Citations

Abstract BACKGROUND First introduced into clinical microbiology laboratories in Europe, MALDI-TOF MS is being rapidly embraced by laboratories around the globe. Although it has multiple applications, its widespread adoption in clinical microbiology relates to its use as an inexpensive, easy, fast, and accurate method for identification of grown bacteria and fungi based on automated analysis of the mass distribution of bacterial proteins. CONTENT This review provides a historical perspective on this new technology. Modern applications in the clinical microbiology laboratory are reviewed with a focus on the most recent publications in the field. Identification of aerobic and anaerobic bacteria, mycobacteria, and fungi are discussed, as are applications for testing urine and positive blood culture bottles. The strengths and limitations of MALDI-TOF MS applications in clinical microbiology are also addressed. SUMMARY MALDI-TOF MS is a tool for rapid, accurate, and cost-effective identification of cultured bacteria and fungi in clinical microbiology. The technology is automated, high throughput, and applicable to a broad range of common as well as esoteric bacteria and fungi. MALDI-TOF MS is an incontrovertibly beneficial technology for the clinical microbiology laboratory.

ABSTRACT Many viruses utilize cell adhesion molecules of the immunoglobulin superfamily as receptors. In particular, viruses of different classes exploit nectins. The large DNA viruses, herpes simplex and pseudorabies viruses, use ubiquitous nectins 1 and 2. The negative-strand RNA virus measles virus (MeV) uses tissue-specific nectin-4, and the positive-strand RNA virus poliovirus uses nectin-like 5 (necl-5), also known as poliovirus receptor. These viruses contact the BC, C′C″, and FG loops on the upper tip of their receptor's most membrane-distal domain. This location corresponds to the newly defined canonical adhesive interface of nectins, but how viruses utilize this interface has remained unclear. Here we show that the same key residues in the BC and FG loops of nectin-4 govern binding to the MeV attachment protein hemagglutinin (H) and cell entry, nectin-4 homodimerization, and heterodimerization with nectin-1. On the other hand, residues in the C′C″ loop necessary for homo- and heterotypic interactions are dispensable for MeV-induced fusion and cell entry. Remarkably, the C′C″ loop governs dissociation of the nectin-4 and H ectodomains. We provide formal proof that H can interfere with the formation of stable nectin-1/nectin-4 heterodimers. Finally, while developing an alternative model to study MeV spread, we observed that polarized primary pig airway epithelial sheets cannot be infected. We show that a single amino acid variant in the BC loop of pig nectin-4 fully accounts for restricted MeV entry. Thus, the three loops forming the adhesive interface of nectin-4 have different roles in supporting MeV H association and dissociation and MeV-induced fusion. IMPORTANCE Different viruses utilize nectins as receptors. Nectins are immunoglobulin superfamily glycoproteins that mediate cell-cell adhesion in vertebrate tissues. They interact through an adhesive interface located at the top of their membrane-distal domain. How viruses utilize the three loops forming this interface has remained unclear. We demonstrate that while nectin-nectin interactions require residues in all three loops, the association of nectin-4 with the measles virus hemagglutinin requires only the BC and FG loops. However, we discovered that residues in the C′C″ loop modulate the dissociation of nectin-4 from the viral hemagglutinin. Analogous mechanisms may support cell entry of other viruses that utilize nectins or other cell adhesion molecules of the immunoglobulin superfamily as receptors.

ABSTRACT Small colony variants (SCVs) are naturally occurring subpopulations of bacteria. The clinical characteristics and treatment outcomes of patients with prosthetic joint infection (PJI) caused by staphylococcal SCVs are unknown. This study was a retrospective series of 113 patients with staphylococcal PJI, with prospective testing of archived sonicate fluid samples. SCVs were defined using two-investigator review. Treatment failure was defined as (i) subsequent revision surgery for any reason, (ii) PJI after the index surgery, (iii) prosthesis nonreimplantation due to ongoing infection, or (iv) amputation of the affected limb. There were 38 subjects (34%) with SCVs and 75 (66%) with only normal-phenotype (NP) bacteria. Subjects with SCVs were more likely to have been on chronic antimicrobials prior to surgery ( P = 0.048), have had prior surgery for PJI ( P = 0.03), have had a longer duration of symptoms ( P = 0.0003), and have had a longer time since joint implantation ( P = 0.007), compared to those with only NP bacteria. Over a median follow-up of 30.6 months, 9 subjects (24%) with SCVs and 23 (32%) with only NP bacteria experienced treatment failure ( P = 0.51). Subjects infected with Staphylococcus aureus were more likely to fail than were those infected with Staphylococcus epidermidis (hazard ratio [HR], 4.03; 95% confidence interval [CI], 1.80 to 9.04). While frequently identified in subjects with PJI and associated with several potential predisposing factors, SCVs were not associated with excess treatment failure compared to NP infections in this study, where they were primarily managed with two-stage arthroplasty exchange. IMPORTANCE Bacteria with the small colony variant (SCV) phenotype are described in small case series as causing persistent or relapsing infection, but there are insufficient data to suggest that they should be managed differently than infection with normal-phenotype bacteria. In an effort to investigate the clinical importance of this phenotype, we determined whether SCVs were present in biofilms dislodged from the surfaces of arthroplasties of patients with staphylococcal prosthetic joint infection and assessed the clinical outcomes associated with detection of SCVs. We found that prosthetic joint infection caused by SCV staphylococci was associated with a longer duration of symptoms and more prior treatment for infection but not with an increased rate of treatment failure, compared to infection caused by normal-phenotype staphylococci.

A technique for the prevention of staphylococcal adhesion by electrical current exposure was investigated. Teflon coupons were exposed to a continuous flow of 10 3 cfu/ml Staphylococcus epidermidis with or without 2000 microA DC electrical current delivered by electrodes on opposite sides of a coupon, touching neither each other nor the coupon. A mean 3.46 (SD, 0.20) and 5.70 (SD, 1.03) log 10 cfu/cm 2 were adhered to the non-electrical current exposed coupons after 4 h and 24 h, respectively. A mean 2.46 (SD, 0.31) and 1.47 (SD, 0.73) log 10 cfu/cm 2 were adhered after 4 h and 24 h with exposure to 2000 microA electrical current delivered by graphite electrodes. A mean 2.21 (SD, 0.14) and 0.55 (SD, 0.00) log 10 cfu/cm 2 were adhered after 4 h and 24 h with exposure to 2000 microA electrical current delivered by stainless steel electrodes. Electrical current may be useful in the prevention of staphylococcal adhesion to biomaterials.

ABSTRACT The detection of pathogens associated with gastrointestinal disease may be important in certain patient populations, such as immunocompromised hosts, the critically ill, or individuals with prolonged disease that is refractory to treatment. In this study, we evaluated two commercially available multiplex panels (the FilmArray gastrointestinal [GI] panel [BioFire Diagnostics, Salt Lake City, UT] and the Luminex xTag gastrointestinal pathogen panel [GPP] [Luminex Corporation, Toronto, Canada]) using Cary-Blair stool samples ( n = 500) submitted to our laboratory for routine GI testing (e.g., culture, antigen testing, microscopy, and individual real-time PCR). At the time of this study, the prototype (non-FDA-cleared) FilmArray GI panel targeted 23 pathogens (14 bacterial, 5 viral, and 4 parasitic), and testing of 200 μl of Cary-Blair stool was recommended. In contrast, the Luminex GPP assay was FDA cleared for the detection of 11 pathogens (7 bacterial, 2 viral, and 2 parasitic), but had the capacity to identify 4 additional pathogens using a research-use-only protocol. Importantly, the Luminex assay was FDA cleared for 100 μl raw stool; however, 100 μl Cary-Blair stool was tested by the Luminex assay in this study. Among 230 prospectively collected samples, routine testing was positive for one or more GI pathogens in 19 (8.3%) samples, compared to 76 (33.0%) by the FilmArray and 69 (30.3%) by the Luminex assay. Clostridium difficile (12.6 to 13.9% prevalence) and norovirus genogroup I (GI)/GII (5.7 to 13.9% prevalence) were two of the pathogens most commonly detected by both assays among prospective samples. Sapovirus was also commonly detected (5.7% positive rate) by the FilmArray assay. Among 270 additional previously characterized samples, both multiplex panels demonstrated high sensitivity (>90%) for the majority of targets, with the exception of several pathogens, notably Aeromonas sp. (23.8%) by FilmArray and Yersinia enterocolitica (48.1%) by the Luminex assay. Interestingly, the FilmArray and Luminex panels identified mixed infections in 21.1% and 13.0% of positive prospective samples, respectively, compared to only 8.3% by routine methods.

AbstractBackgroundLow HCV has recently been qualified by the European Medicines Agency as a biomarker for enrichment of clinical trials in predementia stages of Alzheimer's disease. For automated methods to meet the necessary regulatory requirements, it is essential they be standardized and their performance be well characterized.MethodsThe within‐image and between‐field strength reproducibility of automated hippocampal volumetry using the Learning Embeddings for Atlas Propagation (or LEAP) algorithm was assessed on 153 Alzheimer's Disease Neuroimaging Initiative subjects.ResultsTests/retests at 1.5 T and 3 T, and a comparison between 1.5 T and 3 T, yielded average unsigned variabilities in HCVs of 1.51%, 1.52%, and 2.68%. A small bias between field strengths (mean signed difference, 1.17%; standard deviation, 3.07%) was observed.ConclusionsThe measured reproducibility characteristics confirm the suitability of using automated magnetic resonance imaging analyses to assess HCVs quantitatively and to represent a fundamental characterization that is critical to meet the regulatory requirements for using hippocampal volumetry in clinical trials and health care.

AbstractBackgroundRegulatory qualification of a biomarker for a defined context of use provides scientifically robust assurances to sponsors and regulators that accelerate appropriate adoption of biomarkers into drug development.MethodsThe Coalition Against Major Diseases submitted a dossier to the Scientific Advice Working Party of the European Medicines Agency requesting a qualification opinion on the use of hippocampal volume as a biomarker for enriching clinical trials in subjects with mild cognitive impairment, incorporating a scientific rationale, a literature review and a de novo analysis of Alzheimer's Disease Neuroimaging Initiative data.ResultsThe literature review and de novo analysis were consistent with the proposed context of use, and the Committee for Medicinal Products for Human Use released an opinion in November 2011.ConclusionsWe summarize the scientific rationale and the data that supported the first qualification of an imaging biomarker by the European Medicines Agency.

Staphylococcus aureusis capable of causing a spectrum of human illnesses. During seriousS. aureusinfections, the staphylococcal pathogen-associated molecular patterns (PAMPs) such as peptidoglycan, lipoteichoic acid, and lipoproteins and even intactS. aureus, are believed to act in conjunction with the staphylococcal superantigens (SSAg) to activate the innate and adaptive immune system, respectively, and cause immunopathology. However, recent studies have shown that staphylococcal PAMPs could suppress inflammation by several mechanisms and protect from staphylococcal toxic shock syndrome, a life-threatening systemic disease caused by toxigenicS. aureus. Given the contradictory pro- and anti-inflammatory roles of staphylococcal PAMPs, we examined the effects ofS. aureus-derived molecular patterns on immune responses driven by SSAgin vivousing HLA-DR3 and HLA-DQ8 transgenic mice. Our study showed that neitherS. aureus-derived peptidoglycans (PGN), lipoteichoic acid (LTA), nor heat-killedStaphylococcus aureus(HKSA) inhibited SSAg-induced T cell proliferationin vitro. They failed to antagonize the immunostimulatory effects of SSAgin vivoas determined by their inability to attenuate systemic cytokine/chemokine response and reduce SSAg-induced T cell expansion. These staphylococcal PAMPs also failed to protect HLA-DR3 as well as HLA-DQ8 transgenic mice from either SSAg-induced toxic shock or pneumonia induced by a SSAg-producing strain ofS. aureus.