Int J Clin Exp Pathol 2014;7(8):4837-4846 www.ijcep.com /ISSN:1936-2625/IJCEP0001116

Original Article Clinicopathological features of pulmonary with cryptococcal titan cells: a comparative analysis of 27 cases

Jing-Mei Wang1,4, Qiang Zhou1, Hou-Rong Cai2, Yi Zhuang2, Yi-Fen Zhang1, Xiao-Yan Xin3, Fan-Qing Meng1, Ya-Ping Wang4

1Department of Pathology, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China; 2Department of Respiratory Medicine, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China; 3Department of Radiology, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China; 4Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Nanjing University Medical School, Nanjing, China Received June 15, 2014; Accepted July 28, 2014; Epub July 15, 2014; Published August 1, 2014

Abstract: In addition to the typical size, can enlarge its size to form titan cells during infec- tion, and its diameter can reach up to 100 μm. Clinical reports about cryptococcal titan cells are rare. Most studies focus on aspects of animal models of infection with titan cells. Herein, we report the clinical and imaging character- istics and histopathologic features of 3 patients with titan cells and 27 patients with pathogens of typical size, and describe the morphological characteristics of titan cells in details. Histologically, 3 patients with titan cells show ne- crosis, fibrosis and macrophage accumulation. The titan cells appear in necrotic tissue and between macrophages, and have thick wall with unstained halo around them and diameters range from 20 to 80 μm with characteristic of narrow-necked single budding. There are also organisms with typical size. All 27 patients with normal pathogens show epithelioid granulomatous lesions. There is no significantly difference in clinical and imaging feature between the two groups. Cryptococcus neoformans exhibits a striking morphological change for the formation of titan cells during pulmonary infection, which will result in misdiagnosis and under diagnosis. The histopathological changes may be new manifestation, which need to be further confirmed by the study with animal models of infection and the observation of more clinical cases. Careful observation of the tissue sections is necessary.

Keywords: Cryptococcus neoformans, titan cells, cryptococcosis, pulmonary infection, histopathology

Introduction pressant and the high prevalence of the im- mune deficiency diseases such as AIDS. Cryptococcus neoformans (CN) is an opportunistic pathogen, and also capable of Recent studies have revealed a dramatic mor- infecting immunocompromised and immuno- phological change of CN for the formation of competent individuals. Cryptococcal infection pathogenic giant cells - titan cells, which size can lead to pneumonia, meningitis and skin reach up to 100 μm in diameter [1-3]. Currently, lesions for human beings. Pulmonary crypto- the studies about cryptococcal titan cells are coccosis (PC) results from inhalation of fugal mainly confined to aspects of animal models of cells with subsequent lung infection and pneu- infection [2-4], and the research of pathogene- monia. Because the symptoms and radiograph- sis is still in the early stage, the clinical reports ic findings in PC are variable and nonspecific, are rare and with no clinical and pathologic PC is often misdiagnosed as ordinary pneumo- description in details. Herein, we report 3 PC nia, lung cancer and diffuse lung disease. In cases with titan cells, and analyze retrospec- recent years, the incidence of PC is rising due tively the clinical, radiological and pathological to the abuse of broad-spectrum antibiotics, the data of 27 PC cases with pathogens of typical wide use of cytotoxic agents and immunosup- size (ordinary PC patients) in order to further Cryptococcal titan cells in patients with pulmonary cryptococcosis understand the disease and its clinicopatho- days to 8 months. All patients had no head- logical features. aches, vomiting and other symptoms of menin- geal irritation. Admission diagnosis found pneu- Patients and methods monia, diffuse lung disease, tuberculosis, lung cancer, metastatic cancer and occupying le- All cases (n = 30) were hospitalized patients (n sions, and one patient was diagnosed with = 21) and patients for consultation (n = 9) in acute lymphoblastic leukemia with pulmonary Affiliated Drum Tower Hospital of Nanjing fungal infection. University Medical School from 2004 to 2013. These cases include 3 PC cases with titan cells All patients showed negative results for HIV on and 27 ordinary PC cases. We collected clinical admission examination. Fungal culture showed information and imaging data of all patients. a negative result for sputum.

All specimens were submitted for standard his- Radiographic examination tological processing. Tissue sections were stain- ed by hematoxylin and eosin (HE). Special stain- Chest radiographic examination for cases with ing methods, including Gomori methenamine titan cells exhibited nodules, consolidation, or silver/Grocott’s silver (GMS) and periodic acid- ground-glass attenuation and patchy shadows. Schiff (PAS), were performed to evaluate these All lesions were involved in bilateral lung, and findings further. subpleural lesions were visible. One of the cases was accompanied with cavitary lesions. Results Enhanced CT scan showed a negative result. Pertinent clinical, image and histopathological The chest X-ray/initial CT scan findings of 20/27 features of 3 cases with cryptococcal titan cells ordinary PC patients were collected. Nine are summarized in Table 1. patients showed pulmonary nodules/masses, Clinical information and most of nodules had poorly defined mar- gins. Seven patients were observed to have Three PC cases with titan cells aged 43, 56 and patchy airspace consolidation. Four patients 72 years respectively, including 2 males and 1 exhibited a mixed pattern with nodules and female. Both chief complaints were cough and patchy reticular opacity. Other associated find- expectoration, which lasted for 1-3 months. ings included cavitation (n = 2) and pleural effu- Underlying diseases in 2 patients were autoim- sion (n = 1). The majority of patients had lesions mune liver disease, Sjogren’s syndrome and in the peripheral lung zone. Thirteen patients idiopathic thrombocytopenia. Both were medi- had the lesions involving unilateral lung, 7 cated with immunosuppressant drugs for years. patients had the lesions with bilateral lung Another case without underlying disease firstly involvement. Single lobe involvement was complained of hemoptysis and fever with the found in 13 patients, of whom 7 patients highest temperature of 39.6°C. involved the lower lobe. CT scan showed mild enhancement in only one case. Twenty-seven ordinary PC patients include 19 males and 8 females with a mean age of 53 Macroscopic and histopathologic features years (24 to 80 years). Excepting 11 cases with- out underlying disease, other patients had suf- All samples of PC cases with titan cells were fered from malignant tumor, Sjogren’s syn- CT-guided percutanous transthoracic needle drome, chronic bronchitis, tuberculosis, dia- biopsy (PTNB) pulmonary specimens, with case betes, hypertension, cerebral infarction and 1 followed by lobectomy. Two cases had similar smoking for decades. Three patients had a his- histological features, and showed lots of necro- tory of exposure to pigeons or parrots, and 2 sis, fibrosis and monocyte-macrophage aggre- patients had traveled abroad. Fifteen cases gation by HE staining. There were large amounts had abnormal chest radiograph during routine of pathogens surrounded by a transparent physical examination, including 7 patients unstained area within necrosis, even at lower being treated for malignancy. The other patients magnification. Pathogens appeared as round or (n = 12) complained of cough, sputum, fever, oval fungal organisms with variable size, shell- chest pain and tightness, which lasted for 4 shaped, blue-gray or red dye thick-wall at high

4838 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis

Table 1. Summary of the clinical, radiological and histopathological findings of 3 PC cases with titan cells Pa- Age/gen- Underlying Disease; Medica- Initial Proffered Diagnostic Follow- Chief Complaint Image Features Histopathologic Features Treatment tient der tion History Diagnosis Procedure up 1 72/male Autoimmune liver disease, Cough and Multiple nodules with leaf sign Occupying lesions PTNB, Lots of necrotic tissue, fibrosis Anti-fungal Remis- Sjogren’s syndrome; expectoration for in upper right and lower left lung lobectomy and macrophages accumula- agents com- sion Methylprednisolone 1 month lobe, some beneath the pleura, no tion with a large number of bined with enhancement titan cells lobectomy 2 43/male No underlying disease; Antibiot- Cough, hemopty- Initially, ground-glass opacity in right Pneumonia PTNB Lots of necrotic tissue, fibrosis No treat- Lost ics and hormone therapy sis and fever for 3 lung; then, consolidation in bilateral and macrophages accumula- ment follow-up months lower lobe with cavitation and pleu- tion with a large number of ral effusion titan cells 3 56/female Idiopathic thrombocytopenia; Cough and expec- Multiple nodules and patchy shad- Occupying lesions PTNB Necrotizing epithelioid granu- Anti-fungal Remis- Prednisone and Tripterygium toration for more ows in bilateral middle and lower lomas with organizing pneu- agents only sion than 2 months lobes, some beneath the pleura, no monia with a few titan cells enhancement PTNB, percutanous transthoracic needle biopsy.

4839 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis

Figure 1. Case 1. A. Large amounts of titan cells surrounded by peripheral halo within the necrotic tissue even at lower magnification (HE×100, HE×400 [inset A]). B. Many macrophages accumulated with indistinct vacuoles, and fibrosis with minor lymphocyte infiltration (HE×100). C. PAS staining outlined the fungal cells with thick and red dyed walls within necrosis, and diameters ranged from 20 to 80 µm (×400). D. GMS staining revealed typical narrow- necked budding, as red arrows denoted (×400). magnification Figure( 1A). Many macrophages Multinuclear giant cells, neutrophils and eosin- accumulated with vacuoles of variable size sur- ophils were not observed, In the necrotic zone rounding the necrosis, and fibrosis with minor and the surrounding alveolar space, many lymphocyte infiltration also could be seen round or oval fungal pathogens of varying sizes (Figures 1B and 2A). PAS and GMS staining of can be observed at lower magnification, which the section outlined the fungal cells with red manifested as larger size, thick and red-stained dyed and brown-black dyed walls within necro- wall, shell-like shape and unstained transpar- sis (Figures 1C and 2B) and between macro- ent zone around them and were more apparent phages (Figure 2C), and showed single typical by PAS staining. The specimens from case 3 narrow-necked budding, expressing as a nor- showed epithelioid granuloma with organizing mal size cell attached to a titan cell (Figures 1D pneumonia and small focal necrosis, and a lot and 2B). The organisms were strikingly differ- of macrophages infiltrated under fibrotic back- ent in size. The titan cells size could reach up to ground around the necrotic foci (Figure 3A), 80 µm, and the small cells were 2 to 20 µm in and a handful of pathogenic giant cells shown diameter (Figures 1 and 2). The specimens by special staining were observed (Figure 3B). from case 1 with followed pulmonary segmen- However, the pathogens in multinuclear giant tectomy showed necrotic foci of varying sizes cells of epithelioid granuloma had typical size by microscope, which were surrounded by of common fungi. fibers and collagen with large amounts of mac- rophage reactions, while focal lymphocytic infil- The specimens in 27 ordinary PC patients were tration was found in the edge of the fibers. selected from PTNB in 15 cases, pulmonary

4840 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis

Figure 2. Case 2. A. Monocyte-macro- phage accumulation accompanied with fibrosis and focal necrosis (HE×100). B. PAS staining exhibited the pathogenic giant cells and small cells with viable size, and red arrow denoted the typical budding pattern (×400). C. PAS staining showed the titan cells between macro- phages (×400).

Figure 3. Case 3. A. The specimen of PTNB showed epithelioid granulomatous inflammation with small focal necro- sis (HE×100). B. Two titan cells were observed by GMS staining, and more pathogenic small cells were seen (×400). lobectomy/segmentectomy in 8 cases, thora- to 4.5 cm, and gray, grayish yellow or taupe and coscopic resection of lung lesions in 3 cases solid section, most of which were found at sub- and transbronchial lung biopsy in one case. pleural region of the lung. Histological sections Gross examination in 8 patients undergoing findings in 27 PC patients showed epithelioid pulmonary lobectomy/segmentectomy showed granulomatous lesions, among them, 21 patien- as follows: intact pleura on the surface of the ts were found to be accompanied by non-necro- lung, and single or multiple lung nodules with tizing epithelioid granuloma, 6 cases by necro- the maximum diameter ranging from millet size sis including 3 cases with abscess (Figure 4A),

4841 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis

Figure 4. A. Tissue section of an ordinary PC patient exhibited epithelioid granulomatous lesions with focal necrosis, and pathogens can be observed vaguely (HE×100). B. PAS staining appeared the pathogens as small cells of 4-7 μm in diameter in macrophages and multinuclear giant cells (×400). C. GMS staining showed that the sizes of pathogens were varied with diameters ranged from 2 to 20 μm in an ordinary PC case (×400).

11 cases by organizing pneumonia. Granulo- ited an improved and stable condition after matous lesions were composed by epithelioid treatment. Among 27 ordinary PC patients, 8 cells, fibrocytes, fibroblasts, multinuclear giant cases undergoing lobectomy/segmentectomy cells, lymphocytes and a few plasmocytes. On and 3 cases undergoing thoracoscopic resec- HE section, some round or oval pathogens of tion of lung lesions are in good condition cur- varying numbers may be seen indistinctly in rently after orally taking antifungal agents. multinuclear giant cells, epithelioid cells, tissue Except for four patients who were lost to follow- space and necrotic tissues. Orginisms were up, 4 patients with malignancy continued to shown as the vesicle shape or pale pink appear- progress, and the remaining cases exhibited ance, and around which translucent halo was improved and stable condition after anti-fungal found. PAS and GMS staining showed that the treatment. pathogens were 4-7 µm small cells in diameter and have typical narrow-necked budding Discussion (Figure 4B). Moreover, the size of the patho- As shown in the majority of journal literatures gens in one case was varying with the diameter and reference books in pathology, the average of 2-20 µm (Figure 4C). diameter of the CN cells was 4-7 μm with vary- Follow-up ing sizes (range: 2-20 μm), and single narrow- necked budding was often found. Most of Follow-up visit was conducted by calling and pathogens were surrounded by one layer of consulting the archival data of the patients. polysaccharide capsule with varying thickness, The follow-up visit lasted from 6 to 80 months. and the cell wall can be well displayed by PAS and GMS staining [5]. Excepting occasional In 3 PC patients with titan cells, case 2 was lost reports of CN pseudohyphal form [6-8], it was to follow-up because he discharged from hospi- shown that CN had three morphological and tal voluntarily, and the other two patients exhib- phenotypic variations: changes in capsule

4842 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis structure, changes in capsule size, and chang- cal small cells with the diameter of 2-20 µm es in the total size of the cell which can be could also be observed. These pathogens were achieved by the formation of cryptococcal titan similar to the morphology of the cryptococcal cells or microforms [9]. Only a few reports titan cells described in literatures [2, 3, 21]. showed that cryptococcal titan cells were found The specimens from case 1 who underwent the in the clinical samples [10, 11]. Titan cells were followed lobectomy also exhibited similar histo- found firstly in the lung, brain and cerebrospinal logical changes, and no epithelioid granulo- fluid of patients with cryptococcosis with the mas. The specimens from case 3 showed diameter of 40-60 µm and even more then 60 necrotic epithelioid granulomas with organizing µm. However, in the culture condition in vitro, pneumonia, but a few pathogenic giant cells these pathogens would revert to the small cells were observed, while the majority of pathogens with common forms. were small cells. The cryptococcal titan cells can be displayed obviously even by HE staining, Morphologically, the most significant features which is related to obvious increase of the total of cryptococcal titan cells are that cell volume cell size and significant thickening of cell wall increases strikingly, their diameter can reach and capsule. The distinct necrosis may be up to 100 μm and daughter cells with normal caused by the fact that the pathogenic giant size are reproduced by budding [2, 3]. The cap- cells cannot be swallowed by macrophages, but sules of titan cells are more dense than those the accumulated macrophages can release of typical cells, and the thickness of the cell large amounts of inflammatory mediators/cyto- wall can be up to 2-3 μm while typical cell wall kines to cause necrosis of the cells and tissues. thickness is 50-100 nm. Titan cells are mono- It is notable that multinuclear giant cells could nuclear polyploidy, while the typical cells are not be observed when large amounts of titan haploid. Titan cells are not easy to be swal- cells were appeared, comparing with epitheli- lowed by macrophages for the larger volume oid granulomatous lesions in ordinary PC [12]. patients. It is may be a new manifestation.

The majority of patients with PC need to be con- The diagnosis of PC due to cryptococcal titan firmed by the biopsy or surgical intervention cells for the 3 patients we described is based [13-16]. The histological characteristic of PC is on several histological features. First, the sizes changeable due to different immune states of of the pathogens observed in the 3 patients are the patients [5, 15, 17, 18]. In immunocompro- within the reported size range that the maxi- mised patients and immunocompetent individ- mum diameter of titan cells can be up to 100 uals, the histopathology often display as the μm. Second, the cell wall and capsule of the granulomatous lesions. There are few patients titan cells is dense and thick and the CN of typi- in whom organizing pneumonia is the main his- cal size can also be observed by HE and special tological change [19]. In patients with early or staining. Third, definite single narrow-necked severe immunosuppression, the histologic pat- budding can be identified, and no other fungal terns emerged as minimal inflammation with forms such as endospores, hypha and pseudo- plenty of extracellular organisms that efface hypha. In addition, titan cells are found not only the tissue architecture of lung [20]. The sec- in the necrotic tissues, but also between the tions in all 27 ordinary PC patients in this paper macrophages, moreover, all the 3 patients do showed epithelioid granulomas with/without not receive the treatment with antifungal necrosis and organizing pneumonia, and the agents when undergoing diagnostic biopsy. pathogens was composed of 4-7 µm small cells Therefore, it means that the morphological vari- in diameter in the majority of patients. All 3 ation is not a degenerative phenomenon sec- patients with titan cells exhibited necrosis, ondary to morphological adaptation and necrot- fibrosis and mononuclear macrophage aggre- ic tissues, or long-term antifungal treatment. It gation. Two patients were found to have large is in accord with the morphology of cryptococ- amounts of cryptococcal titan cells of varying cal titan cells described in literatures [3, 21], sizes in the degenerated necrotic tissues and and the diagnosis of PC thus be determined. between macrophages. Their diameters were of 20-80 µm. Moreover, the narrow-necked sin- The clinical and imaging manifestations of PC gle budding with small daughter cells and typi- are diverse, and it often involves a variety of

4843 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis lung diseases, especially malignancy [22]. The and requires 40°C to produce adiaspores, clinical presentation of PC are associated with which are 10 to 25 µm in diameter. Paraco- the immune status of the patient, and may be ccidioides brasiliensis are round or oval totally asymptomatic and also the severe pneu- cells with the diameter of from 3 to 30 µm and monia with respiratory failure [23]. The most occasionally as great as 60 µm. It is often iden- common symptoms include cough, expectora- tified that single and multiple narrow-necked tion, fever, chest pain, dyspnea, night sweat blastoconidia attached to the parent cell in a and hemoptysis [13, 24, 25]. The imaging man- “teardrop” budding by GMS staining. Cocci- ifestations are nonspecific, and include single dioides immitis and posadasii can or multiple pulmonary nodules/masses, seg- expand to form giant spherules with the diam- mental consolidation, diffuse interstitial infil- eter of 30-150 µm. The spherule can evolve a trates, cavitation and diffused mixed lesions large amount of endospores. The mature spher- [13, 24, 26]. The PC patients with titan cells ules would rupture and release endospores complained mainly of cough and expectoration into the infected tissues. The spherules and in this report, and 15 ordinary PC patients did endospores may appear simultaneously. The not exhibited obvious clinical symptoms, while diameter of endospores is generally 2-5 μm. the remaining 12 patients presented with non- GMS and PAS staining can display endospores specific clinical manifestations. Imaging exami- and immature spherules. Rhinosporidium see- nation showed no significant difference beri may result in Rhinosporidiosis. The sporan- between the PC patients with titan cells and gia are round or oval with clear boundary of ordinary PC patients, and lesions both involved wall, and their diameters can reach up to 200- the sub-pleural area. Nevertheless, the clinical 350 μm, so that they can be seen with the and imaging manifestations of PC patients with unaided eye in tissue sections. The mature titan cells need to be further explored. endospores are 8 to 10 μm in diameter, and released into the surrounding tissues following Cryptococcal titan cells detected in the clinical the rupture of the sporangia. specimens may result in a misdiagnosis. The patients infected by other pathogens with giant After CN spores or desiccated yeast cells are forms in fungus have rarely been reported. The inhaled and lodged into the lung from the envi- pathogens mainly include , ronment, the majority of organisms may be Blastomyces dermatitidis, genus Emmonsia, swallowed and eliminated by the mononuclear Paracoccidioides brasiliensis, Coccidioides po- phagocyte of host. However, formation of titan sadasii and [23, cells may make the pathogens escape and 27-32]. Giant blastoconidia can be seen in very adapt the host immune response, so that they few cases with Candida albicans infection with can survive for long time in the host to cause their diameters of up to 30 µm, and such infec- pulmonary infection [1]. However, the mecha- tions are common in immunocompromised nism for the formation titan cells is unclear. patients receiving the treatment with cytotoxic Many studies indicated that it is a phenomenon drug. The diameter of the pathogens in necrotic of morphological adaptation induced by the tissues and abscess can reach up to 20-40 μm interactions between the pathogens and host in case of Blastomyces dermatitidis infection, cells [1-3]. Thermal effect may be one of factors and HE sections showed that the yeast forms causing gigantism of the yeast cells [10]. The had thick, doubly refractile cell walls and promi- yeast cells isolated from human brain abscess nent nuclear content; meanwhile, the organism and then cultured at 25°C in vitro were the was black by GMS staining and exhibited broad- small cells, while the diameter of such cells can based buding. Genus Emmonsia can be giant be about 25 μm at 35°C. DNA replication with- cells and cause Adiaspiromycosis in human. out concomitant cell fission may be a potential The adiaspore size ranged from 25 to 400 µm mechanism for cell size increase and may for Emmonsia crescens. With the HE staining, cause that the cells contain mononuclear with the thick walls of the adiaspores appear to be polyploidy [2, 3]. Moreover, it may be related to composed of two layers: a narrow eosinophilic cAMP/protein kinase A (PKA) signaling pathway outer layer and a chitinous thicker inner hyaline [2], some g-protein coupled receptors (such as layer. The adiaspores are usually empty. Gpr5, Ste3a), and transcription factor Rim101 Emmonsia parva infection in human is rare, may be also involved in the process [33]. In

4844 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis addition, the formation of titan cells may be a Address correspondence to: Dr. Fan-Qing Meng, common response to phagocytic cells through Department of Pathology, Affiliated Drum Tower sensing of phospholipids in the membrane of Hospital of Nanjing University Medical School, 321 the pathogens [34]. Zhongshan Road, Nanjing, Jiangsu 210008, China. Tel: (86)-25-83105177; Fax: (86)-25-83105987; The intracellular cryptococcal cells were easier E-mail: [email protected]; Dr. Ya-Ping Wang, to disseminate to CNS compared to extracellu- Department of Medical Genetics, Jiangsu Key lar pathogens [35]. The daughter cells repro- Laboratory of Molecular Medicine, Nanjing University duced by titan cells have a stronger virulence Medical School, Nanjing 210093, China. Tel: (86)- and resistance to oxidative/nitrosative stress 25-83686495; Fax: (86)-25-83686451; E-mail: [2, 3]. Hence, titan cell production inhibits pha- [email protected] gocytosis to promote CN survival and enhance dissemination of CNS [36]. Garcia-Rodas con- References sidered that the formation of titan cells was related to a reduction in permeability consis- [1] Kronstad JW, Attarian R, Cadieux B, Choi J, tent with an increase in capsule density, which D’Souza CA, Griffiths EJ, Geddes JM, Hu G, may lead to decrease of the penetrability of Jung WH, Kretschmer M, Saikia S, Wang J. Ex- antifungal agents [4]. In 3 PC patients with titan panding fungal pathogenesis: Cryptococcus cells, case 1 exhibited a poor response to oral breaks out of the opportunistic box. Nat Rev antifungal agents after diagnostic procedure of Microbiol 2011; 9: 193-203. PTNB, and then had stable condition after [2] Zaragoza O, García-Rodas R, Nosanchuk JD, receiving lobectomy combined with antifungal Cuenca-Estrella M, Rodríguez-Tudela JL, Casa- agents, which indicated the resistance of titan devall A. Fungal cell gigantism during mamma- lian infection. PLoS Pathog 2010; 6: cells to antifungal agents. Case 3 and most of e1000945. ordinary PC patients who only received antifun- [3] Okagaki LH, Strain AK, Nielsen JN, Charlier C, gal therapy obtain a better efficacy. This result Baltes NJ, Chrétien F, Heitman J, Dromer F, may provide a clinical evidence for the resis- Nielsen K. Cryptococcal cell morphology af- tance of cryptococcal titan cells to antifungal fects host cell interactions and pathogenicity. agents. Therefore, during cryptococcal infec- PLoS Pathog 2010; 6: e1000953. tion, titan cells in large numbers may have dual [4] Garcia-Rodas R, Casadevall A, Rodriguez-Tude- effects: resulting in resistance to antifungal la JL, Cuenca-Estrella M, Zaragoza O. Crypto- agents on one hand, and preventing the dis- coccus neoformans capsular enlargement and semination of the pathogens into the CNS on cellular gigantism during Galleria mellonella the other hand. infection. PLoS One 2011; 6: e24485. [5] Guarner J, Brandt ME. Histopathologic diagno- Our cases highlight that cryptococcal titan cells sis of fungal infections in the 21st century. Clin should be kept in mind during differential diag- Microbiol Rev 2011; 24: 247-80. nosis when we observe organisms of giant cells [6] Bava J, Solari R, Isla G, Troncoso A. Atypical in tissue sections. Titan cells might cause mis- forms of Cryptococcus neoformans in CSF of diagnosis and underdiagnosis. Because of the an AIDS patient. J Infect Dev Ctries 2008; 2: rare clinical cases of the cryptococcal infection 403-5. with titan cells, the morphological changes and [7] Williamson JD, Silverman JF, Mallak CT, Chris- its effects on the clinical and pathological pro- tie JD. Atypical cytomorphologic appearance of Cryptococcus neoformans: a report of five cas- cesses need further investigation in the animal es. Acta Cytol 1996; 40: 363-370. models of infection and observation of more [8] Neilson JB, Ivey MH, Bulmer GS. Cryptococcus clinical cases. neoformans: pseudohyphal forms surviving Acknowledgements culture with Acanthamoeba polyphaga. Infect Immun 1978; 20: 262-266. This work was supported by Medical Science [9] Zaragoza O. Multiple Disguises for the Same and Technology Development Program Fund of Party: The Concepts of Morphogenesis and Nanjing City (YKK10071). Phenotypic Variations in Cryptococcus neofor- mans. Front Microbiol 2011; 2: 181. Disclosure of conflict of interest [10] Love GL, Boyd GD, Greer DL. Large Cryptococ- cus neoformans isolated from brain abscess. J None. Clin Microbiol 1985; 22: 1068-1070.

4845 Int J Clin Exp Pathol 2014;7(8):4837-4846 Cryptococcal titan cells in patients with pulmonary cryptococcosis

[11] Cruickshank JG, Cavill R, Jelbert M. Cryptococ- [26] Zinck SE, Leung AN, Frost M, Berry GJ, Muller cus neoformans of unusual morphology. Appl NL. Pulmonary cryptococcosis: CT and patholo- Microbiol 1973; 25: 309-312. gic findings. J Comput Assist Tomogr 2002; 26: [12] Okagaki LH, Nielsen K. Titan cells confer pro- 330-334. tection from phagocytosis in Cryptococcus [27] Alasio TM, Lento PA, Bottone EJ. Giant blasto- neoformans infections. Eukaryot Cell 2012; conidia of Candida albicans. A case report and 11: 820-826. review of the literature. Arch Pathol Lab Med [13] Brizendine KD, Baddley JW, Pappas PG. Pul- 2003; 127: 868-871. monary cryptococcosis. Semin Respir Crit Care [28] Haque AK, McGinnis MR. Fungal Infections. In: Med 2011; 32: 727-734. Tomashefski JF, Cagle PT, Farver CF, Fraire AE, [14] Kauffman CA. Diagnosis of in editors. Dail and Hammar’s Pulmonary Pathol- immunosuppressed patients. Curr Opin Infect ogy, Volume I: Nonneoplastic Lung Disease. Dis 2008; 21: 421-425. 3rd ed. New York: Springer; 2008. pp. 349- [15] Jarvis JN, Harrison TS. Pulmonary cryptococco- 425. sis. Semin Respir Crit Care Med 2008; 29: [29] Anstead GM, Sutton DA, Graybill JR. Adiaspiro- 141-150. causing respiratory failure and a re- [16] Wu TT, Wang HC, Yang PC, Kuo SH, Luh KT. Pul- view of human infections due to Emmonsia monary cryptococcosis: manifestations in the and spp. J Clin Microbiol 2012; era of acquired immunodeficiency syndrome. J 50: 1346-1354. Formos Med Assoc 1999; 98: 621-626. [30] Pelegrin I, Ayats J, Xiol X, Cuenca-Estrella M, [17] Nakamura S, Izumikawa K, Seki M, Kakeya H, Jucgla A, Boluda S, Fernandez-Sabe N, Rafe- Yamamoto Y, Yanagihara K, Miyazaki Y, Kohno cas A, Gudiol F, Cabellos C. Disseminated adi- S. Pulmonary cryptococcosis in late pregnancy aspiromycosis: case report of a liver transplant and review of published literature. Mycopatho- patient with human immunodeficiency infec- logia 2009; 167: 125-131. tion, and literature review. Transpl Infect Dis [18] Gazzoni AF, Severo CB, Salles EF, Severo LC. 2011; 13: 507-514. Histopathology, serology and cultures in the [31] Sun Y, Bhuiya T, Wasil T, Macias A, Wasserman diagnosis of cryptococcosis. Rev Inst Med Trop PG. Fine needle aspiration of pulmonary adias- Sao Paulo 2009; 51: 255-9. piromycosis: a case report. Acta Cytol 2007; [19] Kessler AT, Al Kharrat T, Kourtis AP. Cryptococ- 51: 217-221. cus neoformans as a cause of bronchiolitis ob- [32] England DM, Hochholzer L. Adiaspiromycosis: literans organizing pneumonia. J Infect Che- an unusual fungal infection of the lung. Report mother 2010; 16: 206-9. of 11 cases. Am J Surg Pathol 1993; 17: 876- [20] Shibuya K, Coulson WF, Wollman JS, Wakaya- 886. ma M, Ando T, Oharaseki T, Takahashi K, Naoe [33] Okagaki LH, Wang Y, Ballou ER, O’Meara TR, S. Histopathology of cryptococcosis and other Bahn YS, Alspaugh JA, Xue C, Nielsen K. Cryp- fungal infections in patients with acquired im- tococcal titan cell formation is regulated by G- munodeficiency syndrome. Int J Infect Dis protein signaling in response to multiple stimu- 2001; 5: 78-85. li. Eukaryot Cell 2011; 10: 1306-1316. [21] Zaragoza O, Nielsen K. Titan cells in Cryptococ- [34] Chrisman CJ, Albuquerque P, Guimaraes AJ, cus neoformans: cells with a giant impact. Curr Nieves E, Casadevall A. Phospholipids trigger Opin Microbiol 2013; 16: 409-413. Cryptococcus neoformans capsular enlarge- [22] Su CT, Chen LK, Tsai YF, Kuo CJ, Lu CL, Chen ment during interactions with amoebae and HY. Disseminated cryptococcosis with pulmo- macrophages. PLoS Pathog 2011; 7: e100- nary and marrow involvement mimicking radio- 2047. logical features of malignancy. J Chin Med As- [35] Charlier C, Nielsen K, Daou S, Brigitte M, Chre- soc 2004; 67: 89-92. tien F, Dromer F. Evidence of a role for mono- [23] Shirley RM, Baddley JW. Cryptococcal lung dis- cytes in dissemination and brain invasion by ease. Curr Opin Pulm Med 2009; 15: 254-260. Cryptococcus neoformans. Infect Immun [24] Chang WC, Tzao C, Hsu HH, Lee SC, Huang KL, 2009; 77: 120-127. Tung HJ, Chen CY. Pulmonary cryptococcosis: [36] Crabtree JN, Okagaki LH, Wiesner DL, Strain comparison of clinical and radiographic char- AK, Nielsen JN, Nielsen K. Titan cell production acteristics in immunocompetent and immuno- enhances the virulence of Cryptococcus neo- compromised patients. Chest 2006; 129: 333- formans. Infect Immun 2012; 80: 3776-3785. 340. [25] Nadrous HF, Antonios VS, Terrell CL, Ryu JH. Pulmonary cryptococcosis in nonimmunocom- promised patients. Chest 2003; 124: 2143- 2147.

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