Diagnosis of Infectious Diseases- a Cytopathologist’s Perspe

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CLINICAL MICROBIOLOGY REVIEWS,0893-8512/98/$04.000

Apr. 1998, p. 341365 Vol. 11, No. 2

Copyright 1998, American Society for Microbiology

Diagnosis of Infectious Diseases: a Cytopathologists PerspectiveCELESTE N. POWERS*

Department of Pathology, SUNY Health Science Center at Syracuse, Syracuse, New York 13210

INTRODUCTION .......................................................................................................................................................341PROCEDURES AND TECHNIQUES......................................................................................................................342

Sampling Procedures..............................................................................................................................................342Cytopreparatory Techniques .................................................................................................................................343Stains ........................................................................................................................................................................344

Ancillary Techniques ..............................................................................................................................................345COMMON PATHOGENS .........................................................................................................................................345

Contaminants ..........................................................................................................................................................345Bacteria ....................................................................................................................................................................346

Mycobacteria .......................................................................................................................................................346Helicobacter pylori ................................................................................................................................................347Actinomyces spp. ..................................................................................................................................................347Nocardia spp. .......................................................................................................................................................348Legionella spp. .....................................................................................................................................................348

Parasites...................................................................................................................................................................348

Trichomonas vaginalis..........................................................................................................................................348Giardia lamblia ....................................................................................................................................................350Strongyloides stercoralis........................................................................................................................................350Echinococcus granulosus......................................................................................................................................350Cryptosporidium parvum......................................................................................................................................352

Fungi.........................................................................................................................................................................352Pneumocystis carinii.............................................................................................................................................352Candida spp. ........................................................................................................................................................353Cryptococcus neoformans .....................................................................................................................................354Blastomyces dermatitidis ......................................................................................................................................354Histoplasma capsulatum.......................................................................................................................................354Coccidioides immitis .............................................................................................................................................354Aspergillus spp. ....................................................................................................................................................356Zygomycetes.........................................................................................................................................................358

Viruses ......................................................................................................................................................................358

Herpes simplex virus ..........................................................................................................................................359Cytomegalovirus ..................................................................................................................................................359Respiratory viruses .............................................................................................................................................359Human papillomavirus.......................................................................................................................................359Human polyomavirus .........................................................................................................................................360

CONCLUSION............................................................................................................................................................361REFERENCES ............................................................................................................................................................362

INTRODUCTION

This review will explore the utility of cytopathology in thediagnosis of infectious disease, with emphasis on the detectionand identification of common microorganisms in various cyto-logic specimens. Cytologic techniques of specimen procure-ment, staining, and rapid identification of infectious agents willbe 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 themedical literature of the 1800s. One of the first reported nee-dle aspirations has been traced to the mid-1880s (166). Perhaps

the first report that suggests the potential of needle aspirationin the diagnosis of infectious disease is a 1904 study by Griegand Gray (50, 60). In this study, needle aspirations of lymphnodes from patients with sleeping sickness revealed motiletrypanosomes. 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 pro-cedure occurred during the 1930s, primarily at the MemorialSloan Kettering Cancer Center in New York, where Martinand Ellis (100) and later Stewart (151) developed significantexperience in this technique. Further refinement of the fine-needle method, using small-diameter needles, took place inEurope following World War II. Concurrent with the devel-opment of FNA came the introduction and advancement ofother cytologic methods for the detection of cervical and lungcancer (42, 116). Since then, the role of diagnostic cytology hasexpanded tremendously. The availability of a variety of rapid,

* Mailing address: SUNY Health Science Center at Syracuse, 750East Adams St., Syracuse, NY 13210. Phone: (315) 464-7161. Fax:(315) 464-4267. E-mail: [email protected].

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safe, and cost-effective techniques and stains often places cy-topathology in the forefront of diagnostic evaluations, includ-ing 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 ofthree mechanisms: exfoliation, abrasion, or aspiration (Table1). The resultant specimens often are prepared by slightlydifferent methods, which if performed improperly may yieldinconclusive or misleading results. Degeneration, shearing,and distortion, as well as air drying, are the most commonproblems that render specimens virtually uninterpretable. Thecytopreparatory techniques discussed below are routinely usedin cytopathology laboratories; for additional information, thereader is directed to the Manual of Cytotechnology (82).

Exfoliative cytology relies on the presence of cells that areshed spontaneously into body fluids such as effusions obtained

from pleural, pericardial, or peritoneal cavities. Collection ofcells immersed in these fluids is considered only a minimallyinvasive procedure with little risk of complication. However,cellular degeneration may be a problem if the specimen is notcollected, fixed, transported, and prepared in the appropriatemanner. The volume of material collected can range from 1 to2 ml of cerebrospinal fluid (CSF) to 1 liter or more of ascites.While procedures may vary slightly depending upon the labo-

ratory, there are two basic practices: immediate fixation of thespecimen and rapid transport of the fresh specimen to thecytopathology laboratory (99). Immediate fixation and/or pro-cessing avoids further specimen degeneration. Sputum andurine collection is a noninvasive procedure that is also consid-ered exfoliative cytology. Although bronchoscopy and FNAtechniques have surpassed sputum analysis in diagnostic accu-racy, it still remains the simplest method for examination of therespiratory tract. Urine, by virtue of ease of collection, remainsone of the most common exfoliative specimens.

Abrasive techniques include endoscopic brushing, as well asmanual scraping. In most cytopathology laboratories, the Pap

TABLE 1. Cytologic methods and fixation and processing techniques

Method Examples Fixation Processing MicroscopyCommon

microorganisms

Exfoliative Sputum Immediate or Sac-comanos (Car-bowax/alcohol)

Blender Smears Candida spp., As-pergillus spp.,Cryptococcus spp.,

Blastomyces spp.Fresh Pick and smear

Urine Fresh or refriger-ated

Cytocentrifuge, mem-brane filtration

Cytospin preparations Candida spp., CMV,polyomavirus

Effusions Fresh or refriger-ated

Cytocentrifuge, cell block,membrane filtration

Cytospin preparations,smears, cell blocksections

CSF Fresh Cytocentrifuge, mem-brane filtration

Cytospin preparations C. neoformans, vi-ruses, T. gondii

Abrasive Pap smear Immediate fixation(alcohol)

Direct smear Smear Candida spp., Acti-nomyces spp.,Trichomonas spp.,HPV, CMV, HSV

Tzanck preparation Air dry Direct smear Smear Herpesviruses

Endoscopic brushings Immediate fixationor air dry

Direct smear Smear Body site dependent

Brush rinse Cytocentrifuge Cytospin preparations Fungi, viruses, para-sites

Aspiration Superficial: breast,thyroid, salivarygland, lymph node,etc.

Immediate fixationor air dry

Smear Smear Body site dependent(fungi, viruses,bacteria, para-sites)

Needle rinse Cytocentrifuge, cell block Cytospin preparations,cell block sections

Radiologically guided:lung, liver, pan-creas, kidney, etc.

Immediate fixationor air dry

Smear Smear Body site dependent(fungi, viruses,bacteria, para-sites)

Needle rinse Cytocentrifuge, cell block Cytospin preparations,cell block sections

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smear is the most common specimen of this type. A variety ofmembers of the normal flora, as well as infectious agents, canbe readily identified on these smears. Another classic exampleof abrasive cytology is the Tzanck preparation (158). This rapidand easy technique samples the base of dermal or mucocuta-neous vesicles and is often used to diagnose herpes simplex

virus (HSV) and varicella-zoster virus. Pap smears and Tzanckpreparations are submitted to the laboratory as fixed or air-

dried slides, respectively. One of the most technically demand-ing procedures in this category of abrasive cytology is endo-scopic sampling. The replacement of rigid endoscopes withflexible fiber-optic endoscopes has resulted in the ability to

visualize and directly sample greater portions of the respiratoryand gastrointestinal tracts by abrasive techniques of brushing,

washing, and lavage, as well as by needle aspiration biopsyprocedures. These specimens are usually semisolid and need tobe fixed or processed immediately to prevent artifacts. Tech-nically, washings and lavages can be categorized as either anexfoliative or abrasive technique: cells are shed into saline

which has been used to remove cells from the respiratory orgastrointestinal tract surface. A variety of bacteria, fungi, and

viruses can be identified in these specimens. A differentialdiagnosis is often generated prior to cytologic examination,

based predominantly on clinical history and the location of thelesion.The final technique, and perhaps the most powerful, is FNA,

which uses a small-gauge (usually 22- to 25-gauge) needle andnegative pressure to withdraw cells or fluid from a mass (72, 87,90, 148). Alternatively, this technique may be used withoutnegative pressure (capillary action will draw the specimen intothe hub of the needle) (175). FNA of superficial targets may beperformed by any physician with training in aspiration tech-niques (147, 148). It requires no sophisticated imaging proce-dures 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 aradiologist to direct placement of the needle by using com-puted tomography, ultrasonography, or other imaging tech-niques. With these techniques, masses in deep organs havebecome increasingly accessible to aspiration. Selection of thesize and type of needle is based on the location and potentialconsistency of the mass, as well as on aspirator experience.Many radiologists select large (18- to 20-gauge) needles; how-ever, when these needles are used, this procedure should notbe termed fine-needle aspiration biopsy (125). FNA biopsytraditionally uses 22-gauge or, preferably, smaller needles andpresents a much lower risk of the complications associated withthe use of large-gauge needles, such as hemorrhage, needletract seeding, and pneumothorax (19, 125). Direct smears areprepared from material obtained from FNA; however, needlerinses for cytospin preparations or cell blocks may also beprepared when appropriate.

Sampling procedures can be performed either by cyto-pathologists or by clinicians often in conjunction with cytotech-nologists. The direct involvement of a cytologist allows speci-mens to be triaged during the procedure, resulting in increaseddiagnostic accuracy and decreased complications. Variousrapid stains can be used on either air-dried or alcohol-fixedmaterial for immediate specimen evaluation. This is very ad-

vantageous since it not only determines specimen adequacy butalso results in the most appropriate selection of cytoprepara-tory and staining methods. Infectious agents can usually beidentified by cytomorphology with routine or special stains. Apreliminary or on site interpretation is particularly useful if

an infectious process is suspected, since an additional sterilesample can be obtained immediately for the microbiology lab-oratory. Specific organism classification and antimicrobial sus-ceptibility testing are definitely within the purview of the mi-crobiology laboratory (31, 170).

Cytopreparatory Techniques

Technical expertise begins, rather than ends, with specimencollection. As in clinical microbiology laboratories, cytopathol-ogy laboratories have guidelines for procurement, transporta-tion, and processing of specimens. Since light microscopy is thestandard for evaluation of cytologic specimens, cytoprepara-tory techniques center around placement of material on glassmicroscope slides. Specimens obtained by abrasion and aspi-ration techniques may be prepared directly by application ofthe material to glass slides (smears) followed by appropriatefixation. The traditional fixative for cytologic specimens is al-cohol. Usually this is 95% ethanol, but 100% methanol or 80%isopropanol may be used. Alcohol fixation causes cells toshrink because alcohol removes intracellular water. Alcohol isa coagulative fixative that results in sharp nuclear detail, butcytoplasmic features may be less well defined. Specimens allo-

cated for staining by Papanicolaous method require immedi-ate immersion in an alcohol fixative. If there is even a shortdelay, air drying occurs. Air-drying artifact is one of the mostcommon problems faced by a cytopathology laboratory, par-ticularly when specimens are obtained by inexperienced per-sonnel. Since air drying may render a specimen uninterpret-able, constant reinforcement of proper technique is necessary.

Another common problem, particularly with FNA specimens,is excessive blood. Clotting of even a small volume of bloodgreatly interferes with good smear preparation and subsequentinterpretation (126). The fibrin in clotting blood entraps cells,resulting in heavy and uneven staining and distortion of thesmear pattern. When direct smears are prepared without con-sideration of the blood present, the material inevitably coversa large area of the slide. In addition, cells and/or microorgan-isms, if present, are separated and diluted by the blood. Smearsshould be made only from the part of the sample contained inthe needle and its hub, which is rich in cellular material. Theblood which has been drawn into the aspiration syringe may beplaced entirely into a small tube of 10% buffered formalin andprocessed as a cell block.

When a specimen is largely fluid, additional preparation isrequired. Before transport, an equal volume of 50% alcoholcan be mixed with the specimen to provide initial fixation.Concentrations of alcohol higher than 50% should not be usedsince they result in coagulation of the cells, making subsequentspecimen preparation difficult. Alternatively, the specimen canbe submitted fresh and unfixed. This is best accomplished byimmediate transport to the laboratory; if that is not possible,the fluid can be refrigerated until transport. Good cell preser-

vation will be retained for at least 48 h. Sterility is not required

for routine cytology specimens. Refrigerated specimens arerarely overgrown by bacteria or fungi. Semisolid material fromaspiration and endoscopy specimens may be processed like afluid by rinsing the specimen into saline or cell culture mediumfor subsequent cytocentrifugation.

Cytocentrifugation can be used for liquid specimens. Thequantity and quality of the fluid, as well as the clinical impres-sion, will determine the specific centrifugation and stainingmethods. The purpose of both manual smear and cytocentrifu-gation techniques is to place a thin, uniform layer of sample onthe slide. For smaller volumes (5 ml), cytocentrifugation ofthe specimen directly onto glass slides (i.e., cytospin prepara-

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tions) can be performed (57). This results in an even distribu-tion of material over a limited area of the slide, usually 25 to50%. Larger volumes, usually obtained from effusions or as-cites, need preliminary centrifugation to consolidate the spec-imens into manageable volumes from which cytospin prepara-tions can be prepared. Cytospin preparations are the closestequivalent to the reproducibility of tissue sections and, as such,are preferred over smears for ancillary studies such as specialstains and immunochemistry. The number of slides prepared isdetermined by the amount of specimen and the clinical im-pression. When an infectious etiology is suspected, slides con-taining fixed but unstained material may be made if special

stains are required.Cell blocks can be quite valuable in exfoliative and aspira-

tion cytology (68). Material obtained for a cell block is pro-cessed like a small tissue biopsy specimen. Needle rinses thatcontain minute fragments of tissue can be centrifuged, and asmall button of tissue is obtained. This specimen is then fixed,paraffin embedded, sectioned, placed onto glass slides, andstained. Occasionally, the specimen is composed of loose,semiliquid material held together by fibrin. This loose clotmay also be processed as a cell block. In FNA, small tissuefragments or microcores are sometimes found in material ex-pressed from the needle hub or lodged within the needle tip.Evaluation of cell block sections from these minibiopsy spec-imens and clots is generally additive and/or supportive tothe smear impression. Cell blocks also provide reproducible

sections for ancillary studies that may include special stains formicroorganisms. With the reference section stained by theconventional histologic stain, hematoxylin and eosin (H&E),the location of any suspicious structure or probable organismcan be identified on the H&E slide and then compared withthe special stains.

Stains

The Papanicolaou (PAP) stain remains the traditional andmost frequently utilized stain for cytologic specimens. How-ever, the increasing popularity of FNA as a primary diagnosticprocedure has demonstrated the utility and adaptability of

other stains such as the Romanowsky stains and H&E. Withthe exception of Pap smears, which are always stained by Pa-panicolaous method, personal preference and experience willdictate which stains are used on other cytologic specimens, andmore than one stain can be used. The PAP stain, which en-hances nuclear detail, is often used in conjunction with one ofthe Romanowsky stains. Romanowsky stains are usually airdried rather than fixed immediately. This results in cellularswelling and loss of nuclear detail; however, cytoplasmic andbackground details are accentuated (Table 2). Surgical pathol-ogists with limited experience in cytopathology may be morecomfortable with the H&E stain, particularly with aspirate

material. Both PAP and H&E stains require rapid smear prep-aration and immediate alcohol fixation to avoid air-drying ar-tifacts. This is sometimes problematic when inexperience re-sults in delayed or improper smear preparation.

Smears designated for staining by the conventional Papani-colaou method are spray-fixed with or submerged directly in95% ethanol, methanol, or isopropanol. While immersion inalcohol often results in some degree of cell loss, care must alsobe taken with aerosol-spray fixatives to prevent freezing arti-facts that result from the propellant (71). A recently developedrapid PAP stain method helps to overcome problems withfixation and cell loss. This method has the advantage of beingperformed on air-dried smears, so that rapid preparation andfixation are less critical. Air drying also enhances the adher-ence of the cells to the slides, increasing cell recovery. The

smears are rehydrated briefly in normal saline, which tends tolyse erythrocytes, thereby reducing the obscuring effect of par-ticularly bloody smears. In addition to excellent preservation ofcell structure, this rapid method still preserves the nucleardetail expected with any PAP stain (172).

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

The term Romanowsky stain encompasses a variety of

TABLE 2. Comparison of DQ and PAP stains

Characteristic Properties DQ staina PAP stain

Technique Fixation Air dry Immediately in alcoholCell loss Minimal ModerateStain time 20 to 30 s 5 to 10 min (rapid stain, 2 min)Microscopy Examine wet Coverslip needed

Background Blood No effect Obscures detailInflammation Limited effect Obscures detailNecrosis Poor detail Limited effect

Cellular detail Cytoplasmic detail Excellent LimitedCytoplasmic products Excellent LimitedNuclear detail Limited ExcellentMitoses Satisfactory Excellent

Infectious elements or microorganisms Bacteria Good Variable to poorMycobacteria Negative images Not visibleFungi

Hyphae Variable to poor Good to variableYeasts Variable to good Variable to good, excellent for C. immitis

Viral inclusionsCytoplasmic (CMV) Excellent SatisfactoryNuclear (CMV, HSV) Limited Excellent

a A Romanowsky stain.



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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 stainsfor FNA specimens because of the vivid metachromatic stain-ing of certain cytoplasmic products, stromal, and backgroundelements (126, 141, 176). In addition, since this stain is appliedto air-dried smears, it reduces the effects of poor technique andincreases cell yield.

A commonly used Romanowsky stain is the DQ stain. Thereare three steps to this stain: fixation in methanol, staining ineosin Y, followed by staining in methylene blue. The proceduretakes less than 20 s to perform, although staining times may

vary depending on the thickness of the smear or cytospinpreparation. In addition to its rapidity, it is very difficult tooverstain smears. Although understaining is common, it is eas-ily corrected by reimmersion into the methylene blue stain.The DQ stain was originally developed for uniformly thin pe-ripheral blood smears. However, its utility in rapid cytologicdiagnosis is now well established (115, 126, 143).

Ancillary Techniques

Immunocytochemistry, electron microscopy (EM), and,most recently, molecular diagnostic techniques may be of valuein selected cases to refine a diagnosis, typically of a neoplasticprocess. However, when routine evaluation of cytologic spec-imens suggests an infectious etiology, ancillary studies may alsobe useful. This does not abrogate the need to obtain additionalsterile 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, areused 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 beperformed on smears, cytospin preparations, or sections froma cell block. Morphology alone or in conjunction with specialstains usually allows general categorization of the potentialpathogen (e.g., bacterium, fungus, or virus) and often leads toa definitive identification (e.g., Helicobacter pylori, Cryptococ-

cus neoformans, or cytomegalovirus [CMV]). However, formore specific classification, referral to the microbiology labo-ratory is required.

In the past, EM has been instrumental in the diagnosis of awide variety of infectious, nonneoplastic, and neoplastic dis-eases. However, EM has given way to more sophisticated,sensitive, and rapid immunodiagnostic techniques, particularlyin the diagnosis of viral infections. For immunodiagnostic tech-niques, cell block material is the most suitable because of theability to generate multiple sections. When possible, a separatespecimen should be taken for these studies and the materialshould be placed directly into buffered 10% formaldehyde.Cell blocks can be prepared from cytology material fixed inalcohol or Saccomanos fixative. Since cell blocks are not al-

ways available, cytospin preparations are an alternative andmay be prepared directly from the fluid specimen or needlerinse. Cytospin preparations may be air-dried or fixed in 95%ethanol. In either case, slides for immunocytochemistry may bestored frozen for several weeks. Material can be preserved in asimilar fashionfor moleculardiagnosticmethods including fluo-rescent in situ hybridization and PCR. Recent advances inPCR have allowed the detection of infectious agents in cyto-logic material (4, 44, 45, 48, 83, 86, 162). Although the quantityof material may be a consideration in certain circumstances,most immunodiagnostic and molecular techniques today haveprotocols adapted to cytologic specimens.

COMMON PATHOGENS

The major roles of the microbiology laboratory are the iso-lation, identification, classification, and susceptibility testing ofmicroorganisms. The role of the cytopathology laboratory isalmost exclusively diagnostic, i.e., to suggest or identify thepresence of an infectious agent. In recent years, in large mea-sure as a result of the increasing number of immunocompro-mised patients, cytologic techniques are often the first choicefor detection of infectious agents. The cytomorphologic ap-pearances of commonly encountered microorganisms are de-scribed below. Regardless of the type of cytology specimen, thepresence of acute or chronic inflammation, abundant macro-

phages or multinucleated giant cells with or without granulo-mas, and necrosis are features that should alert the cytologistto the possibility of an infectious process.

Contaminants

The presence of exogenous structures that mimic a variety ofpathogens can pose a challenge, particularly to the novice.Fibers, suture, talc, and starch granules may be seeded in thespecimen or collection devices during the sampling procedure.Clues that help distinguish these structures from microorgan-isms include their haphazard arrangement and lack of internalstructure. Many of these contaminants are birefringent in po-larized light. There is usually a limited or absent inflammatoryresponse. Occasionally, iron-encrusted collagen fibers are sam-pled from areas of prior hemorrhage. These fibers may resem-

ble hyphal structures; however, their true nature is revealed when GMS stains are negative and iron stains are positive(157). Respiratory specimens seem particularly vulnerable tocontamination, especially with pollen grains, which resembledimorphic fungi. Inorganic and organic fibers and vegetablematerial, either aspirated or as contaminants in sputum spec-imens, may resemble fungal hyphae. Alternaria sp., usually anonpathogenic airborne fungus, is an occasional stain contam-inant. It is easily recognized by its large, brown, septatedconidia and, when present, its septate hyphae with 90 branch-ing (132) (Fig. 1). Fungal contamination may occur when stainsare not filtered regularly. This contamination is usually distin-

FIG. 1. Alternaria sp. in a Pap smear with prominent acute inflammation. Acommon contaminant of cytologic specimens, Alternaria sp. is easily identified byits brown, septated conidia. PAP stain; magnification, 400.



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guished from true infection when only one stain reveals themicroorganism.

Bacteria

In most cases, the cytopathology laboratory is limited inbacterial identification to morphological and Gram stain char-acteristics. There are several types of cytologic specimens in

which bacteria are routinely encountered. Chief among them isthe Pap smear, particularly when taken during the second halfof the menstrual cycle. These smears often have an abundanceof Doderleins bacilli (lactobacilli) that metabolize the glyco-gen in intermediate and parabasal squamous cells. This processis termed cytolysis. The resultant cellular debris, in conjunction

with the sheer number of lactobacilli, may obscure the cervicalsquamous and glandular cells and often limits the interpreta-tion of the Pap smear. Other specimens in which bacteria ofthe normal flora can be seen and occasionally interfere withdiagnosis include sputum specimens, gastrointestinal brush-ings, and FNA specimens of the abdominal organs and pros-tate. A needle introduced into the last two, normally sterile,sites may pick up members of the bowel flora if the colon istraversed.

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 stain-

ing patterns. The significance the cytologist places on the pres-ence of bacteria is directly related to the body site and clinicalinformation provided with the specimen. For example, bacteriaare frequently encountered in urinary tract specimens, and,

while their presence is always reported, the diagnosis of aurinary tract infection requires clinical correlation (Fig. 2).Conversely, more significance is attached to the presence ofbacteria or microorganisms in material from a bone aspiration(55, 99). In this situation, additional stains would be used toconfirm a diagnosis of osteomyelitis. Cytologic specimens fromscrape preparations, swabs, and other collection methods areuseful for the detection of bacteria in abscesses, ulcers, fistulas,

and wounds (13, 20, 54, 74, 77). While Gram stain of thecytologic specimen will yield some information, these speci-mens are more appropriately sent to the microbiology labora-tory for further characterization. Five bacterial species havebeen selected to illustrate the utility and limitations of diag-nostic cytopathology (Table 3).

Mycobacteria. The resurgence of mycobacterial infections isdue in large measure to the increasing incidence of immuno-compromised patients. Infections by Mycobacterium tuberculo-

sis and mycobacteria other than M. tuberculosis not only

present as pulmonary infections but increasingly are implicatedin the etiology of lymphadenopathy (17, 105, 145, 156). Inmany instances, when the patient is known to be immunocom-promised or has had prior mycobacterial infections, the indexof suspicion for infection is high and additional specimensshould be obtained for culture and special stains. Althoughroutine cytologic stains do not stain the actual bacilli, theremay be significant clues that suggest the presence of theseorganisms (36, 128, 133). If granulomas, plump histiocytes,and/or necrosis is identified, particularly in aspirates (Fig. 3),acid-fast staining is usually performed (130). Mycobacteriacannot be identified on PAP stains. While the DQ stain doesnot stain the individual organisms per se, it does outline them.The unstained bacilli appear as slender, straight or slightlycurved, colorless rods highlighted against a dirty blue-grey

background. Thus, the terms negative images or ghost ba-cilli are used as descriptors (Fig. 4) (10, 103, 149). This char-acteristic negative image is presumably the result of hydropho-bic interactions of the water-based DQ stain with the lipid

within the cell walls of the bacilli (126). The identification ofthese negative images within histiocytes and in the backgroundis highly suggestive of mycobacteria. Lowering the substagecondenser often increases the refractility of the bacilli andenhances their identification when they are scattered in thesmear background (Fig. 5). However, it has been reported thatpatients being given antimycobacterial therapy with clofazi-mine may show crystal formation within macrophages that

FIG. 2. Bacteria in an ileal conduit specimen. Both cocci and bacilli are readily identified with Romanowsky stains. Ileal conduit specimens are prone todegeneration and bacterial overgrowth. DQ stain; magnification, 400.



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simulates these negative images (139). Acid-fast stains can beperformed on smears, cytospin preparations, and cell blocksections and should be used for confirmation. The identifica-tion 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 mycobacte-rial infections are from aspirations of superficial, mediastinal,and abdominal lymph nodes. In these aspirates, the presenceof numerous organisms both within distended histiocytes andscattered in the background tends to be from mycobacterialinfections other than tuberculosis. Although material is usuallyobtained for culture or fluorescence microscopy, PCR can nowbe used for the rapid detection and identification of M. tuber-

culosis (44, 48, 86).

Helicobacter pylori. The association between gastric and du-odenal ulcers, mucosa-associated lymphomas, and H. pylori hasincreased the need for detection of these organisms. H. pylorimay be identified in endoscopic brushing specimens as well asin touch or imprint preparations from small gastric biopsy

specimens (39). Cytologic specimens often reveal aggregates ofglandular cells, with or without atypia, in a background ofacute and chronic inflammation. The small, curved organismsare found, often in linear arrangements, close to the apicalsurface of the glandular cells. The Giemsa stain or DQ stainreadily stains this tiny (1- to 3-m), spiral-shaped organism(38, 174). The organism is closely associated with mucin, often

with the long axis of the bacteria oriented parallel to the mucusstrands (135). H. pylori can also be visualized with PAP, Gram,Warthin-Starry, and Dieterle stains (39, 110).

Actinomyces spp. Actinomyces spp. are gram-positive bacteriathat are frequently present in tonsillar crypts; they may beidentified in aspirates from the head and neck and other bodysites, as well as in gynecologic and respiratory specimens (58,97, 122, 123, 138). In cytology specimens, the organismspresent as fragments and tangles of slender filaments branch-ing at acute angles, usually in a background of suppurativeinflammation. Sulfur granules, accumulations of these organ-isms that show a very dark granular center with peripherallyradiating filaments, are numerous (66). Necrosis and abscessformation may also accompany these infections (Fig. 7) (37).Occasionally, respiratory specimens show small clusters of fil-amentous bacteria that may represent oral contamination orinfection by an Actinomyces sp. or by Nocardia spp., which aremorphologically similar to Actinomyces spp. Actinomyces or-ganisms are slightly larger (1 to 1.5 m in diameter, 20 to 70m in length) than Nocardia spp. and branch at acute angles.Most cytologic and histologic stains, including silver stains, canbe used to identify Actinomyces spp.; however, unlike Nocardiaspp., Actinomyces spp. are not acid fast when stained withmodified acid-fast stains. Other useful special stains include

PAS and a modified Gram stain.Actinomyces spp. can also be seen in cervicovaginal smears

and are usually associated with intrauterine contraceptive de-vices, foreign bodies, and vaginal pessaries (64, 65). While thediagnosis ofActinomyces in Pap smears is usually not challeng-ing, hematoidin crystals and even dense clumps of spermato-zoa occasionally resemble sulfur granules, while mucin andfibrin threads and tangles may mimic the slender filaments of

Actinomyces spp. Septate hyphae causing eumycotic mycetomaand nonbranching bacilli or cocci causing botryomycosis mayform sulfur granule colonies that can be differentiated fromactinomycotic granules with GMS and Gram stains.

FIG. 3. Granuloma and necrosis in a lung FNA specimen. Granulomas com-posed of epithelioid histiocytes with elongated or footprint nuclei are oftenassociated with infections. When granulomas are identified, there should be athorough search for microorganisms such as M. tuberculosis. DQ stain; magnifi-cation, 200.

TABLE 3. Staining characteristics of selected bacterial genera

GenusStaining witha: Other

techniquesPAP Romanowsky H&E Gram Acid fast Silver

Mycobacterium GMS PCR(negative images)

Legionella b c Dieterle, W-S DFAmodified

Actinomyces GMS DFAHelicobacter Dieterle, W-SNocardia b d GMS DFA

a DFA, direct fluorescent antibody; W-S, Warthin-Starry; , does not stain; , visible with stain.b Stains best with Giemsa.cL. micdadei will stain acid-fast positive.d Stains best with Fites silver stain.



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Nocardia spp. Nocardia spp. have enough morphologic andstaining differences to allow reasonably accurate classificationand differentiation from Actinomyces spp. Nocardia spp. areprimarily opportunistic pulmonary pathogens. Nocardia pneu-monia occasionally simulates a mass; therefore, it may be en-countered in FNA specimens. In these specimens, Nocardiaspp. may be difficult to identify due to a background of chronicinflammation, suppurative inflammation, and necrosis. Unfor-tunately, this background is rather nonspecific and is commonto a variety of infections that include mycobacterial and fungal

species, as well as neoplastic processes. Although larger indiameter than mycobacteria, Nocardia spp. are narrower than

Actinomyces spp. and fungal hyphae. The slender filaments (0.5to 1.0 m in diameter; 10 to 20 m in length), usually branchat right angles rather than acute angles. Sulfur granules are notpresent. Appearing as a sheath or tangle of filamentous bac-teria, Nocardia spp. are stained best by the Romanowskystains, GMS, and modified acid-fast stains (Fites stain). Both

Actinomyces and Nocardia spp. may be detected by the directfluorescent-antibody test (124).

Legionella spp. Although rarely identified in cytologic mate-rial, Legionella is stained by DQ, Giemsa, Gram, and silver

stains such as Warthin-Starry and Dieterle stains and poorlystained by Gram, PAP, and H&E stains (11, 43, 170). Theseaerobic, motile bacteria appear as slender, short rods that areoften present within the cytoplasm of neutrophils and macro-phages (163). Legionella infection can be accompanied by anacute inflammatory response. The presence of numerous neu-trophils and fibrin makes identification of this organism diffi-cult even when there is clinical suspicion. Confirmation of

Legionella spp. by direct fluorescent-antibody testing is recom-mended (84).

Parasites

There are numerous, scattered, single case reports and smallseries that discuss the identification or diagnosis of a variety of

parasites in cytologic specimens. The recognition of previouslyrare parasitic infections has increased dramatically since theadvent of AIDS and immunosuppressive therapies. The para-sites discussed below represent a few of the more commonlyencountered organisms in the cytopathology laboratory. As

with bacterial infections, recognition of a parasite is highlydependent on the available clinical information and good cy-topreparatory and staining techniques.

Trichomonas vaginalis. Trichomonas vaginalis is usually sus-pected clinically in patients who present with a green-yellow,malodorous discharge. Visualization of small punctate hemor-rhages on the cervix is also a strong indicator of this parasite.

FIG. 4. Mycobacteria presenting as negative images in a mediastinal FNA specimen. The background of Romanowsky stains is typically dark blue to purple. Theslender 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 presentas scattered negative images in the smear background. Lowering the substagecondenser increases the refractility of the mycobacteria and their likelihood ofdiscovery. Erythrocytes stain grey-green. DQ stain; magnification, 264.



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Although the clinician may perform wet mount or hanging-drop slides to detect this motile organism, its presence is usu-ally confirmed when the Pap smear is evaluated. The typicalcytologic presentation is that of scattered individual or small

clusters of pear-shaped organisms that have a slight cyano-philic tinge, faint eccentric nuclei, and fine acidophilic gran-ules. Occasionally, flagella are visible at higher magnification(Fig. 8). Aggregates of neutrophils and degenerating squamous

FIG. 6. Mycobacteria in a cervical lymph node FNA specimen. Acid-fast stains can be performed on most cytologic specimens. The presence of abundant organismssuggests mycobacterial species other than M. tuberculosis. Acid-fast stain; magnification, 800.

FIG. 7. Actinomyces sp. in a lung FNA specimen. This organism is commonly encountered in respiratory and gynecologic specimens, often as scattered haphazardcollections of grey, slender filamentous organisms. PAP stain; magnification, 400.



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cells are seen throughout the smear background. A nonbranch-ing filamentous bacillary structure, formerly referred to as Lep-tothrix, is often associated with T. vaginalis infection (22).

Giardia lamblia. The gastrointestinal parasite Giardia lam-blia is occasionally encountered in washings, brushings, or as-pirates of the gastrointestinal tract, particularly the duodenum,stomach, and pancreas. There are also rare reports of thisorganism being identified in bronchoalveolar lavage and peri-toneal fluid specimens (24, 150). G. lamblia has quite a char-acteristic cytomorphology, making diagnosis relatively easy

when the organism is present (Fig. 9) (39). Trophozoites of thisprotozoan are flat, pear-shaped flagellates that are bilaterallysymmetrical with four pairs of flagella and two nuclei contain-ing large central karyosomes (152).

Strongyloides stercoralis. Although Strongyloides stercoralis isan intestinal nematode, larvae occasionally spread to the re-

spiratory tract. This may result in clinical symptoms rangingfrom cough to hemoptysis and pulmonary infiltrates (168). The

filariform larvae of S. stercoralis may also be encountered inrespiratory specimens, the result of a superinfection in immu-nosuppressed individuals. The organism is eosinophilic onPAP stains and ranges between 400 to 500 m in length incytologic preparations (Fig. 10). Its internal structure, a closedgullet, and V-shaped notched tail help distinguish this parasitefrom cotton and synthetic fiber contaminants that are occa-sionally encountered. Occasionally, a negative image on air-dried, DQ stained slides is obtained when the larvae move orare dislodged from their original position before staining (Fig.11).

Echinococcus granulosus. Echinococcus granulosus is encoun-tered in FNAs of the liver and rarely of other body sites (8, 53,73, 104). Infections in humans are typically the result of expo-sure to dogs infected with this tapeworm. Larval forms, thecausative agent of echinococcosis (also known as hydatid dis-

FIG. 8. T. vaginalis in a Pap smear. This pear-shaped, flagellated parasite isoften associated with slender bacillary structures. Scattered debris, bacteria, andneutrophils contribute to the hazy background. PAP stain; magnification, 660.

FIG. 9. G. lamblia in a pancreatic FNA specimen. These distinctive parasiteswere aspirated from a pancreatic cyst. Note the prominent, symmetric nuclei anddelicate flagella. PAP stain; magnification, 860. Courtesy of Barbara Centeno.

FIG. 10. S. stercoralis in a bronchial washing specimen. The filariform larvaeof S. stercoralis stain brightly eosinophilic with PAP stain. Internal structures(gullet) help distinguish it from contaminants. The associated bronchial cells maybe used as a size reference. PAP stain; magnification, 264. Courtesy of EllenGreenebaum.

FIG. 11. S. stercoralis in a bronchial washing specimen. Romanowsky stainsrequire air-dried slides. The parasite may shift or be dislodged from its originalposition before the slides are stained. This creates a negative image. DQ stain;magnification, 352. Courtesy of Ellen Greenebaum.



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ease), invade intestinal walls and gain access to the blood-stream. The organisms come to reside in the liver, creating theradiologic appearance of a single or multilocular cyst (32). It

was previously believed that aspiration of presumptive hydatidcysts was exceedingly dangerous due to dissemination of thedisease and the potential for anaphylaxis when fluid was re-leased into the surrounding tissue (125, 134). However, morerecent studies have determined that this danger is substantiallydecreased by the use of fine needles (93, 104). Aspirates of the

cyst fluid are dark brown, resembling anchovy paste. In ad-dition to inflammatory cells (including eosinophils), scolices

with attached hooklets (Fig. 12) and detached hooklets areoften present in the dense, granular background (5, 12, 79, 98).Occasionally hooklets stain faintly or not at all by the PAPstain or Romanowsky stains, but they can still be visualized asrefractile structures against the background debris, especially ifthe substage condenser is lowered (Fig. 13).

Amebic abscesses may also occur as cavitary hepatic lesions

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. Detachedhooklets (center) are often poorly staining and are identified primarily by their refractility. PAP stain; magnification, 1,000.



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that yield dark brown granular material when aspirated. Thismaterial usually does not contain the parasites, since amebaeare located predominantly in the wall of the abscess cavity. Inthis instance, the indirect hemagglutination serologic testand/or immunoperoxidase testing on aspirate material is usefulto confirm the cytologic impression (88).

Cryptosporidium parvum. Cryptosporidium parvum is usuallyencountered in gastrointestinal washings, brushings, and bi-opsy specimens. Although there is no tissue damage, theseopportunistic pathogens can cause fairly severe watery diar-rhea in children and immunocompromised patients. Crypto-sporidia can be seen either as scattered free-lying organisms oras spherical structures aligned on the luminal surface of glan-dular intestinal cells. Acute inflammation is usually present(Fig. 14) (142, 153). The small (2- to 4-m) cysts of these

coccidial protozoans often appear stippled when stained withRomanowsky stains. This organism can also be seen withH&E, PAP, and modified acid-fast stains.

Fungi

With rare exceptions, most fungi are diagnosed in cytologyspecimens by their morphologic rather than staining charac-teristics (Table 4). This approach, combined with the properclinical information, is quite accurate for the majority of fungalorganisms encountered. The presence of hyphal and/or yeaststructures begins a mental algorithm that uses size, shape,budding, and branching characteristics to narrow the differen-

tial diagnosis. Although infrequently used immunologic tech-niques are available to specifically identify a variety of fungi(112, 114).

Pneumocystis carinii. Pneumocystis carinii has been difficultto classify and is currently considered a fungus. It is one of themost common causes of nonbacterial pneumonia in infants andimmunocompromised patients. Fortunately, it has a character-istic clinical and radiologic presentation, and so cytologic spec-imens are usually sent with a specific request for identificationof this organism. Before the success of direct fluorescencemethods, bronchoalveolar lavage was the traditional procedurefor obtaining a high-yield specimen (56, 85). Although P. ca-

rinii has been identified in a wide variety of specimens, it is stillidentified primarily in respiratory material (bronchoalveolarlavage fluid, washings, brushings, and sputum) and in lymphnode aspirates during fulminant infections.

A distinctive feature ofP. carinii in cytologic preparations isthe 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 out-lines of the cysts (59, 61). Although most cytologists are famil-iar with the appearance of this organism on PAP stain, thisstain does not specifically stain the organism. Confirmatorysilver stains may be used to document the presence of cysts.Silver stains often yield characteristic single or paired dots orthickenings resembling parentheses in the cyst, which helpdistinguish the cysts from a background of stained erythrocytes

(Fig. 15) (118, 137). Cysts range from 5 to 8 m, are usuallyrounded, crescentic, or cup shaped, and contain six to eightsmall (1- to 2-m) ovoid trophozoites. While the cyst walls maybe refractile in routine stains, the DQ, Wright, and Gram-Weigert stains have an advantage over the PAP stain becausethey stain not only the foamy alveolar casts but also the indi-

vidual trophozoites (40, 125). This results in a distinctive stip-pled appearance of the casts (Fig. 16). Lowering the substagecondenser causes refractility of the cysts and accentuates thisappearance. Thus, the DQ stain can be used as a simple andrapid diagnostic stain. Other rapid stains and techniques, suchas immunoperoxidase, have been developed for the detection

FIG. 14. C. parvum in a gastrointestinal brushing specimen. These small,round parasites can be seen intimately associated with the apical surface ofreactive glandular cells (arrow). PAP stain; magnification, 900. 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.

TABLE 4. Staining characteristics of common fungi

FungusStaining witha:

PAP Romanowsky H&E GMS PAS

Candida spp. Aspergillus spp. Variable Zygomycetes Poor PoorYeasts other than

Candida spp.b c

a PAS, periodic acid-Schiff; , visible with stain; , does not stain.b See Table 5 for selected organisms.c Yeasts stain with variable intensity with Romanowsky stains.



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of P. carinii in respiratory specimens and may be used if conventional methods give negative results (9, 26, 35, 131, 152,169).

Candida spp. Small budding yeasts (3 to 4 m) andpseudohyphae are typically generically identified as Candidaspp. This organism is probably the most frequently encoun-tered fungus in cytologic specimens, is often identified in Papsmears, and is present in urine and sputum specimens mostly

as a contaminant. Although not considered significant whenseen in these specimens, it can be an opportunistic pathogen indebilitated patients, particularly when found in the oral cavity(causing thrush). When it is encountered in lower respiratorytract specimens, it should be reported as a possible pathogen.When Candida spp. are encountered on Pap smears andesophageal brushings, they are often seen as hyphal spearspiercing clusters of squamous cells (Fig. 17) in a background of

FIG. 16. P. carinii in a bronchoalveolar lavage specimen. Individual trophozoites are stained dark purple within the lighter-staining alveolar cast. This distinctivestippling is seen with Romanowsky-stained preparations. This makes DQ stain a good choice for a rapid staining procedure. DQ stain; magnification, 1,000.

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.



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acute inflammation. PAP and DQ stains are adequate to illus-trate their morphology, although if necessary, their identity can

be confirmed with silver stains. The DQ stain is particularlyuseful for urine and sputum specimens, in which both hyphaeand yeast forms are highlighted as darkly stained objects ad-mixed with inflammatory cells and debris (Fig. 18).

Fungi that present primarily as yeast forms are challengingbecause of the tremendous overlap in their clinical, radiologic,and cytologic presentations. However, there are usuallyenough clues for the cytopathologist to accurately distinguishamong the four most common of these fungi: Histoplasma

capsulatum, Cryptococcus neoformans, Blastomyces dermatiti-dis, and Coccidioides immitis (Table 5). These organisms aremost commonly identified in respiratory specimens, lymphnodes, and CSF of immunocompromised patients (77, 78).

Cryptococcus neoformans. Cryptococcal infection is usuallysubacute or chronic and is associated primarily with pulmonary

and/or central nervous system (CNS) involvement, althoughother organs and sites can be affected (155, 159). In aspirationspecimens this organism is usually abundant, found individu-ally or as clusters with accentuated capsular halos (144, 164,167). Acute inflammation and necrosis may obscure the organ-ism, which can stain variably. In DQ-stained preparations, thepurple yeasts with accentuated clear halos against the darkpurple background give the smear a punched-out appearance(Fig. 19) (127). In CSF specimens, Cryptococcus may resembleerythrocytes or even starch granules and as a result may beoverlooked (Fig. 20). A useful cytologic feature is the appear-ance of the teardrop-shaped, narrow-based budding ofC. neo-

formans. At times, the daughter bud will not detach and rep-etition of budding yields small chains of daughter progeny (Fig.21). Diagnosis of C. neoformans still rests primarily on itscytomorphology, with confirmation, as necessary, by specialstains. One of the smallest fungi, this yeast typically rangesfrom 5 to 10 m in diameter but may vary from 2 to 20 m.Occasionally, when these yeasts are engulfed by histiocytes or

when they are nonencapsulated, they are mistaken for H. cap-

sulatum. Special stains may be performed to confirm the cyto-logic impression. Silver stains, such as GMS, stain the organismitself, while mucicarmine, PAS, and alcian blue stain the mu-copolysaccharide capsule (95).

Blastomyces dermatitidis. Yeast-like spherical cells of Blasto- myces dermatitidis are generally larger than those of crypto-cocci (8 to 20 m) 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 identifyon cytologic preparations, chiefly because they are not numer-ous and are often out of the plane of focus. The latter problemis due to their rigid cell walls, which resist compression by acoverslip (Fig. 22). To compound matters, acute and/or gran-ulomatous inflammation of different degrees is present in most

specimens. Although most often identified extracellularly,these organisms can also be engulfed by macrophages. Cyto-morphology is usually sufficient for diagnosis; however, fluo-rescence techniques can be used for confirmation (154).

Histoplasma capsulatum. Histoplasma capsulatum is one ofthe smallest dimorphic fungi and is found predominantly in-tracellularly, as foamy or vacuolated structures within the cy-toplasm of histiocytes. Like the other yeasts, H. capsulatum ismost often encountered as the cause of pulmonary infection,but disease can also be systemic. Macrophages with engulfedorganisms are typically present in a background of granuloma-tous inflammation that can also include necrosis, acute inflam-matory cells, and lymphocytes (113, 146, 160). If present insmall 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 usu-ally in stark contrast to the pale-staining cytoplasm (Fig. 23).At times the organism can retract from its wall, creating a clearspace that mimics the C. neoformans capsule (Fig. 24). If thedifferential diagnosis includes leishmaniasis, recognition of anucleus and kinetoplast in the organism and the absence ofbudding will exclude histoplasmosis (6, 92, 120). Confirmatorystains with GMS should be performed when this organism ispresumptively identified.

Coccidioides immitis. Coccidioides immitis is one of the larg-est of the dimorphic fungi and is particularly associated withboth acute and chronic respiratory infections in the southwest-

FIG. 18. Candida sp. in sputum. Both yeast and hyphal forms are darklystained with Romanowsky stains and are easily identified at scanning magnifi-cations. Pseudo-septations are often colorless and are in sharp contrast to thedark hyphal forms. DQ stain; magnification, 680.

TABLE 5. Cytologic features of selected yeast genera

CriterionCharacteristic for:

Candida spp. Cryptococcus spp. Blastomyces spp. Histoplasma spp. Coccidioides spp.

Size (m) 34 515 820 25 5100 (mean, 1040)Shape Oval Round-oval, clear capsule Spherical, double-

contoured wallOval, endocellular Spherical, refractile

Budding Broad-basedpseudohyphae

Narrow based Broad based Single, narrow Endospores

PAP stain Cyanophilic Pale, cyanophilic Blue-green Dark outline OrangeophilicDQ stain Dark blue-purple Blue-purple Pale blue Dark blue Colorless or light blueSpecial stain GMS, PASa GMS, mucicarmine, alcian blue GMS, PAS GMS, PAS PAS, GMS

a PAS, Periodic acid-Schiff.



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ern United States. It may be accompanied by mild acute,chronic, and/or granulomatous inflammation and is commonlyseen within macrophages (Fig. 25, left). This organism presentsas endospores within thick-walled mature spherules that can

vary tremendously in size. C. immitis can resemble a variety ofother fungi, pollen grains, and even inorganic contaminants(49, 52, 69, 129). Endospores are not visible in immature spher-ules; combined with the thick wall of C. immitis, this oftenresults in an erroneous identification as B. dermatitidis. Whenmature, spherules ofC. immitis contain numerous small (2- to

5-m) endospores. Once the endospores are separated fromthe spherule, they may resemble H. capsulatum or Toxoplasma

gondii, and a careful search for older, folded, and fracturedspherules is necessary (30). Conversely, when these olderspherules are seen without intact, mature spherules, they maybe overlooked as inorganic contaminants. This organism is veryeasy to detect in PAP-stained preparations because of its or-angeophilic staining (Fig. 25, right). The spherules stain lightlyor not at all with the DQ stain, although with experience, thislack of staining is a useful clue. PAS with a light green coun-terstain is a useful confirmatory stain, since the spherules staina vivid magenta against a pale green background. Like Aspergil-

lus spp., chronic respiratory infections with C. immitis can

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 darklystained 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 indistinctcapsules. These yeasts may appear as a contaminant to the neophyte, particularlyif there is accompanying peripheral blood contamination. PAP stain; magnifica-tion, 1,000.

FIG. 21. C. neoformans in a lung FNA specimen. Mucicarmine, used todetect capsular acid mucopolysaccharides, is an excellent confirmatory stain.Occasionally there is incomplete separation of daughter progeny, resulting inchains of yeast cells. Dust-filled alveolar macrophages are also present. Muci-carmine stain; magnification, 760.



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result in the formation of lung cavities or nodules. Rarely,these nodules are exposed to air, resulting in the developmentof mycelial forms of C. immitis with barrel-shaped arthro-conidia. One of the problems associated with recognition ofthis rare phenomenon is the misinterpretation of the mycelialforms as a second infectious agent, such as an Aspergillus spp.,particularly since multiple infections are common in immuno-suppressed individuals.

Aspergillus spp. When a cytologic specimen contains hyphalstructures that are relatively large (3 to 6 m in diameter),septate, with regular, progressive dichotomous branching at45 angles, an Aspergillus sp. should be immediately suspected.

These organisms, often opportunistic pathogens, are usuallyidentified in respiratory specimens and rarely found in othersites (14). The organisms commonly present as tangled clustersof branched hyphae that are accompanied by acute inflamma-tion, necrosis, and cellular debris. Aspergillus spp. stain fairly

well with PAP and H&E stains, which clearly delineate thehyphal septations (Fig. 26). The organisms also stain with DQ;however, the staining is often extreme, i.e., either too dark todistinguish internal structure or too light so that the fungusblends into the dirty background. Septations are often color-less, creating a negative image that contrasts with the darklystained hyphae (Fig. 27). Sheaves or rosettes of needle-like,

FIG. 22. B. dermatitidis in sputum. The characteristic broad-based buddingand double-contoured cell wall help differentiate this yeast from C. neoformans.PAP stain; magnification, 630. Courtesy of Barbara Benstein.

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 withRomanowsky 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. capsulatumcells are usually detected as intracellular organisms in the cytoplasm of macro-phages and histiocytes. PAP stain; magnification, 830. Courtesy of DavidMensi.



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birefringent crystals, representing calcium oxalate, may beidentified, particularly in respiratory tract specimens (46, 96).

Infection with Aspergillus spp. can cause a necrotizing pneu-monitis with central cavitation, which presents radiologically as

a cavitary lesion. The immediate concern in patients, particu-larly smokers, is the possibility that this mass may represent asquamous cell carcinoma with central necrosis. Thus, FNA isoften selected as the diagnostic procedure of choice. A diag-

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 othercontaminants, 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.

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 isolatedtangles of branching, septated hyphae. PAP stain; magnification, 1,000.



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nosis of infection by FNA will spare the patient a more invasiveprocedure. Although malignant squamous cells do not resem-ble fungal hyphae, active infection by Aspergillus spp. can causethe surrounding lung parenchyma to become quite atypical.Severely reactive cells may mimic carcinoma, which can resultin a false-positive diagnosis with potentially serious conse-quences for the patient (140). Unlike most FNAs, which at-tempt to sample the center of a mass to maximize the recoveryof material, FNAs of cavitary lesions are directed away fromthe presumed necrotic center in favor of the peripheral viablelesional tissue. In aspergillomas, this is precisely where viablefungus will be located. Recognition of these branched fungalstructures can still be difficult if necrotic debris and inflamma-tion are also present. Even if close scrutiny reveals no organ-isms, in the absence of a positive diagnosis of carcinoma it isrecommended to perform staining with silver stains to com-pletely exclude the possibility of an infectious agent. Occasion-ally when the fungus cavity is exposed to the air, fruiting bodies

with conidia can be detected in cytologic specimens; their pres-ence helps confirm the presence ofAspergillus spp. and can beused in further identification.

Zygomycetes. The zygomycetes (phycomycetes) include Mu-cor, Absidia, and Rhizopus spp. and are characterized by their

wide (3- to 25-m) ribbon-like hyphae that branch irregularlyand are pauciseptate (Fig. 28). These organisms are opportu-nistic pathogens that have a propensity for the head and neckregion (orbit, paranasal sinuses, and palate), in addition to thelungs and skin. Because these infections are often located closeto the CNS and have a rapid, potentially fatal course, animmediate diagnosis is extremely useful to clinicians. Like As-

pergillus spp., the zygomycetes are most often encountered inaspiration specimens (23). Usually associated with prominentacute inflammation and necrosis, these organisms can be over-looked on DQ stains and are best seen on PAP or H&E stains.Silver stains are generally used for confirmation.

Viruses

Although individual viruses cannot be detected by light mi-croscopy, their cytopathic effects on infected cells are often

visible, even in routine cytologic preparations. The recognitionof nuclear and/or cytoplasmic inclusions provides a fairly spe-cific indication of viral infection, while perinuclear halos andciliocytophthoria and detachment of apical cytoplasm with cil-iary tufts represent more subtle clues (Fig. 29) (117, 121).

Regenerative/reparative atypia often accompanies these morespecific cellular alterations. The most common problem is mis-interpretation of these cytologic features as carcinoma. Clinical

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.

FIG. 28. Mucor sp. in sputum. Staining of zygomycetes is variable with thePAP stain. In this specimen, the broad, nonseptate eosinophilic hyphae arealmost translucent and are partially obscured by inflammatory cells. PAP stain;magnification, 256.



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and radiologic correlation is still a necessity. In cytologic prep-arations, the three most commonly recognized viral infectionsare those caused by HSV, CMV, and human papillomavirus(HPV) (51).

Herpes simplex virus. HSV is a common pathogen in Papsmears and is most prevalent in young, sexually promiscuouspatients. It is also frequently found in gastrointestinal andrespiratory specimens from immunocompromised patients (80,161). Because the key cytologic feature of HSV is the conspic-uous nuclear changes seen in infected cells, the PAP stain isthe most desirable cytologic stain for diagnosis. Nuclear alter-ations may present as enlarged degenerated nuclei withsmudged or homogenized ground-glass or slate-grey nuclei(Cowdry B nuclei) (Fig. 30). Alternatively, large, multinucle-ated cells may be present. Their nuclei are typically molded,

each containing a prominent eosinophilic inclusion (Cowdry Anuclei) 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 suggestsinclusions. This appearance, combined with multinucleationand nuclear enlargement, suggests a viral cytopathic effect,most probably due to HSV (Fig. 32). While routine stains mayprovide indirect evidence for HSV, confirmation can beachieved by a variety of means, including culture, immunocy-tochemistry, immunofluorescence, and in situ hybridizationtechniques (18, 111).

Cytomegalovirus. CMV is also a common problem in immu-nocompromised 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 cytologicsample. In contrast to HSV, CMV has both nuclear and cyto-plasmic inclusions and can be identified with both PAP andDQ stains. As the name implies, infected cells are often dra-matically enlarged compared to their uninfected counterparts.The prominent, often eosinophilic, owls-eye nuclear inclu-sion with marginated chromatin that results in a halo effect isbest visualized with PAP stain (Fig. 33). The numerous smallcytoplasmic inclusions are stained bright magenta with DQ andare easily identified even at scanning magnifications (Fig. 34).With practice, nuclear changes can be identified with the DQstain; however, these can also be misinterpreted as changessecondary to cancer (165). Like HSV, culture and moleculartechniques may be used to confirm the presence of CMV (70,171).

Respiratory viruses. Numerous other viruses have been as-sociated with respiratory infections; they include adenovirus,respiratory syncytial virus, influenza and parainfluenza viruses,and measles virus. While a variety of cytologic features such asmultinucleation, smudged nuclei, and even rather generic nu-

clear and cytoplasmic inclusions may be seen, these infectionsare almost never diagnosed outright on the basis of cytologicspecimens unless there is strong supportive clinical and labo-ratory data (2, 67, 119, 177). Viral cultures, immunofluores-cence, and DNA probes represent more specific and reliabletechniques for the diagnosis of these infections as appropriate(41, 75, 102).

Human papillomavirus. Once the distinctive cytologic man-ifestations of HPV were described and subsequently linked tothe development of dysplasia and its progression to squamouscell carcinoma of the cervix, the identification of HPV becameextremely important. Sampling of the cervical transformation

FIG. 29. Ciliocytophthoria in a bronchial washing specimen. Viral infectionsof the respiratory tract, particularly adenovirus infections, produce alterations inthe bronchial epithelium. Ciliocytophthoria is the detachment of the apicalcytoplasm, terminal bar, and cilia from columnar respiratory cells (arrow). PAPstain; magnification, 586. Courtesy of Barbara Benstein.

FIG. 30. HSV in a Pap smear. Ground-glass nuclei, chromatin margin-ation, and multinucleation are indicative of HSV infection. Cellular enlarge-ment, while nonspecific, is also present. PAP stain; magnification, 900.

FIG. 31. HSV in a Pap smear. Prominent, single, eosinophilic inclusions areconspicuous in these infected cells. PAP stain; magnification, 950.



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zone, which is most vulnerable to this infection, is an integralpart of the Pap smear (15, 16, 106108). The detection of HPVin Pap smears rests predominantly on the identification of cellsknown as koilocytes, derived from the Greek word koilosmeaning hollow or cavity (89). Recognition is based on threefeatures: marked density of the cytoplasm peripheral to thecavity, amphophilic cytoplasm, and enlarged hyperchromaticnucleus (Fig. 35) (106). Other cytologic features that are oftenassociated with HPV infection include bi- and multinucleation,plaques or aggregates of parakeratosis and hyperkeratosis, and

anucleate squames (63). Although other infections, includingfungal (Candida spp.), protozoal (T. vaginalis), and bacterial

infections, may cause small perinuclear or inflammatory ha-los, these structures can usually be distinguished from koilo-cytes, which are larger with abnormal nuclei. Several types ofHPV have been identified, some of which are associated withthe development of high-grade squamous intraepithelial le-sions and squamous carcinoma. Although not considered cost-effective for screening of at-risk populations, molecular diag-nostic techniques such as immunoperoxidase staining, in situhybridization, and PCR are available for viral typing (28, 63,136).

Human polyomavirus. Human polyomavirus, like HPV, is amember of the Papovaviridae family and is sporadically iden-tified in urine specimens. This virus can infect normal individ-uals as well as immunocompromised patients. Although many

FIG. 32. HSV in a bronchoalveolar lavage specimen. Romanowsky-stained preparations accentuate cellular enlargement, even in the presence of obscuringinflammation. Multinucleation and nuclear inclusions appear as subtle nuclear shading. DQ stain; magnification, 400.

FIG. 33. CMV in a Pap smear. Although rare, CMV may be encountered inPap smears. The typical cytologic features of cellular enlargement, prominentintranuclear inclusions, and occasional binucleation are present in this specimen.Cytoplasmic inclusions are often indistinct with PAP stains. PAP stain; magni-fication, 950.

FIG. 34. CMV in a bronchial washing specimen. Romanowsky-stained prep-arations are useful when CMV is suspected. Cytoplasmic inclusions stain a deepmagenta and, when combined with cell enlargement and intranuclear owls-eyeinclusions, allow rapid recognition of infected cells at scanning magnifications.Intranuclear inclusions are visible on DQ stains but are not as distinct as thosestained with PAP stain. DQ stain; magnification, 740.



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patients are asymptomatic, some present with hematuria. Mostinfections tend to resolve spontaneously (91, 109). Infectedcells can vary from few to abundant, and they occur as solitarycells rather than as cell clusters. They have enlarged nuclei

with nuclear inclusions that almost fill the nucleus, leaving onlythin rims or halos (Fig. 36). These large, opaque inclusionshave been misinterpreted as a feature of cancer. As a result,these cells have been termed decoy cells (29, 34). Also in thedifferential diagnosis is CMV. In contrast to CMV, cells in-

fected with polyomavirus are smaller, with thinner halos, lessabundant cytoplasm, and no cytoplasmic inclusions. Immuno-diagnostic technique can be used to confirm this diagnosis (7).

CONCLUSION

The utility of cytologic specimens in the diagnosis of infec-tious disease has been well established during the last 50 years.While morphology continues to be the mainstay of diagnosticcytopathology, recent developments in immunocytochemistryand molecular diagnostics allow not only rapid but also specificdiagnosis and classification of many infectious agents. How-ever, the cytologic identification of microorganisms, no matterhow specific, is not intended to replace microbiologic tech-niques. Rather, cytology should be viewed as a means of rapidinitial recognition of an infectious process by using safe, cost-effective methods of specimen procurement (Table 6).

In contrast to alternative procedures, such as the surgicalbiopsy, the cytologic techniques described in this review areminimally invasive; they are also less expensive and have morerapid turnaround times. In most cytopathology laboratories,routine preparation, staining, and diagnosis of nongynecologicspecimens can be done within a 24- to 48-h period. Specimensmay be processed much more rapidly, within minutes, if adiagnosis is needed emergently.

From the patients viewpoint, the collection of most cyto-logic specimens is a relatively minor procedure, which is oftenaccomplished with a reasonable amount of privacy and littlediscomfort. Many specimens are obtained in FNA clinics or

other outpatient settings. FNA, lumbar puncture, and paracen-tesis can be performed with minimal or no anesthesia and havefew, if any, side effects. From the clinicians and hospital staffsperspective, rapid identification of an infectious agent can di-rect patient management, allow earlier therapeutic interven-tion, and permit rapid initiation of infection control proce-dures if necessary.

Contamination of equipment and personnel is often a con-cern with presumptive or confirmed infectious cases. Cytologicspecimens can be fixed and stained immediately with verylimited exposure to the infectious material. Contamination oflaboratory equipment, such as microtomes and cryostats for

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.

FIG. 36. Human polyomavirus in a urine specimen. A solitary renal tubularcell with an enlarged, smudged nucleus consistent with polyomavirus infection is

visible. PAP stain; magnification, 600.



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frozen-section diagnosis of infectious cases, can be avoided iftechniques such as touch or imprint cytology are used on sur-gical specimens. In many instances, FNA can be used, evenintraoperatively, to obtain material for rapid microscopic anal-

ysis and microbiologic culture, contaminating only the dispos-able needle used for the procedure.

Rapid or immediate identification of presumptive organismsin cytologic specimens also allows additional, sterile materialto be obtained. It also yields information that allows the mi-crobiology laboratory to evaluate specimens, selecting the mostappropriate stains, media, and tests necessary for classificationof the infectious agent. This selectivity is efficient and cost-effective. Conversely, following the preliminary interpretation,

personnel in the microbiology laboratory can communicatetheir particular needs to the cytologist, suggesting the mostappropriate means of collection and transport of specimensselected for microbiologic evaluation.

In summary, the usefulness of the cytopathology laboratoryin the identification of infectious disease is directly related toits acceptance by clinicians and the clinical microbiology lab-oratory. While it is important to have cytologists skilled in theperformance and interpretation of FNA and other cytologictechniques, diagnostic accuracy is greatly enhanced by commu-nication. Current and accurate clinical, radiologic, and labora-tory data must be available to the cytologist; conversely, con-cerns, suspicions, or preliminary interpretations of infectiousagents should be discussed with clinicians, infectious diseaseconsultants, and/or the microbiology laboratory staff.

REFERENCES

1. Abdallah, P. W., J. B. D. Mark, and T. C. Merigan. 1976. Diagnosis ofcytomegalovirus pneumonia in compromised hosts. Am. J. Med. 61:326332.

2. Abreo, F., and T. Bagby. 1991. Sputum cytology in measles infection: a casereport. Acta Cytol. 35:719721.

3. Adams, H. G. 1991. A clinicians perspective, p. 920. In J. F. Silverman(ed.), Guides to clinical aspiration biopsy. Infectious and inflammatorydiseases and other nonneoplastic disorders. Igaku Shoin, New York, N.Y.

4. Adams, V., C. Moll, M. Schmid, C. Rodrigues, R. Moos, and J. Briner. 1996.Detection and typing of human papillomavirus in biopsy and cytologicalspecimens by polymerase chain reaction and restriction enzyme analysis: amethod suitable for semiautomation. J. Med. Virol. 48:161170.

5. Agarwal, P. K., N. Husain, and B. N. Singh. 1989. Cytologic findings in

aspiration hydatid fluid. Acta Cytol. 33:652654.6. Akhtar, M., M. Bakry, S. M. H. Qadri, and M. A. Ali. 1991. Diagnosis of

cutaneous leishmaniasis by fine-needle aspiration biopsy. Report of a case.Diagn. Cytopathol. 7:172177.

7. Akura, K., M. Hatakenaka, K. Kawai, M. Takenaka, and K. Kato. 1988.Use of immunocytochemistry on urinary sediments for the rapid identifi-cation of human polyomavirus infection. A case report. Acta Cytol. 32:247251.

8. Allen, A. R., and C. D. Fullmer. 1972. Primary diagnosis of pulmonaryechinococcosis by the cytologic technique. Acta Cytol. 16:212216.

9. Amin, M. B., E. Mezger, and R. J. Zarbo. 1992. Detection ofPneumocystiscarinii. Comparative study of monoclonal antibody and silver staining.Am. J. Clin. Pathol. 98:1318.

10. Ang, G. A., W. M. Janda, R. M. Novak, and L. Gerardo. 1993. Negativeimages of mycobacteria in aspiration biopsy smears from the lymph node ofa patient with acquired immunodeficiency syndrome (AIDS). Report of acase and a review of the literature. Diagn. Cytopathol. 9:325328.

11. Arnow, P. M., and P. Gardner. 1983. Usefulness of the Gram stain inLegionnaires disease. Clin. Microbiol. Newsl. 5:125127.

12. Ascoli, V., A. Teggi, and F. Nardi. 1990. Hydatid cyst: primary diagnosis byfine-needle aspiration biopsy. Diagn. Cytopathol. 6:4448.

13. Ashton, P. R. 1983. Infectious organisms in cytologic material. Lab. Med.14:227233.

14. Austin, P., A. Dekker, and J. S. Kennerdall. 1983. Orbital aspergillosis:report of a case diagnosed by fine needle aspiration. Acta. Cytol. 27:166169.

15. Ayre, J. E. 1949. The vaginal smear: precancer cells studies using amodified technique. Am. J. Obstet. Gynecol. 58:12051219.

16. Ayre, J. E. 1960. Role of the halo cell in cervical cancerigenesis: a virusmanifestation in premalignancy? Obstet. Gynecol. 13:735738.

17. Bailey, T. M., M. Akhtar, and M. A. Ali. 1985

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