Role of FNB Cytology in the Diagnosis of Infectious Diseases

Associate Professor Andrew FieldNotre Dame Medical School, Sydney

Binford Dammin Society and Papanicolaou Society of Cytopathology Joint Companion Meeting

USCAP, San Diego, 2014

Acknowledgements for microphotographs and cases for photography

• Dr William Geddie, Toronto, in ‘Cytohistology of Lymph Nodes’, by A.Field and W.Geddie, Cambridge University Press, 2014: based on an algorithmic approach to FNB of lymph nodes including infections

• Dr Pamela Michelow, NHLS, Johannesburg• Dr Matthew Zarka, Mayo Clinic, Phoenix• Dr Daneshod, India• Dr Peter Poganyema, Budapest• Dr Hanna, Iran

Role of FNB in diagnosis of infectious diseases

• FNB has been widely used in cases where the FNB is done to diagnose malignancy but infection is found

• Now, increasing use of FNB where infection is the most likely diagnosis, partly due to an increase in immune compromised patients including cancer, transplantation and HIV eg. skin, lymph nodes, lung

• And FNB increasingly used in developing countries, where infection is the most important cause of morbidity and death, leading to a massive increase in the role of FNB in the diagnosis of infections

Benefits of FNB in diagnosis of infections

• Minimally invasive, inexpensive, well tolerated by patient, as an outpatient or clinic procedure

• Provides rapid accurate and often specific diagnoses including infections that cannot or difficult to culture

• Provides immediate triage for selection of the most diagnostic and cost effective ancillary tests

• Provides material for full range of ancillary tests

Role of FNB in diagnosis of infectious lesions in developing countries

• FNB is a very powerful tool in medically resource poor countries with under resourced histopathology and few microbiology labs, and huge infectious disease loads

• Eg young adult with mid anterior cervical neck mass often treated with anti TB treatment, on spec. But FNB can DD TB/granulomatous lymphadenitis, reactive LN, high grade lymphomas including Hodgkins, Burkitts and DLBCL, metastatic carcinomas and a branchial cyst with just a Giemsa stained smear done at the bed side

• FNB empowers the clinician to make the correct diagnosis to benefit the patient and the medical system

• Role of FNB accentuated by the AIDS epidemic, as in developed countries

Place of FNB in diagnostic sequence• FNB ideally suited with palpable or US or CT detected masses to

be the first procedure, especially if the integration of history (including Immune compromised/HIV) and physical findings suggest an infectious lesion

• In deep lesions located by US or CT, or EUS or EBUS, FNB with ROSE is the first line diagnostic approach, supplemented by core biopsy

• Role dependent on site and experience of pathologists

• ALL LESIONS SHOULD BE CONSIDERED AS POSSIBLE INFECTIOUS LESIONS AND A ROUTINE TECHNIQUE SHOULD BE USED TO MINIMIZE THE RISK OF INFECTION TO THE OPERATOR and ROSE used to TRIAGE THE CASE

FNB of specific sites in possible infectious cases

• Head & Neck: FNB is first diagnostic procedure; site suggests DD

• Preauricular, upper cervical, submental: acute or chronic sialadenitis, salivary gland neoplasm, LN lesions (TB, reactive, metastasis, specific infections); rapid staining and assesssment to triage for selection of ancillary testing

• Midcervical: LN lesions, branchial cysts, rarely paragangliomas

• Lower cervical and midline: thyroid (including uncommon viral, TB, fungal infections), LN lesions, rare parathyroid

• Posterior triangle: LN lesions, schwannomas• Occipital: LN lesions (including Toxoplasmosis),

trichilemmal cysts

Thyroid infectious lesions• Subacute granulomatous thyroiditis• Bacterial: suppurative• Viral• TB• Fungal: rare cases of Aspergillus spp, Candida spp,

Hisptoplasma capsulatum, Cryptococcus neoformans, Coccidiodes, Pneumocystis jirovecii, and Pseudallescheria

FNB of skin and palpable subcutaneous lesions

• Pyogenic abscesses, metastases, TB, Kaposi’s sarcoma (HIV)• Superficial and deep fungal infections: mycetomas

(Phialophera parasticus, Sporothrix schenkii, Cladosporium spp, Exophiala jeanselmei, Microsporum canis) and disseminated (Candida spp, Aspergillus spp, Cryptococcus neoformans, Histoplasmosis, Blastomycosis)

• Actinomycetomas• Rhinosporidiosis (protozoan rather than fungus)• Filariasis, Myaisis, Leishmaniasis, Leptosporosis• Leprosy (tuberculoid, borderline, lepromatous, plus neuritic

and cystic lesions)• Postop cellulitic wound and skin infections and Buruli ulcer

Endoscopic Bronchial Ultra Sound • EBUS: standard procedure for initial diagnosis of central lung lesions and

LN adjacent pharynx, trachea, bronchi and mediastinum• EUS: standard for pancreatic and hepatic lesions, and LN adjacent

esophagus, stomach, duodenum, porta hepatis, pancreas, celiac plexus and rectum

• EBUS stages lung cancers by assessing posterior mediastinal LN including subcarinal nodes and left and where accessible right adrenal

• FNB supplemented by core biopsy, plus diagnostic and prognostic immunohistochemistry and molecular studies eg EGFR and KRAS.

• FNB with direct real-time US of LN: US accuracy 80% in detecting malignancy or other abnormality, but with cytology, close to 100% and allows diagnosis of lesion

EBUS: Advantages• Minimally invasive and cost effective outpatient procedure • Diagnose malignancy and infections in previously inaccessible LN in

mediastinum in upto 100% and apply molecular testing, PCR and cultures• Avoid mediastinoscopy: invasive, general anaesthesia, higher cost, higher

complication rate, only anterior mediastinum• CT guided percutaneous FNB only for peripheral lung lesions• Reduce unnecessary thoracotomies for inoperable lung cancers by 60%:

restrict thoracotomy to lung cancers with ipsilateral hilar or FNB negative LN

• Diagnose inflammatory or infectious causes of mediastinal lymphadenopathy (sarcoidosis and mycobacterial and fungal infections) particularly in immune compromised patients

• Complication rate: less than 0.1% (bacteremia, abscess formation, striking larger vessels, hemorrhage) especially elderly

EBUS: Disadvantages• Cost of the equipment and training respiratory physician • Requires careful handling of specimen: very important as the length

of the needle and time in the LN can produce FNB specimen clotting • Technique: once in the lymph node the stylet is withdrawn and the

needle moved rapidly backwards and forwards 5 to 10 times, through LN node and any abnormality with MINIMAL ASPIRATION (minimize blood contamination; aspiration increases blood contamination, and is of doubtful benefit in increasing material)

• ROSE: establish adequacy and provide provisional diagnose to triage for cost effective selection of ancillary testing eg cultures, RPMI for flow cytometry and cell block for IP

ROSE at EBUS FNB: granulomas• At ROSE granulomas needs extra passes for:• Direct smears for ZN and GMS stains• Cell block, culture, PCR for mycobacteria and fungi • Flow cytometry (exclude lymphoma in cases associated with

follicular hyperplasia)• Cell block IHC for keratins, melanoma, HL markers• FNB of mediastinal lymph nodes with ROSE in the clinical

and CXR setting of possible sarcoid, can replace core biopsies and transbronchial biopsies of lung in the diagnosis of sarcoid (recent published research from St Vincents)

Lung• Percutaneous FNB for peripheral masses and EBUS for

more common central lesions and range of infections• Tuberculosis• Gram negative (especially IC) and gram positive

abscesses• Fungal (Aspergillus spp, mucormycosis, cryptococcosis,

histoplasmosis, Pneumocystis jirovecii, coccidioidomycosis, paragonamiasis)

• CMV• Parasites (amebiasis, hydatid disease, microfilariasis)

FNB of Lymph Nodes: low power pattern recognition & high power cell type assessment:

At LP use pattern recognition then HP cell type assessment ask THREE QUESTIONS:

1. Are there tissue fragments present? 2. Are the dispersed cells lymphoid? 3. If the dispersed cells are lymphoid, then using cell type assessment, do

small lymphocytes predominate? Notea. ‘small lymphocytes’: benign reactive LN and does not refer to ‘small

lymphoid cells’ of SLL/CLL, MCL, FL etc.b. ‘tissue fragments’: fragments of epithelial cells of metastatic carcinomas

or follicle germinal center tissue fragments of reactive germinal centers or follicular center cell tissue fragments of follicular nodules in FL, or granulomas in TB, fungal infections or sarcoid, or granulation tissue fragments in suppuration or rarely epithelial and nevoid cell inclusions

Lymph Node: Suppuration & Suppurative Granulomatous

(a) Benign (Histology: suppurative lymphadenitis, with/without specific infection)

i. Acute suppurative lymphadenitisii. Filariasisiii. Pneumocystis jiroveciiv. Cat scratch diseasev. Tularemiavi. Lymphogranuloma venereumvii. Fungal lymphadenitisviii. Leishmaniasis

(b) Malignant (Histology: Suppuration with metastatic carcinoma)i. Rare metastatic carcinomasii. Rare Hodgkin Lymphoma

Lymph Node: Granulomatous Pattern(a) Benign: (Histology: Granulomatous lymphadenitis with/ without specific infection)

i. Mycobacterial lymphadenitisii. Sarcoidosisiii. Foreign bodyiv. Fungal infectionsv. Schistosomiasis

(b) Malignant: (Histology: Malignancy with a granulomatous reaction)

i. Granulomatous reaction to metastatic carcinoma, most typically squamous cell carcinoma

ii. Granulomatous reaction to lymphomas and Hodgkin lymphoma

Liver• Suppurative/pyogenic abscesses

(gram negative eg Klebsiella spp, Escherichia coli, and staphylococcus and streptococcus)

• TB abscesses• Amebiasis• Hydatid disease (? Steroid, albendazole cover)• Fungal (including cryptococcus, Aspergillus spp,

Candida spp)

FNB of other sites• Axilla, femoral, inguinal: LN lesions, including suppurative and

granulomatous infections (Lymphogranuloma venereum and Granuloma inguinale in inguinal region), Kaposi’s sarcoma in HIV population

• Breast: first procedure for abscesses, suppurative (bacteria) and granulomatous (brucellosis, TB). DD: rare necrotizing carcinomas with suppuration

• Testis and scrotum: TB orchitis and epididymitis• Bone, skeletal muscle and soft tissue: eg. osteomyelitis

(suppurative or TB), psoas and para vertebral abscesses, discitis• Spleen: TB, leishmaniasis, leptospirosis VS lymphoma, cysts• Cerebral cortex: FNB via burr holes or brain squashes:

toxoplasmosis, CMV, fungal VS primary or metastatic tumors• Eye: choroiditis, uveitis and retinitis; culture, PCR

Technical considerations 1

• Every FNB should be approached on basis that may be infectious

• Best guide is history and clinical examination supplemented by imaging

• Each FNB operator should have single safe technique for all FNB; gloves, gowns, glasses, adequate space, double gloving in HIV cases; ‘down the syringe’ technique for needle removal.

Technical considerations 2• Cystic or suppurative lesions: closed system of needle,

syringe, syringe holder/aspirator• Needle by itself: more sensitive, solid lesions, LN, skin

(bent needle); repeat FNB of residuum after ‘cyst’ aspiration

• 23, 25 or 27G (22G helps in draining and sampling abscesses eg breast, lymph nodes, spleen, assisting treatment)

• Culture diagnostic yield is greater with more material ie. second complete pass rather than rinsing needle and syringe

Technical considerations 3• Rapid on site evaluation (ROSE) or immediate

provisional reporting: clues from clinical history and site and the macroscopic smear features eg pus: creamy, green with offensive odour and slippery smear

• But immediate airdrying with Giemsa staining essential to triage case for cost effective selection of ancillary tests AND immediate diagnosis of viral cytopathic effects (eg CMV, herpes) and organisms that cannot be cultured (eg Pneumocystis jirovecii)

Technical considerations 4• Smearing technique to split the sample onto several slides• Both airdried Giemsa (for immediate assessment, and better

for bacteria, fungal hyphae, yeasts, negative image of mycobacteria, background, LN) and alcohol fixed Pap stains (better for nuclear details, CMV)- no single stain perfect

• Extra airdried slides for auramine, ZN, Gram, GMS• Material for cultures (mandatory), PCR, direct IF, flow

cytometry, gene rearrangement studies, phylogenetic analysis of nuclear subunit RNA sequence alignments and EM

• Destained Pap or H&E can be used for GMS, DiPAS• Repeat FNB is a simple procedure, but requires patient recall

Technical considerations 5: Cell Block • Produce a cell block in every FNB from normal saline rinse

of needle and syringe or from separate FNB pass • H&E, DiPAS, GMS, ZN, auramine• IHC (full range of markers for metastases, lymphomas ) • EM (eg microsporidia) on cell block or material placed

directly into glutaraldehyde for resin embedding• Direct inoculation of FNB pass into culture bottles especially

for TB and anaerobic bacteria in liver abscesses• Pus with confirming cultures, or granulomatous LN with

positive ZN is adequate, but ‘necrosis’ or pus with no confirmed organism is not adequate to exclude malignancy eg FNB lung yielding pus is not sufficient to exclude SCC or other malignancy

FNB Suppurative Pattern Criteria 1• Creamy grey viscous, smoothly smeared • Proteinaceous granular or amorphous sometimes stringy or

fibrinous background with shadowy degenerate cells• Variable, usually plentiful neutrophils showing degrees of

degeneration• Variable number of histiocytes, macrophages and

lymphocytes, depending on site• Target site components eg. epithelium infiltrated by

neutrophils with varying epithelial atypia• Bacteria and fungi in background or in macrophages• Viral cytopathic effects in nuclei• Other: fragments of hair in pilonidal sinus abscesses;

vegetable matter in lung aspiration pneumonia

FNB Suppurative Pattern Criteria 2• Suppuration common in FNB of LN and other sites in

children/young adults; lung and all sites including skin in IC• Usually associated with Streptococcus spp, Staphylococcus

aureus, Gram negatives including Klebsiella spp and Echerichia coli, and fungi such as Aspergillus spp, cryptococcus and Candida spp, especially in IC

• In immune compromised patients especially with neutropenia FNB may yield only a thin proteinaceous background with some histiocytes and the organism has to be found

• In FNB of HIV patients LN, mycobacteria may be present in the background serum and macrophage cytoplasm as negative images, with scant neutrophils

• Catscratch and some fungal infections in LN FNB produce apoptotic necrosis or suppurative granulomas and histiocytes

FNB Suppurative Pattern 3• Overdiagnosis of malignancy can occur with epithelial atypia

in suppuration: look for acute inflammation, low N:C ratio, uniform nuclear changes and nucleoli rather than pleomorphism (enlarged nuclei, hyperchromatic but not irregular chromatin, prominent nucleoli, ‘predictable’ cell to cell) eg subareolar chronic abscesses with squamous metaplasia; mid cervical inflamed branchial cysts.

• Diagnosis of malignancy requires malignant features in well preserved epithelial nuclei

• Underdiagnosis of malignancy also can occur eg. necrotic metastatic carcinoma in cervical LN associated with inflammation

Granulomatous Pattern(a) Benign:(Histology: Granulomatous lymphadenitis with/ without specific infection)

i. Mycobacterial lymphadenitisii. Schistosomiasisiii. Sarcoidosisiv. Foreign bodyv. Fungal infections

(b) Malignant: (Histology: Malignancy with a granulomatous reaction)

i. Granulomatous reaction to metastatic carcinoma, most typically squamous cell carcinoma

ii. Granulomatous reaction to lymphomas and Hodgkin lymphoma

FNB Granulomatous Pattern Criteria Key cytological criteria• Low to moderate cellularity• Epithelioid histiocytes (elongated/bent nuclei, fine

chromatin, small nucleoli, low N:C, pale copious cytoplasm) singly or in granulomas

• Lymphocytes and MNGC in variable number• Neutrophils in some cases• Necrosis in some cases• Fungi on GMS• Mycobacteria on ZN or auramine or IF of Pap stain• Culture mandatory, PCR, sensitivity testing

M.tuberculosis• FNB has become the first diagnostic test for cervical LN and EBUS

for mediastinal LN or lung lesions in endemic regions or where there is high clinical suspicion

• Diagnosis based on:1. presence of epithelioid granulomas, or 2. presence of epithelioid granulomas with caseous necrosis, or 3. presence of caseous necrosis without granulomas, or 4. mycobacteria are seen on ZN stain• FNB provides material for cultures, drug sensitivities, PCR, and

excludes other specific infections (leishmaniasis, fungal infections), as well as, HL, high grade lymphomas (Burkitt Lymphoma) and metastatic carcinomas

• FNB limitation: not all cases will show granulomas, caseation or mycobacteria. AFB present in Ziehl-Neelsen or variant stains (Khinhoum, modified Wade-Fite), in up to 70% of cases with necrosis (less with granulomas)

Diagnosis of M.tuberculosis• Culture traditionally ‘gold standard’ but are positive in

around 70% of cases and rely on adequate specimens and laboratory growth is slow

• Combining FNB cytology, ZN, cultures and where necessary core biopsy can make the diagnosis in upto 95% of cases

• Routine ZN or variant• Autofluoresence of Pap stained mycobacteria increases

sensitivity (wide band blue excitation filter (450-480nm))• PCR (slightly higher sensitivity than culture) is relatively

expensive and requires significant laboratory infrastructure, but is useful diagnostic adjunct if available.

• Other PCR tests showing great promise

FNB in diagnosis of Mycobacterium leprae

• Endemic in developing world• FNB highly diagnostic (blanched skin technique),

with Fite stain and culture DD cutaneous TB with its epithelioid granulomas and caseous necrosis

• FNB can classify and correlate with Ridley and Jopling histo classificaton

• Nerve abscesses/neuritic leprosy occur

FNB in diagnosis of Mycobacterial leprae 2

• Spectrum of lesions:• Tuberculoid: mod/high cellularity, epithelioid

granulomas, plentiful lymphocytes, few AFB• Borderline: moderate cellularity, histiocytes, AFB, many

lymphocytes• Lepromatous: marked cellularity, foamy macrophages,

neutrophils, lymphocytes, plentiful AFB• Reactional: high cellularity, foamy macrophages,

negative image bacilli, neutrophils, foamy background

FNB fungal infections

• Granulomatous or suppurative granulomas or even suppuration alone in IC and HIV patients, with negative image or variably stained fungi in Giemsa and Pap, depending on fungus and degree of degeneration

Cryptococcosis• Key Cytologic Features• Suppurative pattern: plentiful neutrophils admixed with mixed

lymphoid cells• Varying degrees of suppurative necrosis and granulomatous

inflammation with distinct granulomas and histiocytes in some cases• 5 to 15 micron yeasts, mucicarmine positive, Giemsa negative

mucopolysaccharide ‘halo-like’ capsule and Giemsa positive body and surrounding serum

• Narrow necked budding• Autofluoresence• Immune compromised patients may lack the capsule (DD

histoplasmosis) and development of well formed epithelioid granulomas

• Neutropenic patients may lack neutrophils

Histoplasmosis• Histoplasma capsulatum: endemic in central USA, central and

southern Americas, Ganges river region, central and western Africa, and disseminates to lung, LN, breast, adrenals, spleen, subcutis

• Mediastinal lymphadenopathy with calcifications mimics TB and carcinoma

• Dimorphic fungus, has 3 to 5 micron oval pale yeasts with single, variable narrow-based budding

• Positive in the GMS, and negative in the Giemsa and Pap stains (negative image in the cytoplasm of macrophages

• DD also includes leishmaniasis with its amistigotes.

Aspergillosis• Aspergillus fumigatus and other species: worldwide, commonly

found in upper respiratory tract and paranasal sinuses, lung and mediastinal lymph nodes and disseminates especially in immune compromised to skin, thyroid and central nervous system.

• Key Cytologic Features:• Acutely branching septate hyphae around 7 microns in diameter • Aggregates of hyphae: very weakly staining in Pap and Giemsa

(due to degeneration) or as negative images amid necrosis or suppuration; stain well in the Grocott Methenamine Silver

• Suppurative with or without granulomas

Specific fungal infections FNB 3

Candidiasis (mainly C.albicans): WW, gut, skin commensal, disseminates in IC (AIDS defining);

• dimorphic buddding 3 to 6 micron yeast, and thin pseudohyphae, rare true hyphae; Giemsa, Pap, GMS

Dermatophytes (Microsporum, Trichophyton): WW, parasitic fungi of skin and hair; subcutaneous mycetomas, branching hyphae, GMS, suppuration

• Pneumocystis jirovecii

Pneumocystis jirovecii• WW• pneumonia especially in AIDS, disseminates to

LN, spleen, BM; • 5 to 8 micron cupshaped cysts, faintly birefringent, • dark green nuclei in Pap, black comma shaped

single or double nuclei in GMS; • foamy eosinophilic (Pap) or purple (Giemsa)

aggregates contain cysts; cannot culture; DD RBC from cysts in GMS

Pneumocystis• Key Cytologic Features• Variable cellularity• Lymphocytes, plasma cells, histiocytes, often suppurative• Irregular frothy aggregates of cysts • Rounded and helmet-shaped 5 to 8 micron cysts with faintly

birefringent walls: stain poorly in the Giemsa and Pap and strongly in the GMS stain; nuclei green in the Pap and black in the GMS

• False positive diagnosis of red cells in overheated GMS • P. jiroveci cannot be cultured: diagnosis relies on cytology

assisted by PCR, but P.jiroveci can be found in normal lungs

Blastomycosis• Blastomyces dermatiditis: endemic in USA, especially

the southeastern parts, Canada and Mexico, Middle East, Africa and India

• Causes pneumonitis, spreading to bones, skin, thyroid, genitourinary tract, CNS and spleen

• Dimorphic fungusKey Cytologic Features:

• Suppuration with or without necrotizing granulomas• 8 to 20 micron diameter, broad-based budding yeasts,

with thick Pap and GMS positive, faintly birefringent walls

Giemsa

Blastomycosis

broad based budding

GMS Blastomycosis

Coccidiodomycosis• Coccidioides immitis: endemic in southwestern USA, Mexico,

Central and southern Americas, and disseminates to lung, LN, skin, soft tissue, bones, joints, and meninges

• Key Cytologic Features:• Suppuration with or without granulomas and eosinophilic debris• Refractile, thick-walled spherules or cysts, 20 to 120 micron in

diameter, containing multiple 2 to 4 micron endospores• Pap, GMS and DiPAS positive spherules• Rarely coccidiodomycosis can form thin branching septate

hyphae. • Plant contamination should be excluded.

Giemsa

40 micron spherule in Coccidioidomycosis

PPa p 50 micron spherule

Papanicolaou

stain:

spherule in Coccidioidomycosis

with endospores

FNB in specific fungal infections

• Sporotrichosis (Sporothrix schenkii): India, Sth Africa, Australia, Americas; ‘rose thorn disease’ in skin, disseminates in IC to lung, bone, CNS, joints;

• oval to cigar yeasts in macrophages and background necrosis and suppuration; GMS, Pap

Pap Sporotrichosis

oval yeasts in

macrophage

Giemsa

Sporotrichosis

oval yeasts in macrophage

GMS Sporotrichosis

oval yeasts

FNB of parasites 1• Filariasis (Dirofilaria immitis and Wucheria bancrofti):

WW, esp tropics, India, Nepal, Polynesia; • skin, thyroid, epididymis, spermatic cord, LN, breast;

pulmonary nodules with D.immitis in USA;• slender coiled microfilaria• gravid female worms containing eggs and free eggs; • MNGC, eosinophils, and epithelioid histiocytes adherent

to filaria; Pap, Giemsa• Paragonimiasis (Paragonimus spp): Asia; lung nodules;

adult worm with species specific eggs; suppuration; Pap, Giemsa

FNB of parasites 2Leishmania spp: Mediterranean, near East, Asia; • cutaneous or visceral, especially in IC to spleen,

LN, liver, eye, breast; • 1-2micron amistigote containing nucleus and rod-

like kinetoplast, in macrophages, neutrophils or free;

• granulomas, or suppuration with secondary infection; Giemsa, Pap.

• DD: TB, toxoplasmosis

Leishmaniasis• Key Cytologic Features• Moderate to high cellularity• Variable suppurative pattern with epithelioid granulomas

or single histiocytes, MNGC, plasma cells, mast cells, with amistigotes in the background

• Plentiful rounded macrophages with central nuclei and cleared cytoplasm containing plentiful blue (G) amistigotes each with a pink rod shaped kinetoplast; the Pap stains the amistigotes to a lesser degree

• Occasional large dendritic cells with plentiful amistigotes in cytoplasm

• Suppuration especially in children, who present with painful cervical lymphadenopathy.

FNB of parasites 3• Toxoplasmosis (Toxoplasma gondii): coccidian

protozoan; WW • unilateral cervical LN, pneumonitis, encephalitis,

congenital transmission• follicular hyperplasia, tiny syncytial granulomas,• rare 3-4micron crescentic tachyzoites with dot

nuclei or similar bradyzoites in round cysts• Giemsa: tachyzoites autofluoresce; IP stains (no

culture) • DD lymphoma by flow cytometry

FNB of parasites 4Hydatid disease (Echinococcus granulosus): sheep

farming in Sth Africa, Australia, central Europe, USA, New Zealand, Sth America

• lung, liver, CNS, kidney, spleen, soft tissue, salivary glands

• scolices with refractile hooklets, and fragments of laminated thick wall,

• granular background with granulomas or suppuration, or secondary infection

Cyticercosis (Taenia solum

FNB of parasites 4Hydatid disease (Echinococcus granulosus)Cyticercosis (Taenia solum): tropics, SE Asia, India• skin, muscle, eye, CNS, tongue soft tissue swelling• 1cm cyst with single 1mm scolex with 130-

170micron hooklets, larval cuticle, calcareous corpuscles

• eosinophils, neutrophils, histiocytes, MNGC, granulomas DD hydatid disease with multiple scolices

FNB of parasites 5Rhinosporidiosis (Rhinosporidium seeberi):

tropical; nose, conjunctiva, trachea, nasopharynx; • large spherules/sporangia containing multiple

7micron endospores; • granulomatous; Pap, Giemsa; no cultureEntamoeba histolytica: tropics; GIT, liver,

disseminated; • round/oval basophilic 18-20 micron trophozoites,

with single nucleus and ingested red blood cells; • suppuration, abscesses with scant amebae; Pap,

Giemsa, DiPAS, IF

Schistosomiasis• Second commonest tropical disease to malaria in the world• Flatworms (blood flukes) Schistosoma hematobium (urinary tract disease),

Schistosoma mansoni and Schistosoma japonicum (intestinal disease)• Freshwater snail host• Cercariae (worm stage) invade the skin, travel through lymphatics and blood

to the lungs, and then eggs disseminate producing delayed hypersensitivity granulomatous response, usually in GIT or genitourinary tracts (DD is often colorectal or bladder cancer, respectively)

• Schistosomiasis causes pericolic and mediastinal LN• Granulomatous reaction containing eggs in various stages of degeneration;

suppuration only with secondary infection

Myaisis

• Insect vector (eg botfly) injects eggs that mature into larva in human host skin

• Eg cutaneous myaisis due to Dipteran

FNB of other bacteriaActinomyces spp: WW; • anaerobic commensal in oropharynx, GIT, female genital

tract; intraabdominal with surgery, stents, EUS; breast, pancreas, subcutis (actinomycetoma);

• acutely branching, filamentous, nonseptate 1-1.5micron diameter aggregated filaments;

• suppuration with “sulfur granules” or cotton “wool clusters”; Giemsa, Gram and GMS positive, AFB negative

Nocardia spp

FNB of other bacteria• Actinomyces spp:

• Nocardia spp: WW; • pulmonary and disseminates to subcutis, CNS; • right angle branched 0.5-1mm diameter, 10-

20micron long filaments; • suppuration, necrosis; • Giemsa, ZN positive and auramine (yellowgreen

IF)

Giemsa

Nocardia

ZN Nocardia

FNB of other bacteriaCatscratch disease (Bartonella henselae): cervical or axillary

LN plus systemic in IC; • bacilli on Warthin Starry• apoptotic necrosis, epithelioid granulomas +/-suppuration; • Bartonella quintana causes bacillary angiomatosis in HIV in

skin

Buruli ulcer (Mycobacterium ulcerans): rural Africa, Asia, Americas, Australia;

• skin, subcutis, bone; • AFB similar to TB, ZN, auramine IF • suppuration +/- granulomas with ulceration

FNB of virusesCytomegalovirus: WW; • GIT, eye and in IC disseminates to lung, kidney,

CNS, pancreas, salivary gland• nuclear ‘owls eye’ inclusion with marginated

chromatin, AND coarse cytoplasmic granular inclusions

• suppuration • Giemsa and Pap (nuclear inclusion); IP• Cytolytic effect immediately diagnostic

Herpes simplex

FNB in Immune Compromised Patients 1• High level of suspicion of infectious lesions in transplant

and oncology patients, long term steroids, chronic auto immune diseases, and HIV (pre HAART and now)

• Emerging, opportunistic and multiple (eg lung FNB may reveal CMV and aspergillus and bacteria) infections

• Typical infectious patterns representing interplay between infection and immune state, may not be present; culture is mandatory

• Routine protocols should include extra slides for ZN, Grams, GMS

FNB in Immune Compromised Patients 2

• Flow cytometry and cytogenetics for atypical lymphoid material

• Disseminated fungal infections to skin and LNs (Aspergillus spp, Candida spp, cryptococcus)

• Negative image mycobacteria in background serum and macrophage cytoplasm

• Bacillary angiomatosis (Bartonella henselae and B. quintana) produces spindle cells, neutrophils and histiocytes; Warthin Starry stains organism

• Kaposi’s sarcoma in HIV patients: HHSV8 infection in endothelial cells

FNB diagnosis of Kaposi’s sarcoma 2

• Mycobacterial infections can coexist in LN• Commonest AIDS defining tumour, now less

common in LN and skin in HAART patients (SCC and Lymphomas)

• Human herpes virus-8 infected endothelial cells• LN shows subcapsular rim of KS, with LN

showing varying stages of HIV disease: follicular hyperplasia to involution with plasma cells, few lymphocytes in watery background

FNB diagnosis of Kaposi’s sarcoma

Cytological diagnostic criteria:• Low to high cellularity• Irregular tissue fragments haphazardly arranged spindle cells• Elongated, blunt ended spindle cells, with mildly

pleomorphic hyperchromatic nuclei with small nucleoli• Poorly defined cytoplasm in tissue fragments, delineated by

pink stroma in Giemsa• Single spindle cells in background• Plasmacytoid lymphocytes, plasma cells, tingible body

macrophagesTissue fragments resemble epithelioid granulomas, but spindle cells lack indented nuclei seen in epithelioid granulomas and no MNG

Role of FNB in diagnosis of infections:• Crucial role in developed countries and great potential as a

diagnostic tool in infrastructure-poor medical systems• Diagnosis of primary and secondary malignancies in breast,

thyroid, skin, lymph nodes and bone and soft tissues, and liver and other deeper sites with imaging

• Diagnosis of endemic, emerging and opportunistic infections, including pyogenic bacteria, TB, fungi, cat scratch, toxoplasmosis, disseminated aspergillus, rhinosporidiosis, cryptococcosis and leprosy

• Provides material for ROSE, cultures, drug sensitivity and ancillary tests (flow cytometry, cytogenetics)

Role of FNB in diagnosis of infections:

• Diagnosing infections, Kaposi’s sarcoma, high grade lymphomas and metastatic carcinomas in HIV, oncology and transplant patients,

• Minimal equipment: needles, syringes, syringe holder, staining jars and Giemsa or H&E or PAP stains

• FNB needs only basic laboratory space and easily transported equipment to city and rural clinics

• FNB is rapid, inexpensive and well tolerated by patients• The major challenge in establishing FNB services is to

train cytopathologists and cytologists

Acknowledgements for microphotographs and cases for photography

• Dr William Geddie, Toronto, in ‘Cytohistology of Lymph Nodes’, by A.Field and W.Geddie, Cambridge University Press, 2014: based on an algorithmic approach to FNB of lymph nodes including infections

• Dr Pamela Michelow, NHLS, Johannesburg• Dr Matthew Zarka, Mayo Clinic, Phoenix• Dr Daneshod, India• Dr Peter Poganyema, Budapest• Dr Hanna, Iran