Cytopathology of Infectious and Inflammatory Diseases

A 59-year-old man presented with sudden onset of a painful swelling in the neck. A tender, soft, fluctuant, and slightly erythematous, 5 × 4 cm mass was palpated in the level II region of the left neck. Two smaller, soft, and tender nodules were noted along the cervical lymph node chain. Teeth were in good condition, and no recent dental procedures were reported. The patient was a prison inmate and a smoker, and the clinicians were concerned about tuberculous lymphadenitis and SCC. An FNA was performed and yielded approximately 3 mL of creamy, white-gray material. The Romanowsky- and Papanicolaou-stained slides are shown in Fig. 4.6 . Because the mass was aspirated by the cytopathologists, the patient was both interviewed and examined by the team that was to process and interpret the FNA results. Having the opportunity to talk with and examine the patient is optimal for high-quality FNA interpretation. The clinical findings of relatively rapid onset, erythema, and tenderness were suggestive of infection rather than neoplasia, and a pyogenic bacterial infection was favored. The rapid Romanowsky-stained slide performed onsite (see Fig. 4.6A ) confirmed the presence of a purulent exudate composed largely of neutrophils and macrophages. Dark blue cocci, both intracellular and extracellular, were seen. Because of these findings, an aliquot of the exudate was submitted for bacterial cultures.

Even if infection with Staphylococcus or possibly other aerobic pyogenic cocci is suspected, abscesses should always be cultured for both aerobic and anaerobic bacteria. In addition, if there is adequate material, we habitually submit material (as described earlier) for fungi and acid-fast bacilli determinations. Although it might be argued that this is not cost effective, we believe that it is better, especially if the FNA was obtained under image guidance, to err on the side of a relatively inexpensive overkill. Neutrophils may predominate in some fungal infections, as well as mycobacterial infections, in an immunocompromised host (as discussed later). Culture of the FNA from this patient grew methicillin-resistant S. aureus (MRSA). Culture with request for sensitivity testing of bacterial infections is essential because often the antibiotic sensitivity spectrum cannot be predicted on the basis of either morphology or isolated species. Fibrinopurulent exudate is also seen in the Papanicolaou-stained slide (see Fig. 4.6B ). Bacteria can often be seen as red- or gray-colored structures in Papanicolaou-stained slides (see Fig. 4.6B , inset); however, the contrast between the organisms and the background is not as great as with Romanowsky-stained preparations. By Gram stain—and again we prefer the classic method as used by microbiology laboratories over the tissue Gram stains—one can discriminate between gram-positive and gram-negative bacteria, as demonstrated in Fig. 4.7 .

The differential diagnosis of FNA yielding turbid fluid containing neutrophils includes noninfectious conditions, and the cytopathologist must be aware of these. Two common mimics of infectious lymphadenitis are developmental cysts of the neck and cystic degeneration within metastatic SCC. Fig. 4.8 illustrates these conditions, from two patients with level II neck masses. In Fig. 4.8A , rapid Romanowsky-stained FNA, there is inflammation composed of both neutrophils and giant cell macrophages. Focally (inset), atypical cells are seen. This process may be misinterpreted as an abscess. In Papanicolaou-stained slides, malignant keratinizing cells were seen, and metastatic SCC was diagnosed. Fig. 4.8B shows Papanicolaou-stained FNA from the second neck mass. Neutrophilic inflammation and benign-appearing squamous cells are seen, and the case was interpreted as consistent with an inflamed branchial cleft cyst, which was later confirmed by histology. Although, in our experience, bacterial cultures are typically negative, neutrophils may be abundant in developmental cyst fluid, particularly with branchial cleft cysts. Metastatic SCC, especially from primary in the tonsil or other parts of Waldeyer ring, is often cystic in nature, and neutrophils may be seen in necrotic SCC from any site. A clue to the correct diagnosis is an abundance of coagulative necrosis that should stimulate a search for neoplastic cells. A prominent granulomatous inflammatory reaction, especially in keratinizing SCC, may also be seen, as demonstrated by the giant cell reaction in Fig. 4.8A .

A 25-year-old man with no significant medical history came to the otolaryngology clinic complaining of a swelling in the right jaw. He had undergone extraction of his right third mandibular molar 7 months previously and had noticed progressive swelling in that region thereafter. The patient was a nonsmoker and had recently tested HIV seronegative. A prior CT of the neck showed a 3 × 2 × 1 cm, necrotic-appearing mass in the left neck in the region of the hyoid bone, and physical examination revealed a 4 × 3, firm, mildly tender, right-sided mass just inferior to the angle of the mandible. Approximately 2 mL of thick, white, creamy material was aspirated. Neutrophils were seen in an onsite rapid Romanowsky–stained slide, and material was submitted for cultures, as described previously. Again, the clinical findings are useful: the patient's age and the lesion's temporal relation to tooth extraction are evidence against neoplasia, and he had no known immunosuppressive disease. Papanicolaou-stained preparations showed a neutrophilic exudate and several interspersed, grainlike structures ( Fig. 4.9A ). On high-power magnification, the grains were found to consist of aggregates of bacteria; individual, filamentous bacilli could be seen extending outward from the granules (see Fig. 4.9B ). At the interface between some of the granules and the inflammatory exudate, there was a yellow-colored band surrounding the granule. This band is a Splendore-Hoeppli phenomenon (SHP).

The SHP is seen in H&E staining as club-shaped or bandlike eosinophilic structures surrounding either microorganisms or foreign bodies. Its composition and etiology have been debated but are thought to represent a host reaction including glycoprotein and, in some instances, antigen-antibody complexes. SHP coats the bacteria at the interface and forms club-shaped structures that appear yellow in Papanicolaou stain and dark pink in H&E ( Fig. 4.10 ). The combination of granules of filamentous bacteria and SHP is highly suggestive of an actinomycotic mycetoma, and anaerobic culture is indicated. In such cases it is mandatory to communicate this suspicion to the microbiology laboratory personnel, so that the anaerobic culture bottle will be incubated for at least 1 week before being discarded. Mycetomas are infections that manifest as tumorlike masses and are characterized by the presence of grainlike or granulelike structures composed of tangled aggregates of microorganisms. Mycetomas can be caused by actinomycetes, other types of bacteria (botryomycosis), or fungi (eumycotic mycetomas). Like other types of mycetoma, actinomycotic mycetomas are chronic lesions, and draining sinuses and extensive fibrosis are common.

The following two caveats should be considered:

• 1.

  Fibrosis may predominate over purulent exudate and may prevent successful diagnosis by FNA.

• 2.

  Proliferating fibroblasts and reactive squamous atypia may result in misdiagnosis as malignancy.

In the case of this patient, both Actinomyces meyeri and Actinomyces odontolyticus were cultured from FNA material placed into an anaerobic blood culture bottle. In our experience, actinomycetes, like mycobacteria, are not easily recognized in Romanowsky-stained preparations. We usually recognize actinomycosis by the granules present in the Papanicolaou-stained material. Gram stain, especially the Gram-Weigert, is important for confirmation that the granule is composed of branching gram-positive bacilli ( Fig. 4.11 ). It should be noted that actinomycetes and other bacteria can be seen in GMS-stained material; however, bacterial staining may require a longer time devoted to the silver deposition step and could result in increased nonspecific background staining.

The clinical and cytologic findings described in this case are greatly in favor of actinomycosis; however, botryomycosis and nocardiosis are part of the differential diagnosis. Gram staining helps to rule out the former, and a modified acid-fast stain, the latter. Although nocardial mycetomas are common in tropical countries, in the United States, nocardiosis typically manifests as a necrotizing pneumonia in an immunosuppressed patient ( Fig. 4.12 ). M. fortuitum complex (MFC) infections in AIDS patients may manifest as necrotizing, neutrophil-rich cervical neck abscesses, and the morphology of MFC is difficult or impossible to differentiate from that of Nocardia species. On Gram stains, both appear as branching, beaded, gram-positive bacilli and are at least weakly acid fast. MFC may not fluoresce with routine auramine fluorochrome staining and, like Nocardia , sometimes prefers a modified stain to show off its acid-fast qualities ( Fig. 4.13 ).

Neither the pseudoactinomycotic radiate granules (PAMRGs) seen in cervical Papanicolaou (Pap) tests from pregnant women nor the cocklebur- or rhomboid-shaped crystals of hematoidin are indicative of actinomycosis, and these should not be confused with mycetomas ( Fig. 4.14 ). Although PAMRGs, like Actinomyces , have been described in people with intrauterine devices, our experience since the 1990s is that these are predominantly seen in pregnant women, as described by Zaharopoulos and colleagues. PAMRGs have features similar to the Splendore-Hoeppli reaction ; however, they are not associated with an underlying microorganism. Hematoidin, a golden-brown, cocklebur- or rhomboid-shaped structure, has been found to be a byproduct of erythrocyte degradation and consists of golden pigment, akin to bilirubin, and lipid. Hematoidin is distinct from both actinomycosis and PAMRG and is often found in cavities containing old blood. In our experience, aspirates from pancreatic pseudocysts and organizing hematomas are good places to find hematoidin crystals ( Fig. 4.15 ). We have rarely seen them in cervicovaginal smears. PAMRGs are far more common in the latter.

Neutrophils are an important first line of defense against certain fungi, including Candida , Aspergillus , and zygomycetes (mucormycosis agents). In nonneutropenic patients, invasive infections with these fungi elicit a neutrophil-rich inflammatory cell reaction. Because of the importance of granulocytes as a defense mechanism, invasive candidiasis, zygomycosis, and aspergillosis are potential complications of severe neutropenia. All three have propensities to invade blood vessels, and infarcts with coagulative necrosis are common complications of the invasive forms of these fungal infections. The cytologic features of these fungi are discussed in the sections dealing with cytodiagnosis of infections in immunocompromised patients.

CT monitoring for hepatocellular carcinoma in a Cambodian man with hepatitis C disclosed a liver mass that was sampled by FNA. Fig. 4.16 illustrates the findings in an onsite rapid Romanowsky stain. ROSE revealed the presence of necrosis and a large number of reddish granules, consistent with degenerating eosinophils, and many Charcot-Leyden crystals. For this reason, cancer was preliminarily rejected, and suspicion turned to parasites. The preliminary diagnosis was hepatic eosinophilic pseudotumor, and visceral larva migrans was included in the final differential diagnosis. Subsequent serotesting for Toxocara was positive, and the nodule regressed with therapy.

Necrosis, necrotic eosinophils, and Charcot-Leyden crystals in a solitary liver nodule, especially from a child or from an adult with recent foreign travel, makes one suspect visceral larva migrans. Nonhuman ascarid larvae are the most common culprits; however, the actual nematode larva is rarely seen, even in histologic preparations. A portion of larva surrounded by abundant eosinophils is seen in Fig. 4.17 . This is a histologic section taken from another case of visceral larva migrans–associated hepatic pseudotumor that was resected without presurgical biopsy or FNA. Nodular collections of eosinophils can also be found in Hodgkin disease and other lymphomas, hypereosinophilic syndrome, and carcinomas, most notably gastric adenocarcinoma. Again, careful attention to patients' clinical history and laboratory findings are essential.

Fig. 4.18 illustrates the bronchial brush findings from an elderly woman who had a pulmonary infiltrate and a history of therapy for uterine cancer. Eosinophils and abundant mucin were found, and a GMS stain was performed over one of the Papanicolaou-stained slides. Mucin, eosinophils, and fungi are consistent with an allergic fungal mucus plug, the differential of which is discussed in the following paragraphs. Fig. 4.19 illustrates an aspiration of sphenoid sinus contents from a 6-year-old boy with a history of allergic rhinitis and multiple paranasal sinus opacifications and cranial nerve palsies. Note the presence of granular debris, necrotic eosinophils, and Charcot-Leyden crystals. Elsewhere in the preparations, fungal hyphae were found, and the patient proved to have a noninvasive condition known as allergic fungal sinusitis. Fig. 4.20 is an aspirate from an ethmoid sinus of another patient with allergic fungal sinusitis. Note the presence of hyaline, cyanophilic hyphae admixed with degenerating eosinophils. Elsewhere in the sample, mucin admixed with relatively intact eosinophils were seen. The fungi proved to be dematiaceous by melanin stain (see Fig. 4.20 , inset).

These three cases illustrate the diagnostic significance of allergic mucin. Allergic mucin, composed of a mixture of mucin, cellular debris, eosinophils, and Charcot-Leyden crystals, is found within the conducting airways in allergic airway diseases. The hallmark of Aspergillus (and other fungal-associated) noninvasive allergic disease is allergic mucin. If one sees allergic mucin and fungi in an FNA or bronchial brushing (see Fig. 4.18 ), as in the case of the elderly woman, allergic bronchopulmonary aspergillosis (ABPA) must be ruled out, and communication with the clinical team is recommended. In 2014, ABPA was redefined by clinical parameters that include history of asthma or cystic fibrosis with deterioration of lung function, immediate Aspergillus species skin test reactivity, increased serum immunoglobulin E (IgE) ≥1000 ng/mL, increased Aspergillus species–specific IgE and IgG antibodies, and chest radiographic infiltrates. Peripheral blood eosinophilia, mucus plugs containing fungus, central bronchiectasis, and Aspergillus serum precipitins can be considered additional criteria.

Bronchial mucoid impaction that demonstrates the presence of allergic mucin and fungi but lacks the other criteria for ABPA may also occur. In the case of the elderly woman, no fungus-specific antibodies were found. Allergic mucin, containing fungal organisms, most commonly dematiaceous fungi, is also seen in allergic fungal sinusitis. This condition is similar to ABPA in that the fungi are noninvasive. Allergic fungal sinusitis symptoms are caused by massive mucus impaction within the paranasal sinuses. The impaction may cause compression of cranial nerves and symptoms suspicious for neoplasia. Evacuation of contents of involved sinuses typically yields grumous material that has been described as foul-smelling and having the consistency of peanut butter. Cytologists should be familiar with the microscopic features of allergic mucin to help to establish the diagnosis of allergic fungal disease and rule out both neoplasia and invasive fungal disease.

Peripheral blood eosinophilia and eosinophils admixed with granulomatous inflammation can be associated with chronic, disseminated coccidioidomycosis and may portend poor prognosis. Figs. 4.21 and 4.22 demonstrate the FNA and lymph node biopsy findings, respectively, in a young man from Mexico who presented with lower extremity lymphedema, eosinophilia, and a history of an unknown type of fungal infection. Granulomatous inflammation and abundant eosinophils were seen, with very focal fungal spherules (not illustrated) present in the biopsy sample. Cultures of lymph node grew C. immitis . Special attention should be paid to the patient's history, to the country of origin or travel, and always to the culture results when inflammation is present.

A 73-year-old man had complained of weight loss and cough. By CT, he was found to have a thick-walled cavitary mass in the right upper lung and two smaller masses near the pleura. Clinicians were concerned about cavitary SCC. A bronchial brushing slide contained the findings illustrated in Fig. 4.26A . Note the loosely formed epithelioid cells with interspersed granular material, indicative of caseating granuloma. Scanty acid-fast bacilli were seen on a Kinyoun stain performed over a Papanicolaou-stained brushing slide. Culture of the respiratory material from bronchoscopy grew Mycobacterium avium complex (MAC), and the cavity wall markedly thinned after therapy; however, 1 year later, a dependent, bulbous mass appeared within the base of the cavity. FNA revealed the presence of branching septate hyphae, and the diagnosis rendered was fungus ball arising in a preexisting cavity.

A 41-year-old woman also presented with cough and chest CT interpreted as suspicious for bronchogenic carcinoma. Abundant caseous material with reactive squamous metaplasia was seen (see Fig. 4.26B ). Acid-fast bacilli were seen on Kinyoun stain performed over Papanicolaou-stained slides of bronchial brushing and bronchial washing samples, but culture of bronchoscopic material was not performed. One must keep in mind that mycobacterial infections can mimic neoplasia and that granular amorphous material in an FNA, bronchial brush, or transbronchial fine needle aspiration (TBNA) sample should lead one to request acid-fast (and fungal) stains as well as cultures. Caseation may, at times, be difficult to distinguish from coagulative tumor necrosis; however, outlines of necrotic neoplastic cells can usually be seen in the latter. Note that had ROSE been performed during bronchoscopy and caseous-like necrosis or granuloma been identified, the clinicians would have been instructed to culture bronchial brushing and washings. The following two caveats should be kept in mind:

• 1.

  Reactive squamous metaplasia can lead to misdiagnosis of SCC in cases of infections. We have seen markedly atypical squamous metaplasia associated with tuberculosis and actinomycosis.

• 2.

  MAC cannot be accurately distinguished from M. tuberculosis complex (MTC) by acid-fast stain, and MAC infections are not confined to severely Tcell–immunosuppressed patients. Culture is required for identification of MOTT, and culture or presumptive identification by molecular methods, such as RNA amplification for MTC. Mycobacterium kansasii has a distinct morphology that can allow for presumptive diagnosis in some cases, and MFC closely resembles Nocardia spp., as mentioned previously. The comparative acid-fast staining features of MTC, MAC, and M. kansasii are shown in Fig. 4.27 . All slides were taken from cytologic samples.

Fig. 4.28 illustrates the TBNA and transbronchial biopsy findings from a man with AIDS and mediastinal lymphadenopathy. Lymphoma was suspected. Note the presence of spindle-shaped cells that could be misinterpreted as an Epstein-Barr–associated smooth muscle tumor or Kaposi sarcoma. The spindle cells in this type of pseudotumor have immunohistochemical features of macrophages ; in an AIDS patient, they are highly suspicious for mycobacterial infection. MTC, MAC, and M. kansasii have all been isolated from patients with so-called spindle cell granulomas. In this case, very long, coarsely beaded, acid-fast bacilli with curved ends are seen. Their shape has been compared to a shepherd's crook, although a Christmas candy cane might be a better simile. This is the classic appearance of M. kansasii and is the only type of mycobacteria for which we can usually make a presumptive species identification on the basis of morphology. MFC also has a relatively unique appearance; however, as previously stated, differentiation from Nocardia is problematic.

A 37-year-old HIV-seronegative man was referred for evaluation of an abnormal chest radiograph. CT showed multiple bilateral pulmonary opacities 1 to 4 cm in diameter ( Fig. 4.29 ). An atypical infectious process was favored, and a CT-guided FNA was performed. Findings are illustrated in Fig. 4.30 . This patient was immunocompetent, and epithelioid macrophages, giant cells, lymphocytes, and plasma cells were present, consistent with granulomatous inflammation; however, the macrophage cytoplasm had a vesiculated rather than granular appearance. To best see the intracytoplasmic structures, the substage condenser must be closed. This gives fungi a refractile appearance and makes them much easier to see. On high-power examination, one sees relatively round structures that are clear to slightly pink. The fungi lack abundant capsular material, as is common in infections with this pathogen in immunocompetent or mildly immunosuppressed patients. Note the considerable variation in size, a very important clue to the diagnosis! These organisms typically vary from 5 to 20 µm in diameter. This is well demonstrated in the GMS stain ( Fig. 4.31 ). Also note the thin cell wall and round shape, which are typical of Cryptococcus . Cryptococcus infection in T cell–immunosuppressed patients is discussed in the next section.

One should be aware that cryptococci are easily overlooked in H&E- and Papanicolaou-stained preparations. They may also be mistaken for phagocytized erythrocytes or lipid ( Fig. 4.32 ). Remember to close the substage condenser! Paucicapsular forms of Cryptococcus often stain weakly with mucicarmine. Fontana-Masson melanin stain is recommended in these cases. Remember, the marked variation in diameter of cryptococcal yeast forms helps one to distinguish this fungus from both erythrocytes and other yeastlike pathogens.

A 21-year-old male student from eastern Louisiana underwent bronchoscopy because of a right lower lung infiltrate, chills, and fever that failed to respond to therapy for community-acquired pneumonia. The man had no history of alcohol or drug abuse and was HIV negative. His bronchial brushing material is illustrated in Fig. 4.34 . Note the combination of epithelioid macrophage aggregates and neutrophils (see Fig. 4.34A ). This type of inflammatory pattern and the clinical history should generate a search for dimorphic fungi. In Fig. 4.34B, one sees the characteristic features of B. dermatitidis . In tissue, B. dermatitidis is seen as yeastlike cells that are 8 to 15 µm in length and relatively uniform in size (in contrast to Cryptococcus, Coccidioides spherules, and Paracoccidioides ); the cell wall is thick and double contoured, and the endoplasm is typically retracted so as to leave a clear zone between the cell wall and the internal structures (see Fig. 4.33A and D ). Under very high magnification, multiple nuclei can sometimes be seen as tiny dots, but these may be difficult to discern in Papanicolaou and H&E stains. Broad-based budding is typical of Blastomyces and is useful if found (see Fig. 4.33A ). The main differential includes P. brasiliensis and C. immitis (see Fig. 4.33 ). Because these organisms have different geographic distributions, knowing the travel history or region of birth of the patient is extremely helpful. P. brasiliensis is thick-walled like B. dermatitidis , but it has narrow-necked budding, and multiple buds are common. The characteristic “ship's wheel” budding pattern is lovely and diagnostic if you can find it, but it may be difficult to find. A very useful differentiating feature is the marked variability in size that is found in P. brasiliensis compared with B. dermatitidis . The greatest diagnostic pitfall is the immature spherule of C. immitis , which may closely resemble B. dermatitidis and P. brasiliensis (see Fig. 4.33 ). The Paracoccidioides yeast forms illustrated here derive from a lung section rather than a cytologic specimen and are shown for the sake of comparison.

A 26-year-old man who had been working in the Texas Rio Grande Valley presented with fever and several fluctuant, subcutaneous masses. Chest radiography revealed diffuse interstitial infiltrates. FNA was performed from a 3 × 3 cm subcutaneous mass overlying the fifth lumbar vertebra. Several milliliters of gray-colored, thick material were aspirated. A Papanicolaou-stained slide of aspirate is illustrated in Fig. 4.35 . Spherules of markedly varying sizes are seen in a background of necrosis, giant cells, and neutrophils. Endosporulation is seen within some of the spherules. Note that immature and empty spherules that lack endosporulation can readily be mistaken for pollen grains, Blastomyces , or Paracoccidioides . Cutaneous and subcutaneous lesions of Blastomyces, Paracoccidioides, and Coccidioides typically represent disseminated pulmonary disease; however, in some cases, pulmonary lesions are not apparent. This was a fulminant case, and neutrophils and necrosis are prominent, although epithelioid macrophages and giant cells are also seen.

This patient tested negative for HIV. In patients with coccidioidomycosis and advanced AIDS, extensive necrosis, myriad organisms, and variable numbers of neutrophils and macrophages are seen. Granulomas are absent ( Fig. 4.36 ). Similar histologic reactions to Paracoccidioides, Blastomyces, and Sporothrix have been reported in AIDS patients.

A 37-year-old diabetic man presented with isolated solitary pulmonary and renal lesions. Sputum, urine, and kidney FNA contained zygomycete fungi. Urine and kidney FNA findings are seen in Fig. 4.41 . Note the presence of neutrophils and broad, well-stained, ribbonlike hyphae typical of zygomycetes. This patient proved to have locally invasive intrabronchial zygomycosis with an isolated mycotic renal abscess. Cultures of both sites grew Rhizopus spp. This case illustrates an unusual, indolent presentation of zygomycosis in a nonneutropenic patient who had diabetes mellitus. Cytologic examination could only diagnose zygomycosis. Histologic studies of the resected lung lesion demonstrated chronic, intrabronchial zygomycosis, a condition analogous to chronic necrotizing aspergillosis. The kidney lesion was also resected, and no evidence of other infected sites was found subsequently.