24The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 25 brought with it rapid development of both molecular and serologic assays for identification of 26 COVID-19 infections. While Food and Drug Administration (FDA) emergency use authorization 27 (EUA) is required for clinical application of SARS-CoV-2 molecular tests, submission for EUA 28 is currently a voluntary process for manufacturers of serologic assays. The absence of FDA 29 oversight of serologic tests is concerning, given that the commercially available serologic assays 30 are highly variable, differing in their format, the antibody class detected, the targeted antigen and 31 the acceptable specimen types. An added complication is the lack of a clear understanding for 32 how such assays should be utilized and what the reported results ultimately indicate, or perhaps 33 more importantly, what they do not indicate. Here, we provide a brief summary of the 34 performance of a number of serologic assays reported in the literature, comment on what we do 35 and do not know regarding our immune response to SARS-CoV-2, and provide a number of 36 scenarios for which serologic testing will play a role in during our global response to this 37 pandemic. 38 39 40 41

Despite recent advances in diagnostic technology, microscopic examination of stool specimens remains central to the diagnosis of most pathogenic intestinal protozoa. Microscopy is, however, labor-intensive and requires a skilled technologist. New, highly sensitive diagnostic methods have been developed for protozoa endemic to developed countries, including Giardia lamblia (syn. G. intestinalis/G. duodenalis) and Cryptosporidium spp., using technologies that, if expanded, could effectively complement or even replace microscopic approaches. To date, the scope of such novel technologies is limited and may not include common protozoa such as Dientamoeba fragilis, Entamoeba histolytica, or Cyclospora cayetanensis. This minireview describes canonical approaches for the detection of pathogenic intestinal protozoa, while highlighting recent developments and FDA-approved tools for clinical diagnosis of common intestinal protozoa.

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Development of severe gastric diseases is strongly associated with those strains of Helicobacter pylori that contain the cag pathogenicity island (PAI) inserted into the chromosome. The cag PAI encodes a type IV secretion system that translocates the major disease-associated virulence protein, CagA, into the host epithelial cell. CagA then affects host signaling pathways, leading to cell elongations and inflammation. Since the precise mechanism by which the CagA toxin is translocated by the type IV secretion system remained elusive, we used fusion proteins and immunoprecipitation studies to identify CagA-interacting secretion components. Here we demonstrate that CagA, in addition to other yet-unidentified proteins, interacts with CagF, presumably at the inner bacterial membrane. This interaction is required for CagA translocation, since an isogenic nonpolar cagF mutant was translocation deficient. Our results suggest that CagF may be a protein with unique chaperone-like function that is involved in the early steps of CagA recognition and delivery into the type IV secretion channel.

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.

b Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species-and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications. Recent decades have seen advances in automation of traditional phenotypic and biochemical methods for microbial identification (ID), and advances in sequencing and the proliferation of genomic data hold great promise for further improvements. The development of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has brought microbial diagnostics to another cusp of rapid development. The speed and low cost of bacterial identification by MALDI-TOF MS make it an attractive technology in the clinical microbiology laboratory, and it has shown promise for identification of Gram-positive cocci (2, 6, 8), enteric and nonfermenting Gram-negative rods (11,21,24), HACEK organisms (10), anaerobes (14,17,19,20,31), and broad cohorts of clinically relevant bacteria (3,4,22,27,30).Commercial MALDI-TOF systems identify a broad range of microorganisms based on analysis of unique "fingerprints" of abundant proteins from whole cells or cellular extracts (15,23,26,28). These profiles are searched against databases of reference spectra, and similarity scores for the top database matches are used to determine the identification of unknown isolates. As observed previously, a systematic evaluation of scoring criteria on diverse isolates could improve results (2,10,25,27,29). Identification may be complicated when multiple species-or genus-level matches are among the top 10 results. Most current publications on the MALDI Biotyper system (Bruker Daltonics, Billerica, MA) do not address these complicated situations; however, one example where this problem is addressed is the use of the "10% rule," which stat...

We report three cases of infection due to the Gram-negative rod Ignatzschineria (Schineria) indica involving bacteremia and the urinary tract. Two cases were clearly associated with maggot infestation, and the third could conceivably have had unrecognized maggot infestation of the urinary tract. We believe these cases to be the first I. indica infections reported in association with maggot infestation and myiasis. CASE REPORTSC ase 1 is a 64-year-old homeless male who presented to the emergency department at the University of Louisville Hospital with the chief complaint of a painful left foot. His pertinent medical history included a motor vehicle accident 2 months prior to this admission, at which time he sustained lacerations to the dorsal aspect of the first three digits of his left foot. Because of his social situation, he had been unable to treat his wounds or change the dressings since the accident. He complained of extreme pain in the foot, which was exacerbated with any movement or pressure. He reported no other symptoms or past medical history. On physical examination, his left foot was edematous and erythematous surrounding the bandages. Following removal of the dressings, the wound revealed malodorous lacerations located on the dorsum of the foot and along the border of digits 1, 2, and 3 which expressed serous drainage. Maggots were observed in the wound and between the digits. All pedal pulses were palpable. Vital signs and the remainder of the physical examination were unremarkable.Laboratory studies revealed a normal white blood cell count (8,800/l with 64.2% granulocytes), an elevated erythrocyte sedimentation rate (ESR [57 mm/h]) and elevated C-reactive protein (CRP) level (1.06 mg/dl). X ray of the left foot demonstrated mild dorsal soft tissue swelling with no acute fracture or dislocation. However, magnetic resonance imaging (MRI) of the foot showed a fracture of the third middle phalanx with adjacent soft tissue defect. The clinical impression was osteomyelitis, although not seen on imaging, and the patient was started on empirical ampicillin-sulbactam (3 g intravenous [i.v.] every 6 h [q6h]) and vancomycin (1.25 g i.v., q12h). His wounds were redressed wet to dry with Dakin's solution, and the necrotic tissue was debrided with removal of the maggots. Despite conservative treatment, the third digit was considered unsalvageable, and the patient was taken to surgery for amputation of the digit. Histopathology noted skin ulceration and prominent acute and chronic inflammation extending to the soft tissue margin. On the second day postadmission, two aerobic blood cultures were positive for nonhemolytic Gram-negative short plump rods. The isolate produced a "yellowish" pigment on blood agar. The oxidase and indole tests were, respectively, positive and negative. The organism was identified as Alcaligenes faecalis (97% probability) (RapidID NF Plus; Remel, Lenexa, KS). Attempts to perform susceptibility testing were unsuccessful due to the organism's not growing in the Microscan Gram-negative pane...

The development and implementation of highly multiplexed molecular diagnostic tests have allowed clinical microbiology laboratories to more rapidly and sensitively detect a variety of pathogens directly in clinical specimens. Current US Food and Drug Administration-approved multiplex panels target multiple different organisms simultaneously and can identify the most common pathogens implicated in respiratory viral, gastrointestinal, or central nervous system infections. This review summarizes the test characteristics of available assays, highlights the advantages and limitations of multiplex technology for infectious diseases, and discusses potential utilization of these new tests in clinical practice.