Klebsiella pneumoniae

:

Mucoid, encapsulated K. pneumoniae:

Plump GNR, white spaces around rods = encapsulated (polysaccharide capsule does not stain)

On MacConkey agar, it is a very mucoid organism. Sticks together – mucoid capsule. See stringy-looking material – is capsule-related.

Tuberculosis (Mycobacterium tuberculosis): • Epidemiology: kills > 3 million per year; #1 cause of death worldwide due to any infectious agent • Acid-fast bacilli – red with blue background • Slow-growing aerobes (14-28 days!) • Spread mostly by aerosolized droplets • Can involve virtually every organ, most importantly lungs • Typically associated with granuloma formation

Acid-fast organism. Stains red on Ziehl-Neelsen acid fast stain or modified Kinyoun stain:

Cell Wall Virulence Factors: • Cord Factor: glycolipid that causes TB to grow in serpentine cords – more virulent strains • Lipoarabinomannan (LAM): polysaccharide similar to endotoxin – inhibits macrophage activation by interferon-γ – also induces macrophages to secrete TNF-α (causes fever, weight loss & tissue damage) & IL-10 which suppresses T-cell proliferation • Complement activation: on surface of TB may opsonize organism & facilitate its uptake by macrophages • TB Heat Shock Protein: highly immunogenic & could play role in autoimmune reactions induced by TB • Cell wall lipids and carbohydrates enhance virulence by interfering with phagolysosomal fusion, allowing intracellular survival of TB (inside of macrophages)
Test q: A 59M AIDS patient living in a nursing home develops fever and a cough. A sputum specimen shows acid-fast bacteria in serpentine cords. This is most likely: Mycobacterium tuberculosis.

Above: TB – serpentine cord formation.

Test q: “Cording” is a microscopic description of bacterial growth. This term is most associated with: Mycobacterium tuberculosis. Test q: A 50y/o immigrant from Mexico is being treated for a chronic pneumonia. The causative agent exhibits cord factor and lipoarabinomannan. This infection was acquired from: another human. Test q: A 9y/o boy has had a chronic cough and fever for the past month. A chest radiograph shows enlargement of hilar lymph nodes and bilateral pulmonary nodular interstitial infiltrates. A sputum sample contains acid-fast bacilli. A transbronchial biopsy specimen shows granulomatous inflammation within the lung, marked by the presence of Langhans giant cells. Which of the following mediators is most likely to contribute to giant cell formation and granulomas? Interferon-γ.

Host Factors: • Infants and elderly more susceptible • Socioeconomic factors more important than ethnic/racial factors • Relative risk for U.S. non-whites is 10X the risk for whites • Relative risk for homeless (white or non-white) is 150X higher

Predisposing Diseases: • Diabetes mellitus • Alcoholism or malnutrition • Chronic lung disease, esp. silicosis (inhaling silica) • Immunosuppression, esp. AIDS • (Any debilitating or immunosuppressive condition) Primary Tuberculosis (1st infection with TB - no prior exposure): 1. Mid lung field granuloma (Ghon lesion) 2. Hilar lymph node granuloma These two form the Ghon complex – found in 95% of primary TB. 3. Miliary TB - pulmonary artery – lung - pulmonary vein – systemic – involvement of GI, adrenals, etc. 4. Cavitary TB 5. Chronic pulmonary infection These last three make up 5% of primary TB. Primary Tuberculosis (1st infection with TB - no prior exposure): – Inhaled TB proliferate in alveolar macrophages in lung periphery & are transported back to hilar lymph nodes – Controlled in 95% by T cell-mediated immune response - demonstrated by a positive purified protein derivative (PPD) skin test – indicates sensitivity, not active disease – Fibrosis and calcification in the primary lung lesion with concomitant spread to hilar lymph nodes produces the Ghon complex – Figure: Left – granuloma, had been there a long time in left lower lobe. Has calcium in the periphery, has been walled-off, lots of connective tissue. Right – hilar lymph nodes are also involved = GHON COMPLEX

T-Cells in Tuberculosis: – CD4+ helper T cells secrete interferon-gamma, which activates macrophages to kill intracellular TB via reactive nitrogen intermediates (NO, NO2) and is associated with epithelioid cell granuloma formation and removal of TB – CD8+ suppressor T cells kill macrophages that are infected with TB, resulting in formation of caseating granulomas (delayed-type hypersensitivity rxns)
Test q: A 32F has had a chronic cough w/fever for the past month. On phys exam, she has a temperature of 37.5C, and on auscultation of the chest, crackles are heard in all lung fields. A chest radiograph shows many small, ill-defined nodular opacities in all lung fields. A transbronchial biopsy specimen shows interstitial infiltrates w/lymphocytes, plasma cells, and epithelioid macrophages. Which of the following infectious agents is the most likely cause of this appearance? Mycobacterium tuberculosis. REPEATED x2 (2007, #28 – answer was Histoplasma capsulatum, and TB was not a choice. However, it is Robbins Ch.2 #7, and the answer is M. tuberculosis.)

Epithelioid (L) & Caseating (R) Granulomas:

Left: epithelioid cell granuloma – see middle of lesion – not necrotic, more hyalinized. See multi-nucleated giant cell in the periphery. Also long, stringy cells in the periphery – epithelioid histiocytes. Look in outermost periphery of the whole thing, see lots of lymphocytes and macrophages accumulated. Right: caseating granuloma. Notice 2-3 multi-nucleated macrophages, but the center differs because it is necrotic (looks like cheese).

Secondary Tuberculosis (rare): – May occur one year or 50 years after primary TB, i.e. anytime! – Two varieties: – Reactivation TB (95%) – Reinfection TB (5%) – Active infection in previously sensitized individual - reactivated – Major cause of tissue damage in TB = granulomas that fail to control TB – Granulomas often in lung apices (more oxygen), but may widely disseminate hematogenously throughout lungs and to kidneys, meninges & other organs – Cavities from caseating granulomas may rupture into blood vessels, causing further hematogenous spread, or into airways, releasing infectious TB during a cough or sneeze – highly infectious, requires surgery and multiple drugs to treat adequately – In AIDS: – Poorly formed disseminated granulomas – GI tract and lymph nodes – – – – Apices of lung More necrosis, less lymphatics Miliary TB (More) cavitary – likely because of additional necrosis, more devastating than primary TB

Test q: In secondary or reactivation tuberculosis, involvement is more pronounced in the: Right and left apices, Test q: The lesion of reinfection, reactivation, or adult tuberculosis characteristically begins in which anatomic site? Subapical region of a lung. (Other choices: Hilar lymph node, lower lobe of the lung, major bronchus, random subpleural site)

Cavitary lesion in the right upper lobe. Can see flat line: air-fluid level. Organism likes highly-aerobic environment (apices of lung)

Multiple cavities, cheesy necrosis, upper lobe-involved area.

Miliary Tuberculosis: – Hematogenous spread to many organs – Tiny yellow-white granulomas resemble millet seeds – Via pulmonary artery to lungs – Via pulmonary veins to bone marrow, liver, spleen, kidney, adrenals, prostate, heart
Big granulomas to the right, smaller ones to the left, lots of necrosis within. Miliary TB throughout lungs – little white dots, resemble bird seed.

Clinical Features: – Primary – usually asymptomatic – Secondary – insidious onset of fever, night sweats, weight loss, productive cough with blood streaked sputum or hemoptysis – Prognosis and course vary depending on extent of disease, underlying health, other factors – Antibiotic therapy – generally effective excluding: – neglected cases – emerging highly drug resistant strains “TB or not TB?” – Direct acid-fast stain on sputum or tissue – Culture in liquid Middlebrook medium (7-10 days) – High pressure liquid chromatography (HPLC) – Direct Amplified Probes – No serologic (antibody) tests available Histoplasmosis (Histoplasma capsulatum): – Epidemiology: – dimorphic fungus – found in Indiana and along Ohio and Mississippi Rivers – acquired by inhalation of dust from soil contaminated with bird or bat droppings (think of who might be at risk) – Granulomatous disease – organisms bind complement receptors on macrophages and reproduce within phagolysosomes; intracellular 2-4 µm yeasts – Clinical presentations & anatomic pathology are strikingly similar to tuberculosis
Test q: A 23F AIDS patient presents to the ED w/fever, cough, and a 25lb weight loss. A bronchoalveolar lavage (BAL) is performed and a silver stain (GMS) of the lung washing shows small, budding yeasts seen only inside macrophages. The yeasts are 2-3 microns in diameter. A urine antigen test is positive. Diagnosis? Histoplasma capsulatum. Test q: A 50M has a chronic cough w/a low-grade fever. A chest radiograph shows bilateral, scattered, 0.3-to-2cm nodules in the upper lobes and hilar adenopathy. A fine-needle aspirate of one of the nodules shows inflammation with mononuclear cells, including macrophages that, with GMS stain, show intracellular, 2- to 5-µm, rounded, yeast-like organisms. Which of the following infectious diseases is most likely to produce these findings? Histoplasmosis.

Giemsa stain of BAL sample:

GMS stain:

Shows two macrophages w/dots inside (yeast).

Notice narrow-neck buds of yeast.

Coccidioidomycosis (Coccidioides immitis): – Epidemiology – dimorphic fungus – especially common in southwest/western US and Mexico – Acquired by inhalation of infective arthroconidia – Granulomatous disease that resembles tuberculosis – Pathology – 20-60 µm spherules filled with endospores in sections of lung lesions or other anatomic sites

 Coccidioides immitis: Form at top is arthroconidia form that you inhale – ball-shaped structures that readily get into the air. Biggest form – balloon filled w/spheres = tissue form. Below: Tissue form – H&E.

Spherules filled w/endospores. Some will be empty – outside will stain w/GMS, can be mistaken (overlap in size) w/Blasto.

Spherules that have released their contents – When two endospores come in contact, can look like budding yeast but don’t confuse!

INFECTIONS WITH GRAM-POSITIVE PYOGENIC BACTERIA AND ANAEROBES

11/9/10

OBJECTIVES: • Understand how infections with gram-positive bacteria and anaerobes are acquired • Discuss the pathogenesis of infections by gram-positive bacteria and anaerobes, including important predisposing factors • Know the pathology associated with these infections • Understand how to properly select, collect, & transport specimens for diagnosis of anaerobic infections • Know how these infections are diagnosed in the laboratory EXAMPLE QUESTION: A common characteristic of infections involving anaerobes is a/an: A. putrid odor of a wound Old test q – repeated twice B. superficial skin lesion C. clinical sepsis with positive aerobic culture only D. acute pharyngitis E. sudden onset of vomiting 4 hours after eating at a Chinese restaurant
Test q: Which of the following clinical clues would be most consistent w/infection involving anaerobes? Putrid odor of an acute, ulcerated gingival lesion.

Staphylococcal Infections: Staphylococcus aureus • Gram-positive cocci; grape-like clusters (see in upper right)  • Facultative anaerobes; form catalase and coagulase • Grow both in air and anaerobic conditions – more rapidly in environments w/oxygen. • Acute (“pyogenic”) inflammation, often with abscesses • Abscess def: “localized collection of pus (neutrophils) in a cavity formed by disintegration of tissues” Staphylococcal Infections Include: • Superficial Skin & Soft Tissue Infections (SSTIs) • Wound Infections • Deep Tissue Abscesses – lung, liver, etc. • Blood stream infections (eg., bacteremia; endocarditis – s. aureus can destroy valves) • Pneumonia Septic arthritis; acute bacterial osteomyelitis Food Poisoning (within hours – preformed toxin  vomiting) Acute Enterocolitis – associated w/patients receving antibiotics (like C. difficile) Scalded Skin Syndrome

• • • •

Test q: A 29M drug abuser has a community-acquired pneumonia. Pus aspirated from a lung abscess shows gram-positive cocci are in clusters within cells and single cocci extracellularly. Diagnosis? Staphylococcus aureus.

Pathogenesis – S. aureus Virulence Factors: • Catalase. H2O2 to H2O probably counteracts PMN killing by splitting toxic oxygen radicals – look for bubbling • Coagulase. Converts fibrinogen to fibrin  clot formation (?? WBCs penetrate fibrin poorly ??) • Hyaluronidase. Hydrolyses connective tissue matrix • β-lactamases. Hydrolyse β-lactam antibiotics (penicillin, others) • Leukocidin. Makes pores in membranes of PMNs (eg., Panton-Valentine leukocidin [PVL] – associated w/community-acquired MRSA) • Exfoliatins. Staphylococcal scalded skin syndrome • Superantigens. Toxins produced by S. aureus that stimulate T-cell cytokine (e.g., TNF & IL-1) release systemically, and cause both toxic shock syndrome (TSS) & food poisoning • TSS; was originally associated with hyperabsorpent tampons that became colonized with S. aureus during use; TSS can also be caused by S. aureus in infected surgical sites. • TSS is characterized by hypotension (shock), renal failure, coagulopathy, liver disease, respiratory distress, generalized erythematous rash, & localized tissue necrosis at infected site. • Note: TSS can also be caused by Streptococcus pyogenes • Food poisoning; Superantigens produced by S. aureus also cause vomiting, presumably by affecting the CNS or enteric nervous system (more to follow on food poisoning next lecture)
Test q: The Panton-Valentine Leukocidin (PVL) Toxin Gene is present in: Community-acquired MRSA. (Other choices: Nosocomial MRSA, Nosocomial C. difficile; Community-acquired C. difficile; Enterococcus fecalis) Test q: Within the past 24hr, a 26y/o previously healthy woman has developed a high fever and generalized diffuse erythematous macular rash resembling a sunburn. She is menstruating, and her menstrual cycles are regular. She has nausea, vomiting, abdominal pain, diarrhea, myalgias, sore throat, headache, and dizziness. On phys exam, her temp is 39.4C, pulse 101/min, resp 18/min, and BP 90/40 mmHg. She has oropharyngeal and conjunctival hyperemia. The vaginal mucosa is erythematous. A tampon is present in the vagina vault. She is disoriented, but there are no neurologic deficits. Lab findings show Hgb 13.5, hematocrit 41.4%, platelet count 100,000/mm3, WBC count 11,200/mm3, glucose 70mg/dL, creatinine 2.5 mg/dL, total bilirubin 2.4 mg/dL, AST 82 U/L, and ALT 29 U/L. A chest radiograph shows no abnormal findings. She receives supportive therapy of nafcillin w/clindamycin and improves, but skin and mucous membrane desquamation is noted 10 days later. These findings are most likely produced by an exotoxin elaborated by which of the following organisms? Staphylococcus aureus.

Furuncle (Boil): • Tender, subcutaneous inflammatory nodule – extends to dermis • Tend to form on back of neck, axillae, face, groin, thighs, buttock (where hair is) • Risk factors: neutrophil defects, obesity (get in skin folds, under breasts), steroids, occlusive dressings, diabetes • Healthy people with recurrent furunculosis are often nasal carriers of S. aureus Carbuncle: • Series of coalescent furuncles that express pus from multiple tracts – more severe • Can o ccur on the back of the neck • Rx: incision & drainage plus antibiotics carbuncle Impetigo: a primary pyoderma seen in children 2-to-5 years of age • Staphylococcal or Streptococcal • Confined to superficial epidermis – very superficial infection. • Skin ulcerates.

furuncle

 Impetigo (all three figures)

Staphylococcal pneumonia: Recent Case - Frozen Section: Right lower lobe abscess/ Low power H&E

Higher power H&E: High power view – nothing but neutrophils. In some areas, even the neutrophils are being destroyed, leaving holes/spaced/edematous areas.

Staphylococcal Scalded Skin Syndrome  • Caused by exfoliative toxins (serine proteases) of S. aureus • Cells in granular layer of epidermis split apart; skin desquamates • Neither staphylococci nor leucocytes in involved epidermis • Protective antibodies develop, skin becomes intact again
Proportion of S. aureus Nosocomial Infections Resistant to Oxacillin (MRSA) Among Intensive Care Unit Patients,1989-2001:

Methicillin Resistant Staphylococcus aureus (MRSA): • Healthcare-Associated (HA): HIGHLY RESISTANT (all betalactams & multiple other antimicrobials)! • Requires special susceptibility testing (pH, time, temp, etc.) • Nursing Homes, Nosocomial Infections • Requires isolation of patients • Community-Associated (CA) MRSA: often resistant to only betalactam agents & erythromycin • CA-MRSA have become the most frequent cause of skin & soft tissue infections presenting to emergency departments
Test q: Both hospital-acquired MRSA and community-acquired MRSA express: changes in Penicillin-binding protein.

Laboratory Identification: • Staphylococcus aureus, the most virulent member of the genus is COAGULASE POSITIVE. • The other > 30 species of Staphylococcus (eg., S. epidermidis, & S. saprophyticus – associated w/UTI) are COAGULASE NEGATIVE. Coagulase Positive Coagulase Negative

Coagulase: Prothrombin-Like Substance Able to Clot Human or Rabbit Plasma: Slide Coagulase Test: Tube Coagulase Test:

Negative – looks smooth. Positive – agglutinates – bound coagulase.

Tube coagulase – clots.

Screening for MRSA: Real-Time PCR (Smart Cycler System) – Real-time PCR to look for MecA gene. Real-time PCR uses fluorescence to detect target-specific PCR products as they accumulate in “real-time” during the PCR amplification process in a single reaction tube. Useful for detecting MRSA carriers.

 Figure: Biggest colonies (yellow, lots of hemolysis) – S. aureus. Little tiny colony in background – mixed culture – S. pyogenes.

CATALASE TEST  • H2O2  H2 + O2 (bubbling) • Slide catalase • Genus Staphylococcus is Catalase Positive • Genera Streptococcus & Enterococcus – (formerly Group D strep) are Catalase Negative
Test q: Staphylococcus aureus is described as: Catalase (+); Coagulase (+). Test q: A 24M laborer develops two small abscesses on his right arm. The abscesses are surgically incised and drained. The pus is cultures aerobically and grows gram-positive cocci. The bacteria show beta-hemolysis on blood agar. The bacteria are catalase-positive and coagulasenegative. Identify: Coagulase-negative staphylococcus. (Other choices: Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae, and Anaerobic streptococci.)

Streptococcal Infections (Group A – S. pyogenes): • Facultative anaerobe, Gram + cocci in pairs or long chains • Remember, staph is in clumps. Strep is in a “strip” – long chain. • Beta-hemolytic – complete hemolysis around the colonies • Infections include “strep. throat”, tonsillitis, erysipelas, impetigo, cellulitis, wound infections, scarlet fever, necrotizing fasciitis, septicemia, & pneumonia. • • Acute cellulitis: lesion may spread or remain localized – assoc’d w/abundant neutrophils, spread within the tissue. Several virulence factors
• Test q: A characteristic shared by both Staphylococcus aureus and Streptococcus pyogenes is their ability to cause: Impetigo.

ß-hemolysis (clear)

α -hemolysis (green)

Test q: A 24M medical student presents to the Wishard ED w/fever, lymphadenopathy, and pharyngitis. The Latex agglutination screening test of the throat swab for Streptococcus pyogenes is negative. Culture of the swab grows Group A, B-hemolytic, gram-positive streptococci. Diagnosis? Streptococcus pyogenes.

 Figure: Group A Strep. pharyngitis & tonsillitis (the major antecedent of poststreptococcal rheumatic fever and glomerulonephritis [also follows skin infections involving s. pyogenes]) Dark red back of throat. Notice exudate on swollen tonsils – sometimes tonsils can swell so much that it impedes breathing.

Above: Diabetic lower extremity – widespread on skin and beneath skin = streptococcal cellulitis. Spreading infection not w/abscess formation.

Streptococcal Erysipelas:
Can attain aspirate of skin lesion – do Gram stain. This Gram stain shows abundance of large objects (neutrophils in various stages of disintegration) and chains of streptococci. Butterfly rash, find neutrophils infiltrating. Bright red, confluent rash – localized group A strep infection. Can be on trunk, under the arms – but when in these locations, is a small, punctate rash (unlike this picture).

Why is Group A Strep. so Virulent? • M–protein - M+ strains = resistant to phagocytosis • Erythrogenic Toxin (Pyrogenic Exotoxin) - rash of scarlet fever • Streptokinase/fibrinolysin (breakdown fibrin) - Spreading infections, e.g. cellulitis, erysipelas • Hyaluronidase (breaks down ground substance) - Spreading infections • Other Exotoxins (fever, shock, tissue damage)

Other Streptococcal Infections: • S. pneumoniae - pneumonia & meningitis • Group B - (S. agalactiae) - neonatal sepsis and meningitis. • Viridans streptococci - includes many species (e.g., S. mitis, S. sanguis, S. salivarius, S.intermedius, S.mutans, and others) - the most frequent streptococcal causes of subacute bacterial endocarditis. • Enterococcus faecalis, E. faecium & others) mixed wound infections; septicemia; endocarditis; vancomycin resistance (faecium, faecalis less so)

S. pneumoniae– most important cause of community-acquired bacterial pneumonia. S. mutans– important in tooth decay.

Gram-positive Rods: Bacillus Clostridium (anaerobe) Corynebacterium Listeria Propionibacterium (anaerobe) Erysipelothrix Lactobacillus Actinomyces Nocardia

• Top picture: rod w/hole in it – “sporeforming rod” = bacillus and clostridium species only (could be bacillus of anthrax, clostridium of gas gangrene, etc.) • Middle group: diphtheroids – Chinese characters, or could line up like picket fence row. • Lactobacillus – women UG tract, oropharynx of healthy people – rod chains • Actinomyces/Nocardia - thin rods that elongate and branch

Test q: A 35M farmer develops several skin ulcers several days after butchering a cow. Gram stain shows large Gram-positive rods. The causative agent grows aerobically and produces spores. Diagnosis? Bacillus anthracis. (Other choices: Clostridium difficile, Clostridium perfringens, Listeria monocytogenes, E. coli)

Diphtheria: Toxin Mediated Disease Caused by Corynebacterium diphtheriae: • Highly contagious, airborne droplet spread or skin shedding • Bacteria (Gram-positive rods) at local mucosal site • Pseudomembrane; Local infection of pharynx, larynx or trachea  (skin/rare) – do not remove it! (Patient could asphyxiate, bleed, etc.) • Hyperplastic lymph nodes of neck  Systemic Effects of Diphtheria Toxin (Phage Encoded Toxin Gene Necessary for Toxin Production): • Fatty myocardial degeneration – lots of inflammation within the heart  • Polyneuritis with myelin degeneration • Degeneration of liver, kidney, muscle – but organism is not found within these sites – only in airway. • Death caused by resp. obstruction & myocard. insufficiency • Dx: Clinical suspicion; culture + toxin tests ≥ 1 wk
Test q: A 23F has had increasing delirium for two days and is admitted to the hospital. On phys exam, she has acute pharyngitis w/an overlying dirty-white, rough mucosal membrane. Paresthesias w/decreased vibratory sensation are present in the extremities. On auscultation, there is an irregular cardiac rhythm. A chest radiograph shows cardiomegaly. A Gram stain of the pharyngeal membrane shows numerous small, gram-positive rods in a fibrinopurulent exudate. Which of the following is the most likely mechanism for development of cardiac disease in this patient? Elaboration of exotoxin. (Other choices: Microabscess formation, Endotoxin-mediated hypotension and shock; Vasculitis w/thrombosis and infarction; Granulomatous inflammation) REPEATED x2

Listeriosis: • L. monocytogenes: small GPRs (often resemble cocci) • Most listeriosis is foodborne – Disseminates hematogenously – Affinitiy of Listeria for CNS & placenta • Other modes of transmission – Mother to child transplacentally, or infected birth canal, cross-infection in neonatal nurseries – Veterinarians & farmers at risk from aborted calves & infected poultry. – Much more common than diphtheria. Food-borne illness. Carried in intestinal tracts of mammals – cattle, sheep, etc. • CNS infection or septicemia in immunocompromised hosts Gram-stain: Umbrella motility:
Test q: A newborn male becomes listless, feeds poorly, and the mother thinks he is afebrile. On phys exam, the pediatrician suspects meningitis. The baby undergoes a spinal tap. Protein is elevated, glucose is minimally decreased, and both lymphocytes and neutrophils are present. The gram stain is non-rewarding, but culture grows a gram (+) bacillus that exhibits tumbling or umbrella motility. Diagnosis? Listeria monocytogenes. (2009 #85 – the lettering is off, so it is incorrectly keyed as “S. agalactiae”)

Pathogenesis: L. monocytogenes, a Facultative Intracellular Pathogen, Can Grow in Macrophages & Epithelial Cells: • Entry into nonphagocytic cells is mediated by leucine rich proteins (internalins), which interact with glycoprotein receptors (E-cadherin) on surface of host cells • Once inside a host cell, a bacterial exotoxin (listeriolysin-O) & two phospholipases aid Listeria to escape phagolysosomal killing • In host cell cytoplasm, Listeria replicates, produces a surface protein (ActA) which induces actin polymerization, & propels itself into adjacent cells (eg, macrophages, enterocytes) • Entry into macrophages following passage through intestinal mucosa carries the bacteria to liver and spleen, leading to disseminated disease.

Listeriosis: Pathology: • Listerial septicemia leads to widespread focal necroses and microabscesses in various organs. • Numerous monocytes/macrophages and T cells may appear at periphery of these lesions and migrate inward to transform them into granulomas. • Facultative parasite – likes to live in macrophages. Infections By Anaerobic Bacteria:
 Infections by anaerobic bacteria: Bacteria carried around in our micro-flora – mouth, colon, UG tracts – can cause brain, head/neck, sinusitic, oropharyngeal infections (tooth abscess), could be infections from head-to-toe.

Adrenal Lesion:

Major Anaerobes Encountered in Wounds and Abscesses: • Bacteroides fragilis group • Pigmented Prevotella/Porphyromonas group – GNRs in mouth, black/brown colonies (accumulate iron precursors) • Fusobacterium spp. (e.g., F. nucleatum) • Peptostreptococcus spp. (the anaerobic cocci) • Streptococcus intermedius • Actinomyces (e.g., A. israelli) • Propionibacterium acnes • Eubacterium spp. • Clostridium spp. (C. perfringens, C. septicum) Characteristics of Anaerobes: • Cause endogenous infections (except clostridia, which also cause exogenous infections) • Require compromised tissue in a compromised host • Often involved in mixed infections • Growth enhanced by ↓ oxygen tension & ↓ Eh (red-ox potential) Predisposing Factors – poor blood supply, decreased oxygen tension in tissue: • Trauma - surgery or injury • Malignancy • Vascular insufficiency • Steroids, immunosuppressant & cytoxic drugs • Diabetes & other debilitating diseases • Prior antimicrobial agents Some Examples of Clinically Significant Anaerobe Infections: • Brain abscess • Actinomycosis • Anaerobic dental/oral infections (Vincent’s disease) • Anaerobic pleuropulmonary infections: aspiration pneumonia, abscess, thoracic empyema • Intraabdominal sepsis • Gas gangrene • Non-clostridial crepitant cellulitis Anaerobe abscess 

Important members of microbiota.

Abscess can break down and seed CSF:

Draining Actinomyces lesion:

Sulfur granules of actinomyces:

Bacteroides fragilis (pleomorphic GNRs)

Extended all the way thru the mandible.

Gram stain of tissue. See branching filaments staining Gram+, surrounded by serum proteins (pink around it).

Actinomyces vs Nocardia in Pulmonary Specimens: Occasionally, filaments of actinomyces and nocardia can look alike – branching filaments. “WEAKLY ACID FAST” = NOCARDIA – very fine red-staining acid-fast branching rods. Bottom left – Actinomyces only stains blue (not acid-fast)

Test q: A 42M w/HIV has had a fever and cough for the past month. On phys exam, his temp is 37.5C. On auscultation of the chest, decreased breath sounds are heard over the right posterior lung. A chest radiograph shows a large area of consolidation w/a central air-fluid level involving the right middle lobe. A transbronchial biopsy specimen contains gram-positive filamentous organisms that are weakly acid fast. The patient’s course is further complicated by empyema and acute onset of a headache. A head CT scan shows a 4cm discrete lesion of the right hemisphere w/ring enhancement. Which of the following infectious agents is most likely to produce these findings? Nocardia asteroides.

Cavitary lesion in the lung – anaerobic abscess. Looks like TB, can get air-fluid level. Gangrenous appendix: ruptured in the gut.

Filled w/pus.

Gram stain pus  can see thin, faintlystaining GNRs. Fusobacterium nucleatum – thin-pointed, GNRs. Big blobs – disintegrating neutrophils.  Patient developed intra-abdominal abscess 2wk after appendix removed, drained 500mL of purulent exudate. Had very putrid odor – bad fecal smell = anaerobes. Requires surgeon, antibiotics.

Hunter w/Shotgun Wound to Lower Extremity
(subsequently received hyperbaric oxygen Rx):

Direct Microscopic Examination:

Amputated around the knee – treated w/very high dose Penicillin G. Gas gangrene.

Above (left): H&E stained section revealing necrosis, gas spaces and myriads of rods. Above (right): Gram’s stain reveals necrotic background, gas spaces and relatively large, “box-car” shaped gram-positive rods; absence of spores all consistent with Clostridium perfringens myonecrosis.

Crepitant (Anaerobic) Cellulitis: • Seen mostly in diabetic patients. • Characteristically involves subcutaneous tissues or retroperitoneal tissues. • Muscle and fascia not involved. • Nonsporeforming anaerobes & other bacteria were involved in this case. Not all lesions w/gas in tissue are gas gangrene. This anaerobic cellulitis was caused by mixture: Gram-neg. Bacteroides fragilis, E. coli (aerobe), and cocci (typical of enterococcus species).

Anaerobic Bacteria – Abscesses: • Usually polymicrobial involving mixed anaerobes, facultative anaerobes or aerobes (e.g. Nocardia) • Head & neck - Prevotella/Porphyromonas, or Fusobacterium mixed with S. aureus or a Streptococcus spp. • Fusobacterium necrophorum, an oral commensal, causes Lemierre syndrome, an infection of the lateral pharyngeal space with septic jugular vein thrombosis • Lung abscess - anaerobes normally found in oral cavity e.g., Prevotella/Porphyromonas, Fusobacterium, & Peptostreptococcus • Abdominal abscesses - Bacteroides fragilis, Peptostreptococcus, Clostridia, often mixed with E. coli. Clostridial Infections: • Clostridium species = anaerobic, gram-positive sporeforming anaerobes • C. perfringens and C. septicum cause muscle necrosis in wounds (gas gangrene) or Clostridial cellulitis, food poisoning or bowel necrosis in neutropenic patients – C. perfingens α-toxin (phospolipase C) degrades lecithin in cell membranes; destroys red cells & muscle cells – Histologically, gangrene shows gas bubbles, myonecrosis, hemolysis (double zone), & thrombosis; resembles an infarct but lacks inflammatory cells. • C. tetani in wounds or umbilical stump of newborns causes tetanus by releasing tetanospamin that causes spastic paralysis by blocking release of γ-aminobutyric acid neurotransmitter that is responsible for inhibiting motor neuron activitiy. • C. botulinum grows in contam. foods & releases neurotoxin that causes descending flaccid paralysis of respiratory & skeletal muscle (foodborne botulism). Botulinal neurotoxin binds irreversibly to periph nerve endings (eg, neuromuscular junctions), blocking release of acetylcholine. In contrast, aerobic spore-forming bacteria = Bacillus.

Test q: A 55y/o construction worker suffers a superficial puncture that evolves into a painful wound in his lower left leg. The wound is opened in the operating room and a biopsy taken. Touch preparations are Gram-stained and show myonecrosis, large clear spaces, and large Gram-positive rods. Diagnosis? Clostridium perfringens. Test q: While repairing a fence on his farm, a 40M cuts the skin over his shin. The wound heals w/o any complications. Four days later, he develops muscle spasms of the face and extremities. These spasms worsen to the point of severe contractions. Which of the following actions by a toxin is responsible for the clinical features in this case? Cleavage of synaptobrevin in synaptic vesicles of neurons. (Robbins: This man has tetanus. The contamination of a wound w/Clostridium tetani can result in the elaboration of a potent neurotoxin. This toxin is a protease that cleaves synaptobrevin , a major transmembrane protein of the synaptic vesicles of the inhibitory neurons.)

Tetanus: Spastic Paralysis in a Soldier (L) and Newborn (R):

↑ Soldier dying of tetanus. Strongest muscle groups take over (back muscles). Neonatal tetanus. ↑ Foodborne Botulism: Infant Botulism: Acute Flaccid Paralysis

Family died – ate home-canned green beans.

Infant botulism: Most common form of botulism in US. Ingest spores, germinate in distal part of small intestine/colon – toxin gets absorbed through colon – peripheral nerve ending dysfuction. Blocks release of Ach. Descending muscle paralysis – starts w/cranial nerves (head control, suck reflex)  breathing (intercostal muscles, diaphragm). Doesn’t clear until new nerve endings have sprouted.
Test q: A 3-week-old infant from San Diego, whose pacifier had been repeatedly dipped in honey, developed constipation followed by signs of descending flaccid paralysis, and had to be placed on a ventilator. The most likely diagnosis is: Infant botulism.

Clinical Clues to Infections Involving Anaerobes: • Putrid odor • Location in proximity to mucosal surface • Gas in tissue or necrotic tissue • Septic thrombophlebitis • Underlying malignancy • Diabetic foot infections • Infections following surgery, trauma (bites) • Sulfur granules -- black discoloration of tissue • Clinical sepsis with negative blood cultures Laboratory Diagnosis: Important considerations: • Selection of appropriate specimens (not contaminated with normal flora) • Rejection of inappropriate specimens • Cleansing of surfaces (e.g., with iodine) to avoid normal flora when collecting samples • Aspirates (needle & syringe) and biopsies are best • Swabs not desirable: 1) tiny volume, 2) tend to dry out, 3) easily contaminated, 4) undue exposure of anaerobes to oxygen – needle/syringe has less exposure to oxygen

Needle/syringe technique – inject pus into tube, transport it back to lab. CO2 or nitrogen gas is in the tube.

48-72 hr of anaerobic incubation required before colonies can be seen on agar plates (most anaerobes). Can move things in and out w/o contaminating inside of chamber. Use catalysts to grow anaerobes. Use dissecting microscope to see distinct features of colonies.

Clostridium perfringens: • Double zone of hemolysis; sheep blood agar • Lecithinase activity; egg yolk agar • Spores rarely seen • Nonmotile • GLC: Acetic & Butyric

Above (left): Double zone of hemolysis: big complete clearing beneath colony – due to theta-toxin. Area of partial clearing due to alpha-toxin (phospholipase C that chews holes in membrane as in gas gangrene). Above (middle): Egg yolk agar – opaque zone w/colony on the surface. Illustration of lecithinase activity. Above (right): Gram stain, variable sizes and shapes of GPRs (box-car-shaped GPR) – lack of spores but classified as spore-former (especially in food-borne forms)

Fusobacterium nucleatum:

Fusobacterium nucleatum:

Colonies 1-2mm diameter. Look under dissecting microscope, have little specks of birefringent material in them (speckled opalescence).

Needle-pointed GNRs on gram stain.

Example of a Miniaturized Biochemical System for Rapid 2-to-4h Identification of Anaerobes  (Rapid test of pre-formed enzymes)

INFECTIOUS DISEASES OF THE GASTROINTESTINAL TRACT Objectives: • Understand how gut barriers protect the host from intestinal infectious diseases. • Describe the ways in which intestinal pathogens are spread to humans. • Discuss the pathogenesis of specific gastrointestinal infections. • Describe the major pathologic features of specific gastrointestinal infections. • Know how infectious diseases of the GI tract are diagnosed in the laboratory.

11/12/10

Example Question: A 12 year old child, who visited a fast food restaurant 3 days ago, developed bloody diarrhea, thrombocytopenia, and renal failure. The most likely cause is: A. Rotavirus B. Escherichia coli O157:H7 Old test q – repeated twice C. Shigella sonnei D. Staphylococcus aureus E. Salmonella species
Test q: A 10M who ate hamburger at a fast food restaurant 3 days ago developed bloody diarrhea, thrombocytopenia, and renal failure. The most likely cause is: Escherichia coli 0157:H7.

Infectious Diseases of the Gastrointestinal Tract: • Second only to common cold • Large nuisance and economic impact • High mortality in 3rd world countries • Est. > 200 million people worldwide have diarrhea in any 24 hour period Gut Barriers to Infectious Microorganisms: • Gastric acidity • Bile • Pancreatic enzymes (e.g., trypsin) • Mucus • Intestinal motility • Immune defenses (e.g., secretory IgA) • Normal flora Gastric Acid: • Gastrin is a hormone that stimulates secretion of gastric acid • Destroys bacteria; ↓ susceptibility to infection • Patients with gastric resection and achlorhydria have ↑ susceptibility to infection • Bicarbonate p.o. ↑ susceptibility to infection (ie., experimental Shigella, Escherichia coli and cholera) • Nonenveloped viruses (e.g., rotavirus and Norwalk virus) resistant to gastric juices • Gastric juices kill most enveloped viruses
Test q: A 40F exhibits watery diarrhea, midepigastric pain and shallow ulcers in the duodenum. Antacids and cimetidine give no relief. What serum test is likely to be elevated? Gastrin.

Normal Bacterial Bowel Flora May Help Prevent Acquisition of Diarrheal Disease: • Flora (syn. = microbiota) competes with pathogens for nutrients • Short-chain fatty acids inhibit pathogens. • Prior antibiotic Rx lowers infective dose of Salmonella typhi (experimental model studies) “Food poisoning” related to preformed toxins in food: • Staphylococcal; 2- 4 hr incubation • Foodborne botulism; incubation 2h-8d (avg. 12- 48h) – but vomiting not apparent • Bacillus cereus food poisoning; vomiting toxin incubation pd. 1- 6; diarrheal toxin incubation pd. 6- 24h – FRIED RICE. 5 • Clostridium perfringens = >10 vegetative bacilli/g of food, spores form in small bowel (germinate release toxin); 9 -15h incubation pd.
Test q: A 42M develops GI distress about 5hr after eating fried rice at a restaurant specializing in oriental cuisine. The most likely etiology is: Bacillus cereus. Test q: A 29M purchased a soft drink and a hot dog from a street vendor. He develops gastric cramping and vomiting 3hr later. It is most likely that he was experiencing the effects of a toxin produced by: Staphylococcus aureus.

Other Pathogenetic Mechanisms Used by Enteropathogenic Bacteria: 1. Vibrio cholerae and toxigenic E. coli multiply inside mucous layer overlying gut epithelium ⇒ exotoxins 2. Shigella, Salmonella, and Campylobacter invade intestinal mucosa & lamina propria ⇒ inflammation, ulceration, bloody stools 3. S. typhi damages mucosa ⇒ Peyer patches ⇒ mesenteric lymph nodes ⇒ blood stream ⇒ systemic infection. Causes typhoid fever. Fungal Infections of GI Tract: Mainly in Immunocompromised Patients: • Candida predilection for squamous epithelium – Oral thrush – Esophagitis • But also disseminates to – Stomach – Lower GI tract – Systemic organs Figures: Localized inflammation of squamous cell epithelium by Candida  Lower picture – shows pseudohyphae of Candida. Mechanisms Cont.: Intestinal Parasites: • Cysts of protozoa resist stomach acid • Trophs of Giardia lamblia attach to epithelial brush border • Cryptosporidia form gametes & spores inside enterocytes • E. histolytica attaches, invades & ulcerates colonic mucosa • Intestinal helminths, when in large numbers, obstruct gut &/ or invade bile ducts (Ascaris) • Hookworms suck blood from villi ⇒ Fe deficiency anemia • Diphyllobothrium latum competes for & depletes vitamin B12 Viral Enteritis and Diarrhea: • Rotavirus = major cause of childhood diarrhea • Diarrhea in infants can ⇒ severe dehydration & metabolic acidosis ⇒ requiring hospitalization • Norovirus (Norwalk-like virus) = epidemic gastroenteritis with diarrhea, nausea & vomiting (both children & adults) + • Viral diarrhea caused by ⇓ absorption of Na & H2O in small bowel • Pathology nonspecific: blunting & destruction of villus epithelial cells, crypt hyperplasia, & mixed inflammation in lamina propria • Dx: EIA tests; reverse transcription (RT)-PCR; sequencing Norovirus Outbreaks in the News: • CDC estimates 23 million cases of acute norovirus gastroenteritis • ~ 50% of all foodborne outbreaks of gastroenteritis now due to norovirus • The most common settings for norovirus outbreaks are: – 10% cruiseships – 13% schools – 23% nursing homes – 36% restaurants • These outbreaks continue to be in the news… • Most outbreaks of norovirus illness arise through direct contamination of food by a food handler. Bacillary Dysentery (Shigellosis): • Colon invaded by nonmotile, lactose neg. GNRs • Shigella sonnei (most US cases); S. flexneri (gay males); S. dysenteriae (rare in US; outbreaks in Latin America, Africa & Asia); S. boydii (India) • Most patients children < 5 yo • Spread (problem in day care centers, families): feces, food, fingers, fomites, water; infecting dose < 100 cfu (cholera, E. coli, & Salmonella require millions) st • Fever, abdominal cramps, & watery diarrhea at 1 , then pain & bloody, low-volume stools

Below: Red dots (organisms) associated w/crypts of Lieberkuhn make their way into lamina propria (then normally stop, not deeper than that) – on occasion, makes it into blood (bacteremia).

May have toxic CNS symptoms, febrile, flecks of pus in the stool.
Test q: Several children at a local daycare center developed diarrhea as did several adult workers. Severe abdominal cramps and bloody diarrhea were noted in most patients. Microscopic exam of stool revealed blood and neutrophils. The most likely etiologic agent is: Shigella sonnei.

Massive amount of organisms w/in a crypt. On other side of enteric On other side of enteric epithelial area, see myriads of bacteria deeper in the lamina propria. May
or may not be lots of inflammatory cells.

Figure: Lighter areas – ulceration, sloughing of hyperemic damaged mucosa  Campylobacter Enteritis: • Campylobacter jejuni. A motile, comma-shaped (like Vibrio spp.), gram-negative rod; grows best in 510% oxygen (microaerophilic), & at 42ο C. • Reservoir. GI tracts of many animals (esp. beavers) – their excreta may contaminate soil, water, or foods. • Diarrhea (often bloody) with abdominal pain & fever follows a 2-10 d (usually 2-5 d) incubation pd. • Pathology – inflammation of entire gut from jejunum to anus. There may be colonic crypt abscesses (as in ulcerative colitis) along with hyperemia, & infiltrates of PMNs, lymphocytes & plasma cell in lamina propria. • Rare septicemia.

Above: Markedly abnormal colon – edematous, foci of hemorrhage, necrotic epithelium, tremendous edema, looks almost like muscularis is necrotic. Campylobacter enteritis in its most severe form.

Yersinia Enteritis: • Yersinia enterocolitica – infrequent cause of diarrhea & abdominal pain in US (common in Northern Europe, S. America, Africa & Asia). • Incubation pd. 1-10 d, usually 4-6 d • Foodborne outbreaks (e.g., chocolate milk in New York; pasteurized milk in Tennessee; raw pork intestines [chitterlings] in Atlanta & Baltimore). • Can grow at refrigerator temperatures – can grow in ice cream, dairy products, etc. • Pathology – involves terminal ileum & colon. Intestinal lesions similar to typhoid. Also, enlarged mesenteric lymph nodes with “stellate abscesses” similar to lympogranumloma venereum or cat-scratch disease.

Above: Background – lymphocytes in periphery, then curved pale zone near the middle – stellate necrosis.

Test q: A 25M medical student visited relatives on a farm where he drank unpasteurized milk and ate chitterlings. On return to Indianapolis he develops fever and abdominal pain. The peripheral blood smear shows a left shift and a WBC count of 30,000/mL. The patient undergoes appendectomy. The appendix is normal but regional lymph nodes show stellate abscesses. Diagnosis? Yersenia enterocolitica.

Note: Child who has signs and symptoms of acute appendicits – may operate and see normal appendix but enlarged mesenteric lymph node nearby. This organism is capable of causing mesenteric adenitis/terminal ileitis – shows localized invasiveness of terminal ileum or cecal area.

Nontyphoidal Salmonella Infections: • Incubation pd: 6h-10 d, usually 6-48 h • Diarrhea, often with fever and abdominal cramps • Food vehicles: – Eggs – Ice Cream – Turkey – Chicken – Beef – Ham – Pork – Misc. (e.g., Mexican food, baked foods, potato salad)

Pathology of Nontyphoidal Salmonella Infections: • Food poisoning – colonic mucosa eroded; mixed inflammation in lamina propria; prompt recovery • Enteric fevers – due to S. typhimurium, & S. choleraesuis; severity intermediate between food poisoning and typhoid; • Salmonella bacteremia with or without intestinal lesions; also may be localized infections (e.g., osteomyelitis associated with sickle cell disease).

Test q: Which one of the following predisposes to osteomyelitis in patients w/Salmonella infections? Sickle cell disease. Test q: A 20M has sudden onset of severe abdominal and back pain and dyspnea. His medical history indicates similar episodes over a 12yr period. One year ago, he had osteomyelitis of the left hip; the bone culture was positive for Salmonella enteritidis. On phys exam, his lungs are clear to auscultation, but he has tachycardia. Palpation of the abdomen reveals diffuse tenderness w/rigidity of abdominal musculature but no apparent masses. A CT scan of the chest shows prominent pulmonary veins but no infiltrate. An abdominal CT scan shows the presence of multiple 0.5-to-1.0-cm stones in the gallbladder, a very small spleen, and prominent hepatic veins. An abdominal radiograph shows no free air. CBC shows hemoglobin 10.2 g/dL, hematocrit 30.9%, 3 3 3 MCV 99 µm , RDW 22, platelet count 189,300/mm , and WBC count 6320/mm . Which of the following additional lab test findings is most likely to be reported? Haptoglobin decreased. (Other choices: Amylase increased, Calcium increased, Plasma free hemoglobin decreased, and Triglyceride decreased.)

Typhoid fever: Typhoid fever: Caused by Salmonella typhi  Bloodstream infection  whole body. Intestinal bleeding due to hemorrhage in Peyer’s patches, fever, diarrhea, headache, rose spots on abdomen, confusion, weakness, bronchitis, pneumonia.
Test q: A 37M visiting a 3 world country drinks from a focally contaminated source of water. During the next week he has gradually increasing fever, anorexia, myalgia, and headache. He subsequently develops a maculopapular rash on his abdomen and his fever increases to 104F rd w/abdominal pain and splenomegaly. During the 3 week, his condition rapidly deteriorates w/intestinal bleeding, shock, and death. An autopsy reveals ulcerations overlying the Peyer patches of the small intestine, one of which is perforated. Which of the following is the most likely diagnosis? Typhoid fever. (Other choices: Amebiasis, Cholera, Cryptosporidiosis, Giardiasis)
rd

Above (left): Terminal ileum – Peyer’s patches enlarged, may ulcerate, hemorrhage (circled). Ulcer goes in direction of fecal flow – not transverse. Can lead to hemorrhagic cecitis (left side). Above (middle): Profound lymphocytic infiltrate (because in lymphatic area of distal small bowel, Peyer’s patch). Penetrating ulcer – on its way down through layers of the bowel – can penetrate muscle in terminal ileum. Develop peritonitis (often lethal). Above (right): Typhoid nodule in liver. Typhoid circulates around in the bloodstream. Facultatively parasitic organism – loves to parasitize macrophages (like listeria, TB, brucella, etc.) but does not have to. May start out as a microabscess, macrophages move in, get loose granulomas – not w/giant cells. Typhoid nodule – loose granuloma (here, in liver).
Test q: In typhoid fever, the distribution of the ulcers found in the small intestine are described as: linear.

People can continue to be stool culture positive for S. typhi for months/year – if a year +, defined as typhoid carrier. Requires cholesystectomy – almost always have cholelithiasis with it (gall stones) 

Cholera: • Illness caused by enterotoxin of Vibro cholerae • Toxin activates adenylate cyclase in small bowel mucosa ⇒ secretion of Cl & H2O ⇒ massive secretory diarrhea • Stools resemble “rice water.” • No invasion or morphologic alteration of mucosal cells. • At autopsy, victims are dehydrated (wrinkled skin, sunken eyes and cheeks).
Test q: A 25M medical student joins a medical missionary trip to South America. Shortly after arrival he develops watery diarrhea. Stool exam reveals no RBCs or WBCs. The most likely etiology for his illness is: Vibrio cholerae. Test q: A 25F has sudden onset of severe profuse, watery diarrhea. Over the next 3 days, she becomes severely dehydrated. Lab studies of the diarrheal fluid shows microscopic flecks of mucus but no blood and no WBCs. The woman is hospitalized and receives IV fluid therapy for 1week. Diagnosis? Cholera.

Vibrio-shaped organism. Looks like campylobacter – comma-shaped rod 

Escherichia coli Enteric Infections: • Enterotoxigenic: Cholera-like toxin (heat labile toxin), no invasion; source = food or H2O ⇒ traveler’s diarrhea • Enteroinvasive E. coli: dysentery; involves colon resembles shigellosis; source = food, H2O, person-to-person • Enteroadherent (or “enteropathogenic”) E. coli: pili adhere to receptors  diarrhea; common “O” groups (eg., O55, 111, 125); source = food, water ⇒ diarrhea, infants, toddlers Enterohemorrhagic E. coli O157:H7: • Mechanism: Shiga-like toxin, no invasion. • Have developed Shiga-toxin immunoassay • Source: undercooked beef products … • Clinical/pathologic features: • Hemorrhagic diarrhea & colitis • Hemolytic uremic syndrome (Chapter 20) Figure: E. coli O157:H7 Infection  Hemorrhagic – stool reflects what is going on in the colon. Invasiveness (severe or not) w/a lot of blood in stool. In most severe cases, necrosis. In least severe, not much inflammation or necrosis. Thrombocytopenia, kidney failure at the same time. Lab Diagnosis of Enteric Bacteria @ CPL: • Stool specimens routinely cultured for Salmonella, Shigella, C. jejuni (with Campy-BAP at 42ο) & E. coli 0157:H7 on MacConkey-Sorbitol) • Special request required forYersinia (cold enrichment; MAC or SS at 25ο) & Vibrio (TCBS agar)  MacConkey Sorbitol TCBS   Far left: MacConkey’s sorbutol – selective media. Capable of showing multi-colored colonies – aids in getting to diagnosis quickly.

 Contaminated spinach recall

Entamoeba histolytica – amoebic dysentery: • Cysts ingested, excyst in intestine, trophs invade colon • E. histolytica pathogenic; E. dispar nonpathogenic • Disease: dysentery, colitis, ameboma, abscess • Pathogenesis: attachment to colon, lysis of cells, invasion of bowel wall • Dx: collect stool for “o” & “p” exam • Rx: metronidazole • Flask-shaped ulcers in the colon • Erythrophagocytosis- amoebae ingest RBCs
Test q: A 50F returns from a missionary trip to Honduras. She became ill during the trip and continues to show fever, diarrhea, and abdominal cramping. She dies after a prolonged illness and at autopsy, ulcers are noted in the colon. The ulcers have a narrow neck and broad base (flaskshaped). Diagnosis? Entamoeba histolytica. Test q: A 60M has persistent bloody diarrhea, abdominal cramps, and fever for the past week. A stool sample is positive for occult blood. Sigmoidoscopic exam shows mucosal ulceration in the cecum. Microscopic exam of a colonic biopsy specimen shows flask-shaped mucosal ulcers. Which of the following infectious organisms is most likely to produce these findings? Entamoeba histolytica.

Amebiasis of the Colon:  Left: Flask-shaped ulcer of E. Histolytica. Right: cannot see ulcers as well.

Left: lab thought it was precancerous lesion. Closer look (right): amoebaforms – got mistaken for macrophages. Key: Red cells in cytoplasm.

E. Histolytica: Do not see liver abscess rd much in the US, but common in 3 world countries – can be difficult to diagnose 

Giardia lamblia: • Most common GI parasite in US • Cysts not killed by chlorine; endemic in unfiltered public water supplies, & streams • Frequent in daycare centers, institutions for retarded, & male homosexuals (but not ⇑ disease in patients with AIDS) • Incubation pd. avg. 1 wk; anorexia, nausea, acute diarrhea, cramping pain, flatulence, abdominal distension; may be wt. loss, malabsorbtion & mushy foul-smelling stools • Wet-mount: pear-shaped trophozoites with “falling leaf motility”
Test q: A 27F medical student returns from a medical missionary trip w/severe abdominal cramping and diarrhea. Stool samples are non-diagnostic; WBCs are not present. She eventually has upper endoscopy w/a duodenal biopsy. H&E stains reveal intact intestinal villi and “leaf-shaped” microorganisms coating the mucosal surface w/no evidence of invasion. Diagnosis? Giardia lamblia. Test q: A 50M returns from a hiking trip to Colorado. Stool exam is negative for WBCs. Exam for ova and parasites shows pear-shaped trophozoites w/”falling leaf motility”. Diagnosis? Giardia lamblia.

Morphology of Giardia lamblia:

Giardiasis: Light Microscopy & Scanning EM:

May flatten villi, associates very closely with/attaches to the mucosa – has suctorial disk that makes depressions on mucosal surface. Pear-shaped trophozoites. Falling leaf motility.

Infections in the Compromised Host: Objectives: • Understand the underlying causes that predispose to opportunistic infections • Know which microorganisms are associated with particular defects in the body’s defense system Compromised, Immunocompromised or Immunosuppressed: • A patient with a defect in one or more of the body’s natural defense mechanisms that predisposes to infection • Immunological, metabolic, biological, physical

11/12/10 Defects That Predispose to Infections: 1. Granulocytopenia 2. Dysfunction of Cell Mediated Immunity 3. Dysfunction of Humoral Immunity 4. Splenectomy 5. Obstruction (eg. lung) 6. Central Nervous System (CNS) dysfunction 7. Medical procedures and devices 8. Loss of normal flora (microbiota) 9. Diabetics 10. Medical Immune Supression (eg. Remicade)

1. Granulocytopenia or Defects in Phagocytosis: • PMN count of 500-1000/µL or less • Linear risk down to zero • Duration of neutropenia • Bacteria - E. coli, K. pneumoniae, CNS (coagulase-negative staphylococci) **Also MRSA and Pseudomonas** • Yeast - Candida • Molds - Aspergillus, Mucor Aspergillosis: • Aspergillus fumigatus and A. flavus • Transplant patients and diabetics • Relatively rare in AIDS* (counterintuitive) • Especially transplant patients with neutropenia; blood vessel invasion • Morphologic identification or culture • Narrow uniform hyphae with septae; 45 degree angle branching Septate hyphae of Aspergillus (Giemsa) 
Test q: Presumptive identification of Aspergillus species in tissue biopsies requires demonstration of: Septate hyphae.

 Can see crosswalls (septae). Branch dichotomously – every time they branch, there is a Y-shape. Aspergillus fumigatus fruiting bodies  Aspergillum (vesicle) produced in nature – we inhale the conidia on these structures.

Note: Coenocytic = aseptate. Used on boards.

2. Cellular Immune Dysfunction: • Primary - DiGeorge syndrome (thymic hypoplasia), X-linked agammaglobulinemia of Bruton, other • Secondary (more common) - aging, malnutrition, malignancy, viral diseases • Bacteria – TB, Legionella, Listeria, Salmonella • Fungi - Aspergillus, Candida, Histoplasma, Cryptococcus • Protozoa - Cryptosporidium, Toxoplasma • Viruses - CMV, EBV, HSV, Varicella Rule of thumb: If you don’t have humoral immunity (not making antibodies)  bacterial infections If you don’t have cellular immunity  viral infections and fungal infections HIV and Cells of The Immune System: • Decreased CD4+ T cells • B cells are ineffective at making antibody – not directed against anything in particular. • Cytotoxic T cells cannot recognize or kill viruses • Reduced interleukin-2 and gamma-interferon; killer cells and macrophages ineffective
Test q: During heterosexual intercourse, seminal fluid containing HIV contacts vaginal squamous mucosa. The virions are captured by cells, which then tranpost the virus via lymphatics to the regional lymph nodes. Within the germinal centers of these lymph nodes, the virions infect CD4 lymphocytes and proliferate, causing CD4 lysis w/release of more virions, which are then taken up on the surface of cells having Fc receptors, allowing continued infection by HIV of more CD4 cells passing through the nodes. Which of the following types of cells is most likely to capture HIV onto its surface via Fc receptors? Follicular dendritic cell. (Other choices: B cells, CD8 cell, Langhans giant cell, Macrophage) REPEATED x2

Clinical AIDS: • CD4+ T cells <200/µL • ~7-10 years to reach this point • Risk for developing opportunistic infections and neoplasms greatly increases (majority of deaths) • Extent of viremia (viral load) also indicates HIV progression: HIV-1 RNA levels increase as immunologic containment of HIV fails Viral load is more important than CD4 count.
Test q: Opportunistic infections in HIV-infected patients are most likely to occur when the CD4 lymphocyte count is: <200/uL.

Typical Course of HIV Infection:

CD4 count starts high, gradually falls throughout the course of AIDS. Viral load (yellow) is originally very high, falls to a low level, plateaus for many years (5-7 years), then the CD4 count drops, viral load goes up  opportunistic infections. Opportunistic and AIDS-Associated Infections: • Pseudomonas • • Legionella • • M. avium-intracellulare • • M. tuberculosis • CMV Candida Cryptococcus Pneumocystis

Bacterial, virla, and fungal.

Pneumocystis jirovecii (formerly carinii): • Infection in AIDS, transplant/heme-onc, children with protein-calorie malnutrition (rare) • Commonly the 1st opportunist in AIDS • Pneumonia when CD4 count below 200/µL • Direct morphologic identification: • Direct fluorescent antibody (DFA) • Nonculturable • Life cycle unclear – cyst form contains 8 trophozoites • Fungus because of cell wall composition; ribosomal sequencing • Some still consider it protozoan • Multiple forms (cyst and trophozoite) Parasite name w/fungal DNA

Cysts

Trophozoites

Above: Use different stains to see different forms: GMS (silver) stain – will see cysts, some have dots, some look like side of a teacup, some have grooves, but no budding. Giemsa stain – see trophozoites.

Alveolar wall inflamed, thickened. Alveolar space – bubbly pink, filled w/microorganisms.

Silver stain – dots, grooves, sideways tea cups

Giemsa stain – see cysts but wall does not stain – only see the forms inside.

Test q: A 42y/o HIV-positive patient w/a CD4 count of 60/µL presents w/bilateral diffuse pulmonary infiltrates on chest x-ray. A GMS (Gomori’s methenamine silver) stain of BAL (bronchoalveolar lavage) fluid reveals 4-6µm extracellular, round or cup-shaped organisms. Many small, pink-purple dots are seen w/Giemsa staining. The most likely diagnosis is infection with: Pneumocystis jiroveci

Mycobacterium avium-intracellulare: • Especially AIDS patients - CD4+ <60/µL • Most originate in GI (gastrointestinal) tract; some lung • Enlarged lymph nodes, liver, spleen • High organism load • Granulomas, lymphocytes, tissue destruction rare • NO cord factor
Figure: Mycobacterium avium infection in the spleen - gray nodules are granulomas. Each gray nodule is a colony.

Mycobacterium avium in macrophages (acid-fast stain) 

Acid fast stain – can see macrophages w/red bacilli inside – STUFFED full.

Cytomegallic Inclusion Disease - Cytomegalovirus (CMV): • Herpesvirus • Esophagitis, colitis, hepatitis, chorioretinitis (cause blindness), meningoencephalitis • In AIDS: almost always accompanied by Pneumocystis • Morphologic identification: • Large infected cells with intranuclear and cytoplasmic inclusions (Cowdry B) • Owl’s eye nuclei • Shell Vial culture for 24-48 hour results
Test q: A 31F AIDS patient develops pneumonia despite prophylaxis w/Septra. A bronchoalveolar lavage (BAL) is performed. A Giemsa stain shows enlarged pneumocytes w/apparent viral inclusion within the nucleus and cytoplasm. Diagnosis? Cytomegalovirus. Test q: A 22y/o kidney transplant patient is hospitalized w/a complaint of cough, fever, and shortness of breath. Chest x-ray reveals pneumonia. The bronchoalveolar lavage revealed only normal flora on routine culture but histology of an open lung biopsy showed enlarged cells w/owl’s eye nuclei and cytoplasmic inclusions. The most likely diagnosis is: Cytomegalovirus.

Above (left): Pulmonary specimen – congested alveolar septum. BIG cell – see dark outline of the nucleus – inclusion is blob inside. Owl’s eye inclusion. Above (right): Smear from BAL – can see normal-size cells. Compare to big cell – infected by CMV. Can see viral inclusion in the nucleus (right) and lots of little ones in the cytoplasm (right).

Cryptococcosis: • Cryptococcus neoformans • Found in bird droppings (especially pigeon) and soil • Latex agglutination of serum or CSF (high sensitivity and specificity – because of Crypto’s thick capsule) • Culture – brown on bird/niger seed agar • 50% of infected patients have no known immune defect • About 10% of AIDS patients • Usually little immune response (cryptic); encapsulated • Can see it both ways – In kidney, we saw glomeruli stuffed full but no inflammation. In the bone marrow, there were granulomas • Central nervous system, pneumonia Histo – chicken, blackbirds, bats Crypto – pigeons

Test q: A 35M who received a kidney transplantation was being treated w/cyclosporine, azathioprine, and high doses of corticosteroids. While on this regimen, the patient began to experience headaches and became lethargic. A clinical diagnosis of meningoencephalitis was made. He died seven days later. Autopsy showed gelatinous meningeal exudates, and on sectioning of the brain, multiple small cyst-like areas were seen. Microscopic exam showed areas containing rounded structures w/a prominent capsule that stained brightly w/mucicarmine. Which of the following tests would have been most useful for diagnosis of this condition during life? Exam of CSF w/latex agglutination. REPEATED x2 (Other choices: India Ink stain on blood; Brain biopsy specimen stained for viral inclusions; Culture of CSF for Streptococcus pneumonia; PCR assay to detect Epstein-Barr virus genome in lymphocytes isolated from CSF)