Maximizing Reflexive HCV RNA Testing of HCV Antibody-Reactive Samples Within United States Public Health Laboratories.

Hepatitis C virus (HCV) infection can cause significant morbidity and mortality if left untreated and diagnosis is the first step in any treatment regimen. In the United States, a two-step algorithm is recommended for detection of current HCV infection. The algorithm consists of an HCV antibody screening test followed by a supplemental test for HCV RNA if the HCV antibody test is reactive. To assess HCV testing practices and identify associated challenges, the Association of Public Health Laboratories (APHL) conducted a survey on HCV diagnostics practices of US public health laboratories. Additionally, APHL and the Centers for Disease Control and Prevention's (CDC) Division of Viral Hepatitis (DVH) convened a two-day meeting of HCV subject matter experts (SMEs) to identify opportunities for improvement in diagnosis of current HCV infection. Automatic reflexive HCV RNA testing of HCV antibody-reactive specimens was identified as a high priority target area by HCV SMEs and as a gap in laboratory practice by the APHL survey, which found that only 54% of respondent laboratories always automatically reflexed or referred an Ab-reactive specimen to an HCV RNA test. To facilitate accurate diagnosis and ensure that patients are not lost to follow up, laboratories and public health programs should work to ensure that the entire HCV testing algorithm (i.e., antibody and nucleic acid testing) can be completed with a sample(s) collected during a single patient visit.

High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome.

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.

Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis.

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 (stx1 and stx2) (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.

Bacteremia caused by Microbacterium binotii in a patient with sickle cell anemia.

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.

Hazenella coriacea gen. nov., sp. nov., isolated from clinical specimens.

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 23436(T) ( = DSM 45707(T) = LMG 27204(T)).

State requirements for rabies-related animal control.

OBJECTIVE: Rabies postexposure prophylaxis is an important secondary prevention step but is unnecessary if the exposing animal is not rabid. Effective rabies-related animal control (RRAC) requirements enforced by animal control officers (ACO) are an alternative step to reduce the number of rabies exposures and postexposure prophylaxes. The purpose of this study was to describe the variability of requirements for RRAC by statutes and regulations across the United States. METHODS: Current state laws and regulations pertaining to rabies and animal control were reviewed and assessed for 3 primary RRAC activities related to obtaining animals that have potentially exposed humans to rabies, that have been potentially exposed to rabies, or that show signs of rabies. Animal control infrastructure was assessed on the basis of the requirement for, and authority granted to, ACOs for conducting these RRAC activities. State Public Health Veterinarians, State Veterinarians with the Departments of Agriculture, and/or State Epidemiologists were contacted for verification and assistance with interpretation of laws and regulations. RESULTS: Twenty-three states and the District of Columbia authorize specific actions related to all 3 RRAC activities. Twenty-four states have laws and regulations that do not clearly address at least 1 of the RRAC activities or limit the authority to domestic animals. Three states have laws or regulations that address RRAC nonspecifically or leave the requirements to localities. Eleven states mandate the placement of ACOs with authority over domestic and wild animals, 7 states require ACOs for control of domestic animals only, and 32 states and the District of Columbia have no statewide requirements for ACOs. DISCUSSION: Only 9 states have legal requirements for ACOs with authority over wild and domestic animals and RRAC that addresses all 3 primary RRAC activities. Consequently, RRAC requirements may represent an incompletely tapped rabies prevention mechanism.

Clinical and demographic features associated with infections with extended-spectrum beta-lactamase-producing Escherichia coli in a health system in Maine, 2017.

In this case-case control study, we identified receipt of ß-lactam antibiotics and older age as independently associated with increased infection risk with ESBL-producing Escherichia coli among residents aged 20-88 years in a rural Maine hospital system where the infection prevalence of antibiotic-resistant E. coli is low.

Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

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 (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) 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.

The Use of a Shared Services Model for Mycobacteriology Testing: Lessons Learned.

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.

Implementation and performance of the BioFire FilmArray® Blood Culture Identification panel with antimicrobial treatment recommendations for bloodstream infections at a midwestern academic tertiary hospital.

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.

Real-time PCR and pyrosequencing for differentiation of medically relevant Bartonella species.

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.

Common pathways for receptor-mediated ingestion of Escherichia coli and LDL cholesterol by Entamoeba histolytica regulated in part by transmembrane kinase 39.

The single-celled parasite, Entamoeba histolytica, is an enteric pathogen that ingests bacteria and host cells. Inhibition of phagocytosis renders the parasite avirulent. The ligand/receptor interactions that allow E. histolytica to phagocytose are not well understood. We hypothesised that E. histolytica trophozoites might accomplish ingestion through the utilisation of a scavenger receptor for cholesterol. Here we show that acetylated low density lipoprotein cholesterol was phagocytosed by amoebae via receptor mediated mechanisms. Acetylated low density lipoprotein cholesterol competitively inhibited by 31 ± 1.3% (P < 0.005) the ingestion of Escherichia coli, but not erythrocytes and Jurkat T lymphocytes, suggesting a partially redundant phagocytic pathway for E. coli and cholesterol. Inducible expression ofa signalling-dead dominant-negative version of E. histolytica transmembrane kinase 39 inhibited ingestion of E. coli by 55 ± 3% (P < 0.005) but not LDL particles. We concluded that ingestion of E. coli was regulated by TMK39 and partially shared the acetylated low density lipoprotein cholesterol uptake pathway.

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis.

Entamoeba histolytica contains a large and novel family of transmembrane kinases (TMKs). The expression patterns of the E. histolytica TMKs in individual trophozoites and the roles of the TMKs for sensing and responding to extracellular cues were incompletely characterised. Here we provide evidence that single cells express multiple TMKs and that TMK39 and TMK54 likely serve non-redundant cellular functions. Laser-capture microdissection was used in conjunction with microarray analysis to demonstrate that single trophozoites express more than one TMK gene. Anti-peptide antibodies were raised against unique regions in the extracellular domains of TMK39, TMK54 and PaTMK, and TMK expression was analysed at the protein level. Flow cytometric assays revealed that populations of trophozoites homogeneously expressed TMK39, TMK54 and PaTMK, while confocal microscopy identified different patterns of cell surface expression for TMK39 and TMK54. The functions of TMK39 and TMK54 were probed by the inducible expression of dominant-negative mutants. While TMK39 co-localised with ingested beads and expression of truncated TMK39 interfered with trophozoite phagocytosis of apoptotic lymphocytes, expression of a truncated TMK54 inhibited growth of amoebae and altered the surface expression of the heavy subunit of the E. histolytica Gal/GalNAc lectin. Overall, our data indicates that multiple members of the novel E. histolytica TMK family are utilised for non-redundant functions by the parasite.