Mycopathologia

, Volume 174, Issue 1, pp 87–92

Cryptococcus gattii Infection in an Immunocompetent Patient from Southern Italy

Authors

    • Department of Biomedical Science and Human Oncology, Section of Hygiene“Aldo Moro” University of Bari
  • Ferry Hagen
    • Department of Yeast and Basidiomycete ResearchCBS-KNAW Fungal Biodiversity Centre
    • Canisius Wilhelmina Hospital
  • Cecilia Fico
    • Department of Infectious DiseaseTriggiano Hospital
  • Nunzia Lopatriello
    • Laboratory of Clinical PathologyTriggiano Hospital
  • Teun Boekhout
    • Department of Yeast and Basidiomycete ResearchCBS-KNAW Fungal Biodiversity Centre
  • Maria Teresa Montagna
    • Department of Biomedical Science and Human Oncology, Section of Hygiene“Aldo Moro” University of Bari
Article

DOI: 10.1007/s11046-011-9493-8

Cite this article as:
Iatta, R., Hagen, F., Fico, C. et al. Mycopathologia (2012) 174: 87. doi:10.1007/s11046-011-9493-8
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Abstract

Cryptococcus gattii has becoming more prevalent in temperate climate zones, during the past decades. We describe a C. gattii serotype B infection in an immunocompetent Italian patient with sclerosing cholangitis. The patient traveled once to Eastern Canada and otherwise no other countries than Italy were visited. Molecular analysis revealed that the C. gattii isolate belong to genotype AFLP4/VGI and has mating-type α which is the most common genotype in the Mediterranean environment. The C. gattii strain was found to be closely related, but not identical, to other C. gattii strains from the Mediterranean area.

Keywords

Cryptococcus gattiiCryptococcosisMolecular typeItalyInfection

Introduction

Cryptococcus neoformans and Cryptococcus gattii are encapsulated basidiomycetous yeasts that can cause life-threatening meningoencephalitis. Whereas C. neoformans occurs worldwide and primarily affects individuals with an impaired immunity, such as HIV-positive patients, C. gattii occurs mainly in apparently immunocompetent hosts and is predominant in tropical and subtropical regions [1, 2]. However, the distribution pattern of C. gattii has changed by an outbreak in the temperate climate of Vancouver Island, Canada, that expanded further into the Pacific Northwest [35]. Cryptococcus gattii is becoming more prevalent in the temperate climate of Europe as well [69]. The clinical occurrence of C. gattii is more restricted than that of C. neoformans which has a global distribution [10, 11].

Cryptococcus neoformans and C. gattii can be distinguished by serotype (A, D and AD for the former, B and C for the latter) using different typing techniques such as RAPD [12, 13], PCR fingerprinting [14], RFLP [15], AFLP fingerprinting [16] and DNA sequencing [1719].

Within C. neoformans two varieties have been distinguished, namely var. grubii and var. neoformans. Cryptococcus gattii can be subdivided into five genotypic groups designated AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV and AFLP10/VGIV [20]. Cryptococcus gattii AFLP4/VGI is the most widespread molecular type and accounts for most clinical and environmental isolates in Australia [2, 21]. Cryptococcus gattii AFLP6/VGII is mostly restricted to Northern and Western Australia, and is more common in South America, the Pacific Northwest and British Columbia (Canada). Genotype AFLP5/VGIII is commonly reported from South America, but occurs also in the United States and Central America, while AFLP7/VGIV has been reported from Africa and South and Central America [2, 5, 10, 22].

In 1995, the first C. gattii infection has been described in Apulia, a South Eastern Italian region. The host was a HIV-positive patient who lived in a rural area near a fauna park with exotic animals and Eucalyptus camaldulensis trees [23]. Nineteen years later, we report a second case caused by C. gattii serotype B in the same Italian region isolated from an apparently immunocompetent host. This C. gattii isolate belonged to genotype AFLP4/VGI but possessed mating-type α.

Molecular typing of a global set of C. gattii isolates demonstrated that AFLP4/VGI is the most prevalent genotype in the Mediterranean area [7, 20, 23]. Therefore, in countries where this fungus is non-endemic, clinicians should be aware about the possible emergence of this pathogen in both healthy and immunocompromised hosts.

Case

A 37-year-old man, born in Canada, Toronto and lifetime resident of the Metropolitan area of Bari in the Apulia region in Southern Italy, was admitted to the medical facility on March 23, 2009 complaining of a persistent headache, fever and malaise for 10 days. In 2002, he was diagnosed with sclerosing cholangitis and at that time was treated with ursodeoxycholic acid (900 mg/day). At the time of admission, X-ray and a CT-scan revealed a 4 cm rounded opacity in the lower lobe of the left lung (Fig. 1a). The primary diagnosis was a bacterial pneumonia and, consequently he was treated with antibiotics. After 4 days, the clinical manifestations did not improve, a lumbar puncture was performed and examination of cerebrospinal fluid (CSF) showed predominantly lymphocytic pleocytosis (lymphocytes 111/μl) with low levels of glucose (20 mg/dl) and high protein concentration (77.8 mg/dl). Further blood tests revealed a lymphopenia with a CD4-count of 600/mm3.
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Fig. 1

Progression of the C. gattii infection, in the left lower lobe of the lung, based on CT-scan and X-ray. a CT-scan made at 23rd March 2009, showing evidence of a 4 cm rounded opacity (white arrow); b chest X-ray imaging made at 20th April 2009, revealing a decrease of the round mass lesion (white arrow); c 21st May 2009, chest X-ray after the surgical resection indicates the absence of the nodule (white arrow)

An Indian ink stain was performed, demonstrating the presence of encapsulated yeast cells similar to Cryptococcus spp. The CSF and serum cryptococcal latex agglutination test (CALAS, Meridian Diagnostics, Cincinnati, OH) was positive showing titers of 1:128 and 1:1,024, respectively. A culture from CSF was performed on canavanine-glycine-bromothymol blue (CGB) medium. After 5 days of incubation at 25°C, the CGB medium showed a change in color from yellow to blue indicating the presence of C. gattii. In vitro antifungal susceptibility testing was performed and the isolate was found to be susceptible to FLZ, VOR, AMB and 5FC. All bacteriological and viral tests were negative, including those for tuberculosis and HIV.

On April 4, the patient was initially treated with intravenous FLZ (800 mg/day) for 2 weeks, followed by liposomal AMB and 5FC (3, 100 mg/kg/day, respectively) for 2 weeks. During the antifungal treatment, a chest x-ray imaging revealed a decrease of rounded mass lesion to 1 cm (Fig. 1b). Because of renal toxicity/nephropathy by AMB and 5FC and improvement of his symptoms, the treatment was changed successively to 400 mg FLZ maintenance and the patient was discharged to continue treatment at home.

After 1 month, he was readmitted at the hospital presenting again with persistent headache, fever and malaise. On May 14, a chest CT-scan revealed a 4 cm rounded nodule in the left lung. On May 21, a lung surgical resection was performed and a chest X-ray showed the surgical outcomes (Fig. 1c). The histopathology of the tissue confirmed the presence of yeast cells similar to Cryptococcus sp. The patient was started on maintenance FLZ therapy (400 mg/day) and he was discharged from the hospital. On October 3, a chest X-ray and CT-scan showed a 3 cm nodule in the left lung which indicated presumably a Cryptococcus relapse. The serum cryptococcal antigen was positive with a titer of 1:512. The patient was treated with liposomal AMB and 5FC (3 mg/kg/day, 100 mg/kg/day, respectively) for 2 weeks followed by FLZ (400 mg/day). On January 21, 2010, a pulmonary CT-scan revealed no regression of the rounded opacity. The cryptococcal antigen serum and liquor titers were 1:32 and 1:4, respectively. The patient was treated with VOR 6 mg/kg i.v. 2 QD for 1 day, followed by 4 mg/kg 2 QD for 2 weeks and then an analogous oral dosage for 3 months. At follow-up at May 21, 2010 he was completely recovered. A follow-up chest X-ray revealed that the lung nodules were completely vanished.

The Cryptococcus isolate (CBS11747) was genotyped using amplified fragment length polymorphism (AFLP) fingerprint analysis [16]. The mating-type was analyzed by using a PCR that amplifies either the mating-type a or α STE12-allele, as previously described [25]. Multi-locus sequence typing (MLST) was performed and included the ten nuclear loci CAP10, CAP59, GPD1, IGS1, LAC1, MPD1, PLB1, SOD1, TEF1α and URA5 [26, 27]. Sequences were deposited in Genbank and are accessible via accession numbers HQxxxxx–HQxxxxx. For phylogenetic analyses, the seven loci previously used by Fraser et al. (2005) were included, while the CAP59, SOD1 and URA5 loci were sequenced for future comparison based on the recently launched consensus MLST [26, 27]. A bootstrapped Maximum Likelihood analysis was performed to compare the strain from the current case report with C. gattii strains used in two previous population studies [7, 27]. These molecular investigations revealed that the strain belonged to C. gattii genotype AFLP4/VGI mating-type α that was based on a seven-loci MLST analysis genetically closely related, but different, from environmental and human C. gattii AFLP4/VGI isolates that were previously found in the Mediterranean area (Fig. 2) [7, 27]. After diagnosis of the C. gattii infection the patient was questioned about his travel history and in 2007 he visited Toronto and Montreal (Canada) for a month.
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Fig. 2

The phylogenetic relatedness of the clinical Italian C. gattii strain CBS11747 was compared to other European and globally collected genotype AFLP4/VGI strains using a seven-loci Multi-Locus Sequence Typing approach as previously proposed by Fraser et al. [27] and including also European Mediterranean strains studied in a recent European population study [7]. An unrooted 1,000× bootstrapped Maximum Likelihood phylogenetic analysis, based on the Hasegawa–Kishino–Yano model, was performed. The percentage of trees in which the associated taxa clustered together is shown next to the branches (only when highly supported, e.g., ≥75%). The clinical Italian C. gattii strain is highlighted in boldface

Discussion

Most C. gattii infections in humans have been reported from tropical and subtropical areas, mainly Australia and the South Pacific, but also South America, the USA, and Sub-Saharan Africa [28]. Only few infections have been reported from countries with a moderate temperate climate. In Europe, autochthonous C. gattii infections in humans and animals have been described, especially in Italy, Greece and Spain [6, 7, 9, 23, 29]. Moreover, infections in European residents were found to be acquired outside Europe [30, 31].

Recently, it has been suggested that this fungus may have become adapted to local Mediterranean environmental conditions [7, 24]. This is important, because as C. gattii appears to have a greater propensity to infect immunocompetent hosts than C. neoformans [2] and may occur in more patients in the future in this region. The findings of this pathogen in the environment, including the first environmental isolation, of Italy, as well as the publication of several case reports of autochthonous C. gattii infections demonstrate that it is also able to occur in temperate regions and it probably has a more widespread distribution in the Mediterranean area [69, 23]. Currently, two C. gattii genotypes are known to occur in this area: AFLP4/VGI and AFLP6/VGII [25]. Published and unpublished data show that most of the European C. gattii isolates belong to genotype AFLP4/VGI [7], (Hagen F, Boekhout T, Iatta R, Montagna MT, unpublished data). Only two recent clinical cases involving C. gattii in Greece were found to be caused by genotype AFLP6/VGII strains [18]. However, these strains had a different MLST genotype than those causing the ongoing Canadian outbreak [21].

The first C. gattii serotype B isolations from clinical, veterinary and environmental sources have been described in Apulia, a South East Italian region, in 1995. The host was an immunocompromised patient with HIV infection who lived in a rural area near a fauna park with exotic animals, some of them imported from Australia, and that was planted with Eucalyptus camaldulensis trees [8]. Detailed genotyping studies demonstrated that all the isolates were AFLP4/VGI and mating-type a (Hagen F, Boekhout T, Iatta R, Montagna MT, unpublished data).

After 19 years, a second C. gattii serotype B infection is reported. The isolate possessed genotype AFLP4/VGI and mating-type α, which represents the most common molecular type that is known to have an association with Ceratonia siliqua and Eucalyptus trees [7, 8, 20, 24].

In addition, according to the MLST scheme as proposed by Fraser et al. [27], our C. gattii strain is closely related, but not identical, to clinical, veterinary and environmental isolates from Spain and two environmental Portuguese strains [7, 27].

These and our findings support the hypothesis that C. gattii may occur more widely distributed in the Mediterranean area. The fact that the patient was infected with C. gattii AFLP4/VGI suggests that he was infected in his living area, rather than during his visit to Canada. In non-endemic countries, infections by C. gattii are likely not recognized because the species identification requires phenotypic and/or molecular tests that are not always available in microbiological laboratories. Therefore, further data on the environmental, veterinary and clinical occurrence of this pathogen are needed to reveal the presence of C. gattii in the Mediterranean area and in Europe in general.

Copyright information

© Springer Science+Business Media B.V. 2011