Introduction

The widespread use of FNB for both palpable and impalpable lesions where the clinical and imaging differential diagnosis (DD) includes malignancy and sometimes infection has led to the diagnosis of infectious lesions. But with the increase in the number of immune compromised (IC) patients in developed countries, particularly due to the HIV epidemic and cancer therapy, FNB increasingly is used to assess skin, lymph node, lung and other sites where infection is the main diagnostic expectation.^1,2 FNB can make the diagnosis of many specific infections on cytomorphology and special stains, and it also delivers material for the full array of ancillary studies including cultures, PCR and DNA analysis of other infections. The rapid diagnosis of infections, some of which cannot be cultured, can facilitate immediate treatment.

Now, with the gradual spread of FNB into the developing world where infections are the leading cause of morbidity and mortality, there has been a rapid if patchy increase in the use of FNB to make specific infectious diagnoses. FNB is minimally invasive, inexpensive, acceptable to patients, provides a rapid accurate diagnosis and immediate triaging for the most cost-effective selection of special stains and ancillary tests, and provides material for culture and the full range of ancillary tests. FNB is a very powerful diagnostic tool, especially where surgical resources and histopathological and microbiological laboratories are in short supply and grossly underfinanced.³ A cervical lymph node in a young adult that would, in subSaharan Africa, be treated expectantly as TB, can have an FNB that may show tuberculosis, metastatic carcinoma, lymphoma, reactive lymph node or branchial cyst. The FNB empowers the clinician to make the correct, often infectious, diagnosis, provides the best possible outcome for the patient and saves the medical system – which may be severely stressed by the AIDS epidemic, patient numbers and inadequate funding – the cost of surgical biopsy, hospital bed occupancy and inappropriate treatment

Place of FNB in the investigative sequence

The role of FNB is dependent on the body site and the facilities and expertise of personnel at particular institutions. In both the developed world and the four-fifths of the world where medical resources are limited, an inexpensive outpatient FNB can triage and, in the vast majority of cases, make a definitive diagnosis of any palpable lesion. FNB is the most efficient, least invasive and most cost-effective investigative procedure, particularly in cases where an infectious process is expected based on an integration of patient history and examination. This is exemplified by patients with suspected or known immune deficiency due to cancer, therapy or HIV disease.^4,5

When dealing with imaging-detected deep lesions and impalpable superficial lesions, percutaneous FNB using ultrasound or CT guidance and endoscopic and endoscopic bronchial ultrasound-directed FNB are the mainstay of tissue diagnosis, supported when necessary by core biopsy. As interventional radiology, especially ultrasound, extends into the developing world, FNB of impalpable lesions will increase.

Specific sites

In the head and neck region after completion of a history and examination, which may include endoscopy of the upper aerodigestive tract, FNB of palpable lumps is the first line of investigation in all patients, especially the IC. The exact site of the lesion suggests the DD. In the preauricular, upper cervical and submental regions, acute or chronic sialadenitis,^6,7 salivary gland neoplasms and a full range of lymph node lesions including reactive nodes, metastatic tumor, mycobacterial infection, lymphoma or other specific infection have to be considered. Rapid staining and immediate assessment of FNB slides is very powerful in this setting to direct selection of ancillary testing.

In the mid-cervical region, lymph node lesions including metastatic carcinoma, possible lymphoma, mycobacterial infections and non-specific infections are most common, but paragangliomas and large, often inflamed, tender and even fixed branchial cysts should always be considered.

In the lower cervical and adjacent midline region, lesions of the thyroid, lymph nodes and rarely the parathyroids form the differential diagnosis. Specific infections of the thyroid due to tuberculosis, viruses and, less commonly, Aspergillus spp, Candida spp, Histoplasma capsulatum, Cryptococcus neoformans, Coccidioides, Pneumocystis jirovecii blastomycosis and Pseudallescheria have been reported in increasing numbers.⁸ In the posterior triangle lymph nodes and schwannomas, and in the suboccipital area lymph nodes with specific infections including toxoplasmosis, and trichilemmal cysts and other skin lesions need to be considered.

In the axilla, femoral and inguinal regions lymph nodes may contain metastatic tumor, suppurative or granulomatous infections, non-specific reactive processes and lymphomas. Lymphogranuloma venereum and Granuloma inguinale should be considered in the groin.

Skin nodules and palpable subcutaneous lesions are easily sampled by FNB. Pyogenic abscesses and infections such as tuberculosis⁹ are easily differentiated from tumor metastases, and, in the HIV population, from Kaposi’s sarcoma. Both superficial and deep fungal infections can be diagnosed by FNB and, especially in the IC, may represent disseminated fungal infection due to Aspergillus spp, Cryptococcus neoformans, histoplasmosis, blastomycosis,¹⁰ sporotrichosis and Candida spp,¹¹ or one of the increasing number of other reported fungal infections producing subcutaneous nodules or mycetomas, including, Phialophera parasiticus, Sporothrix schenkii, Cladosporium spp, Exophiala jeanselmei¹² and Microsporum canis.¹³ Actinomyces producing actinomycetomas, and rhinosporidiosis,¹⁴ filariasis,¹⁵ myaisis,¹⁶ leishmaniasis and leptosporosis all occur in the skin. The tuberculoid, borderline and lepromatous variants of leprosy, as well as neuritic and cystic lesions, can all be diagnosed.¹⁷ FNB of cellulitic skin to diagnose surgical wound skin infections¹⁸ and Buruli ulcers¹⁹ by direct smears and culture have been recommended. Skin nodules due to myaisis are rare but eminent FNB targets (Fig. 18.1).

Fig. 18.1 Cutaneous myaisis

Dipteran larva in background of pus from 23-gauge FNB of skin abscess (HE, ×600).

(Courtesy Dr Peter Pogany, Budapest, Hungary)

FNB without prior imaging is the first-line diagnostic approach for suspected breast abscesses yielding suppuration and a wide range of common bacteria and other agents including brucellosis²⁰ and can assist in the drainage of the abscess. Cultures are mandatory. Repeat FNB is required if a significant palpable or ultrasound lesion remains after treatment because very occasional high-grade carcinomas can be associated with obscuring necrosis, neutrophilic infiltrate or multinucleated giant cells (MNGC) and a careful assessment of any inflammatory smear is required to ensure these unusual but rare carcinomas are not missed. Breast FNB may produce granulomatous material where mycobacterial and other infections must be excluded by culture.²¹

Lesions in the testis and scrotum including mycobacterial orchitis and epididymitis can be sampled by FNB to allow differentiation from neoplasms with no risk of a neoplasm seeding in the FNB track and jeopardising possible surgery.²²

FNB of bone, soft tissue and skeletal muscle lesions,²³ when palpable or under ultrasound direction, is a straightforward procedure and the diagnosis of osteomyelitis due to tuberculosis²⁴ or other infectious agents can be made and confirmed on culture.²⁵

FNB of deep and impalpable lesions found on imaging diagnostic work-up usually involve a DD of malignancy, and FNB is the most rapid, least invasive and inexpensive test available to diagnose specific infections by smears, special stains, cultures and other ancillary tests.

In most developed countries, FNB of central lung lesions, mediastinal, hilar, coeliac, peripancreatic and porta hepatis lymph nodes and pancreatic lesions is now performed under endoscopic ultrasound or endoscopic bronchial ultrasound direction. Percutaneous FNB of peripheral lung, liver and splenic lesions also yield infectious diagnoses, and may be the diagnostic test of choice, for example in paediatric peripheral lung lesions.²⁶

In the liver, amebic and tuberculous abscesses, schistosomiasis²⁷ and pyogenic abscesses due to fungi, staphylococcus, streptococcus, and Gram-negative bacteria including Klebsiella spp and Escherichia coli can be diagnosed. The use of albendazole and steroids as cover for the potential but very low risk of anaphylactic shock due to spillage of hydatid cyst contents during FNB should be considered if hydatid disease is suspected in an FNB of any site.²⁸

In the pancreas, tuberculosis and infections diagnosed by EUS FNB have been increasingly reported in cases suspected of carcinoma.²⁹ EUS-FNB also accesses the adrenal glands, where suspected metastases have been proven to be tuberculosis, cryptococcosis and disseminated histoplasmosis.³⁰

In the lung, FNB is frequently used to distinguish tuberculosis or Gram-negative or -positive bacterial abscesses from carcinomas. Aspergillosis, mucormycosis,³¹ candidiasis and cryptococcosis, as well as, P.jirovecii in the HIV population, and CMV, coccidioidomycosis³² and parasites including paragonamiasis,³³ amebiasis, hydatid disease and microfilariasis have all been reported.

In cases of splenomegaly and investigation of splenic lesions found on imaging, FNB is best performed under ultrasound guidance. Diagnoses include tuberculosis, lymphoma and other lesions but uncommon infections such as leishmaniasis should be considered.³⁴

FNB of cerebral cortex lesions via burr holes can be used to diagnose toxoplasmosis, CMV and fungal infections³⁵ in the DD with metastases and primary brain tumors.³⁶

Choroiditis, uveitis and retinitis can be diagnosed by FNB performed by an experienced ophthalmologist, ideally using 25 or 27-gauge needles, and the fluid can be used for cultures, PCR and viral serology studies.

Technical considerations

Every FNB should be approached on the basis that there may be an infectious cause and that the patient may suffer from a transferable infection. The best guide is a good clinical history and examination. Every FNB operator should have, and teach, a single safe technique to use in all FNBs they perform. Gloves, and in cases of HIV patients double gloving, are recommended to cut down the potential of needle stick injuries. When removing the needle from the syringe to put air into the syringe once the FNB is finished, the hand that is to remove the needle should be brought down from the wrist of the hand holding the syringe holder, to the barrel of the syringe and then to the hub of the needle, rather than bringing this hand up to the needle point. Optical or safety glasses, gowns, uncluttered bench areas for placing needles and syringes during the procedure, and adequate clinic space are required.

When a cystic lesion or abscess is suspected it is recommended that a needle on a syringe should be used, creating a ‘closed’ system to avoid infective fluid spurting from the needle hub, and to assist in obtaining several milliliters of pus for culture. But using the needle by itself gives more sensitivity for FNB of palpable lesions, and can be used for a second pass of any residual mass after draining a cyst, or for solid and small lesions, especially skin nodules. Needles of 22 to 27 gauge are recommended for lymph nodes and skin lesions. If pus or necrosis is found and material is needed for extra slides, stains and cultures, a 22 gauge or 23 gauge will produce more material. The diagnostic yield of the cultures is higher with more material, so repeat needling using all the material for culture is recommended rather than just saline rinses of needle and syringe.³⁷

The macroscopic appearances of the FNB smears may give clues to the infectious nature of the material, with pus having its own pale creamy green color, distinctive slippery feel on smearing and possible purulent odor. However, the best use of FNB in all situations is to have immediate slide smearing, staining and assessment by a cytopathologist or experienced cytotechnician, to confirm adequacy of material, provide a provisional diagnosis and to triage the case immediately for ancillary tests. In many cases, rapid diagnosis of a specific organism or the immediate diagnosis of an organism, such as P. jirovecii, that cannot be cultured, assists patient care, and facilitates the cost-effective selection of ancillary tests.

Ideally, both Giemsa and Papstain should be prepared routinely by specimen splitting of the material on the slide or separate FNB passes. Giemsa is better for bacteria, negative images of mycobacteria and fungal hyphae, yeasts and CMV cytoplasmic inclusions, and Papstain is better for some fungal hyphae and yeasts, and viral intranuclear inclusions. If infection is suspected or the immediate provisional reports suggest infection, extra air-dried slides for the auramine, ZN, Gram and methenamine silver stains (GMS) should be made immediately, and material taken for cultures, PCR, direct immunofluorescence (IF), flow cytometry and gene rearrangement studies (if lymphoma is a DD), and ultimately, in some organisms, phylogenetic analysis based on nuclear small subunit RNA sequence alignments. No single stain or protocol will diagnose all infections and culture. Destained H&E- or Pap-stained slides can be used for GMS and other special stains, and repeat FNB recommended.

A cell block should be prepared in almost all cases from saline rinses of the needle or ideally from separate needling, for H&E, special stains, immunoperoxidase studies (IP) and electron microscopy where required. In TB or HIV endemic areas or where the patient is thought to be IC, direct inoculation into culture bottles can be used.³⁸ In cases such as liver abscesses where anaerobes are expected, pus should be placed directly into appropriate microbiologic containers. If the DD is lymphoma, especially in lymph nodes, lungs or spleen, FNB material should be placed into normal saline, Hanks medium or similar for flow cytometry and cytogenetic and gene rearrangement studies.

Immune compromised patients

FNB is extremely useful in the ever-increasing number of IC patients, including patients on long-term steroids, undergoing treatment for solid and hematologic malignancies, solid organ transplant recipients and HIV-positive persons.^4,5 The successful role of FNB has continued into the era of highly active antiretroviral therapy (HAART).³⁹ The typical inflammatory reactions to common infectious agents may be both deficient and atypical, and unusual and opportunistic infections may be present, making culture of FNB material mandatory.⁴ More than one infection may be present, for example CMV and bacterial infections in transplant patients.

Extra slides routinely should be prepared for Gram, auramine, ZN and GMS, and material taken for cultures for AFB, fungi and bacteria. A cell block should be prepared, and flow cytometry included in the work-up if lymphoma is suspected.

In these patients, suppurative lymphadenitis suggests not only a bacterial infection, but also disseminated fungal infection, most commonly Aspergillus spp, Cryptococcus neoformans or Candida spp infection. Granulomatous lymphadenitis raises the differential diagnosis of tuberculosis or possibly sarcoidosis. In Giemsa-stained smears from IC patients, mycobacterial infection may produce large numbers of plump histiocytes with cross-hatched cytoplasm representing ‘negative image’ bacilli, which may also be seen in the background serum. If neutrophils are admixed with spindle cells and histiocytes, bacillary angiomatosis should be considered, and the causative organism, Bartonella henselae and Bartonella quintana, demonstrated using Warthin-Starry stain.

Contraindications

There are no contraindications to FNB of a palpable lesion where infection is suspected. Contraindications at deep sites in infectious cases such as the spleen are the same, if any, as for FNB for suspected malignancy.

Complications

Complications of FNB for infectious cases are the same as FNB for suspected malignancy in all body sites, e.g. a pneumothorax following FNB of lung.

Criteria for adequate smear

The diagnosis of infections by FNB occurs in the setting of FNB of a lesion suspected of malignancy where infection may or may not have been considered, or in the setting where infection is the leading provisional diagnosis based on clinical and imaging findings.

When a FNB yields suppurative or necrotic inflammatory material confirming the clinical and imaging findings, this is diagnostic, especially when special stains reveal a specific organism and this is confirmed on cultures or PCR. However, if a neoplasm is expected and pus or even granulomatous material is found, repeat FNB and further investigation such as core biopsy should be considered, for example an FNB of a mid-cervical mass producing bland squamous debris and suppuration consistent with a branchial cyst, or a FNB of a perihilar lymph node producing granulomatous material consistent with tuberculosis may be diagnostic of a benign infectious process, but FNB of a lung lesion producing necrosis with negative special stains and cultures usually warrants repeat FNB to exclude a necrotic squamous cell or other carcinoma.

Suppurative inflammation

Pyogenic infection in FNB is most commonly seen in lymph nodes in the head and neck and other regions in children, teenagers and young adults and in the lung, but suppuration is common in FNB from IC patients from all sites particularly the skin.

Suppuration is usually associated with Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumoniae and Gram-negative bacteria including Klebsiella spp and Escherichia coli, as well as fungal infections such as Aspergillus spp, Cryptococcus neoformans and Candida spp, especially in IC patients.

In the mid-cervical region of the neck, a tender hot red mass raises a DD of inflamed lymph node, inflamed branchial cyst or possibly metastatic squamous cell carcinoma. The clinical site of the lesion, age of the patient and other history help to differentiate these lesions and repeat passes (up to 3 or 4) will usually provide an adequate sample. A diagnosis of malignancy should be made only in the presence of preserved squamous nuclei with malignant features, and not on pyknotic nuclei in degenerate squames in a suppurative or granulomatous background. Finally, a trial of antibiotic treatment with a repeat FNB of any residual mass may make the diagnosis. In the breast, squamous metaplasia in inflamed subareolar ducts (subareolar recurring abscess or Suska’s disease) can give rise to cytological atypia and worrying clinical signs of Paget’s disease or carcinoma. Correlation of imaging, clinical and cytological findings is essential.

In IC patients with neutropenia, a thin proteinaceous background may contain scant or no neutrophils and an organism must be sought with special stains and cultures. In AIDS patients, there may be plentiful plump histiocytes with cell debris and neutrophils resembling acute bacterial infections, but plentiful curved bacilli will be seen as negative images in the background and cross-hatched in macrophage cytoplasm (See Fig. 18.5). Culture and drug sensitivity testing is required.

In fungal and cat scratch disease,⁴⁰ suppurative granulomas with neutrophils interspersed among the epithelioid histiocytes in the granulomas may be present (Fig. 18.2).

Fig. 18.2 Suppurative granuloma

Neutrophils infiltrate epithelioid granuloma consisting of epithelioid histiocytes in background of neutrophils (Giemsa, ×600).

Granulomatous inflammation

Clinical

The commonest cause of granulomatous cytomorphology, and the diagnosis which must always be excluded, is that of mycobacterial infections. These are endemic worldwide and the prevalence of infections including atypical mycobacterial infections has increased with the HIV epidemic in almost all countries, especially India and sub-Saharan Africa. In developed countries, increased intercontinental travel, migration and increased numbers of IC patients of all types have fostered the resurgence of mycobacterial infections.

Criteria

♦ Low to moderate cellularity,

♦ Epithelioid histiocytes (modified histiocytes with elongated, bent or centrally indented footprint nuclei with fine, even chromatin, small or inconspicuous nucleoli and considerable, sometimes eccentric, pale cytoplasm) are present singly or syncytially aggregated in granulomas (cohesive tissue fragments of epithelioid histiocytes) (Fig. 18.3),

♦ Small lymphocytes in variable but usually low number in lymph nodes because of the diffuse nature of granulomatous process,

♦ Multinucleated giant cells with irregularly arranged nuclei that may aggregate or be peripherally arranged in a horseshoe in the granular or finely vacuolated copious pale cytoplasm (Langhan’s giant cells),

♦ Neutrophils in some cases,

♦ Necrotic debris, depending on cause,

♦ Eosinophilic sharply delineated masses containing few cells; first reported in tuberculosis,

♦ Fungi on methenamine stain,

♦ Mycobacteria on ZN, Fite or auramine stain.

Fig. 18.3 Granuloma

Epithelioid histiocytes with elongate, curved, indented nuclei form a granuloma (Pap, ×400).

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Culture for fungi and mycobacteria with drug sensitivity testing is mandatory.

Problems and differential diagnosis

Mycobacterium tuberculosis infection may produce granulomas consisting of epithelioid histiocytes with or without Langhan’s MNGC and caseous necrosis, but in some cases may produce only caseous necrosis (Fig. 18.4). In IC patients, especially AIDS patients, FNB of lymph nodes may show neutrophils, a variable number of lymphocytes, plasmacytoid lymphocytes and plasma cells, depending on the stage of HIV lymphadenopathy. But where TB is endemic and HIV-positive patients present without the very low CD4 counts achieved in AIDS patients in developed countries in the era before HAART, typical granulomas can be seen.

Fig. 18.4 Tuberculosis

Epithelioid granuloma with adjacent necrosis (Giemsa, ×400).

(Courtesy Dr William Geddie, Toronto, Canada)

Negative images of mycobacteria may be seen in the background serum and cytoplasm of histiocytes in the Giemsa stain (Fig. 18.5), and the beaded 3–4-micron bacilli stain in the ZN (Fig. 18.6A) and auramine (Fig. 18.6B) stains and autoflouresce in the Papstain.⁴¹

Fig. 18.5 Negative image of curved beaded atypical mycobacteria in histiocyte cytoplasm and serum background; note tendency for several bacilli to attach end to end (HIV-positive patient) (Giemsa, ×1000).

(Courtesy Dr William Geddie, Toronto, Canada)

Fig. 18.6 (A) Curved beaded mycobacteria in Ziehl Neelsen stain (ZN, ×1000); (B) Mycobacteria in Auramine stain (Auramine, ×1000).

Caseating necrosis can be extensive in FNBs of lymph nodes and lung and may be the only material on the slides in M. tuberculosis cases, where the DD is necrotizing metastasis from SCC or a cavitating lung primary.

Mycobacteria may be very scant in FNB material, limiting the usefulness of AFB stains on direct smears and cell blocks (see below). The auramine IF stain allows rapid scanning of direct smears and multiple sections of cell block material, and PCR and ELISA stains increase the detection rate.

Sarcoidosis has distinctive rounded epithelioid granulomas infiltrated by lymphocytes without necrosis, but its diagnosis requires negative AFB stains, mycobacterial cultures and PCR with the correct clinical presentation and serology (Fig. 18.7).⁴²

Fig. 18.7 (A) Cohesive rounded sarcoid granuloma with occasional lymphocytes (Giemsa,

×400); (B) Rounded sarcoid granuloma (Pap, ×400).

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Fungal infections can be associated with granulomas and the yeasts and hyphae may be seen as negative images or variably stained in the Giemsa and Papstain depending on the specific fungus and the degree of degeneration of the hyphae. Cryptococcus may have no granulomas, especially in IC patients, and the 5–15-micron round yeasts may be seen within the cytoplasm of macrophages and MNGC or in the background with narrow-necked budding, a mucicarmine-positive and Giemsa-negative ‘halo-like’ capsule, and variably stained body (Fig. 18.8).^43-45 In profoundly IC AIDS patients, there may be no capsule and the distinction from Histoplasma capsulatum relies on culture, as both stain with Giemsa. Histoplasmosis has largely intracellular smaller 2– 5-micron round yeasts showing variable budding in the GMS, and in Papstains appear as ‘holes’ in macrophage cytoplasm (Fig. 18.9).^46-48

Fig. 18.8 (A) Cryptococcus yeasts with occasional narrow-necked budding and negative-staining capsule delineated by round body and surrounding serum (Giemsa, ×600); (B) Cryptococcus in mucicarmine stain with positive capsule and occasional narrow- necked budding (Muc, ×600).

Fig. 18.9 Punched out ‘holes’ in cytoplasm of multinucleated histiocyte represent 3– 5-micron yeasts of Histoplasma capsulatum (Giemsa, ×600).

(Courtesy Dr William Geddie, Toronto, Canada)

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In Southeast Asia and China, Penicillium marneffei, a rapidly emerging opportunistic infection in HIV-positive patients, has 2–4-micron yeasts in macrophages and sausage shaped yeasts in the extracellular background, showing septa in the GMS due to their division by binary fission, not budding.^49,50 The budding yeasts of Sporothrix schenkii are cigar shaped (Fig. 18.10),^51,52 and Paracoccidioides brasiliensis, endemic in Central and South America, has variably sized yeasts with pathognomonic multiple budding.^33,53

Fig. 18.10 (A, B, C) Sporotrichosis: oval and elongate yeasts in macrophages and in the GMS in the background (Pap, Giemsa, Grocott Methenamine silver, ×1000).

(Courtesy Dr Rene Gerhard, Sao Paolo, Brazil)

Blastomycosis produces large 8–20-micron yeasts with broad-based budding and a thick double-contoured, weakly birefringent wall featuring inner and outer wall staining (Fig. 18.11).^8,10,54 Blastomycosis shares a distribution with coccidioidomycosis which has much larger 20–120-micron refractile, thick-walled spherules, some of which contain 2–4-micron endospores (Fig. 18.12),³² which have to be differentiated from leishmaniasis, whose amistigotes contain nuclei and kinetoplasts (Fig. 18.13), and the crescentic tachyzoites containing dot nuclei of toxoplasmosis (Fig. 18.14A). In lymph node FNBs, toxoplasmosis is associated with follicular hyperplasia and discrete small syncytial epithelioid granulomas (Fig. 18.14B), and the tachyzoites in cysts and the background are rarely seen. Confirmatory diagnosis can be made by serology.

Fig. 18.11 (A, B) Blastomycosis: 10– 12-micron yeasts showing broad based budding (Giemsa and GMS, ×1000).

(Courtesy Dr David Carter, USA)

Fig. 18.12 (A, B, C) Coccidiodomycosis: 60-micron spherule with faintly birefringent wall containing 2–4-micron endospores (Giemsa, Pap, GMS, ×1000).

(Figures 12A, 12C Courtesy Dr William Geddie, Toronto, Canada; Figure 12B Courtesy Dr Matthew Zarka, Scottsdale, Arizona)

Fig. 18.13 Leishmania

(A) Leishman-Donovan bodies in background and in macrophage cytoplasm (Giemsa, ×400); (B) Leishman-Donovan bodies in macrophage cytoplasm at high power (Pap, ×1000).

(Courtesy Dr Khosrow Daneshbod, Shiraz, Iran)

Fig. 18.14 Toxoplasmosis

(A) brachyzoites in macrophage cytoplasm (Giemsa, ×600); (B) Syncytial epithelioid minute granuloma in reactive lymphoid background.

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Candida spp have thin, branched pseudo-hyphae and 3– micron yeasts,⁵⁵ while Aspergillus spp have 5–7-micron diameter septate hyphae showing acute angle branching (Fig. 18.15)^56,57 and Mucormycosis has thicker, irregular 2– 50-micron, ribbon-like nonseptate hyphae and variable but often 90 degree branching (Fig. 18.16).³² Aspergillosis can be associated with birefringent needle-like calcium oxalate crystals⁵⁸ and marked metaplastic squamous atypia in the margins of cavitating lung lesions.

Fig. 18.15 Aspergillosis

(A) Negative image and faintly stained branching aggregated crisscrossing hyphae in Pap stain (Pap, ×400); (B) Negative image and pale linear staining in aggregated hyphae (Giemsa, ×400); (C) Acutely branching hyphae (GMS, ×400).

Fig. 18.16 Mucormycosis.

Specific infections

Specific infections are described in Tables 18.1 and 18.2, which emphasize the morphological diagnosis in direct smears and special stains. For a more detailed discussion of their microbiology, please refer to standard texts.⁵⁹

Table 18.1 Fungi

Table 18.2 Parasites, mycobacteria, other bacteria and viruses

Fig. 18.18 Pneumocystis

(A) granular foamy aggregate of 5-micron cup-shaped cysts (Pap, ×400); (B) Aggregated cup-shaped cysts with dot nuclei (GMS, ×400).

Fig. 18.19 Filariasis due to Dirofilaria repens

(A) With multiple microfilariae in background of neutrophils, with blunt heads and tapered rear ends (insert) (Pap, ×200); (B) Aggregated eggs (Pap, ×400); (C) Adult gravid female in background of eosinophils, from FNB of ‘malignant’ breast lesion (Pap, ×200).

(Figures 19A, 19B Courtesy Dr Khosrow Daneshbod, Shiraz, Iran; Figure 19C Courtesy Dr Roy, Mumbai, India)

Fig. 18.20 (A) Hydatid scolex, showing hooklets and sucker (Pap, ×1000); (B) Hydatid laminated membrane. (C) Hooklet (Pap, ×400).

Fig. 18.21 Cysticercosis

Intact larva including scolex with hooklets (sickle shaped) and attached wall fragment (HE, ×200).

(Courtesy Dr Harsh Mohan, Chandigash, India)

Fig. 18.22 Actinomyces

(A) Aggregated filaments which can form macroscopic “sulphur granules” amid neutrophils (Giemsa, x400); (B) Aggregated filaments (Gram, ×1000).

(Figure 22B Courtesy Dr William Geddie, Toronto, Canada)

Fig. 18.23 Nocardia

(A) Filaments in background of pus (Giemsa, ×1000); (B) Filaments (ZN, ×1000).

(Courtesy Dr William Geddie, Toronto, Canada)

Fig. 18.24 CMV intranuclear inclusion in lung (Pap, ×1000).

Mycobacterial infections include M. tuberculosis, M. avium complex, M. leprae and M. ulcerans (Buruli’s ulcer).

Tuberculosis is endemic across most countries, and with the increase in IC patients and the HIV epidemic there has been a resurgence of TB in the developed world and the development of multi-resistant strains.⁶⁰ FNB of all sites offers a rapid, inexpensive and efficient outpatient clinic diagnostic method based on cytomorphology (see Figs 18.3-18.5) and the Ziehl-Neelsen (see Fig. 18.6A) and auramine (see Fig. 18.6B) stains, and the FNB delivers material for culture in traditional Lowenstein-Jensen or other media or PCR or immunohistochemistry.^38,61 This is highlighted by the role of FNB in children with cervical lymphadenopathy.⁴¹

Extrapulmonary TB most commonly presents with head and neck lymphadenopathy where the DD is often malignancy – for example, intraparotid lymph nodes can present as a neoplasm – and in the skin,⁹ bone,²⁴ vertebral and paravertebral regions,²³ testes and epididymis²² and pancreas.⁶² Mycobacterium avium complex is the commonest nontuberculous mycobacterium and the commonest cause of cervical lymphadenopathy in immune competent children, and may disseminate from lungs and gut to involve lymph nodes and skin in HIV-positive patients.⁶³

Positive cytology is usually defined as granulomas, MNGC, caseation and mycobacterial visualization, but there is a wide range of cytomorphological patterns on smears varying from the classic features of epithelioid granulomas with Langhan’s giant cells and caseous necrosis, to mostly or solely caseous necrosis, and to tuberculous abscess with plentiful neutrophils, degenerating epithelioid cells and a watery necrotic background. The cytology pattern and number of AFB on ZN stain depends on the patient’s immune status, and in cases where FNB produces only caseous necrosis the percentage showing AFB on the ZN stain may be as low as 7%.⁶⁴ M. avium complex especially may yield histiocytes and some neutrophils in a watery background with a variable number of AFB in HIV-positive patients.⁶³ Culture or PCR is mandatory for confirmation, speciation and drug resistance, but these tests are often not available in TB-endemic developing world areas, where there is reliance on demonstrating AFB.

Other techniques have been put forward including the modified bleach method for ZN staining,⁶⁴ the IF auramine-rhodamine stain, immunostaining of direct cytological smears,⁶⁵ PCR on DNA scraped and processed from dried unstained cytology smears,⁶⁶ and the ELISA test,⁶⁷ most of which can achieve positive rates in the range 70% to 85%.

But the combination of cytomorphology, ZN stain and autofluorescence on Papstain can achieve concordance with culture in around 70% cases,⁴¹ and culture remains the mainstay of diagnosis and drug sensitivity testing.⁶¹

Extrapulmonary TB in the breast presents as a mass, discharging sinuses, cold abscess or non-healing ulcers, raising a DD of carcinoma. AFB stains and cultures, however, may not be positive, and other causes of granulomatous mastitis such as Brucella melitenses, fungi and organizing fat necrosis should be excluded by cultures and PCR.²⁰

FNB of the skin and other lesions of leprosy due to Mycobacterium leprae, which is endemic across the developing world, are highly diagnostic, especially if skin is blanched by gently lifting and squeezing the site to be aspirated to reduce bleeding and the Fite stain and cultures are used.¹⁷ The DD is cutaneous tuberculosis with its epithelioid granulomas and caseous necrosis.⁹ There is a spectrum of lesions:

The FNB can classify most cases to match the Ridley and Jopling histological classification of leprosy. Soft tissue swellings with nerve abscesses in neuritic leprosy show abundant caseous necrosis and many degenerate neutrophils in a thin proteinaceous background with a negative AFB stain.⁶⁹

Kaposi’s sarcoma

This neoplasm is caused by human herpesvirus-8 and is derived from infected endothelial cells. It is an AIDS-defining illness and one of the commonest neoplasms seen in homosexual AIDS patients, although in the HAART era, its incidence has decreased and been overtaken by lymphomas and squamous mucosal lesions. In lymph nodes, the spindle cell proliferation involves the subcapsular and other sinuses, and the remaining lymph node shows the various stages of HIV infection, from follicular hyperplasia through to involution. Nodal involvement should be considered in patients with tender lymphadenopathy, low CD3 count, associated infections and Kaposi’s sarcoma skin lesions.⁷⁰

Criteria for diagnosis (Fig. 18.17)

♦ Low cell yield; irregular tissue fragments of haphazardly arranged spindle cells,

♦ Elongated, blunt-ended, mildly irregular hyperchromatic nuclei with inconspicuous nucleoli,

♦ Poorly defined cytoplasm, which in some cases in the Giemsa stain is delineated by metachromatic stroma between the cells,

♦ Single spindle cells with bipolar or long and wispy cytoplasm,

♦ Plasmacytoid lymphocytes, plasma cells, lymphocytes and plentiful tingible-body macrophages.

Fig. 18.17 (A) Tissue fragment of Kaposi’s sarcoma, showing crowded, spindle shaped cells with hyperchromatic, blunt-ended nuclei (Giemsa, x200); (B) Tissue fragment of spindle cells, with poorly defined cytoplasm Giemsa, x400); (C) Single spindle cells with cigar-shaped bland nuclei, and granular chromatin (Pap, x400) Kaposi’s sarcoma.