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)