Cytopathology provides a rapid, inexpensive, and valuable means to diagnose and manage a wide range of infectious diseases.1 Moreover, many ancillary techniques, including special stains, immunocytochemistry, and molecular studies, can be applied to cytology samples, thereby increasing specificity and sensitivity. Consequently, cytology samples can provide timely and specific diagnoses of infections and help classify certain microorganisms. The emergence of various opportunistic infections in transplant recipients and AIDS patients has greatly increased the demand for minimally invasive and rapid diagnostic methods such as cytology. We are also witnessing a resurgence of formerly rare infections owing to the increasing incidence of immunocompromised patients.
The lungs are a common site of infectious disease. An accurate diagnosis of a pulmonary infection by means of cytology can be lifesaving. Sputum, lung FNA, bronchoscopic brushing or washing, BAL, and pleural effusion evaluation can all help provide a fast, cost-effective, and noninvasive diagnosis of pleuropulmonary infection. The optimal diagnostic method depends on the location and radiologic findings of the suspected infection. For lesions that are centrally located and involve major airways, sputum samples and bronchial brushings and washings may yield diagnostic material, whereas a BAL may be more useful for assessing infectious processes in peripheral lung regions. BAL remains the diagnostic procedure of choice for detecting opportunistic infections in immunocompromised hosts. Several infections may manifest as localized lesions mimicking malignancy (for example, tuberculoma, cryptococcoma, histoplasmosis-related lung nodule with associated mediastinal or hilar lymphadenopathy). Transbronchial or ultrasound-guided FNA permits such endobronchial lesions or hilar lymph nodes to be sampled, and radiologically guided FNA can be used when subpleural regions need to be aspirated that cannot be easily assessed by bronchoscopy.
A variety of microorganisms can infect the lungs including viruses, bacteria, fungi, and parasites. However, identifying some of these microorganisms based solely on their cytomorphologic appearance can be challenging, and hence ancillary studies may be required. In many cases, the implicated pathogen will depend on the clinical setting (for example, community-acquired pneumonia, nosocomial infection, or an immunocompromised host). Equally important when evaluating these cases is the host’s response to organisms (for example, abscess formation, necrotizing granulomas, or eosinophilia), which often provides important diagnostic clues. The presence of calcium oxalate crystals, which are strongly birefringent under polarized light, are highly suggestive of aspergillosis. Inflammatory atypia and/or repair reactions that accompany certain infections, such as cavitary fungal lesions or ulceration, may result in a false-positive diagnosis of malignancy. In cases with worrisome reactive epithelial or mesenchymal repair, there is often a continuum of changes from cells with benign features to those with a reactive or repair appearance.
Viruses commonly infect the respiratory tract. While not all viral infections cause cytopathic changes, in many cases the cytologic features of viral infection can be diagnostically helpful:
- Herpes simplex/zoster virus —Cowdry type A and B inclusions.
- Cytomegalovirus—large intranuclear and small cytoplasmic inclusions.
- Adenovirus—intranuclear inclusions (smudge cells) and ciliocytophthoria.
- Respiratory syncytial virus—syncytial giant cells.
- Parainfluenza—syncytial giant cells with large cytoplasmic inclusions and ciliocytophthoria.
- Measles—multinucleated giant cells with cytoplasmic and intranuclear Cowdry type A inclusions.
Herpetic inclusions can be seen in metaplastic squamous cells (when inflammation is centered around airways) or multinucleated giant cells (within necroinflammatory debris with the interstitial form of infection). These changes need to be distinguished from CMV infection, which rarely causes multinucleation (Fig. 1 PDF, 239 KB). The number of cells showing viral cytopathic changes varies with the severity of infection or as a result of patients receiving prophylactic antiviral therapy. Ancillary studies such as immunohistochemistry, viral culture, and molecular tests may be required in some cases.
Bacteria are another common cause of pulmonary infection. Bacterial pneumonia may be lobar, lobular, or present in an atypical fashion (for example, mass-like or interstitial disease). Necrotizing pyogenic infections may cause an abscess. Although a Gram stain in such cases can be performed, the etiology of bacterial pneumonia is best established by correlating findings with microbiology studies. Oral actinomyces that contaminate sputum and bronchial specimens need to be distinguished from true infection, where bacteria are often associated with abundant neutrophils. Gram-positive filamentous actinomyces found on FNA are unlikely to be due to contamination. Nocardia are also filamentous bacteria, but unlike actinomyces they tend to be beaded, exhibit right-angle branching, and are weakly positive with an acid-fast stain. The main finding with mycobacterial infection is granulomatous inflammation. The differential diagnosis includes other microorganisms that cause necrotizing granulomatous inflammation and that are AFB positive (for example, Nocardia, Rhodococcus, and Legionella micdadei) as well as noninfectious causes of granulomatous lung disease such as sarcoidosis.
Pulmonary fungal infections can be diagnosed by means of exfoliative cytology or FNA. Fungal infections are frequently associated with a granulomatous or necroinflammatory reaction. The morphology of fungi may vary with the organism, stage of infection, and prolonged antifungal exposure:
- Candida: pseudohyphae, rarely true hyphae, and/or narrow neck “teardrop”-shaped budding yeast (2–10 µm).
- Histoplasma: narrow neck budding yeast (3–5 µm).
- Blastomyces: broad neck budding yeast with a thick cell wall (5–15 µm).
- Cryptococcus: yeast with thick capsules (5–20 µm).
- Coccidioides: thick-walled spherules (10–80 µm) filled with endospores (2–5 µm).
- Aspergillus: regular septate hyphae with 45° branching (3–6 µm wide) and fruiting bodies.
- Mucor: ribbon-like, nonseptate hyphae with 90° branching (6–50 µm wide).
Cytopathologists are regularly called upon to rule out Pneumocystis pneumonia (or pneumocystosis), caused by the yeast-like fungus Pneumocystis jirovecii (formerly P. carinii). Specimens used to diagnose pulmonary pneumocystis include sputum (where induced sputum is more sensitive than expectorated samples), BAL (which is more invasive but has a greater diagnostic yield), and, for intubated patients, tracheal aspirates. While foamy alveolar casts that contain cysts are typically seen, in some specimens only organisms within macrophages may be seen. P. jirovecii cysts, best visualized with silver stains such as GMS, are usually present in aggregates which helps to differentiate them from Histoplasma or Cryptococcus, which typically do not aggregate. Budding does not occur with P. jirovecii; however, adjacent or overlapping cysts may sometimes mimic budding organisms. With histoplasmosis one usually finds numerous intracellular yeasts within macrophages, but when cells get disrupted these microorganisms may also be located extracellularly. Invasive Cryptococcus is another pathogen that has become increasingly important among HIV-positive and transplant patients. Cryptococcal yeasts are often surrounded by thick capsules (Fig. 2 PDF, 205 KB) that stain positively with mucicarmine, alcian blue, and colloidal iron stains. In Cryptococcus pneumonia organisms may rarely be intracellular (Fig. 3 PDF, 627 KB), and in these cases are often overlooked.
Pulmonary disease caused by parasites occurs when the lungs are involved during their life cycle. Infection is often associated with eosinophilia. A number of parasitic organisms may infect the respiratory tract:
- Protozoa: Toxoplasma gondii, Entamoeba histolytica, Cryptosporidium, Microsporidia.
- Nematoda (roundworms): Dirofilaria immitis, Filaria sp. (Wuchereria bancrofti, Onchocerca volvulus, etc.), Strongyloides stercoralis.
- Cestoda (tapeworms): Echinococcus granulosus, E. multilocularis.
- Trematoda (flukes): Paragonimus sp. (westermani, africanus, mexicanus, etc.), Schistosoma sp. (mansoni, japonicum, haematobium).
The most common cestode pathologic to the lung is Echinococcus granulosus, which manifests as a hydatid cyst (Fig. 4 PDF, 282 KB). Strongyloides stercoralis may be seen in cytology laboratories that receive respiratory specimens from patients who are immunosuppressed. Their filariform larvae (large 400–500 µm worms with notched tails and a short buccal cavity) need to be distinguished from the larval forms of Ascaris lumbricoides and hookworms. Toxoplasmosis is yet another infection that may occur in immunocompromised patients. Therefore, it is important that respiratory specimens from these patients be carefully examined for small (5–7 µm) crescent-shaped trophozoites. Such parasitic organisms are best identified with a Giemsa-stained preparation or an immunohistochemical stain, if available.
1. Pantanowitz L, Michelow P, Khalbuss WE. Cytopathology of Infectious Diseases. New York, NY: Springer; 2011.
Dr. Pantanowitz is associate professor of pathology and director of the FNA Clinic and Dr. Khalbuss is associate professor of pathology and director of cytopathology and the cytopathology fellowship, UPMC Shadyside Hospital, Department of Pathology, University of Pittsburgh. Dr. Khalbuss is a member of the CAP Cytopathology Committee.
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