Infectious Disease Pathology p1-30

Introduction to Infectious Disease

11/1/10

NOTE: This question should have gone in the endocrine study guide – N. meningitidis is a cause of WaterhouseFriderichsen syndrome (adrenal insufficiency resulting from bilateral adrenal hemorrhage): Test q: A 23F, previously healthy, died suddenly after complaining of a mild sore throat the previous day. At autopsy, her adrenal glands are enlarged, and there are extensive bilateral cortical hemorrhages. Infection w/which of the following organisms best accounts for these findings? Neisseria meningitidis. REPEATED x4 (2009 #46, answer is incorrectly keyed as “Histoplasma capsulatum”)

Sample Question: An increase in the number of cases of tuberculosis in New York in the 1980s was due to: A. multiply drug-resistant M. tuberculosis B. resistance to INH C. genes for capsule production D. virulent strains from Tailand E. more homeless and AIDS patients

Sample question 2: A special stain that will demonstrate bacteria, fungi, parasites and viral inclusions is: A. Gram stain B. Acid-fast stain C. Modified acid-fast stain D. Giemsa stain Old test q – repeated x3 E. Silver (GMS) stain

Old test q – repeated x2

ID Outbreaks in the U.S. Polio Legionnaire’s Disease Toxic Shock Syndrome Lyme Disease Human papillomavirus Tuberculosis

Hantavirus SARS and Anthrax *MRSA *Clostridium difficile * often hospital-acquired

“Emerging” Infectious Diseases: Microorganisms do not change much Human behavior changes a lot PARALYTIC POLIO Poor Sanitation (U.S. in 1800) childhood diarrhea enterovirus infection and immunity paralysis rare

Good Sanitation (U.S. in 1900) children not infected and do not become immune young adults infected receptors on anterior horn cells- paralysis

Red areas exhibit the highest rates of paralytic polio:

Legionnaire’s Disease: • Legionella is present in fresh water ponds, plumbing systems and aerosols • Sporadic cases occur • American Legion convention with many elderly attendees with chronic lung disease • New tools developed to identify Legionella- DFA, urine antigen test and DNA probes • Hyper-chlorination of water in hospitals – preventative mechanism

Legionella DFA:

Toxic Shock Syndrome: • Staphylococcus aureus with TSST-1 toxin • 1980s- Use of hyperabsorbent tampons (RELY brand) caused an epidemic of cases • Toxic shock still seen sporadically caused by both staphylococci and streptococci Rash of Toxic Shock Syndrome:

Gram positive cocci on blood smear:

Lyme Disease: • Borrelia burgdorferi (spirochete) - rash, arthritis, (meningitis and myocarditis) • deer tick and white-tail deer reservoir • rat tick and woodrat reservoir in California • more deer-more ticks-more people • wildlife protection-housing development Rash of Lyme disease:

Spirochetes:

Looks like bulls-eye/target. National Lyme disease risk map w/four categories of risk  Test q: A 30M comes to the physician because he has had joint pain in the right hip and left elbow and a headache for the past week. One month ago, he had similar pain in the left hip and knee, which slowly resolved. He remembers having a ring-like skin rash on his left thigh several months ago after a tick bite. On phys exam, there is joint tenderness but no swelling or deformity of the right hip and left elbow. His heart rate is slightly irregular. Which of the following infectious agents is most likely to produce these findings? Borrelia burgdorferi. Test q: The incidence of Lyme disease in the US has increased partially due to an increase in the population of: White-tailed deer.

Human Papillomavirus: • Causes condyloma, dysplasia and cervical carcinoma • There is a more sexually active population • The incidence of CIS is increasing in the U.S. (but mortality is decreasing) • ** Most new head/neck squamous carcinoma cases in the US are HPV-associated HPV DNA Types 16 and 18: • HPV Infection produces proteins E6 and E7 • p53 protein, RB and p21 protein are inactivated • Apoptosis does not occur and the infected cells are not killed • Dysplasia and cervical cancer may result

Condyloma caused by HPV infection- not malignant ↑ Review HPV questions that popped up on 2 test: Test q: A 35F is diagnosed w/squamous cell carcinoma in situ of the cervix. The five year survival rate for this patient if properly treated is: 100%. REPEATED x2 Test q: A 29F presents for pelvic exam and pap smear. What anatomic site is most likely to yield dysplastic or malignant change? Squamocolumnar junction. Test q: A pap smear test has the LOWEST sensitivity for detection of: Endometrial adenocarcinoma (Other choices: LGSIL, HGSIL, CIS, Invasive squamous cell carcinoma) Test q: HPV infections of the cervix have been increasing every year in the US. The number of cases of CIS is also increasing but the number of cases of invasive cervical cancer is decreasing. What explains this paradox? Screening with pap smear. A 35F patient has a pap smear. The diagnosis is ASC-H. What is the next step? Refer to colposcopy for biopsy Test q: A 45F presents to a family practice doctor w/oral and vaginal thrush. A gynecological exam reveals an ulcerated and bleeding cervix. A biopsy reveals invasive squamous cell carcinoma of the cervix. Which of the following best explains the development of cancer (oncogenesis) in this patient? Protein E6 and E7 production by HPV type 16 or 18. nd

.Tuberculosis:

• • •

Mycobacterium tuberculosis In the U.S. the people at risk are: homeless, elderly in nursing homes, prisoners and AIDS patients If people are poor, malnourished, and have crowded living conditions, tuberculosis will flourish

Test q: An increase in the number of cases of tuberculosis in Indianapolis in 2009 is due to: More nursing home patients and immigrants (Other choices: Multiple drug-resistant M. tuberculosis and M. bovis; Resistance to INH; Genes for capsule production; Virulent strains from Thailand)

Above (left): Reported TB cases US, 1982-2004. Decreased when streptomycin was introduced. Dropped til the 1980s – then we had bad recession and introduction of HIV into the population (went back up). Above (right): Reported TB cases by origin and race/ethnicity, US. U.S. Born: Whites and Blacks most common. Foreign Born: Asians and Hispanics most common. Test q: In the US in 2004, the population at greatest risk for acquiring active tuberculosis is: Foreign-born Asians. (Other choices: US-born whites, Foreign-born whites, US-born Asians, Foreign-born blacks)

Ghon Complex or Primary Complex: (LN and peripheral lesion – macrophages migrate) Tuberculosis is a communicable disease.

 TB in the upper lobecaseous granulomas. Immune system breaks down, then TB reactivates in the upper lobe.

Hantavirus: • 4 corners area (SW USA – Utah, Colorado, Arizona, New Mexico) • flu-like illness to hemorrhagic fever with sudden death (carried by deer mice) – similar to Ebola virus The deermouse and family: • excess rain leads to bumper crop of pinon nuts • deer mice eat nuts and population increases • Navajos harvest pinon nuts Test q: HIV has spread world-wide but Ebola Virus has primarily remained in a single, small geographic area. What is the explanation of this inability to create a pandemic like HIV? Ebola Virus is rapidly symptomatic and usually fatal.

Hantavirus

MRSA and C. difficile: MRSA Wide use beta-lactams Poor handwashing- more patients per nurse/doctor

Clostridium difficile Use of Clindamycin Use of proton pump inhibitors Use of quinolones (gattifloxacin, moxifloxacin) Plus not hand-washing

Emerging Diseases ?? The human population changes behavior Microorganisms do not change behavior Koch’s Postulates: • Microrganism is found in lesions of the disease • Organism is isolated on solid media cultures • Organisms from culture causes lesions in experimental animals • Organisms can be recovered on solid media cultures from lesions in the animals Problems with Koch’s Postulates: • Viruses, Rickettsiae, Chlamydia etc. do not grow on solid media • DNA Probes and Amplified DNA Probes are more sensitive and more specific than culture (on solid media or tissue culture) • Therefore, the “Gold Standard” is often NOT culture Categories of Infectious Agents: • Prions- kuru, C-J Disease, mad cow disease • Viruses • Bacteriophage/Plasmid • Bacteria • Chlamidiae/Rickettsiae/Mycoplasma • Fungi • Parasites (protozoa, worms, ectoparasites)

Microorganisms have to overcome a lot of resistance in order to establish an infection. At every point, they are attacked by different defense systems.  Release and Transmission: • Contact- wound, body fluids or mucosal surfaces (as in STDs) • Cough with aerosol as in TB • Insect vectors as in malaria, Lyme disease • Diseases spread from person to person are said to be contagious or COMMUNICABLE • Diseases acquired in the hospital are NOSOCOMIAL Test q: A nosocomial infection is best described as one that is: acquired in the hospital.

How Do Microorganisms Cause Disease? • Direct contact with cell death (Streptococcus produces cellulase) • Release of toxins that enter the blood and kill target tissue (C. diphtheriae) • The host response, such as abscess (staphylococci) or granuloma (TB), that destroys host tissue Viral Surface Protein Receptors: • EBV- **CD21 (CR2) receptor on B-cells and perhaps macrophages; atypical lymphocytes in blood are mainly CD8+** • Rabies- acetylcholine receptor on neurons • Rhinovirus- ICAM-1 on mucosal cells • HIV- CD4 , CXCR4 or CCR-5 Figure: HIV mechanism of attachment  HIV Kills CD4 (Helper) T- Lymphocytes: • Only 1/100,000 CD4 lymphocytes are infected • Infected cells induce non-infected cells to commit suicide by APOPTOSIS and other mechanisms

 How viruses work: take over host machinery.

Bacterial Injury: • Virulence genes • Salmonella and E. coli have similar virulence genes but E. coli lacks genes for attachment and invasion • Helicobacter pylori and Corynebacterium diphtheria strains that do NOT produce toxins are not pathogenic (most diphtheria strains do not make a toxin)

Bacterial Adhesins: • Lipoteichoic acids on Streptococci bind tightly to blood cells and oral epithelial cells • Pili on GN rods and cocci- proteins at the tips of the pili bind sugars • type I- mannose (UTI) • type P- galactose (pyelonephritis) • type S- sialic acid (meningitis)

Bacterial Targets: • Unlike viruses that invade many cell types bacteria primarily infect epithelial cells (Shigella, E. coli), macrophages (TB) or both (Salmonella, Listeria) • Macrophages- receptors for Ab or C’ • Epithelial Cells- many bacteria bind to integrins such as CR3 (complement iC3b) Bacterial Toxins: Endotoxin, Exotoxin, Enterotoxin Figure: Diphtheria Exotoxin  Fragment B (COOH)- attachment Fragment A (NH2)- enzymatically active Connected by a S-S bridge Toxin produced in upper airway but travels through blood and causes heart damage. Figure (below): Immune Evasion: 1. Inaccessibility- C. difficile in the intestinal lumen – sends its toxin to do the dirty work 2. Block phagocytosis- S. pneumoniae’s mucopolysaccharide capsule 3. Antigenic Variation- influenza virus and rhinovirus 4. Immunosuppression- HIV and EBV

Diagnosing Infectious Diseases: • Special Stains • Laboratory Culture • Nucleic acid probes (DNA probes) • Amplified nucleic acid probes • Clinical History Special Stains for Microorganisms: • Gram Stain- G+ and G- bacteria; Candida • Giemsa Stain- bacteria, fungi (all), viral inclusions, parasites - EVERYTHING • Silver Stain (GMS)- fungi and a few bacteria • (Modified)-Acid-Fast Stain- mycobacteria and Nocardia

GRAM STAIN: • Any fluid or tissue • Screen for or identify bacteria • 30 minute turnaround • GPC, GPR, GNC, GNR and yeast • Poor sensitivity for filamentous fungi, mycobacteria, parasites and viral inclusions Gram-negative bacilli:

Gram stain of Clostridium perfringens

Gram-positive rods.

Gram Stain of Candida albicans

Most fungi do not stain w/Gram stain at all.

Giemsa Stain or Diff-Quick (rapid Giemsa) Stain: • Any tissue or fluid • Works best on smears • Bacteria, fungi, parasites, viral inclusions • Rapid preparation (1 minute) Giemsa stain of perispinal abscess:

Shows many intracellular cocci See neutrophils, bands. Staphylococcus

Giemsa stain of H. capsulatum:

Histoplasma capsulatum. RBCs, macrophage w/yeast inside.

MICROWAVE GMS (Silver) STAIN: • Any tissue or fluid- 30 minutes • All fungi including Pneumocystis • Histoplasma, Cryptococcus, Mucor, Aspergillus • Most sensitive stain for fungi • Poor for bacteria and mycobacteria • O.K. for Nocardia and Actinomyces

Giemsa stain of CMV:

Cytomegallovirus – inclusions (dark areas)

Silver stain of Histoplasma capsulatum:

ACID-FAST STAINS (Kinyoun, Ziel-Neelsen, etc.): • Carbolfucsin or fluorescent(more sensitive) • “modified” AFB for Nocardia- weak acid wash • Mycobacteria, Nocardia, Rhodococcus, Cryptosporidium • Sputum must be decontaminated and concentrated for high sensitivity Fluorescent Acid-Fast Stain:

M. tuberculosis

Acid-Fast Stain (Mycobacterium avium):

Acid-fast stain:

Lymph node biopsy – traditional acid-fast stain (red)

M. tuberculosis - “cording”

Cording – wrap around each other like cords in a rope. BAD sign – high virulent strength. Nocardia

Partially acid-fast and may grow on “TB” media

Filamentous Nocardia:

Nocardia (modified-acid-fast stain):

Gram Stain (Gram positive filaments)

If a question says “WEAKLY ACID FAST” = NOCARDIA. SPECIAL SILVER STAINS: Warthin Starry, Dieterle – spirochetes Treponema pallidum Legionella Bartonella

DIRECT FLUORESCENT ANTIBODY (DFA) STAIN: Sputum or respiratory washings Legionella, Bordetella persussis Herpes virus and others **Currently we are identifying Influenza A by DFA and most are H1N1 (by PCR)

Dieterle/Warthin-Starry Stain:

Fluorescent antibody stain: Herpes-infected cells

Shows Treponema pallidum (syphilis)

Multi-nucleated giant cell – nuclei light up w/antibody to herpes.

Laboratory Culture: • Solid Media- wounds, throat, urine, sputum • Broth- blood and sterile body fluids • Tissue Culture- virus, Chlamydia (rare) • Now, most chlamydia testing is molecular.

DNA Probes: • GC and Chlamydia from urogenital specimens *(including Pap Smears) • ID of bacterial, fungal and mycobacterial growth in broth and on solid media • Candida, Gardnerella and Trichomonas from vaginal discharges

DNA Probe:

If we use a direct DNA probe, we target ribosomal RNA because there are 10,000 copies/cell, while there are very few DNA copies per cell. We see if it hybridizes (HPA, above). Amplified DNA Probes: • PCR, LCR, TMA, SDA • GC and Chlamydia- urogenital swabs or urines • M. tuberculosis in respiratory specimens

:

(TMA – transcription-mediated amplification) (SDA – strand displacement amplification)

SDA:

TMA:

Linear SDA:

Common DNA – blue. Unique DNA to organism – pink. Yellow is what we test for by PCR.

Real-time PCR for MRSA, GBS and C. difficile: • Results in 1-2 hours (short as 45 minutes) • Methicillin-resistant Staphylococcus aureus (MRSA) and Group-B streptococcus/ S. agalactiae (GBS) are FDAapproved tests • C. difficile • Other tests are ASRs (analyte specific reagents) that must be validated in your own laboratory; NO ready-to-use kits Test q: The chief advantage for choosing real-time PCR over direct DNA probe or amplified DNA probes is: decreased turnaround time for results

MRSA: MecA gene

Methylicillin-resistant staph – standard beta-lactam antibiotics don’t work because of altered penicillinbinding protein.

™

IDI-MRSA Provides a Definitive Result

• DNA detection of the SCCmec-orfX junction found only in MRSA provides definitive identification of MRSA • Only molecular method to definitively identify MRSA in specimens which contain methicillin-resistant coagulase negative Staphylococci and mecA negative S. aureus. Normally such a combination would yield false-positives in other PCR methods • Detects both HA-MRSA and CA-MRSA strains (i.e. USA300)

Test q: A 50F is admitted for a right knee replacement. The surgical procedure was a success, but 3 days (72hr) post-operation, the surgical site is red, warm, and swollen w/drainage of pus. Real-time PCR is performed on the pus and is (+) for Staphylococcus aureus w/MecA gene present. This history is consistent with: Nosocominal MRSA. (Other choices: Community-acquired MRSA; S. aureus, methicillin susceptible; Coagulase-negative staphylococcus; Viridans streptococci)

Target Specific Fluorogenic Probe – Molecular Beacon:

Rapid IDI-MRSA/MSSA Assay* - under development

• •

Assay under development, allowing the direct detection of MRSA and MSSA in under 2 hours of laboratory time Plan to validate four different specimen types: • Positive blood bottles, wound swabs, nasal swabs, and rectal swabs

Fluorescence is monitored for every cycle:

The secret to real-time PCR is that the reaction doesn’t have to go to completion – can read during the beginning of the reaction.

Positives and negatives.

Inflammatory Response to Infectious Diseases: • Host Dependent- eg. Greatly reduced in AIDS • PMNs- pyogenic response to many bacteria: staphylococci, streptococci, GNRs • Granulomatous- tuberculosis, histoplasmosis • Lymphocytic – viruses • Bacteria – usually neutrophils (PMNs); liquefactive necrosis; abscess • Fungi – usually macrophages/lymphocytes; granulomas • Virus – usually lymphocytes; may show cytopathic effect (CPE) • Immune Deficient Host- there may be absence of the normal inflammatory response and high numbers of microorganisms (eg. M. avium in AIDS) “All bets are off” Bacterial abscess in the brain: Abscess:

Pus pocket

TB Granuloma:

Destroyed tissue, lots of neutrophils

Lung slice, TB granuloma:

TB granuloma – spontaneously healed. Infection may be walled off w/calcium around it.

Most important mediator is IFN-γ

Kidney filled w/caseating granulomatous inflammation

TB granuloma:

Primary and Secondary TB: 1° – peripheral lesion and lymph node involvement, mid lung fields. Reactivation (2°) – primarily infection in upper lung because TB likes oxygen (and most oxygen is in lung apices) Inflammatory Response: • Cytopathic- viral inclusions in Herpes and CMV or dysplastic changes in HPV • Necrosis- Clostridium perfringens in gas gangrene Koilocytosis of HPV:

Example in HPV – infects cervical epithelial cells  koilocytosis

ID Lab Preview

11/2/10

Special Stains for Microorganisms: • Gram Stain- G+ and G- bacteria; Candida • Giemsa Stain- bacteria, fungi (all), viral inclusions, parasites • Silver Stain (GMS)- fungi and a few bacteria • (Modified)-Acid-Fast Stain- mycobacteria and Nocardia GRAM STAIN: • Any fluid or tissue • Screen for or identify bacteria • 30 minute turnaround • GPC, GPR, GNC, GNR and yeast •

•

GPC = Gram+ cocci; GPR = Gram+ rods; GNC = Gram neg. cocci; GNR = Gram neg. rods

Poor sensitivity for filamentous fungi, mycobacteria, parasites and viral inclusions

Gram negative bacilli:

Gram (+) Clostridium perfringens:

Gram Stain of Candida albicans:

Ocassionally, yeast can be seen w/a Gram stain. Giemsa Stain or Diff-Quick (rapid Giemsa) Stain: • Any tissue or fluid • Works best on peripheral blood smears • Bacteria, fungi, parasites, viral inclusions • Rapid preparation (1 minute) Giemsa stain of a perispinal abscess:

Staphylococcus: see many intracellular cocci.

Giemsa stain:

Histoplasma capsulatum

Giemsa stain of CMV:

Cytomegallovirus: viral inclusions in the nuclei. Also have cytoplasmic inclusions.

MICROWAVE GMS (Silver) STAIN: • Any tissue or fluid- 30 minutes • All fungi including Pneumocystis, Histoplasma, Cryptococcus, Mucor, Aspergillus • Most sensitive stain for fungi • Poor for bacteria and mycobacteria • O.K. for Nocardia and Actinomyces Ribbony appearance of Mucor:

Silver Stain of H. capsulatum:

Cysts of Pneumocystis jiroveci:

Above: Can look like a tea cup from the side – can also have groove in it (top left) or dot in it (middle). Pneumocystis is actually a fungus, but see no budding. Has characteristic forms on Silver stain.

ACID-FAST STAINS (Kinyoun, Ziel-Neelsen, etc.): • Carbolfucsin or fluorescent (more sensitive for Mycobacterium tuberculosis) • “modified” AFB for Nocardia- weak acid wash • Mycobacterium, Nocardia, Rhodococcus, Cryptosporidium • Sputum must be decontaminated and concentrated for high sensitivity Acid fast stain: some organisms can be stained w/certain stains, and if you bleach them w/acid, the stain goes away (this is the way most bacteria are – non-acid fast). If you stain an organism and unsuccessfully try to remove the stain w/acid, the organism is acid-fast. MODIFIED Acid fast used for NOCARDIA. M. tuberculosis in a gastric biopsy:

Fluorescent Acid-Fast Stain of M. tuberculosis

TB shows “cording” (Mycobacteria wrapping around each other); blue organisms are Candida

More sensitive stain.

SPECIAL SILVER STAINS: • Warthin Starry, Dieterle • Treponema pallidum • Legionella • Bartonella

DIRECT FLUORESCENT ANTIBODY STAIN • Sputum or respiratory washings • Legionella, Bordetella persussis, Herpes virus and others • High specificity/Low sensitivity

Silver stains used on spirochetes and other small rods. Dieterle/Warthin-Starry Stain:

Fluorescent antibody stain: Herpes-infected cells

Shows Treponema pallidum (syphilis)

Bronchopneumonia: • Patchy or “hit and miss” • Staphylococcus, GNRs, anaerobes • Aspiration with spread through the airways Lobar is consolidated – non-patchy. Many microorganisms can cause bronchopneumonia.

Bronchopneumonia:

Patchy bronchopneumonia:

* = Pneumonia. Abnormal part is paler – lots of WBCs. Can also see patchy involvement on CXR. Lobar pneumonia: • Involvement of the entire lobe (most of it) • Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae • Encapsulated bacteria • Aspiration then spread through alveolar walls (pores of Kohn) Test q: Bacteria noted to cause lobar pneumonia include: Streptococcus pneumoniae and Klebsiella pneumonia.

Above (left): In lobar pneumonia, have pores that the organisms can squeeze through because they have a capsule. Most bacteria and other organisms cannot. Get involvement of one alveolus  spreads to the next one, to the next one, and so on. Above (middle): Grossly, have entire lobe involved (bottom) and entire lobe uninvolved (top) Above (right): Lobe is completely whited out. No breath sounds heard in this area. Percussion – dullness over area. Bronchopneumonia – would hear crackles.

Paragonimus westermani: • Human lung fluke – one of the few parasites that migrates through the lungs • Granulomatous reaction to the eggs Tremendous granulomatous inflammatory response  Within granulomas, have multi-nucleated histiocytes, rim of lymphocytes/fibrosis. Can see organism’s eggs, too. 

Aspergillus fumigatus (A. flavus): • Opportunistic infection in transplant and hematology/oncology patients • Neutropenia • Narrow, septate hyphae that branch at acute angles (45 degrees or less) Figure: High power – Silver stain (best for these fungi). See cross-walls/septae. Branches at 45* angles or less. 

Aspergillus – hyphae in blood vessel walls:

Vascular invasion is common (w/infarction):

Aspergillus is noted to invade blood vessles – this is a bv that has thrombosed.

Lung w/multiple thrombi in vessels.

Mucormycosis (Rhizipomycosis):: • Mucor and Rhizopus • Diabetic ketoacidosis (DKA), transplant/heme-onc • Nasal sinuses, lung, GI tract, brain • Rhinocerebral mucormycosis – most disastrous complication – grows through bone, sinuses, into brain. • Morphologic identification or culture • Broad irregular ribbon-like hyphae, no septae (aseptate), right-angle branching • Aseptate fungal hyphae (no crosswalls) • Branch at 90 degrees (right angles) • Diabetics (poorly controlled) and transplantation patients • Northern USA – Mucor. Southern USA - Rhizopus • 90° branch coming off. Very folded, ribbony 

Above (left): Involves blood vessles. Looks like jigsaw puzzle – irregular. Donut shapes = hyphae cut in cross section (like a pipe). Above (right): All the donuts are hyphae cut in cross-section. Broad hyphae, no cross-walls. Can see branching over 45°.

Actinomycosis: • Sulfur granules (yellow on gross) • Sulfur granules (pink/blue on H&E) • Filamentous anaerobic bacteria • Gram-positive, acid-fast- negative filamentous bacteria (Actinomyces) • Aspiration • Draining fistulas are common Grows in the mouth. Poor oral hygiene can cause it. Can get “lumpy jaw” or even fistulas growing into the lung or abdomen. Nocardia is also filamentous but AEROBIC. Nocardiosis is acid-fast POSITIVE. Gram-positive, filamentous Actinomyces  At high magnification, see filamentous bacteria that make up the colonies. Test q: A 56M farmer presents to the ED w/cough, fever, weight loss, and a draining fistula in his neck. Grossly, the draining material contains yellow granules. Filamentous bacteria are recovered on anaerobic media. The bacteria are Gram-variable and non-acid-fast. Diagnosis? Actinomyces israelii. Test q: A 70F has a fever and a cough productive of yellow sputum. On phys exam, there is dullness to percussion at the left lung base. A chest radiograph shows areas of consolidation in the left lower lobe. Despite antibiotic therapy, the course of the disease is complicated by abscess formation, and she dies. At autopsy, there is a bronchopleural fistula surrounded by a pronounced fibroblastic reaction. Small, yellow, 1-2mm “sulfur granules” are grossly visible within the area of abscess formation. Which of the following organisms is most likely to produce these autopsy findings? Actinomyces israelii.

Colonies of Actinomyces:

Yellow sulfur granules

Colony of Actinomyces in a bronchiole: Actinomyces colonies on H&E Stain:

H&E Stain

Have orange-pinkish rim around them. Background cells are neutrophils.

Salpingitis: Inflammation/infection of the fallopian tube. • Chlamydia trachomatis • Neisseria gonorrhoeae • Sterility, ectopic pregnancy Chlamydia (C) and gonorrhea (G) do the same things when they infect cervix/fallopian tubes. G is more virulent – will do more damage, more likely to be symptomatic. C – less damage, more likely to be asymptomatic (so more likely to go untreated). Can destroy/scar tubes so much that eggs can’t pass  ectopic pregnancies, problems, sterility.  Fimbriae – should be lined by columnar epithelium w/cilia. In lumen is the pus.

Above: Salpingitis. All the material inside the lumen is pus. Will see lots of pink areas – congested blood vessels.

Abscess w/segmented neutrophils:

Above (left): Lung abscesses. Often see air-fluid level in an abscess (green circle) – see black = air, gray = dense, fluid. Above (middle): Abscess in brain – if chronic, can get fibrotic edge. Above (right): See lots of neutrophils in abscesses – some have 3 lobes. Some undergoing apoptosis – live 4 hours and die by cell suicide.

Appendicitis: • Neutrophils are easiest to see in the muscle and fat • Leukemoid reaction (left shift) is common – high granulocyte count. • Pyuria due to involvement of the ureter Neutrophils:

Pyelonephritis: • Most are ascending (post bladder infection) • E. coli is most common cause bc it’s the most common cause of cystitis – organisms swim upstream to kidney. • High fever, chills , pain • “Thyroidization of the kidney” seen on H&E-stained sections Thyroidization:

Look like thyroid follicles w/blue cells (~parafollicular cells). These are actually tubules in the kidney filled w/protein and lymphocytes.

See glomeruli, so know it’s kidney.

Diphtheria: • Corynebacterium diphtheriae • C. diphtheriae can be normal flora- do toxin assay for diagnosis of diphtheria • Exotoxin destroys myocytes  myocarditis  can cause a fatal arrythmia  heart failure • Abscesses in the myocardium – die of either heart failure or myocarditis Pseudomembrane:  Microscopically, can see bronchus (cartilage) w/pseudomembrane – mixture of inflammatory cells, dead epithelial cells, and microorganisms.

Syphilis: • Obliterative endarteritis seen in primary, secondary and tertiary syphilis – arteries are attacked • Plasma cells surround vasa vasorum • Tertiary- ascending or thoracic aorta is most commonly involved (80%) • Aneurysms in abdominal aorta usually due to atherosclerosis. • Treponema pallidum Chancre on Chest  Aneurysm in ascending aorta:

Inflammation in aorta wall:

Silver stain:

Shows disorganized elastic tissue. Reticular and elastic fibers are destroyed – wall is weakened and balloons out to form aneurysm.

Tuberculosis: • Mycobacterium tuberculosis • Communicable • Necrotizing granulomas (microscopic) is usual but non-necrotizing granulomas are possible • Caseous necrosis (gross) • Ghon complex in lung (primary TB) • Apical disease (secondary/reactivation TB)

Necrotizing granuloma:

* Large granulomas (hilar lymph nodes) ** Small granulomas (miliary TB) Lymphadenopathy – esp. in hilar/central part of lung

Histoplasma capsulatum: • Not communicable • Necrotizing granulomas – look just like TB granulomas. Until you culture or special stain, can’t distinguish. • Small (2-3 microns), budding yeast • Intracellular • Disease almost identical to TB but not communicable. Get it by inhaling conidia from soil. H&E Stain with yeasts of H. capsulatum:

Hard to see yeast.

Coccidioides immitis: “West Coast Histo” • California and Arizona • Necrotizing granulomas • Spherules contain endospores

Giemsa stain:

Sample from blood.

GMS (Silver) stain:

Best seen w/silver stain.

C. immitis Spherules – H&E:

GMS (silver) Stain of C. immitis:

PAS Stain of Coccidioides immitis:

Above: Inflammatory cells outside. Can see endospores in spherule. SPHERULES = COCCIDIOIDES! Test q: A 50y/o resident of Phoenix, Arizona (in 2005, was a resident of Southern Cali) has had a cough that has persisted for one month. On phys exam, his temp is 38.1C. A chest radiograph shows a 3.5-cm opacity w/central cavitation in the right apical region. An open lung biopsy is performed to exclude cancer. Microscopic exam of the biopsy specimen shows caseating granulomatous inflammation containing 60-µm spherules filled w/smaller, rounded structures. Which of the following organisms is most likely to produce these findings? Coccidioides immitis. REPEATED x2 Test q: A 50M who recently moved to Indianapolis from Arizona develops fever, cough, and lymphadenopathy. A fine-needle aspiration of a cervical lymph node shows large (50-75 microns) round bodies. Budding is not seen. Diagnosis: Coccidioides immitis.

Blastomyces dermatitidis: • Large yeast with Broad-Based Buds • Pseudoepitheliomatous hyperplasia mimics (clinically and microscopically) squamous carcinoma in skin and in the larynx – epithelium proliferates, piles up, looks like cancer • Inflammation is mixed: histiocytes with giant cells and abscesses with neutrophils H&E shows broad-based-budding:

GMS (silver) Stain:

Test q: A 55M presents to the Derm clinic w/fever, cough, and a tumor-like growth on his nose. A biopsy of the nose mass is performed and the H&Estained tissue shows pseudo-epitheliomatous hyperplasia but no cancer. A silver (GMS) stain shows large yeasts that are at least 12 microns in diameter and show broad-based buds. A capsule stain (mucicarmine) is negative. Diagnosis? Blastomyces dermatitidis. Test q: Granulomatous inflammation w/numerous polymorphonuclear leukocytes is typical of: Blastomycosis

Pneumocystis pneumonia: • (Pneumocystis carinii pneumonia) or PCP • Pneumocystis jiroveci – new name • Two forms: cysts and trophozoites • Cysts on GMS stain • Trophozoites on Giemsa stain Pneumocystis pneumonia on H&E Stain  H&E – just see a bunch of foamy pink stuff in alveoli.

Giemsa Stain of BAL:

Cysts

Trophozoites

GMS stain:

Pneumocystis cyst with 8 trophozoites. (BAL = bronchoalveolar lavage)

Cysts (“cups, targets, grooves”)

P. jiroveci has cup-saucer shapes, target middle shapes, grooves, but NO BUDDING.

Cryptococcus neoformans: • Variably-sized budding yeast with thick capsule of mucopolysaccharides • India Ink stain (poor sensitivity and specificity) • Latex agglutination on CSF or serum has high sensitivity and specificity • Cryptococcal meningitis 100% fatal if untreated Test q: The optimal test for Cryptococcus neoformans in CSF is: Latex agglutination. Test q: A 25y/o AIDS patient presents in the ED w/a severe headache. You are concerned that he might have cryptococcal meningitis which if untreated, is 100% fatal. The most sensitive and specific test for detection of Cryptococcus neoformans in spinal fluid (CSF) is: Latex agglutination.

C. Neoformans in glomeruli (H&E):

C. Neoformans in bone marrow:

Bone marrow (higher magnification):

Above: Glomeruli filled with little round bodies – always lots of clear space around them because of capsule. GMS Stain- C. neoformans

India Ink: Latex Agglutination: See +/- tests below. Particles coated w/antibody, mixed w/CSF – if C. Neoformans antigen present, cross-link and clump.

Chest wall biopsy. Capsule lots of space.

C. Neoformans in CSF

Polio: • Anterior/motor horns have receptors • Paralytic polio occurs in non-vaccinated adults if infected by the virus Polio: Anterior horns are destroyed. Best seen on low power 

Entameba histolytica: • Flask-shaped ulcers in the colon • Erythrophagocytosis- amoebae ingest RBCs; if so, it is E. histolytica H&E- Flask-shaped ulcer of E. histolytica:

Erythrophagocytosis:  Can find ameobae containing RBCs – erythrophagocytosis.

E. histolytica – see erythrophagocytosis

Strongyloidiasis: • Autoinfection- the entire life cycle can occur in humans if they are immune compromised • Association with HTLV-1 infections The only worm parasite that can go through entire life cycle in the host. No intermediary/secondary host needed. Often patients come in w/pneumonia, are coughing up the organisms. Calcified worms  H&E Stain of Strongyloides (calcified worm)

Adenovirus: • Nucleus only inclusions • Cowdry B type in Adenovirus • Cowdry A type in Herpes H&E Stain with Adenovirus inclusions in hepatocyte nuclei  Big viral inclusions – see big purple blobs in the nuclei. By history, we know it’s adeno (would not necessarily know by this pic).

CMV: • •

Enlarged cells Intranuclear (Cowdry B) and cytoplasmic inclusions are present in some cells

Below: Arrows = CMV-infected cells

CMV infection in lung:

Enlarged (cytomegallic) cells – see outline of nucleus, viral particles are concentrated in one area.

Varicella – in Herpes family, associated w/nerves • Nuclear inclusions • Cowdry B • Ganglia and adrenal medulla • Won’t be able to tell apart from CMV histologically. Involves ganglia. Below: VZV-infected cells and hemorrhage:

Toxoplasmosis: • Brain abscess • “Ring-enhancing” lesions • Cysts

VZV – Adrenal Gland:

Toxoplasmosis: Cysts in brain (abscesses) 

H&E Stain of brain biopsy with Toxoplasma:

Cysts of T. gondii in brain- H&E Stain:

See cyst forms of toxoplasmosis.

Hepatitis: • Lymphocytes in the portal zones • Loss of hepatocytes • Cirrhosis Chronic Hepatitis: Hepatocytes w/inflammatory cells.

Chronic Hepatitis: Plasma cells, lymphocytes.

Get lots of lymphocytes (viral infection) – inflammation around the bile ducts/artery/vein.

Respiratory Infections OBJECTIVES: • Understand how anatomical barriers protect the host from developing respiratory infections • Describe the modes of transmission for specific microorganisms that cause respiratory infections • List major factors that predispose people to respiratory infections • Discuss the pathogenesis of specific respiratory infections • Describe the pathologic features of specific respiratory infections • Know how infectious diseases of the respiratory tract are diagnosed in the laboratory EXAMPLE QUESTION ICAM-1 serves as the receptor for attachment of: A. Mycobacterium tuberculosis B. Influenza viruses C. Haemophilus influenzae D. Rhinoviruses Old test q – repeated x2 E. Histoplasma capsulatum

11/8/10

Topics for today’s lecture: • Influenza • Bacterial pneumonia • Bronchopneumonia • Lobar pneumonia • Selected pathogens and diagnostic considerations • Tuberculosis • Histoplasmosis • Coccidioidomycosis Intro: Respiratory Tract Infections: • Among the most common and least preventable of all infectious diseases • A diverse array of infections ranging from common colds in previously well persons to life-threatening pneumonia in patients with other severe problems • Caused by a wide variety of microbial agents – will be talking about mostly acute but mycobacterium is more chronic. • Antimicrobial resistance is increasing • Diagnostic methods are inadequate but evolving • Community acquired pneumonia is common • 5.6 million people (est.) annually in US resulting in 1.3 million hospital admissions per year • And in those over age 65, the number one cause of death from infectious diseases • Nosocomial (hospital acquired) pneumonia is the leading cause of death from infection in US hospitals; its occurrence prolongs hospital stays about 8 days • Cost of pneumonia care in US hospitals estimated to be over $40 billion Viral Respiratory Infections: • Frequent, contagious, spread by droplets; range from common cold to pneumonia • May damage bronchial epithelium, obstruct airways & lead to bacterial superinfection • Upper Respiratory: rhinitis, sinusitis, otitis media, pharyngitis & tonsillitis • Lower Respiratory: laryngotracheobronchitis, bronchiolitis, interstitial pneumonia, & pleuritis • Most important: Rhinoviruses & Influenza viruses Rhinoviruses: • 60% of common colds due to rhinoviruses: other causes = coronavirus 15% (also is agent of SARS - Severe Acute Respiratory Syndrome), influenza virus, parainfluenza virus, respiratory syncytial virus (RSV), adenovirus, & enterovirus (1-10% of colds) • Rhinoviruses: picornavirus family (small RNA viruses with single stranded RNA genome) • >100 serotypes • Rhinovirus binds intercellular adhesion molecule (ICAM-1) on respiratory epithelial cells & induces mucus secretion via bradykinin release • Dx by clinical findings, but bx of nasal mucosa would show: abundant mucin/mucosal edema, lymphocytes & plasma cells (nonspecific) Test q: A 24M has a fever and runny nose, sneezing, and coughing that have worsened over the past four days. The symptoms abate, and he has sequelae (on 2005 test, he has no sequelae). This infection is most likely to be promoted by binding of which of the following organisms to intercellular adhesion molecule-1 (ICAM-1)? Rhinovirus, REPEATED x2 Test q: Most respiratory viral infections are accompanied by an interstitial infiltrate of: Lymphocytes.

Influenza Viruses: • Single-stranded RNA • Types A, B, or C • Subtypes (H1 - H3: N1 or N2) determined by viral hemagglutinin & neuraminidase in lipid envelope – e.g., H3N2, H1N1 (swine flu 2009) • Viruses are spread person to person by airborne droplets or contact with contaminated hands or surfaces • Major mode of prevention? • Epidemics through mutations of hemagglutinin (H) & neuraminidase (N) that allow virus to escape host antibodies – Antigenic drift: minor antigenic change due to point mutations – Antigenic shift: major antigenic change (both H and N replaced) due to genetic reassortment between animal & human influenza A viruses (but not B or C) • Pandemics due to novel influenza viruses: 1918, 1947, 1957, 1968, 1968, 1977, and 2009 – 1918 Spanish flu killed 20-40 million world wide – As of 11-1-09, pandemic H1N1 2009 was in > 199 countries & overseas territories, & had infected > 482,300 & killed ~ 6000

Test q: The H5N1 stain of avian influenza A in Southeast Asia led to over 85 human cases w/over 200 deaths within the past year. There are reports that the virus has the potential to jump from birds to humans, and it has been found in pigs in China, raising fears that they could serve as a “mixing bowl” making it easier for the virus to mutate and spread to humans. This “mixing” which would result in a major antigenic change (both H and N replaced) is termed: Antigenic shift. Test q: The following is the mechanism by which influenza causes disease: Mutations in hemagglutinin and neuraminidase allow escape from host antibodies. REPEATED x2

Influenza: More Characteristics: • Clinical: rapid onset of fever, chills, headache, myalgias, arthralgias, dyspnea, cough & anorexia • Pathology: nonspecific mucosal hyperemia, lymphomonocytic and plasmacytic infiltrate in submucosa, mucus overproduction • Pneumonia: persons with underlying heart & lung disease at risk • Clearance of infection - occurs when cytotoxic T cells kill virus-infected cells • Host antibodies to H and N prevent future infection with that specific virus • Lab dx: swab of nasopharynx, throat swab, combined nose/throat swab, nasal washes, or bronchial lavages – virus isolation/culture – gold standard but takes 3-7 d – viral Ag detection by direct FA (fluorescent antibody); sensitivity at CPL ~ 85-90%; takes 4 h – multiplex RT-PCR (reverse transcription PCR) also used at CPL; more sensitive; but slower than FA; takes overnight – Rapid Ag point-of-care tests are the least sensitive/false negs; takes 15 min • Rx: Tamiflu (oestelamivir) and Relenza (zanamivir) effective in 1st 24-48h vs influenza A (neuraminidase inhibitors of both influenza A & B) Autopsy findings in lungs of a patient who died (Fall 09) of H1N1/09

Above: Tremedous mucin outpouring in the lumen – Mucosa is flattened, eroded, ulcerated. Monocytic infiltrate – lymphocytes, macrophages

Routes By Which Bacteria Get Into Lungs: • Aspiration of contaminated oropharyngeal contents (e.g. pneumococci, GNRs, anaerobes) • Inhalation (M. tuberculosis, Legionella, plague) • Bacteremia • Direct extension into lungs Factors That Predispose To Bacterial Pneumonia: Age (extremes) Cystic fibrosis Alcohol Debility in general Anesthesia Edema; congestion Marrow transplant (CHF) Malignancy Cerebrovascular illness Chemotherapy; Immune deficiencies immunosuppression Obstruction COPD Viral infections Splenic dysfunction Cigarette smoking Ventilator use Cirrhosis

1

2

3

4

Very severe pneumonia, involved most lobes, was bilateral 1. Bronchus – in the lumen is mucoid material w/lots of cells. Epithelial cells have fallen apart – sloughed. 2. Marked congestion and hemorrhage. Much cellular activity – including macrophages (very active there), lymphocytes. 3. Alveoli filled w/edema fluid. Alveolar septae are widened, filled w/RBCs 4. See foci of acute inflammation, PMNs in the mix. These findings show variation – in immunocompromised host, can have chronic findings that you don’t see in others.

Community-Acquired Pneumonia: Pathogens: • Streptococcus pneumoniae • Haemophilus influenzae • Moraxella catarrhalis • Staphylococcus aureus • Legionella spp. • Enterobacteriaceae (e.g., Klebsiella pneumoniae) • Pseudomonas aeruginosa • Viruses, Mycoplasma, Chlamydophila

Hospital-Acquired Pneumonia: • Staphylococcus aureus: MRSA > MSSA • Gram-negative rods – Enterobacteriaceae (Klebsiella spp., Escherichia coli, and others) – Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp. • Legionella spp. • Anaerobes (aspiration) • Viruses (Influenza, RSV, Parainfluenza

Morphology of Bacterial Pneumonia: • 2 frequently overlapping GROSS morphologic patterns – Bronchopneumonia – Lobar pneumonia • Caused by a variety of Gram-positive and Gram-negative bacteria Bronchopneumonia • Patchy exudative consolidation of lung parenchyma • Gross: dispersed, elevated, focal firm areas • Microscopic: acute neutrophilic suppurative exudate filling air spaces and airways at level of bronchi and bronchioles (with or without lots of edema fluid or RBCs) • Aggressive disease may result in abcess • Organization may result in fibrous scarring

Comparison of Bronchopneumonia and Lobar Pneumonia:

Lobar pneumonia: • Involves large portion of lobe or entire lobe • Bacteria spread alveolus-to-alveolus through pores of Köhn • Most common cause = pneumococcus (Streptococcus pneumoniae), but occas. K. pneumoniae, staphylococci, streptococci, H. influenzae • “Classic” sequence of stages portray natural history of uncomplicated lobar pneumonia – but infrequently seen because of antibiotic therapy “Classic” stages of Lobar Pneumonia: • Stage 1 st – Congestion and inflammatory edema – 1 24 hours • Stage 2 – Red hepatization (consolidation) – looks like liver tissue – Gross: red, firm, liver-like – Micro: confluent exudate with neutrophils and RBCs • Stage 3 – Grey hepatization – blood removed, abundant neutrophils, fibrin mixed together – Gross: gray-brown – Microscopic: rbcs disintegrate leaving fibrinosuppurative exudate • Stage 4 – Resolution – enzymatic and cellular degradation – normal structure restored Resolution is less likely in Klebsiella pneumonia.

Lobar pneumonia: Somewhere between congestion and red hepatization. Alveolus full of neutrophils, engorgement of alveolar septal vessels w/RBCs

 Gray hepatization (bottom portion) – consolidated, confluent appearance. Top right – congestion. Red hepatization – looks like liver. Neutrophils, exudate.

Complications of Lobar Pneumonia: • Abscess • Pleuritis and empyema – thoracic cavity pus • Organizing pneumonia (leaves residual fibrosis) • Bacteremia and sepsis • Infarct Laboratory Diagnosis of Pneumonia: • Sputum specimen collected and sent to lab • Direct smears for Gram stain • Specimen plated on various media Sputum being prepared for Gram stain

Diagnosis of Pneumonia due to Streptococcus pneumoniae: • Gram-stained sputum containing many neutrophils and typical Gram-positive, lancet-shaped diplococci supports diagnosis of pneumococcal pneumonia • But remember, S. pneumonia is part of oropharyngeal microbiota in 20% of adults • Its isolation from blood cultures is more specific but less sensitive than sputum culture; only 25-30% of patients have positive blood cultures Test q: A 68F from a nursing home develops fever and shortness of breath. Percussion reveals dullness over the entire right lower lobe w/other lung fields normal. A sputum specimen reveals neutrophils and Gram-positive cocci. You suspect: Streptococcus pneumoniae.

Streptococcus pneumoniae: Sputum Gram Stain

Streptococcus pneumoniae:

See Gram positive cocci in the background.

Big red objects in the background – neutrophils. Little objects stained dark – look like two little footballs w/pointed ends together = pneumococci.

Streptococcus pneumoniae: colonies  Blood agar plate, organisms more flat on the surface. Surface drops when they begin to autolyze – start to self-destruct. Show zone of partial hemolysis.

Infections with Haemophilus influenzae: • Respiratory – Life threatening epiglottitis - submucosal inflammatory edema may obstruct airway < 24h after onset; generally children 2-4 yrs – Otitis media, chronic bronchitis, bronchopneumonia – Pathology: dense fibrin-rich exudates of neutrophils Acute Otitis Media:

Fibrinosuppurative exudate on H&E:

Bulging tympanic membrane – acute OM – both Haemophilus and S. Pneumoniae are common.

H. influenzae: Acute Purulent Bronchitis:

Bronchus disrupted by neutrophilic exudate sloughing of the mucosa

Diagnosis of H. influenzae Infection: • Tiny gram-negative coccobacilli in gram-stained smears of sputum or CSF • Culture on chocolate agar - requires X & V factors for growth • Rapid I.D. -- e.g., latex agglutination or DNA probe

H. influenzae: Gram-Negaitve Rods in Sputum:

Test q: A 28y/o immigrant from Haiti is seen in the Wishard ED for cough and fever. Sputum shows an encapsulated gram-negative rod. Hemin and NAD are required for growth. Diagnosis? Haemophilus influenzae.

Pseudomonas aeruginosa Infection: • P. aeruginosa necrotizing pneumonia • With necrotizing vasculitis • Leads to necrosis in lungs – small cavities in lung tissue become necrotic w/lots of acute inflammation. • Common in hospital-acquired pneumonia, especially in ICU patients and the immunocompromised. • Small, GNR. Produces complete hemolysis on blood agar – green colonies, smells fruity. Get purple haze of bacteria on H&E – can see it around vessels (below):

Small GNR:

Hemolysis on blood agar: