Wound-Healing Markers Revealed by Proximity Extension Assay in Tears of Patients following Glaucoma Surgery.

Tears are a constantly available and highly valuable body fluid collectable by non-invasive techniques. Although it can give information on ocular status and be used for follow-ups, tear analysis is challenging due to the low amount of sample that is available. Proximity extension assay (PEA) allows for a sensitive and scalable analysis of multiple proteins in a single run from a one-µL sample, so we applied this technique and examined the amount of 184 proteins in tears collected at different time points after trabeculectomy. The success rate of this surgical intervention highly depends on proper wound healing; therefore, information on the process is indispensable. We observed significantly higher levels of IL-6 and MMP1 at the early time points (day one, two, and four) following trabeculectomy, and the protein amounts went back to the level observed before the surgery three months after the intervention. Patients with or without complications were tested, and proteins that have roles in the immune response and wound healing could be observed with altered frequency and amounts in the cases of patients with complications. Our results highlight the importance of inflammation in wound-healing complications, and at the same time, indicate the utility of PEA in tear analysis.

Performance of Vitek 2 for Antimicrobial Susceptibility Testing of Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia with Vitek 2 (2009 FDA) and CLSI M100S 26th Edition Breakpoints.

The performances of Vitek 2 AST-GN69 and AST-XN06 cards were compared to Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) for 99 isolates of Pseudomonas aeruginosa, 26 Acinetobacter baumannii isolates, and 11 Stenotrophomonas maltophilia isolates. In total, 15 antimicrobials were evaluated, with 11 for P. aeruginosa, 14 for A. baumannii, and 2 for S. maltophilia Categorical agreement (CA) was assessed using both Vitek 2 breakpoints and 2016 CLSI M100S 26th edition breakpoints. The essential agreement values for P. aeruginosa, A. baumannii, and S. maltophilia were 99.5%, 99.2%, and 100%, respectively. The CA values for P. aeruginosa, A. baumannii, and S. maltophilia were 94.1%, 92.7%, and 95.5%, respectively, by the Vitek 2 breakpoints, and 93.4%, 92.3%, and 95.5%, respectively, by the CLSI breakpoints. Overall, the Vitek 2 performance was comparable to that of BMD using both Vitek 2 breakpoints and 2016 CLSI M100S 26th edition breakpoints. Improved performance was noted for the reformulated piperacillin-tazobactam and imipenem found on the AST-GN69 card, with no very major or major errors noted when using the CLSI breakpoints.

Right-Sizing Technology in the Era of Consumer-Driven Health Care.

Technology for modern clinical and public health microbiology laboratories has evolved at an impressive rate over the last two decades. Contemporary diagnostics can rapidly provide powerful data that can impact patient lives and support infectious disease outbreak investigations. At the same time, dramatic changes to health care delivery are putting new pressures on a system that is now focusing on patient-centric, value-driven, convenient care. For laboratories, balancing all these demands in a cost-contained environment remains a challenge. This article explores the current and future directions of diagnostics in our dynamic health care environment.

Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011.

BACKGROUND: Mucormycosis is a fungal infection caused by environmentally acquired molds. We investigated a cluster of cases of cutaneous mucormycosis among persons injured during the May 22, 2011, tornado in Joplin, Missouri. METHODS: We defined a case as a soft-tissue infection in a person injured during the tornado, with evidence of a mucormycete on culture or immunohistochemical testing plus DNA sequencing. We conducted a case-control study by reviewing medical records and conducting interviews with case patients and hospitalized controls. DNA sequencing and whole-genome sequencing were performed on clinical specimens to identify species and assess strain-level differences, respectively. RESULTS: A total of 13 case patients were identified, 5 of whom (38%) died. The patients had a median of 5 wounds (range, 1 to 7); 11 patients (85%) had at least one fracture, 9 (69%) had blunt trauma, and 5 (38%) had penetrating trauma. All case patients had been located in the zone that sustained the most severe damage during the tornado. On multivariate analysis, infection was associated with penetrating trauma (adjusted odds ratio for case patients vs. controls, 8.8; 95% confidence interval [CI], 1.1 to 69.2) and an increased number of wounds (adjusted odds ratio, 2.0 for each additional wound; 95% CI, 1.2 to 3.2). Sequencing of the D1-D2 region of the 28S ribosomal DNA yielded Apophysomyces trapeziformis in all 13 case patients. Whole-genome sequencing showed that the apophysomyces isolates were four separate strains. CONCLUSIONS: We report a cluster of cases of cutaneous mucormycosis among Joplin tornado survivors that were associated with substantial morbidity and mortality. Increased awareness of fungi as a cause of necrotizing soft-tissue infections after a natural disaster is warranted.

Evaluation of Surrogate Disk Tests for Detection of Ciprofloxacin and Levofloxacin Resistance in Clinical Isolates of Salmonella enterica.

Detection of fluoroquinolone resistance in Salmonella enterica has become increasingly difficult due to evolving resistance mechanisms to this antimicrobial class in this organism. We evaluated two quinolone disks and five fluoroquinolone disks for their ability to act as a surrogate agent for the detection of fluoroquinolone resistance in a collection of 136 S. enterica isolates, including 111 with intermediate or resistant ciprofloxacin MICs mediated by a variety of resistance mechanisms. Ciprofloxacin, ofloxacin, and pefloxacin disks detected all isolates resistant to ciprofloxacin (0% very major error) and yielded false resistance (major error) in 8, 4, and 12% of susceptible isolates, respectively. Ciprofloxacin and pefloxacin provided clearer differentiation of susceptible and resistant isolates.

Comparison of the AmpliVue, BD Max System, and illumigene Molecular Assays for Detection of Group B Streptococcus in Antenatal Screening Specimens.

The performances of the AmpliVue, BD Max, and illumigene group B Streptococcus (GBS) nucleic acid amplification tests (NAATs) were compared to that of enriched culture for detection of GBS in antenatal screening specimens. Two hundred specimens were tested simultaneously with the NAATs, following 18 to 24 h of Lim broth enrichment; 15% of specimens were culture positive for GBS, whereas 31.5% were positive by at least one NAAT. All three NAATs were more sensitive (sensitivity, 90.9 to 100%) than culture (sensitivity, 53.6%).

Performance of Vitek 2 for antimicrobial susceptibility testing of Enterobacteriaceae with Vitek 2 (2009 FDA) and 2014 CLSI breakpoints.

Vitek 2 (bioMérieux Inc., Durham, NC) is a widely used commercial antimicrobial susceptibility test system. We compared the MIC results obtained using the Vitek 2 AST-GN69 and AST-XN06 cards to those obtained by CLSI broth microdilution (BMD) for 255 isolates of Enterobacteriaceae, including 25 isolates of carbapenem-resistant Enterobacteriaceae. In total, 25 antimicrobial agents were examined. For 10 agents, the MIC data were evaluated using two sets of breakpoints: (i) the Vitek 2 breakpoints, which utilized the 2009 FDA breakpoints at the time of the study and are equivalent to the 2009 CLSI M100-S19 breakpoints, and (ii) the 2014 CLSI M100-S24 breakpoints. There was an overall 98.7% essential agreement (EA). The categorical agreement was 95.5% (CA) using the Vitek 2 breakpoints and 95.7% using the CLSI breakpoints. There was 1 very major error (VME) (0.05%) observed using the Vitek 2 breakpoints (cefazolin) and 8 VMEs (0.5%) using the CLSI breakpoints (2 each for aztreonam, cefepime, and ceftriaxone, and 1 for cefazolin and ceftazidime). Fifteen major errors (MEs) (0.4%) were noted using the Vitek 2 breakpoints and 8 (0.5%) using the CLSI breakpoints. Overall, the Vitek 2 performance was comparable to that of BMD for testing a limited number of Enterobacteriaceae commonly isolated by clinical laboratories. Ongoing studies are warranted to assess performance in isolates with emerging resistance.

Performance of Etest and disk diffusion for detection of ciprofloxacin and levofloxacin resistance in Salmonella enterica.

We compared Etest and disk diffusion to broth microdilution for the detection of fluoroquinolone resistance in 135 typhoidal and nontyphoidal serovars of Salmonella. Categorical agreements for the ciprofloxacin and levofloxacin Etests were 89.6 and 83.7%, respectively. Disk diffusion categorical agreements were 88.2 and 93.3%, respectively. Only minor errors were observed.

Real-time PCR assays for genotyping of Cryptococcus gattii in North America.

BACKGROUND: Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen. METHODS: We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human. RESULTS: Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA. CONCLUSIONS: The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies.

A large community outbreak of blastomycosis in Wisconsin with geographic and ethnic clustering.

BACKGROUND: Blastomycosis is a potentially life-threatening infection caused by the soil-based dimorphic fungus Blastomyces dermatitidis, which is endemic throughout much of the Midwestern United States. We investigated an increase in reported cases of blastomycosis that occurred during 2009-2010 in Marathon County, Wisconsin. METHODS: Case detection was conducted using the Wisconsin Electronic Disease Surveillance System (WEDSS). WEDSS data were used to compare demographic, clinical, and exposure characteristics between outbreak-related and historical case patients, and to calculate blastomycosis incidence rates. Because initial mapping of outbreak case patients' homes and recreational sites demonstrated unusual neighborhood and household case clustering, we conducted a 1:3 matched case-control study to identify factors associated with being in a geographic cluster. RESULTS: Among the 55 patients with outbreak-related cases, 33 (70%) were hospitalized, 2 (5%) died, 30 (55%) had cluster-related cases, and 20 (45%) were Hmong. The overall incidence increased significantly since 2005 (average 11% increase per year, P < .001), and incidence during 2005-2010 was significantly higher among Asians than non-Asians (2010 incidence: 168 vs 13 per 100 000 population). Thirty of the outbreak cases grouped into 5 residential clusters. Outdoor activities were not risk factors for blastomycosis among cluster case patients or when comparing outbreak cases to historical cases. CONCLUSIONS: This outbreak of blastomycosis, the largest ever reported, was characterized by unique household and neighborhood clustering likely related to multifocal environmental sources. The reasons for the large number of Hmong affected are unclear, but may involve genetic predisposition.

Pseudo-outbreak of Lecanicillium and Acremonium species in orthopedic surgery patients.

Acremonium species cause a variety of human infections, while Lecanicillium species have not been reported as human pathogens. We describe a pseudo-outbreak involving both organisms, highlighting the role and limitations of molecular methods in the characterization of rare fungal isolates. Repeated isolation of these fungi from patient tissue samples raises concerns about exogenous contamination in the hospital environment.

Murine visceral leishmaniasis: IgM and polyclonal B-cell activation lead to disease exacerbation.

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post-infection there is a marked B-cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post-infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti-leishmania response. Although B-cell-deficient JhD BALB/c mice are relatively resistant to infection, neither B-cell-derived IL-10 nor B-cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JhD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non-specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JhD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post-infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL-10 or IFN-gamma). Overall these studies indicate that polyclonal B-cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.

Novel electronic biosensor for automated inoculum preparation to accelerate antimicrobial susceptibility testing.

A key predictor of morbidity and mortality for patients with a bloodstream infection is time to appropriate antimicrobial therapy. Accelerating antimicrobial susceptibility testing from positive blood cultures is therefore key to improving patient outcomes, yet traditional laboratory approaches can require 2-4 days for actionable results. The eQUANT-a novel instrument utilizing electrical biosensors-produces a standardized inoculum equivalent to a 0.5 McFarland directly from positive blood cultures. This proof-of-concept study demonstrates that eQUANT inocula prepared from clinically significant species of Enterobacterales were comparable to 0.5 McF inocula generated from bacterial colonies in both CFU/ml concentration and performance in antimicrobial susceptibility testing, with ≥ 95% essential and categorical agreement for VITEK2 and disk diffusion. The eQUANT, combined with a rapid, direct from positive blood culture identification technique, can allow the clinical laboratory to begin antimicrobial susceptibility testing using a standardized inoculum approximately 2-3 h after a blood culture flags positive. This has the potential to improve clinical practice by accelerating conventional antimicrobial susceptibility testing and the resulting targeted antibiotic therapy.

Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2.

Herpes simplex virus (HSV) type 2 infection occurs primarily at the genital mucosal surfaces and is a leading cause of ulcerative lesions. Despite the availability of animal models for HSV-2 infection, little is known regarding the mechanism of immune induction within the vaginal mucosa. Here, we examined the cell types responsible for the initiation of protective Th1 immunity to HSV-2. Intravaginal inoculation of HSV-2 led to a rapid recruitment of submucosal dendritic cells (DCs) to the infected epithelium. Subsequently, CD11c(+) DCs harboring viral peptides in the context of MHC class II molecules emerged in the draining lymph nodes and were found to be responsible for the stimulation of IFNgamma secretion from HSV-specific CD4(+) T cells. Other antigen-presenting cells including B cells and macrophages did not present viral peptides to T cells in the draining lymph nodes. Next, we assessed the relative contribution to immune generation by the Langerhans cells in the vaginal epithelium, the submucosal CD11b(+) DCs, and the CD8alpha(+) lymph node DCs. Analysis of these DC populations from the draining lymph nodes revealed that only the CD11b(+) submucosal DCs, but not Langerhans cell-derived or CD8alpha(+) DCs, presented viral antigens to CD4(+) T cells and induced IFNgamma secretion. These results demonstrate a previously unanticipated role for submucosal DCs in the generation of protective Th1 immune responses to HSV-2 in the vaginal mucosa, and suggest their importance in immunity to other sexually transmitted diseases.

Utility of a Luminex-based assay for multiplexed, rapid species identification of Candida isolates from an ongoing candidemia surveillance.

A Candida-specific Luminex-based assay with 11 probes was employed for multiplexed, rapid identification of 1182 Candida sp. isolates that were received as part of an ongoing population-based surveillance. All the Candida isolates were previously identified by a combination of methods, including phenotype and sequence analysis. Results showed that the Luminex assay was an attractive alternative to reference methods, as it is rapid, yields correct species identification, and is user friendly.

Comparative analysis of cytokine profiles of glaucomatous tears and aqueous humour reveals potential biomarkers for trabeculectomy complications.

Glaucoma is a multifactorial neurodegenerative disease that causes impaired vision and, in advanced cases, blindness. The increasing prevalence of glaucoma due to an ageing population has necessitated the identification of suitable biomarkers for the early detection of the disease. Aqueous humour (AH) has been proposed as a source of biomarkers, but it can only be collected using a minor, yet invasive surgical intervention. Tears, however, are constantly available and can be collected any time via noninvasive methods. In order to examine the utility of tear as a surrogate for aqueous humour in biomarker development, we compared the levels of 27 cytokines and chemokines in paired samples of tear and aqueous humour using a Luminex multiplex immunobead-based technique. Significantly higher levels of cytokines in tear compared to aqueous humour were detected suggesting that tear and aqueous humour are not identical in terms of inflammation response. Furthermore, the levels of IFN-γ, GM-CSF and IL-5 in tear were significantly lower in patients who developed complications after one year, but no statistically significant changes in cytokine levels were observed in aqueous humour. These three molecules may have potential as predictive biomarkers for the appearance of late flap-related complications of trabeculectomy.

Longitudinal Changes in Corneal Cell and Nerve Fiber Morphology in Young Patients with Type 1 Diabetes with and without Diabetic Retinopathy: A 2-Year Follow-up Study.

Purpose: We have previously used in vivo corneal confocal microscopy (IVCCM) to demonstrate significant alterations in the corneal epithelial cells, stromal keratocytes, and subbasal nerves in young patients with type 1 diabetes mellitis (T1DM), especially those with diabetic retinopathy (DR). We have evaluated the change in corneal cellular and subbasal nerve morphology over 2 years in young patients with T1DM with or without DR. Methods: A total of 19 patients with T1DM, without (n = 12) and with (n = 7) DR and 19 age- and sex-matched healthy control subjects underwent quantification of corneal cellular and subbasal nerve plexus morphology by using IVCCM at baseline and after 2 years. Results: There was no significant change in corneal basal epithelial, posterior stromal keratocyte, or endothelial cell densities over 2 years. However, there was a significant reduction in corneal nerve branch (P = 0.03) and total nerve branch density (P = 0.04) in patients without DR and a significant reduction in corneal nerve fibre density (P = 0.004) in those with DR. Conclusions: IVCCM can detect a progressive loss of corneal nerve fibers in young patients with T1DM and may allow the identification of individuals at risk of neuropathy progression for more active risk factor reduction.

Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms.

Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness among patients with diabetes. The appearance and the severity of the symptoms correlate with the duration of diabetes and poor blood glucose level management. Diabetic retinopathy is also categorized as a chronic low-level inflammatory disease; the high blood glucose level promotes the accumulation of the advanced glycation end products and leads to the stimulation of monocytes and macrophages. Examination of protein level alterations in tears using state-of the art proteomics techniques have identified several proteins as possible biomarkers for the different stages of the diabetic retinopathy. Some of the differentially expressed tear proteins have a role in the barrier function of tears linking the diabetic retinopathy with another eye complication of diabetes, namely the diabetic keratopathy resulting in impaired wound healing. Understanding the molecular events leading to the eye complications caused by hyperglycemia may help the identification of novel biomarkers as well as therapeutic targets in order to improve quality of life of diabetic patients. BIOLOGICAL SIGNIFICANCE: Diabetic retinopathy (DR), the leading cause of blindness among diabetic patients can develop without any serious symptoms therefore the early detection is crucial. Because of the increasing prevalence there is a high need for improved screening methods able to diagnose DR as soon as possible. The non-invasive collection and the relatively high protein concentration make the tear fluid a good source for biomarker discovery helping the early diagnosis. In this work we have reviewed the administration of advanced proteomics techniques used in tear biomarker studies and the identified biomarkers with potential to improve the already existing screening methods for DR detection.

Quantitative body fluid proteomics in medicine - A focus on minimal invasiveness.

Identification of new biomarkers specific for various pathological conditions is an important field in medical sciences. Body fluids have emerging potential in biomarker studies especially those which are continuously available and can be collected by non-invasive means. Changes in the protein composition of body fluids such as tears, saliva, sweat, etc. may provide information on both local and systemic conditions of medical relevance. In this review, our aim is to discuss the quantitative proteomics techniques used in biomarker studies, and to present advances in quantitative body fluid proteomics of non-invasively collectable body fluids with relevance to biomarker identification. The advantages and limitations of the widely used quantitative proteomics techniques are also presented. Based on the reviewed literature, we suggest an ideal pipeline for body fluid analyses aiming at biomarkers discoveries: starting from identification of biomarker candidates by shotgun quantitative proteomics or protein arrays, through verification of potential biomarkers by targeted mass spectrometry, to the antibody-based validation of biomarkers. The importance of body fluids as a rich source of biomarkers is discussed. SIGNIFICANCE: Quantitative proteomics is a challenging part of proteomics applications. The body fluids collected by non-invasive means have high relevance in medicine; they are good sources for biomarkers used in establishing the diagnosis, follow up of disease progression and predicting high risk groups. The review presents the most widely used quantitative proteomics techniques in body fluid analysis and lists the potential biomarkers identified in tears, saliva, sweat, nasal mucus and urine for local and systemic diseases.

Early Corneal Cellular and Nerve Fiber Pathology in Young Patients With Type 1 Diabetes Mellitus Identified Using Corneal Confocal Microscopy.

PURPOSE: The aim of this study was to quantify epithelial, stromal, and endothelial cell density, and subbasal nerve morphology in young patients with type 1 diabetes mellitus with and without diabetic retinopathy. METHODS: A total of 28 young patients (mean age, 22.86 ± 9.05 years) with type 1 diabetes, with (n = 18) and without (n = 10) retinopathy, and 17 age-matched healthy control subjects (mean age, 26.53 ± 2.43 years) underwent corneal confocal microscopy (CCM). RESULTS: We found significantly lower epithelial (P < 0.0001) and endothelial (P = 0.001) cell densities and higher keratocyte cell density (P = 0.024) in patients with type 1 diabetes compared to controls. Significantly lower corneal nerve fiber density (P = 0.004), nerve branch density (P = 0.004), total nerve branch density (P = 0.04), and nerve fiber length (P = 0.001), and greater nerve fiber width (P = 0.04) were observed in patients with type 1 diabetes compared to control subjects. Significantly lower epithelial (P < 0.001) and endothelial (P = 0.02) cell densities, nerve branch density (P = 0.02), and nerve fiber length (P = 0.04), and significantly higher keratocyte cell density (P = 0.02) were found in patients with type 1 diabetes without retinopathy compared to control subjects. CONCLUSIONS: Corneal confocal microscopy identifies corneal cellular and small nerve fiber pathology in young patients with type 1 diabetes without retinopathy, which increases in severity in those with retinopathy. Corneal confocal microscopy appears to have considerable use as an imaging biomarker for early subclinical pathology in young patients with type 1 diabetes mellitus.