MMG 465 Learning Objectives PDF

Learning Objectives MMG 465 FS23 1. Diagnosis of Infectious Diseases 1.1. Describe the purpose of specimen direct microscopy reporting. Provide examples of the critical information provided by direct microscopy results. Direct microscopy can help identify the pathogen whether it...

Learning Objectives MMG 465 FS23 1. Diagnosis of Infectious Diseases 1.1. Describe the purpose of specimen direct microscopy reporting. Provide examples of the critical information provided by direct microscopy results. Direct microscopy can help identify the pathogen whether it is Gram positive, or Gram negative you can then go from there. In clinical micro light and fluorescent microscopes are used, electron not so much. Can do Wet prep or Fixed and stained: Gram stain, Acid fast, Acridine orange, Trichrome, Giemsa, Methylene blue, Lactophenol cotton blue, Calcofluor white. 1.2. Select appropriate stains for direct specimen microscopic examination and pure cultures a. Gram Stain- used for general bacterial identification. Differentiation b/t Gram positive and Gram negative. b. Acid Fast Stain- used for suspected Mycobacterium spp. (tuberculosis). Have unique cell wall composition. c. Acridine Orange Stain- rapid detection of low bacterial counts in specimen. Fluorescent stain that has high sensitivity. 1.3. Interpret isolated colony and direct specimen Gram and Acid Fast Stain slides. Isolated colony and direct specimen Gram and Acid-Fast stain slides can be interpreted by observing the color, shape, and arrangement of the microorganisms, which help determine their Gram reaction (positive or negative) and their ability to retain the acid- fast stain (for Mycobacteria). 1.4. Compare direct specimen Gram stain results and primary isolation suspect colonies to determine if the two results agree Comparing direct specimen Gram stain results with primary isolation suspect colonies involves checking for consistency in morphology, Gram reaction, and other characteristics to confirm whether the same organism is present in both the specimen and isolated colonies. 1.5. Describe the intended use and components of primary inoculation media used for bacterial culture. a. Non-selective: allows different types of microorganisms to grow without discrimination (i.e., Nutrient broth, tryptone soy broth, All culture agar). b. Differential media: used to differentiate b/t two closely related microorganisms (MAC- contains lactose and pH indicator if it is utilized, BAP- pH indicator for different types of hemolysis). c. Selective Media: used to inhibit growth of other microorganisms (MAC- contains bile salts that inhibit growth of most Gram-positive bacteria, HEK- bile salts inhibit  growth of gram-positive organisms, MSA- utilizes high salt concentration to inhibit the growth of most bacteria except for staphylococci). d. Enrichment: will grow a wide range of microorganisms, including fastidious organisms (CHOC). 1.6. Explain the basis of and given and image visually differentiate between alpha (α), beta, (β), and non-hemolytic or gamma (γ) hemolysis of colonies grown on sheep blood agar (BAP). Alpha hemolysis: greenish-brown color of colonies, partial damage to RBCs Beta hemolysis: yellow colonies, RBCs are completely lysed. Gamma hemolysis: non hemolytic, no color change of colonies, opaque/whitish. No lysis of RBC. 1.7. Provided images use accepted terms to describe colony morphology. Form (circular, filamentous, irregular), elevation (flat, raised, convex, or umbonate), margin (entire, undulate, lobate, or curled), surface (smooth, glistening, rough, wrinkled), texture (dry, mucoid, brittle, viscid), size, opacity, color, odor, hemolysis. 1.8. Describe the principles and application of common immunologic assay used to diagnose infectious diseases a. Enzyme immune assay: This assay uses enzymes linked to antibodies to detect the presence of antigens or antibodies, providing a color change or fluorescence to indicate a positive result, commonly used for diagnosing infections like HIV or hepatitis. b. Direct fluorescent antibody assay: This test uses fluorescently labeled antibodies to detect specific antigens directly in patient specimens, helping to diagnose infections like influenza or respiratory viruses. c. Indirect fluorescent antibody assay: This method detects antibodies in patient samples by using a fluorescently labeled secondary antibody, which binds to antibodies in the specimen, useful in diagnosing diseases like syphilis or Lyme disease. 1.9. Describe the principles, procedures and clinical laboratory applications of polymerase chain reaction, real time PCR, MALDI TOF, and whole genome sequencing Polymerase chain reaction (PCR): PCR amplifies specific DNA sequences to detect microorganisms at the genetic level, widely used to identify pathogens like bacteria, viruses, and fungi in clinical specimens. Real-time PCR: A variation of PCR that quantifies DNA amplification in real-time, allowing for more precise measurement of pathogen load, frequently used in viral load testing, such as for HIV or COVID-19. MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight): A mass spectrometry technique used to rapidly identify microorganisms by analyzing their protein profiles, improving pathogen identification in clinical microbiology.  Whole Genome Sequencing (WGS): This technique sequences the entire genome of a pathogen, providing detailed information on its genetic makeup, helping in epidemiological tracking and antibiotic resistance profiling. KEY TERMS Columbia colistin-naladixic acid agar (CNA) Differential stain Chocolate agar Simple stain Antibody titer Mordant IgG Antigen IgM Antibody Substrate Conjugate Analyte Alpha hemolysis Conjugate Beta hemolysis Matrix Gamma hemolysis Ionization Blood agar plate (BAP) Primer MacConkey agar Taq polymerase Thioglycolate broth Denature Meuller-Hinton agar Anneal Hektoen agar Amplicon Xylose lysine desoxycholate agar (XLD) Taqman probe Ct value Learning Objectives Quality Management in the Clinical Microbiology Laboratory 1. Explain how pre-examination, examination, and post-examination phase factors impact test quality. Pre-examination: wrong sample, mislabeling, not enough of sample, Examination: Expired reagent, faulty machine, not enough reagent added, wrong temp of water bath, contamination  Post-examination: misread results, misread antibody testing… 2. Analyze testing scenarios to identify sources of error at each phase of testing and create actions to prevent the error from reoccurring. To analyze testing scenarios and identify sources of error at each phase of testing (pre- analytical, analytical, and post-analytical), one must first examine factors such as sample collection, handling, and storage (pre-analytical), equipment calibration, reagent quality, and technician technique (analytical), and data interpretation, reporting, or follow-up (post-analytical). Once errors are identified, corrective actions could include improving training, standardizing protocols, implementing quality control measures, ensuring proper equipment maintenance, and verifying the accuracy of results before reporting to prevent recurrence of the error. 3. Identify the 12 Quality System Essentials: organization, personnel, equipment, purchasing/inventory, process control, information, management, documents and records, occurrence management, assessment, process improvement, customer service, facilities and safety 4. Explain how the quality of clinical microbiology processes are assured through verification, validation, quality control and proficiency testing. Verification: confirmation that an assay is performing as it is intended to perform. Validation: establishment of assay performance specifications for the first time Quality Control (QC): Routine procedures to monitor and maintain the accuracy and reliability of testing, such as using known controls and calibrators with each test run to detect any drift or issues in reagents or equipment. Proficiency testing: External assessments in which laboratories receive unknown samples and are evaluated on their ability to correctly identify and report results, ensuring competency and consistency in laboratory performance. 5. Calculate the sensitivity and specificity of a diagnostic test. Sensitivity = (true positive)/(true positive + false negative) x 100 Specificity = (True negative)/(true negative + false positive) x 100 Key terms False positive False negative True positive True negative Verification Validation Quality control Proficiency testing Audit Learning Objectives Safety in the Clinical Microbiology Laboratory 1. Define Good Microbiology Laboratory Practices Having chemical safety (SDS, inventory, training, fume hoods, PPE), having compressed gases secured and stored in well-ventilated area, fire safety (alarm, evacuation, contain, extinguish), electrical safety (don’t overload circuits, never bypass safety equipment, use only sound equipment and cords), avoid accidents due to falling/lifting, have infection controls, and avoid repetitive motion injuries (multi-pipette). 2. Define Standard Precautions and apply to situations in the clinical microbiology laboratory. Standard precaution: extends universal precautions to include all body fluids and secretions (except sweat) regardless of whether blood is present. -Treat everything as if it were infectious. Apply to situations: hand-washing, use latex or nitrile gloves, mask or face shield if there is risk to aerosols, long-sleeved lab coats and cover legs and feet, dispose of sharps appropriately (slides in sharps), clean and disinfect surfaces that are potentially contaminated. 3. Identify laboratory design features, personal protective devises and equipment used at Biosafety Levels 1, 2, 3 and 4. BSL-1: Design control includes easily cleaned benches and closable doors. Equipment is none. PPE with or without gloves. BSL-2: Design control is lockable doors and windows, impermeable benchtops and floors. Equipment includes class 1 of 2 BSC biohazard sign, mechanical pipetting device, autoclave near lab, handwashing sink, and eye wash. PPE includes front closing lab coat, latex or equivalent gloves, and face shield. BSL-3: Design control is anteroom and negative air pressure. Equipment includes pass through autoclave and centrifuge with closeable caps. PPE includes back closing and wrist cuff lab coat, N95 or PAPR mask and eye protection. BSL-4: Design control is shower in, shower out anteroom, and air lock. Equipment is class 3 BSC. PPE is positive pressure suits. 4. Identify non-biological safety hazards in the clinical microbiology laboratory. Non-biological safety hazards in the clinical microbiology laboratory include chemical hazards (e.g., exposure to toxic reagents like formaldehyde, alcohol, and disinfectants), physical hazards (e.g., slips, trips, and falls, as well as ergonomic hazards from improper workstation setups), and electrical hazards (e.g., faulty equipment or electrical malfunctions). Additionally, radiation exposure from diagnostic imaging or UV light sources and potential fire hazards from flammable chemicals or equipment also pose risks. 5. Describe the each physical process listed below and determine if it is microbial killing completeness. Identify laboratory or hospital situations where each method is recommended. a. Incineration: Burning materials at very high temperatures (800C to 1000C) to destroy microorganisms, including hazardous waste, biological samples, and contaminated materials. Recommended situation: Disposal of needles, syringes, biohazardous waste, and other infectious material. b. Autoclaving: Pressurized steam at temperatures around 121C for 15-20 minutes to sterilize equipment, media, and waste. Used for sterilizing glassware, surgical instruments, and certain contaminated waste in hospitals or microbiology labs.  c. Dry heat: burning to ashes (ex: flaming incineration). Uses hot air (typically 160-180C) in a controlled environment to kill microorganisms. Recommended for sterilizing materials thst may be damaged by moisture, such as glassware, metal instruments, and powders. d. Ionizing radiation: (Gamma rays or X-rays) damages microbial DNA, rendering it incapable of reproduction, thus sterilizing materials. Recommended for sterilizing heat-sensitive medical supplies, drugs, vaccines, and food products, especially when other sterilization methods are not feasible. e. Filtration: exclude microbe from gas or liquid (example: filter sterilize heat-sensitive media). Involves passing liquids or gases through a filter with pores small enough to physically remove microorganisms, typically for heat-sensitive materials. Recommended for sterilizing heat-sensitive liquids (culture media, vaccines) or for air filtration in biosafety cabinets and isolation rooms. 6. Describe the mode of action agents for control of microorganisms listed below. Describe the situation in which the agent should be used and determine if it represents a sterilizing or disinfecting agent. a. Alcohols: MOD: denature proteins, dissolve lipids (ex; ethanol, isopropanol). This is used as a disinfectant. b. Aldehydes: MOD: react with proteins (formaldehyde, glutaraldehyde). This is used as disinfectant and sterilization. c. Phenolics: MOD: denature proteins, dissolve lipids (phenol, carbolic acid). This is used as disinfectant agent. d. Quaternary ammonium compounds: MOD: detergent that disrupts cell membrane. This is used as a disinfectant agent. 7. List the steps and activities in a risk assessment -Identify hazards and risks -evaluate risks -determine controls -implement controls -review effectiveness of controls Learning Objectives Antimicrobial Resistance and Susceptibility Testing 1. Describe the mode of action and the cellular targets of the antimicrobial classes/agents. -Cell wall synthesis inhibitors: stop bacterium from putting wall together (B-lactams, Glycopeptides, Fosfomycin, Bacitracin, Alafosfalin) -DNA gyrase inhibitors: prevents DNA from being unwinded so bacterium is unable to get to DNA (Quinolones and Coumermycin antibiotics) -Inhibition of DNA-dependent RNA polymerase: Different target (rifamycin) -Cell membrane synthesis disruptors: (cell membrane is made of phospholipids- (Lipolipids) -Folate synthesis inhibitors (Sulfonamides) -RNA synthesis inhibitors: stops from making messenger protein (Ansamycines) -Protein synthesis (30S and 50S inhibitors): stop bacteria from making proteins (Tetrcyclines, Aminoglycosides, Macrolides, Lincosamides, Amphenicols, Pleuromutilins, Oxazolidnones) 2. Define and compare intrinsic and acquired mechanisms of resistance to antimicrobial agents. Intrinsic: part of their genome -all strains of the species lack target -outer membrane prevents uptake -thin or no cell wall -enzymes that destroy the antibiotic -Efflux pump Acquired: bacteria gain it -mutation/alteration in target -destruction or alteration of antibiotic via enzyme (bacteria pass around genes more easily than eukaryotes -efflux pump -decreased uptake (porin mutation, cell wall precursor mutation) 3. Explain the mechanisms by which microbes acquire and disseminate antimicrobial resistance genes. Efflux pump: Active transport systems that expel antimicrobial agents from the cell, reducing their intracellular concentrations and effectiveness. Blocked penetration: Alteration or loss of porins in the cell membrane prevents antibiotics from entering the bacterial cell. Inactivation of enzymes: Bacteria produce enzymes (e.g., beta-lactamases) that break down or modify the antimicrobial agent, rendering it ineffective.  Target modification: Mutations or modifications in the antimicrobial agent’s target site (e.g., ribosomal RNA or enzymes) reduce the drug’s binding affinity, making it ineffective. 4. Explain and the molecular basis for inducible resistance and how this impacts rapid antimicrobial resistance test results. Inducible resistance: resistance that is not constitutively present. It requires something to induce its activity (upregulate genes). This impacts rapid antimicrobial resistance test results because although it may be in the gene it may not be expressed to where we could see it. Inducible resistance refers to the resistance that is not always expressed but can be triggered under certain conditions, typically through exposure to the antimicrobial agent (e.g., the presence of an antibiotic that activates specific resistance genes). This means that the resistance might not be detectable in routine testing until it is induced, leading to false- negative results in rapid antimicrobial resistance tests until the resistance is triggered. Therefore, results from such tests may not fully represent the true antimicrobial susceptibility of the organism. 5. Explain the principle of the following tests. Provided images, interpret results 1. Kirby-Bauer disk diffusion: In this method, antimicrobial-impregnated paper disks are placed on an agar plate inoculated with the test microorganism. The antibiotic diffuses radially, and the zone of inhibition (clear area around the disk) indicates the effectiveness of the antibiotic. Measure the diameter of the inhibition zone to determine if the microorganism is susceptible, intermediate, or resistant based on established zone size breakpoints. 2. Minimum inhibitory concentration tests: The MIC is the lowest concentration of an antimicrobial agent that prevents visible growth of the microorganism in liquid culture. The MIC is determined by observing the concentration at which bacterial growth is inhibited, often reported in µg/mL. The organism is classified as susceptible, intermediate, or resistant based on this concentration. 3. Broth dilution: Serial dilutions of an antimicrobial agent are prepared in liquid broth, and the test microorganism is inoculated into each dilution. The lowest concentration without visible growth indicates the MIC. Like MIC testing, broth dilution quantifies antimicrobial activity and provides a numerical value for susceptibility. 4. Gradient strip diffusion (E-test)Agar dilution: This test uses a plastic strip with a gradient of antimicrobial concentrations, which is placed on an agar plate inoculated with the microorganism. The point where the growth inhibition intersects with the strip indicates the MIC. The MIC is read directly from the strip where the bacterial growth is inhibited, allowing for precise determination of antibiotic resistance. 5. D test: Erythromycin elbows to say there is a problem (line forms between two discs looking like a capital D). The D-test detects inducible clindamycin resistance in Staphylococcus species. It involves placing an erythromycin disk near a clindamycin disk on an agar plate. If the bacteria exhibit resistance to clindamycin, a "D-shaped" zone of inhibition forms, indicating the presence of resistance mechanisms. A "D" shape indicates inducible resistance to clindamycin, which means clindamycin should not be used in treatment. 6. Modified Carbapenam Inhibition Test (mCIM): mCIM is used to detect carbapenemase-producing organisms. A suspected carbapenem-resistant organism is cultured on an agar plate with a carbapenem disk and a meropenem disk placed near it. A lack of inhibition or "drop" in inhibition near the meropenem disk indicates carbapenemase production. A positive result indicates that the microorganism produces carbapenemases, conferring resistance to carbapenem antibiotics. 7. Commercial automated systems: These systems, like VITEK and BD Phoenix, automate the process of antimicrobial susceptibility testing by utilizing pre- loaded panels of antibiotics to determine the MIC or susceptibility profile of bacterial isolates based on turbidity or color changes in the media. Results are automatically generated and interpreted by the system, providing a comprehensive susceptibility profile in a short period. 8. Nitrocephin disk method: This test detects beta-lactamase production. A disk impregnated with nitrocephin, a chromogenic substrate, is placed on a bacterial culture. If the bacteria produce beta-lactamase, the nitrocephin is hydrolyzed, resulting in a color change. A color change (yellow to pink) indicates beta-lactamase production, confirming resistance to beta-lactam antibiotics. 6. Identify the Kriby-Bauer and broth dilution procedure modifications required to detect methicillin and vancomycin resistance in Staphylococcus aureus. Methicillin Resistance (MRSA): -Kirby-Bauer Modification: For detecting methicillin resistance in Staphylococcus aureus, the standard Kirby-Bauer disk diffusion test uses an oxacillin disk instead of a penicillin disk, as oxacillin is more stable and reflects methicillin resistance more accurately. The testing conditions, including incubation at 35-37°C for 24 hours, and the use of specific zone size breakpoints for oxacillin, are essential to correctly interpret the results. -Broth Dilution Modification: In broth dilution testing, oxacillin (or cefoxitin, an alternative) is used instead of penicillin to determine the minimum inhibitory concentration (MIC). If the MIC is above a certain threshold (usually ≥ 4 µg/mL), the isolate is considered resistant. The presence of resistance may be confirmed by detecting the mecA gene, which encodes the altered penicillin-binding protein (PBP2a). Vancomycin Resistance (VISA or VRSA): -Kirby-Bauer Modification: For vancomycin resistance, the vancomycin disk is used. The interpretive criteria for the zone of inhibition are adjusted for vancomycin, as the concentration required to inhibit Staphylococcus aureus may be higher than that for other antibiotics. -Broth Dilution Modification: For vancomycin MIC determination, vancomycin is tested in broth dilution, and resistance is indicated if the MIC exceeds 2 µg/mL, as isolates with an MIC of ≥ 2 µg/mL are classified as vancomycin-intermediate S. aureus (VISA). True vancomycin resistance (VRSA) is less common and is characterized by an MIC ≥ 16 µg/mL. 7. Describe the mechanisms of resistance and diagnosis for the following emerging problem is antimicrobial resistance: 1. Candida auris: Mechanisms of Resistance: Candida auris has emerged as a multidrug-resistant pathogen, with resistance mechanisms including mutations in the ergosterol biosynthesis pathway (affecting azoles), efflux pumps (affecting polyenes and azoles), and reduced drug permeability. Some strains also have intrinsic resistance to echinocandins due to mutations in the Fks1 gene. Diagnosis: Diagnosis is performed by culture, with Candida auris often requiring specialized media and incubation conditions for proper identification. Molecular testing, such as PCR, and MALDI-TOF mass spectrometry can help confirm the identity of C. auris and detect resistance markers. Antifungal susceptibility testing (e.g., broth microdilution) is essential for confirming resistance patterns. 2. Extended spectrum beta lactamases: Mechanisms of Resistance: ESBLs are enzymes produced by bacteria that hydrolyze extended-spectrum cephalosporins (such as ceftriaxone and cefotaxime) and monobactams (e.g., aztreonam). They are often produced by Escherichia coli and Klebsiella pneumoniae. ESBL-producing organisms may also exhibit co-resistance to other classes of antibiotics, such as aminoglycosides and fluoroquinolones,  through additional resistance mechanisms (e.g., plasmid-mediated resistance). Diagnosis: Detection of ESBL production can be performed using phenotypic tests like the double disk synergy test (using clavulanate with a cephalosporin) or broth microdilution methods. Molecular methods (PCR) can identify bla-CTX-M, bla-SHV, or bla-TEM genes, which encode common ESBLs. 3. Carbapenem resistant Enterobacterales: Mechanisms of Resistance: Carbapenem resistance in Enterobacterales (which includes E. coli, K. pneumoniae, Enterobacter species) is most commonly due to the production of carbapenemases, enzymes that hydrolyze carbapenems. The main types of carbapenemases include KPC (Klebsiella pneumoniae carbapenemase), NDM (New Delhi metallo-beta-lactamase), VIM (Verona integron-encoded metallo-beta-lactamase), and OXA-48. Resistance may also be mediated by porin loss and efflux pumps. Carbapenem-resistant organisms can be identified by disk diffusion (using carbapenem disks) or broth microdilution for MIC determination. The presence of carbapenemase production can be confirmed using molecular tests (e.g., PCR for carbapenemase genes) or phenotypic tests like the carbapenem inactivation method (CIM), which evaluates the organism's ability to degrade carbapenem antibiotics. CRE infections can also be detected with modified Hodge tests or by using commercial systems capable of identifying carbapenemase-producing organisms. Can detect CRE resistance using CarbaNP or mCIM. 8. Explain the use and development of a hospital cumulative antibiogram. Antibiogram looks at local trends and tells us general empire therapy. Help us see what local population looks like. Organisms greater than or equal to 30 isolates. Limitations include no MICs, resistance and efficacy may vary by patient age (could be looking at wrong patient population), and don’t identify multidrug resistant organisms. 9. Recognzie examples of inappropriate pathogen-drug combinations and identify where these combinations can be found. Key Terms Break point Nitrocephin Horizontal gene transmission Non-susceptible Antibiogram Intermediate Minimum inhibitory concentration Resistant Zone of inhibition Extended spectrum β-lactamase Learning Objectives Infections of the Central Nervous System 1. Describe the anatomy of the central nervous system and how it provides both a defense mechanism and a therapeutic challenge. The central nervous system has meninges that is protected by bone. CSF flows down spine and then back up the posterior. 2. List the normal microbiota of the CNS. The normal microbiota of the CNS should have no normal flora. 3. Describe the glucose and protein levels and cell count/types expected in normal CSF and CSF from persons with bacterial, viral, fungal and parasitic infections. Bacterial: the appearance will be cloudy yellow. Glucose level will be less than 40 mg/dL. Protein count will be 100-500 mg/dL. WBC count will be 1000-5000 cells/mm^3. The primary WBC type would be Neutrophil. Viral: Appearance is cloudy. Glucose level is greater than 45 mg/dL. Protein is greater than 200 mg/dL. WBC count is 50-1000 cells/mm^3. The primary WBC type is lymphocyte. Fungal: The appearance is slightly cloudy. The glucose is less than 40 mg/dL. Protein is greater than 45 mg/dL. The WBC count is 20-500 cells/mm^3. The primary WBC type is mononuclear. Parasitic: The appearance is clear. The glucose levels are normal/low. The protein level is normal/elevated. The WBC count is 500-200 cells/mm^3. The primary WBC type is eosinophils greater than 10% and neutrophils. Prion (TSE): The appearance is clear. The glucose is normal. The protein level is 90-100 mg/dL. The WBC count is 0-20 cells/mm^3. 4. Differentiate between meningitis, encephalitis and brain abscess. Page 898 in textbook. Meningitis: Meningitis is the inflammation of the meninges, the protective membranes covering the brain and spinal cord. Caused by bacterial, viral, fungal, or parasitic infections, as well as non-infectious conditions like autoimmune diseases. The most common pathogens for bacterial meningitis are Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Diagnosis is typically confirmed by lumbar puncture with cerebrospinal fluid (CSF) analysis, revealing an elevated white blood cell count (pleocytosis), increased protein levels, and decreased glucose in bacterial meningitis. Encephalitis: Encephalitis is inflammation of the brain parenchyma (the brain tissue itself), often involving the gray matter. caused by viral infections, with herpes simplex virus (HSV) being the most frequent pathogen, followed by other viruses like arboviruses (e.g., West Nile virus, Japanese encephalitis), varicella-zoster virus (VZV), and enteroviruses. MRI or CT scans may show evidence of brain inflammation, while lumbar puncture can reveal elevated white blood cell counts in CSF. PCR testing of CSF is crucial to identify viral pathogens like HSV. Antiviral therapy (e.g., acyclovir for HSV encephalitis) is often used, along with supportive care. For non-viral causes, antibiotics or antifungals may be required.  Brain abscess: hematogenous or continuous spread, surgery or trauma. Solid organ transplant and immunosuppression. CSF culture usually negative. Need imaging + biopsy to see. 5. Identify the most common bacterial causes of meningitis in newborn, children, adolescents and young adults and older adults. Newborn (64): Streptococcus pneumoniae 6. Identify primary isolation media routinely used for isolation of the most common bacterial pathogens infecting the CNS. Choc and blood agar? 7. Describe the antigens used in the meningitis, Hib, and pneumococcal vaccines. i. Meningococcal vaccine: Protects against Neisseria meningitidis. Antigens: Uses polysaccharides or conjugated polysaccharides from capsules. Serogroup A,C, W, and Y. Serogroup B: recombinant proteins or surface antigens to stimulate immune response. Types: Conjugate vaccines and serogroup B. vaccine. j. Haemophilus influenza Type b (Hib) Vaccine: antigens are based on capsular polysaccharide with a substance called polyribosylribitol phosphate (PRP). Types: Conjugate vaccines that use PRP antigen. k. Pneumococcal vaccine: Polysaccharides from the capsule of Streptococcus pneumoniae, with conjugate vaccines (PCV13) for children and polysaccharide vaccines (PPSV23) for adults and high-risk individuals. 8. Identify the groups at highest risk of infection, transmission route, disease, laboratory methods for culture and identification of bacterial causes of meningitis. a. Neisseria meningitides Risk groups: US infants, adolescents and young adults. Close contact setting (households, dormitories, daycare centers). Transmission: Respiratory droplets Disease: Meningitis and Sepsis Lab methods: CSF culture on CHOC and BAP → Fastidious, Gram-negative diplococci, oxidase (+), PCR, Antigen detection b. Listeria monocytogenes Risk groups: Newborns and elderly (>65 years), pregnant women and unborn child Transmission: Ingestion (foodborne) and vertical transmission Disease: Meningitis (associated with encephalitis or bacteremia), Sepsis and focal infections (abscesses). Death rate in known infections >25% Lab methods: CSF culture on BAP → GPR, Catalase (+), subtle beta hemolysis, umbrella motility at 25C, CAMP + c. Haemophilus influenzae Risk: Infants and children (esp those under 5 years) Transmission: Respiratory droplets  Disease: Meningitis, pneumonia, epiglottitis and otitis media Lab methods: CSF culture on CHOC (requires factor V+X) → Gram negative coccobacilli, X and V factor test (requires both), PCR d. Streptococcus pneumoniae Risk: Leading cause of meningitis in children 60 years. Transmission: Respiratory droplets Disease: Meningitis and Sepsis Lab methods: BAP → GPC in pairs or chains, alpha hemolysis (green zone of hemolysis), Optochin S, Bile solubility test (lyse), Latex agglutination or PCR e. Streptococcus agalactiae Risk: Newborns, Screen pregnant women Transmission: Vertical transmission Disease: Neonatal meningitis, pneumonia and sepsis, invasive disease Lab methods: BAP → GPC in pairs or chains, narrow zone of beta-hemolysis, Catalase (-), CAMP (+), PCR, Bacitracin R. culture sensitivity is enhanced by selective broth media (Lim broth) and subculture to solid media. Use Carrot broth (selective and differential) for screening moms. 9. Identify the groups at highest risk of infection, transmission route, disease, laboratory diagnostic method for viral causes of encephalitis and meningitis a. Herpes viruses Risk: Neonates (serious infections seen in infants born to mothers with genital herpes- Encephalitis and brain damage). Immunocompromised, elderly. Transmission: HSV-1: direct contact. HSV-2: Sexual contact, vertical transmission. Disease: Herpes simplex encephalitis (HSE) and neonatal herpes. Diagnostics: PCR, elevated WBC, normal glucose, elevated protein. b. Enteroviruses (Coxsackievirus, Echovirus, Poliovirus, EV 68) Risk: Infants and young children, immunocompromised individuals. Transmission: Fecal-oral, body fluid, waterborne Disease: Aseptic (viral) meningitis, encephalitis, polio, hand foot and mouth, viral conjunctivitis, rarely myocarditis Diagnostics: PCR, Viral culture  c. Rabies virus Risk: Animal handlers, people with exposure to wildlife, unvaccinated individuals Transmission: Zoonotic, bite or scratch Disease: Rabies encephalitis, hallucination, insomnia, confusion, tingling at bite Diagnostics: DFA on brain tissue (post-mortem for animals), PCR, Serologic testing, post-mortem brain tissue d. West Nile virus Risk: Elderly individuals (>60 years), immunocompromised Transmission: Mosquito bite, blood transfusion or organ transplant Disease: West Nile encephalitis or meningitis (fever, headache, confusion, muscle weakness, neurologic defects) Diagnostics: IgM EIA, Viral culture and neutralization, NAAT 10. Identify the mammals most frequently infected with the rabies virus. Bats mainly, then raccoons, skunks, squirrels, etc. 11. Explain the role in human infection control and methods of detection of rabies virus in animal tissue. Infection Control: Post-exposure prophylaxis (PEP) with rabies vaccination and rabies immune globulin (RIG) following animal bites, particularly from suspected rabid animals. Rabies surveillance: Monitoring and reporting rabid animals, particularly in endemic regions.Vaccination programs: Ensuring domestic animals (e.g., dogs, cats) are vaccinated, and controlling stray animal populations. Methods of Detection in Animal Tissue: Direct Fluorescent Antibody (DFA) test on brain tissue: The most common and reliable test for rabies virus detection in animals. PCR: Can be used for detecting rabies virus RNA in saliva, corneal tissue, or brain tissue. Histopathology: Identification of Negri bodies in brain tissue (pathognomonic for rabies). 12. Identify the human prion diseases and how prions are transmitted to humans. Human Prion Diseases (also known as Transmissible Spongiform Encephalopathies (TSEs)): Creutzfeldt-Jakob disease (CJD): The most common prion disease in humans, can be sporadic, hereditary, or acquired. Variant CJD (vCJD): Associated with bovine spongiform encephalopathy (BSE), or "mad cow disease," transmitted through consumption of infected beef products. Gerstmann-Sträussler-Scheinker syndrome: A hereditary prion disease. Fatal Familial Insomnia (FFI): A rare hereditary prion disease. Kuru: Historically seen in the Fore people of Papua New Guinea, transmitted via ritualistic cannibalism.  Transmission: Consumption of infected animal tissue (e.g., vCJD through BSE-contaminated beef). Medical procedures: Contaminated surgical instruments, dura mater grafts, or hormone preparations (rare). Hereditary transmission: Genetic mutations in the prion protein gene (PRNP). Prion diseases are not transmitted through casual human-to-human contact. 13. Identify the groups at highest risk of infection, transmission route, disease, laboratory diagnostic methods for fungal causes of meningitis. a. Cryptococcus neoformans Risk: Immunocompromised Transmission: Inhalation, no person to person Life cycle: Yeast form is inhaled into the lungs, can spread hematogenous to the brain and meninges Disease: Cryptococcal meningitis Diagnostics: BAP or Sabouraud dextrose agar (cream colored colonies), India ink stain (encapsulated yeast cells, narrow based budding yeast, globose and oblong), Antigen test, Bird seed agar (brown colonies). b. Cryptococcus gattii Risk: Healthy can get, but immunocompromised are at higher risk. Transmission: Inhalation of spores in tropical and subtropical regions Diagnostics: Same as above, but distinguish based on genotyping 14. Identify the groups at highest risk of infection, transmission, life cycle, disease and laboratory diagnostic methods for parasitic causes of meningitis and encephalitis. a. Naegleria fowleri Risk: Young, healthy individuals often swimming or diving Transmission: Inhalation of contaminated water, through nose Life cycle: Amoeba enter nasal passage, travels to brain via olfactory nerve, and causes brain inflammation Disease: Headache, fever, nausea, and altered mental status. b. Acanthamoeba spp. Risk: Contact lens wearers and immunocompromised Transmission: Inhalation or direct contact with contaminated water or soil. Warm fresh, brackish and sea water and soil, biofilms in pools, hot tubs and HVAC systems. Diagnostics: Trophozoites and cysts found in human CSF and brain tissue. Trophs have spiny projections called “ acanthopodia.” PCR or culture. Karyosome is large, there is no peripheral chromatin.  c. Toxoplasma gondii Risk: Immunocompromised, pregnant women (vertical transmission) Transmission: Ingestion of oocyst, vertical transmission Diagnostics: PCR, IgG and IgM antibodies d. Trypanosoma brucei African Sleeping Sickness Highest risk: Residents of sub-Saharan Africa Transmission: Tsetse fly bite Life cycle: Parasite enters the bloodstream, migrates to CNS Disease: Sleeping sickness, swollen lymph nodes, CNS involvement with sleep disturbances Diagnostics: Blood smear looking for trypanosomes and PCR Key Terms Encephalitis Disseminated Intravascular Coagulation Meningitis d-dimer test Abscess Kuru Nuchal rigidity Bradyzoite Petechia Tachyzoite Purpura Blood-brain barrier Primary Amoebic meningioencephalitis Tsetse fly Blood Stream Infections Learning Objectives 1. Identify host innate and adaptive immune defenses that prevent or eliminate microbial infections of the blood stream. Innate: Skin, mucus membranes, amylase, phagocytic cells (e.g., neutrophils and macrophages) Adaptive: Antibody production, complement, Tc cells 2. Identify the ports of entry for microbes gain access to the blood stream. Seeding from other site of infection (pneumonia, UTI, etc.), indwelling devices (e.g., CLABSI- central line), or invasive procedures. Mouth, anus, vagina, cuts, abscesses, surgical incision sites. 3. List the normal microbiota of the blood stream. None if healthy and under normal conditions 4. Explain how bacteria in the blood cause disease and describe symptoms of blood stream infections. Shivering, fever, chills, pain/discomfort, clammy/sweaty, confusion, pale, sleepy 5. Identify populations with the highest risk of blood stream and cardiovascular infections. -Pre-existing infection -Extremes of age (babies and old people) -Immunosuppression (chronic diseases like diabetes, Cancer, or HIV) -Major surgery or injury (risk) -Prolonged hospital stay (nosocomial infections) -Genetic (heart murmurs, etc.) - Cardiovascular anatomical defect or defect in immune system -Persons who inject drugs (PWID) - primary immunodeficiencies, inject drugs (insulin, testosterone, not only recreational) 6. Describe patient preparation and collection of an acceptable blood specimen for bacterial culture (timing, volume, site preparation) and predict the impact on test results when specimens are improperly collected. Sample: whole blood Site disinfectant: 2% chlorhexidine gluconate of 2% iodine. Povidone iodine requires 1-2 minutes of contact time before the draw. How many to collect: Adults- 2-4 sets (40-80mL) – 1 set=1 blood culture Where to collect: Venipuncture (peripheral catheter if no venous access) or catheter (if catheter-associated infection is suspected). 7. Describe the content and role of blood culture media and additives. Contain media and nutrients to support growth of several bacterial organisms, an anticoagulant, and something to inactivate antibiotics. 8. Describe the blood culture workflow. Specimen collection: Site prep alcohol plus iodine or chlorhexidine disinfection. Collect 2-4 sets of blood culture one after the other, from different sites (adults).  Incubation: Bottles loaded onto a continuous monitoring blood culture system. Incubated until the blood flags positive, or 5 days (at 5 days, considered negative and removed from the system). Anaerobes grow slowly. Identification: Disinfect diaphragm, extract growth with safety device or needle and syringe. Rapid identification direct from sample (BioFire or Verigene). Gram stain → Subculture to CHOC, MAC, and BAP → Biochemical identification. 9. Identify the laboratory method of diagnosis for the most common infectious agents that cause healthcare acquired and community onset blood stream infections, including: 1. the HACEK group Commonly cause blood infection Grouped together based on their ability to cause endocarditis. Fastidious, Gram negative. Colonize respiratory mucosa. Include Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella 2. Leptospira Less common cause of Blood stream infection Zoonosis transmission, animal urine persists in soil and water. Spiral shaped bacteria. Biphasic disease: Septicemic phase and immune phasic. 3. Escherichia coli Commonly found in healthcare acquired blood infections and community-onset infections, and commonly causes bloodstream infections. 4. Klebsiella pneumoniae Health-care associated infections esp in ICU. Lactose fermentation on MAC and Urease +. PCR and AST. 5. Haemophilus influenzae CHOC, PCR, latex agglutination. 6. Streptococcus pneumoniae GPC in pairs or chains, alpha hemolysis on BAP. Optochin S, and bile solubility test, PCR, antigen detection. 7. Staphylococcus aureus Community onset and healthcare- associated. GPC in clusters. Coag positive. MRSA. 8. Staphylococcus epidermidis Nosocomial bacteremia in immunocompromised patients. CoNS. Biofilm formation, device-related infections. 9. Coagulase negative Staphylococcus Catheter-related infections and prosthetic devices. Biofilm formation on medical devices. 10. Enterococcus faecium UTI and endocarditis. GPC in pairs or chains. VRE. 11. Enterococcus faecalis Bacteremia and endocarditis. GPC in pairs or chains. Resistance profiling for vancomycin and other antibiotics. 12. Bacteroides fragilis group  Grown anaerobically, often in polymicrobial infections. GNR. 10. Interpret blood culture results in the context of culture results and patient risk factors for infection. Blood culture results must be interpreted in the context of clinical symptoms, patient history, and risk factors. Multiple positive blood cultures suggest a true bloodstream infection, especially when the same pathogen is isolated from multiple sites. A single positive culture of a typically contaminant organism (e.g., coagulase- negative Staphylococci) may indicate contamination or a true infection, especially if the patient has risk factors like indwelling devices. Susceptibility testing helps determine appropriate treatment, especially for multi- drug resistant organisms like MRSA, VRE, and ESBL-producing E. coli. Consider clinical context (e.g., neutropenic patients, intravenous drug users, recent surgeries) when interpreting results. 11. Describe the lifecycle, vector, disease symptoms and outcomes, laboratory diagnosis and prevention of human infections with Plasmodium, Babesia, Leishmania, and Trypanosoma cruzi and Trypanosoma brucei. Plasmodium spp. (Malaria) Lifecycle: Vector- Mosquito. Parasite enters blood stream, infects liver cells, then red blood cells, causing cyclic fevers. Asexual development = schizogony and sexual development = sporogony. Symptoms: fever, chills, sweats, headache, anemia, splenomegaly. Can lead to cerebral malaria, organ failure, and death. Diagnosis: Blood smear of plasmodium trophozoites. Babesia spp. (Babesiosis) Lifecycle: Vector – Ixodes ticks (same as Lyme disease). Tick transmits the parasite into human blood, where it infects red blood cells. Symptoms: fever, chills, fatigue, anemia, splenomegaly. Diagnostics: Blood smear identifying Babesia trophozoites in RBCs, Maltese cross. PCR detection. Prevention: Tick control, avoid tick bites, prompt removal of ticks.  Leishmania spp. (Leishmaniasis) Lifecycle: Vector – sandfly. Promastigotes are transmitted into the skin, infecting macrophages and other cells. Symptoms: Skin lesions (ulcers) at bite site. Visceral: fever, hepatosplenomegaly, weight loss, anemia. Diagnosis: Skin biopsy and PCR. Prevention: Bed nets, vector control, insect repellent. Trypanosoma cruzi (Chagas Disease) Lifecycle: Vector - Kissing bugs. The parasite enters through the skin or mucous membranes, often from bites or feces. Symptoms: Acute- Fever, swelling at site of infection (Chagoma), lymphadenopathy. Chronic – Heart failure, megacolon, megaesophagus. Diagnosis: Blood smear of trypomastigotes in blood. IgG Abs for T.cruzi. Trypanosoma brucei (African Sleeping Sickness) Lifecycle: Vector – Tsetse fly. Parasite enters bloodstream and crosses into the CNS, causing encephalitis. Symptoms: fever, swollen lymph nodes, CNS involvement. Diagnosis: Blood smear of Trypanosomes. Prevention: vector control, sleeping nets, and early diagnosis and treatment. Key Terms Bacteremia Septicemia Septic Shock Fungemia Viremia Parasitemia Transient bacteremia Multisystem organ failure Endocarditis CRP Procalcitonin Systemic Inflammatory Response (SIRS) Indwelling catheter Trypanosome Giemsa stain Schizongony Sporogony Sporozoite Merozoite Gametocyte Paroxysm Cerebral malaria Recrudescence Relapse Definitive host Intermediate host Hemoflagellate Kinetoplast Cardiomegaly Chagoma Reduviid/triatome/kissing bug Urinary Tract Infections 1. Describe the male and female urinary tract anatomy and host defenses that prevent and control infections. Female: Kidneys → Ureters → bladder → urethra (4cm) Male: Kidneys → ureters → bladder → prostate → urethra (20cm) Host defenses that prevent and control infections: ureter peristalsis, voiding, epithelial cell shedding 2. Contrast the symptoms of cystitis and pyelonephritis.  Cystitis: infection of the bladder. Symptoms: strong, persistent urge to urinate, dysuria (burning sensation), passing frequent and small amounts of urine, can be cloudy (WBCs and epithelial cells), red or brown -Uncomplicated: infection in a normal, unobstructed genitourinary tract in healthy, non- pregnant, pre-menopausal adult women -Complicated: infection with presence of anatomical or functional urologic abnormality, a catheter, comorbidities, pregnancy, or infection in post-menopausal women. UTI in men. Pyelonephritis: infection of the kidneys. Flank pain, fever, chills, nausea, vomiting, frequency, urgency, burning, white blood cells or casts in urine, sepsis, shock, death 3. Identify routes of transmission of urinary pathogens and explain how high risk groups acquire infection. Routes of transmission: fecal-perianal-urethral contamination, introduction from catheter, hematogenous High risk groups: Institutionalized Care: -In-dwelling catheters >80% -Multi-resistant organisms -Chronic diseases, structural, function Pregnant Women: -Hormonal changes -Enlarging uterus -Asymptomatic bacteria increases risk of pyelonephritis -Increased incidence: premature labor, low birth weight babies Pediatric: -65 Years: -Men have enlarged prostate (obstruction)  -Decreased mobility -Increased catheterization 4. Identify the laboratory method of diagnosis for the most common community acquired and health care associated infectious agents responsible for UTIs including species-level identification of: 1. Staphylococcus saprophyticus→ Gram (+) cocci in clusters → Catalase (+) → Oxidase (-) → Coagulase (-) → Novobiocin R 2. Enterobacterales → Gram negative rods, anaerobic → Motile at 37C → Oxidase (-) → Ferments glucose → Reduce nitrate to nitrite 5. Provide the primary isolation media routinely used for each type of urine specimen. Clean catch and catheter: BAP, CNA/PEA, MAC Suprapubic: BAP, MAC, CAN/PEA, anaBAP, Thio 6. Describe patient preparation and specific uses of urine specimen collections options and the advantages and disadvantages of each. Voided midstream (clean catch): self-collection, disinfection of genitalia, contamination likely, no preservative in sterile container Catheter: Tube for injecting or removing fluids. Indwelling: disinfect port and purge initial flow, don’t use urine in collection bag. External female catheter specimens are unacceptable. Suprapubic bladder aspiration: sterile but invasive. The only urine specimen suitable for anaerobic culture. 7. Correlate urine dipstick results, direct microscopy, Gram stain microscopy of urine specimens, and semi-quantitative culture results. 8. Recognize typical normal microbiota growth of clean catch and suprapubic collected urine specimens. Suprapubic should have no normal microbiota. 9. Describe the inoculation technique used for semi-quantitative urine culture. Inoculate plate with 0.001mL loop (1µL) or 0.01 mL loop (10µL) for sterile collected specimens. Touch and drag straight down and then multiple perpendicular streaks back and forth covering entire plate. 10. Calculate the concentration of bacteria in urine provided the inoculum volume and number of colonies. Number of colonies / loop volume = CFU / mL  11. Interpret the relevance of bacterial growth (CFU/mL and identification) from clean catch, catheter and suprapubic urine specimens. 12. Describe the lifecycle including intermediate host, transmission, disease symptoms and outcome, and laboratory diagnosis of Schistosoma haematobium. Lifecycle: Cercariae released by snail into water and free-swimming, penetrate skin, cercariae loses tails during penetration and become schistosomulae, circulation, migrate to portal blood in liver and mature into adults, paired adult worms migrate to mesenteric venules of bowel/rectum (laying eggs that circulate to the liver and shed in stools) or venous plexus of bladder. Intermediate host: Cercariae Transmission: Direct penetration of skin by free-living cercariae Disease symptoms and outcome: bladder fluke, bilharzia, Katayama fever Symptoms: hematuria and anemia, stunted growth, fibrosis of bladder and ureter, kidney damage, vaginal bleeding and painful intercourse, bladder cancer Laboratory diagnosis: Antibody detection, ova in urine (prominent terminal spine) Key Terms Cystitis Pyelonephritis Urinary catheter Suprapubic Clean catch urine Pyuria Hematuria Dysuria Fluke Trematode Cercaria Miracidium Schistosomes Learning Objectives Upper Respiratory Tract Infections 1. Describe the anatomy of the upper respiratory tract. Nose, nasal cavity, sinuses, oral cavity, pharynx, larynx 2. Recognize typical colony morphology and species of the normal microbiota of the upper respiratory tract. -Gram positive aerobes: Streptococcus mitis Group (including St. pneumoniae), other viridians strep, beta hemolytic strep (non- Group A), non-hemolytic strep, Coagulase negative staph, Micrococcus spp., Diphtheroids. Anaerobes: Bacteroides spp., Fusobacterium spp., Prevotella spp., Porphyromonas spp. Gram negative aerobes: Neisseria spp., Eikenella spp., Capnocytophaga spp.Occasionally present: Haemophilous influenzae, Haemophilous parainfluenzae, Peptostreptococcus sp., Actinomycetes, Staphylococcus aureus (from hospitals), Mycoplasma. 3. Identify the anatomy, innate and acquired immunity that protect the respiratory tract. Innate immunity: Nasal hairs, mucociliary escalator, lysozyme, mechanical clearing (coughing, sneezing, swallowing). Acquired immunity: IgA, complement, macrophages 4. Describe the collection technique and appropriate materials for collecting upper respiratory tract samples. Nasal swab: anterior nares or mid-turbinate. Nasopharyngeal swab: all the way up nose and to back of throat. Oropharyngeal swab: avoid teeth and tongue, brush along tonsils. 5. Identify media used to isolate the most common bacteria cause pharyngitis. BAP and CNA 6. Identify typical colonies of Streptococcus pyogenes amongst normal microbiota in a photo of a throat swab culture on BAP. Beta-hemolysis = Strep pyogenes  Beta hemolysis = clearing 7. Identify typical colonies of Haemophilus influenzae amongst normal microbiota in a photo of a sputum specimen culture on Chocolate agar. Non-hemolytic small colonies = H. influenziae 8. Identify the diseases, laboratory method of diagnosis (including media, where appropriate) for the most common infectious agents associated with upper respiratory tract infections, including: 1. Streptococcus pyogenes → Diseases: bacterial pharyngitis, acute sinusitis. Lab method for diagnosis: Gram pos cocci, beta-hemolytic, catalase (-), PYR (+), CAMP (-), Bacitracin S 2. Bordetella pertussis → Diseases: pertussis. Lab methods: Gram neg coccobacilli, Bordet-Gengou agar, Regan-Lowe agar, doesn’t grow on BAP or MAC 3. Bordetella parapertussis → Diseases: Pertussis. Lab method: Gram negative coccobacilli, Bordet-Gengou agar, Regan-Lowe agar, Growth on BAP (gray- brown colonies) 4. Corynebacterium diphtheriae → Disease: pharyngitis (diphtheria). Lab method: Gram pos rod, Tinsdale with black colonies and brown halos, beta- hemolytic, catalase (+), nonmotile 5. Haemophilus influenzae → Disease: Acute sinusitis, epiglottitis, otitis media. Lab method: Gram variable coccobacilli, non-hemolytic, X and V factors, grows on CHOC, oxidase (+)  6. Streptococcus pneumoniae → Disease: acute sinusitis, otitis media. Lab methods: Gram (+) cocci, alpha hemolysis, catalase (negative), PYR (-), 6.5% NaCl (-), Optochin S 7. Rhinoviruses: Rhinitis, common cold, viral pharyngitis. No testing and no treatment. Can lead to otitis media, sinusitis, and bronchitis. 8. Coronaviruses: Diseases: Viral pharyngitis and can lead to cause common cold 9. Adenovirus: Diseases: Viral pharyngitis and can lead to cause common cold Key Terms Pneumonia Sinusitis Aspiration pneumonia Bronchitis Colonization Epiglottitis Otitis media Pertussis Diphtheria Scarlet fever Rheumatic fever Glomerulonephritis Learning Objectives MMG 465 FS23 Lower Respiratory Tract Infections 1.1. Identify the anatomical barriers, innate and acquired immunity that protect the respiratory tract. Trachea (windpipe), Bronchi, Bronchioles, Alveoli. Innate: consists of mucosal secretions, macrophages and neutrophils. Acquired: adaptive responses like antibodies and T-cells. 1.2. Describe patient preparation, process and materials used to collect sputum, bronchial wash and nasopharyngeal swab specimens. Identify the pathogens targeted in each specimen type. Patient preparation for sputum, bronchial wash, and nasopharyngeal swab collection includes instructing patients to cough or use saline for deep lung specimens, and targets pathogens like Mycobacterium tuberculosis, Streptococcus pneumoniae, and viruses, depending on the specimen. 1.3. Determine the acceptability of a sputum specimen based on Gram stain results. A sputum specimen is acceptable if it contains predominantly lower respiratory tract cells (e.g., neutrophils) and few or no epithelial cells, as observed under Gram stain. >10 squamous epithelial cells (SECs) per low power field (LPF) = reject 1.4. Compare laboratory safety design and PPE, specimen collection and processing, primary isolation media, culture conditions, identification, for routine bacterial causes of pneumonia and tuberculosis. Safety design and PPE for pneumonia and tuberculosis differ in handling, with tuberculosis requiring higher isolation precautions (e.g., N95 masks), while routine bacterial cultures use media like blood agar, MacConkey agar, and incubation at 35- 37°C. 1.5. Identify the primary isolation media used for routine sputum culture. BAP, CHOC, MAC 1.6. Identify the diseases and laboratory method of diagnosis (including specimen processing and specialty media, where appropriate) for the most common infectious agents that cause of lower respiratory tract infection, including: a. Mycobacterium tuberculosis → Disease: Tuberculosis; Diagnosis: Sputum acid-fast bacillius (AFB) smear, culture on Lowenstein-Jensen or Middlebrook agar and Thio broth, PCR. b. Streptococcus pneumoniae → Disease: Pneumoniae, meningitis; Diagnosis: blood agar culture, Gram (+) cocci, alpha hemolysis, catalase (negative), PYR (-), 6.5% NaCl (-), Optochin S, bile solubility, MALDI-TOF c. Haemophilus influenzae → Disease: Pneumonia, epiglottitis, otitis media; Diagnosis: Choc → Gram variable coccobacilli, non-hemolytic, X and V factors, grows on CHOC, oxidase (+), latex agglutination d. Legionella pneumophila →Disease: Legionnaires’ disease; Buffered charcoal yeast extract (BCYE) agar, urine antigen test or DFA e. Staphylococcus aureus → Disease: Pneumoniae, skin infections, septicemia; Diagnosis: Blood agar, Gram pos cocci in clusters, beta hemolysis (strong), mannitol salt agar, catalase (+), coagulase (+) f. Mycoplasma pneumoniae →Disease: Atypical pneumoniae (walking pneumoniae); Diagnosis: SP4 broth: “fried egg” colony. Media changes from pink to yellow. NAT g. Chlamydia pneumonia → Disease: Atypical pneumonia; Diagnosis: PCR, serology (IFA or ELISA), culture on McCoy cells. h. Chlamydophilia psittaci → Disease: Psittacosis; Diagnosis: PCR, serology (IFA), culture in McCoy cells. i. Pseudomonas aeruginosa→ Disease: Hospital-acquired pneumoniae, cystic fibrosis infections; Diagnosis: BAP, MAC, Oxidase (+), PCR, Gram negative aerobic rods j. Klebsiella pneumoniae →Disease: Pneumoniae, aspiration pneumoniae; Diagnosis: BAP, MAC, mucoid morphology, Gram negative rod, catalase (+), oxidase (-), citrate (+), gas (+), H2S (-), indole (-), Nitrate reduction (+), A/A, urease (+), VP (+) k. Enterobacter spp. → Disease: Healthcare-associated pneumoniae, urinary tract infection; Diagnosis: BAP, MAC, PCR, Gram negative rods, oxidase (-), indole (-), facultative anaerobes l. Nocardia braziliensis → Disease: Nocardiosis (lung infection, abscesses);Diagnosis: BAP, Sabouraud dextrose agar, acid-fast staining, urease (+), Gram positive bacilli, positive acid-fast staining, PCR m. Influenza A and B viruses: Influenza; Diagnosis: Rapid antigen tests, PCR, viral cultures in cells lines n. Adenovirus: Respiratory infections, pneumonia; Diagnosis: PCR, viral culture, antigen detection (EIA) o. Respiratory syncytial virus → Disease: Bronchiolitis, pneumoniae in infants; Diagnosis: PCR, rapid antigen tests, viral culture. p. Human metapneumovirus → Disease: Respiratory infections, pneumoniae; Diagnosis: PCR, viral culture, rapid antigen test. q. SARS-CoV-2 → Disease: COVID-19; Diagnosis: PCR, antigen tests, biral culture (BSL- 3)  r. Histoplasma capsulatum → Disease: Histoplasmosis, Reactivation infection possible in AIDS Opportunistic infection; Diagnosis: Culture on Sab agar, antigen detection, Yeast in phagocytes (narrow-based budding yeast), tuberculate macroconidia. s. Coccidioides immitis → Disease: Coccidioidomycosis (Valley fever) found in Four corners region SW US, Lab-acquired infection so use BSC; Diagnosis: Spherule containing endospores, Barrel-shaped arthroconidia. Culture on Sab dex. t. Blastomyces dermatiditis →Disease: Blastomycosis, seen in Mississippi and Ohio river; Diagnosis: broad-based budding yeast, looks like egg, oval conidia on delicate conidiophores (Lollipops, Balloons). u. Aspergillus spp. → Disease: Aspergillosis (lung, invasive), Allergic bronchopulmonary aspergillosis (grows in balls in sinus), disseminated (esp. in immunocompromised); Hyaline mold (no melanin). Diagnostics: green with white apron. Phialides look like vials, vesicles, metulae. 1.7. Compare the chronicity of infection, laboratory culture and susceptibility testing, safety and treatment strategy differences between Mycobacterium tuberculosis and other common causes of pulmonary infection. Mycobacterium tuberculosis has a slower growth rate, requires specialized culture media (e.g., Lowenstein-Jensen), and needs longer susceptibility testing and stricter biosafety precautions compared to other bacterial pathogens like Streptococcus pneumoniae. Tuberculosis is a BSL-3 that does acid-fast staining. Uses Lowenstein- Jensen media that grows for 4-6 weeks to get positive and stays incubated for negatives up to 12 weeks. It also uses a thio broth that is 1-2 weeks earlier than solid media. We now have the interferon-gamma test. 1.8. Compare the microbial causes of community onset, chronic and healthcare associated pneumonia. Microbial causes of pneumonia differ based on onset and setting: community-acquired pneumonia often involves Streptococcus pneumoniae and Mycoplasma pneumoniae, healthcare-associated pneumonia may involve Pseudomonas aeruginosa and Klebsiella pneumoniae, and chronic infections often involve Mycobacterium tuberculosis or Nocardia species. Key Terms Neuraminidase Bronchial lavage Antigenic shift Hemoptysis Antigenic drift Hemaglutinin Latent tuberculosis Active tuberculosis Mantoux or tuberculin skin test Granuloma Interferon Gamma Response assay Caseous necrosis Learning Objectives MMG 465 FS23 Skin and Soft Tissue Infections 1.10. Identify the location and names of skin layers, hair follicles and sweat glands.  Skin consists of three main layers: epidermis (outer layers), dermis (middle layer), and subcutaneous tissue (hypodermis- deep layer). Hair follicles are located in the dermis, and sweat glands are found in the dermis and subcutaneous tissue. 1.11. Describe and name the predominant aerobic and anaerobic normal microbiota of the skin Aerobic: Staphylococcus epidermidis, other CoNS, S. aureus, Corynebacterium spp. (diphtherioids- log jams). Anaerobes: Cutibacterium sp. and Peptostreptococcus sp. 1.12. Identify the diseases and laboratory method of diagnosis for the most common infectious agents that cause skin and soft tissue infections, including: a. Staphylococcus aureus Disease: Skin abscesses, impetigo, cellulitis, folliculitis, necrotizinf fasciitis, toxic shock syndrome. Diagnosis: Blood or wound culture: GPC in clusters → Coagulase +, MSA (yellow colonies, ferment mannitol), PCR to identify MRSA. b. Streptococcus pyogenes Disease: impetigo, erysipelas, cellulitis, necrotizing fasciitis, TSS Diagnosis: GPC in chains, beta-hemolysis on BAP, rapid antigen test, Lancefield testing c. Vibrio vulnificus Disease: Cellulitis, necrotizing fasciitis, wound infections (especially after exposure to seawater). Diagnosis: Blood or wound culture: GN curved rods. Thiosulfate-citrate-bile salts- sucrose (TCBS) agar shows yellow colonies (ferment sucrose). PCR. d. Pseudomonas aeruginosa Disease: Burn wound infections, folliculitis (hot tubs), chronic wounds, cellulitis. Diagnosis: Blood or wound culture: GNR, oxidase positive. Beta-hemolytic on BAP, fluorescent green pigment on agar. Grape like odor. MAC: lactose non-fermenter. e. Eikenella corrodens Disease: Human bite wounds, fist fights (clenched fist injury), periodontal infections, cellulitis. Diagnosis: GNR, growth on BAP, corroding appearance. Fermentation patterns. f. Pasturella sp. Disease: Cat or dog bite infections, cellulitis, abscesses Diagnosis: GN coccobacilli. Non-hemolytic on BAP, oxidase positive. g. Capnocytophaga canimorsus Disease: Dog bite infections, sepsis (in immunocompromised hosts) Diagnosis: GNR (long and slender rods), culture on BAP and CHOC h. Clostridium perfringens Disease: Gas gangrene, necrotizing fasciitis, food poisoning Diagnosis: GPR (spore forming), double-zone hemolysis on BAP. Anaerobic culture and PCR for toxin genes. i. Cutibacterium acnes Disease: Acne vulgaris, prosthetic joint infections, endocarditis (rarely) Diagnosis: Typically slow-growing, non-hemolytic on BAP j. Mycobacterium leprae Disease: Leprosy (Hansen’s disease): skin lesion, nerve damage, disfiguring deformities. Diagnosis: Acid-fast bacilli on skin biopsy. PCR. Ziehl-Neelsen stain. k. Mycobacterium ulcerans Disease: Buruli ulcer – chronic skin ulcerations with necrosis. Diagnosis: PCR and acid-fast stain from tissue samples or biopsy. l. Human papillomavirus (HPV) Disease: Warts, genital warts, cutaneous papilloma, skin cancer (squamous cell carcinoma) Diagnosis: Visual inspection, PCR for high-risk strains, histopathology for koilocytes. m. Actinomyces isrealii Disease: Actinomycosis – chronic abscesses, draining sinuses, often in the face, jaw, or abdominal region. Diagnosis: GP branching filaments. Anaerobic culture on BAP, Sulfur granules found in abscess. n. Sarcoptes scabiei var. hominis Disease: Scabies – itchy skin rash, burrows, inflammatory response Diagnosis: Skin scraping of mites and eggs. Wood’s lamp – can highlight affected areas. o. Leishmania spp. Disease: Cutaneous leishmaniasis, visceral leishmaniasis Diagnosis: Skin biopsy see amastigotes in macrophages, PCR p. Candida Disease: Candidiasis: oral thrush, vaginal infections, cutaneous candidiasis, onychomycosis. Diagnosis: Yeast cells with budding. Culture of Sabouraud agar or Chromagar. PCR q. Sporothrix schenkii Disease: Sporotrichosis. Cutaneous and subcutaneous tissue. Rose Gardeners Disease. Skin ulcers and nodules, often after thorn pricks or animal exposure. Possible spread to multiple nodules along lymph channels.  Diagnosis: Culture on SAB agar, forms cigar-shaped yeast. Thermal dimorphic fungus. Oval to cigar shaped budding yeast. Mold at 30C. Septate hyphae with daisy wheel or rosette pattern. r. Fusarium Disease: Fusariosis – fungal infection of the skin, nails, or eyes, often in immunocompromised individuals, but can be seen in healthy individuals. Frequently seen in mycotic keratitis. Fungemia. Localized skin infections due to trauma and water/soil inoculation (farming). Diagnosis: Culture on SAB agar or potato dextrose agar, look for fusiform conidia. Tissue- look for hyaline septate hyphae that branch at acute and right angles. Culture hyaline, banana-shaped multicellular macroconidia with foot at base. Can be seen in clover pack like from Horton Hears a Who. s. Malessezia furfur Disease: Pityriasis versicolor (tinea versicolor): Hypo or hyperpigmented skin lesions. Diagnosis: Wood’s lamp (affected skin may fluoresce yellow-green. KOH prep – yeast cells and spaghetti and meatball appearance, short septate hyphae and budding yeast. Culture on SAB agar. t. Piedraia hortae Disease: Black Piedra – hard, black nodules in scalp hair. Dark brown gritty nodules on hair shaft. Associated with poor personal hygiene. Superficial mycoses. Diagnosis: Dark, hard nodules on hair seen under microscope. Culture on SAB agar. u. Hortaea werneckii Disease: Tinea nigra: Brown or black patches on palms or soles. Diagnosis: KOH prep: presence of yeast cells with dark hyphae. Culture on SAB agar. v. Trichosporon spp. Disease: Can be found as normal skin biota and isolated from animals and soil. White Piedra occurs on hir shaft and is characterized by soft mycelial mat surrounding hair of scalp, face, and pubic region. Opportunistic systemic infections. Can be fatal in immunocompromised hosts. Diagnosis: Grow rapidly on primary fungal media and produce arthroconidia, hyphae, and blastoconidia. Colonies are straw-to-cream colored and yeastlike. Absence of carbohydrate fermentation, use of potassium nitrate, assimilation of sugars, and urease positive. w. Trichophyton sp. Disease: Infect hair, skin, and nails. Cause various forms of “ringworm.” Tinea. Dermatophyte.  Diagnosis: Clinical, Wood’s lamp (bright green fluorescence), microscopy wet mount with 10% KOH or NaOH or Calcifluor white, culture. Cigar-shaped with smooth thin walls. x. Microsporum sp. Disease: Infect hair, skin, and nails. Cause various forms of “ringworm.” Tinea. Diagnosis: Septate macroconidia. Spindle shaped. y. Epidermophyton sp. Disease: Infect hair, skin, and nails. Cause various forms of “ringworm.” Tinea. Diagnosis: Smooth thin walled produced in clusters growing directly from hyphae, septate. Greenish-brown with suede-like surface, tufts of growth. Reverse side is deep yellow-brown. 1.13. Explain where and how folliculitis, boils and carbuncles develop. Folliculitis, boils, and carbuncles develop when hair follicles become infected, typically by Staphylococcus aureus. Folliculitis is a superficial infection, boils are deeper abscesses, and carbuncles are multiple interconnected boils. 1.14. Provide the mechanisms by which bacteria enter into deep tissue spaces and cause infections. Bacteria can enter deep tissue spaces through breaks in the skin, such as cuts, abrasions, or surgical wounds. Once they breach the epidermis, bacteria can spread via the bloodstream (hematogenous spread), along nerve or lymphatic pathways, or directly invade deeper tissues through soft tissue spaces. 1.15. Identify the skin infections mediated by toxins. Skin infections mediated by toxins include staphylococcal scalded skin syndrome (SSSS), caused by Staphylococcus aureus toxins. SSSS has a staph exfoliation toxin which targets epidermal cells, leading to desquamation (peeling layer), this affects infants and immunocompromised. Scarlet fever -develops subsequent to strep infection in throat or skin -Strep pyogenes (GAS)  -Antibitoic treatment with amoxicillin Toxic shock syndrome (TSS), which can be caused by both Staphylococcus aureus and Streptococcus pyogenes. 1.16. Provide the underlying risks for diabetic foot infections and list the typical cause(s) of this infection. Monomicrobial: S. aureus, Streptococci Polymicrobial: skin flora, anaerobes, Enterobacterales, Pseudomonas Risk factors: foot trauma, inadequate blood glucose control, walking barefoot. Peripheral neuropathy (unnoticed injury). Vascular damage (impaired blood flow). Treatment: wound care, debridement, amputation, antibiotics Prevention: monitoring for injury, footwear, glucose control 1.17. Provide the typical causes of animal and human bite infections. Human bite (oral flora): Streptococcus anginosus group, S. aureus, Eikenella corrodens, Fusobacterium nucleatum, Prevotella melaninogenica Dog bite: Capnocytophaga canimorsus, Pasteurella, Bacteroides, Fusobacterium, Prevotella Cat bite (deep puncture): Pasteurella multocida, Bacteroides, Fusobacterium, Porphyromonas 1.18. Provided images of fungal pathogens listed above, provide identification. Key Terms Pressure ulcer Diabetic foot wound Carbuncle Furuncle Boil Folliculitis Erysipelas Erysipeloid Impetigo Polymicrobial Cellulitis Necrotizing Fasciitis Gangrene Desquamation Mycetoma  Learning Objectives Genital and Sexually Transmitted Infections 1.1. Describe the characteristics of the normal microbiota of the female and male genital tracts. Reproductive age female vagina: Lactobacillus spp., Corynebacteria spp. (diphtheroids)- log jams, Candida spp., Streptococcus spp., Gardernella vaginalis Male urethra: Staphylococcus epidermidis, Corynebacterium spp. (diphtehroids), Various anaerobes 1.2. List effective methods of preventing sexually transmitted diseases. Abstinence, vaccination (HPV), reduce numbers of sex partners, mutual monogamy, male condoms, prophylaxis (PreP) 1.3. List the method of laboratory diagnosis, disease and populations with the highest rates of infections and describe the diseases for microbial causes of STIs including: a. Neisseria gonorrhoeae Disease: Gonorrhea (urethritis, cervicitis, pelvic inflammatory disease, proctitis, pharyngitis, and conjunctivitis). Laboratory Diagnosis: Gram stain (for urethral or cervical samples), culture on Thayer-Martin agar, PCR or NAAT (nucleic acid amplification testing). High-Risk Populations: Sexually active adolescents and young adults, particularly in high-prevalence communities or those with multiple sexual partners. Part of normal flora in females. b. Chlamydia trachomatis Disease: Chlamydia (urethritis, cervicitis, pelvic inflammatory disease, conjunctivitis, neonatal pneumonia). Laboratory Diagnosis: NAAT, PCR, or direct fluorescent antibody (DFA) test on urine or genital samples. High-Risk Populations: Sexually active adolescents, young adults, especially females, and individuals with multiple sexual partners. c. Treponema pallidum Disease: Syphilis (primary: painless ulcer, secondary: rash, mucosal lesions, tertiary: organ damage).  Laboratory Diagnosis: Serologic tests (RPR or VDRL for screening, FTA-ABS or TPPA for confirmatory testing), darkfield microscopy (for primary syphilis). High-Risk Populations: Men who have sex with men (MSM), individuals with multiple sexual partners, and people living with HIV. d. Haemophilus ducreyi Disease: Chancroid (painful genital ulcers, swollen inguinal lymph nodes). Laboratory Diagnosis: Gram stain (shows Gram-negative coccobacilli), culture on enriched media. High-Risk Populations: Sexually active individuals in regions where chancroid is endemic (e.g., sub-Saharan Africa and Southeast Asia). e. Herpes simplex viruses 1 and 2 Disease: Herpes simplex virus infection (HSV-1 commonly causes oral lesions, HSV-2 causes genital lesions; both can cause neonatal herpes). Laboratory Diagnosis: PCR, viral culture, direct fluorescent antibody (DFA) test, or serology for HSV-specific IgG/IgM antibodies. High-Risk Populations: Individuals with a history of multiple sexual partners, MSM, and individuals with HIV. f. Human papillomavirus Disease: Genital warts, cervical dysplasia, and cervical cancer (certain high-risk strains). Laboratory Diagnosis: Pap smear (cytology), HPV DNA testing (for high-risk strains), and colposcopy with biopsy. High-Risk Populations: Sexually active women, particularly those with multiple partners, immunocompromised individuals, and MSM. g. Human immunodeficiency virus Disease: Acquired immunodeficiency syndrome (AIDS) (chronic infection, opportunistic infections, immune system failure). Laboratory Diagnosis: HIV antibody/antigen tests (ELISA), Western blot, and HIV RNA PCR for viral load and genotyping. High-Risk Populations: MSM, intravenous drug users, individuals with multiple sexual partners, and people with other STIs. h. Trichomonas vaginalis Disease: Trichomoniasis (vaginal discharge, itching, dysuria in women; often asymptomatic in men). Laboratory Diagnosis: Wet mount microscopy (motile trichomonads), PCR, or antigen detection. High-Risk Populations: Sexually active women, particularly those with multiple sexual partners or other STIs. i. Candida spp. Disease: Candidiasis (vulvovaginal candidiasis in women, oral thrush, and systemic infections in immunocompromised individuals). Laboratory Diagnosis: KOH preparation (for yeast cells), culture on Sabouraud agar, PCR, or Candida-specific antibody tests.  High-Risk Populations: Women of reproductive age, especially those with diabetes, pregnancy, or antibiotic use; immunocompromised individuals (e.g., HIV, cancer patients). 1.4. List STIs for which vaccinations are available and recommended in the US. HPV (Human Papillomavirus) and Hepatitis B 1.5. Identify the sexually transmitted pathogens that can be transmitted to fetuses in utero. Provide methods of prevention and diagnosis. Syphilis, HIV, Rubella, Cytomegalovirus (CMV), and Herpes simplex virus (HSV). Prevention methods include vaccination, antiviral therapy, and early screening; diagnosis involves serological testing and PCR. 1.6. Provide the microbes and outcomes of infection in pregnant women, the fetus, and the neonate. Group B Streptococcus (neonatal sepsis), Toxoplasma gondii (congenital toxoplasmosis), Listeria monotcytogenes (preterm birth, neonatal infection), HIV-1, Syphilis, CMV. Outcomes vary from miscarriage to developmental delays. 1.7. Describe the outcomes of congenital syphilis infection and how it is prevented. Congenital syphilis can result in stillbirth, premature birth, or developmental issues. Prevention involves screening and treating pregnant women with penicillin during pregnancy. Can get false positive in 1-2% of population with RPR (pregnant women in this population). Need confirmatory testing. 1.8. Describe the outcome for the neonate and prevention of fetus/infant infection caused by maternal infection with N. gonorrhoeae, C. trachomatis, Streptococcus agalactiae and Treponema pallidum. N. gonorrhoeae: Neonatal conjunctivitis; prevent with erythromycin eye ointment. C. trachomatis: Neonatal conjunctivitis, pneumonia; prevent with screening and treatment during pregnancy. S. agalactiae: Neonatal sepsis, meningitis; prevent with penicillin during labor. T. pallidum: Congenital syphilis; prevent with penicillin treatment during pregnancy. 1.9. Define “clue cells” and their significance when observed microscopically in KOH suspensions of vaginal secretions. Clue cells are epithelial cells covered with bacteria (typically Gardnerella vaginalis), indicating bacterial vaginosis when observed in KOH suspensions of vaginal secretions. 1.10. Describe the microscopic bodies observed in wet prep and KOH prep from vaginal swabs from women with bacterial vaginosis, candidiasis and trichomoniasis. Bacterial vaginosis: Clue cells and a shift in microbial flora (overgrowth of anaerobes) Candidiasis: Pseudohyphae and yeast cells Trichomoniasis: Trichomonas protozoa, which are motile 1.11. Define bacterial vaginosis, its cause, and the microbes associated with this dysbiosis.  Bacterial vaginosis is a condition caused by an imbalance in vaginal flora, with overgrowth of Gardnerella vaginalis and other anaerobes, leading to symptoms like odor, discharge, and irritation. 1.12. Describe and interpret the tests used to diagnose and monitor HIV anti-retroviral therapy (ART). Following the CDC HIV algorithm. Diagnosis of HIV starts with HIV-1/2 antigen/antibody combination assay such as an EIA, then is positive and differentiation immunoassay is done. If HIV-1 is negative or indeterminate an NAT is done. And if the HIV test is positive ART therapy is started. For infants (