Rapid Identification of Microorganisms to Enhance Clinical Care

At the conclusion of this presentation the participant will be able to: • Interpret rapid blood culture identification reports • Learn expected turn-around-times for positive culture results with identification and/ or antimicrobial susceptibility. • Learn how to locate antibiogram guides and empiric treatment tables Presentation: 20 minute

HIV Genotyping Cost Analysis with Follow-up as an Indicator

HIV-1 genotype (GHIV), HIV-1 Integrase (HIV1I) and HIV-1 Trophile (HIV1T) assays are sendout tests that incur a significant financial burden on the laboratory when ordered on inpatients who do not receive follow-up clinic visits. For these assays to be utilized in guiding antiretroviral therapy, the patient must receive follow-up. It will reduce the sendout budget by restricting these tests to the outpatient clinic setting

Workflow Modifications and Addition of MALDI-TOF Technology Significantly Improved Turn-Around-Time to Identification of Common Urine Pathogens

Background: In order to improve the identification of common aerobic urine cultures as well as antimicrobial susceptibility testing (AST) setup at an Academic Medical Center, work-flow modifications and MALDI-TOF technology were incorporated. Previously, the majority of species identification was achieved with conventional identification/antimicrobial susceptibility combo panels. All urine cultures, regardless of laboratory receipt time, were previously read once per day on 1st shift. Methods: The initial workflow modification involved addition of a 2nd shift urine culture reading. Urine specimens received from 8:00 AM to 4:00 PM were read on 1st shift, while urine specimens received from 4:00 PM to 8:00 AM were read on 2nd shift. Additionally, urine cultures were sorted into categories: no growth (NG) at 24 hours, no growth at \u3c24 hours, single colonies of growth, multiple colonies of growth, and potential contaminants. No growth cultures were signed out at 24 hours. No growth cultures at \u3c 24 hours were reincubated to be read on subsequent shift. Cultures with growth were set aside as either single colony types or multiple colony types. Cultures of probable contaminants were signed out. Once cultures were sorted, the isolated colonies underwent MALDI-TOF analysis (Bruker) and antimicrobial susceptibility testing (AST) as appropriate. Individual technologists setup the MALDITOF target plate map and spotted the associated target plate. AST was setup at the same time. The MALDI-TOF was then operated by a central technologist and results reported by the original technologist reading the culture. Results: Retrospective pre-workflow (September-November 2013) and post-workflow (May, June, October 2014) modification turn-around-times were compared for 16 commonly isolated pathogens. These pathogens consisted of common urine pathogens as noted in Table 1. Staphylococcus aureus was previously identified in our laboratory by a positive coagulase test and not included in this analysis. The average turn-around-times, standard deviations and the p-values for each organism are indicated in Table 1. Conclusion: Converting from conventional identification methods to MALDI-TOF, in conjunction with workflow modifications such as a 2nd culture reading, notably improved urine culture turn-around-time for identification and AST

Turn-Around-Time Improvements for Positive Blood Cultures from Incorporation of Workflow Modifications

Background: Emergence of direct from positive blood culture bottle identification (ID) methods reveal opportunities for improving bacterial ID and select resistance marker detection turn-around-times. Each system has various advantages and disadvantages; each institution must select the method/s that best fit the laboratory and patient needs. Here we elucidate improvements in 24 hour workflow through incorporating multiple rapid technologies for positive blood culture ID into a 24 hour algorithm. Methods: MALDI-TOF (Bruker) analysis with sepsityper extraction (aerobic Gram-positive and anaerobic bacteria); MALDI-TOF analysis with serum separator tube concentration (Gram-negative bacteria); and a FilmArray Blood Culture Panel (Biofire) were utilized. MALDI was utilized on 1st shift for single bacterium positives. FilmArray was performed on 2nd and 3rd shift for aerobic bottles and on 1st shift for gram-positive cocci in clusters and Candida. We examined all events during our pre-modification (September-November 2013) and post-modification (late-December 2014-March 2015) time periods and defined an event as the first positive blood culture for a patient within the examined data period. The Antimicrobial Stewardship Pharmacist (ASP) was notified with identifications and also KPC carbapenemase positives, to implement a carbapenem-resistant Enterobacteriaceae (CRE) empiric treatment algorithm. For KPC positives (CRE) a custom minimum inhibitory concentration (MIC) panel was utilized, replacing a standard susceptibility panel and Etests. Finally, 2nd shift began susceptibility setup on subcultured bloods that had turned positive from 11 p.m.-6 a.m. Results: Pre- and post- workflow modification average turn-around times (TAT) and p-values are shown in the Table. Detection of either the KPC or the mecA marker significantly improved the TAT needed for phenotypic detection of carbapenem or methicillin resistance. KPC was detected in 3 Enterobactericeae. Conclusions: Improvements to patient care are to be determined, but strong collaboration with ASP is anticipated to make a significant impact on patient outcomes. Of note, while having a universal Staphylococcus species target is useful, it can lead to complications with multi-species positive bottles. With the universal Staphylococcus species target, it is not possible to differentiate between a mixed coagulase negative Staphylococcus species (CNSS) versus Staphylococcus aureus when both are present as the CNSS may harbor the mecA target, preventing adequate treatment. Furthermore, a Staphylococcus lugdunensis specific marker would be clinically useful

Directed Carbapenemase Testing Is No Longer Just for Enterobacterales: Cost, Labor, and Workflow Assessment of Expanding Carbapenemase Testing to Carbapenem-Resistant \u3cem\u3eP. aeruginosa\u3c/em\u3e

Molecular carbapenem-resistance testing, such as for the presence of carbapenemases genes, is commonly implemented for the detection of carbapenemase-producing Enterobacterales. Carbapenemase-producing P. aeruginosa is also associated with significant morbidity and mortality, although; prevalence may be underappreciated in the United States due to a lack of carbapenemase testing. The present study sought to compare hands-on time, cost and workflow implementation of carbapenemase gene testing in Enterobacterales and P. aeruginosa isolates versus sending out isolates to a public health laboratory (PHL) for testing to assess if in-house can provide actionable results. The time to carbapenemase gene results were compared. Differences in cost for infection prevention measures were extrapolated from the time of positive carbapenemase gene detection in-house versus PHL. The median time to perform carbapenemase gene testing was 7.5 min (range 5–14) versus 10 min (range 8–22) for preparation to send isolates to the PHL. In-house testing produced same day results compared with a median of 6 days (range 3–14) to receive results from PHL. Cost of in-house testing and send outs were similar ($46.92 versus$40.53, respectively). If contact precautions for patients are implemented until carbapenemase genes are ruled out, in-house testing can save an estimated $76,836.60 annually. Extension of in-house carbapenemase testing to include P. aeruginosa provides actionable results 3–14 days earlier than PHL Standard Pathway testing, facilitating guided therapeutic decisions and infection prevention measures. Supplemental phenotypic algorithms can be implemented to curb the cost of P. aeruginosa carbapenemases testing by identifying isolates most likely to harbour carbapenemases

Cerebrospinal Fluid Cytokine and Chemokine Patterns in Central Nervous System Infections, Hemorrhage and Neoplasms

Cytokines and chemokines are soluble proteins that act as regulators of cellular functions throughout the body. Cytokines and chemokines released in the setting of various CNS disorders appear in the CSF compartment where determination of their levels can provide insight into pathogenic processes such as neuroinflammation. We utilized the Millipore HCYTOMAG 60K assay/kit/system to perform multiplex profiling of 42 different cytokines/chemokines in the CSF of patients with a variety of distinct CNS disease processes, including infection, hemorrhage and neoplasia. CNS infections included viral (Chronic Parechovirus type 3 (HPeV3), Enterovirus (EV) 68, Adenovirus, JC virus, West Nile virus), bacterial (Mycobacterium tuberculosis, Borrelia burgdorferi, Propionibacterium acnes, Staphylococcus epidermidis, Streptococcus sp.), fungal (Cryptococcus neoformans) and single celled parasite (Toxoplasma gondii). CSF specimens negative for infectious organisms in noninflammatory conditions were selected as controls. Additional non-infectious samples tested were obtained from patients with subarachnoid hemorrhage (SAH) and following surgery for glioblastoma. The glioblastoma samples were noteworthy in having negligible elevations in the cytokines/chemokines tested. CSF from patients with SAH was elevated in only MCP-1/CCL2. Distinct patterns of cytokine/chemokine expression were detected for each infectious patient population. Picornavirus infections HPeV3 and EV68 were associated with increased levels of the monocyte chemoattractant protein MCP-1/CCL2 when compared to non-infectious, non-inflammatory samples. In contrast to chronic HPeV3 infection, EV68 encephalitis was associated with increased CSF levels of additional cytokines; CCLX1, IL-4 and IL-7. Adenovirus infection was associated with markedly higher levels of fractalkine in CSF when compared to any of the other non-inflammatory, infectious, hemorrhage or tumor cases. CSF from a Mycobacterium tuberculosis infection demonstrated increased levels of a greater variety of cytokines/chemokines than any of the other groups tested. Patterns of cytokine/chemokine expression in the CNS reveal characteristics of the host innate response that provide insight into the disease process and potential targets for therapeutic intervention

Case Report Diagnostic Challenges of Cryptococcus neoformans in an Immunocompetent Individual Masquerading as Chronic Hydrocephalus

Cryptococcus neoformans can cause disseminated meningoencephalitis and evade immunosurveillance with expression of a major virulence factor, the polysaccharide capsule. Direct diagnostic assays often rely on the presence of the cryptococcal glucuronoxylomannan capsular antigen (CrAg) or visualization of the capsule. Strain specific phenotypic traits and environmental conditions influence differences in expression that can thereby compromise detection and timely diagnosis. Immunocompetent hosts may manifest clinical signs and symptoms indolently, often expanding the differential and delaying appropriate treatment and diagnosis. We describe a 63-year-old man who presented with a progressive four-year history of ambulatory dysfunction, headache, and communicating hydrocephalus. Serial lumbar punctures (LPs) revealed elevated protein (153-300 mg/dL), hypoglycorrhachia (19-47 mg/dL), lymphocytic pleocytosis (89-95% lymphocyte, WBC 67-303 mg/dL, and RBC 34-108 mg/dL), and normal opening pressure (13-16 cm H 2 O). Two different cerebrospinal fluid (CSF) CrAg assays were negative. A large volume CSF fungal culture grew unencapsulated C. neoformans. He was initiated on induction therapy with amphotericin B plus flucytosine and consolidation/maintenance therapy with flucytosine, but he died following discharge due to complications. Elevated levels of CSF Th1 cytokines and decreased IL6 may have affected the virulence and detection of the pathogen

Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy.</p> <p>Methods</p> <p>Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis.</p> <p>Results</p> <p>Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from <it>in vivo </it>animal models.</p> <p>Conclusion</p> <p>Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine.</p> <p>Trial Registration</p> <p>The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.</p

Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the <it>srv </it>mutant (MGAS5005Δ<it>srv</it>) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the <it>srv </it>mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δ<it>srv </it>to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δ<it>srv </it>was due only to the constitutive production of SpeB or to other changes inherent in the <it>srv </it>mutant strain. To address this question, we constructed a Δ<it>srv</it>Δ<it>speB </it>double mutant through allelic replacement (MGAS5005Δ<it>srv</it>Δ<it>speB</it>) and tested its ability to form biofilms <it>in vitro</it>.</p> <p>Findings</p> <p>Allelic replacement of <it>speB </it>in the <it>srv </it>mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.</p> <p>Conclusions</p> <p>The constitutive production of SpeB by the <it>srv </it>mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of <it>speB</it>/SpeB.</p

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms