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Objectives: Public health laboratories (PHLs) provide essential services in the diagnosis and surveillance of diseases of public health concern, such as tuberculosis. Maintaining access to high-quality laboratory testing is critical to continued disease detection and decline of tuberculosis cases in the United States. We investigated the practical experience of sharing tuberculosis testing services between PHLs through the Shared Services Project. Methods: The Shared Services Project was a 9-month-long project funded through the Association of Public Health Laboratories and the Centers for Disease Control and Prevention during 2012-2013 as a one-time funding opportunity to consortiums of PHLs that proposed collaborative approaches to sharing tuberculosis laboratory services. Submitting PHLs maintained testing while simultaneously sending specimens to reference laboratories to compare turnaround times. Results: During the 9-month project period, 107 Mycobacterium tuberculosis complex submissions for growth-based drug susceptibility testing and molecular detection of drug resistance testing occurred among the 3 consortiums. The median transit time for all submissions was 1.0 day. Overall, median drug susceptibility testing turnaround time (date of receipt in submitting laboratory to result) for parallel testing performed in house by submitting laboratories was 31.0 days; it was 43.0 days for reference laboratories. The median turnaround time for molecular detection of drug resistance results was 1.0 day (mean = 2.8; range, 0-14) from specimen receipt at the reference laboratories. Conclusions: The shared services model holds promise for specialized tuberculosis testing. Sharing of services requires a balance among quality, timeliness, efficiency, communication, and fiscal costs.

The FilmArray® Blood Culture Identification (BCID) panel was recently implemented at a midwestern academic tertiary care hospital to provide rapid identification (ID) of common pathogens from positive blood cultures. This study evaluated the clinical performance of the BCID panel compared to culture-based ID methods. One hundred thirty-eight monomicrobial and 8 polymicrobial blood cultures were evaluated during the 30-day study resulting in the ID of 152 total organisms by culture with 115 organisms correctly identified using the BCID panel. The BCID panel had sensitivities of 80.4% (115/152) for all organisms identified during the study and 94.6% (115/122) when considering only on-panel organisms. BCID panel specificity was 100%. Implementation of the BCID panel was coupled with the development of empiric therapy recommendations for bloodstream infections by the antimicrobial stewardship team. Based on this study, the FilmArray® BCID panel is a rapid and reliable test for the detection of common bloodstream pathogens, and therapeutic decisions can be based upon panel results.

NK cells are key effectors of innate immunity and host survival during cytomegalovirus (CMV) infection. Innate murine CMV (MCMV) resistance in MA/My mice requires Ly49H/m157-independent H-2k-linked NK cell control. Here we show that replacement of MA/My H-2kwith C57L H-2bsusceptibility genes led to a remarkable loss of innate virus immunity, though NK gamma interferon was induced in H-2band H-2kstrains shortly after infection. Thus, H-2bgenes expressed in C57L or MA/My.L-H2bare sufficient in alerting NK cells to intrusion but fail to support NK restraint of viral infection. In addition, novel H-2 recombinant strains were produced and utilized in a further refinement of a critical genetic interval controlling innate H-2k-linked MCMV resistance. Importantly, this analysis excluded the gene interval from Kkclass I through class II. The responsible gene(s) therefore resides in an interval spanning Dkclass Ia and more-distal major histocompatibility complex (MHC) nonclassical class Ib genes. Recently, the NK activation receptor Ly49P and MHC class I Dkproteins were genetically implicated in MCMV resistance, in part because Ly49P-expressing reporter T cells could specifically bind Dkmolecules on MCMV-infected mouse embryonic fibroblasts (MEFs). However, as we found that H-2kinnate resistance differs in the C57L or MA/My backgrounds and because MCMV very efficiently downregulates H-2kclass I proteins in L929 cells and primary MEFs shortly after infection, a Ly49P/Dkmodel should not fully explain H-2k-linked MCMV resistance.

The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides , Salmonella spp., Vibrio spp., Vibrio cholerae , Yersinia enterocolitica , enteroaggregative E. coli , enteropathogenic E. coli , enterotoxigenic E. coli , Shiga-like toxin-producing E. coli ( stx 1 and stx 2 ) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli , Cryptosporidium spp., Cyclospora cayetanensis , Entamoeba histolytica , Giardia lamblia , adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens ( n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.

Microbacterium species are non-spore-forming, Gram-positive rods rarely associated with human disease. In this report, we describe the first case of bacteremia caused by Microbacterium binotii in a patient with sickle cell anemia. The utility of using 16S rRNA gene sequence analysis along with phenotypic methods for identification is shown.

Single-particle cryo-electron microscopy has the immense advantage over crystallography in being able to image frozen-hydrated biological complexes in their “native” state, in solution. For years the ribosome has been the benchmark sample for particles without symmetry. It has witnessed steady improvement in resolution from the very first single-particle 3D reconstruction to today’s reconstructions at near-atomic resolution. In this study, we describe the different steps of sample preparation, data collection, data processing, and modeling that led to the 5A structure of the T. brucei ribosome [32]. A local resolution estimation demonstrates the extent to which resolution can be anisotropic and pinpoints regions of higher heterogeneity or structural flexibility. This study also shows an example of misuse of spatial frequency filters leading to overfitting of the data and the artifacts that can be observed in the resulting density map.

A Gram-staining-positive, endospore-forming rod was isolated independently from clinical specimens in New York State, USA, once in 2009 and twice in 2011. The three isolates had identical 16S rRNA gene sequences and, based on their 16S rRNA gene sequence, are most closely related to the type strains of Laceyella sediminis and L. sacchari (94.6 % similarity). The partial 23S rRNA gene sequences of the three strains were also 100 % identical. Maximum-likelihood phylogenetic analysis suggests that the new isolates belong to the family Thermoactinomycetaceae . Additional biochemical and phenotypic characteristics of the strains support the family designation and suggest that the three isolates represent a single species. In each of the strains, the predominant menaquinone is MK-7, the diagnostic diamino acid is meso-diaminopimelic acid and the major cellular fatty acids are iso-C15 : 0, anteiso-C15 : 0 and iso-C13 : 0. The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, four unknown phospholipids, four unknown aminophospholipids and an unknown lipid. It is proposed that the novel isolates represent a single novel species within a new genus, for which the name Hazenella coriacea gen. nov., sp. nov. is proposed. The type strain of Hazenella coriacea is strain 23436T ( = DSM 45707T = LMG 27204T).

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in virtually all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart, owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids, unicellular eukaryotic parasites often infectious to humans. Here we present a high-resolution cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that causes African sleeping sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large expansion segments and ribosomal-protein extensions leading to the formation of four additional inter-subunit bridges. We also find additional rRNA insertions, including one large rRNA domain that is not found in other eukaryotes. Furthermore, the structure reveals the five cleavage sites of the kinetoplastid large ribosomal subunit (LSU) rRNA chain, which is known to be cleaved uniquely into six pieces, and suggests that the cleavage is important for the maintenance of the T. brucei ribosome in the observed structure. We discuss several possible implications of the large rRNA expansion segments for the translation-regulation process. The structure could serve as a basis for future experiments aimed at understanding the functional importance of these kinetoplastid-specific ribosomal features in protein-translation regulation, an essential step towards finding effective and safe kinetoplastid-specific drugs.

Multiple Bartonella species cause disease in humans. Although fast and accurate species differentiation could inform effective treatment interventions, species-level diagnosis of Bartonella infections is not typical. Here we describe a real-time PCR and pyrosequencing based algorithm for rapid differentiation of at least 11 medically relevant Bartonella spp.

Effective diagnostic tools are essential in order to combat disease caused by the parasite Entamoeba histolytica . In this study, we compared the commercially available Ridascreen Entamoeba test (R-Biopharm) and the E. histolytica II test (Techlab), and we found that the E. histolytica II test detects E. histolytica infections more accurately.

Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMKΔ932). Expression of the carboxy-truncation of PATMKΔ932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection., Author Summary There is a highly ordered process by which the parasite Entamoeba histolytica interacts with human cells. Adherence via a parasite lectin is followed in seconds by killing, with only the corpse and not a living cell ingested by the ameba. This process is so central to pathogenesis that clinicians use the presence of ingested erythrocytes to identify E. histolytica and distinguish it from harmless commensal amebae of the gut. We hypothesized that identification of molecules involved in the ingestion of the corpse might provide insight into how amebae cause colitis. We identified a member of the transmembrane kinase family as an early component of the phagosome. Inhibition of this kinase blocked red cell ingestion and prevented amebae from colonizing and invading the gut. There was no impact on dominant-negative parasites to cause liver abscess, suggesting the pathogenesis program differs between anatomic sites. Future studies of the transmembrane kinanse in erythrophagocytosis may provide insight into how amebae colonize and invade the gut, with the ultimate goal of preventing disease.

Ribosomes, the protein factories of living cells, translate genetic information carried by messenger RNAs into proteins, and are thus involved in, virtually, all aspects of cellular development and maintenance. The few available structures of the eukaryotic ribosome reveal that it is more complex than its prokaryotic counterpart owing mainly to the presence of eukaryote-specific ribosomal proteins and additional ribosomal RNA insertions, called expansion segments. The structures also differ among species, partly in the size and arrangement of these expansion segments. Such differences are extreme in kinetoplastids. Here, we present a high-resolution (∼5 A) cryo-electron microscopy structure of the ribosome of Trypanosoma brucei, the parasite that is transmitted by the tsetse fly and that which causes African Sleeping Sickness. The atomic model reveals the unique features of this ribosome, characterized mainly by the presence of unusually large eukaryotic expansion segments and ribosomal protein extensions...

Eukaryotic 80S ribosomes of known structure are far more complex than their 70S bacterial counterparts. Those from Saccharomyces cerevisiae, Tetrahymena thermophila, and Triticum aestivum, for example, bear insertions of ribosomal RNA (rRNA) called expansion segments (ES) and additional ribosomal proteins. The ribosomes of the kinetoplastid Trypanosoma brucei, though, are especially fascinating: structurally and their other kinetoplastids’ ribosomes bear very large ESs, as well as smaller ESs, and protein extensions. Additionally, T. brucei ribosomes require novel protein factors for maturation, although they do not require several eukaryotic initiation factors or a recycling factor. As a species, T. brucei is fascinating not only in terms of structure, but also in terms of gene expression and even public health: the species is responsible for the incurable, terminal human African Trypanosomiasis (sleeping sickness); and during post-transcriptional regulation, a single common RNA segment called a splice l...