Diagnosis of Infectious Diseases: a Cytopathologist’s Perspective

Abstract

This review explores the role of the cytopathology laboratory in the detection and presumptive identification of microorganisms. Sample procurement by exfoliation, abrasion, and aspiration techniques, as well as a variety of cytopreparatory and staining methods, is reviewed. Emphasis is placed on the utility of fine-needle aspiration as a rapid, safe, and cost-effective diagnositic procedure. The role of rapid interpretation and specimen triage is also discussed. Cytomorphologic features and staining characteristics are presented for a spectrum of microorganisms potentially encountered in the cytopathology laboratory. Pitfalls in diagnosis and the usefulness of special stains and ancillary techniques are also evaluated. The importance of communication, collaboration, and clinical correlation is stressed.

This review will explore the utility of cytopathology in the diagnosis of infectious disease, with emphasis on the detection and identification of common microorganisms in various cytologic specimens. Cytologic techniques of specimen procurement, staining, and rapid identification of infectious agents will be discussed. In particular, the utility of fine-needle aspiration (FNA) as a rapid, cost-effective, and safe diagnostic procedure will be highlighted.

The examination of cells encountered in various body fluids, sputum, and urine can be demonstrated sporadically in the medical literature of the 1800s. One of the first reported needle aspirations has been traced to the mid-1880s (166). Perhaps the first report that suggests the potential of needle aspiration in the diagnosis of infectious disease is a 1904 study by Grieg and Gray (50, 60). In this study, needle aspirations of lymph nodes from patients with sleeping sickness revealed motile trypanosomes. The authors suggested that this procedure would be a more rapid and satisfactory method of diagnosing cases of sleeping sickness than examination of blood (60). Further development of needle aspiration as a diagnostic procedure occurred during the 1930s, primarily at the Memorial Sloan Kettering Cancer Center in New York, where Martin and Ellis (100) and later Stewart (151) developed significant experience in this technique. Further refinement of the fine-needle method, using small-diameter needles, took place in Europe following World War II. Concurrent with the development of FNA came the introduction and advancement of other cytologic methods for the detection of cervical and lung cancer (42, 116). Since then, the role of diagnostic cytology has expanded tremendously. The availability of a variety of rapid, safe, and cost-effective techniques and stains often places cytopathology in the forefront of diagnostic evaluations, including the diagnosis of infectious disease (3, 13, 21, 27, 33, 76, 81, 101).

PROCEDURES AND TECHNIQUES

Sampling Procedures

Material for cytologic evaluation may be procured by one of three mechanisms: exfoliation, abrasion, or aspiration (Table 1). The resultant specimens often are prepared by slightly different methods, which if performed improperly may yield inconclusive or misleading results. Degeneration, shearing, and distortion, as well as air drying, are the most common problems that render specimens virtually uninterpretable. The cytopreparatory techniques discussed below are routinely used in cytopathology laboratories; for additional information, the reader is directed to the Manual of Cytotechnology (82).

TABLE 1.

Cytologic methods and fixation and processing techniques

Method	Examples	Fixation	Processing	Microscopy	Common microorganisms
Exfoliative	Sputum	Immediate or Saccomano’s (Carbowax/alcohol)	Blender	Smears	Candida spp., Aspergillus spp., Cryptococcus spp., Blastomyces spp.
		Fresh	Pick and smear
	Urine	Fresh or refrigerated	Cytocentrifuge, membrane filtration	Cytospin preparations	Candida spp., CMV, polyomavirus
	Effusions	Fresh or refrigerated	Cytocentrifuge, cell block, membrane filtration	Cytospin preparations, smears, cell block sections
	CSF	Fresh	Cytocentrifuge, membrane filtration	Cytospin preparations	C. neoformans, viruses, T. gondii
Abrasive	Pap smear	Immediate fixation (alcohol)	Direct smear	Smear	Candida spp., Actinomyces spp., Trichomonas spp., HPV, CMV, HSV
	Tzanck preparation	Air dry	Direct smear	Smear	Herpesviruses
	Endoscopic brushings	Immediate fixation or air dry	Direct smear	Smear	Body site dependent
		Brush rinse	Cytocentrifuge	Cytospin preparations	Fungi, viruses, parasites
Aspiration	Superficial: breast, thyroid, salivary gland, lymph node, etc.	Immediate fixation or air dry	Smear	Smear	Body site dependent (fungi, viruses, bacteria, parasites)
		Needle rinse	Cytocentrifuge, cell block	Cytospin preparations, cell block sections
	Radiologically guided: lung, liver, pancreas, kidney, etc.	Immediate fixation or air dry	Smear	Smear	Body site dependent (fungi, viruses, bacteria, parasites)
		Needle rinse	Cytocentrifuge, cell block	Cytospin preparations, cell block sections

Exfoliative cytology relies on the presence of cells that are shed spontaneously into body fluids such as effusions obtained from pleural, pericardial, or peritoneal cavities. Collection of cells immersed in these fluids is considered only a minimally invasive procedure with little risk of complication. However, cellular degeneration may be a problem if the specimen is not collected, fixed, transported, and prepared in the appropriate manner. The volume of material collected can range from 1 to 2 ml of cerebrospinal fluid (CSF) to 1 liter or more of ascites. While procedures may vary slightly depending upon the laboratory, there are two basic practices: immediate fixation of the specimen and rapid transport of the fresh specimen to the cytopathology laboratory (99). Immediate fixation and/or processing avoids further specimen degeneration. Sputum and urine collection is a noninvasive procedure that is also considered exfoliative cytology. Although bronchoscopy and FNA techniques have surpassed sputum analysis in diagnostic accuracy, it still remains the simplest method for examination of the respiratory tract. Urine, by virtue of ease of collection, remains one of the most common exfoliative specimens.

Abrasive techniques include endoscopic brushing, as well as manual scraping. In most cytopathology laboratories, the Pap smear is the most common specimen of this type. A variety of members of the normal flora, as well as infectious agents, can be readily identified on these smears. Another classic example of abrasive cytology is the Tzanck preparation (158). This rapid and easy technique samples the base of dermal or mucocutaneous vesicles and is often used to diagnose herpes simplex virus (HSV) and varicella-zoster virus. Pap smears and Tzanck preparations are submitted to the laboratory as fixed or air-dried slides, respectively. One of the most technically demanding procedures in this category of abrasive cytology is endoscopic sampling. The replacement of rigid endoscopes with flexible fiber-optic endoscopes has resulted in the ability to visualize and directly sample greater portions of the respiratory and gastrointestinal tracts by abrasive techniques of brushing, washing, and lavage, as well as by needle aspiration biopsy procedures. These specimens are usually semisolid and need to be fixed or processed immediately to prevent artifacts. Technically, washings and lavages can be categorized as either an exfoliative or abrasive technique: cells are shed into saline which has been used to remove cells from the respiratory or gastrointestinal tract surface. A variety of bacteria, fungi, and viruses can be identified in these specimens. A differential diagnosis is often generated prior to cytologic examination, based predominantly on clinical history and the location of the lesion.

The final technique, and perhaps the most powerful, is FNA, which uses a small-gauge (usually 22- to 25-gauge) needle and negative pressure to withdraw cells or fluid from a mass (72, 87, 90, 148). Alternatively, this technique may be used without negative pressure (capillary action will draw the specimen into the hub of the needle) (175). FNA of superficial targets may be performed by any physician with training in aspiration techniques (147, 148). It requires no sophisticated imaging procedures but relies on skilled palpation and manual dexterity. A variety of body sites are amenable to superficial FNA: breast, salivary gland, thyroid, lymph nodes, skin, and soft tissue. Small masses (<1 cm) may be difficult to sample adequately.

Radiologically guided, or deep, FNA typically requires a radiologist to direct placement of the needle by using computed tomography, ultrasonography, or other imaging techniques. With these techniques, masses in deep organs have become increasingly accessible to aspiration. Selection of the size and type of needle is based on the location and potential consistency of the mass, as well as on aspirator experience. Many radiologists select large (18- to 20-gauge) needles; however, when these needles are used, this procedure should not be termed fine-needle aspiration biopsy (125). FNA biopsy traditionally uses 22-gauge or, preferably, smaller needles and presents a much lower risk of the complications associated with the use of large-gauge needles, such as hemorrhage, needle tract seeding, and pneumothorax (19, 125). Direct smears are prepared from material obtained from FNA; however, needle rinses for cytospin preparations or cell blocks may also be prepared when appropriate.

Sampling procedures can be performed either by cytopathologists or by clinicians often in conjunction with cytotechnologists. The direct involvement of a cytologist allows specimens to be triaged during the procedure, resulting in increased diagnostic accuracy and decreased complications. Various rapid stains can be used on either air-dried or alcohol-fixed material for immediate specimen evaluation. This is very advantageous since it not only determines specimen adequacy but also results in the most appropriate selection of cytopreparatory and staining methods. Infectious agents can usually be identified by cytomorphology with routine or special stains. A preliminary or “on site” interpretation is particularly useful if an infectious process is suspected, since an additional sterile sample can be obtained immediately for the microbiology laboratory. Specific organism classification and antimicrobial susceptibility testing are definitely within the purview of the microbiology laboratory (31, 170).

Cytopreparatory Techniques

Technical expertise begins, rather than ends, with specimen collection. As in clinical microbiology laboratories, cytopathology laboratories have guidelines for procurement, transportation, and processing of specimens. Since light microscopy is the standard for evaluation of cytologic specimens, cytopreparatory techniques center around placement of material on glass microscope slides. Specimens obtained by abrasion and aspiration techniques may be prepared directly by application of the material to glass slides (“smears”) followed by appropriate fixation. The traditional fixative for cytologic specimens is alcohol. Usually this is 95% ethanol, but 100% methanol or 80% isopropanol may be used. Alcohol fixation causes cells to shrink because alcohol removes intracellular water. Alcohol is a coagulative fixative that results in sharp nuclear detail, but cytoplasmic features may be less well defined. Specimens allocated for staining by Papanicolaou’s method require immediate immersion in an alcohol fixative. If there is even a short delay, air drying occurs. Air-drying artifact is one of the most common problems faced by a cytopathology laboratory, particularly when specimens are obtained by inexperienced personnel. Since air drying may render a specimen uninterpretable, constant reinforcement of proper technique is necessary. Another common problem, particularly with FNA specimens, is excessive blood. Clotting of even a small volume of blood greatly interferes with good smear preparation and subsequent interpretation (126). The fibrin in clotting blood entraps cells, resulting in heavy and uneven staining and distortion of the smear pattern. When direct smears are prepared without consideration of the blood present, the material inevitably covers a large area of the slide. In addition, cells and/or microorganisms, if present, are separated and diluted by the blood. Smears should be made only from the part of the sample contained in the needle and its hub, which is rich in cellular material. The blood which has been drawn into the aspiration syringe may be placed entirely into a small tube of 10% buffered formalin and processed as a cell block.

When a specimen is largely fluid, additional preparation is required. Before transport, an equal volume of 50% alcohol can be mixed with the specimen to provide initial fixation. Concentrations of alcohol higher than 50% should not be used since they result in coagulation of the cells, making subsequent specimen preparation difficult. Alternatively, the specimen can be submitted fresh and unfixed. This is best accomplished by immediate transport to the laboratory; if that is not possible, the fluid can be refrigerated until transport. Good cell preservation will be retained for at least 48 h. Sterility is not required for routine cytology specimens. Refrigerated specimens are rarely overgrown by bacteria or fungi. Semisolid material from aspiration and endoscopy specimens may be processed like a fluid by rinsing the specimen into saline or cell culture medium for subsequent cytocentrifugation.

Cytocentrifugation can be used for liquid specimens. The quantity and quality of the fluid, as well as the clinical impression, will determine the specific centrifugation and staining methods. The purpose of both manual smear and cytocentrifugation techniques is to place a thin, uniform layer of sample on the slide. For smaller volumes (<5 ml), cytocentrifugation of the specimen directly onto glass slides (i.e., cytospin preparations) can be performed (57). This results in an even distribution of material over a limited area of the slide, usually 25 to 50%. Larger volumes, usually obtained from effusions or ascites, need preliminary centrifugation to consolidate the specimens into manageable volumes from which cytospin preparations can be prepared. Cytospin preparations are the closest equivalent to the reproducibility of tissue sections and, as such, are preferred over smears for ancillary studies such as special stains and immunochemistry. The number of slides prepared is determined by the amount of specimen and the clinical impression. When an infectious etiology is suspected, slides containing fixed but unstained material may be made if special stains are required.

Cell blocks can be quite valuable in exfoliative and aspiration cytology (68). Material obtained for a cell block is processed like a small tissue biopsy specimen. Needle rinses that contain minute fragments of tissue can be centrifuged, and a small button of tissue is obtained. This specimen is then fixed, paraffin embedded, sectioned, placed onto glass slides, and stained. Occasionally, the specimen is composed of loose, semiliquid material held together by fibrin. This loose “clot” may also be processed as a cell block. In FNA, small tissue fragments or microcores are sometimes found in material expressed from the needle hub or lodged within the needle tip. Evaluation of cell block sections from these “minibiopsy specimens” and “clots” is generally additive and/or supportive to the smear impression. Cell blocks also provide reproducible sections for ancillary studies that may include special stains for microorganisms. With the reference section stained by the conventional histologic stain, hematoxylin and eosin (H&E), the location of any suspicious structure or probable organism can be identified on the H&E slide and then compared with the special stains.

Stains

The Papanicolaou (PAP) stain remains the traditional and most frequently utilized stain for cytologic specimens. However, the increasing popularity of FNA as a primary diagnostic procedure has demonstrated the utility and adaptability of other stains such as the Romanowsky stains and H&E. With the exception of Pap smears, which are always stained by Papanicolaou’s method, personal preference and experience will dictate which stains are used on other cytologic specimens, and more than one stain can be used. The PAP stain, which enhances nuclear detail, is often used in conjunction with one of the Romanowsky stains. Romanowsky stains are usually air dried rather than fixed immediately. This results in cellular swelling and loss of nuclear detail; however, cytoplasmic and background details are accentuated (Table 2). Surgical pathologists with limited experience in cytopathology may be more comfortable with the H&E stain, particularly with aspirate material. Both PAP and H&E stains require rapid smear preparation and immediate alcohol fixation to avoid air-drying artifacts. This is sometimes problematic when inexperience results in delayed or improper smear preparation.

TABLE 2.

Comparison of DQ and PAP stains

Characteristic	Properties	DQ staina	PAP stain
Technique	Fixation	Air dry	Immediately in alcohol
	Cell loss	Minimal	Moderate
	Stain time	20 to 30 s	5 to 10 min (rapid stain, 2 min)
	Microscopy	Examine “wet”	Coverslip needed
Background	Blood	No effect	Obscures detail
	Inflammation	Limited effect	Obscures detail
	Necrosis	Poor detail	Limited effect
Cellular detail	Cytoplasmic detail	Excellent	Limited
	Cytoplasmic products	Excellent	Limited
	Nuclear detail	Limited	Excellent
	Mitoses	Satisfactory	Excellent
Infectious elements or microorganisms	Bacteria	Good	Variable to poor
	Mycobacteria	Negative images	Not visible
	Fungi
	Hyphae	Variable to poor	Good to variable
	Yeasts	Variable to good	Variable to good, excellent for C. immitis
	Viral inclusions
	Cytoplasmic (CMV)	Excellent	Satisfactory
	Nuclear (CMV, HSV)	Limited	Excellent

^aA Romanowsky stain. 

Smears designated for staining by the conventional Papanicolaou method are spray-fixed with or submerged directly in 95% ethanol, methanol, or isopropanol. While immersion in alcohol often results in some degree of cell loss, care must also be taken with aerosol-spray fixatives to prevent freezing artifacts that result from the propellant (71). A recently developed rapid PAP stain method helps to overcome problems with fixation and cell loss. This method has the advantage of being performed on air-dried smears, so that rapid preparation and fixation are less critical. Air drying also enhances the adherence of the cells to the slides, increasing cell recovery. The smears are rehydrated briefly in normal saline, which tends to lyse erythrocytes, thereby reducing the obscuring effect of particularly bloody smears. In addition to excellent preservation of cell structure, this rapid method still preserves the nuclear detail expected with any PAP stain (172).

The use of a rapid staining protocol allows the quality of the stain, the smear, and the specimen to be checked immediately. In many cases, a diagnosis is made on the basis only of these rapid stains. If necessary, additional material can be obtained and/or evaluated for treatment with other stains and ancillary studies that aid in a more specific, final diagnosis (25, 115).

The term “Romanowsky stain” encompasses a variety of stains derived from the Giemsa stain; they include the May-Grunwald-Giemsa (MGG), Wright-Giemsa (WG), and Diff-Quik (DQ) stains. Many cytologists prefer Romanowsky stains for FNA specimens because of the vivid metachromatic staining of certain cytoplasmic products, stromal, and background elements (126, 141, 176). In addition, since this stain is applied to air-dried smears, it reduces the effects of poor technique and increases cell yield.

A commonly used Romanowsky stain is the DQ stain. There are three steps to this stain: fixation in methanol, staining in eosin Y, followed by staining in methylene blue. The procedure takes less than 20 s to perform, although staining times may vary depending on the thickness of the smear or cytospin preparation. In addition to its rapidity, it is very difficult to overstain smears. Although understaining is common, it is easily corrected by reimmersion into the methylene blue stain. The DQ stain was originally developed for uniformly thin peripheral blood smears. However, its utility in rapid cytologic diagnosis is now well established (115, 126, 143).

Ancillary Techniques

Immunocytochemistry, electron microscopy (EM), and, most recently, molecular diagnostic techniques may be of value in selected cases to refine a diagnosis, typically of a neoplastic process. However, when routine evaluation of cytologic specimens suggests an infectious etiology, ancillary studies may also be useful. This does not abrogate the need to obtain additional sterile material when an infectious process is suspected.

Perhaps the most routine ancillary study is the special stain. These stains, often performed by the histology laboratory, are used to identify bacteria (Gram and Warthin-Starry), fungi (Gomori methenamine silver [GMS] and periodic acid-Schiff [PAS]), and acid-fast organisms (Ziehl-Neelsen) and can be performed on smears, cytospin preparations, or sections from a cell block. Morphology alone or in conjunction with special stains usually allows general categorization of the potential pathogen (e.g., bacterium, fungus, or virus) and often leads to a definitive identification (e.g., Helicobacter pylori, Cryptococcus neoformans, or cytomegalovirus [CMV]). However, for more specific classification, referral to the microbiology laboratory is required.

In the past, EM has been instrumental in the diagnosis of a wide variety of infectious, nonneoplastic, and neoplastic diseases. However, EM has given way to more sophisticated, sensitive, and rapid immunodiagnostic techniques, particularly in the diagnosis of viral infections. For immunodiagnostic techniques, cell block material is the most suitable because of the ability to generate multiple sections. When possible, a separate specimen should be taken for these studies and the material should be placed directly into buffered 10% formaldehyde. Cell blocks can be prepared from cytology material fixed in alcohol or Saccomano’s fixative. Since cell blocks are not always available, cytospin preparations are an alternative and may be prepared directly from the fluid specimen or needle rinse. Cytospin preparations may be air-dried or fixed in 95% ethanol. In either case, slides for immunocytochemistry may be stored frozen for several weeks. Material can be preserved in a similar fashion for molecular diagnostic methods including fluorescent in situ hybridization and PCR. Recent advances in PCR have allowed the detection of infectious agents in cytologic material (4, 44, 45, 48, 83, 86, 162). Although the quantity of material may be a consideration in certain circumstances, most immunodiagnostic and molecular techniques today have protocols adapted to cytologic specimens.

COMMON PATHOGENS

The major roles of the microbiology laboratory are the isolation, identification, classification, and susceptibility testing of microorganisms. The role of the cytopathology laboratory is almost exclusively diagnostic, i.e., to suggest or identify the presence of an infectious agent. In recent years, in large measure as a result of the increasing number of immunocompromised patients, cytologic techniques are often the first choice for detection of infectious agents. The cytomorphologic appearances of commonly encountered microorganisms are described below. Regardless of the type of cytology specimen, the presence of acute or chronic inflammation, abundant macrophages or multinucleated giant cells with or without granulomas, and necrosis are features that should alert the cytologist to the possibility of an infectious process.

Contaminants

The presence of exogenous structures that mimic a variety of pathogens can pose a challenge, particularly to the novice. Fibers, suture, talc, and starch granules may be seeded in the specimen or collection devices during the sampling procedure. Clues that help distinguish these structures from microorganisms include their haphazard arrangement and lack of internal structure. Many of these contaminants are birefringent in polarized light. There is usually a limited or absent inflammatory response. Occasionally, iron-encrusted collagen fibers are sampled from areas of prior hemorrhage. These fibers may resemble hyphal structures; however, their true nature is revealed when GMS stains are negative and iron stains are positive (157). Respiratory specimens seem particularly vulnerable to contamination, especially with pollen grains, which resemble dimorphic fungi. Inorganic and organic fibers and vegetable material, either aspirated or as contaminants in sputum specimens, may resemble fungal hyphae. Alternaria sp., usually a nonpathogenic airborne fungus, is an occasional stain contaminant. It is easily recognized by its large, brown, septated conidia and, when present, its septate hyphae with 90° branching (132) (Fig. 1). Fungal contamination may occur when stains are not filtered regularly. This contamination is usually distinguished from true infection when only one stain reveals the microorganism.

FIG. 1.

Alternaria sp. in a Pap smear with prominent acute inflammation. A common contaminant of cytologic specimens, Alternaria sp. is easily identified by its brown, septated conidia. PAP stain; magnification, ×400.

Bacteria

In most cases, the cytopathology laboratory is limited in bacterial identification to morphological and Gram stain characteristics. There are several types of cytologic specimens in which bacteria are routinely encountered. Chief among them is the Pap smear, particularly when taken during the second half of the menstrual cycle. These smears often have an abundance of Doderlein’s bacilli (lactobacilli) that metabolize the glycogen in intermediate and parabasal squamous cells. This process is termed cytolysis. The resultant cellular debris, in conjunction with the sheer number of lactobacilli, may obscure the cervical squamous and glandular cells and often limits the interpretation of the Pap smear. Other specimens in which bacteria of the normal flora can be seen and occasionally interfere with diagnosis include sputum specimens, gastrointestinal brushings, and FNA specimens of the abdominal organs and prostate. A needle introduced into the last two, normally sterile, sites may pick up members of the bowel flora if the colon is traversed.

Routine stains of cytologic specimens can reveal bacteria. Although bacteria may be stained red or blue by the PAP stain, this cannot be correlated to gram-positive and -negative staining patterns. The significance the cytologist places on the presence of bacteria is directly related to the body site and clinical information provided with the specimen. For example, bacteria are frequently encountered in urinary tract specimens, and, while their presence is always reported, the diagnosis of a urinary tract infection requires clinical correlation (Fig. 2). Conversely, more significance is attached to the presence of bacteria or microorganisms in material from a bone aspiration (55, 99). In this situation, additional stains would be used to confirm a diagnosis of osteomyelitis. Cytologic specimens from scrape preparations, swabs, and other collection methods are useful for the detection of bacteria in abscesses, ulcers, fistulas, and wounds (13, 20, 54, 74, 77). While Gram stain of the cytologic specimen will yield some information, these specimens are more appropriately sent to the microbiology laboratory for further characterization. Five bacterial species have been selected to illustrate the utility and limitations of diagnostic cytopathology (Table 3).

FIG. 2.

Bacteria in an ileal conduit specimen. Both cocci and bacilli are readily identified with Romanowsky stains. Ileal conduit specimens are prone to degeneration and bacterial overgrowth. DQ stain; magnification, ×400.

TABLE 3.

Staining characteristics of selected bacterial genera

Genus	Staining witha:						Other techniques
	PAP	Romanowsky	H&E	Gram	Acid fast	Silver
Mycobacterium	−	+	−	−	++	GMS	PCR
		(negative images)
Legionella	−	+b	−	+	−c	Dieterle, W-S	DFA
				modified
Actinomyces	+	+	+	+	−	GMS	DFA
Helicobacter	+	+	+	+	−	Dieterle, W-S
Nocardia	−	+b	−	+	+d	GMS	DFA

^aDFA, direct fluorescent antibody; W-S, Warthin-Starry; −, does not stain; +, visible with stain. 

^bStains best with Giemsa. 

^cL. micdadei will stain acid-fast positive. 

^dStains best with Fite’s silver stain. 

Mycobacteria.

The resurgence of mycobacterial infections is due in large measure to the increasing incidence of immunocompromised patients. Infections by Mycobacterium tuberculosis and mycobacteria other than M. tuberculosis not only present as pulmonary infections but increasingly are implicated in the etiology of lymphadenopathy (17, 105, 145, 156). In many instances, when the patient is known to be immunocompromised or has had prior mycobacterial infections, the index of suspicion for infection is high and additional specimens should be obtained for culture and special stains. Although routine cytologic stains do not stain the actual bacilli, there may be significant clues that suggest the presence of these organisms (36, 128, 133). If granulomas, plump histiocytes, and/or necrosis is identified, particularly in aspirates (Fig. 3), acid-fast staining is usually performed (130). Mycobacteria cannot be identified on PAP stains. While the DQ stain does not stain the individual organisms per se, it does outline them. The unstained bacilli appear as slender, straight or slightly curved, colorless rods highlighted against a dirty blue-grey background. Thus, the terms “negative images” or “ghost bacilli” are used as descriptors (Fig. 4) (10, 103, 149). This characteristic negative image is presumably the result of hydrophobic interactions of the water-based DQ stain with the lipid within the cell walls of the bacilli (126). The identification of these negative images within histiocytes and in the background is highly suggestive of mycobacteria. Lowering the substage condenser often increases the refractility of the bacilli and enhances their identification when they are scattered in the smear background (Fig. 5). However, it has been reported that patients being given antimycobacterial therapy with clofazimine may show crystal formation within macrophages that simulates these negative images (139). Acid-fast stains can be performed on smears, cytospin preparations, and cell block sections and should be used for confirmation. The identification of mycobacteria is not limited to respiratory specimens (Fig. 6) (47, 62, 74, 94, 173). Indeed, in my experience, the majority of rapid or on-site diagnoses of potential mycobacterial infections are from aspirations of superficial, mediastinal, and abdominal lymph nodes. In these aspirates, the presence of numerous organisms both within distended histiocytes and scattered in the background tends to be from mycobacterial infections other than tuberculosis. Although material is usually obtained for culture or fluorescence microscopy, PCR can now be used for the rapid detection and identification of M. tuberculosis (44, 48, 86).

FIG. 3.

Granuloma and necrosis in a lung FNA specimen. Granulomas composed of epithelioid histiocytes with elongated or “footprint” nuclei are often associated with infections. When granulomas are identified, there should be a thorough search for microorganisms such as M. tuberculosis. DQ stain; magnification, ×200.

FIG. 4.

Mycobacteria presenting as negative images in a mediastinal FNA specimen. The background of Romanowsky stains is typically dark blue to purple. The slender bacilli are outlined as negative images or ghosts by the stain. DQ stain; magnification, ×400.

FIG. 5.

Mycobacteria in a lung FNA specimen. These bacilli may be present as scattered negative images in the smear background. Lowering the substage condenser increases the refractility of the mycobacteria and their likelihood of discovery. Erythrocytes stain grey-green. DQ stain; magnification, ×264.

FIG. 6.

Mycobacteria in a cervical lymph node FNA specimen. Acid-fast stains can be performed on most cytologic specimens. The presence of abundant organisms suggests mycobacterial species other than M. tuberculosis. Acid-fast stain; magnification, ×800.

Helicobacter pylori.

The association between gastric and duodenal ulcers, mucosa-associated lymphomas, and H. pylori has increased the need for detection of these organisms. H. pylori may be identified in endoscopic brushing specimens as well as in touch or imprint preparations from small gastric biopsy specimens (39). Cytologic specimens often reveal aggregates of glandular cells, with or without atypia, in a background of acute and chronic inflammation. The small, curved organisms are found, often in linear arrangements, close to the apical surface of the glandular cells. The Giemsa stain or DQ stain readily stains this tiny (1- to 3-μm), spiral-shaped organism (38, 174). The organism is closely associated with mucin, often with the long axis of the bacteria oriented parallel to the mucus strands (135). H. pylori can also be visualized with PAP, Gram, Warthin-Starry, and Dieterle stains (39, 110).

Actinomyces spp.

Actinomyces spp. are gram-positive bacteria that are frequently present in tonsillar crypts; they may be identified in aspirates from the head and neck and other body sites, as well as in gynecologic and respiratory specimens (58, 97, 122, 123, 138). In cytology specimens, the organisms present as fragments and tangles of slender filaments branching at acute angles, usually in a background of suppurative inflammation. Sulfur granules, accumulations of these organisms that show a very dark granular center with peripherally radiating filaments, are numerous (66). Necrosis and abscess formation may also accompany these infections (Fig. 7) (37). Occasionally, respiratory specimens show small clusters of filamentous bacteria that may represent oral contamination or infection by an Actinomyces sp. or by Nocardia spp., which are morphologically similar to Actinomyces spp. Actinomyces organisms are slightly larger (1 to 1.5 μm in diameter, 20 to 70 μm in length) than Nocardia spp. and branch at acute angles. Most cytologic and histologic stains, including silver stains, can be used to identify Actinomyces spp.; however, unlike Nocardia spp., Actinomyces spp. are not acid fast when stained with modified acid-fast stains. Other useful special stains include PAS and a modified Gram stain.

FIG. 7.

Actinomyces sp. in a lung FNA specimen. This organism is commonly encountered in respiratory and gynecologic specimens, often as scattered haphazard collections of grey, slender filamentous organisms. PAP stain; magnification, ×400.

Actinomyces spp. can also be seen in cervicovaginal smears and are usually associated with intrauterine contraceptive devices, foreign bodies, and vaginal pessaries (64, 65). While the diagnosis of Actinomyces in Pap smears is usually not challenging, hematoidin crystals and even dense clumps of spermatozoa occasionally resemble sulfur granules, while mucin and fibrin threads and tangles may mimic the slender filaments of Actinomyces spp. Septate hyphae causing eumycotic mycetoma and nonbranching bacilli or cocci causing botryomycosis may form sulfur granule colonies that can be differentiated from actinomycotic granules with GMS and Gram stains.

Nocardia spp.

Nocardia spp. have enough morphologic and staining differences to allow reasonably accurate classification and differentiation from Actinomyces spp. Nocardia spp. are primarily opportunistic pulmonary pathogens. Nocardia pneumonia occasionally simulates a mass; therefore, it may be encountered in FNA specimens. In these specimens, Nocardia spp. may be difficult to identify due to a background of chronic inflammation, suppurative inflammation, and necrosis. Unfortunately, this background is rather nonspecific and is common to a variety of infections that include mycobacterial and fungal species, as well as neoplastic processes. Although larger in diameter than mycobacteria, Nocardia spp. are narrower than Actinomyces spp. and fungal hyphae. The slender filaments (0.5 to 1.0 μm in diameter; 10 to 20 μm in length), usually branch at right angles rather than acute angles. Sulfur granules are not present. Appearing as a sheath or tangle of filamentous bacteria, Nocardia spp. are stained best by the Romanowsky stains, GMS, and modified acid-fast stains (Fite’s stain). Both Actinomyces and Nocardia spp. may be detected by the direct fluorescent-antibody test (124).

Legionella spp.

Although rarely identified in cytologic material, Legionella is stained by DQ, Giemsa, Gram, and silver stains such as Warthin-Starry and Dieterle stains and poorly stained by Gram, PAP, and H&E stains (11, 43, 170). These aerobic, motile bacteria appear as slender, short rods that are often present within the cytoplasm of neutrophils and macrophages (163). Legionella infection can be accompanied by an acute inflammatory response. The presence of numerous neutrophils and fibrin makes identification of this organism difficult even when there is clinical suspicion. Confirmation of Legionella spp. by direct fluorescent-antibody testing is recommended (84).

Parasites

There are numerous, scattered, single case reports and small series that discuss the identification or diagnosis of a variety of parasites in cytologic specimens. The recognition of previously rare parasitic infections has increased dramatically since the advent of AIDS and immunosuppressive therapies. The parasites discussed below represent a few of the more commonly encountered organisms in the cytopathology laboratory. As with bacterial infections, recognition of a parasite is highly dependent on the available clinical information and good cytopreparatory and staining techniques.

Trichomonas vaginalis.

Trichomonas vaginalis is usually suspected clinically in patients who present with a green-yellow, malodorous discharge. Visualization of small punctate hemorrhages on the cervix is also a strong indicator of this parasite. Although the clinician may perform wet mount or hanging-drop slides to detect this motile organism, its presence is usually confirmed when the Pap smear is evaluated. The typical cytologic presentation is that of scattered individual or small clusters of pear-shaped organisms that have a slight cyanophilic tinge, faint eccentric nuclei, and fine acidophilic granules. Occasionally, flagella are visible at higher magnification (Fig. 8). Aggregates of neutrophils and degenerating squamous cells are seen throughout the smear background. A nonbranching filamentous bacillary structure, formerly referred to as Leptothrix, is often associated with T. vaginalis infection (22).

FIG. 8.

T. vaginalis in a Pap smear. This pear-shaped, flagellated parasite is often associated with slender bacillary structures. Scattered debris, bacteria, and neutrophils contribute to the hazy background. PAP stain; magnification, ×660.

Giardia lamblia.

The gastrointestinal parasite Giardia lamblia is occasionally encountered in washings, brushings, or aspirates of the gastrointestinal tract, particularly the duodenum, stomach, and pancreas. There are also rare reports of this organism being identified in bronchoalveolar lavage and peritoneal fluid specimens (24, 150). G. lamblia has quite a characteristic cytomorphology, making diagnosis relatively easy when the organism is present (Fig. 9) (39). Trophozoites of this protozoan are flat, pear-shaped flagellates that are bilaterally symmetrical with four pairs of flagella and two nuclei containing large central karyosomes (152).

FIG. 9.

G. lamblia in a pancreatic FNA specimen. These distinctive parasites were aspirated from a pancreatic cyst. Note the prominent, symmetric nuclei and delicate flagella. PAP stain; magnification, ×860. Courtesy of Barbara Centeno.

Strongyloides stercoralis.

Although Strongyloides stercoralis is an intestinal nematode, larvae occasionally spread to the respiratory tract. This may result in clinical symptoms ranging from cough to hemoptysis and pulmonary infiltrates (168). The filariform larvae of S. stercoralis may also be encountered in respiratory specimens, the result of a superinfection in immunosuppressed individuals. The organism is eosinophilic on PAP stains and ranges between 400 to 500 μm in length in cytologic preparations (Fig. 10). Its internal structure, a closed gullet, and V-shaped notched tail help distinguish this parasite from cotton and synthetic fiber contaminants that are occasionally encountered. Occasionally, a negative image on air-dried, DQ stained slides is obtained when the larvae move or are dislodged from their original position before staining (Fig. 11).

FIG. 10.

S. stercoralis in a bronchial washing specimen. The filariform larvae of S. stercoralis stain brightly eosinophilic with PAP stain. Internal structures (gullet) help distinguish it from contaminants. The associated bronchial cells may be used as a size reference. PAP stain; magnification, ×264. Courtesy of Ellen Greenebaum.

FIG. 11.

S. stercoralis in a bronchial washing specimen. Romanowsky stains require air-dried slides. The parasite may shift or be dislodged from its original position before the slides are stained. This creates a negative image. DQ stain; magnification, ×352. Courtesy of Ellen Greenebaum.

Echinococcus granulosus.

Echinococcus granulosus is encountered in FNAs of the liver and rarely of other body sites (8, 53, 73, 104). Infections in humans are typically the result of exposure to dogs infected with this tapeworm. Larval forms, the causative agent of echinococcosis (also known as hydatid disease), invade intestinal walls and gain access to the bloodstream. The organisms come to reside in the liver, creating the radiologic appearance of a single or multilocular cyst (32). It was previously believed that aspiration of presumptive hydatid cysts was exceedingly dangerous due to dissemination of the disease and the potential for anaphylaxis when fluid was released into the surrounding tissue (125, 134). However, more recent studies have determined that this danger is substantially decreased by the use of fine needles (93, 104). Aspirates of the cyst fluid are dark brown, resembling “anchovy paste.” In addition to inflammatory cells (including eosinophils), scolices with attached hooklets (Fig. 12) and detached hooklets are often present in the dense, granular background (5, 12, 79, 98). Occasionally hooklets stain faintly or not at all by the PAP stain or Romanowsky stains, but they can still be visualized as refractile structures against the background debris, especially if the substage condenser is lowered (Fig. 13).

FIG. 12.

E. granulosus in a liver FNA specimen. Intact organisms and fragments of scolices and hooklets may be recovered from aspirates of echinococcal cysts. These organisms stain eosinophilic with PAP stain. Hooklets are often refractile. PAP stain; magnification, ×400. Courtesy of Terri L. Johnson.

FIG. 13.

E. granulosus in a liver FNA specimen. Aspirated hydatid cyst fluid occasionally contains abundant granular debris that can obscure organisms. Detached hooklets (center) are often poorly staining and are identified primarily by their refractility. PAP stain; magnification, ×1,000.

Amebic abscesses may also occur as cavitary hepatic lesions that yield dark brown granular material when aspirated. This material usually does not contain the parasites, since amebae are located predominantly in the wall of the abscess cavity. In this instance, the indirect hemagglutination serologic test and/or immunoperoxidase testing on aspirate material is useful to confirm the cytologic impression (88).

Cryptosporidium parvum.

Cryptosporidium parvum is usually encountered in gastrointestinal washings, brushings, and biopsy specimens. Although there is no tissue damage, these opportunistic pathogens can cause fairly severe watery diarrhea in children and immunocompromised patients. Cryptosporidia can be seen either as scattered free-lying organisms or as spherical structures aligned on the luminal surface of glandular intestinal cells. Acute inflammation is usually present (Fig. 14) (142, 153). The small (2- to 4-μm) cysts of these coccidial protozoans often appear stippled when stained with Romanowsky stains. This organism can also be seen with H&E, PAP, and modified acid-fast stains.

FIG. 14.

C. parvum in a gastrointestinal brushing specimen. These small, round parasites can be seen intimately associated with the apical surface of reactive glandular cells (arrow). PAP stain; magnification, ×900.

Fungi

With rare exceptions, most fungi are diagnosed in cytology specimens by their morphologic rather than staining characteristics (Table 4). This approach, combined with the proper clinical information, is quite accurate for the majority of fungal organisms encountered. The presence of hyphal and/or yeast structures begins a mental algorithm that uses size, shape, budding, and branching characteristics to narrow the differential diagnosis. Although infrequently used immunologic techniques are available to specifically identify a variety of fungi (112, 114).

TABLE 4.

Staining characteristics of common fungi

Fungus	Staining witha:
	PAP	Romanowsky	H&E	GMS	PAS
Candida spp.	+	+	+	+	−
Aspergillus spp.	+	Variable	+	+	+
Zygomycetes	+	Poor	+	±	Poor
Yeasts other than Candida spp.b	+	±c	+	+	+

^aPAS, periodic acid-Schiff; +, visible with stain; −, does not stain. 

^bSee Table 5 for selected organisms. 

^cYeasts stain with variable intensity with Romanowsky stains. 

Pneumocystis carinii.

Pneumocystis carinii has been difficult to classify and is currently considered a fungus. It is one of the most common causes of nonbacterial pneumonia in infants and immunocompromised patients. Fortunately, it has a characteristic clinical and radiologic presentation, and so cytologic specimens are usually sent with a specific request for identification of this organism. Before the success of direct fluorescence methods, bronchoalveolar lavage was the traditional procedure for obtaining a high-yield specimen (56, 85). Although P. carinii has been identified in a wide variety of specimens, it is still identified primarily in respiratory material (bronchoalveolar lavage fluid, washings, brushings, and sputum) and in lymph node aspirates during fulminant infections.

A distinctive feature of P. carinii in cytologic preparations is the presence of ill-defined, amorphous or foamy alveolar casts. These casts are a pale, almost translucent, green on PAP, slightly eosinophilic on H&E, and pale to dark purple on DQ; they represent numerous superimposed circlets that are outlines of the cysts (59, 61). Although most cytologists are familiar with the appearance of this organism on PAP stain, this stain does not specifically stain the organism. Confirmatory silver stains may be used to document the presence of cysts. Silver stains often yield characteristic single or paired dots or thickenings resembling parentheses in the cyst, which help distinguish the cysts from a background of stained erythrocytes (Fig. 15) (118, 137). Cysts range from 5 to 8 μm, are usually rounded, crescentic, or cup shaped, and contain six to eight small (1- to 2-μm) ovoid trophozoites. While the cyst walls may be refractile in routine stains, the DQ, Wright, and Gram-Weigert stains have an advantage over the PAP stain because they stain not only the foamy alveolar casts but also the individual trophozoites (40, 125). This results in a distinctive stippled appearance of the casts (Fig. 16). Lowering the substage condenser causes refractility of the cysts and accentuates this appearance. Thus, the DQ stain can be used as a simple and rapid diagnostic stain. Other rapid stains and techniques, such as immunoperoxidase, have been developed for the detection of P. carinii in respiratory specimens and may be used if conventional methods give negative results (9, 26, 35, 131, 152, 169).

FIG. 15.

P. carinii in a bronchoalveolar lavage specimen. Although DQ stains can be used to identify trophozoites, silver stains, which stain the cyst wall, remain the traditional confirmatory stain. GMS stain; magnification, ×1,000.

FIG. 16.

P. carinii in a bronchoalveolar lavage specimen. Individual trophozoites are stained dark purple within the lighter-staining alveolar cast. This distinctive stippling is seen with Romanowsky-stained preparations. This makes DQ stain a good choice for a rapid staining procedure. DQ stain; magnification, ×1,000.

Candida spp.

Small budding yeasts (3 to 4 μm) and pseudohyphae are typically generically identified as Candida spp. This organism is probably the most frequently encountered fungus in cytologic specimens, is often identified in Pap smears, and is present in urine and sputum specimens mostly as a contaminant. Although not considered significant when seen in these specimens, it can be an opportunistic pathogen in debilitated patients, particularly when found in the oral cavity (causing thrush). When it is encountered in lower respiratory tract specimens, it should be reported as a possible pathogen. When Candida spp. are encountered on Pap smears and esophageal brushings, they are often seen as hyphal spears piercing clusters of squamous cells (Fig. 17) in a background of acute inflammation. PAP and DQ stains are adequate to illustrate their morphology, although if necessary, their identity can be confirmed with silver stains. The DQ stain is particularly useful for urine and sputum specimens, in which both hyphae and yeast forms are highlighted as darkly stained objects admixed with inflammatory cells and debris (Fig. 18).

FIG. 17.

Candida sp. in sputum. The pseudohyphae of Candida often pierce clusters of squamous cells. Background inflammation is usually present. PAP stain; magnification, ×1,000.

FIG. 18.

Candida sp. in sputum. Both yeast and hyphal forms are darkly stained with Romanowsky stains and are easily identified at scanning magnifications. Pseudo-septations are often colorless and are in sharp contrast to the dark hyphal forms. DQ stain; magnification, ×680.

Fungi that present primarily as yeast forms are challenging because of the tremendous overlap in their clinical, radiologic, and cytologic presentations. However, there are usually enough clues for the cytopathologist to accurately distinguish among the four most common of these fungi: Histoplasma capsulatum, Cryptococcus neoformans, Blastomyces dermatitidis, and Coccidioides immitis (Table 5). These organisms are most commonly identified in respiratory specimens, lymph nodes, and CSF of immunocompromised patients (77, 78).

TABLE 5.

Cytologic features of selected yeast genera

Criterion	Characteristic for:
	Candida spp.	Cryptococcus spp.	Blastomyces spp.	Histoplasma spp.	Coccidioides spp.
Size (μm)	3–4	5–15	8–20	2–5	5–100 (mean, 10–40)
Shape	Oval	Round-oval, clear capsule	Spherical, double-contoured wall	Oval, endocellular	Spherical, refractile
Budding	Broad-based pseudohyphae	Narrow based	Broad based	Single, narrow	Endospores
PAP stain	Cyanophilic	Pale, cyanophilic	Blue-green	Dark outline	Orangeophilic
DQ stain	Dark blue-purple	Blue-purple	Pale blue	Dark blue	Colorless or light blue
Special stain	GMS, PASa	GMS, mucicarmine, alcian blue	GMS, PAS	GMS, PAS	PAS, GMS

^aPAS, Periodic acid-Schiff. 

Cryptococcus neoformans.

Cryptococcal infection is usually subacute or chronic and is associated primarily with pulmonary and/or central nervous system (CNS) involvement, although other organs and sites can be affected (155, 159). In aspiration specimens this organism is usually abundant, found individually or as clusters with accentuated capsular halos (144, 164, 167). Acute inflammation and necrosis may obscure the organism, which can stain variably. In DQ-stained preparations, the purple yeasts with accentuated clear halos against the dark purple background give the smear a punched-out appearance (Fig. 19) (127). In CSF specimens, Cryptococcus may resemble erythrocytes or even starch granules and as a result may be overlooked (Fig. 20). A useful cytologic feature is the appearance of the teardrop-shaped, narrow-based budding of C. neoformans. At times, the daughter bud will not detach and repetition of budding yields small chains of daughter progeny (Fig. 21). Diagnosis of C. neoformans still rests primarily on its cytomorphology, with confirmation, as necessary, by special stains. One of the smallest fungi, this yeast typically ranges from 5 to 10 μm in diameter but may vary from 2 to 20 μm. Occasionally, when these yeasts are engulfed by histiocytes or when they are nonencapsulated, they are mistaken for H. capsulatum. Special stains may be performed to confirm the cytologic impression. Silver stains, such as GMS, stain the organism itself, while mucicarmine, PAS, and alcian blue stain the mucopolysaccharide capsule (95).

FIG. 19.

C. neoformans in a cervical lymph node FNA specimen. This organism is seen as small, purple yeast forms surrounded by large, clear capsules in a darkly stained background of blood, lymphocytes, and debris. This punched-out staining pattern is appreciated best with Romanowsky stains. DQ stain; magnification, ×1,000.

FIG. 20.

C. neoformans in CSF. C. neoformans can vary in size, with indistinct capsules. These yeasts may appear as a contaminant to the neophyte, particularly if there is accompanying peripheral blood contamination. PAP stain; magnification, ×1,000.

FIG. 21.

C. neoformans in a lung FNA specimen. Mucicarmine, used to detect capsular acid mucopolysaccharides, is an excellent confirmatory stain. Occasionally there is incomplete separation of daughter progeny, resulting in chains of yeast cells. Dust-filled alveolar macrophages are also present. Mucicarmine stain; magnification, ×760.

Blastomyces dermatitidis.

Yeast-like spherical cells of Blastomyces dermatitidis are generally larger than those of cryptococci (8 to 20 μm) and have refractile double-contoured walls. Broad-based budding is a useful criterion to differentiate B. dermatitidis from the other dimorphic yeasts. Although larger, cells of B. dermatitidis are sometimes very difficult to identify on cytologic preparations, chiefly because they are not numerous and are often out of the plane of focus. The latter problem is due to their rigid cell walls, which resist compression by a coverslip (Fig. 22). To compound matters, acute and/or granulomatous inflammation of different degrees is present in most specimens. Although most often identified extracellularly, these organisms can also be engulfed by macrophages. Cytomorphology is usually sufficient for diagnosis; however, fluorescence techniques can be used for confirmation (154).

FIG. 22.

B. dermatitidis in sputum. The characteristic broad-based budding and double-contoured cell wall help differentiate this yeast from C. neoformans. PAP stain; magnification, ×630. Courtesy of Barbara Benstein.

Histoplasma capsulatum.

Histoplasma capsulatum is one of the smallest dimorphic fungi and is found predominantly intracellularly, as foamy or vacuolated structures within the cytoplasm of histiocytes. Like the other yeasts, H. capsulatum is most often encountered as the cause of pulmonary infection, but disease can also be systemic. Macrophages with engulfed organisms are typically present in a background of granulomatous inflammation that can also include necrosis, acute inflammatory cells, and lymphocytes (113, 146, 160). If present in small numbers, H. capsulatum cells can be very difficult to visualize on PAP and H&E stains. The DQ stain is useful because it stains these organisms a dark purple, which is usually in stark contrast to the pale-staining cytoplasm (Fig. 23). At times the organism can retract from its wall, creating a clear space that mimics the C. neoformans capsule (Fig. 24). If the differential diagnosis includes leishmaniasis, recognition of a nucleus and kinetoplast in the organism and the absence of budding will exclude histoplasmosis (6, 92, 120). Confirmatory stains with GMS should be performed when this organism is presumptively identified.

FIG. 23.

H. capsulatum in a bronchoalveolar lavage specimen. This is one of the smallest yeasts encountered in cytologic specimens. It is stained dark purple with Romanowsky stains and may be difficult to differentiate from Leishmania spp. DQ stain; magnification, ×1,000. Courtesy of David Mensi.

FIG. 24.

H. capsulatum in a bronchoalveolar lavage specimen. H. capsulatum cells are usually detected as intracellular organisms in the cytoplasm of macrophages and histiocytes. PAP stain; magnification, ×830. Courtesy of David Mensi.

Coccidioides immitis.

Coccidioides immitis is one of the largest of the dimorphic fungi and is particularly associated with both acute and chronic respiratory infections in the southwestern United States. It may be accompanied by mild acute, chronic, and/or granulomatous inflammation and is commonly seen within macrophages (Fig. 25, left). This organism presents as endospores within thick-walled mature spherules that can vary tremendously in size. C. immitis can resemble a variety of other fungi, pollen grains, and even inorganic contaminants (49, 52, 69, 129). Endospores are not visible in immature spherules; combined with the thick wall of C. immitis, this often results in an erroneous identification as B. dermatitidis. When mature, spherules of C. immitis contain numerous small (2- to 5-μm) endospores. Once the endospores are separated from the spherule, they may resemble H. capsulatum or Toxoplasma gondii, and a careful search for older, folded, and fractured spherules is necessary (30). Conversely, when these older spherules are seen without intact, mature spherules, they may be overlooked as inorganic contaminants. This organism is very easy to detect in PAP-stained preparations because of its orangeophilic staining (Fig. 25, right). The spherules stain lightly or not at all with the DQ stain, although with experience, this lack of staining is a useful clue. PAS with a light green counterstain is a useful confirmatory stain, since the spherules stain a vivid magenta against a pale green background. Like Aspergillus spp., chronic respiratory infections with C. immitis can result in the formation of lung cavities or nodules. Rarely, these nodules are exposed to air, resulting in the development of mycelial forms of C. immitis with barrel-shaped arthroconidia. One of the problems associated with recognition of this rare phenomenon is the misinterpretation of the mycelial forms as a second infectious agent, such as an Aspergillus spp., particularly since multiple infections are common in immunosuppressed individuals.

FIG. 25.

C. immitis in sputum. (Left) The spherules of C. immitis are often colorless or poorly stained with Romanowsky stains and may resemble pollen or other contaminants, particularly when seen inside large multinucleated giant cells. Endospores are not present within fractured spherules. DQ stain; magnification, ×1,000. (Right) The organism often stains orangeophilic with PAP stains. Its thick, refractile walls are often easily recognized at scanning magnifications. PAP stain; magnification, ×1,000.

Aspergillus spp.

When a cytologic specimen contains hyphal structures that are relatively large (3 to 6 μm in diameter), septate, with regular, progressive dichotomous branching at 45° angles, an Aspergillus sp. should be immediately suspected. These organisms, often opportunistic pathogens, are usually identified in respiratory specimens and rarely found in other sites (14). The organisms commonly present as tangled clusters of branched hyphae that are accompanied by acute inflammation, necrosis, and cellular debris. Aspergillus spp. stain fairly well with PAP and H&E stains, which clearly delineate the hyphal septations (Fig. 26). The organisms also stain with DQ; however, the staining is often extreme, i.e., either too dark to distinguish internal structure or too light so that the fungus blends into the dirty background. Septations are often colorless, creating a negative image that contrasts with the darkly stained hyphae (Fig. 27). Sheaves or rosettes of needle-like, birefringent crystals, representing calcium oxalate, may be identified, particularly in respiratory tract specimens (46, 96).

FIG. 26.

Aspergillus sp. in a lung FNA specimen. Although this organism is often associated with necrosis and cellular atypia; it may also appear as large isolated tangles of branching, septated hyphae. PAP stain; magnification, ×1,000.

FIG. 27.

Aspergillus sp. in a bronchial washing specimen. This organism stains variably with Romanowsky stains; the septations vary from colorless to indistinct. Alveolar macrophages are seen in the background. DQ stain; magnification, ×1,000.

Infection with Aspergillus spp. can cause a necrotizing pneumonitis with central cavitation, which presents radiologically as a cavitary lesion. The immediate concern in patients, particularly smokers, is the possibility that this mass may represent a squamous cell carcinoma with central necrosis. Thus, FNA is often selected as the diagnostic procedure of choice. A diagnosis of infection by FNA will spare the patient a more invasive procedure. Although malignant squamous cells do not resemble fungal hyphae, active infection by Aspergillus spp. can cause the surrounding lung parenchyma to become quite atypical. Severely reactive cells may mimic carcinoma, which can result in a false-positive diagnosis with potentially serious consequences for the patient (140). Unlike most FNAs, which attempt to sample the center of a mass to maximize the recovery of material, FNAs of cavitary lesions are directed away from the presumed necrotic center in favor of the peripheral viable lesional tissue. In aspergillomas, this is precisely where viable fungus will be located. Recognition of these branched fungal structures can still be difficult if necrotic debris and inflammation are also present. Even if close scrutiny reveals no organisms, in the absence of a positive diagnosis of carcinoma it is recommended to perform staining with silver stains to completely exclude the possibility of an infectious agent. Occasionally when the fungus cavity is exposed to the air, fruiting bodies with conidia can be detected in cytologic specimens; their presence helps confirm the presence of Aspergillus spp. and can be used in further identification.

Zygomycetes.

The zygomycetes (phycomycetes) include Mucor, Absidia, and Rhizopus spp. and are characterized by their wide (3- to 25-μm) ribbon-like hyphae that branch irregularly and are pauciseptate (Fig. 28). These organisms are opportunistic pathogens that have a propensity for the head and neck region (orbit, paranasal sinuses, and palate), in addition to the lungs and skin. Because these infections are often located close to the CNS and have a rapid, potentially fatal course, an immediate diagnosis is extremely useful to clinicians. Like Aspergillus spp., the zygomycetes are most often encountered in aspiration specimens (23). Usually associated with prominent acute inflammation and necrosis, these organisms can be overlooked on DQ stains and are best seen on PAP or H&E stains. Silver stains are generally used for confirmation.

FIG. 28.

Mucor sp. in sputum. Staining of zygomycetes is variable with the PAP stain. In this specimen, the broad, nonseptate eosinophilic hyphae are almost translucent and are partially obscured by inflammatory cells. PAP stain; magnification, ×256.

Viruses

Although individual viruses cannot be detected by light microscopy, their cytopathic effects on infected cells are often visible, even in routine cytologic preparations. The recognition of nuclear and/or cytoplasmic inclusions provides a fairly specific indication of viral infection, while perinuclear halos and ciliocytophthoria and detachment of apical cytoplasm with ciliary tufts represent more subtle clues (Fig. 29) (117, 121). Regenerative/reparative atypia often accompanies these more specific cellular alterations. The most common problem is misinterpretation of these cytologic features as carcinoma. Clinical and radiologic correlation is still a necessity. In cytologic preparations, the three most commonly recognized viral infections are those caused by HSV, CMV, and human papillomavirus (HPV) (51).

FIG. 29.

Ciliocytophthoria in a bronchial washing specimen. Viral infections of the respiratory tract, particularly adenovirus infections, produce alterations in the bronchial epithelium. Ciliocytophthoria is the detachment of the apical cytoplasm, terminal bar, and cilia from columnar respiratory cells (arrow). PAP stain; magnification, ×586. Courtesy of Barbara Benstein.

Herpes simplex virus.

HSV is a common pathogen in Pap smears and is most prevalent in young, sexually promiscuous patients. It is also frequently found in gastrointestinal and respiratory specimens from immunocompromised patients (80, 161). Because the key cytologic feature of HSV is the conspicuous nuclear changes seen in infected cells, the PAP stain is the most desirable cytologic stain for diagnosis. Nuclear alterations may present as enlarged degenerated nuclei with smudged or homogenized “ground-glass” or slate-grey nuclei (Cowdry B nuclei) (Fig. 30). Alternatively, large, multinucleated cells may be present. Their nuclei are typically molded, each containing a prominent eosinophilic inclusion (Cowdry A nuclei) that has given rise to the term, “eggs in a basket” (Fig. 31). HSV produces no cytoplasmic inclusions. Occasionally, HSV is detected in Romanowsky-stained preparations from immunocompromised patients. While the nuclear detail is not crisply delineated, subtle shading within the nucleus suggests inclusions. This appearance, combined with multinucleation and nuclear enlargement, suggests a viral cytopathic effect, most probably due to HSV (Fig. 32). While routine stains may provide indirect evidence for HSV, confirmation can be achieved by a variety of means, including culture, immunocytochemistry, immunofluorescence, and in situ hybridization techniques (18, 111).

FIG. 30.

HSV in a Pap smear. “Ground-glass” nuclei, chromatin margination, and multinucleation are indicative of HSV infection. Cellular enlargement, while nonspecific, is also present. PAP stain; magnification, ×900.

FIG. 31.

HSV in a Pap smear. Prominent, single, eosinophilic inclusions are conspicuous in these infected cells. PAP stain; magnification, ×950.

FIG. 32.

HSV in a bronchoalveolar lavage specimen. Romanowsky-stained preparations accentuate cellular enlargement, even in the presence of obscuring inflammation. Multinucleation and nuclear inclusions appear as subtle nuclear shading. DQ stain; magnification, ×400.

Cytomegalovirus.

CMV is also a common problem in immunocompromised patients, especially renal transplant recipients (1). This virus can affect almost any body site, and, as a result, virally infected cells can appear in almost any type of cytologic sample. In contrast to HSV, CMV has both nuclear and cytoplasmic inclusions and can be identified with both PAP and DQ stains. As the name implies, infected cells are often dramatically enlarged compared to their uninfected counterparts. The prominent, often eosinophilic, “owl’s-eye” nuclear inclusion with marginated chromatin that results in a halo effect is best visualized with PAP stain (Fig. 33). The numerous small cytoplasmic inclusions are stained bright magenta with DQ and are easily identified even at scanning magnifications (Fig. 34). With practice, nuclear changes can be identified with the DQ stain; however, these can also be misinterpreted as changes secondary to cancer (165). Like HSV, culture and molecular techniques may be used to confirm the presence of CMV (70, 171).

FIG. 33.

CMV in a Pap smear. Although rare, CMV may be encountered in Pap smears. The typical cytologic features of cellular enlargement, prominent intranuclear inclusions, and occasional binucleation are present in this specimen. Cytoplasmic inclusions are often indistinct with PAP stains. PAP stain; magnification, ×950.

FIG. 34.

CMV in a bronchial washing specimen. Romanowsky-stained preparations are useful when CMV is suspected. Cytoplasmic inclusions stain a deep magenta and, when combined with cell enlargement and intranuclear “owl’s-eye” inclusions, allow rapid recognition of infected cells at scanning magnifications. Intranuclear inclusions are visible on DQ stains but are not as distinct as those stained with PAP stain. DQ stain; magnification, ×740.

Respiratory viruses.

Numerous other viruses have been associated with respiratory infections; they include adenovirus, respiratory syncytial virus, influenza and parainfluenza viruses, and measles virus. While a variety of cytologic features such as multinucleation, smudged nuclei, and even rather generic nuclear and cytoplasmic inclusions may be seen, these infections are almost never diagnosed outright on the basis of cytologic specimens unless there is strong supportive clinical and laboratory data (2, 67, 119, 177). Viral cultures, immunofluorescence, and DNA probes represent more specific and reliable techniques for the diagnosis of these infections as appropriate (41, 75, 102).

Human papillomavirus.

Once the distinctive cytologic manifestations of HPV were described and subsequently linked to the development of dysplasia and its progression to squamous cell carcinoma of the cervix, the identification of HPV became extremely important. Sampling of the cervical transformation zone, which is most vulnerable to this infection, is an integral part of the Pap smear (15, 16, 106–108). The detection of HPV in Pap smears rests predominantly on the identification of cells known as koilocytes, derived from the Greek word “koilos” meaning hollow or cavity (89). Recognition is based on three features: marked density of the cytoplasm peripheral to the cavity, amphophilic cytoplasm, and enlarged hyperchromatic nucleus (Fig. 35) (106). Other cytologic features that are often associated with HPV infection include bi- and multinucleation, plaques or aggregates of parakeratosis and hyperkeratosis, and anucleate squames (63). Although other infections, including fungal (Candida spp.), protozoal (T. vaginalis), and bacterial infections, may cause small perinuclear or “inflammatory” halos, these structures can usually be distinguished from koilocytes, which are larger with abnormal nuclei. Several types of HPV have been identified, some of which are associated with the development of high-grade squamous intraepithelial lesions and squamous carcinoma. Although not considered cost-effective for screening of at-risk populations, molecular diagnostic techniques such as immunoperoxidase staining, in situ hybridization, and PCR are available for viral typing (28, 63, 136).

FIG. 35.

HPV in a Pap smear. HPV is identified predominantly in Pap smears; the hallmark of HPV infection is the detection of koilocytes. The large, irregular, hollow cavity in conjunction with nuclear enlargement and hyperchromasia and frequent binucleation are cytologic features of HPV effect. PAP stain; magnification, ×1,000.

Human polyomavirus.

Human polyomavirus, like HPV, is a member of the Papovaviridae family and is sporadically identified in urine specimens. This virus can infect normal individuals as well as immunocompromised patients. Although many patients are asymptomatic, some present with hematuria. Most infections tend to resolve spontaneously (91, 109). Infected cells can vary from few to abundant, and they occur as solitary cells rather than as cell clusters. They have enlarged nuclei with nuclear inclusions that almost fill the nucleus, leaving only thin rims or halos (Fig. 36). These large, opaque inclusions have been misinterpreted as a feature of cancer. As a result, these cells have been termed decoy cells (29, 34). Also in the differential diagnosis is CMV. In contrast to CMV, cells infected with polyomavirus are smaller, with thinner halos, less abundant cytoplasm, and no cytoplasmic inclusions. Immunodiagnostic technique can be used to confirm this diagnosis (7).

FIG. 36.

Human polyomavirus in a urine specimen. A solitary renal tubular cell with an enlarged, smudged nucleus consistent with polyomavirus infection is visible. PAP stain; magnification, ×600.

CONCLUSION

The utility of cytologic specimens in the diagnosis of infectious disease has been well established during the last 50 years. While morphology continues to be the mainstay of diagnostic cytopathology, recent developments in immunocytochemistry and molecular diagnostics allow not only rapid but also specific diagnosis and classification of many infectious agents. However, the cytologic identification of microorganisms, no matter how specific, is not intended to replace microbiologic techniques. Rather, cytology should be viewed as a means of rapid initial recognition of an infectious process by using safe, cost-effective methods of specimen procurement (Table 6).

TABLE 6.

Utility of cytology in the work-up of patients with infectious diseases

Facet of cytopathology	Advantages
Specimen procurement	Rapid and minimally invasive
	Minimal contamination
	Cost-effective
Specimen triage	Rapid assessment
	Additional material for cultures and special stains, immunodiagnostics, and molecular diagnostics
Patient perception	Rapid with minimal discomfort
	Reasonable cost
	Privacy
Clinician perception	Rapid turnaround time
	Assist in patient management, infection control procedure, and early therapy
Cytopathologist perception	Rapid diagnostic procedure, especially when combined with ancillary techniques, immunocytochemistry, and molecular diagnostics

In contrast to alternative procedures, such as the surgical biopsy, the cytologic techniques described in this review are minimally invasive; they are also less expensive and have more rapid turnaround times. In most cytopathology laboratories, routine preparation, staining, and diagnosis of nongynecologic specimens can be done within a 24- to 48-h period. Specimens may be processed much more rapidly, within minutes, if a diagnosis is needed emergently.

From the patient’s viewpoint, the collection of most cytologic specimens is a relatively minor procedure, which is often accomplished with a reasonable amount of privacy and little discomfort. Many specimens are obtained in FNA clinics or other outpatient settings. FNA, lumbar puncture, and paracentesis can be performed with minimal or no anesthesia and have few, if any, side effects. From the clinicians’ and hospital staff’s perspective, rapid identification of an infectious agent can direct patient management, allow earlier therapeutic intervention, and permit rapid initiation of infection control procedures if necessary.

Contamination of equipment and personnel is often a concern with presumptive or confirmed infectious cases. Cytologic specimens can be fixed and stained immediately with very limited exposure to the infectious material. Contamination of laboratory equipment, such as microtomes and cryostats for frozen-section diagnosis of infectious cases, can be avoided if techniques such as touch or imprint cytology are used on surgical specimens. In many instances, FNA can be used, even intraoperatively, to obtain material for rapid microscopic analysis and microbiologic culture, contaminating only the disposable needle used for the procedure.

Rapid or immediate identification of presumptive organisms in cytologic specimens also allows additional, sterile material to be obtained. It also yields information that allows the microbiology laboratory to evaluate specimens, selecting the most appropriate stains, media, and tests necessary for classification of the infectious agent. This selectivity is efficient and cost-effective. Conversely, following the preliminary interpretation, personnel in the microbiology laboratory can communicate their particular needs to the cytologist, suggesting the most appropriate means of collection and transport of specimens selected for microbiologic evaluation.

In summary, the usefulness of the cytopathology laboratory in the identification of infectious disease is directly related to its acceptance by clinicians and the clinical microbiology laboratory. While it is important to have cytologists skilled in the performance and interpretation of FNA and other cytologic techniques, diagnostic accuracy is greatly enhanced by communication. Current and accurate clinical, radiologic, and laboratory data must be available to the cytologist; conversely, concerns, suspicions, or preliminary interpretations of infectious agents should be discussed with clinicians, infectious disease consultants, and/or the microbiology laboratory staff.