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Contents
Pages vii-xi

Forthcoming Issues
Page xii

2020: A Year for Clinical Microbiology
Nicole D. Pecora, Matthew A. Pettengill
Pages xiii-xiv

Modern Blood Culture: Management Decisions and Method Options
Mark D. Gonzalez, Timothy Chao, Matthew A. Pettengill
Pages 379-392

Panels and Syndromic Testing in Clinical Microbiology
Jennifer Dien Bard, Erin McElvania
Pages 393-420

Practical Aspects and Considerations When Planning a New Clinical Microbiology Laboratory
Dwight J. Hardy
Pages 421-431

Update on Susceptibility Testing: Genotypic and Phenotypic Methods
Romney M. Humphries
Pages 433-446

Clinical Pathogen Genomics
Andrew Cameron, Jessica L. Bohrhunter, Samantha Taffner, Adel Malek, Nicole D. Pecora
Pages 447-458

Coronavirus Detection in the Clinical Microbiology Laboratory: Are We Ready for Identifying and Diagnosing a Novel Virus?
Katharine Uhteg, Karen C. Carroll, Heba H. Mostafa
Pages 459-472

Update on Biosafety and Emerging Infections for the Clinical Microbiology Laboratory
Michael A. Pentella
Pages 473-482

Point-of-Care Testing in Microbiology
Linoj Samuel
Pages 483-494

Update in Pediatric Diagnostic Microbiology
James J. Dunn, Paula A. Revell
Pages 495-508

Antimicrobial Stewardship: What the Clinical Laboratory Needs to Know
Diana Alame, Bryan Hess, Claudine El-Beyrouty
Pages 509-520

Fellowship Training for the Future Clinical Microbiology Laboratory Director
Bobbi S. Pritt, Carrie A. Bowler, Elitza S. Theel
Pages 521-533

Novel Assays/Applications for Patients Suspected of Mycobacterial Diseases
Niaz Banaei, Kimberlee A. Musser, Max Salfinger, Akos Somoskovi, Adrian M. Zelazny
Pages 535-552

Food Safety Genomics and Connections to One Health and the Clinical Microbiology Laboratory
Marc W. Allard, Jie Zheng, Guojie Cao, Ruth Timme, … Eric W. Brown

Update in Infectious Disease Diagnosis in Anatomic Pathology
Alvaro C. Laga
Pages 565-585

Blood Banking and Transfusion Medicine Challenges During the COVID-19 Pandemic
Andy Ngo, Debra Masel, Christine Cahill, Neil Blumberg, Majed A. Refaai
Pages 587-601

Measuring the Serologic Response to Severe Acute Respiratory Syndrome Coronavirus 2: Methods and Meaning
Nicole D. Pecora, Martin S. Zand
Pages 603-614

The complement system plays a crucial role in the innate defense against common pathogens. Activation of complement leads to robust and efficient proteolytic cascades, which terminate in opsonization and lysis of the pathogen as well as in the generation of the classical inflammatory response through the production of potent proinflammatory molecules. More recently, however, the role of complement in the immune response has been expanded due to observations that link complement activation to adaptive immune responses. It is now appreciated that complement is a functional bridge between innate and adaptive immune responses that allows an integrated host defense to pathogenic challenges. As such, a study of its functions allows insight into the molecular underpinnings of host-pathogen interactions as well as the organization and orchestration of the host immune response. This review attempts to summarize the roles that complement plays in both innate and adaptive immune responses and the consequences of these interactions on host defense.

Phagocytosis is a cellular process for ingesting and eliminating particles larger than 0.5 μm in diameter, including microorganisms, foreign substances, and apoptotic cells. Phagocytosis is found in many types of cells and it is, in consequence an essential process for tissue homeostasis. However, only specialized cells termed professional phagocytes accomplish phagocytosis with high efficiency. Macrophages, neutrophils, monocytes, dendritic cells, and osteoclasts are among these dedicated cells. These professional phagocytes express several phagocytic receptors that activate signaling pathways resulting in phagocytosis. The process of phagocytosis involves several phases: i) detection of the particle to be ingested, ii) activation of the internalization process, iii) formation of a specialized vacuole called phagosome, and iv) maturation of the phagosome to transform it into a phagolysosome. In this review, we present a general view of our current understanding on cells, phagocytic receptors and phases involved in phagocytosis.

Phagosome maturation. The nascent phagosome gets transformed into a microbicidal vacuole, the phagolysosome, by sequential interactions with vesicles from the endocytic pathway. The process can be described in three stages of maturation: early (A), late (B), and phagolysosome (C). In this process, composition of the membrane changes to include molecules that control membrane fusion, such as the GTPases Rab5 and Rab7. The phagolysosome becomes increasingly acidic by the action of a proton-pumping V-ATPase and acquires various degradative enzymes, such as cathepsins, proteases, lysozymes, and lipases (scissors). EEA1, early endosome antigen 1; LAMP, lysosomal-associated membrane protein; NADPH, nicotinamide adenine dinucleotide phosphate oxidase.

Cells can die as a consequence of being phagocytosed by other cells — a form of cell death that has been called phagotrophy, cell cannibalism, programmed cell removal and primary phagocytosis. However, these are all different manifestations of cell death by phagocytosis (termed ‘phagoptosis’ for short). The engulfed cells die as a result of cytotoxic oxidants, peptides and degradative enzymes within acidic phagolysosomes. Cell death by phagocytosis was discovered by Metchnikov in the 1880s, but was neglected until recently. It is now known to contribute to developmental cell death in nematodes, Drosophila and mammals, and is central to innate and adaptive immunity against pathogens. Cell death by phagocytosis mediates physiological turnover of erythrocytes and other leucocytes, making it the most abundant form of cell death in the mammalian body. Immunity against cancer is also partly mediated by macrophage phagocytosis of cancer cells, but cancer cells can also phagocytose host cells and other cancer cells in order to survive. Recent evidence indicates neurodegeneration and other neuropathologies can be mediated by microglial phagocytosis of stressed neurons. Thus, despite cell death by phagocytosis being poorly recognized, it is one of the oldest, commonest and most important forms of cell death.

The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.

Model of hepatitis C virus particles (lipo-viral particles) secreted from cells. A: Most of the lipo-viral particles (LPV)’s membrane is a lipid monolayer. A bilayer-containing region is where the viral envelope proteins (i.e., E1 and E2) are inserted. Viral envelope proteins may also exist in the phospholipid monolayer membrane. Though the precise structure has not yet been determined, LVPs are believed to have multiple copies of Apo-E and less Apo-A1 molecules but only one Apo-B100 molecule (left). Some LPVs may not have Apo-B (right). Within the phospholipid monolayer, there are the core proteins wrapping the viral RNA genome and neutral lipids (e.g., cholesterol esters and triglycerides); B: Serum lipoproteins are possibly associated with hepatitis C virus particles in different ways. HCV: Hepatitis C virus.
Model of cell-free virus entry into hepatocytes. Hepatitis C virus (HCV) lipo-viral particles (LPVs) may be captured by DC-SIGN on the dendritic cells or L-SIGN on the endothelium in the sinusoidal space. After transfer to Space of Disse, HCV LPVs could attach to the hepatocytes through interacting with highly sulfated heparan sulfate proteoglycans, low-density lipoprotein receptor and scavenger receptor class B type 1 (1). This attachment allows the engagement of LPVs to cluster of differentiation 81 (CD81) and then induces the epidermal growth factor receptor receptor signaling (2). Lateral diffusion of the CD81–HCV complexes results in the association of CD81–HCV with Claudin-1 (3) and then OCLIN (4). Formation of the HCV–CD81–CLDN1–OCLIN complex allows viral particles internalized through clathrin-dependent endocytosis (5). Endosomal acidification induces the fusion of viral particles possibly through E1 and leads to the release of the viral genomic RNA into cytosol (6). HSPGs: Heparan sulfate proteoglycans; HCV: Hepatitis C virus; EGFR: Epidermal growth factor receptor; LDLR: Low-density lipoprotein receptor.
Hepatitis C virus protein translation. A: Translation of hepatitis C virus (HCV) genomic RNA is regulated by the internal ribosome entry site in the 5’-untranslated region (5’UTR) along with a short segment of the core gene sequence, the cis-acting replication element in the NS5B coding region and by the entire 3’UTR. Binding sites for eIF3 and 40S were marked. The start and stop codons for protein translation are marked by black squares, while two recognition sites on the 5’UTR for miR122 are marked by black rectangles; B: Polyprotein is co- and post-translationally cleaved by host or viral proteases to yield the structural proteins (core, E1 and E2) and the nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins). Core, E1 and E2 are processed by cellular signal peptidase (filled arrowhead). Mature core protein will be generated after further cleavage by signal peptide peptidase (empty arrowhead)[317]. The NS2/NS3 junction site is cleaved by the NS2–NS3 auto-protease[318] (empty arrow), and the remaining nonstructural proteins are processed by the NS3/4A proteinase[319] (filled arrow); C: All of the HCV proteins are associated with endoplasmic reticulum directly or indirectly[320]; D: Then, NS3, NS4A, NS4B, NS5A and NS5B proteins will form the replication complex. Core and NS5A proteins will be transferred to lipid droplets, while E1 and E2 proteins will stay in the assembly sites. IRES: Internal ribosome entry site; 5’UTR: 5’-untranslated region.

A proposed model for hepatitis C virus assembly. Lipid droplets (LDs) are surrounded by endoplasmic reticulum (ER)[321]. LDs with a hepatitis C virus (HCV) core and NS5A proteins are close to replication sites [double membrane vesicle (DMV)] and assembly sites. HCV genomic RNA synthesized by the replication complex (NS3–NS5B proteins) in the DMVs will be transferred by NS5A and NS3-4A proteins and encapsidated by the core proteins to form the nucleocapsid. Then, the HCV nucleocapsid will interact with glycoproteins E1/E2 in the assembly sites and bud into the ER lumen. Both Apo-B-dependent and -independent mechanisms are possibly involved in HCV particle assembly. One model shows the production of a fused form of HCV with very-low-density lipoproteins. Another model shows the budding of HCV particles with several apolipoproteins but not Apo-B. Nascent viral particles may be further lipidated by luminal lipoproteins and incorporated with exchangeable apolipoproteins. ER: Endoplasmic reticulum.

More than twenty years of study has provided a better understanding of hepatitis C virus (HCV) life cycle, including the general properties of viral RNA and proteins. This effort facilitates the development of sensitive diagnostic tools and effective antiviral treatments. At present, serologic screening test is recommended to perform on individuals in the high risk groups and nucleic acid tests are recommended to confirm the active HCV infections. Quantization and genotyping of HCV RNAs are important to determine the optimal duration of anti-viral therapy and predict the likelihood of response. In the early 2000s, pegylated interferon plus ribavirin became the standard anti-HCV treatment. However, this therapy is not ideal. To 2014, boceprevir, telaprevir, simeprevir, sofosbuvir and Harvoni are approved by Food and Drug Administration for the treat of HCV infections. It is likely that the new all-oral, interferon-free, pan-genotyping anti-HCV therapy will be available within the next few years. Majority of HCV infections will be cured by these anti-viral treatments. However, not all patients are expected to be cured due to viral resistance and the high cost of antiviral treatments. Thus, an efficient prophylactic vaccine will be the next challenge in the fight against HCV infection.

Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ε RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

(1) Reversible and non-cell-type specific attachment to cellassociated heparan sulfate proteoglycans.
(2) Specific and probably irreversible binding to an unknown hepatocyte-specific preS1-receptor. This step presumably requires activation of the virus resulting in exposure of the myristoylated N-terminus of the L-protein [1].
(3) Two different entry pathways have been proposed: (3A) endocytosis followed by release of nucleocapsids from
endocytic vesicles; (3B) fusion of the viral envelope at the plasma membrane.
(4) Cytoplasmic release of the viral nucleocapsid containing the relaxed circular partially double stranded DNA (rcDNA) with its covalently linked polymerase.
(5) Transport of the nucleocapsid along microtubules. Accumulation of the capsids at the nuclear envelope facilitates interactions with adaptor proteins of the nuclear pore complex.
(6) Possible trapping of the nucleocapsid in the nuclear basket and release of rcDNA into the nucleoplasm. The mechanisms determining the breakdown of the capsid and the release of the viral DNA genome are unsolved [2].
(7) ‘‘Repair” of the incoming rcDNA: Completion of the plus strand of the rcDNA by the viral polymerase. Removal of the polymerase from the 50-end of the minus strand
DNA. Removal of a short RNA-primer used for the DNAplus strand synthesis. Both processes are mediated by cellular enzymes [3].
(8) cccDNA formation by covalent ligation of both DNA strands (reviewed in [3]). The cccDNA molecule is organized as a chromatin-like structure displaying the typical beads-on-a string arrangement consisting of both histone and non-histone proteins (minichromosome) [4]. The lack of cccDNA in artificial host cells (e.g. hepatocytes of HBV transgenic mice) suggests that host specific factors may regulate cccDNA formation.
(9) Transcription. The cccDNA utilizes the cellular transcriptional machinery to produce all viral RNAs necessary for protein production and viral replication. Both host transcription factors, such as CCAAT/enhancer-binding protein (C/EBP) and hepatocyte nuclear factors (HNF) and viral proteins (core, the regulatory X-protein) regulate this process [4] and may modulate viral gene expression by interacting with the viral promoters of the four major overlapping open reading frames (ORFs): (I) the precore/ core gene, coding for the nucleocapsid protein and for the non-structural, secreted, precore protein, the HBeAg;
(II) the polymerase gene coding for the reverse transcriptase, RNase H and terminal protein domains; (III) the
L-,M-, and S-gene, coding for the three envelope proteins, which are synthesized in frame from different promoters; and (IV) the X gene, coding for the small regulatory X-protein. A correlation between viremia levels and the acetylation status of cccDNA-bound histones has been reported [5], indicating that epigenetic mechanisms can regulate the transcriptional activity of the cccDNA.
(10) All 4 major mRNAs utilize a single common polyadenylation signal. Processing of viral RNAs, nuclear export as well as stabilization of the viral RNAs appears to be exclusively mediated by host factors (i.e. La RNA binding protein).
(11) Translation of the pregenomic RNA (pgRNA) to the core protein and the viral polymerase. The regulatory X-protein and the three envelope proteins are translated from the subgenomic RNAs.
(12) Complex formation of the pgRNA (via its epsilon stemloop structure) with the core protein and the polymerase and self-assembly of an RNA-containing nucleocapsid.
(13) Reverse transcription of the pgRNA followed by plusstrand DNA-synthesis within the nucleocapsid. Maturation of the RNA-containing nucleocapsids to DNA-containing nucleocapsids within the cytoplasm.
(14) DNA-containing nucleocapsids can be either re-imported into the nucleus to form additional cccDNA molecules
(14A) or can be enveloped for secretion (14B). The envelope proteins are co-translationally inserted into the ER membrane, where they bud into the ER lumen, and are secreted by the cell, either as 22 nm subviral envelope particles (SVPs) or as 42 nm infectious virions (Dane particles) if they have enveloped the DNA-containing nucleocapsids before budding. During synthesis of the L-protein, the preS-domains remain cytoplasmically exposed and become myristoylated. At some step after preS-mediated nucleocapsid envelopment translocation across the membrane occurs.
(15) Experiments performed using duck hepatitis B revealed that the majority of cccDNA molecules in infected hepatocytes comes from newly synthesized nucleocapsids. 1–50 cccDNA molecules appear to accumulate per cell, though differences in cccDNA dynamics and efficiency of cccDNA accumulation may exist between HBV and the other hepadnaviruses. Both viral and host factors controlling cccDNA formation and pool size are yet poorly defined. A negativefeedback mechanism suppressing cccDNA amplification might involve the L-protein. As HBV polymerase inhibitors do not directly affect the cccDNA, a decrease in cccDNA levels is supposed to derive from the lack of sufficient recycling of viral nucleocapsids to the nucleus, due to inhibition of viral DNA-synthesis in the cytoplasm, and less incoming viruses from the blood [6].
(16) Compared to virions spherical and filamentous SVPs are secreted in a 103–106-fold excess into the blood of infected individuals. SVPs lack a nucleocapsid and are therefore non-infectious.

Gut Microbes. 2022; 14(1): 2055943.

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Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers in developing countries. ETEC is characterized by the ability to produce major virulence factors including colonization factors (CFs) and enterotoxins, that bind to specific receptors on epithelial cells and induce diarrhea. The gut microbiota is a stable and sophisticated ecosystem that performs a range of beneficial functions for the host, including protection against pathogen colonization. Understanding the pathogenic mechanisms of ETEC and the interaction between the gut microbiota and ETEC represents not only a research need but also an opportunity and challenge to develop precautions for ETEC infection. Herein, this review focuses on recent discoveries about ETEC etiology, pathogenesis and clinical manifestation, and discusses the colonization resistances mediated by gut microbiota, as well as preventative strategies against ETEC with an aim to provide novel insights that can reduce the adverse effect on human health.

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