Case report

Primary cutaneous cryptococcosis in an immunocompetent patientdue to Cryptococcus gattii molecular type VGI in Brazil: a casereport and review of literature

Erika Nascimento,1 Maria Em�ılia Nadaletto Bonif�acio da Silva,2 Roberto Martinez1 and

Marcia Regina von Zeska Kress2

1Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, S~ao 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 immuno-

competent 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. neofor-

mans 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, cryp-

tococcosis due to C. gattii occurs in healthy individu-

als, 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

temperate climatic zones.5–8 Molecular determinations

of genetically diverse subgroups within each Crypto-

coccus sp. serotype have been used to reveal the asso-

ciation 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 bud-

ding). 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 Cryptococ-

cus is manifested mainly as central nervous system or

pulmonary disease, associated or not with blood

stream infection. Cutaneous involvement is uncom-

mon and results usually of haematogenous dissemina-

tion. PCC is characterised by a unique skin lesion

associated with positive culture for Cryptococcus

spp. and absence of systemic disease.18 Besides

Correspondence: Marcia Regina von Zeska Kress, School of Pharmaceuti-

cal 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: kress@fcfrp.usp.br

Submitted for publication 21 September 2013

Revised 16 January 2014

Accepted for publication 23 January 2014

© 2014 Blackwell Verlag GmbH doi:10.1111/myc.12176

mycosesDiagnosis,Therapy and Prophylaxis of Fungal Diseases

Cryptococcus species and genotypes, disease onset and

clinical manifestation can be determined by strain vir-

ulence 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 examina-

tion 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

inflammatory process, granuloma formation and the

presence of yeast with a capsule. The Urease test posi-

tive and other mycological tests have identified Crypto-

coccus spp as the yeast colonies isolated from tissue

biopsy.

The genus Cryptococcus was characterised molecu-

larly by the sequencing of the Internal Transcribed

Spacer (ITS) region of the ribosomal DNA.21 Serotype/

mating-type ‘B/Ca’ was determined by PCR amplifica-

tion 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 pro-

duction was determined by the diameter of the capsule

as described by Zaragoza et al. [27] melanin produc-

tion was quantified by the evaluation of laccase activ-

ity 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 clini-

cal 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

(a) (b) (c)

900 bp →

100 bp →

M M

PRI

MERS

M Figure 1 Cryptococcus gattii molecular

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-mat-

ing type ‘B/Ca’ (100 bp); b. Molecular

typing by PCR fingerprinting employing

primers for the minisatellite-specific core

sequence (GACA)4; VGI, Cryptococcus gat-

tii ATCC32269; c. Amplification pattern

of molecular types VGI, VGII, VGII and

VGIV56 employing primers for the mini-

satellite-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 GmbH2

E. Nascimento et al.

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 clini-

cal isolate were Amphotericin B (0.06 lg ml�1), Fluco-

nazole (8.0 lg ml�1), Itraconazole (0.25 lg ml�1),

Voriconazole (0.50 lg ml�1) and 5-Flucitocine

(8.0 lg ml�1).

Discussion

Cryptococcus spp. usually disseminate to the brain to

cause meningoencephalitis. The occurrence of cutane-

ous cryptococcosis lesions (secondary cutaneous cryp-

tococcosis) may be the first clinical manifestation of

disseminated infection, particularly in persons with

immunity deficits.32,33 Direct inoculation into a pre-

existing skin lesion can cause PCC. The proposed crite-

ria 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, expo-

sure to Cryptococcus sp. in outdoor or rural activities

(avian excreta, wood debris, soil, or needle contami-

nated with Cryptococcus sp.) and elderly and immuno-

competent 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 cryptococco-

sis,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.

Sex/age in

years

Main employment

or hobby Site of lesion

Treatment

Outcome

Follow-up

(year)

Geographic

region

Chest

X-rays Reference/year

Antifungal

agent

(dosage

mg/day)

Duration

(months)

M/75 Orchid grower Elbow, arm

and forearm

Itra (400) 11 days Cured - Australia Negative [40]/1997

Itra (200) 3

M/65 Seller Forearm Fluc (450) 45 days Cured - Southern

Brazil

Normal [41]/2002

M/751 Retired

carpenter/handled

Eucalyptus logs

while building a

farmyard fence

Forearm Fluc (400) 5 Cured 1 Southern

Brazil

Normal [42]/2011

M/891 - Forearm Itra (400) 3 Cured 1.5 Southern

Brazil

Normal [43]/2011

M/37 Forklift driver at a

local port

warehouse

Scalp None - Cured - Singapore Normal [44]/2011

M/69 - Forearm Itra (200) 6 Cured 5 Southern

Brazil

- [34]/2012

M/682 Bus driver/former

canaries and

parakeets owner

Forearm Fluc (200) 40 days Cured 1 Southern

Brazil

Pulmonary

emphysema

This case

5-FC, 5-fluorocytosine; AmB, amphotericin B; F, female; Fluc, fluconazole; Itra, itraconazole; Keto, ketoconazole; M, male; ‘-’, not

available.1VGII, molecular type.2VGI/MATa, molecular type/mating type.

© 2014 Blackwell Verlag GmbH 3

C. gattii VGI/a in an immunocompetent patient

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 mating-

type 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 suc-

cessful 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 Van-

couver Island and Northwest Pacific Coast of the Uni-

ted 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 north-

ern 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 MATawas isolated from a skin lesion of an immunocompe-

tent 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 viru-

lent.19,20 Melanin protects fungi from reactive oxy-

gen species and plays a role as an antioxidant.52,53

Another feature that contributes to its virulence is

extracellular phospholipases secretion, causing dam-

age to host cell membranes.53 The Cryptococcus sp.

isolated from the patient skin lesion had a large cap-

sule 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

fungal infections.54 Fluconazole showed in vitro activ-

ity 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 identifica-

tion of the molecular and mating type and virulence

factors is becoming increasingly important to under-

stand the degree to which cryptococcal species influ-

ence the patient immunity profile and clinical disease

course.

Acknowledgments

The authors would like to thank the Fundac�~ao de Am-

paro a Pesquisa do Estado de Sao Paulo (FAPESP) for

the financial support.

conflict of interest

There are none.

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