effective interventions to reduce MRSA infections

Identification of Eastern United States Reticulitermes Termite Species via PCR-RFLP, Assessed Using Training and Test Data

Reticulitermes termites play key roles in dead wood decomposition and nutrient cycling in forests. They also damage man-made structures, resulting in considerable economic loss. In the eastern United States, five species (R. flavipes, R. virginicus, R. nelsonae, R. hageni and R. malletei) have overlapping ranges and are difficult to distinguish morphologically. Here we present a molecular tool for species identification. It is based on polymerase chain reaction (PCR) amplification of a section of the mitochondrial cytochrome oxidase subunit II gene, followed by a three-enzyme restriction fragment length polymorphism (RFLP) assay, with banding patterns resolved via agarose gel electrophoresis. The assay was designed using a large set of training data obtained from a public DNA sequence database, then evaluated using an independent test panel of Reticulitermes from the Southern Appalachian Mountains, for which species assignments were determined via phylogenetic comparison to reference sequences. After refining the interpretive framework, the PCR-RFLP assay was shown to provide accurate identification of four co-occurring species (the fifth species, R. hageni, was absent from the test panel, so accuracy cannot yet be extended to training data). The assay is cost- and time-efficient, and will help improve knowledge of Reticulitermes species distributions

Genomic sequencing capacity, data retention, and personal access to raw data in Europe

Whole genome/exome sequencing (WGS/WES) has become widely adopted in research and, more recently, in clinical settings. Many hope that the information obtained from the interpretation of these data will have medical benefits for patients and—in some cases—also their biological relatives. Because of the manifold possibilities to reuse genomic data, enabling sequenced individuals to access their own raw (uninterpreted) genomic data is a highly debated issue. This paper reports some of the first empirical findings on personal genome access policies and practices. We interviewed 39 respondents, working at 33 institutions in 21 countries across Europe. These sequencing institutions generate massive amounts of WGS/WES data and represent varying organisational structures and operational models. Taken together, in total, these institutions have sequenced ∼317,259 genomes and exomes to date. Most of the sequencing institutions reported that they are able to store raw genomic data in compliance with various national regulations, although there was a lack of standardisation of storage formats. Interviewees from 12 of the 33 institutions included in our study reported that they had received requests for personal access to raw genomic data from sequenced individuals. In the absence of policies on how to process such requests, these were decided on an ad hoc basis; in the end, at least 28 requests were granted, while there were no reports of requests being rejected. Given the rights, interests, and liabilities at stake, it is essential that sequencing institutions adopt clear policies and processes for raw genomic data retention and personal access

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

BACKGROUND: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data was donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. [Results]: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. [Conclusions]: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.This work was supported by funds provided through the Gene Partnership and the Manton Center for Orphan Disease Research at Boston Children’s Hospital and the Center for Biomedical Informatics at Harvard Medical School and by generous donations in-kind of genomic sequencing services by Life Technologies (Carlsbad, CA, USA) and Complete Genomics (Mountain View, CA, USA).Peer Reviewe

Molecular epidemiology of waterborne zoonoses in the North Island of New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science (Epidemiology and Public Health) at Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Palmerston North, New Zealand

Campylobacter, Cryptosporidium and Giardia species are three important waterborne zoonotic pathogens of global public health concern. This PhD opens with an interpretive overview of the literature on Campylobacter, Cryptosporidium and Giardia spp. in ruminants and their presence in surface water (Chapter 1), followed by five epidemiological studies of Campylobacter, Cryptosporidium and Giardia spp. in cattle, sheep and aquatic environment in New Zealand (Chapters 2-6). The second chapter investigated four years of retrospective data on Campylobacter spp. (n=507) to infer the source, population structure and zoonotic potential of Campylobacter jejuni from six high-use recreational rivers in the Wanganui- Manawatu region of New Zealand through the generalised additive model, generalised linear/logistic regression model, and minimum spanning trees. This study highlights the ubiquitous presence of Campylobacter spp. in both low and high river flows, and during winter months. It also shows the presence of C. jejuni in 21% of samples containing highly diverse strains, the majority of which were associated with wild birds only. These wild birds-associated C. jejuni have not been detected in human, suggesting they may not be infectious to human. However, the presence of some poultry and ruminant-associated strains that are potentially zoonotic suggested the possibility of waterborne transmission of C. jejuni to the public. Good biosecurity measures and water treatment plants may be helpful in reducing the risk of waterborne Campylobacter transmission In the third study, a repeated cross-sectional study was conducted every month for four months to investigate the source of drinking source-water contamination. A total of 499 ruminant faecal samples and 24 river/stream water samples were collected from two rural town water catchments (Dannevirke and Shannon) in the Manawatu- Wanganui region of New Zealand, and molecular analysis of those samples was performed to determine the occurrence of Campylobacter, Cryptosporidium, and Giardia spp. and their zoonotic potential. The major pathogens found in faecal samples were Campylobacter (n=225 from 7/8 farms), followed by Giardia (n=151 from 8/8 farms), whereas Giardia cysts were found in many water samples (n=18), followed by Campylobacter (n=4). On the contrary, Cryptosporidium oocysts were only detected in a few faecal (n=18) and water (n=3) samples. Cryptosporidium and Giardia spp. were detected in a higher number of faecal samples from young animals (≤ 3 months) than juvenile and adult animals, whereas Campylobacter spp. were highly isolated in the faecal samples from juvenile and adult ruminants. PCRsequencing of the detected pathogens indicated the presence of potentially zoonotic C. jejuni and C. coli, Cryptosporidium parvum (gp60 allelic types IIA18G3R1 and IIA19G4R1) and Giardia duodenalis (assemblages AII, BII, BIII, and BIV) in cattle and sheep. In addition, potentially zoonotic C. jejuni and Giardia duodenalis assemblages AII, BI, BII, and BIV were also determined in water samples. These findings indicate that these three pathogens of public health significance are present in ruminant faecal samples of farms and in water, and may represent a possible source of human infection in New Zealand. In the fourth study, PCR-sequencing of Cryptosporidium spp. isolates obtained from the faeces of 6-week- old dairy calves (n=15) in the third study were investigated at multiple loci (18S SSU rDNA, HSP70, Actin and gp60) to determine the presence of mixed Cryptosporidium spp. infections. Cryptosporidium parvum (15/15), C. bovis (3/15) and C. andersoni (1/15), and two new genetic variants were determined along with molecular evidence of mixed infections in five specimens. Three main Cryptosporidium species of cattle, C. parvum, C. bovis and C. andersoni, were detected together in one specimen. Genetic evidence of the presence of C. Anderson and two new Cryptosporidium genetic variants are provided here for the first time in New Zealand. These findings provided additional evidence that describes Cryptosporidium parasites as genetically heterogeneous populations and highlighted the need for iterative genotyping at multiple loci to explore the genetic makeup of the isolates. The C. jejuni and C. coli isolates (n=96) obtained from cattle, sheep and water in the third study were subtyped to determine their genetic diversity and zoonotic potential using a modified, novel multi-locus sequence typing method (“massMLST”; Chapter 5). Primers were developed and optimised, PCR-based target-MLST alleles’ amplification were performed, followed by next generation sequencing on an Illumina MiSeq machine. A bioinformatics pipeline of the sequencing data was developed to define C. jejuni and C. coli multi-locus sequence types. This study demonstrated the utility and potential of this novel typing method, massMLST, as a strain typing method. In addition to identifying the possible C. jejuni/coli clonal complexes or sequence types of 68/96 isolates from ruminant faeces and water samples, this study reported three new C. jejuni strains in cattle in New Zealand, along with many strains, such as CC-61, CC-828 and CC-21, that have also been found in humans, indicating the public health significance of these isolates circulating on the farms in the two water catchment areas. Automation of the massMLST method and may allow a cost-effective high-resolution typing method in the near future for multilocus sequence typing of large collections of Campylobacter strains. In the final study (Chapter 6), a pilot metagenomic study was carried out to obtain a snapshot of the microbial ecology of surface water used in the two rural towns of New Zealand for drinking purposes, and to identify the zoonotic pathogens related to waterborne diseases. Fresh samples collected in 2011 and 2012, samples from the same time that were frozen, and samples that were kept in the preservative RNAlater were sequenced using whole-genome shotgun sequencing on an Illumina MiSeq machine. Proteobacteria was detected in all the samples characterised, although there were differences in the genus and species between the samples. The microbial diversity reported varied between the grab and stomacher methods, between samples collected in the year 2011 and 2012, and among the fresh, frozen and RNAlater preserved samples. This study also determined the presence of DNA of potentially zoonotic pathogens such as Cryptosporidium, Campylobacter and Mycobacterium spp. in water. Use of metagenomics could potentially be used to monitor the ecology of drinking water sources so that effective water treatment plans can be formulated, and for reducing the risk of waterborne zoonosis. As a whole, this PhD project provides new data on G. duodenalis assemblages in cattle, sheep and surface water, new information on mixed Cryptosporidium infections in calves, a novel “massMLST” method to subtype Campylobacter species, and shows the utility of shotgun metagenomic sequencing for drinking water monitoring. Results indicate that ruminants (cattle and sheep) in New Zealand shed potentially zoonotic pathogens in the environment and may contribute to the contamination of surface water. A better understanding of waterborne zoonotic transmission would help in devising appropriate control strategies, which could reduce the shedding of Campylobacter, Cryptosporidium, and Giardia spp. in the environment and thereby reduce waterborne transmission

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY challenge

Background: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. Results: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. Conclusions: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge

There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups