mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Case report
Primary cutaneous cryptococcosis in an immunocompetent patient due to Cryptococcus gattii molecular type VGI in Brazil: a case report and review of literature cio da Silva,2 Roberto Martinez1 and Erika Nascimento,1 Maria Emılia Nadaletto Bonifa 2 Marcia Regina von Zeska Kress 1 Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sa~o Paulo, Brazil and 2School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil
Summary
Primary Cutaneous Cryptococcosis is an uncommon infection caused by the yeast Cryptococcus neoformans and C. gattii. Few case reports are available in the literature describing in detail primary cutaneous cryptococcosis due to C. gattii in immunocompetent patients. Herein, we present a case of a 68-year-old immunocompetent male patient with erythematous nodular lesions on the right forearm due to C. gattii mating-type a and molecular type VGI. The virulence factors test was performed for capsule diameter, melanin production and phospholipase activity. In vitro fluconazole testing showed the sensitivity profile of this clinical isolate. In addition, a review of the literature on this subject was carried out and verified that this is the first reported case of VGI in the south-east region of Brazil.
Key words: Cryptococcus gattii, molecular type VGI, primary cutaneous cryptococcosis - PCC, immunocompetent
patient, virulence factors, antifungal therapy.
Introduction Primary cutaneous cryptococosis (PCC) is a rare lesion caused by Cryptococcus neoformans or C. gattii in patients from rural areas, who are exposed to pigeon droppings, soil and wood debris.1 C. neoformans has a worldwide distribution and particularly affects individuals with impaired immunity, mainly owing to HIV infection, cancer chemotherapy and other types of immunosuppression.2 Conversely, cryptococcosis due to C. gattii occurs in healthy individuals, and historically described as restricted to tropical and subtropical areas,3,4 although in the last decade cases and outbreaks has been also observed in Correspondence: Marcia Regina von Zeska Kress, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Av. do Caf e s / n, Ribeirao Preto, SP 14040-903, Brazil. Tel.: +55 16 36020240. E-mail: [email protected] Submitted for publication 21 September 2013 Revised 16 January 2014 Accepted for publication 23 January 2014
© 2014 Blackwell Verlag GmbH
temperate climatic zones.5–8 Molecular determinations of genetically diverse subgroups within each Cryptococcus sp. serotype have been used to reveal the association between geographic origin and clinical manifestations with a particular fungal genotype.9 C. neoformans has the serotypes A, D and AD, and molecular types VNI, VNII, VNIII, VNIV 10 and VNB.11 C. gattii has the serotypes B, C and molecular types VGI, VGII, VGIII and VGIV.12 Cryptococcus sp. is usually haploid and reproduces asexually (i.e. by budding). However, this fungus also possesses a bipolar mating system which consists of the mating types (MAT) a and a.13,14 The majority of environmental and clinical isolates belong to MATa, which are more virulent than MATa.15–17 Cryptococcosis caused by both species of Cryptococcus is manifested mainly as central nervous system or pulmonary disease, associated or not with blood stream infection. Cutaneous involvement is uncommon and results usually of haematogenous dissemination. PCC is characterised by a unique skin lesion associated with positive culture for Cryptococcus spp. and absence of systemic disease.18 Besides
doi:10.1111/myc.12176
E. Nascimento et al.
Cryptococcus species and genotypes, disease onset and clinical manifestation can be determined by strain virulence factors, such as the polysaccharide capsule, melanin production and secretion of enzymes.19,20 The aims of this report were to describe the case of a healthy individual with PCC caused by C. gattii and to characterise genotypically and phenotypically this clinical isolate.
Case report A 68-year-old male, a bus driver from south-east region of Brazil had an erythematous lesion on his right forearm for 50 days, which emerged after an itchy reaction due to an insect bite. Physical examination showed an erythematous and squamous lesion with papules and ulcers covered with scabs across the circumference of the right forearm and absence of both enlarged lymph nodes and systemic changes. The patient admitted to be a chronic smoker and heavy beer drinker. He used to live in an urban area and in the past he kept canaries and parakeets in his house. A serological investigation revealed that the patient was negative for HIV-I (ELISA). Anti-Cryptococcus spp. antibodies were detected by the serum agglutination test (1:128), and counterimmunoelectrophoresis (1:8). The Haemoglobin was 11.9 g dl 1, and blood glucose level was 108 mg dl 1. A chest radiograph showed pulmonary emphysema. The patient was treated daily with 200 mg fluconazole and after 40 days the lesion was healed. Periodic Acid Schiff staining histology of the biopsy of the skin lesion revealed lymphohistiocytic
(a)
(b)
(c)
P R I M E R S
Figure 1 Cryptococcus gattii molecular
M
900 bp →
100 bp →
2
inflammatory process, granuloma formation and the presence of yeast with a capsule. The Urease test positive and other mycological tests have identified Cryptococcus spp as the yeast colonies isolated from tissue biopsy. The genus Cryptococcus was characterised molecularly by the sequencing of the Internal Transcribed Spacer (ITS) region of the ribosomal DNA.21 Serotype/ mating-type ‘B/Ca’ was determined by PCR amplification employing seven pairs of specific primers which are able to amplify all serotypes/mating types of C. neoformans and C. gattii 9,22–26 (Fig. 1 a). Molecular typing was performed by PCR fingerprinting employing primers for the minisatellite-specific core sequence (GACA)412 (Fig. 1 b,c). Thus, the encapsulated yeast isolated from the patient skin lesion was identified as C. gattii serotype B/C, mating-type a, molecular type VGI. Virulence factors and antifungal susceptibility were assessed to better understand this case. Capsule production was determined by the diameter of the capsule as described by Zaragoza et al. [27] melanin production was quantified by the evaluation of laccase activity as described by Pukkila-Worley et al. [28] and extracellular phospholipase activity was determined by the method of Chen et al. [29] The results of the clinical isolate for capsule diameter, melanin production and phospholipase activity were 3.547 lm, OD450 0.173 and 0.79 Pz respectively. Antifungal susceptibility tests were performed according to the recommendations proposed by the Clinical and Laboratory Standards Institute (CLSI) MS27-A3 method.30 The minimum inhibitory
M
M
type VGI and mating-type MATa. a. Genotyping and mating-typing showing PCR amplification for C. gattii serotype ‘B/C’ (900 bp) and C. gattii serotype-mating type ‘B/Ca’ (100 bp); b. Molecular typing by PCR fingerprinting employing primers for the minisatellite-specific core sequence (GACA)4; VGI, Cryptococcus gattii ATCC32269; c. Amplification pattern of molecular types VGI, VGII, VGII and VGIV56 employing primers for the minisatellite-specific core sequence (GACA)4. M = GeneRulerTM 1 kb Plus DNA Ladder (Fermentas). M, GeneRulerTM DNA Ladder Mix (Fermentas); bp, base pairs.
© 2014 Blackwell Verlag GmbH
C. gattii VGI/a in an immunocompetent patient
concentration (MIC) values were interpreted according to clinical breakpoints defined for Candida albicans,31 once the cut-off points for Cryptococcus sp. are not established by the CLSI. MICs values found for the clinical isolate were Amphotericin B (0.06 lg ml 1), Fluconazole (8.0 lg ml 1), Itraconazole (0.25 lg ml 1), and 5-Flucitocine Voriconazole (0.50 lg ml 1) 1 (8.0 lg ml ).
Discussion Cryptococcus spp. usually disseminate to the brain to cause meningoencephalitis. The occurrence of cutaneous cryptococcosis lesions (secondary cutaneous cryptococcosis) may be the first clinical manifestation of disseminated infection, particularly in persons with immunity deficits.32,33 Direct inoculation into a preexisting skin lesion can cause PCC. The proposed criteria for diagnosis of PCC in patients with positive skin lesion culture for C. neoformans serotype D or C. gattii are the absence of systemic lesions, a common unique skin lesion on unclothed body areas, evidence of
previous skin injury, identical body side for prior injury or former skin and cryptococcal lesion, exposure to Cryptococcus sp. in outdoor or rural activities (avian excreta, wood debris, soil, or needle contaminated with Cryptococcus sp.) and elderly and immunocompetent patients.1 The predominant types of lesions in PCC are ulceration, nodule, cellulitis, whitlow and phlegmon1 or have the aspect of an infiltrate plaque.34 Few case reports are available in the literature which describes in detail an initial skin lesion with positive culture for C. gattii in immunocompetent patients. Five of the published case reports refer to the absence of a pre-existing trauma on the lesion site, and after the initial skin lesion, describe the course of the disease followed by the development of systemic cryptococcosis,35–39 which characterises the associated cutaneous cryptococcosis. In contrast, another six case reports describe a skin lesion with positive culture for C. gattii which fit the criteria proposed for the diagnosis of PCC34,40–44 (Table 1). Among these, a case occurred in Australia,40 another occurred in Singapore,44 and four occurred in the southern Brazilian region.34,41–43
Table 1 Clinical and epidemiological characteristics of published case reports of primary cutaneous cryptococcosis due to C. gattii in immunocompetent patients. Treatment
Site of lesion
Antifungal agent (dosage mg/day)
Duration (months)
Elbow, arm and forearm Forearm
Itra (400) Itra (200) Fluc (450)
11 days 3 45 days
Forearm
Fluc (400)
Forearm
Itra (400)
Forklift driver at a local port warehouse -
Scalp
None
Forearm
Itra (200)
Bus driver/former canaries and parakeets owner
Forearm
Fluc (200)
Sex/age in years
Main employment or hobby
M/75
Orchid grower
M/65
Seller
M/751
Retired carpenter/handled Eucalyptus logs while building a farmyard fence -
M/891 M/37
M/69 M/682
Outcome
Follow-up (year)
Geographic region
Chest X-rays
Reference/year
Cured
-
Australia
Negative
[40]/1997
Cured
-
Normal
[41]/2002
5
Cured
1
Southern Brazil Southern Brazil
Normal
[42]/2011
3
Cured
1.5
Normal
[43]/2011
Cured
-
Southern Brazil Singapore
Normal
[44]/2011
Cured
5
-
[34]/2012
Cured
1
Southern Brazil Southern Brazil
Pulmonary emphysema
This case
-
6 40 days
5-FC, 5-fluorocytosine; AmB, amphotericin B; F, female; Fluc, fluconazole; Itra, itraconazole; Keto, ketoconazole; M, male; ‘-’, not available. 1
VGII, molecular type. VGI/MATa, molecular type/mating type.
2
© 2014 Blackwell Verlag GmbH
3
E. Nascimento et al.
The individuals were elderly, except for a 37-year-old patient, and all but one were male (Table 1). The molecular type VGII was determined for the C. gattii strain isolated from skin lesions of immunocompetent patients by Le~ ao et al. [42] and Pasa et al. [43] in the southern region of Brazil. In addition, Pasa et al. [43] demonstrated that the clinical isolate was the matingtype MATa. Diverse molecular methods were employed to define these subgroups. Multilocus sequence typing (MLST) is the method of choice for strain typing by the ‘Genotyping of Cryptococcus neoformans and C. gattii working group I’.9 Despite the new methods, DNA fingerprint is a classic and widely used and successful method for standardisation of Cryptococcus sp. molecular types.45The molecular type VGI of C. gattii represents the more frequently isolated molecular type in Oceania and Asia, and the molecular type VGII in Oceania, North and South America. The molecular type VGII was solely responsible for outbreaks in Vancouver Island and Northwest Pacific Coast of the United States. The more rarely found molecular types VGIII and VGIV were, respectively, found in Oceania, North and South America, and in Africa.45,46 In Brazil, the molecular type VGI is absent in the northern region and present in the southern region, but in lower frequency than VGII in the same region. The molecular type VGII is present in high prevalence in the Brazilian northern region. The molecular type VGIII is found in all Brazilian regions, but in low frequency, and VGIV was not found.47 In our case, C. gattii molecular type VGI and mating-type MATa was isolated from a skin lesion of an immunocompetent elderly male patient and is a probable case of PCC. Yeast cells with a large polysaccharide capsule inhibit phagocytosis and suppress cellular and humoral immunity,48 and are more virulent than capsule-free cells.49 In C. neoformans and C. gattii, melanin is synthesised during infection 50,51 and mutants that do not produce melanin are less virulent.19,20 Melanin protects fungi from reactive oxygen species and plays a role as an antioxidant.52,53 Another feature that contributes to its virulence is extracellular phospholipases secretion, causing damage to host cell membranes.53 The Cryptococcus sp. isolated from the patient skin lesion had a large capsule and was melanin, and phospholipase producer. These virulence factors probably contributed to the fungal survival and penetration across the damaged skin and subcutaneous tissue. On the other hand, the alcoholism presented by the patient eventually facilitated the cryptococcal infection, as in other
4
fungal infections.54 Fluconazole showed in vitro activity against the clinical isolate, and the MIC value is equal to the epidemiological cut-off value for C. gattii molecular type VGI.55 A short antifungal therapy with the azolic drug resulted in the patient forearm lesion clinical cure. Few clinical cases report the relationship regarding the C. gattii virulence factors, and clinical outcome of the patient. The identification of the molecular and mating type and virulence factors is becoming increasingly important to understand the degree to which cryptococcal species influence the patient immunity profile and clinical disease course.
Acknowledgments ~o de AmThe authors would like to thank the Fundacßa paro a Pesquisa do Estado de Sao Paulo (FAPESP) for the financial support.
conflict of interest There are none.
References 1
2
3
4 5
6
7
8
9
10
Neuville S, Dromer F, Morin O, Dupont B, Ronin O, Lortholary O. Primary cutaneous cryptococcosis: a distinct clinical entity. Clin Infect Dis 2003; 36: 337–47. doi:10.1086/345956. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS–100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995; 8: 515–48. Kwon-Chung KJ, Bennett JE. High prevalence of Cryptococcus neoformans var. gattii in tropical and subtropical regions. Zentralbl Bakteriol Mikrobiol Hyg A 1984; 257: 213–8. Ellis DH, Pfeiffer TJ. Natural habitat of Cryptococcus neoformans var. gattii. J Clin Microbiol 1990; 28: 1642–4. MacDougall L, Kidd SE, Galanis E et al. Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest. USA. Emerg Infect Dis 2007; 13: 42–50. doi:10.3201/eid1301. 060827. Byrnes EJ III, Marr KA. The Outbreak of Cryptococcus gattii in Western North America: Epidemiology and Clinical Issues. Curr Infect Dis Rep 2011; 13: 256–61. doi:10.1007/s11908-011 -0181-0. Marr KA. Cryptococcus gattii as an important fungal pathogen of western North America. Expert Rev Anti Infect Ther 2012; 10: 637– 43. doi:10.1586/eri.12.48. Iatta R, Hagen F, Fico C, Lopatriello N, Boekhout T, Montagna MT. Cryptococcus gattii infection in an immunocompetent patient from Southern Italy. Mycopathologia 2012; 174: 87–92. doi:10.1007/ s11046-011-9493-8. Meyer W, Aanensen DM, Boekhout T et al. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol 2009; 47: 561–70. doi:10.1080/ 13693780902953886. Meyer W, Marszewska K, Amirmostofian M et al. Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on
© 2014 Blackwell Verlag GmbH
C. gattii VGI/a in an immunocompetent patient
11
12
13 14
15
16
17
18
19
20
21 22
23
24
25
26
27
28
29
30
which to base a detailed epidemiological survey. Electrophoresis 1999; 20: 1790–9. doi:10.1002/(SICI)1522-2683(19990101)20:8% 3c1790:AID-ELPS1790%3e3.0.CO;2-2. Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG. Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics 2006; 172: 2223–38. doi:10.1534/genetics. 105.046672 Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis 2003; 9: 189–95. doi:10.3201/eid0902.020246. Kwon-Chung KJ. A new genus, filobasidiella, the perfect state of Cryptococcus neoformans. Mycologia 1975; 67: 1197–200. Kwon-Chung KJ. Morphogenesis of Filobasidiella neoformans, the sexual state of Cryptococcus neoformans. Mycologia 1976; 68: 821–33. Chowdhary A, Randhawa HS, Boekhout T, Hagen F, Klaassen CH, Meis JF. Temperate climate niche for Cryptococcus gattii in Northern Europe. Emerg Infect Dis 2012; 18: 172–4. doi:10.3201/eid1801. 111190. Moore TD, Edman JC. The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol 1993; 13: 1962–70. Halliday CL, Bui T, Krockenberger M, Malik R, Ellis DH, Carter DA. Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia. J Clin Microbiol 1999; 37: 2920–6. Pau M, Lallai C, Aste N, Atzori L. Primary cutaneous cryptococcosis in an immunocompetent host. Mycoses 2010; 53: 256–8. doi:10. 1111/j.1439-0507.2009.01707.x. Rhodes JC, Polacheck I, Kwon-Chung KJ. Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans. Infect Immun 1982; 36: 1175–84. Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med 1996; 184: 377–86. Innis MA. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 1990. Lin X, Nielsen K, Patel S, Heitman J. Impact of mating type, serotype, and ploidy on the virulence of Cryptococcus neoformans. Infect Immun 2008; 76: 2923–38. doi:10.1128/IAI.00168-08. Okabayashi K, Kano R, Watanabe T, Hasegawa A. Serotypes and mating types of clinical isolates from feline cryptococcosis in Japan. J Vet Med Sci 2006; 68: 91–4. D’Souza CA, Hagen F, Boekhout T, Cox GM, Heitman J. Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals. Curr Genet 2004; 46: 92–102. doi:10.1007/s00294-004-0511-y. Chaturvedi S, Rodeghier B, Fan J, McClelland CM, Wickes BL, Chaturvedi V. Direct PCR of Cryptococcus neoformans MATalpha and MATa pheromones to determine mating type, ploidy, and variety: a tool for epidemiological and molecular pathogenesis studies. J Clin Microbiol 2000; 38: 2007–9. Escandon P, Quintero E, Granados D, Huerfano S, Ruiz A, Castaneda E. Isolation of Cryptococcus gattii serotype B from detritus of Eucalyptus trees in Colombia. Biomedica 2005; 25: 390–7. Zaragoza O, Fries BC, Casadevall A. Induction of capsule growth in Cryptococcus neoformans by mammalian serum and CO(2). Infect Immun 2003; 71: 6155–64. Pukkila-Worley R, Gerrald QD, Kraus PR et al. Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade. Eukaryot Cell 2005; 4: 190– 201. doi:10.1128/EC.4.1.190-201.2005. Chen SC, Wright LC, Santangelo RT et al. Identification of extracellular phospholipase B, lysophospholipase, and acyltransferase produced by Cryptococcus neoformans. Infect Immun 1997; 65: 405–11. CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition, CLSI document M27-A3.: Clinical and Laboratory Standards Institute, Wayne; 2008.
© 2014 Blackwell Verlag GmbH
31
32 33 34
35
36
37 38
39
40
41
42
43
44
45 46
47
48 49
50
51
52
De Bedout C, Ordonez N, Gomez BL et al. In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii. Rev Iberoam Micol 1999; 16: 36–9. Sarosi GA, Silberfarb PM, Tosh FE. Cutaneous cryptococcosis. A sentinel of disseminated disease. Arch Dermatol 1971; 104: 1–3. Negroni R. Cryptococcosis. Clin Dermatol 2012; 30: 599–609. doi:10.1016/j.clindermatol.2012.01.005. Marques SA, Bastazini I Jr, Martins AL et al. Primary cutaneous cryptococcosis in Brazil: report of 11 cases in immunocompetent and immunosuppressed patients. Int J Dermatol. 2012; 51: 780–4. doi:10.1111/j.1365-4632.2011.05298.x. Riddell RJ, Entwisle BR. Cryptococcal granulomata of the skin in a patient with pulmonary and cerebral cryptococcosis. Australas J Dermatol 1969; 10: 100–8. Severo LC, Berta EZI, Londero AT. Cutaneous cryptococcosis due to Cryptococcus neoformans var. gattii. Rev Iberoam Micol. 2001; 18: 200–1. Xiujiao X, Ai’e X. Two cases of cutaneous cryptococcosis. Mycoses 2005; 48: 238–41. doi:10.1111/j.1439-0507.2005.01079.x. Dora JM, Kelbert S, Deutschendorf C et al. Cutaneous cryptococccosis due to Cryptococcus gattii in immunocompetent hosts: case report and review. Mycopathologia 2006; 161: 235–8. doi:10.1007/ s11046-006-0277-5. Tilak R, Prakash P, Nigam C, Tilak V, Gambhir IS, Gulati AK. Cryptococcal meningitis with an antecedent cutaneous Cryptococcal lesion. Dermatol Online J 2009; 15: 12. Hamann ID, Gillespie RJ, Ferguson JK. Primary cryptococcal cellulitis caused by Cryptococcus neoformans var. gattii in an immunocompetent host. Australas J Dermatol 1997; 38: 29–32. Lacaz Cda S, Heins-Vaccari EM, Hernandez-Arriagada GL et al. Primary cutaneous cryptococcosis due to Cryptococcus neoformans var. gattii serotype B, in an immunocompetent patient. Rev Inst Med Trop Sao Paulo 2002; 44: 225–8. Leao CA, Ferreira-Paim K, Andrade-Silva L et al. Primary cutaneous cryptococcosis caused by Cryptococcus gattii in an immunocompetent host. Med Mycol 2011; 49: 352–5. doi:10.3109/13693786. 2010.530697. Pasa CR, Chang MR, Hans-Filho G. Post-trauma primary cutaneous cryptococcosis in an immunocompetent host by Cryptococcus gattii VGII. Mycoses 2012; 55: e1–3. doi:10.1111/j.1439-0507.2011. 02058.x. Lingegowda BP, Koh TH, Ong HS, Tan TT. Primary cutaneous cryptococcosis due to Cryptococcus gattii in Singapore. Singapore Med J 2011; 52: e160–2. Meyer WT. L. Genotyping of the Cryptococcus neoformans/C. gattii species complex. Australian. Biochemist. 2010; 41: 5. Cogliati M. Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An atlas of the molecular types. Hindawi Publishing Corporation Scientifica 2013; 2013: 1–23. Trilles L, Lazera Mdos S, Wanke B et al. Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil. Mem Inst Oswaldo Cruz 2008; 103: 455–62. Cherniak R, Sundstrom JB. Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infect Immun 1994; 62: 1507–12. Fromtling RA, Shadomy HJ, Jacobson ES. Decreased virulence in stable, acapsular mutants of cryptococcus neoformans. Mycopathologia 1982; 79: 23–9. Nosanchuk JD, Valadon P, Feldmesser M, Casadevall A. Melanization of Cryptococcus neoformans in murine infection. Mol Cell Biol 1999; 19: 745–50. Rosas AL, Nosanchuk JD, Feldmesser M, Cox GM, McDade HC, Casadevall A. Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents. Infect Immun 2000; 68: 2845–53. Abegg MA, Cella FL, Faganello J, Valente P, Schrank A, Vainstein MH. Cryptococcus neoformans and Cryptococcus gattii isolated from the excreta of psittaciformes in a southern Brazilian zoological garden. Mycopathologia 2006; 161: 83–91. doi:10.1007/ s11046-005-0186-z.
5
E. Nascimento et al.
53
54
55
6
Karkowska-Kuleta J, Rapala-Kozik M, Kozik A. Fungi pathogenic to humans: molecular bases of virulence of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Acta Biochim Pol 2009; 56: 211–24. Byrnes EJ III, Bartlett KH, Perfect JR, Heitman J. Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals. Microbes Infect 2011; 13: 895–907. doi:10.1016/j.micinf.2011.05. 009. Espinel-Ingroff A, Aller AI, Canton E et al. Cryptococcus neoformansCryptococcus gattii species complex: an international study of wild-
56
type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole. Antimicrob Agents Chemother 2012; 56: 5898–906. doi:10.1128/ AAC.01115-12. Pedroso RS, Ferreira JC, Lavrador MA, Maffei CM, Candido RC. Evaluation of the experimental inoculation of Cryptococcus albidus and Cryptococcus laurentii in normal mice: virulence factors and molecular profile before and after animal passage. Mycopathologia 2009; 168: 59–72. doi:10.1007/s11046-0099202-z.
© 2014 Blackwell Verlag GmbH
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Case report
Primary cutaneous cryptococcosis in an immunocompetent patient due to Cryptococcus gattii molecular type VGI in Brazil: a case report and review of literature cio da Silva,2 Roberto Martinez1 and Erika Nascimento,1 Maria Emılia Nadaletto Bonifa 2 Marcia Regina von Zeska Kress 1 Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sa~o Paulo, Brazil and 2School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil
Summary
Primary Cutaneous Cryptococcosis is an uncommon infection caused by the yeast Cryptococcus neoformans and C. gattii. Few case reports are available in the literature describing in detail primary cutaneous cryptococcosis due to C. gattii in immunocompetent patients. Herein, we present a case of a 68-year-old immunocompetent male patient with erythematous nodular lesions on the right forearm due to C. gattii mating-type a and molecular type VGI. The virulence factors test was performed for capsule diameter, melanin production and phospholipase activity. In vitro fluconazole testing showed the sensitivity profile of this clinical isolate. In addition, a review of the literature on this subject was carried out and verified that this is the first reported case of VGI in the south-east region of Brazil.
Key words: Cryptococcus gattii, molecular type VGI, primary cutaneous cryptococcosis - PCC, immunocompetent
patient, virulence factors, antifungal therapy.
Introduction Primary cutaneous cryptococosis (PCC) is a rare lesion caused by Cryptococcus neoformans or C. gattii in patients from rural areas, who are exposed to pigeon droppings, soil and wood debris.1 C. neoformans has a worldwide distribution and particularly affects individuals with impaired immunity, mainly owing to HIV infection, cancer chemotherapy and other types of immunosuppression.2 Conversely, cryptococcosis due to C. gattii occurs in healthy individuals, and historically described as restricted to tropical and subtropical areas,3,4 although in the last decade cases and outbreaks has been also observed in Correspondence: Marcia Regina von Zeska Kress, School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Av. do Caf e s / n, Ribeirao Preto, SP 14040-903, Brazil. Tel.: +55 16 36020240. E-mail: [email protected] Submitted for publication 21 September 2013 Revised 16 January 2014 Accepted for publication 23 January 2014
© 2014 Blackwell Verlag GmbH
temperate climatic zones.5–8 Molecular determinations of genetically diverse subgroups within each Cryptococcus sp. serotype have been used to reveal the association between geographic origin and clinical manifestations with a particular fungal genotype.9 C. neoformans has the serotypes A, D and AD, and molecular types VNI, VNII, VNIII, VNIV 10 and VNB.11 C. gattii has the serotypes B, C and molecular types VGI, VGII, VGIII and VGIV.12 Cryptococcus sp. is usually haploid and reproduces asexually (i.e. by budding). However, this fungus also possesses a bipolar mating system which consists of the mating types (MAT) a and a.13,14 The majority of environmental and clinical isolates belong to MATa, which are more virulent than MATa.15–17 Cryptococcosis caused by both species of Cryptococcus is manifested mainly as central nervous system or pulmonary disease, associated or not with blood stream infection. Cutaneous involvement is uncommon and results usually of haematogenous dissemination. PCC is characterised by a unique skin lesion associated with positive culture for Cryptococcus spp. and absence of systemic disease.18 Besides
doi:10.1111/myc.12176
E. Nascimento et al.
Cryptococcus species and genotypes, disease onset and clinical manifestation can be determined by strain virulence factors, such as the polysaccharide capsule, melanin production and secretion of enzymes.19,20 The aims of this report were to describe the case of a healthy individual with PCC caused by C. gattii and to characterise genotypically and phenotypically this clinical isolate.
Case report A 68-year-old male, a bus driver from south-east region of Brazil had an erythematous lesion on his right forearm for 50 days, which emerged after an itchy reaction due to an insect bite. Physical examination showed an erythematous and squamous lesion with papules and ulcers covered with scabs across the circumference of the right forearm and absence of both enlarged lymph nodes and systemic changes. The patient admitted to be a chronic smoker and heavy beer drinker. He used to live in an urban area and in the past he kept canaries and parakeets in his house. A serological investigation revealed that the patient was negative for HIV-I (ELISA). Anti-Cryptococcus spp. antibodies were detected by the serum agglutination test (1:128), and counterimmunoelectrophoresis (1:8). The Haemoglobin was 11.9 g dl 1, and blood glucose level was 108 mg dl 1. A chest radiograph showed pulmonary emphysema. The patient was treated daily with 200 mg fluconazole and after 40 days the lesion was healed. Periodic Acid Schiff staining histology of the biopsy of the skin lesion revealed lymphohistiocytic
(a)
(b)
(c)
P R I M E R S
Figure 1 Cryptococcus gattii molecular
M
900 bp →
100 bp →
2
inflammatory process, granuloma formation and the presence of yeast with a capsule. The Urease test positive and other mycological tests have identified Cryptococcus spp as the yeast colonies isolated from tissue biopsy. The genus Cryptococcus was characterised molecularly by the sequencing of the Internal Transcribed Spacer (ITS) region of the ribosomal DNA.21 Serotype/ mating-type ‘B/Ca’ was determined by PCR amplification employing seven pairs of specific primers which are able to amplify all serotypes/mating types of C. neoformans and C. gattii 9,22–26 (Fig. 1 a). Molecular typing was performed by PCR fingerprinting employing primers for the minisatellite-specific core sequence (GACA)412 (Fig. 1 b,c). Thus, the encapsulated yeast isolated from the patient skin lesion was identified as C. gattii serotype B/C, mating-type a, molecular type VGI. Virulence factors and antifungal susceptibility were assessed to better understand this case. Capsule production was determined by the diameter of the capsule as described by Zaragoza et al. [27] melanin production was quantified by the evaluation of laccase activity as described by Pukkila-Worley et al. [28] and extracellular phospholipase activity was determined by the method of Chen et al. [29] The results of the clinical isolate for capsule diameter, melanin production and phospholipase activity were 3.547 lm, OD450 0.173 and 0.79 Pz respectively. Antifungal susceptibility tests were performed according to the recommendations proposed by the Clinical and Laboratory Standards Institute (CLSI) MS27-A3 method.30 The minimum inhibitory
M
M
type VGI and mating-type MATa. a. Genotyping and mating-typing showing PCR amplification for C. gattii serotype ‘B/C’ (900 bp) and C. gattii serotype-mating type ‘B/Ca’ (100 bp); b. Molecular typing by PCR fingerprinting employing primers for the minisatellite-specific core sequence (GACA)4; VGI, Cryptococcus gattii ATCC32269; c. Amplification pattern of molecular types VGI, VGII, VGII and VGIV56 employing primers for the minisatellite-specific core sequence (GACA)4. M = GeneRulerTM 1 kb Plus DNA Ladder (Fermentas). M, GeneRulerTM DNA Ladder Mix (Fermentas); bp, base pairs.
© 2014 Blackwell Verlag GmbH
C. gattii VGI/a in an immunocompetent patient
concentration (MIC) values were interpreted according to clinical breakpoints defined for Candida albicans,31 once the cut-off points for Cryptococcus sp. are not established by the CLSI. MICs values found for the clinical isolate were Amphotericin B (0.06 lg ml 1), Fluconazole (8.0 lg ml 1), Itraconazole (0.25 lg ml 1), and 5-Flucitocine Voriconazole (0.50 lg ml 1) 1 (8.0 lg ml ).
Discussion Cryptococcus spp. usually disseminate to the brain to cause meningoencephalitis. The occurrence of cutaneous cryptococcosis lesions (secondary cutaneous cryptococcosis) may be the first clinical manifestation of disseminated infection, particularly in persons with immunity deficits.32,33 Direct inoculation into a preexisting skin lesion can cause PCC. The proposed criteria for diagnosis of PCC in patients with positive skin lesion culture for C. neoformans serotype D or C. gattii are the absence of systemic lesions, a common unique skin lesion on unclothed body areas, evidence of
previous skin injury, identical body side for prior injury or former skin and cryptococcal lesion, exposure to Cryptococcus sp. in outdoor or rural activities (avian excreta, wood debris, soil, or needle contaminated with Cryptococcus sp.) and elderly and immunocompetent patients.1 The predominant types of lesions in PCC are ulceration, nodule, cellulitis, whitlow and phlegmon1 or have the aspect of an infiltrate plaque.34 Few case reports are available in the literature which describes in detail an initial skin lesion with positive culture for C. gattii in immunocompetent patients. Five of the published case reports refer to the absence of a pre-existing trauma on the lesion site, and after the initial skin lesion, describe the course of the disease followed by the development of systemic cryptococcosis,35–39 which characterises the associated cutaneous cryptococcosis. In contrast, another six case reports describe a skin lesion with positive culture for C. gattii which fit the criteria proposed for the diagnosis of PCC34,40–44 (Table 1). Among these, a case occurred in Australia,40 another occurred in Singapore,44 and four occurred in the southern Brazilian region.34,41–43
Table 1 Clinical and epidemiological characteristics of published case reports of primary cutaneous cryptococcosis due to C. gattii in immunocompetent patients. Treatment
Site of lesion
Antifungal agent (dosage mg/day)
Duration (months)
Elbow, arm and forearm Forearm
Itra (400) Itra (200) Fluc (450)
11 days 3 45 days
Forearm
Fluc (400)
Forearm
Itra (400)
Forklift driver at a local port warehouse -
Scalp
None
Forearm
Itra (200)
Bus driver/former canaries and parakeets owner
Forearm
Fluc (200)
Sex/age in years
Main employment or hobby
M/75
Orchid grower
M/65
Seller
M/751
Retired carpenter/handled Eucalyptus logs while building a farmyard fence -
M/891 M/37
M/69 M/682
Outcome
Follow-up (year)
Geographic region
Chest X-rays
Reference/year
Cured
-
Australia
Negative
[40]/1997
Cured
-
Normal
[41]/2002
5
Cured
1
Southern Brazil Southern Brazil
Normal
[42]/2011
3
Cured
1.5
Normal
[43]/2011
Cured
-
Southern Brazil Singapore
Normal
[44]/2011
Cured
5
-
[34]/2012
Cured
1
Southern Brazil Southern Brazil
Pulmonary emphysema
This case
-
6 40 days
5-FC, 5-fluorocytosine; AmB, amphotericin B; F, female; Fluc, fluconazole; Itra, itraconazole; Keto, ketoconazole; M, male; ‘-’, not available. 1
VGII, molecular type. VGI/MATa, molecular type/mating type.
2
© 2014 Blackwell Verlag GmbH
3
E. Nascimento et al.
The individuals were elderly, except for a 37-year-old patient, and all but one were male (Table 1). The molecular type VGII was determined for the C. gattii strain isolated from skin lesions of immunocompetent patients by Le~ ao et al. [42] and Pasa et al. [43] in the southern region of Brazil. In addition, Pasa et al. [43] demonstrated that the clinical isolate was the matingtype MATa. Diverse molecular methods were employed to define these subgroups. Multilocus sequence typing (MLST) is the method of choice for strain typing by the ‘Genotyping of Cryptococcus neoformans and C. gattii working group I’.9 Despite the new methods, DNA fingerprint is a classic and widely used and successful method for standardisation of Cryptococcus sp. molecular types.45The molecular type VGI of C. gattii represents the more frequently isolated molecular type in Oceania and Asia, and the molecular type VGII in Oceania, North and South America. The molecular type VGII was solely responsible for outbreaks in Vancouver Island and Northwest Pacific Coast of the United States. The more rarely found molecular types VGIII and VGIV were, respectively, found in Oceania, North and South America, and in Africa.45,46 In Brazil, the molecular type VGI is absent in the northern region and present in the southern region, but in lower frequency than VGII in the same region. The molecular type VGII is present in high prevalence in the Brazilian northern region. The molecular type VGIII is found in all Brazilian regions, but in low frequency, and VGIV was not found.47 In our case, C. gattii molecular type VGI and mating-type MATa was isolated from a skin lesion of an immunocompetent elderly male patient and is a probable case of PCC. Yeast cells with a large polysaccharide capsule inhibit phagocytosis and suppress cellular and humoral immunity,48 and are more virulent than capsule-free cells.49 In C. neoformans and C. gattii, melanin is synthesised during infection 50,51 and mutants that do not produce melanin are less virulent.19,20 Melanin protects fungi from reactive oxygen species and plays a role as an antioxidant.52,53 Another feature that contributes to its virulence is extracellular phospholipases secretion, causing damage to host cell membranes.53 The Cryptococcus sp. isolated from the patient skin lesion had a large capsule and was melanin, and phospholipase producer. These virulence factors probably contributed to the fungal survival and penetration across the damaged skin and subcutaneous tissue. On the other hand, the alcoholism presented by the patient eventually facilitated the cryptococcal infection, as in other
4
fungal infections.54 Fluconazole showed in vitro activity against the clinical isolate, and the MIC value is equal to the epidemiological cut-off value for C. gattii molecular type VGI.55 A short antifungal therapy with the azolic drug resulted in the patient forearm lesion clinical cure. Few clinical cases report the relationship regarding the C. gattii virulence factors, and clinical outcome of the patient. The identification of the molecular and mating type and virulence factors is becoming increasingly important to understand the degree to which cryptococcal species influence the patient immunity profile and clinical disease course.
Acknowledgments ~o de AmThe authors would like to thank the Fundacßa paro a Pesquisa do Estado de Sao Paulo (FAPESP) for the financial support.
conflict of interest There are none.
References 1
2
3
4 5
6
7
8
9
10
Neuville S, Dromer F, Morin O, Dupont B, Ronin O, Lortholary O. Primary cutaneous cryptococcosis: a distinct clinical entity. Clin Infect Dis 2003; 36: 337–47. doi:10.1086/345956. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS–100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995; 8: 515–48. Kwon-Chung KJ, Bennett JE. High prevalence of Cryptococcus neoformans var. gattii in tropical and subtropical regions. Zentralbl Bakteriol Mikrobiol Hyg A 1984; 257: 213–8. Ellis DH, Pfeiffer TJ. Natural habitat of Cryptococcus neoformans var. gattii. J Clin Microbiol 1990; 28: 1642–4. MacDougall L, Kidd SE, Galanis E et al. Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest. USA. Emerg Infect Dis 2007; 13: 42–50. doi:10.3201/eid1301. 060827. Byrnes EJ III, Marr KA. The Outbreak of Cryptococcus gattii in Western North America: Epidemiology and Clinical Issues. Curr Infect Dis Rep 2011; 13: 256–61. doi:10.1007/s11908-011 -0181-0. Marr KA. Cryptococcus gattii as an important fungal pathogen of western North America. Expert Rev Anti Infect Ther 2012; 10: 637– 43. doi:10.1586/eri.12.48. Iatta R, Hagen F, Fico C, Lopatriello N, Boekhout T, Montagna MT. Cryptococcus gattii infection in an immunocompetent patient from Southern Italy. Mycopathologia 2012; 174: 87–92. doi:10.1007/ s11046-011-9493-8. Meyer W, Aanensen DM, Boekhout T et al. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol 2009; 47: 561–70. doi:10.1080/ 13693780902953886. Meyer W, Marszewska K, Amirmostofian M et al. Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on
© 2014 Blackwell Verlag GmbH
C. gattii VGI/a in an immunocompetent patient
11
12
13 14
15
16
17
18
19
20
21 22
23
24
25
26
27
28
29
30
which to base a detailed epidemiological survey. Electrophoresis 1999; 20: 1790–9. doi:10.1002/(SICI)1522-2683(19990101)20:8% 3c1790:AID-ELPS1790%3e3.0.CO;2-2. Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG. Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var. grubii (serotype A), including a unique population in Botswana. Genetics 2006; 172: 2223–38. doi:10.1534/genetics. 105.046672 Meyer W, Castaneda A, Jackson S, Huynh M, Castaneda E. Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis 2003; 9: 189–95. doi:10.3201/eid0902.020246. Kwon-Chung KJ. A new genus, filobasidiella, the perfect state of Cryptococcus neoformans. Mycologia 1975; 67: 1197–200. Kwon-Chung KJ. Morphogenesis of Filobasidiella neoformans, the sexual state of Cryptococcus neoformans. Mycologia 1976; 68: 821–33. Chowdhary A, Randhawa HS, Boekhout T, Hagen F, Klaassen CH, Meis JF. Temperate climate niche for Cryptococcus gattii in Northern Europe. Emerg Infect Dis 2012; 18: 172–4. doi:10.3201/eid1801. 111190. Moore TD, Edman JC. The alpha-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene. Mol Cell Biol 1993; 13: 1962–70. Halliday CL, Bui T, Krockenberger M, Malik R, Ellis DH, Carter DA. Presence of alpha and a mating types in environmental and clinical collections of Cryptococcus neoformans var. gattii strains from Australia. J Clin Microbiol 1999; 37: 2920–6. Pau M, Lallai C, Aste N, Atzori L. Primary cutaneous cryptococcosis in an immunocompetent host. Mycoses 2010; 53: 256–8. doi:10. 1111/j.1439-0507.2009.01707.x. Rhodes JC, Polacheck I, Kwon-Chung KJ. Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans. Infect Immun 1982; 36: 1175–84. Salas SD, Bennett JE, Kwon-Chung KJ, Perfect JR, Williamson PR. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J Exp Med 1996; 184: 377–86. Innis MA. PCR protocols: a guide to methods and applications. San Diego: Academic Press, 1990. Lin X, Nielsen K, Patel S, Heitman J. Impact of mating type, serotype, and ploidy on the virulence of Cryptococcus neoformans. Infect Immun 2008; 76: 2923–38. doi:10.1128/IAI.00168-08. Okabayashi K, Kano R, Watanabe T, Hasegawa A. Serotypes and mating types of clinical isolates from feline cryptococcosis in Japan. J Vet Med Sci 2006; 68: 91–4. D’Souza CA, Hagen F, Boekhout T, Cox GM, Heitman J. Investigation of the basis of virulence in serotype A strains of Cryptococcus neoformans from apparently immunocompetent individuals. Curr Genet 2004; 46: 92–102. doi:10.1007/s00294-004-0511-y. Chaturvedi S, Rodeghier B, Fan J, McClelland CM, Wickes BL, Chaturvedi V. Direct PCR of Cryptococcus neoformans MATalpha and MATa pheromones to determine mating type, ploidy, and variety: a tool for epidemiological and molecular pathogenesis studies. J Clin Microbiol 2000; 38: 2007–9. Escandon P, Quintero E, Granados D, Huerfano S, Ruiz A, Castaneda E. Isolation of Cryptococcus gattii serotype B from detritus of Eucalyptus trees in Colombia. Biomedica 2005; 25: 390–7. Zaragoza O, Fries BC, Casadevall A. Induction of capsule growth in Cryptococcus neoformans by mammalian serum and CO(2). Infect Immun 2003; 71: 6155–64. Pukkila-Worley R, Gerrald QD, Kraus PR et al. Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade. Eukaryot Cell 2005; 4: 190– 201. doi:10.1128/EC.4.1.190-201.2005. Chen SC, Wright LC, Santangelo RT et al. Identification of extracellular phospholipase B, lysophospholipase, and acyltransferase produced by Cryptococcus neoformans. Infect Immun 1997; 65: 405–11. CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard-third edition, CLSI document M27-A3.: Clinical and Laboratory Standards Institute, Wayne; 2008.
© 2014 Blackwell Verlag GmbH
31
32 33 34
35
36
37 38
39
40
41
42
43
44
45 46
47
48 49
50
51
52
De Bedout C, Ordonez N, Gomez BL et al. In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii. Rev Iberoam Micol 1999; 16: 36–9. Sarosi GA, Silberfarb PM, Tosh FE. Cutaneous cryptococcosis. A sentinel of disseminated disease. Arch Dermatol 1971; 104: 1–3. Negroni R. Cryptococcosis. Clin Dermatol 2012; 30: 599–609. doi:10.1016/j.clindermatol.2012.01.005. Marques SA, Bastazini I Jr, Martins AL et al. Primary cutaneous cryptococcosis in Brazil: report of 11 cases in immunocompetent and immunosuppressed patients. Int J Dermatol. 2012; 51: 780–4. doi:10.1111/j.1365-4632.2011.05298.x. Riddell RJ, Entwisle BR. Cryptococcal granulomata of the skin in a patient with pulmonary and cerebral cryptococcosis. Australas J Dermatol 1969; 10: 100–8. Severo LC, Berta EZI, Londero AT. Cutaneous cryptococcosis due to Cryptococcus neoformans var. gattii. Rev Iberoam Micol. 2001; 18: 200–1. Xiujiao X, Ai’e X. Two cases of cutaneous cryptococcosis. Mycoses 2005; 48: 238–41. doi:10.1111/j.1439-0507.2005.01079.x. Dora JM, Kelbert S, Deutschendorf C et al. Cutaneous cryptococccosis due to Cryptococcus gattii in immunocompetent hosts: case report and review. Mycopathologia 2006; 161: 235–8. doi:10.1007/ s11046-006-0277-5. Tilak R, Prakash P, Nigam C, Tilak V, Gambhir IS, Gulati AK. Cryptococcal meningitis with an antecedent cutaneous Cryptococcal lesion. Dermatol Online J 2009; 15: 12. Hamann ID, Gillespie RJ, Ferguson JK. Primary cryptococcal cellulitis caused by Cryptococcus neoformans var. gattii in an immunocompetent host. Australas J Dermatol 1997; 38: 29–32. Lacaz Cda S, Heins-Vaccari EM, Hernandez-Arriagada GL et al. Primary cutaneous cryptococcosis due to Cryptococcus neoformans var. gattii serotype B, in an immunocompetent patient. Rev Inst Med Trop Sao Paulo 2002; 44: 225–8. Leao CA, Ferreira-Paim K, Andrade-Silva L et al. Primary cutaneous cryptococcosis caused by Cryptococcus gattii in an immunocompetent host. Med Mycol 2011; 49: 352–5. doi:10.3109/13693786. 2010.530697. Pasa CR, Chang MR, Hans-Filho G. Post-trauma primary cutaneous cryptococcosis in an immunocompetent host by Cryptococcus gattii VGII. Mycoses 2012; 55: e1–3. doi:10.1111/j.1439-0507.2011. 02058.x. Lingegowda BP, Koh TH, Ong HS, Tan TT. Primary cutaneous cryptococcosis due to Cryptococcus gattii in Singapore. Singapore Med J 2011; 52: e160–2. Meyer WT. L. Genotyping of the Cryptococcus neoformans/C. gattii species complex. Australian. Biochemist. 2010; 41: 5. Cogliati M. Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An atlas of the molecular types. Hindawi Publishing Corporation Scientifica 2013; 2013: 1–23. Trilles L, Lazera Mdos S, Wanke B et al. Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil. Mem Inst Oswaldo Cruz 2008; 103: 455–62. Cherniak R, Sundstrom JB. Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infect Immun 1994; 62: 1507–12. Fromtling RA, Shadomy HJ, Jacobson ES. Decreased virulence in stable, acapsular mutants of cryptococcus neoformans. Mycopathologia 1982; 79: 23–9. Nosanchuk JD, Valadon P, Feldmesser M, Casadevall A. Melanization of Cryptococcus neoformans in murine infection. Mol Cell Biol 1999; 19: 745–50. Rosas AL, Nosanchuk JD, Feldmesser M, Cox GM, McDade HC, Casadevall A. Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents. Infect Immun 2000; 68: 2845–53. Abegg MA, Cella FL, Faganello J, Valente P, Schrank A, Vainstein MH. Cryptococcus neoformans and Cryptococcus gattii isolated from the excreta of psittaciformes in a southern Brazilian zoological garden. Mycopathologia 2006; 161: 83–91. doi:10.1007/ s11046-005-0186-z.
5
E. Nascimento et al.
53
54
55
6
Karkowska-Kuleta J, Rapala-Kozik M, Kozik A. Fungi pathogenic to humans: molecular bases of virulence of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. Acta Biochim Pol 2009; 56: 211–24. Byrnes EJ III, Bartlett KH, Perfect JR, Heitman J. Cryptococcus gattii: an emerging fungal pathogen infecting humans and animals. Microbes Infect 2011; 13: 895–907. doi:10.1016/j.micinf.2011.05. 009. Espinel-Ingroff A, Aller AI, Canton E et al. Cryptococcus neoformansCryptococcus gattii species complex: an international study of wild-
56
type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole. Antimicrob Agents Chemother 2012; 56: 5898–906. doi:10.1128/ AAC.01115-12. Pedroso RS, Ferreira JC, Lavrador MA, Maffei CM, Candido RC. Evaluation of the experimental inoculation of Cryptococcus albidus and Cryptococcus laurentii in normal mice: virulence factors and molecular profile before and after animal passage. Mycopathologia 2009; 168: 59–72. doi:10.1007/s11046-0099202-z.
© 2014 Blackwell Verlag GmbH
