|Products Used to Facilitate Antibody Identification|
Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable.
|Significance of Reactions at Different Phases of Testing|
Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases: after immediate spin, after incubation at 37°C, and after the addition of antihuman globulin (AHG) and centrifugation. Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting (IgG). It will also help to distinguish between antibodies that are clinically significant and not significant. Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR) or hemolytic disease of the newborn (HDN) are usually IgG and react best in the AHG phase.Readings can be done at all three phases if a tube method is used. If a gel method is used, readings are done only at AHG. Immediate spin: Antibodies reacting in this phase tend to be cold reactive. They are usually IgM class and not clinically significant (with the exception of the A and B antibodies). 37°: Antibodies that react in this phase include strong IgM or IgG antibodies. After incubation, the tubes are examined for the presence of hemolysis. If complement was bound during incubation then hemolysis could be seen. NOTE: This reaction would only occur in serum samples. If EDTA plasma samples are used for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for complement to be activated. AHG:Antibodies reacting in this phase are considered clinically significant. They are usually warm reactive and IgG.
|The bacterial species shown growing on 5% sheep blood agar was recovered from the spun sediment of a midstream urine specimen after 24 hours incubation at 35C. Each of the following tests would be useful in supporting the presumptive identification of Enterococcus species except: (Choose all that apply)||View Page|
A 63-year-old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture. A second blood culture was drawn from the opposite arm, with 10 mL of blood being placed into each an aerobic and an anaerobic bottle, following customary practice. The complete blood count revealed a hemoglobin of 15.8 mg/dL, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy. Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted. A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.
|The Gram stain shown in the image was prepared from a positive anaerobic blood culture bottle after 36 hours incubation. Based on the morphology of the bacterial cells (some with spores, noted by the blue arrows), what the most likely identification?||View Page|
The growth observed on the anaerobic blood agar plate after 48 hours incubation (see upper image), revealed a spreading colony. The spreading nature of the colony is better observed in the lower image. No growth was observed on subcultures incubated aerobically indicating that this isolate is truly an anaerobe (although aerotolerance studies would be needed for confirmation). The spreading nature of the colony and the lack of hemolysis are highly suggestive of Clostridium septicum. However, biochemical confirmation is necessary.
|S. anginosus ("milleri") CO2|
Enhanced growth under CO2 incubation is an additional clue to the identification of S. anginosus ("milleri"). Note in the image the increased growth of the colonies grown on the plate incubated under CO2, compared to those incubated in ambient air (O2).
|Beta hemolytic colonies grew from the blood culture bottle after 18 hours incubation (see image). Which of following tests would be helpful in making a preliminary identification? (Choose all that apply)||View Page|
|Shown in the image is a close-in view of the colony growth after 48 hours incubation. What are the possible presumptive identifications suggested by the colonies observed? (Choose all that apply)||View Page|
Image of the surface of blood agar after 24 hours incubation at 35C in 10% CO2, on which are growing tiny, translucent, gray colonies surrounded by a narrow zone of "soft" beta hemolysis. There was no growth on the MacConkey plate.
Image of the surface of a 5% sheep blood agar plate after 48 hours incubation at 35 degrees C in 10% CO2. The colonies shown are small, flat, entire, dull gray, and show superficial pitting of the agar (see yellow arrows). A slight discoloration of the agar surrounding the colonies is seen. A bleach-like odor is detected. Similar growth was seen on a chocolate agar plate set up in parallel. Growth was not observed on the MacConkey plate.
|Performance of a Mixing Study|
Step TwoThe next step is an immediate re-run of the PT and/or aPTT test with the newly created sample mixture. The results should be documented on a worksheet to compare to the original PT and/or aPTT tests.Step ThreeThe sample that has been made for the mixing study consisting of the pooled normal plasma and the patient plasma should also be incubated to rule-out any slow reacting inhibitors. To do this the "mixed" specimen is incubated at 37°C for 1 - 2 hours or as long as required by your laboratory's procedure. A set of control tubes should also be incubated at the same time as the mixed sample tube. A pure patient plasma sample and a pure pooled normal plasma sample will serve as the controls in this procedure. You may incubate all 3 tubes together in a water bath or heat block. Refer to the image to the right. The incubation of these controls will account for the heat-labile state of some coagulation factors which will be discussed again.After the incubation phase, the PT and/or aPTT tests should then be repeated once more. If any coagulation inhibitors were present in the patient sample, the incubation phase would have given the ideal temperature and time for the antibody-coagulation factor reaction to take place. This is especially helpful in the case of anti-factor VIII inhibitors since they are often slow acting or weak inhibitors.
|Interpreting the Mixing Study Results|
Interpretation patterns for mixing study results include the following scenarios: If the results of the mixing study show correction for both the immediate and incubated PT/aPTT tests, the patient most likely has a factor deficiency (or multiple factor deficiencies). If the results of the mixing study show no correction in either the immediate or incubated PT/aPTT, the patient may have a coagulation inhibitor, most likely a lupus anticoagulant. If the results of the mixing study show correction for the immediate PT/aPTT results, but no correction in the incubated PT/aPTT, the patient may have a slow-acting inhibitor such as anti-factor VIII.Note: if the control tubes also show prolongation after incubation, there may have been a problem with the heat-labile factors in the sample losing their activity. If this is the case, the mixing study should be repeated.
| Initial aPTTImmediate aPTT mixing studyIncubated aPTT mixing study 63 sec. (normal range 21-34 seconds)26 sec.65 sec.Has the aPTT been corrected by the mix? Is a factor deficiency or a coagulation inhibitor the more likely cause of the patient's prolonged aPTT?||View Page|
|The Key Benefits: Improved Sensitivity of Detection|
There are three key benefits that molecular methods can offer, in contrast to traditional culture methods:Improved sensitivity of detectionImproved specificity of identificationReduced turnaround timeImproved sensitivity of detectionSuccessful cultivation or detection of an organism depends on many factors, including the:Ability of the organism to survive transport Fastidious nature of the organism/its ability to grow in available culture media/systems Number of organisms present in a specimen Ability of a staining/culture system to visualize/recover low numbers of organisms Sensitivity of non-culture (antigen detection) methodsOrganisms that have been shown to be very labile are difficult to cultivate even when they are present in significant numbers. The immediacy of transport, plating, and incubation are critical factors that frequently cannot be controlled in a positive direction.Even under the ideal transport conditions, some organisms may require culture media and conditions that are not routinely available in every laboratory, which reduces the likelihood of successful cultivation.
|The Key Benefits: Reduced Turnaround Time|
Organisms that are either present in very low numbers, or that possess a characteristically slow growth rate, may require an extended incubation before they are detected in culture. Amplification and/or detection of unique sequences of either DNA or RNA provide for a more timely identification. This is true whether they are applied to the specimen for direct detection, or in some cases, to culture positive specimens for culture confirmation/identification.Even for organisms that are easy to recover and identify (example: Staphylococcus aureus) various molecular methods offer the ability for either direct detection in clinical material, or more rapid identification that would greatly aid in treatment and/or clinical management decisions.
|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.
|Molecular Versus Culture - Pros and Cons|
Traditional culture methods for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) employing mannitol salt and/or blood agar for cultivation, can take up to 72 hours for isolation and identification, depending on the identification procedures utilized. Concurrent with the development of molecular assays, improvements in culture methods have also been achieved. CHROmagar™ media, specific for MRSA, are employed by many laboratories. These media are both selective and differential, containing chromogenic substrates. MRSA strains utilize the substrates to produce colonies of a specific and characteristic color, minimizing the need for additional identification procedures.Initially these agars required 48 hours of incubation; newer formulations require only 24 hours incubation.Given the reduced incubation and identification requirements, what are the pros and cons of the molecular assays? Cost per test will be greater with the molecular assays as compared to culture methods. Will molecular methods provide for a more efficient workflow and significant improvement in availability of results? To some extent, this will depend on how they can be implemented within each different laboratory setting. Both of the previously described molecular assays require manual specimen preparation and extraction before the sample is placed into the instrument. This hands-on work may actually be greater than the effort expended in swabbing and streaking a culture plate. How much an obstacle this is for implementation will depend on both the volume of testing and the staff available. In a high volume setting, this will be a greater factor.Will tests be performed as specimens come in, or will specimens be accumulated and batched? If controls are required with each run, batching is desirable to reduce this cost. If testing will occur in batches, how many batches can be performed in one day? This will be heavily influenced by the capacity of the instrument. (For example, a single Smart Cycler unit can run up to 16 samples; multiple units would be needed in a high volume lab.) Can they be set up on more than one shift? The greater the number and frequency of batches that can be run, the greater improvement in turnaround time can be realized. Given these variables, implementation of a molecular assay for MRSA is not a given in each laboratory.
|Previous Methodologies: Culture and Cell Cytotoxicity Neutralization Assay (CCNA)|
CultureBacterial culture, utilizing selective/differential media, is an effective method for recovering Clostridium difficile. Its drawbacks are the length of time required (up to four days), as well as the inability to distinguish toxigenic strains from non-toxigenic strains. Positive cultures require follow-up testing for the ability to produce toxin.Cell Cytotoxicity Neutralization Assay (CCNA) This assay detects the presence of C. difficile toxin in fecal samples. A filtrate of stool sample is prepared and inoculated onto sensitive tissue culture cells. Typically human fibroblast cells are utilized; if toxin is present in the filtrate, it causes the fibroblasts to round up in a characteristic cytopathic effect. To verify that the cytopathic effect is caused by C. difficile toxin (and not some other toxic component or viral agent), the filtrate is also inoculated in parallel onto a second set of tissue culture cells, to which C. difficile specific anti-toxin has been added. Absence of cytopathic effect in the second set of cell cultures provides evidence that the cellular changes in the first set were caused by C. difficile toxin. Although CCNA is considered a gold standard for the detection of C. difficile toxin, it is labor intensive, requires the use of cell cultures, and requires at least 48 hours of incubation.
|Several methods of detection are available for the detection of Clostridium difficile in clinical samples. Which methods have the capability for detection in less than 48 hours? (Choose all that apply.)||View Page|
|Cell Cytotoxicity Neutralization Assay (CCNA)|
CCNA was developed to detect the presence of C. difficile toxin in fecal samples.In this assay, a filtrate of stool sample is prepared and inoculated onto sensitive tissue culture cells. Typically human fibroblast cells are utilized; if toxin is present in the filtrate, it causes the fibroblasts to round up in a characteristic cytopathic effect.To verify that the cytopathic effect is in fact caused by C. difficile toxin (and not by some other toxic component or viral agent) the filtrate is also inoculated in parallel onto a second set of tissue culture cells, to which C. difficile specific anti-toxin has been added.Absence of the cytopathic effect in the second set of cell cultures provides evidence that the cellular changes in the first set were caused by C. difficile toxin.Although CCNA is considered a gold standard for the detection of C. difficile toxin, it is labor intensive, requires the use of cell cultures, and requires at least 48 hours incubation.
|Various methods have been employed for detection of C. difficile disease: cultivation of the organism, cell cytotoxin neutralization assays, and enzyme immunoassays have all been among the staples of diagnostic approaches. Which statements are accurate characterizations of these assays?||View Page|
|Identification of Enterococcus Species From Clinical Cultures|
Gram stain: Gram-positive cocci in singles, pairs, or chains; cells can be ovoid to coccobacillaryColony morphology: On blood agar after 24 hours of incubation, colonies are nonhemolytic or alpha hemolytic (rare strains may be beta hemolytic), and approximately 1 to 2 mm in diameter.Catalase: NegativePresumptive identification: Growth on bile esculin agar and in 6.5% salt broth are two characteristics that have commonly been used to identify enterococcus species to the genus level. A positive esculin in combination with a positive PYR reaction is another approach to presumptive identification.Species identification:E. faecalis and E. faecium are usually easily identified by most commercial systems. Successful identification of the other species on these systems may vary. With respect to vancomycin intermediate or resistant strains, two key characteristics are motility and pigment. E. casseliflavus is both motile and possesses a yellow pigment; E. gallinarum is also motile but non-pigmented. E. faecalis and E. faecium demonstrate neither characteristic.
|Susceptibility Testing of Enterococci|
All susceptibility testing for enterococci should follow the standards defined by CLSI. Selection of drugs for testing will follow the same criteria defined previously. Because the enterococci possess many intrinsic resistance factors, there are many antibiotics that should not be tested (or if tested, suppressed from the final report). CLSI document M100 defines all applicable criteria. Both Disk Diffusion (Kirby Bauer) and broth dilution (MIC) methods are commonly employed, utilizing the following testing conditions:Medium: MHA for disk diffusion; CAMHB for broth dilution(supplemented to 50 ug/mL for daptomycin) Inoculum: Growth method or direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 + 2°C; ambient air. Disk diffusion; 16 to 18 hours; Dilution methods; 16 to 20 hours. All methods: 24 hours for vancomycin.
|High Level Aminoglycoside Resistance (HLAR) Screening|
HLAR is another significant acquired resistance factor. Since the standard approach for treating systemic infections is a combination of a cell wall targeted antibiotic with an aminoglycoside, assessment of resistance to both classes of antibiotics is important. High level resistance to aminoglycosides will negate the synergistic effect of combined therapy with either penicillin or vancomycin.Standard susceptibility methods (either disk diffusion or broth dilution) will not detect HLAR patterns, unless the protocol incorporates testing at increased concentrations of gentamicin and/or streptomycin. CLSI document M100-S23 outlines recommended protocols for screening for HLAR.Gentamicin HLARDisk diffusion MHA agar; 120 µg gentamicin disk; standard inoculum; standard incubation temperature (35 +/- 2° C; ambient air), 16-18 hours incubation durationInterpretationResistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution BHI broth; 500 µg/mL gentamicin; standard inoculum; standard incubation conditions, incubation duration 24 hours.Interpretation Any growth equates to resistant.Streptomycin HLARDisk diffusion MHA agar; 300 µg streptomycin disk; standard inoculum; standard incubation conditions, incubation duration16 - 18 hours.InterpretationResistant = 6 mm Inconclusive = 7-9 mm Susceptible > 10 mmBroth microdilution BHI broth; 1000 µg/mL streptomycin; standard inoculum; standard incubation conditions; incubation duration 24-48 hours. If susceptible at 24 hours, reincubate and re-read at 48 hours.Interpretation Any growth equates to resistant.Clinical correlation A resistant result indicates that synergistic effects will not be achieved between the indicated aminoglyocside and the cell wall active agent (eg, ampicillin, penicillin, or vancomycin).A susceptible result indicates that synergistic effects are possible.
|With regards to identifying resistance in enterococci, which general statements are true?||View Page|
When a clinical isolate is presumptively identified as S. aureus, susceptibility testing will be performed by either the standardized disk diffusion (Kirby-Bauer) or broth dilution (MIC) methods, using the following testing conditions as recommended by the Clinical and Laboratory Standards Institute (CLSI):MediumMHA for disk diffusionCation-adjusted Mueller-Hinton broth (CAMHB) + 2% NaCL for oxacillin, methicillin, and nafcillinCAMHB supplemented up to 50 µg/mL calcium for daptomycin InoculumDirect colony suspension-- Inoculum from an 18-24 hour non-selective agar plate used to prepare a direct inoculum equivalent to a 0.5 McFarland Standard. Incubation35° C (Testing at temperatures above 35° C may not detect MRSA); 24 hours for oxacillin, methicillin, nafcillin, and vancomycin.
|Which of the following scenarios represents appropriate detection of MRSA by the Kirby Bauer method?||View Page|
|Several saprophytic, hyaline molds have microscopic characteristics that mimic the mold forms of the dimorphic fungi (Histoplasma capsulatum, Blastomyces dermatitidis, etc.). Each of the following can be used to differentiate the saprophytic from the dimorphic fungi EXCEPT:||View Page|
|Of the following dematiaceous fungi, the black, suede-like colony illustrated here, reaching no larger than the size of a dime after 7 days incubation, most likely can be identified as:||View Page|
|The dematiaceous colony illustrated here grew to a diameter of 3 - 4 cm in 5 days. The dematiaceous fungus that can be ruled out is:||View Page|
|The dematiaceous conidium shown in this image was obtained from a tiny portion of dark colony that grew to maturity in six days. Spores incubated in a saline mount for four hours developed germ tubes from both terminal cells. The features observed confirm the identification of:||View Page|
|The dimorphic fungus that may produce black, yeast-like colonies after prolonged incubation at 37°C is:||View Page|
|Arrange the fungal species that are listed in the drop-down box according to length of time of recovery in primary culture; from most rapid to the slowest.||View Page|
|The colonies shown in the upper image were obtained on blood agar from a sputum specimen after 10 days incubation at 30°C. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The diagnosis is:||View Page|
|The growth of the colonies shown in the upper image was obtained on blood agar from a sputum specimen after 8 days of incubation at 30°C. The lower image is a photomicrograph of a lactophenol blue mount made from a portion of the colony. The diagnosis is:||View Page|
|This image illustrates a lactophenol blue mount of a mold recovered after 7 days incubation of brain heart infusion broth. The large macroconidia suggests the mold form of Histoplasma capsulatum. However, there is the possibility that this mold represents its saprophytic counterpart, which is:||View Page|
|This image illustrates a lactophenol blue mount prepared from a mold recovered after 7 days incubation on brain heart infusion broth. The individual microconidia, each borne by a delicate conidiophore, suggests the mold form of Blastomyces dermatitidis. However, there is the possibility that this mold represents its saprophytic counterpart, which is called:||View Page|
|Procedures for the rapid culture confirmation of suspected colonies of B. dermatitidis, C. immitis and H. capsulatum recovered from clinical specimens include:||View Page|
|Match the names of each of the species of yeast listed with its associated phenotypic property that is helpful in establishing a species identification.||View Page|
|The colonies illustrated in this photograph were recovered from a blood culture after 48 hour incubation at 30°C. The most likely source for the septicemia is:||View Page|
Culture Characteristics:Growth may be noted as soon as eight hours after inoculation and occurs on most routine media, including sheep blood agar (SBA), chocolate agar (CHOC), and routine blood culture media Does not grow on MacConkey (MAC) agarColony Morphology on SBA at 35°C, 18-24 hours:Flat or slightly raised, gray to white with a "ground glass" appearance Described as "tenacious" or "sticky" like petroleum jelly, shown in the top image B. anthracis is NOT hemolytic, while B. cereus is hemolyticCharacteristic Features:After 18 hours of incubation on SBA at 35°C, the slightly undulate margin may show curling, displaying a so-called "Medusa head" or described as comma-shaped protrusions, shown in the lower image
Culture Characteristics: Slow growth on sheep blood agar (SBA) and chocolate (CHOC) Growth in commercial blood culture systems, but may require extended incubation Enriched atmosphere with CO2may enhance growth of some strains If Brucella is expected or suspected, extend incubation up to seven days Colony Morphology on SBA at 35oC: Visible growth may take 48-72 hours Small, convex, and glistening Non-hemolytic
|Which of the following organisms display the characteristic "Medusa head" on sheep blood agar (SBA) after 18 hours of incubation at 35°C?||View Page|
|Category A Agents: Reasons Why They May be Used to Create Public Health Emergencies|
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