Surgical Pathology for Microbiologists

Surgical Pathology for Microbiologists

Michael DiMaio, M.D. Gastrointestinal Pathology Fellow Stanford University Medical Center Goals/Objectives

• Understand the concept of surgical pathology from specimen collection to diagnosis and reporting • Recognize histologic features of infectious disease pathology and ancillary techniques to help identify specific agents of disease • Understand the limitations of surgical pathology with regard to microbiologic diagnosis and recognize pitfalls in accurate diagnosis Talk Outline

1. What is surgical pathology: ‐ surgery (biopsy) Æ glass slides 2. What kinds of infections can we diagnose on routine slides 3. Other tools/studies we perform 4. Case examples that demonstrate the importance of surgical pathology

Limitations of surgical pathology peppered throughout the talk Zabriskie Point –Death Valley National Park, CA

Part 1 – What is Surgical Pathology?

• Gross and microscopic examination of tissue specimens obtained from biopsy or surgery

• Purpose is to make a definitive diagnosis, most commonly in the context of cancer

• Biopsies to rule out infection are not as common and if they are performed – Often part of the specimen is sent for culture if infection is suspected

• With regard to microbiology Æ Surgical pathology allows you to see the organisms and the disease process it is causes – In the microbiology lab, you just have the bug outside of its native environment

• Autopsy pathology –same techniques for the purposes of understanding the mechanism of death

Process of surgical pathology

• Gross examination +/‐ frozen section examination • Grossing • Tissue processing, sectioning, staining • Microscopic analysis • Reporting and communicating results Examples of Gross Examination

Total gastrectomy specimen –with ulcer Whipple specimen –with pancreatic cyst

Grossing and Tissue Preparation

http://grossing‐technology.com/ Processing and Staining

• Formalin • Alcohol dehydration • Xylene • Paraffin Frozen section video Slide staining

Eosin, dehydration with ethanol, clearing with xylene

Hematoxylin and bluing reagent

Microscopic examination Queen Elizabeth National Park –Uganda Part 2 –Infections that we diagnose

• Viral • Bacterial • Fungal • Parasitic H&E Clues to infectious etiologies (without special stains)

• Inflammation • Identification of organisms on routine sections (rare) • Cellular changes including nuclear atypia • Necrosis

• We rarely make definitive species specific diagnoses in surgical pathology and always suggest correlation with microbiologic cultures – This is often possible when an infectious etiology is suspected at the time of surgery or biopsy (some material will be sent separately) – When infection was not suspected and is only later seen on the glass slides, species specific diagnosis is much more difficult • Ancillary techniques –paraffin scrolls, nuclei acid extraction, ribosomal sequencing and/or PCR may be helpful

Viral infections (3 examples)

• Requires careful, high power examination of cytologic and nuclear features – Nuclear enlargement with inclusions – Chromatin margination – Formation of syncitia with nuclear molding

Herpes Simplex Virus

Features: dense inflammation, characteristic cytologic features, (3 Ms) – molding, margination, multinucleation http://www.auanet.org/education/modules/pathology/penis‐infections/herpes‐simplex.cfm Cytomegalovirus (CMV)

http://www.pathology.washington.edu/about/education/gallery/infections/Aspergillus_d_ppt.jpg • Features: http://www.pathologyoutlines.com/images/lungcmv1.jpg – cytomegaly – intranuclear inclusions (Cowdry type B) – cytoplasmic inclusions

Adenovirus

http://info.fujita‐hu.ac.jp/~tsutsumi/photo/photo093‐2.htm http://info.fujita‐hu.ac.jp/~tsutsumi/photo/photo093‐4.htm

• Adenoviral infection of lung typically produces any combination of – (1) ulcerative bronchiolitis with karyorrhexis – (2) neutrophilic pneumonia – (3) acute intrapulmonary necrosis with hemorrhage – (4) diffuse alveolar damage • Infected cells can exhibit intranuclear inclusions or smudgy nuclei

Adenovirus hepatitis

• In the liver, “pox‐like” granulomas are seen – punctate spots of necrosis with macrophages – look for adenoviral inclusions at the border of the lesion Bacterial infections (3 examples)

• Intense acute inflammation, often with abscess formation • Granulomas (especially mycobacterial infections) • Identification of organisms on modified Gram stain for paraffin embedded tissue – Brown‐Brenn stain • Chronic inflammation Bacterial pneumonia –Autopsy photos

http://library.med.utah.edu/WebPath/LUNGHTML/LUNGIDX.html Abscess cavity with necrosis

• Often seen with Staphylococcal pneumonia, Klebsiella

http://library.med.utah.edu/WebPath/LUNGHTML/LUNGIDX.html http://www.microscopyu.com/staticgallery/pathology/lobarpneumonia10x04.html H&E section Tissue Gram Stain

Diagnostic Pathology of Infectious Disease, 1st Ed. 2010 http://www.newcomersupply.com/media/products/main/4255‐gram‐100x‐oil‐p5.jpg

Mycobacterial Infection

http://depts.washington.edu/ghivaids/reslimited/case3/discussion.html http://pathologyoutlines.com/wick/tuberculous%20lymphadenitis%20gross.jpg • Patient with cervical lymphadenopathy and subsequent excisional lymph node biopsy Necrotizing granulomatous lymphadenitis

• Zonal pattern –necrosis, epithelioid histiocytes, lymphocytic cuff Osteomyelitis

• Definition ‐ Infection of bone or bone marrow space – hematogenous spread – direct inoculation from trauma • Histologic clues of osteomyelitis – neutrophils, plasma cells, lymphocytes with necrotic bone and reactive new bone formation – fibrosis and capillary proliferation in late stages Actinomycosis

• Cervicofacial disease that causes abscess, fistulae, draining sinus tracts – Actinomyces is normally present in the mouth but causes disease when present in deep tissues of the mouth and jaw – Often associated with dental work, poor oral hygiene, facial trauma, radiation therapy

• Actinomyces osteomyelitis – Rare infection, but commonly occurs in the mandible – Commonly seen in the setting of either osteoradionecrosis or patients http://intranet.tdmu.edu.ua/data/kafedra/internal/stomat_hir/classes_stud/en/stomat/ on bisphosphonate therapy

Actinomyces osteomyelitis

• pleomorphic branching, filamentous, gram‐positive bacteria that frequently aggregate into sulfur granules, exhibit the Splendore‐Hoeppli phenomenon Fungal infections (3 examples)

• Molds are easier (generally) to identify than yeast forms on routine H&E sections – Exceptions – Candida in the esophagus, Coccidiomycosis • Fungi can elicit both acute and granulomatous inflammation • Often associated with tissue necrosis Pulmonary fungal infection

http://library.med.utah.edu/WebPath/LUNGHTML/LUNG030.html http://library.med.utah.edu/WebPath/LUNGHTML/LUNG041.html Coccidiodomycosis

http://www.azdhs.gov/diro/borderhealth/bids/valley‐fever.htm

http://www.webpathology.com/image.asp?case=374&n=2 • Higher magnification shows granulomas with multiple intact thick‐walled, non‐budding spherules 20 to 60 µm in diameter within giant cells. Coccidioidomycosis is endemic in Southwest and western United States. Primay infection is asymptomatic in almost 90% of individuals. Some may develop fever, cough, pleuritis, lung lesions and skin lesions

Aspergillosis

Low power ‐ Fungus ball (aspergilloma) seen in a lung bronchus.

The fungus stains deeply basophilic. High power –radiating hyphae with 45 deg branching and septae http://library.med.utah.edu/WebPath/LUNGHTML/LUNG044.html Esophagitis

http://www.naspghan.org/wmspage.cfm?parm1=698 Candida Esophagitis

http://www.gastrointestinalatlas.com/English/Esophagus/Candida_Esophagitis/candida_esophagitis.html

• Squamous mucosa with superficial debris and fungal organisms invading the mucosa Parasitic infections (3 examples) • Acute inflammation with eosinophils • Often see the parasitic organisms, which are larger than bacteria, yeast • Sometimes no inflammation

Eosinophils

http://www.images.missionforvisionusa.org/anatomy/uploaded_images/WebAller http://www.studyblue.com/notes/note/n/chapter‐14/deck/1325315 gic‐739068.jpg

Liver biopsy

http://edurston.files.wordpress.com/2010/01/hepatosplenomegaly1.jpeg Schistosomiasis S. mansoni in liver tissue

Portal eosinophilic infiltrate with granulomas containing ova with characteristic lateral spine; dense periportal fibrosis

http://dpd.cdc.gov/dpdx/HTML/ImageLibrary/Schistosomiasis_il.htm Adult forms in tissue

http://dpd.cdc.gov/dpdx/HTML/ImageLibrary/Schistosomiasis_il.htm Schistosomiasis Life Cycle Multiple brain lesions

http://www.med.uc.edu/neurorad/webpage/bfa.html • Multiple ring enhancing lesions are present throughout both cerebral hemispheres, with associated marked edema. • Differential diagnosis: metastases, infection, lymphoma Cyst aspiration Brain biopsy Æ Toxoplasma

http://www.ajnr.org/content/25/2/270/F3.large.jpg http://info.fujita‐hu.ac.jp/~tsutsumi/photo/photo126‐8.htm

Multiple toxoplasma cysts (open arrow), probable neuronal cells with round, dark nuclei (arrowhead), and additional trophozoites in the extracellular space after the cysts rupture (straight arrow).

Patient with chronic diarrhea Æ small bowel biopsy Normal Patient

http://en.wikipedia.org/wiki/File:Giardiasis_duodenum_high.jpg http://www.flickr.com/photos/40764007@N08/4072450806/ Giardia lamblia/intestinalis

Light microscopy –Variable villous blunting, possibly increased inflammatory cells, organisms are teardrop (pear) shaped with paired nuclei between villi

Giardia lifecycle Elephant seals –Año Nuevo State Park, Pescadero, CA Part 3

• What happens when you (the pathologist) or the treating physician is worried about an infectious agent but you don’t see an organism? Special (histochemical) Stains • Purpose of special stains is to help identify organisms that are difficult or impossible to identify on routine stains. • Takes advantage of a biochemical property of the organism to create colorimetric reaction – Often uses a counterstain for contrast

• Examples of stains – PAS/PAS‐d – GMS – Alcian yellow/Giemsa – AFB/Fite – Warthin‐Starry – Mucicarmine – Fontana‐Masson PAS and PAS‐diastase stains

• PAS –Periodic acid‐Schiff stain – Stains polysaccharides and glycoproteins and glycolipids – Periodic acid oxidizes the sugar – Schiff reagent imparts a magenta color • PAS‐d (predigestion with diastase) –used for fungal identification – Diastase enzyme digests glycogen inherent to cell cytoplasm – Mucins and glycated compounds should not be affected PAS stain PAS‐diastase stain

Glycogen‐rich urothelial carcinoma Candida esophagitis

Arch Pathol Lab Med 1995; 119:79‐81 http://alf3.urz.unibas.ch/pathopic/e/getpic‐fra.cfm?id=005154 PAS‐d stain of Coccidiodomycosis

http://dermpathexpert.com/imagelib/sitebuilder/misc/show_image.html?linkedwidth=560&linkpath=htt p://dermpathexpert.com/sitebuildercontent/sitebuilderpictures/Coccidioidomycosis/.pond/SI097726Diss eminatedCoccidioidomycosisPASD40x5.jpg.w560h422.jpg&target=_self&title=Coccidioidomycosis

GMS stain • Gomori (Grocott) methenamine silver – stains the polysaccharides of the fungal cell

Aspergillus morphology Blastomyces morphology

http://lh4.ggpht.com/_9CH2B0dPDo8/S5lZ5JI21ZI/AAAAAAAAAbo/4JwOke3gq54/s640/Aspergillis%20pre http://moon.ouhsc.edu/kfung/IACP‐OLP/APAQ‐Images/N1‐IF‐01‐05.gif sumptive%20hawk%20trachea.JPG

GMS stain of coccidiodomycosis

http://dermpathexpert.com/imagelib/sitebuilder/misc/show_image.html?linkedwidth=560&linkpath=htt p://dermpathexpert.com/sitebuildercontent/sitebuilderpictures/Coccidioidomycosis/.pond/SI097726Diss eminatedCoccidioidomycosisGMS40x.jpg.w560h422.jpg&target=_self&title=Coccidioidomycosis

Alcian yellow stain • Alcian stain highlights mucin and the toluidine blue counterstain will highlight surface organisms • Background Æ Pale Blue

http://www.mikrol.ru/news‐media/image‐galleries/specimen‐preparation/life‐science/index.htm http://www.medschool.lsuhsc.edu/pathology/pathist/surgpath/special%20stains/pag es/page2.htm

Acid fast stains

• High lipid content of mycolic acids and fatty acids in the cell wall of certain organisms, which causes them to bind and retain basic dye carbol‐fuchsin even after strong decolorization

• Ziehl‐Neelsen (classic): common method; bacteria stain bright red due to retention of carbol‐fuchsin dye; background is methylene blue counterstain; uses hydrochloric acid to decolorize – Used for Mycobacteria, Cryptosporidia, Isospora

• Fite stain (modified for delicate organisms): combines peanut and vegetable oil with xylene to minimize exposure of cell wall to solvents and protect delicate acid‐fastness of certain organisms. Uses sulfuric acid to decolorize – Used for M. leprae (leprosy) and Rhodococcus Examples of acid‐fast stains

Mycobacterium avium‐complex Cryptosporidium oocysts

http://library.med.utah.edu/WebPath/HISTHTML/STAINS/STAIN018.html http://people.upei.ca/sgreenwood/assets/images/Cryptosporidium_oocysts‐acid_fast‐40X1.jpg 3 year‐old girl, colectomy of for megacystis‐microcolon intestinal hypoperistalsis syndrome Acid‐fast stain –Mycobacterium spp. Warthin‐Starry

• Silver stain that highlights spirochetes as well as

Helicobacter organisms

• Oral Syphilis Oral lesions may represent the

primary stage of infection.

Warthin‐Starry stain highlights

spirochete organsims consistent

with Treponema

http://www.pathology.pitt.edu/lectures/gi/esoph‐b/03.htm Mucicarmine stain

• Stains the polysaccharide capsule

• Histoplasma capsulatum, Candida, and Coccidioides are not detected with mucicarmine

Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #962.

Fontana‐Masson

• Useful in the detection of capsule‐deficient Cryptococcus

• Stain reacts with melanin in the cell wall and is not specific to the capsule

• May stain other dematiaceous fungi

• Most yeast forms have a characteristic thick, polysaccharide capsule

Photo from Indian J Pathol Microbiol, 2011;54(1):216‐8. Immunohistochemistry and In situ hybridization • Helps identify organisms that are: – not easily seen (rare CMV inclusions) – cannot be seen on H&E (EBV, treponema) • Provides serologic confirmation of the organisms – all nuclear inclusions are not the same • Controls must be performed – Positive controls with known organisms – Negative controls to evaluate for endogenous pigments (hemosiderin/melanin) or peroxidases Immunohistochemistry Process

• Start with tissue on an unstained slide • Perform antigen retrieval

http://bitesizebio.com/13392/troubleshooting‐immunohistochemistry/ Immunohistochemistry Process

• Choose primary antibody • Add peroxidase‐conjugated secondary antibody • Add substrate

http://bitesizebio.com/13392/troubleshooting‐immunohistochemistry/ Challenges with Immunohistochemistry

• Incomplete blocking Examples of Immunohistochemistry

Nuclear stain for ER Cytoplasmic stain for keratin Immunohistochemistry for Toxoplasma

http://info.fujita‐hu.ac.jp/~tsutsumi/photo/photo126‐5.htm • Brown staining indicates reactivity for the cysts (left) as well as dispersed tachyzoites in the brain Treponemal immunohistochemistry

• Different chromogens are available

http://wwwnc.cdc.gov/eid/article/6/5/00‐0507‐f2.htm In situ hybridization

• Involves nucleic acid hybridization of complimentary sequences instead of protein‐antibody binding

• Most commonly used to detection of EBV (EBV encoded RNA)

http://www.nature.com/modpathol/journal/v24/n8/fig_tab/modpathol201162f3.html Limitations of surgical pathology

• No, we cannot send for culture • No, we cannot perform sensitivities • Maybe we can perform species identification – PCR from formalin fixed tissues Sheikh Zayed Grand Mosque –Abu Dhabi, UAE Part 4 –Case Examples

• 3 cases where surgical pathology is crucial in making a microbiologic diagnosis 5 year‐old female with history of acute leukemia, on chemotherapy, now with a red lesion in the hard palate

Biopsy is performed and shows inflammation with fungal organisms.

Imaging shows infection involving the facial sinuses and extending to the skull base

ENT plans to perform surgical debridement in the operating room

Frozen section performed

10x power 200x power Permanent section GMS stain Follow‐up • Permanent sections show non‐septate (or sparsely septate) broad hyphae suggestive of Mucor (zygomycosis)

• Zygomycetes invade vascular tissues, commonly causing infarction of the infected tissue. Mortality rates are high. Surgical debridement or surgical resection, as well as antifungal therapy, are usually required.

• Multiple frozen sections were performed on this case as the surgeons tracked the fungus to the skull base. Frozen sections were used to identify the extent of disease and determine where the resection could stop. 56 year‐old female with chronic abdominal pain, refractory to medical therapy • Endoscopy shows non‐specific gastritis • Biopsy is performed to determine the etiology

http://www.gastrointestinalatlas.com/English/Stomach/Gastritis/gastritis.html Biopsy

Normal gastric antral mucosa Area of gastritis – Lamina propria expanded by lymphocytes and plasma cells, foci of active neutrophilic inflammation http://www.pathguy.com/histo/078.htm Immunohistochemistry for Helicobacter spp. Helicobacter gastritis

• Chronic H. pylori gastritis affects 2/3 of the world population – Eradicated with “triple therapy” antibiotics • H. pylori is a strong risk factor gastric cancer • Causes gastric and duodenal ulcers • Causes MALT lymphoma Diagnostic methods

• Non‐invasive – Serology – Stool antigen – Urea breath test with radiolabeled urea • Invasive – endoscopy – Culture of biopsy tissue – notoriously difficult – Rapid urease testing – Histologic examination (considered the gold standard) Case 3

• 75 year‐old female with clinical diagnosis of lobar pneumonia, prior to passing away • Autopsy was performed Lung tissue

PAS‐diastase stain Gram stain Diagnosis: Aspiration pneumonia

• Mixed oral flora – streptococci • Organic plant/grain material present • Strongly highlighted by PAS‐d stain for glycated molecules Thank you for listening!