Infectious Information and Courses from MediaLab, Inc.

Under normal circumstances, fluids collected from any enclosed body space such as a cerebrospinal fluid (CSF), pleural, peritoneal, pericardial or synovial fluids, should be sterile. When an infection occurs within a body cavity, the fluid that is collected from that site for diagnostic purposes will potentially have infectious organisms present on the cytospin. There can be several types of organisms demonstrated on the Wright stained preparation.Bacteria and fungus are the most common but it is possible to demonstrate the presence of protozoan parasites as well.Spirochetes and acid fast bacilli (AFB) such as mycobacteria will not stain with Wright stain so they will not be detected even if they are present.Since bronchial alveolar lavages, or BALs, are collected through an open airway it is normal to observe respiratory flora, however yeast and hyphae are never normal in a BAL.It is standard practice before reporting the presence of bacteria in a fluid to correlate/confirm the findings with the microbiology lab.

While the segmented neutrophils are easy to identify in this smear, the large number of cells make it difficult to evaluate this smear for the presence of bacteria or other infectious agents. Remaking the smear with a smaller sample volume or using a larger dilution will make a cytospin that is easier to evaluate.

This is a CSF sample from a patient diagnosed with bacterial meningitis.Note the intracellular and extracellular bacteria present on this smear (see arrows).Notice the white blood cells clumps around the bacteria. This is not simply an artifact of the preparation, but rather a response to the presence of the actual bacteria. WBC clumping can be a great clue to the presence of bacteria, so laboratorians should always scan any clumps present for infectious organisms.

Suppola JP. Kuikka A. Vaara M. Valtonen VV. Comparison of risk factors and outcome in patients with Enterococcus faecalis vs Enterococcus faecium bacteremia. Scandinavian Journal of Infectious Diseases. 30(2):153-7, 1998. The purpose of our study was to determine retrospectively the risk factors for the acquisition of Enterococcus faecalis vs E. faecium bacteremia, as well as the clinical outcomes of these patients. 62 patients with Enterococcus faecalis bacteremia were compared to 31 patients with E. faecium bacteremia. Haematologic malignancies, neutropenia, high-risk source and previous use of aminoglycosides, carbapenems, cephalosporins and clindamycin were significantly associated with E. faecium bacteremia. Instead, urinary catheterization was found to be related to Enterococcus faecalis bacteremia. The mortality rates within 7 d and 30 d were 13% and 27%, respectively, in patients with E. faecalis bacteremia and 6% and 29%, respectively, in patients with E. faecium bacteremia. There was no difference in mortality between E. faecalis and E. faecium bacteremia, nor was there a difference in seriousness of disease at the time of bacteremia. In the subgroups of patients with monomicrobial or clinically significant E. faecalis vs E. faecium bacteremia, the mortality rates were similar to the results of all subjects. Our results do not support the theory that E. faecium would be a more virulent organism than E. faecalis.

Hershow RC. Khayr WF. Smith NL.: A comparison of clinical virulence of nosocomially acquired methicillin-resistant and methicillin-sensitive Staphylococcus aureus infections in a university hospital (University of Illinois at Chicago). Infection Control & Hospital Epidemiology. 13(10):587-93, 1992 OBJECTIVES: To compare the clinical virulence of nosocomially acquired methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) infections in 1989. DESIGN: A retrospective comparison of host factors, in-hospital exposures, sites of infections, and outcomes of patients with nosocomial MRSA and MSSA infections. PARTICIPANTS: Forty-four adult patients with nosocomial S.aureus infections. RESULTS: The 22 MRSA-infected and 22 MSSA-infected persons were similar regarding mean age, gender, underlying diseases, and exposure to surgery. Before developing infection, MRSA-infected persons were more likely to have received antibiotics and to have stayed in the hospital > 2 weeks. Bacteremia was the most common presentation in the MRSA and MSSA groups (55% and 59%, respectively). Infectious complications and death were infrequent in both groups. CONCLUSIONS: MRSA and MSSA strains infect patients with similar demographic features and underlying diseases, but MRSA infections are significantly more common among patients with previous antibiotic therapy and a prolonged preinfection hospital stay. Clinical presentations and outcomes did not differ significantly between the 2 groups. Thus, similar to studies in the early 1980s, our findings do not suggest greater intrinsic virulence of MRSA.

Gelfand MS. Bakhtian BJ. Simmons BP.: Spinal sepsis due to Streptococcus milleri (anginosus): two cases and review. Reviews of Infectious Diseases. 13:559-63, 1991 We have recently cared for two patients with spinal sepsis secondary to infection with Streptococcus milleri (anginosus). One patient had a spinal epidural abscess and the other had meningitis as well as a spinal subdural empyema. A review of the English-language literature revealed only two previously reported cases of spinal epidural abscess due to S. milleri (anginosus) and no cases of spinal subdural empyema due to S. milleri (anginosus). We report two cases of spinal sepsis due to S. milleri (anginosus) and discuss pertinent literature.

Robinson LG. Kourtis AP.: Tale of a toothpick: Eikenella corrodens osteomyelitis. Infection. 28(5):332-3, 2000 Tale of a Toothpick is a case of Eikenella corrodens osteomyelitis in a young woman, that resulted from puncture of her foot with a toothpick. The epidemiology, microbiology, common clinical presentations and therapy of E. corrodens are reviewed. A brief summary of the extent of toothpick injuries and their infectious complications are also presented.

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction, as shown in the table below:ConditionUrine Bilirubin ResultUrine Urobilinogen ResultHemolytic diseaseNegativeIncreasedHepatitic diseasePositive or negativeIncreasedBiliary obstructionPositiveNormal* *Urine chemical reagent strip methods cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction. Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative. Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

In addition to the puncture device, additional equipment may be required when performing a successful dermal puncture.Plastic microcollection devices: Plastic microcollection devices are small plastic tubes designed to collect capillary blood from a dermal puncture wound. Each small collection tube is color-coded in the same manner as blood collection tubes used for venipuncture. The color of the cap of each container tube corresponds to the type of additive inside the tube, most often an anticoagulant. The additive coats the inside of the tube. Examples of microcollection devices are shown below. Heel warmer: It is best practice to warm the heel of an infant with a warming device known as a heel warmer. The heel warmer, when activated, is designed to warm its contents to a standardized temperature. This temperature will be hot enough to effectively warm the heel and facilitate blood flow to the area without causing heat injury to the patient. It is unacceptable to warm a cloth using a microwave. There may be "hot spots" on the cloth that could potentially burn the patient. Keep in mind, what may feel warm to you, the phlebotomist, may feel hot to your patient!Plastic or Mylar-wrapped capillary tube: In some facilities blood from a capillary puncture is collected directly into a capillary tube. These tubes are very delicate and must be used with great caution. As soon as the tube is two thirds to three-fourths filled, one end is sealed to prevent blood from leaking out.Glass microscope slides: In some facilities, the person collecting the capillary specimen may also be required to prepare a blood smear for laboratory examination. A drop of blood is placed directly on a glass slide and spread to create an area for cell examination. If you are required to prepare blood smears, remember that the slide is considered infectious until fixed or stained. It is also important to remember that glass is a sharps hazard. If not used correctly, the glass may cause injury to both the patient and the phlebotomist. Be as cautious with a glass slide containing blood as you are with a contaminated needle. Dispose of glass slides that will not be used for testing in approved sharps containers.Alcohol and gauze pads: Alcohol is the disinfectant of choice for dermal puncture. The alcohol must be allowed to air dry, which will prevent hemolysis of the specimen and discomfort for the patient. A piece of clean or sterile gauze is used to wipe away the first drop of blood. Gauze is also used to apply pressure to the wound after the specimen collection is complete to stop the wound from bleeding.Iodine or other approved cleaning agents may be used as an alternative to alcohol.Bandage: It may be necessary to apply a bandage to the puncture wound on a finger or heel if the site continues to bleed. However, it is NOT recommended to bandage the finger of a child who is 2-years-old or younger since the bandage may become a choking hazard if the child puts that finger in his/her mouth.Personal protective equipment (PPE): All healthcare professionals that may come in contact with blood and/or body fluids while performing a laboratory procedure are required to wear intact gloves. It is against safety guidelines to alter gloves in any way that may compromise the integrity of the gloves. Eye protection, such as safety goggles, is recommended if there is the possibility of a splash of blood while collecting a capillary blood specimen. In many facilities, special gowns are required in some patient areas such as special-care nurseries. Always follow the policies of your facility in regard to PPE.

Molecular diagnostics have begun to play an integral part in clinical laboratory diagnostic testing. Traditionally, molecular diagnostics have been utilized in three major clinical areas: Infectious diseases Genetics Tumor markers These molecular based diagnostic tests, while historically reserved for specialty/reference labs, have recently seen expansion of their utility within the scope of routine clinical laboratories. Molecular based diagnostics can be utilized by small labs as well as large ones, and can be found in virtually every department of the clinical laboratory.

Molecular methodologies offer numerous advantages to the clinical laboratory. These include:Sensitivity: Amplification methodologies are particularly useful in increasing the sensitivity of a methodology and useful in the identification of target molecules of interest that are only present in low concentrations. Specificity: Molecular methods minimize false positive test results by targeting the specific molecule of interest.Turn around time: In comparison with standard traditional culture methods, molecular methodologies usually offer better turn around times from receipt to result reporting.Application: Broader application can be found with molecular methodologies such as infectious diseases, genetic testing, forensics, drug resistance, and tumor marker detection and monitoring.

The reason to chose a particular molecular method can be influenced by disease detection, monitoring, or therapy in certain patient populations. Molecular methodologies can be used to identify alterationsor variations or changes in DNA sequencing that can cause disease. Sequence alterations that are known to cause disease are termed mutations. These changes or mutations can be applied to areas of the clinical laboratory such as infectious disease, paternity, genetic testing, and pharmacogenetics. Some of the more common alterations are:Deletion: A missing nucleotide or other portion of DNA sequence Insertion: An extra DNA nucleotide or other portion of DNA sequence Missense: A nucleotide or sequence substitution that codes for a different amino acidNonsense: A nucleotide substitution that ends in early termination of the protein manufacturing process; usually due to a stop codon.The most common alteration is a single base change or single nucleotide polymorphism (SNP).

Burtis CA, Ashwood ER, Bruns DE, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Inc; 2006.Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved Guideline.CLSI document MM13-A. NCCLS.Wayne, PA: 2006.Clinical and Laboratory Standards Institute (CLSI). Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline. Second ed. CLSI document MM3-A2. NCCLS. Wayne, PA: 2006.

Cervical Cancer: Prevention and Early Detection. American Cancer Society. Available at http://www.cancer.org/docroot/CRI/content/CRI_2_6x_cervical_cancer_prevention_and_early_detection_8.asp. Accessed December 1, 2011. Cervista HPV, Cervista HPV – Invader Technology. HOLOGIC. Available at http://www.cervistahpv.com/laboratory/invadertechnology.html. Accessed December 1, 2011.Chin-Hong PV, Klausner JD. Diagnostic tests for HPV infection. Medical Laboratory Observer. October 2004:10-16.Cobo F, Concha A, Ortiz M. Human papillomavirus (HPV) type distribution in females with abnormal cervical cytology. A correlation with histological study. Virology Journal. 2009;3:60-66.Cox JT, Moriarty AT, CastlePE. Commentary on statement on HPV DNA test utilization. American Journal Clinical Pathology. 2009;131:770-773.HPV Vaccine Information for Clinicians. Centers for Disease Control and Prevention. Available at http://cdc.gov/std/hpv/stdfact-hpv-vaccine-hcp.htm. Accessed December 1, 2011.Human Papillomavirus (HPV) Natural History. American Society for Colposcopy and Cytological Pathology. Available at http://www.asccp.org/hpv_history.shtml. Accessed December 1, 2011.Human Papillomavirus (HPV) Vaccines. National Cancer Institute. Available at http://www.cancer.gov/cancertopics/factsheet/prevention/HPV-vaccine. Accessed December 1, 2011.Human papillomaviruses and Cancer: Questions and Answers. National Cancer Institute Fact Sheet. Available at http://www.cancer.gov/cancertopics/factsheet/risk/hpv. Accessed December 1, 2011.Hybrid Capture 2 Technology. QIAGEN - Sample & Assay Technologies. Available at http://www1.qiagen.com/hpv/hc2technology.aspx. Accessed December 1, 2011.Markowitz LE, Sternberg M, Dunne EF, et al. Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: national health and nutrition examination survey 2003-2004. Infectious Disease. 2009;200:1059-1067.Molecular Diagnostics Fundamentals, Methods, and Clinical Applications. Leal Buckingham and Maribeth L. Flaws. Philadelphia:FA Davis Company, 2007.Schutzbank TE, Jarvis C, Kahmann N, et al. Detection of high-risk papillomavirus DNA with commercial invader-technology-based analyte-specific reagents following automated extraction of DNA from cervical brushings in Thinprep media. Journal of Clinical Microbiology. 2007;45:4067-4069.Solomon D, Papillo JL, Davey DD. Statement on HPV DNA test utilization. American Journal of Clinical Pathology. 2009;131:768-769.Vernick JP, Steigman, CK. The HPV DNA virus hybrid capture assay: what is it—and where do we go from here? Medical Laboratory Observer. Mar 2003:8-13.Voss JS, Kipp BR, Campion MB et al. Comparison of fluorescence in situ hybridization, hybrid capture 2 and polymerase chain reaction for the detection of high-risk human papillomavirus in cervical cytology specimens. Analytical and Quantitative Cytology and Histology. 2009;31:208-216.

At the highest level are the "national" laboratories. Examples would include those operated by CDC, the United States Army Medical Research Institute for Infectious Diseases, and the Naval Medical Research Center. These laboratories have very unique resources to handle highly infectious agents and the ability to identify specific agent strains.

Biological attacks involve bacteria, viruses or natural toxins. The effects of toxins can be immediate but for bacteria and viruses the effects may not be apparent for weeks. A bioterrorist may attack by infecting animals, contaminating food and water, spraying bacteria or viruses into the air. In infections such as smallpox and plague, once a few individuals are infected they can further spread the disease from person to person. An attack could also come from through a building's ventilation system, the mail, or even through exposure to an infected terrorist seeking to spread disease during an infectious stage.

Commercially prepared controls come in either assayed or unassayed forms. Assayed controls are tested by multiple methods before sale, and come with measuring system-specific values that are meant to be used as target values for the laboratory using the controls. Assayed controls:Are more expensive than unassayed controlsAre used to evaluate accuracy and precisionMay only be suitable for specific method systems Unassayed controls have no assigned analyte values provided by the manufacturer. The control values for these materials must be determined by the individual laboratory. Unassayed controls:Are less expensive than assayed controlsAre used to evaluate accuracy and precisionAre not linked to specific method systems Note: although commercially available control materials are screened for hepatitis antigens and HIV antibodies, control materials should still be handled with precautions, since they contain biological materials and could contain infectious agents.

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

Once relegated to the domain of research laboratories, molecular methods for the diagnosis of infectious disease had little, if any place, in a clinical diagnostic laboratory prior to 1985. Procedurally, molecular methods were very complex and required specialized instrumentation and dedicated laboratory space. They were also susceptible, initially, to the influence of variation of technique. Although they represented valuable research tools, and were helpful as esoteric testing for unique clinical situations, their performance characteristics simply did not fit well into most clinical laboratories.Certain pathogens were logical targets for development. Organisms that were of concern for significant patient populations, were difficult to sustain in transport, and/or were difficult to cultivate and detect by traditional methods represented some of the first targets of commercially offered molecular based assays.Sexually transmitted diseases, affecting significant numbers of people, with key pathogens affected by lability in transport or poor sensitivity with traditional cultivation or antigen detection methods, were among the first targets for development.

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.

Most Clostridium infections arise from endogenous sources. That is, many of the Clostridium species that are associated with disease in humans are part of the normal intestinal microflora, which is true of Clostridium difficile.The organism was originally isolated in 1935 as a component of the normal intestinal flora of healthy newborns. It was dubbed difficile because the organism grows slowly and is difficult to culture. Early investigators also noted that the organism produced a potent toxin, but the relationship between C. difficile antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC) was not elucidated until the 1970's. PMC is an inflammatory disease of the colon caused by toxins of Clostridium difficile. Normal intestinal flora is an important factor in host response to an infectious microorganism. Resistance to intestinal infection is significantly reduced when there is a reduction in the normal flora as a result of antibiotic treatment. The most common manifestation of this decreased host resistance is the development of PMC.

As a health care worker, you come into contact with materials that may contain bloodborne pathogens. These are infectious organisms, usually viruses, that live in human blood and body fluids.The bloodborne pathogens that are of greatest concern to health care workers are:Hepatitis B virus (HBV) Human immunodeficiency virus (HIV) Hepatitis C virus (HCV)

Standard precautions mean that all blood and body fluids should be handled as if they are infectious and capable of transmitting disease. Standard precautions apply to: BloodBody fluidsSecretions (except sweat)ExcretionsNon-intact skinMucous membranes

Patients infected with HBV or other bloodborne organisms can appear healthy, so you can't tell whose blood is infectious.So treat all:bloodbody fluidssecretions (except sweat)excretionsnon-intact skinmucous membranes as if they were infectious.

A person with HBV is infective soon after contracting the virus, and the infectious period continues through the acute and chronic illness.

Place blood and other infectious specimens ... first in an appropriate sealed container and then in a secondary red or biohazard-labeled bag. Or place them in the facility-approved tray for transport within the institution.

Even after taking all the proper precautions there is still a small chance of an exposure incident. An Exposure incident occurs when: Blood or another potentially infectious body fluid comes into direct contact with mucous membranes or non-intact skin. Parenteral exposure means: Exposure occurring as a result of piercing the skin barrier through needlesticks, cuts, or abrasions.

As a healthcare worker, you come into contact with bloodborne pathogens. These are infectious organisms, usually viruses, which live in human blood and other potentially infectious body fluids.The most important ones are... Hepatitis B Virus (HBV) Human Immunodeficiency Virus (HIV)

A person infected with HBV can spread the virus soon after the initial infecting incident, and the infectious period continues through the acute and chronic illness.

Work practice controls affect the transport of blood and other potentially infectious materials.

Gloves must be worn: when there is a reasonable chance of exposure to blood, other infectious body fluids, mucous membranes, or nonintact skin, during vascular access procedures, including phlebotomy, or when handling contaminated items or surfaces.

According to federal and international regulations, all personnel who are involved in the packaging and shipping of infectious materials are required to have training. This includes anyone who:Packages, labels, and/or marks the package Is responsible for classifying the materials Is responsible for documenting the package contents on a shipper's declaration for dangerous goods form, air waybill, etc. Signs a shipper's declarationTransports hazardous materials by vehicle, plane, or vesselThe training that is required for individuals who package and ship only category B infectious substances is not as comprehensive as the training required for individuals who may package and ship category A infectious substances.

Training records must include:Employee name Most recent date trained Description of training Description, copy, or location of training materials Name and address of trainerThese records must be maintained throughout employment and 90 days thereafter, according to the US Department of Transportation (DOT). IATA requires repeat training every two years. DOT requires training every three years. Laboratory accrediting agencies require training at the frequency appropriate to the specimen types and distance transported. You will be able to print a certificate when you have completed this course that will certify your completion of training for packaging and shipping Division 6.2 (infectious) materials.

Hazardous material classifications are consistent across all agencies who regulate commercial shipping and are based on criteria developed by the United Nations (UN) Committee of Experts working with the World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC), medical professionals, microbiologists, transportation professionals, and packaging technical experts. These requirements can be found in the 13th and 14th editions of the United Nations Recommendations for the Transport of Dangerous Goods, the 2005 - 2006 edition of the International Civil Aviation Organization Technical Instructions for the Safe Transport of Dangerous Goods by Air (ICAO Technical Instructions), and the International Maritime Organization (IMO) Dangerous Goods Code. The classification system for Division 6.2 Infectious Substances includes two catergories, known simply as Category A and Category B.

A category B infectious substance is not in a form generally capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs. The proper shipping name and Identification number is:Biological substance, Category B, UN 3373

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for:drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies The US Department of Transportation (DOT) has no "Exempt Specimen" classification and there are no DOT guidelines for packaging non-regulated specimens.* According to the DOT, in the U.S., if a package is marked as "Exempt Human/Animal Specimen" the understanding is that it contains no infectious substance. However, both IATA and the US Postal Service (USPS) have these requirements for packaging exempt specimens: Packaging IssueIATAUSPSType of packaging requiredTriple packagingTriple packagingOuter containerOne dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet One dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet Quantity limits: outer containerNone NoneQuantity limits: Primary receptacleNone500 mLQuantity limits: secondary packagingNone500 mL* Non-regulated specimens may become regulated because of preservatives, such as 10% formaldehyde (class 9) or 25% formaldehyde (class 8); or 25% ethanol (class 3).

Triple packaging is required for category A substances.The image on this page illustrates the correct packaging scheme. Packaging must include: A leakproof primary receptacle A leakproof secondary packaging Absorbent material in sufficient quantity to absorb the entire contents. (This is not required if the infectious substance is a solid.) The absorbent material is to be placed between the primary receptacle and the secondary packaging An itemized list of contents, enclosed between the secondary packaging and the outer packaging (preferably enclosed in a zip lock bag). Rigid outer packaging

Packages that contain category A substances must exhibit these labels.Proper shipping name and UN number(Category A label) Include quantity. Do NOT put technical name (organism name) on Proper Shipping Name label or anywhere on the outside of the package.orHazard class 6 infectious substance label that includes this statement:In case of damage or leakage, immediately notify public health authority. In the USA, notify director- CDC Atlanta, GA 1-800-232-0124. UN package certification markOrientation arrows (if greater than 50 mL). However, IATA regulations state that orientation arrows are not needed if the package contains hermetically sealed inner packages of liquid, each containing not more than 500 mL.Contact information (Shipper or Consignee Identification)The contact person (usually the shipper), referred to as the "responsible person" by IATA, must be someone who can be reached 24 hours a day, seven days a week (24/7) and can answer questions about the content of the package. The 24/7 number must reach that person directly and not a pager or answering machine/service. If the contact person that you are listing is the person receiving the specimen, be certain that the person is aware you are listing him/her as the contact person and has consented to it. IATA requirement for labels on outer packagingThe name and address of the shipper and consignee should be on the same surface as the marking for the UN number and proper shipping name when the package size is adequate. The net quantity of dangerous goods must be shown on all packages regardless of the class of the dangerous goods. The lower image on the right shows this in greater detail.

A category A infectious substance is in a form that is capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals when exposure to it occurs. Exposure would occur if the substance were released from its protective packaging and a human or animal came into contact with it. Therefore, it is critical that a category A infectious substance does not end up in the hands of an unauthorized individual who may purposely or unknowingly release the substance from its protective packaging and endanger humans or animals. Being aware of the people that you interact with in the process of packaging and sending category A substances is vital to the safety of the transport and prevention of a health disaster. An outsider with limited access and system knowledge could constitute a threat, but be aware that insiders could also be a threat, e.g., a disgruntled employee or a person who is angry with his or her supervisor or job or the government. Anyone desiring to do harm could potentially seize the opportunity to steal a hazardous material.Follow these precautionary procedures: When you are questioned about an infectious substance that you are packaging for shipment, it is important that you know the person that is asking AND that he or she has a need to know. If you do not know the person and if you are not aware that the person needs to know about the substance that is being shipped, do not answer the questions. You could refer him or her to your supervisor. Watch for unusual behavior. Secure the package until it is picked up. Check the identification of the courier who will be picking up the package. Use an intralaboratory chain of custody procedure if the specimens are tranferred within the facility or system. Track the package once it has been sent to be sure it arrives safely. Notify the Responsible Official or federal authority if the package does not arrive at its destination.

International Air Transport Association. Guidance document: Dangerous Goods Regulations (DGR). 52nd ed. 2011.National Laboratory Training Network. Packaging and shipping Division 6.2 materials. Georgia Public Health Laboratory; 2011. Sentinel laboratory guidelines for suspected agents of bioterrorism: Clinical laboratory bioterrorism readiness plan. Available at: http://stanfordhospital.org/PDF/bioterrorism/labGuidelinesSuspectedAgentsBT.pdf. Accessed January 31, 2011.US Department of Transportation Pipeline and Hazardous Materials Safety Administration. Transporting Infectious Substances Safely. Available at http://www.phmsa.dot.gov/staticfiles/PHMSA/DownloadableFiles/Files/Transporting_Infectious_Substances_brochure.pdf.

Treat all blood & body fluids as if they were infectious.Always wear gloves during vascular access procedures.

Bloodborne pathogens are infectious micro-organisms which live in the bloodstream.You can be exposed to bloodborne pathogens if you are injured with a contaminated needle.You can also be exposed if your mucous membranes, including eyes, mouth, or the inside of your nose come into contact with contaminated body fluids.

Potentially infectious body fluids include: Blood, Semen, Vaginal Secretion, Peritoneal, pericardial and pleural fluids, and Saliva Sweat and tears are not generally considered infectious. It is important to remember that bloodborne pathogens are not transmitted by casual contact, like a handshake.

A reference laboratory within the LRN performs tests to detect and confirm (rule-in) the presence of a threat agent. These labs ensure a timely local response in the event of a terrorist incident. Rather than having to rely on confirmation from laboratories at the Centers for Disease Control (CDC), reference laboratories are capable of producing conclusive results. This allows local authorities to respond quickly to emergencies. A national laboratory is the highest level within the LRN. Examples would include those operated by CDC, the United States Army Medical Research Institute for Infectious Diseases, and the Naval Medical Research Center. These laboratories have very unique resources to handle highly infectious agents and the ability to identify specific agent strains.

Francisella tularensis is a dangerous, highly infectious organism that can cause laboratory-acquired infections. It should NOT be manipulated on an open bench.Catalase: F. tularensis is weakly catalase positive. Catalase testing MUST be performed with extreme caution in a biosafety cabinet (BSC) due to the creation of aerosols. Oxidase: NegativeBeta-lactamase: PositiveUrease: NegativeXV factors: Not required for growthImportant note: F. tularensis is often incorrectly identified on automated identification systems. These systems may key out as Haemophilus influenzae or Actinobacillus species.

Anthrax (B. anthracis): Inhalation of anthrax spores is virtually 100% fatal Spores can remain infectious for decadesBotulism: Most lethal toxic agent known Toxin could be used to contaminate food supplies Can be aerosolized in enclosed areasPneumonic Plague (Y. pestis): Aerosolized in large amounts Short incubation period, usually in less than three days, and invariably fatal without early and effective antimicrobial therapy Untreated, fatality rate exceeds 90% Disease is spread from direct exposure to respiratory droplets of infected humansSmallpox: Highly contagious and deliberate spread by aerosol is extremely infectious Mass panic would be createdTularemia (F. tularensis): Highly contagious and easily spread An aerosol containing as few as 25 organisms can cause infection Easily penetrates the smallest breaks in the skinViral Hemorrhagic Fever: Causes internal and external bleeding and would likely cause great panic and easily spread by direct contact with body fluids or respiratory droplets Outbreak due to bioterrorist attack could lead to mass illness and death

There are numerous applications for real-time PCR in the laboratory for both diagnostic and research purposes. Diagnostic applicationsReal-time PCR can rapidly detect nucleic acids that are diagnostic of infectious diseases, cancers, and genetic abnormalities. Real-time PCR has allowed for viral quantitation of infectious and newly emerging diseases such as influenza A H1N1 subtype. In malignant diseases, real-time PCR can be performed directly on genomic DNA to detect translocation-specific malignant cells. For RNA samples, real-time PCR has become extremely important for the detection and monitoring of HIV, hepatitis C and CMV. Real-time PCR can also be used for array verification and drug therapy efficacy. Research applicationsIn a research setting, real-time PCR is primarily used to measure gene transcription. The technology is commonly used to determine genetic expression of a particular gene over time in response to different pharmacologic agents or environmental conditions and can also be used to compare gene expression in exposed and unexposed individuals. The use of real-time PCR in this manner can help researchers find and detect diagnostic or prognostic indicators to increase the understanding of disease pathogenesis.

Dimmock NJ, Easton AJ, Leppard KN. Introduction to Modern Virology. 6th ed. Malden, MA: Blackwell Publishing; 2007.Foxman B. Molecular Tools and Infectious Disease Epidemiology. San Diego, CA: Elsevier; 2011. Kaplan LA, Amadeo JP. Clinical Chemistry:Theory, Analysis, Correlation. 5th ed. St. Louis, MO: Mosby-Elsevier; 2010.Logan J, Edwards K, Saunders N. Real-Time PCR: Current Technology and Applications. Norfolk, UK: Caister Academic Press; 2009. Ream W, Gellar B, Trempy J, Field K. Molecular Microbiology Laboratory: A Writing-Intensive Course. Boston, MA: Academic Press; 2003. Turgeon ML. Immunology & Serology in LaboratoryMedicine. 3rd ed. St. Louis, MO: Mosby Elsevier; 2003.Walker JM, Rapley, R. Molecular Biology and Biotechnology. Cambridge: The Royal Society of Chemistry; 2009.

The safety of both the phlebotomist and patient is of utmost concern at all times. In the unfortunate event of an accidental needlestick or if you get blood or other potentially infectious materials in your eyes, nose, mouth, or on broken skin, immediately flood the exposed area with water and clean any wound with soap and water or a skin disinfectant if available. Report this immediately to your employer and seek immediate medical attention. It is imperative that the phlebotomist follow facility protocol for reporting the incident. This ensures prompt treatment for the injury. The facility procedure must be followed whether the accidental puncture was from a clean or contaminated needle.The single most important element to prevent an accidental needlestick is for the phlebotomist to fully concentrate during every procedure. Keeping your mind on the task at hand contributes to a successful and safe result.

The basic laboratory skills that you will need to do a semen analysis include: Use of a microscopePerformance of manual cell countsMeasuring volumeMeasuring pHMeasuring viabilityKnowledge of OSHA regulations for handling potentially infectious human fluids

Influenza A viruses, including the 2009 H1N1 strain, are able to survive and maintain infectivity on surfaces for extended lengths of time. Influenza A viruses typically remain infectious for 12 - 48 hours on non-porous surfaces, for 8 - 12 hours on cloth or paper, and for 5 minutes on hands.To reduce spread of the virus from person to person, it is important to discard contaminated items such as tissues and laboratory testing supplies and to wash hands frequently. To eliminate viruses from contaminated surfaces, a number of disinfectants can be used, such as chlorine, hydrogen peroxide, detergents, iodophores, and alcohols. Influenza viruses also can be rapidly inactivated with heat from 167 - 212°F.

Even though blood components are tested rigorously for certain infectious diseases, bacterial, viral, parasitic, and prion pathogens continue to evolve. If they are not detected, they can cause harm to the patient and even death. Donors must pass a medical screening and questionnaire. They are also tested for hepatitis B and C, human immunodeficiency virus (HIV) 1 and 2, human T-cell lymphotrophic virus (HTLV) I and II, West Nile virus and syphilis. The table to the right describes the screening tests performed on all blood donors in the United States. It is not yet possible to eliminate the risk of infectious disease transmission through transfusions. There are many other organisms that may be transfusion-transmitted which are not routinely tested for in the blood supply. These include the Epstein-Barr virus, cytomegalovirus (CMV), bacteria, and parasites such as malaria, Babesia microti, and Trypanosoma cruzi which is responsible for Chagas disease, and prions such as variant Creutzfeldt-Jakob disease (vCJD). Selection of eligible donors is a critical part of ensuring the safety of the blood supply. Donors with certain lifestyles, medical conditions, travel histories, immigration backgrounds, or specific physical findings are deferred, either for a specific period of time or indefinitely. This minimizes the risk that a transmittable agent will be present in the donors blood. Click here to learn more about donor eligibility criteria from the American Red Cross.Click here to learn more about Babesia microti. Click here to learn more about Chagas Disease. Click here to learn more about vCJD.Click here to learn more about malaria.

Measures taken to reduce bacterial contamination of blood components include donor screening, improved skin disinfection, diversion of the first aliquot of blood, and pretransfusion bacterial detection. Screening of donors is done by questioning them about fever occurrence and dental or medical procedures that occurred days before donation. Donors who develop symptoms of an infection may be asked to notify the blood bank. Complete skin disinfection is not possible because of organisms living in places that are inaccessible, such as sebaceous glands and hair follicles. Factors affecting skin disinfection are the type and concentration of antiseptic, use or single or multiple antiseptics, method and steps of application, and contact time. Studies have shown that a two-stage method using a sponge scrub and ampule with tincture of iodine is the most effective method. The AABB recommends an initial 30 second scrub with a 0.7% iodophor solution followed by the application of a 10% iodophor compound, which must be allowed to dry for 30 seconds. To avoid normal flora contamination, blood may be diverted into a satellite bag at the beginning of donation. These bags are developed so that backflow is prevented. Blood contained in the satellite bag is used for blood grouping and infectious disease testing. Blood diversion is not a mandatory practice in the United States. The AABB requires that the transfusion service have a method to detect bacteria in all platelet components. Culture-based methods are used at blood collecting facilities near the time of collection. Hospital-based transfusion services use other less costly non-culture based methods such as gram staining or pH and glucose analysis prior to releasing the product for transfusion. Recently, a qualitative immunoassay for the detection of bacteria in platelets has been developed. This test detects antigens on the cell walls of the bacteria. It has been documented to be more sensitive than other non-culture based methods.

Mycobacterium tuberculosis is spread through infectious droplet nuclei. When a person infected with pulmonary tuberculosis coughs, sneezes, shouts, or sings, the infectious particles are expelled into the air. The risk of infection is related to both concentration of infectious droplet nuclei and duration of exposure. Laboratory workers are at risk when an infectious aerosol is generated while handling liquid cultures, during preparation of frozen sections, and when performing autopsies on infected patients.

All specimens suspected of containing M. tuberculosis (including specimens processed for other microorganisms) should be handled in a Class I or II biological safety cabinet (BSC). Appropriate personal protective equipment (PPE) must be used. At a minimum, this includes gloves and fluid-resistant laboratory coat or gown. Non-aerosol-producing manipulations (eg, preparing direct smears for acid-fast staining when done in conjunction with training and periodic checking of competency) can be performed using biosafety level-2 (BSL-2) practices and procedures, containment equipment, and facilities. BSL-3 practices, safety equipment, and facility design and construction are applicable to microbiology laboratories that work with indigenous or exotic agents with a potential for respiratory transmission, and which may cause serious and potentially lethal infection. If the laboratory is propagating and manipulating cultures for M. tuberculosis, BSL-3 practices, containment equipment, and facilities are required. Barriers include controlled access to the laboratory and ventilation requirements that minimize the release of infectious aerosols from the laboratory. Secondary barriers should include self-closing double-door access and negative airflow into the laboratory. Exhausted air must not be recirculated. Work surfaces must be decontaminated, using the laboratory-approved disinfectant, upon completion of procedures, immediately following a spill, and at the end of the work shift, if the surface was recontaminated since the last cleaning. Laboratory equipment should be routinely decontaminated.Hands must be washed upon completion of work with potentially infectious materials and before leaving the laboratory.

The following pages in this presentation includes a series of white blood cell and platelet abnormalities (nonneoplastic) that may be identified in a peripheral blood smear. Many cases will simulate the practice of a peripheral smear review by a hematology technologist. He or she must assess what responses in patient care may be triggered by the clinician attempting to interpret the reported findings on a peripheral smear.Observations of white blood cell abnormalities in the peripheral blood smear should be reported in order to direct the physician to an immediate specific diagnosis, such as: Atypical lymphocytes, suggesting infectious mononucleosis rather than leukemia Toxic granules in neutrophils, as found in acute infections, or atypical granules suggesting a genetic disorder An unusual mix of cells, such as too many or too few neutrophils, monocytes, or other myeloid cells The presence of giant platelets, myelocytes, or other cells, suggesting a myelodysplastic syndromeIn summary, laboratory data should be presented to clinicians in a user-friendly fashion to promote effective decision making.

The presence of atypical inclusions within the cytoplasm of neutrophils and other leukocytes should lead to a clinical investigation of the setting for these findings. Atypical neutrophil inclusions may be seen in the following disorders: Chediak-Higashi syndrome, May-Hegglin anomaly, Alder-Reilly anomaly, Fechtner , Sebastian, Epstein and Alport-like syndromes and in infectious and toxic conditions (in the form of Dohle bodies).Although a specific entity may not be evident from examination of the peripheral blood alone, it is important that hematology technologists include a comment reporting on the presence of these inclusions or granules. A clinical investigation with further hematologic and genetic studies may then appropriately be considered. Many of the disorders with atypical neutrophil cytoplasmic granules are also associated with platelet abnormalities, particularly giant platelets (lower image). Therefore, when atypical granules are recognized, scanning of the peripheral blood smear for atypical platelets may be revealing. These observations serve as readily identifiable markers for acquired and genetic human maladies, and as a guide for unraveling the reasons for a patient's suffering and impaired health.

A 14-year-old boy came to the physician's office with a sore throat that progressively worsened over a three-day period. His posterior pharynx was swollen, shiny and erythematous. The boy complained of pain on swallowing. His temperature was 98.5°F. A rapid direct streptococcal antigen test was positive. However, his symptoms did not subside over the next two days while on antibiotic therapy. Anorexia and nausea were persistent and compounded by a frontal headache. Cervical lymph nodes became noticeably enlarged. A complete blood count (CBC) was ordered. The results of the CBC were: WBC 11.9 x 109/L ( Reference interval= 3.8 - 9.8 x 109/L) with: 17% segmented neutrophils 5% band neutrophils 72% lymphocytes 6% monocytes All red cell findings were normal. The automated differential flagged for atypical cells, presumptively atypical lymphocytes. A peripheral blood smear was prepared. The image on the right is a representative field from the Wright-Giemsa stained smear (1000X magnification).A rapid qualitative test for infectious mononucleosis was positive. This is a case of group-A streptococcal infection superimposed on infectious mononucleosis. Symptoms subsided in three weeks following completion of the antibiotic therapy.