key: cord-016962-8vjaot6i authors: Pantanowitz, Liron; Leiman, Gladwyn; Garcia, Lynne S. title: Microbiology date: 2011-07-04 journal: Cytopathology of Infectious Diseases DOI: 10.1007/978-1-4614-0242-8_4 sha: doc_id: 16962 cord_uid: 8vjaot6i In order to render an accurate diagnosis, and correctly identify clinically important microorganisms, a good understanding and knowledge of microbiology is essential. This chapter provides a broad overview of microbiology that is relevant to the practicing cytologist. Virology addresses the cytopathic effects caused by viruses and discusses many key infections. Bacteriology covers important bacterial causes of infection including those due to mycobacteria and filamentous bacteria. Mycology deals with common fungi as well as deep mycoses, particularly those caused by invasive and dimorphic fungal organisms. Parasitology highlights the protozoa, apicomplexans, and helminths likely to be seen in cytology samples. Algae are also briefly mentioned. Cytologists are likely to encounter infectious diseases either because a cytology sample was obtained for diagnostic purposes or incidentally when an infectious process or microorganism is discovered in the material they are reviewing. In order to render an accurate diagnosis, and correctly identify clinically important species or microorganisms, a good understanding and knowledge of microbiology is essential. This chapter provides a broad overview of microbiology that is relevant to the practicing cytologist, but is not intended to replace standard microbiology texts. Viruses replicate only inside host cells. Their particles (called • • virions) consist of DNA or RNA and a capsid (coat) that may be surrounded by a lipid envelope. Once they attach to and penetrate cells, they uncoat and replicate so that their progeny may be released following host cell lysis. Viral infection may cause cell death, proliferation, or neoplastic • • transformation (oncogenesis) ( Table 4 .1). Tumor viruses may promote cancer by expression of viral oncoproteins (or oncogenes) and/or inactivation of tumor suppressor genes. In general, viruses are too small to be identified directly by • • light microscopy. Viruses that remain latent often do not cause apparent changes to infected cells. However, several viruses may cause cytopathic changes (Fig. 4 .1) that affect the nucleus (e.g., inclusions, margination, multinucleation), cytoplasm (e.g., koilocytosis, syncytial giant cell formation), and/or entire cell (e.g., cytomegaly, ciliacytopthoria). Recognition of these changes can be life-saving as this would initiate confirmatory studies and/or therapy ( Fig. 4.2) . Superinfection by pyogenic bacteria is a complication of many • • viral infections that may mask subtle viral changes. Papillomaviruses (genus) are nonenveloped viruses that contain • • double-stranded circular DNA molecules that replicate exclusively in skin and/or mucosal keratinocytes. They belong to the Papillomaviridae family. Their genome is divided into an early (E) region that encodes • • genes E1-E7, and a late region (L) that encodes the capsid genes L1 and L2. In oncogenic human papillomavirus (HPV) types, the genes E6 (binds to p53) and E7 (binds to retinoblastoma [Rb] protein) are key players in transforming cells. HPV infection causes various diseases including warts (ver-• • ruca), anogenital lesions (condylomata acuminata, intraepithelial neoplasia) and cancer, epidermodysplasia verruciformis (genodermatosis), oral and laryngeal papillomas, as well as orpharyngeal and conjunctival cancer. Genital HPV infection is covered in greater detail in Chap. 5. Retroviruses are enveloped viruses that belong to the viral family Retroviridae. They are RNA viruses that replicate in host cells using the enzyme reverse transcriptase to produce DNA from its RNA genome. DNA is then incorporated into the host genome. that measure 0.5-5.0 mm in length. Mycoplasma spp. are among the smallest bacteria. Bacteria have a wide range of shapes. Most are spherical (cocci) or rod-shaped (bacilli), but they may also be curved or spiral-shaped (e.g., spirochaetes, Helicobacter pylori). Some bacteria are described as being coccobacilli because they have the ability to exist as a coccus, bacillus, or intermediate form (e.g., Haemophilus influenzae, Rhodococcus equi, Bartonella spp.). Bacteria may also form pairs (e.g., diploids), chains (e.g., Streptococcus), or clusters (e.g., Staphylococcus). Some bacteria may also have flagella. Anerobic bacteria do not need oxygen for growth. Some anerobes • • die when oxygen is present (obligate anerobes), whereas others will utilize oxygen if it is present (facultative anaerobes). They are found in normal flora (e.g., Fusobacterium in the mouth, Bacteroides fragilis in the large bowel, Lactobacillis in the vagina). These bacteria can usually be isolated from abscesses, aspiration pneumonia, empyema, and wounds. Material being collected from sites that do not harbor indigenous flora (e.g., body fluids other than urine and fine needle aspirates) should always be cultured for anerobic bacteria. Bacteria, along with some fungi (mainly • • Candida spp.) and archaea (single-celled microorganisms), make up the normal human flora of the skin, mouth, gastrointestinal tract, conjunctiva, and vagina (lactobacilli). Loss of normal flora may permit the unfavorable growth of harmful pathogens that can lead to infection. Some bacteria form biofilms, which are bacterial aggregates • • embedded within a self-produced matrix (slime). These bacterial clusters, seen associated with amorphous mucoid material, may be encountered in cytology specimens related to catheter infections, Pseudomonas aeruginosa pulmonary infections in cystic fibrosis, middle ear infections, joint prostheses, and dental (gingival) disease. Bacteria can generally be divided into Gram-positive and Mycobacteria are aerobic Gram-positive rod-shaped bacilli • • that are acid-alcohol fast (so-called AFB) with acid fast stains (Fite, Ziehl-Neelsen, Kinyoun, and auramine rhodamine stains). Mycobacterium tuberculosis are strongly acid fast positive (stain deep red), thin, and slightly curved bacilli that measure 0.3-0.6 × 1-4 nm ( Fig. 4 .5). The bacteria of MAI are typically short and cocobacillary like. Beading may be seen in some mycobacteria, which represents nonuniform staining of the bacillus. For example, M. kansasii are characteristically long and broad and exhibit a cross-banded or barred appearance. Acid-fast staining of morphologically similar bacteria such as Nontuberculous mycobacteria (NTM), which include all of the other mycobacteria. These are also known as atypical mycobacteria, mycobacteria other than tuberculosis (MOTT), or environmental mycobacteria. Infections with these mycobacteria are increasingly being seen in immunosuppressed patients. Infection causes lung disease but may also disseminate to involve the hematopoietic system, gastrointestinal tract, as well as skin and soft tissue. While most NTM can be detected microscopically with an acid-fast stain, culture and/or molecular studies may be required to identify these species. Bacteria can be elongated to form filaments (e.g., Actinobacteria, Nocardia, Rhodococcus, Streptomyces, Actinomadura). They can sometimes form complex, branched filaments that morphologically resemble fungal mycelia (mass of branching hyphae). These bacteria are usually part of the normal oral flora. Most infections are acquired by inhalation of the bacteria or via trauma. • • Actinomyces (genus) belong to the Actinobacteria (class of bacteria). Infection (actinomycosis) with these Gram-positive bacteria forms multiple abscesses and sinus tracts that may discharge sulfur granules. Actinomycosis is most frequently caused by Actinomyces israelii. Chlamydia are known to produce human disease: Chlamydia pneumonia (also Chlamydophila pneumoniae and previously known as the TWAR agent). Infection causes pharyngitis, bronchitis, and atypical pneumonia. Less common infections include meningoencephalitis, arthritis, and myocarditis. An association with atherosclerosis and possibly lung cancer has been reported. Chlamydia trachomatis (previously called TRIC agent). This includes three human biovars: trachoma (serovars A, B, Ba or C), urethritis (serovars D-K), and lymphogranuloma venereum (LGV, serovars L1, 2 and 3). Infection causes inclusion conjunctivitis (trachoma), pneumonia in neonates, and sexually transmitted disease in adults (e.g., cervicitis, urethritis, salpingitis, proctitis, epididymitis). Chlamydia psittaci (also called Chlamydophila psittaci) causes respiratory psittacosis and is acquired from birds ( Fig. 4.8) . On the basis of morphologic forms fungi can be divided into Hyphae can produce • • conidia (synonymous with spores). Large complex conidia are called macroconidia. Smaller more simple conidia are termed microconidia. When these conidia are enclosed in a sac (the sporangium) they are called endospores. A sporangium-bearing hypha is referred to as a sporangiophore. Dimorphism ( • • dimorphic fungi) is the condition whereby a fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions (Fig. 4.9 ). Candida • • Candida is a polymorphic fungus that undergoes a yeast-tomycelial transition. In clinical specimens, they produce pseudohyphae (hyphae that show distinct points of constriction resembling sausage links), rarely true septate hyphae, and budding yeast forms (blastoconidia). The yeast-like forms (blastoconidia) are oval and measure • • 3-5 mm in diameter Fig. 4.9 . Fungal morphology. Hyphae may be characterized as (a) pseudohyphae (e.g., Candida spp.), (b) septate (e.g., Aspergillus) or (c) coenocytic (aseptate) hyphae (e.g., Zygomycetes). Conidia (spores) develop from asexual fruiting structures such as (d) a conidiophore or (e) enclosed in a sac called a sporangium, in which case they are then called endospores. Although typically seen extracellularly, intracellular can mimic other small fungi such as Histoplasma. Candida usually exhibit variably sized yeast cells, lack a pseudocapsule, and elicit more of a suppurative reaction than a granulomatous response. • • Candida yeasts form part of the normal flora on the skin and mucous membranes of the respiratory, gastrointestinal, and female genital tracts. They often contaminate cytology samples from these sites. They may also colonize tissue (e.g., after prolonged antibiotic use, prolonged skin moisture, and in patients with diabetes). Infection may result from overgrowth or when introduced into • • the body (e.g., intravenously). Superficial infections include oropharyngeal and vulvovaginal candidiasis (thrush). Candidiasis may also become a systemic illness causing widespread abscesses, endocarditis, thrombophlebitis, endocarditis, eye infections, or involve other organs. • • Candida albicans is clinically the most significant member of this genus. Candida glabrata (previously known as Torulopsis glabrata) is a nondimorphic species (only has a yeast form) (Fig. 4.10 ). Cryptococci are small (5-15 • • mm) pleomorphic (ovoid to spheroid) yeasts that are characterized by often having a thick gelatinlike capsule and demonstrating narrow-based (teardrop-shaped) budding. They have thin walls and are occasionally refractile. Their capsules may have a diameter of up to five times that of the fungal cell, and form a halo on Diff-Quik, Pap, and India ink stains. Smaller (2-5 • • mm) capsule-deficient cryptococci can resemble other organisms with similar microforms (e.g., Histoplasma, Candida, and immature spherules of Coccidioides immitis). In such cases, with careful examination some weakly encapsulated yeasts can still be detected. Loss of capsular material usually elicits an intense inflammatory reaction characterized by suppuration and granulomas. Yeasts usually produce single buds, but multiple buds and even • • Aspergillus genus consists of many mold species. Pathogenic species include Aspergillus fumigatus and Aspergillus flavus. These fungi consist of septate hyphae that branch at 45° angles. Other dichotomous hyphae that may mimic Aspergillus include the hyalinohyphomyces (e.g., Fusarium, Penicillium) and dermatophytes. Species specific conidiophores called fruiting bodies have swollen The zygomycetes belong to the phylum Zygomycota ( (zygospores) and a vegetative mycelium. They have broad, ribbon-like, aseptate hyaline hyphae (coenocytic hyphae) with wide-angle branching. These morphological features are helpful in differentiating the zygomycetes from other fungal agents of infection that may be seen in cytologic specimens (Table 4 .5). In cytology samples hyphal forms may be twisted, collapsed, or • • wrinkled making them hard to evaluate. Moreover, in tissue sections from biopsies or cell block material, folds and creases in the section may cause the hyphae to appear as if they have septae. In respiratory samples, the zygomycetes can be distinguished Dimorphic fungi can exist both as a mold form that consists of • • hyphae (when grown at room temperature outside the host) and as yeast (when grown at body temperature in the host). Therefore, in clinical samples obtained from patients the cytologist will encounter yeasts from these organisms (Table 4 .6). Several such fungal species are potential pathogens. The most well-known species of this genus is Blastomyces dermatitidis, endemic to the United States (especially the southeastern, south central, and midwestern states) and Canada. Infection (blastomycosis) occurs by inhalation of the fungus from its natural soil habitat. Infection may involve virtually any organ including the lungs, skin, bones, and brain ( Fig. 4.15 ). • • Coccidioides. This fungus presents with endospores contained within thick-walled spherules that vary in size (20-150 mm). Endospores measuring 3-5 mm may be seen scattered singly if the spherule ruptures. When free endospores occur within macrophages, they can imitate other intracellular yeasts. The causative agents of infection (coccidioidomycosis) are C. immitis and C. posadasii. These fungi are endemic in American deserts. Infection causes granulomatous and miliary disease affecting largely the lungs. In endemic regions, fungus balls may develop within lung cavities. • • Paracoccidioides. These yeasts measure 5-30 mm in size and are round to oval. Budding is characterized by a central yeast with multiple surrounding daughter buds, that morphologically resembles a "ship's wheel." Infection (paracoccidioidomycosis) is caused by Paracoccidioides brasiliensis, typically found in Brazil and elsewhere in South America. Primary infection (Valley Fever) is usually mild and self-limiting, but may progress into a systemic mycosis producing oral lesions, generalized lymphadenopathy, and miliary pulmonary lesions. Infection can also spread to bones, meninges, and the spleen. • • Histoplasma. This budding yeast is round to oval, on average 1-5 mm in size and observed mainly within macrophages (Fig. 4.16 ), but sometimes also within neutrophils. Narrow based round to oval budding may be noted, but because of their small size buds are often not seen. Intracellular yeasts are usually surrounded by a clear zone (halo). However, with cell disruption organisms may be spilled extracellularly. This fungus is usually found in bird and bat ( • • Penicillium. These fungi produce penicillin. Penicillium marneffei is the only known thermally dimorphic species. In cytology specimens, Penicillium present in the mold phase with septate and branched hyphae represent a contaminant. However, in immunosuppressed patients P. marneffei is an opportunistic infection that causes penicilliosis. There is a particularly high incidence of penicilliosis in AIDS patients from tropical Southeast Asia. Infection after inhalation spreads from the lungs to involve the hematopoietic system and skin. Yeast-like cells are present within macrophages and extracellularly. They are not true yeast cells, but rather arthroconidia. Intracellular "yeasts" measure 2-3 mm in diameter and are round to oval. Because they divide by binary fission, budding is not observed. The extracellular organisms tend to be more elongated, sometimes up to 13 mm, and can have "septae" (crosswalls from binary fission). • • Pneumocystis jirovecii (previously called Pneumocystis carinii) is a yeast-like fungus of the genus Pneumocystis, which is the causative organism of Pneumocystis pneumonia (or pneumocystosis, formerly referred to as PCP). The cysts often collapse forming crescent-shaped bodies. All stages of the life cycle are found within the lung alveoli. Once inhaled, unicellular trophozoites (1-4 mm, Giemsa positive) undergo binary fission to form a precyst (difficult to distinguish by light microscopy) and ultimately develop thick walled cysts (5-8 mm, GMS positive). Spores (eight) form within these cysts, which are eventually released on rupture of the cyst wall. This organism is often seen in the lungs of healthy individuals, • • but is an opportunistic pathogen in immunosuppressed people, especially those with AIDS. Extrapulmonary disease may be seen with advanced HIV infec-• • tion presenting with involvement of the lymph nodes, spleen, liver, bone marrow, gastrointestinal tract, eyes, thyroid, adrenal glands, kidneys, and within macrophages in pleural effusions (Fig. 4.17 ). The dematiaceous (naturally pigmented) group of fungi pro- • • mycosis (also called chromomycosis) and phaeohyphomycosis (or phaeomycotic cyst). Dermatophytes cause infections of the skin and hair (ringworm • • or tinea) as well as the nails (onychomycosis). The three genera that cause these diseases include Microsporum, Epidermophyton, and Trichophyton. A rapid scraping of the nail, skin, or scalp can be used to iden-• • tify characteristic hyphae and sometimes spores associated with squamous cells or within broken hairshafts. Hyalohyphomycosis is the term used to group together inva-• • sive mycotic infections caused by hyaline septate hyphae. This includes species of Aspergillus, Penicillium, Paecilomyces, Acremonium, Beauveria, Fusarium, and Scopulariopsis. They may represent contamination or cause invasive disease in the immunosuppressed host. • • Fusarium hyphae are similar to those of Aspergillus, with septate hyphae that branch at acute and right angles. Sporulation may also occur in tissue with infection (fusariosis). Their macroconidia are crescent-shaped, orangeophilic, and septate structures that measure 80-120 × 3-6 mm in size. Protozoa are unicellular motile organisms. They are tradition- ally divided according to their means of locomotion such as amebae, flagellates, and ciliates. Their life cycle often alternates between trophozoites (feeding-• • dividing stage) and cysts (dormant stage able to survive outside the host). Their characteristics (particularly the nuclei and cytoplasmic inclusions) help in species identification. Ingested cysts cause infection by excysting (releasing trophozoites) in the alimentary tract. Intestinal amebae. Entamoeba histolytica causes amebiasis that may manifest with dysentery, flask-shaped colon ulcers, a colonic ameboma, and possible extraintestinal abscesses that contain anchovy paste-like material within the liver, and infrequently spleen or brain. Several of the amebae such as Entamoeba dispar are harmless and some may be relatively common, such as Entamoeba gingivalis that is usually found in the mouth. Unlike other amebae, the cytoplasm of pathogenic E. histolytica contains ingested red blood cells. Free-living amebae. These include Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri. Acanthamoeba cause granulomatous amebic encephalitis (GAE), contact lensassociated Acanthamoeba keratitis, and skin lesions. Balamuthia also causes GAE. N. fowleri ("brain-eating" ameba) is associated with rapidly fatal primary amebic meningoencephalitis (PAM), most often seen in children swimming in fresh water ponds and rivers during which amebae enter the nasal passages and migrate to the brain via the olfactory nerve. These trophozoites can be seen in CSF specimens, but culture on nonnutrient agar plates seeded with Escherichia coli and/or a flagellation test is required for confirmation. • • Flagellates (Mastigophora) are organisms that have one or more flagella. Giardia. There are approximately 40 species described, but the species that infects humans is Giardia lamblia (also called G. intestinalis or G. duodenalis). This parasite is the most common cause of protozoal gastroenteritis (giardiasis). Trophozoites (9-21 mm long and 5-15 mm wide) are kite (or pear)-shaped and have two nuclei, four pairs of flagella, and two central axonemes running down their middle. Their cysts (8-14 × 7-10 mm) seen in stool specimens are oval, thick walled, and contain four nuclei and multiple curved median bodies. Trichomonas. There are several trichomonad species such as the intestinal Pentatrichomonas hominis which is a nonpathogenic organism. Trichomonas vaginalis is the anaerobic, flagellated protozoan that causes trichomoniasis (Fig. 4.18) . Details of this sexually transmitted infection are covered in Chap. 5. Apart from urogenital infections, T. vaginalis has also been reported to cause pneumonia, bronchitis, and oral lesions. Pulmonary trichomoniasis is usually caused by aspirated Trichomonas tenax (mouth commensal) and less often T. vaginalis infection. An association between flagellated protozoa and asthma has been reported. Leishmania. These parasites are acquired from the sandfly. Depending on the species, they may cause cutaneous (e.g., oriental sore), mucocutaneous (espundia or uta), or visceral (kala-azar) leishmaniasis (Table 4 .7). There are two morphological forms: promastigote (with a flagellum) found in the insect host and an amastigote (without flagella) present in the human host ( Fig. 4.19 ). Diagnostic samples may be procured from skin lesions (cutaneous or mucocutaneous) or bone marrow aspirates (visceral). The morphologic hallmark is the presence of multiple small (2-5 mm) intracellular amastigotes within macrophages (Leishman-Donovan bodies). Amastigotes are spherical to ovoid in shape and have both a nucleus and ovoid or rod-shaped kinetoplast. The differential diagnosis for multiple small organisms within histiocytes includes H. capsulatum (with budding and only intracellular) and toxoplasmosis (more curved and mostly extracellular). Trypanosoma. The major human diseases caused by trypanosomatids are African trypanosomiasis (sleeping sickness) caused by Trypanosoma brucei and American trypanosomiasis (Chagas disease) caused by T. cruzi. Trypomastigotes (30 mm in length) are usually found in peripheral blood. They have an undulating membrane, central nucleus, and kinetoplast at the anterior end. • • Ciliates (Ciliophora) include the parasitic species Balantidium coli, the only member of this phylum known to be pathogenic to humans. The trophozoite is relatively large (50-70 mm), has a ciliated surface, and contains a kidney bean-shaped macronucleolus. The cyst form may occasionally also have cilia. Cilicytophthoria may be sometimes mistaken for these ciliated organisms. The Apicomplexa are a diverse group of protists that includes The microsporidia, at one time a separate group (not coccidian), are now classified with the fungi. • • Cryptosporidium spp. C. parvum is the causative agent of cryptosporidiosis in humans and animals, a major cause of protracted diarrhea in patients with AIDS. Other species that cause human disease include C. hominis. These small (8-15 mm) oval parasites are identified within the brush border of the intestinal epithelium, and are discussed in the chapter on gastrointestinal infections. Ultrastructural studies have shown them to be intracellular but with an extracytoplasmic localization in enterocytes. Modified acid-fast thick-walled oocysts (4-6 mm) may be detected in stool samples. Cryptosporidium can disseminate beyond the intestine, especially in patients with AIDS, to involve the biliary tract, stomach, lungs, middle ear, and pancreas. • • Microsporidia include Enterocytozoon bieneusi and Encephalitozoon intestinalis (previously Septata intestinalis). Although these organisms are now considered fungi, parasitologists still maintain them in most books. In cytology specimens obtained from the small intestine, microsporidia appear as numerous small intracellular organisms within the apical portion of enterocytes. They stain well with Gram and silver stains (e.g., Warthin-Starry). Their spores (1-1.5 mm) may be found with a modified trichrome stain in stool samples as well as urine. • • I. belli and Sarcocystis infections very rarely have trophozoites that are detected. Their oocysts, however, may be seen when excreted in feces. • • T. gondii belongs to the genus Toxoplasma. Although infection can be acquired from the accidental ingestion of infective oocysts from cat feces (definitive host), most infections are acquired from eating infected rare or raw meats. Disease ranges from mild flu-like illness to fatal fetal infections. Latent infection may reactivate in immunosuppressed patients. Organisms may be found in samples from the brain, heart, eye, hematopoietic system, and lungs. Specimens may contain free (extracellular) tachyzoites which are small (3-5 mm), curved (banana-shaped) forms (Fig. 4.20) . When parasites accumulate within macrophages (so-called "bag of parasites") they form a pseudocyst (parasitophorous vacuole) containing bradyzoites. Helminths (parasitic worms) are categorized into three groups: • • cestodes (tapeworms), nematodes (roundworms), and trematodes (flukes) ( (Fig. 4.21) . Infections are usually diagnosed by the characteristics of these different developmental stages. Diagram showing a macrophage with a pseudocyst containing multiple bradyzoites: Note that some of these microorganisms are still crescent shaped. The cysts are usually round in brain tissue, more elongated in muscle, and often very small and hard to identify in lung tissue. (Bottom right) An infected macrophage contains a cluster of bradyzoites (Pap stain, high magnification). Parasites The host response to these parasites includes eosinophilia, acute containing larvae located in several sites such as the brain (neurocysticercosis causes seizures), eye, as well as muscles and subcutaneous tissue (causes painful nodules). These cysts may cause eosinophilia, an inflammatory reaction and eventually they may calcify. Echinococcus. Infection from the accidental ingestion of food or drink contaminated with tapeworm eggs results in hydatid disease, also known as echinococcosis. The larval cysts that develop can be found in virtually any site, grow slowly, and persist for many years until they cause symptoms or are discovered incidentally. Disruption of a cyst containing highly antigenic fluid may result in anaphylactic shock, and for this reason it has been recommended that they should not be biopsied. However, on the basis of published data adverse reactions are rare. Use of a fine gauge needle by a skilled operator is important to prevent fluid leakage during aspiration. (Table 4 .7), but those likely to be seen in cytology specimens are Enterobius vermicularis, Strongyloides stercoralis, and the microfilariae. E. vermicularis (pinworm, also known as the threadworm in the United Kingdom) causes intestinal infestation (enterobiasis). The adult female worm migrates out onto the perianal area at night, and once exposed to oxygen she lays her eggs. This causes perianal pruritis and possibly vaginitis. Occasionally subcutaneous perineal nodules may develop. In such cases, Pap tests may contain an adult worm and/or eggs. The worm has a characteristic pointed tail (Fig. 4.23) . Their eggs are colorless, oval, thin walled, and flattened on one side, S. stercoralis. This worm causes strongyloidiasis. After penetrating the skin, infective larvae pass through the lung (Loeffler syndrome). They are then coughed or swallowed and infest the duodenum. Here new autoinfective larvae may develop (autoinfection) leading to chronic infection. In immunocompromised patients, larvae may penetrate the intestinal wall and the pattern of nuclei in their tail are the main features used to distinguish the various species. Occult filariasis has been diagnosed by many bloody FNA procedures containing microfilariae, worms, or even eggs. Filarial morphology is best appreciated with a Giemsa stain. The background tissue response in cytology aspirates may include eosinophils, neutrophils, chronic inflammation, and even granulomas. • • Trematodes. The flukes are oval or worm-like helminthes that are parasites of molluscs and vertebrates. The liver flukes include Fasciola hepatica and Clonorchis sinensis that result in infestation of the bile ducts and subsequent biliary fibrosis. Infection with C. sinensis is a risk factor for cholangiocarcinoma. Fasciolopis buskii is an intestinal fluke that infests both the bile ducts and duodenum. Paragonimus westermani, the lung fluke, causes lung infestation with pulmonitis. Also included are the schistosomes (blood flukes). Schistosomes. Infection by these trematodes causes schistosomiasis (bilharzia). There are three human schistosome pathogens: Schistosoma mansoni from Africa (Nile delta) which causes intestinal schistosomiasis, S. haematobium from Africa and the Middle East that infests the urinary bladder, and S. japonicum from China and Southeast Asia that primarily involves the liver. S. haematobium infection can lead to squamous cell carcinoma of the bladder. Microscopic identification of eggs in stool or urine specimens, as well as tissue biopsies, can provide a rapid diagnosis. The spines present on schistosome eggs help distinguish these different species. The egg of S. haematobium (oval) has a terminal spine, S. mansoni (oval) has a lateral spine, and S. japonicum (round) has a small knob-like lateral spine. Algae are ubiquitous and include a diverse group of simple • • organisms that range from unicellular to multicellular forms. The main algal groups include the cyanobacteria, green algae, and red algae (e.g., dinoflagellates). Most algae present in cytology samples are from contamination, • • discussed in greater detail in Chap. 15. Organisms may be seen within macrophages or extracellularly. They form spherical sporangia that range in size from 3 to 10 mm, have thick walls, and show no budding. These morular forms often contain endospores arranged symmetrically Ash & Orihel's atlas of human parasitology Cytologic diagnosis of adenovirus bronchopneumonia Amoebiasis: diagnosis by aspiration and exfoliative cytology Left) Diagram of protothecae demonstrating variable morula formation: (Right) A sporangium of P. wickerhamii is shown in which endospores have a moruloid (daisy-like) pattern (Pap stain, high magnification) (image courtesy of Rafael Martinez Girón Diagnostic medical parasitology Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology Role of fine needle aspiration cytology in diagnosis of filarial infestation Kala-azar: liver fine needle aspiration findings in 23 cases presenting with a fever of unknown origin Medically important fungi. A guide to identification Disseminated nocardiosis diagnosed by fine needle aspiration biopsy: quick and accurate diagnostic approach Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution The study of cytopathological aspects induced by human cytomegalovirus infection Atypial cytomorphologic appearance of Cryptococcus neoformans: a report of five cases