Pathology

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Tools for Diagnosis of Infectious Diseases

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Pathology
-Histopathology: Examination of tissues to:
- evaluate type of inflammation and to
- visualize the possible pathogen.
-Examination of tissue for identification of infectious agents is particularly helpful in viral and fungal infections.
Laboratory Diagnosis
-Specimen Staining: Different stains depending on suspected organism (bacteria, fungi, mycobacteria, etc.)
-Culture: Growing the organism in culture media or cell lines followed by methods of identification
Serology
-Acute and convalescent serum samples (helpful to confirm diagnosis)
Amplification Tests
-Several platforms for amplification of DNA or RNA and amplification of signal

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Suppurative (Acute) Inflammation

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Leukocyte infiltration, mainly neutrophils (PMNs)
PMNs are attracted to the site by release of chemoattractants produced by rapidly dividing extracellular bacteria
-Gram positive bacteria: Streptococcus pneumoniae, causing pneumonia
-Gram negative bacteria: N. meningitidis, causing meningitis

Large amount of PMNs results in the formation of pus
The size of exudative lesions vary according to bacterial pathogen involved and the location of the infection

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Mononuclear and Granulomatous Inflammation

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Diffuse, predominantly mononuclear interstitial infiltrates in response to viruses, intracellular bacteria and intracellular parasites

The predominant mononuclear cells depend on the host immune response to the organism (e.g., mostly lymphocytes in Hepatitis B infection, and eosinophils in some parasitic infections)

Granulomatous inflammation occurs when aggregates of altered macrophages form giant cells.

The lesions are usually evoked by the slowly dividing organisms in the presence of T cell-mediated immune response
Histology: Central area of caseation Surrounded by epithelioid histiocytes
lymphocytes and fibroblasts

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Cytopathic-Cytoproliferative Inflammation

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Characteristic of virus-mediated damage
Virus mediated damage to individual cells in the absence of host inflammatory response
Some induce cells to fuse (See figure showing Herpes virus). Epithelial cells detach and produce blisters
Others replicate within cell and cause aggregates that are visible as inclusion bodies (e.g., CMV, RSV, adenovirus)
Viruses can also cause proliferation of epithelial cells and form warts.

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Necrotizing Inflammation

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Usually toxin-mediated
Toxins cause severe tissue damage and cell death (e.g., Clostridium perfringens, Group A Streptococcus)
Few inflammatory cells involved
Viruses may cause necrotic inflammation when host cell damage is widespread and severe (e.g., liver necrosis by Hepatitis virus
Myonecrosis caused by Clostridium perfringens:
-Rapid and severe tissue damage. Few inflammatory cells are involved. Cell death is the dominant feature

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Chronic Inflammation

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May lead either to complete healing or to extensive scarring

The patterns of tissue reaction (acute polymorphonuclear inflammation and chronic inflammation and fibrosis) frequently overlap

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Laboratory Diagnosis of Bacterial Infections

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Direct examination of specimen
-Gram Stain: Gram positive and Gram negative bacteria
-Acid-Fast Staining (Ziehl-Nielsen, Kinyoun) for Mycobacteria species
-Auramine-rhodamine fluorescent stain: Mycobacteria species
-Immunofluorescent staining: eg., Legionella

Examination of tissues (Histopathology):
-Gross Pathology examination
-Microscopy: Modified Gram stain is used in some cases
-In situ hybridization stains used for organisms that do not grow in culture

Culture
-Use of specific solid and liquid media, then apply identification methods

Amplification methods
-DNA or RNA detection

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Culture Media by Clinical Specimen Bacteria

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Blood:
-Blood agar (aerobic and anaerobic), Chocolate plates
Respiratory:
-Blood agar, Chocolate, MacConkey plates
Spinal fluid (CSF):
-Broth, Chocolate, Blood agar plate
Urine:
-Blood agar and MacConkey
Stool:
-MacConkey and other selective agar plates

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ID of facultative gram positive cocci

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Alpha hemolysis
-+bile solubility, optochin: S. pneumoniae
--bile solubility, optochin (susceptible): S. viridans

Beta:
-+bacitracin (susceptible): S. pyogenes
--bacitracin: non group A

Gamma:
-Bile esculin, high salt: enterococcus

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Legionella pneumophila

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Contamination of water supplies
Higher incidence in patients with underlying diseases
Difficult to grow in culture (needs cysteine in agar). Use of BCYE with cystein
Pleomorphic, WEAKLY staining gram-negative rods
Diagnosis by Direct Immunofluorescence, Legionella urinary antigen (serogroup 1), amplification methods and serology
Histopathology: Acute fibrin-purulent exudative pneumonia
with neutrophils and macrophages within the
background fibrinous exudate

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Streptococcus hemolysis

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Beta:
-Group A
-Group B
-Group C, G, or F

Alpha:
-S. pneumoniae

Alpha or non hemolytic:
-S. viridans

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Fungi: Approach to identification

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Eukaryotic cells which can be divided into 2 morphologic groups :
-Yeasts (Candida spp, Cryptococcus spp, Malassezia furfur)
-Moulds: Filamentous fungi forming hyphae.
-Septate: clear or dark
-nonseptate:

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Laboratory Diagnosis of Fungal Infections

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Direct examination of specimen: Hyphae vs yeast
-Lactophenol Blue: Lactophenol acts as clearing agent and aids in preserving fungal structures. Cotton Blue gives color to the structures.

-KOH: Wet mounts of 10% KOH are used to distinguish fungi in thick mucoid specimens or hair/nails. Dissolves proteinaceous tissues, including keratin, and renders them transparent. This enables fungi to be visualized more easily.

-KOH-Calcuofluor White. Calcuofluor binds to the chitin of cell walls of fungi and fluoresces under UV light.
-India ink for detecting capsule of Cryptococcus

Culture: At 37o C development of yeast phase. At 25o C development of mold phase

Antigen detection in clinical specimens: eg., Cryptococcus antigen in CSF, galactomannan in serum for diagnosis of Aspergillosis.

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Stains used for Demonstrating Fungi in Tissues

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Routine: Hematoxylin and eosin (H&E). Best stain to demonstrate host reaction (type of inflammation)

Fungal stains: Useful for detecting Hyphae and yeast cells
-Gomori’s methenamine silver (GMS)
-Periodic acid-Shiff reaction

Mucin stains
-Mucicarmine: Demonstrate mucoid capsule of Cryptococcus

Melanin stains: Confirm presence of melanin in pigmented agents

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H&E

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Stains most fungi violet to purple , although some stain faintly, in mild to moderate contrast to the lighter background tissue
Has advantage of allowing observation of natural pigment of fungi
It is the best to demonstrate host tissue reaction

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GMS

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(Gomori/Grocott methenamine silver) – fungal walls stain black (but so can collagen fibers, rbc’s, etc.)
Fungi (and Pneumocystis) stain black
Drawback is that it often stains fungi too densely to observe structural details

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Zygomycosis vs Aspergillus staining

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H&E: Broad nonseptate
ribbon-like hyphae. Consistent with Zygomycosis. Confirm with culture

GMS stain: Consistent with
Aspergillus spp. Septated hyphae (45o branching) Confirm with culture

Zygomycetes and Aspergillus spp can cause acute inflammation and usually invade vessels causing thrombosis and necrosis

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Comparisons of different yeast cells seen in tissue

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Candida
-Size 3-5 micron
-Growth phase yeast
-Oval yeast cells, pseudohyphae
Histoplasma capsulatum:
-size 2-4 micron
-Growth phase dimorphic
-Often intracellular within WBC, macs
Cryptococcus neoformans:
-size 3-20 micron
-Growth phase yeast
-Yeast of varying size within same tissue specimen, thick capsule
Blastomyces dermatitidis:
-size 3-30 micron
-growth phase dimorphic
-Large yeast cells with thick double wall and broad-based buds
Coccidioides immitis:
-size 10-60 micron spherule, 3-5 micron spores
-growth phase dimorphic
-large thick walled spherules with tiny endospores

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Laboratory Methods for Diagnosis of Viral Infections

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If viral inclusions are seen in tissue, or the clinician is suspecting a viral infection, specimens (BAL, urine, blood) are sent to the lab for culture

In the laboratory, perform inoculation of cell lines followed by direct examination of viral inclusions

Tissue culture (standard and shell vial)
Detection of antigen in clinical specimens
-Direct Fluorescence Assay
-EIA -Enzyme Immuno Assay (eg.: Rapid test for Influenza A/B)

Detection of nucleic acid by amplification tests

Serology. Acute and convalescent serum samples to confirm disease when virus is not able to grow in cell culture

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Diagnosis of Cytomegalovirus (CMV)Pneumonia in Tissue

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CMV causes interstitial pneumonitis, but in some cases can cause inflammatory nodules as shown here (left). CMV produces characteristic intranuclear inclusions surrounded by clear halos (right).

Shell vial culture, traditional viral culture or PCR can confirm the diagnosis

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Immunofluorescent Stains

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Specific antibodies to different viruses are used to identify viruses growing in cell culture

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Respiratory Syncytial Virus (RSV) diagnosis

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What type of test was done? Rapid EIA
What other test could confirm the diagnosis? Molecular test

Histopathologic examination:
-Cytopathic Effect: Tissue section showing giant cells with intracytoplasmic inclusion

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Multiplex PCR: Detection and differentiation of respiratory viruses

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Flu A
-Seasonal A/H1 or H3
-2009 H1N1
Flu B
RSV A or B
Adenovirus
ParaFlu 1, 2, and 3
hMPV
Rhino enterovirus

In CSF
-Enterovirus
-HSV 1/2

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use of Nucleic Acid Testing (NAT)

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has reduced “Window Period” for HIV and HCV.
-From 22 to 11 days with HIV
-From 70 to 1 days with HCV

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Laboratory Diagnosis of Parasitic Infections

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Direct examination of specimen
-Some parasites can be seen macroscopically
-Protozoa and eggs are examined under the microscope
-Wright-Giemsa stain used for detection of blood parasites: Plasmodium spp, Babesia spp.

Direct examination of tissues
-In some infections the parasite can be seen directly inside the tissue.
Depending on site of infection and host response, they can produce chronic inflammation or formation of granuloma.

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Giardia lamblia Infection

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Chronic active duodenitis with increased intraepithelial lymphocytes.
Giardia throphozoites can be seen in the intestinal lumen. Trophozoites of Giardia lamblia have two large nuclei and 4 pair of flagella

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Cryptosporidium parfum

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Diarrhea
4-6 micron oocysts in stool seen with acid fast stain
Spherical structures seen along microvillous border of epithelial cells
Smaller cysts that giardia

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Blood-Borne Protozoa: Babesiosis and Malaria

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RBCs infected with Babesia spp “Maltese cross”
Transmitted by tick bite in Northeastern and upper
Midwestern states

RBCs infected with Plasmodium spp
-Ring forms
-Banana shaped gametocytes.

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Methods of Susceptibility Testing

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Phenotypic test methods
-Minimal inhibitory concentration (MIC) determination
-Differentiation of susceptible, intermediate, and resistant
Genotypic test methods
-Based on the detection of a resistance gene
-mecA -- predict oxacillin resistance in S aureus
-Beta-lactamases----- resistance to beta-lactams
By deduction – “expert rules”
-If mecA positive – then report as resistant to all beta-lactams
-If erythromcyin resistant – then report azithromycin and clarithromycin resistant

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MIC and Breakpoint

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Minimal inhibitory concentration (MIC) :
-The lowest antibiotic concentration required to visually inhibit bacterial growth following an overnight incubation
-This is an In Vitro test and results need to be compared with obtainable antibiotic concentrations in serum and at site of infection
-Correlation of MICs of organisms causing infection with bacterial eradication and clinical success can be made.
Breakpoints:
-Cut off MIC (or zone diameter) for applying interpretation (S, I, or R)

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Interpretation of Susceptibility Results

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Susceptible: Infection may be appropriately treated with dosage of antibiotic recommended for that organism

Intermediate: Implies clinical applicability in body sites where the drug is concentrated

Resistant: Not inhibited by achievable concentrations of agent with normal dosage and /or mechanism of resistance present.

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Susceptibility Testing

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Macrodilution method
Microdilution method (automated)
Kirby-Bauer disc diffusion method
E-test

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What is the process for setting and revising breakpoints?

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Microbiologic data- MIC distribution of wild type or normal population

PK (Pharmacokinetics
-Serum concentration
-Penetration to site of infection
PD (Pharmacodynamics)
-Susceptibility – MIC
-Time above MIC

Clinical studies- outcome in vivo

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Time above MIC

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Correlation of serum pharmacokinetics with MIC (susceptibility) of an organism. The target for efficacy is that concentration should be above the MIC at least for 40% of the time between dosing