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Exercise 6: Clinical Testing of Fungal and Parasitic Microorganisms Eukaryotic microorganisms that are pathogenic are very different from prokaryotic pathogens. Eukaryotic microorganisms have more complex cells with organelles that perform specific functions for the cell and house their DNA within a nucleus. Because humans are also eukaryotic organisms, the way in which eukaryotic microorganisms are diagnosed and treated is very different from bacteria. Most antibiotics do not work to treat these types of infections. Students will be required to think very differently for this laboratory. The gram stain and specialized media will not play a role in diagnosis. Instead, looking for specific structures or life stages associated with eukaryotic microbes is required. In other words, in today's lab, students will be observing diagnostic structures and stages under the microscope. Fungi, protists, and helminths are the most common eukaryotic microorganisms that infect humans, and though they are often viewed by students as not as important as bacterial and viral infections, fungi are common opportunistic pathogens of the immunocompromised, and protists and helminths infect billions of people around the world. Therefore, it is important for students to understand how to diagnose these infections. Fungi are eukaryotic organisms that have unicellular forms called yeast (Figure 1) and multicellular forms called molds (Figure 2). Yeast are more similar to bacteria and can be cultured on specialized media, but during staining, cells appear more ovoid and larger than bacterial cells. They reproduce through an asexual process called budding in which an exact genetic copy is made from the parent and is pushed off from the top of the yeast cell. The most common yeast to infect humans is Candida albicans. Infections are often opportunistic and occur post-antibiotics, during chemotherapy, or in the immunocompromised. Molds, on the other hand, are multicellular and grow via hair-like filaments called hyphae. The hyphae make up the body of the mold (called the mycelium) and are how the mold interacts with the environment. The hyphae are used to extract nutrients and water. Molds reproduce both sexually and asexually. Asexual reproduction in mold requires that the mold produces asexual spores. Spores are environmentally resistant reproductive structures that contain all the genetic material to give rise to a fungus. Asexual spores contain the exact genetic code of the parent and give rise to cloned offspring. Sexual reproduction also occurs in most mold species. This requires the exchange of genetic material between different mating types (similar to males and females) and creates sexual spores. Sexual spores are morphologically different from asexual spores, and both are used in diagnosis. This is because asexual and sexual spores have different shapes and structures based on the type of fungi. In this lab, students will observe fungal yeasts and molds growing in cultures as well as make microscopic observations on yeast cell and spore morphology. Parasitic helminths are a major cause of disease throughout the world, and although many helminthic diseases are not commonly found in the US, this doesn't mean that they are not of great importance. Helminths or parasitic worms are multicellular eukaryotic animals that come in three different taxonomic groupings: trematodes (flatworms), cestodes (tapeworms), and nematodes (roundworms). Transmission of helminths is very diverse. They can spread via arthropod vectors, ingestion of contaminated food and water, and skin penetration. Additionally, parasites are extremely motile and rarely stay at their portal of entry. They are known to migrate around the body, and some enter the bloodstream or encyst in muscle. Thus, diagnosis of helminth infections can be tricky. Diagnosis usually involves searching for the eggs that are being released from the body because each parasite produces eggs that are of a unique shape and size. This requires that physicians have knowledge of the parasite lifecycle and egg morphology. Most often, eggs are released in fecal matter, but they can also be released in sputum. For parasites that reside in the bloodstream and muscle, a blood smear or biopsy, respectively, is required. In this laboratory, students will view both the eggs and the helminth itself of several helminths that are parasitic to humans. Vector-borne protists, on the other hand, tend to be virulent and have the potential to cause death. For example, two highly virulent diseases caused by vector-borne protists are malaria (Plasmodium spp.) and trypanosomiasis (Trypanosoma spp.). Vectors that transmit protistan microorganisms are usually arthropods, including mosquitoes, biting flies, and ticks. There are no free-living stages; the protist must be acquired directly from the vector. Often, the vector deposits the protist directly into the host bloodstream through a bite, leading to a systemic infection. Once the protist has access to the bloodstream, it can infect organs such as the liver, kidneys, and brain. Diagnosis of vector-borne protists is commonly done with a blood smear that is stained to make the parasite visible. In this laboratory, students will observe diagnostic stages of pathogenic protists. Microscopic Protista can be parasitic (live in a host), free-living (do not require a host), or both. For this lab, we will concentrate on protists that cause human infection, many of which have free-living stages. Most protistan parasites fall into two categories of human infection: water-borne infections and vector-borne infections. Water-borne protists are typically ingested with water contaminated with fecal material. They tend to infect the gastrointestinal tract, causing abdominal pain, nausea, and diarrhea. Infections are usually mild. These protists can often live for prolonged periods of time outside of the body because they have the ability to form a cyst stage in which the protist is protected by an environmentally resistant outer coating. Inside a host, water-borne protists are released from their cyst stage and develop into the reproductive feeding stage called the trophozoite. This is the stage that causes disease. However, both stages are often released into the environment via the feces and can, therefore, be diagnostic if found in a fecal culture. Exercise 6: Clinical Diagnosis of Fungal and Parasitic Microorganisms Pre-lab Questions 1. What are the growth forms of fungi? How are they different? 2. What is used to identify different species of molds? 3. What are the two main types of infections caused by protists? 4. How are protists typically diagnosed? 5. What are the three types of helminths? Give an example of each.

Exercise 6: Clinical Testing of Fungal and Parasitic Microorganisms

Eukaryotic microorganisms that are pathogenic are very different from prokaryotic pathogens. Eukaryotic microorganisms have more complex cells with organelles that perform specific functions for the cell and house their DNA within a nucleus. Because humans are also eukaryotic organisms, the way in which eukaryotic microorganisms are diagnosed and treated is very different from bacteria. Most antibiotics do not work to treat these types of infections. Students will be required to think very differently for this laboratory. The gram stain and specialized media will not play a role in diagnosis. Instead, looking for specific structures or life stages associated with eukaryotic microbes is required. In other words, in today's lab, students will be observing diagnostic structures and stages under the microscope.

Fungi, protists, and helminths are the most common eukaryotic microorganisms that infect humans, and though they are often viewed by students as not as important as bacterial and viral infections, fungi are common opportunistic pathogens of the immunocompromised, and protists and helminths infect billions of people around the world. Therefore, it is important for students to understand how to diagnose these infections.

Fungi are eukaryotic organisms that have unicellular forms called yeast (Figure 1) and multicellular forms called molds (Figure 2). Yeast are more similar to bacteria and can be cultured on specialized media, but during staining, cells appear more ovoid and larger than bacterial cells. They reproduce through an asexual process called budding in which an exact genetic copy is made from the parent and is pushed off from the top of the yeast cell. The most common yeast to infect humans is Candida albicans. Infections are often opportunistic and occur post-antibiotics, during chemotherapy, or in the immunocompromised. Molds, on the other hand, are multicellular and grow via hair-like filaments called hyphae. The hyphae make up the body of the mold (called the mycelium) and are how the mold interacts with the environment. The hyphae are used to extract nutrients and water. Molds reproduce both sexually and asexually. Asexual reproduction in mold requires that the mold produces asexual spores. Spores are environmentally resistant reproductive structures that contain all the genetic material to give rise to a fungus. Asexual spores contain the exact genetic code of the parent and give rise to cloned offspring. Sexual reproduction also occurs in most mold species. This requires the exchange of genetic material between different mating types (similar to males and females) and creates sexual spores. Sexual spores are morphologically different from asexual spores, and both are used in diagnosis. This is because asexual and sexual spores have different shapes and structures based on the type of fungi. In this lab, students will observe fungal yeasts and molds growing in cultures as well as make microscopic observations on yeast cell and spore morphology.

Parasitic helminths are a major cause of disease throughout the world, and although many helminthic diseases are not commonly found in the US, this doesn't mean that they are not of great importance. Helminths or parasitic worms are multicellular eukaryotic animals that come in three different taxonomic groupings: trematodes (flatworms), cestodes (tapeworms), and nematodes (roundworms). Transmission of helminths is very diverse. They can spread via arthropod vectors, ingestion of contaminated food and water, and skin penetration. Additionally, parasites are extremely motile and rarely stay at their portal of entry. They are known to migrate around the body, and some enter the bloodstream or encyst in muscle. Thus, diagnosis of helminth infections can be tricky. Diagnosis usually involves searching for the eggs that are being released from the body because each parasite produces eggs that are of a unique shape and size. This requires that physicians have knowledge of the parasite lifecycle and egg morphology. Most often, eggs are released in fecal matter, but they can also be released in sputum. For parasites that reside in the bloodstream and muscle, a blood smear or biopsy, respectively, is required. In this laboratory, students will view both the eggs and the helminth itself of several helminths that are parasitic to humans. Vector-borne protists, on the other hand, tend to be virulent and have the potential to cause death. For example, two highly virulent diseases caused by vector-borne protists are malaria (Plasmodium spp.) and trypanosomiasis (Trypanosoma spp.). Vectors that transmit protistan microorganisms are usually arthropods, including mosquitoes, biting flies, and ticks. There are no free-living stages; the protist must be acquired directly from the vector. Often, the vector deposits the protist directly into the host bloodstream through a bite, leading to a systemic infection. Once the protist has access to the bloodstream, it can infect organs such as the liver, kidneys, and brain. Diagnosis of vector-borne protists is commonly done with a blood smear that is stained to make the parasite visible. In this laboratory, students will observe diagnostic stages of pathogenic protists. Microscopic Protista can be parasitic (live in a host), free-living (do not require a host), or both. For this lab, we will concentrate on protists that cause human infection, many of which have free-living stages. Most protistan parasites fall into two categories of human infection: water-borne infections and vector-borne infections. Water-borne protists are typically ingested with water contaminated with fecal material. They tend to infect the gastrointestinal tract, causing abdominal pain, nausea, and diarrhea. Infections are usually mild. These protists can often live for prolonged periods of time outside of the body because they have the ability to form a cyst stage in which the protist is protected by an environmentally resistant outer coating. Inside a host, water-borne protists are released from their cyst stage and develop into the reproductive feeding stage called the trophozoite. This is the stage that causes disease. However, both stages are often released into the environment via the feces and can, therefore, be diagnostic if found in a fecal culture.

Exercise 6: Clinical Diagnosis of Fungal and Parasitic Microorganisms Pre-lab Questions

1. What are the growth forms of fungi? How are they different?
2. What is used to identify different species of molds?
3. What are the two main types of infections caused by protists?
4. How are protists typically diagnosed?
5. What are the three types of helminths? Give an example of each.

Added by Richard C.

21. You are in charge of a patient recently transferred from a long term-term care facility. You note in the chart that the patient has been catheterized by blood vessel for 8 weeks. The patient has recently developed a high fever (104 degrees fahrenheit) and is exhibiting severe hypotension. Repeated blood cultures are negative for bacterial cells (bacteria are not present in the blood). Which of the following apply: Staphylococcus aureus secretes exoenzymes onto dermal tissues will the release of DAMPs, that activate dendritic cells to secrete IL-1 A CRE strain of E.coli uses attachment pilli to bind patient tissues with lipopolysaccharide (LPS) release that causes neutralizing IgG secretion Pseudomonas aeruginosa forms a capsule in the catheter (IV line) and requirement treatment with Eravacycline (Exerava), and Lefamulin (Xenleta) Staphylococcus epidermidis forms a biofilm in the catheter (IV line) are releases lipoteichoic acids (LTA) into the blood that results in systemic release of tumor necrosis factor alpha from resident macrophage Streptococcus pneumoniae forms a capsule in the blood leading to C5a release and activation of tissue mast cells None of the above apply 22. Which of the following apply to the TORCH test? The test determines infections with teratogens The determines infections with mump’s virus The test determines infection with Group B streptococcus The determines the severity of Hemolytic Disease of the Newborn The test determine the severity of neonatal (newborn) HIV 24. Which of the following occur during an infectious process with the bacterial pathogen Streptococcus pyogenes, an extracellular pathogen that causes strep throat characterized by a severe sore throat and high fever: A secretion of digestive exoenzymes, including proteases, from S. pyogenes and release of IL-1 from resident macrophage and secretion of specific IgA and IgG to remove the pathogen Attachment to the host cell with specific capsomere proteins that can be blocked by neutralizing IgD Outgrowth of reticulate body (RB forms) in the host cell cytoplasm with a requirement of specific tc cells to remove the pathogen Invasiveness characterized by rapid budding of that causes tissues damage with a requirement of Th cells to secrete Il-2 to remove the pathogen The infection was caused by reactivation of the latent genome that will require secretion of specific IgM to remove the pathogen None of the above apply 25. All of the following apply to an enveloped virus that has a broad host range and narrow tissue specificity and causes severe pulmonary edema three days after injection EXCEPT: A presence of a viral genome that is protected by a capsid Presence of a glycoprotein spike that recognize similar a receptor in different species Presence of a capsule and biofilm that protects the virus from complement activation Rapid budding from the infected cell Release of DAMPs that cause to the production of tumor necrosis factor alpha IL-1, Il-6, IL-8 26. Which of the following is TRUE statement regarding the effect of high doses of corticosteroids, such as dexamethasone, on the immune system: Functions to specifically inhibit cyclooxygenase-2 (COX-2) to decrease pain and inflammation Functions to inhibit enzyme phospholipase A2, which decreases cyclooxygenase-2 (COX-2) levels Functions to inhibit enzyme phospholipase A2, which causes lethal disruption of WBC function (WBC death) Functions to inhibit vascular change by blockade of tumor necrosis factor alpha function Functions to inhibit IgG secretion from autoimmune B Functions of decrease secondary responses that contribute to inflammation None of the above are true statements 27. Which of the following adaptive immune system responses will be required to resolve a systemic (blood) infection with Staphylococcus aureus, an extracellular bacterial agent Synthesis and secretion of S. aureus-specific IgM from B (plasma) cells Synthesis and secretion of S. aureus-specific IgA from B (plasma) cells Clonal expansion of the S. aureus-specific macrophage Clonal expansion of the S. aureus-specific Tc cells Increased levels of MHC 1 displaying S. aureus-specific antigen All of the above apply 28. Which of the following apply to B cell recognition and response to antigen: Recognition of antigen displayed by MHC I with secretion cytotoxins Recognition of antigen displayed by MHC II with secretion of cytokines Recognition of antigen in the absence of MHC molecules with secretion Recognition of PAMPs by toll-like receptors with secretion of tumor necrosis factor alpha None of the above apply 29. You are a pediatric specialty nurse caring for a patient who is infected with the virus that causes mumps. The virus infects the parotid salivary glands with acute illness. Which of the following apply during the early, symptomatic stages of illness. The patient has a population of mumps-specific memory cells The patient has no mumps-specific TH, Tc, and B cells The patient has a small number of mumps-specific, TH, Tc, and B cells The patient has a high level of mumps-specific IgE The patient has previously been vaccinated against the mumps virus None of these apply 30. The Gram negative cell wall contains lipopolysaccharide (LPS) as a unique component. All of the following apply to LPS EXCEPT: LPS is found in the outer membrane of Gram negative cells O-linked side chains have unique carbohydrate composition specific for each Gram negative species LPS is a PAMP that can be recognized by toll-like receptors (TLRs) as a foreign molecule for phagocytosis Recognition of individual Gram negative species by specific antibody LPS can be recognized by B cells in the absence of MHC molecules LPS can be a target for specific IgG for ADCC-opsonization All of above apply, no exception 31. Which of the following will be MOST important antibody function(s) in the clearance of a capsule -producing bacterial species by phagocytosis: Complement directed lysis and chemotaxis of neutrophils ADCC-opsonization and agglutination Neutralization and anti-toxin activity against exotoxins Association with mast cells to release histamines, prostaglandin, and leukotrienes Induction of perforin and granzyme B synthesis All of the above are equally important antibody functions 32. All of the following apply to influence A virus infections and the immune response to the virus EXCEPT: Use of hemagglutinin to adsorb to respiratory epithelial cells Requirement for the vaccine to be reformulated yearly due to antigenic drift Global pandemics due to antigen shift Release of perforin and granzyme on infected cells Requirement for IL-2 for resolution of the infection Use of an inactive vaccine preparation that is composed of whole, killed virus All the above apply, no exceptions 33. Vancomycin resistant enterococci (VRE) are a common source of nosocomial infections. All of the following apply to VRE EXCEPT: VRE can be part of the normal flora VRE infections primarily impact elderly patients in the intensive care unit (ICU) VRE is an example of hospital acquired infection (HAI) VRE produce altered tetrapeptide chains VRE infectious cause the release of both PAMPs and DAMPs All of the above apply, no exception 34. Which of the following apply to a patient who has been infected with the Hepatitis C Virus (HCV) for 19 years: Alteration of PAMPs and DAMPs to prevent production of cell adhesion molecules and vascular permeability The patient is experiencing a re-activation of the virus due to immunosuppression caused by inhibition of phagocytosis and complement activation Continual release of HCV from infected cells to body fluids with inhibition of alpha interferon signaling and decrease in MHC-I synthesis Systemic secretion of leukocidins that kill white blood cells Damage due to release of tissue factor from activated macrophage that recognized HCV lipopolysaccharide (LPS) None of the above apply 35. Which of the following are potential hazards associated with vaccines: Attenuated vaccines may revert to wild type (virulent) form Attenuated vaccines may cause fetal damage in pregnant individuals Attenuated vaccine may cause clinical disease in immunosuppressed patients Vaccines may cause allergies due to extraneous materials found in vaccine preparations Vaccines may cause serious side effects, including blood clot formation leading to hospitalization All of the above apply 36. You are a nurse caring for a patient with a pulmonary (lung) mold infection. The mold represents a large, macroscopic pathogen. Which of the following adaptive immune system responses would be MOST effective at controlling the mold infection: Production of a large population of antigen-specific Tc cells to destroy mold cells An increase in host cell production of MHC-I High levels of IgG that promotes agglutination, complement activation, and opsonization High levels of IgA that promote neutralization High levels of IgE that promote vascular change in association with mast cells High levels of IgM that promote complement activation None of the above apply 37. Which of the following contribute to the functional antibody protein structure: TCR CD4 CD8 CD20 Gamma interferon IL-2 None of the above apply 38. Which of the following apply to MHC II molecules: Are found on the surface of antigen presenting cells Are found on the surface of all human cells Are found on the surface of red blood cells Function to present (display) antigen for Tc cells Function to present (display) antigen for B cells None of the above apply 39. Which of the following apply measles virus infection signs and symptoms: Fecal oral transmission into the intestinal tract Use of toxoid vaccine to provide protection for measles virus Development of a maculo-papular skin rash Infection of a fetus during primary response in a pregnant patient Infection of the parotid salivary glands All of the above 40. Which of the following apply to a secondary immune response: A lag time of 5-7 days Symptomatic illness of the patient A large, antigen-specific memory cell population A small number of antigen -specific lymphocytes A large population of the infectious agent None of the above apply

Adi S.

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