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Coagulase Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Coagulase and links to relevant pages within the course.

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Case Studies in Clinical Microbiology
The bacterial cells shown in the image were observed in a smear prepared from the colony shown before. Which of the following tests will help to affirm the identification of Staphylococcus aureus?View Page
The tube coagulase test, shown in this image (upper tube positive), should be performed on all S. aureus-suspicious isolates giving a negative slide coagulase reaction.View Page

Microbiology / Serology Question Bank - Review Mode (no CE)
Which of the following organisms will give a positive coagulase test:View Page
The slide coagulase test is a rapid method for identifying which of the following organisms.View Page
Which of the following is a presumptive test for the identification of Lancefield group A Streptococcus:View Page
Which one of the following tests would be positive in the presence of Klebsiella:View Page
Which two of the following tests are helpful for documenting previous Streptococcal throat and skin infections:View Page

Molecular Methods in Clinical Microbiology
Identification of Staphylococcus aureus with Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH)

Staphylococcus aureus, particularly methicillin resistant strains (MRSA), have represented a likely target for molecular development, particularly in blood cultures. As more institutions implement patient screening protocols for MRSA, replacement of routine culture methods with molecular assays has gained increasing attention.PNA-FISH assays provide for the definitive identification of Staphylococcus aureus from positive blood culture vials. Peptide nucleic acid fluorescent in-situ hybridization is a relatively straight forward procedure that does not involve amplification and has limited equipment requirements. Procedurally it is easy to perform with minimal hands on time.PNA is a synthetic imitator of a nucleic acid sequence in which the backbone is a pseudopeptide rather than a sugar. PNA behaves similarly to DNA and will bind to complementary nucleic acid strands. A PNA probe is constructed, utilizing a complementary, hybridizing sequence for a known nucleic acid target sequence. The probe is typically bound to a fluorescent protein as a means of visualizing/detecting the target. In one commercially available method, once a blood culture vial demonstrates gram-positive cocci in clusters, a drop of the blood culture broth is added to fixation solution on a slide. Heat or methanol is used to fix the smear. After fixation, probe that targets species-specific ribosomal RNA is added to the smear, which is then cover-slipped.Slides are then incubated at 55oC. Post incubation, slides are immersed in a preheated wash solution and coverslips gently removed. After incubation in the wash solution, smears are air dried; a drop of mounting medium is added and the slide is cover-slipped again.The slides are examined with a fluorescent microscope, utilizing specific filters. Green fluorescing cocci in clusters are identified as Staphylococcus aureus. This identification would be available, depending on the routine identification system utilized, potentially 24 hours earlier than the norm.A significant number of blood cultures that demonstrate gram-positive cocci in clusters yield coagulase negative staphylococci (CNS), which represent potential contaminants, rather than significant infection. What is the significance of differentiating blood cultures that contain S. aureus from those that are growing CNS in a much earlier timeframe?Studies have shown that IF the differentiation of CNS from S. aureus is effectively communicated to clinicians and pharmacy/antimicrobial stewardship teams, active assessment can occur utilizing defined exclusion criteria for those patients whose cultures yielded CNS rather than S. aureus. In scenarios where contamination rather than infection is indicated, vancomycin can be discontinued earlier, and length of hospital stay is also shortened. Reduced antibiotic exposure, reduced risk of development of resistance, and reduced cost are all potential benefits.

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Development of Assays

Cepheid was one of the first companies to market an assay for methicillin-resistant Staphylococcus aureus (MRSA), based on its SmartCycler® real-time PCR platform.Molecular detection of methicillin resistance in staphylococci is based on the detection of the mecA gene. However, since coagulase negative staphylococci (CNS) can also possess this gene, discrimination between CNS and MRSA must be achieved by the simultaneous detection of additional gene sequences specific for S. aureus. Cepheid's assay was a multiplex assay that did include targets for six variants of the mecA gene, as well as the S. aureus orfX gene. Despite this, independent investigators documented incidences of both false-positives and false-negatives. The BD GeneOhm™ MRSA assay is another real time assay designed for the SmartCycler® platform. This assay employs molecular beacons for detection. The probe has a hairpin shape, with a fluorophore at one end, and a quencher at the other. In the absence of the target, the hairpin is closed and fluorescence is quenched. In the presence of the target, the hairpin opens when the beacon hybridizes to the target, resulting in the emission of fluorescence, which is measured during each cycle of amplification. Result availability is similar to the Cepheid assay. As with the Cepheid assay, independent investigators documented some incidence of both false positives and false negatives, but noted the advantage of rapid availability of screening results for surveillance purposes.

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What is successful molecular identification of methicillin-resistant Staphylococcus aureus (MRSA) based upon? (Choose the BEST answer)View Page
References

BD GeneOhm™ MRSA [package insert]. Quebec, Qc, Canada: BD Diagnostics; 2009. Available at: http://www.bd.com/geneohm/english/products/pdfs/mrsa_pkginsert.pdf. Accessed February 22, 2012.Bonetta L. Prime time for real-time PCR. Nature Methods. 2005;2:305-312. Available at: http://www.nature.com/nmeth/journal/v2/n4/full/nmeth0405-305.html. Accessed February 22, 2012.Boughton B. Universal PCR Screening for MRSA May Cut Costs, Reduce Infection. In Medscape Medical News. Available at: http://www.medscape.com/viewarticle/708813. Accessed February 22, 2012.CDC Response: A Year in Review. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/yearinreview.htm. Accessed February 22, 2012.Centers for Disease Control and Prevention. Evaluation of Rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus ---United States, 2009. Morbidity and Mortality Weekly Report. August 7, 2009;58(30):826-829. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5830a2.htm. Accessed February 22, 2012.Department of Biochemistry. University at Buffalo, School of Medicine and Biomedical Sciences Website. Available at: http://www.smbs.buffalo.edu/bch/Labs/SinhaLab/Protocols/RT-PCR.pdf. Accessed February 22, 2012.Desjardins M, Guibord C, Lalonde B, Toye B, Ramotar K. Evaluation of the IDI-MRSA Assay for the Detection of Methicillin-Resistant Staphylococcus aureus from Nasal and Rectal Specimens Pooled in Selective Broth. J Clin Microbiol. 2006 April;44(4):1219-1223. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448652/. Accessed February 22, 2012.Eastwood K, Else P, Charlett A, Wilcox M. Comparison C. difficile detection methods. J Clin Microbiol. 2009;doi:10.1128/JCM.01082-09. Available at: http://jcm.asm.org/cgi/content/short/JCM.01082-09v1Farley JE, Stamper PD, Ross T, Cai M, Speser S, Carroll KC. Comparison of the BD GeneOhm Methicillin-Resistant Staphylococcu aureus (MRSA) PCR Assay to Culture by Use of BBL CHROMagar MRSA for Detection of MRSA in Nasal Surveillance Cultures from an At-Risk Community Population. J Clin Microbiol. 2008;46(2):743-746. Available at: http://jcm.asm.org/content/46/2/743.full. Accessed February 22, 2012.Forrest GN, Mehta S, Weeks E, Lincalis DP, Johnson JK, Venezia RA. Impact of Rapid In Situ Hybridization Testing on Coagulase Negative Staphylocci Positive Blood Cultures. J Antimicrob Chemother. 2006;58(1):154-158. Available at: http://jac.oxfordjournals.org/content/58/1/154.full. Accessed February 22, 2012.Garcia LS, Isenberg HD, eds-in-chief. Clinical Microbiology Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2007.Hindiyeh M, Hillyard DR, Carroll KC. Evaluation of the Prodesse Hexaplex Multiplex PCR Assay for Direct Detection of Seven Respiratory Viruses in Clinical Specimens. Am J Clin Pathol. 2001;116:218-224. Available at: http://ajcp.ascpjournals.org/content/116/2/218.full.pdf. Accessed February 22, 2012.Hunt M. Real Time PCR. University of South Carolina School of Medicine Website. Available at: http://pathmicro.med.sc.edu/pcr/realtime-home.htm. Accessed February 22,2012.Interim Guidance for Influenza Surveillance: Prioritizing RT-PCR Testing in Laboratories. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/screening.htm. Accessed February 22, 2012.Interim Guidance for the Detection of Novel Influenza A Virus Using Rapid Influenza Diagnostic Tests. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/guidance/rapid_testing.htm. Accessed February 22, 2012.Levenson D. Molecular Testing for Respiratory Viruses. In Clinical Laboratory News. March 2008: Vol 34, No 3. Washington, DC: AACC Press; 2008. Available at: http://www.aacc.org/publications/cln/2008/mar/Pages/cover1_0308.aspx. Accessed February 22, 2012.Morshed MG, Lee MK, Jorgensen D, Issac-Renton JL. Molecular methods used in clinical laboratory: prospects and pitfalls. FEMS Immunol Med Microbiol. 2007;49:184-191. Available at: http://www.canlyme.com/morshed_pcr.pdf. Accessed February 22, 2012.Paillard F, Hill CS. Direct nucleic acid diagnostics tests for bacterial infectiousdiseases: Streptococcal pharyngitis, pulmonary tuberculosis, vaginitis, chlamydial and gonococcal infections. MLO-online. 2004;10-15. Available at: http://www.mlo-online.com/articles/0104/mlo0104coverstory.pdf. Accessed February 22, 2012.PCR: an outstanding method. Roche Website. Available at: http://www.roche.com/pages/facets/pcr_e.pdf. Accessed February 22, 2012.Persing DH, ed-in-chief.Molecular Microbiology, Diagnostic Principles and Practice. 2nd ed. Washington, DC: ASM Press; 2010.Pfaller MA. Molecular Approaches to Diagnosing and Managing Infectious Diseases: Practicality and Costs. Emerg Infect Dis. 2001;eid0702. Available at: http://wwwnc.cdc.gov/eid/article/7/2/70-0312_article.htm. Accessed February 22, 2012.Rossney AS, Herra CM, Brennan GI, Morgan PM, O'Connell B. Evaluation of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay Using the GeneXpert Real-Time PCR Platform for Rapid Detection of MRSA From Screening Specimens. J Clin Microbiol. 2008;46(10):3285-3290. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566096/. Accessed February 22, 2012.The 2009 H1N1 Pandemic: Summary Highlights, April 2009-April 2010. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/cdcresponse.htm. Accessed February 22, 2012.Timeline of PCR and Roche. Roche Website. Available at: http://molecular.roche.com/About/pcr/Pages/PCRTimeline.aspx. Accessed February 22, 2012.

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Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
Staphylococcus aureus Virulence Factors

S. aureus is the most pathogenic member of the genus Staphylococcus; it possesses several factors that contribute to its virulence: Structural components of its cell wall function as a protective barrier, aid in adherence to mucous membranes, and allow the organism to resist phagocytosis. The production of several different toxins Enterotoxins A, D, F (TSST1) Exfoliative toxin ( causing scalded skin syndrome Cytolytic toxins (causing cell & tissue damage). Production of enzymes Catalase – distinguishes staphylococci from streptococci Coagulase – distinguishes S. aureus from other staphylococci Hyaluronidase & lipase – aid in skin colonization/infection spread Beta-lactamase – breaks down the beta-lactam antibiotics, e.g., penicillins, cephalosporins, carbapenems and monobactams.

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Laboratory Identification of Staphylococcus aureus

A clinical isolate is presumptively identified as Staphylococcus aureus by means of several simple procedures :Gram Stain : Gram-positive cocci, occurring singly, in pairs or "bunches of grapes"Catalase test: Staphylocci are catalase-positive, distinguishes them from Streptococci which are catalase-negative.Coagulase test: S. aureus is coagulase-positive.DNAse Test – S. aureus is DNAse-positiveHeat stable endonuclease – S. aureus is positiveThere are also many commercial kits available for identification of S. aureus based on latex agglutination.

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Assume you perform microbiology for an institution submitting surveillance cultures for MRSA. Which isolate should receive further workup to rule out methicillin (oxacillin) resistance?View Page


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