Abstract

Cryptococcosis is an important systemic mycosis caused by members of the Cryptococcus neoformans species complex. This disease is potentially fatal in various animals, including koalas. We describe the long-term surveillance and treatment of subclinical cryptococcosis and nasal colonization of koalas by Cryptococcus neoformans and C. gattii. Of the 15 animals investigated through the use of samples obtained by nasal swabs, antigen titer measurements, and pathologic examination, C. neoformans was found associated with nine koalas and C. gattii with one animal. Nine koalas showed subclinical disease and one clinical infections and antigenemia. Treatment with fluconazole, itraconazole and amphotericin B upon detection of C. neoformans or C. gattii was not effective. The results of the present study showed that C. neoformans was the predominant species isolated from the nasal swab samples and the fungus might have naturally become associated with the koalas' nasal cavities at Kanazawa Zoological Gardens. The unclear treatment effectiveness might have been caused by a shorter treatment period that is routinely used and unstable itraconazole absorption. This investigation also underscores the need for identifying effective treatment regimens for subclinical cryptococcosis and efficient measures for eradicating C. neoformans and C. gattii in koalas.

Introduction

Cryptococcosis is caused by one of the two fungal species, i.e., Cryptococcus neoformans (serotypes A, D and AD) and C. gattii (serotypes B and C) [1]. In contrast to C. neoformans, which has a global distribution, C. gattii has until recently been regarded as being restricted to tropical and subtropical climates [2,3]. However, isolated cases of infections caused by C. gattii have been identified since 1999 in non-tropical regions [4], and the recovery of C. gattii from koalas and humans has also been reported in Japan [5,6].

While cryptococcosis is a mainly asymptomatic disease [7], it can cause life-threatening infections. The major clinical features of cryptococcosis in koalas caused by the two species are similar and consist of respiratory tract involvement, including pneumonia and upper respiratory tract disease, as well as meningitis and encephalitis [8]. C. gattii is often associated with neurologic sequelae and frequently requires aggressive neurosurgical management [9,10], underscoring the importance of differential diagnosis. Cryptococcosis in the koalas was first reported in 1960 [11], and subsequent studies of infections in these animals have been reported [8,12]. Interestingly, the incidence of cryptococcosis in the koala (Phascolarctos cinereus) in Australia is higher than that in domestic animals and humans [7]. Therefore, cryptococcosis should be regularly monitored especially in the koala.

In this case report, we describe the long-term surveillance of subclinical cryptococcosis and nasal colonization caused by C. neoformans and C. gattii in captive koalas at Kanazawa Zoological Gardens, (Yokohama, Kanagawa, Japan). Our aim in this investigation is to identify the Cryptococcus species recovered from the nasal cavity, the detection rate and the transition of the nasal colonization to isolate Cryptococcus species, and the effectiveness of the treatment for subclinical cryptococcosis.

Case report

Fifteen captive koalas (male, n = 6; female, n = 9) were carefully examined, monitored and treated for cryptococcosis at Kanazawa Zoological Gardens between November 2002 and January 2010. We examined the nasal swabs and cryptococcal antigen levels of living koalas, as well as performing post mortem pathological examinations of the animals.

Nasal swab samples were collected from each gently restrained conscious koala by rubbing a sterile swab over 20 mm of the rostral region of the left and right nasal vestibules, at least once a year from 2002–2005, and more frequently from 2005–2010. The swabs were immediately inoculated onto CHROMagar Candida (CHROMagar, Paris, France) plates containing micafungin (30 μg plate-1), kept at 4°C, and sent to Teikyo University Institute of Medical Mycology, Tokyo for processing. Each of the samples from the koalas and those recovered from the environment was investigated at this same institution.

For yeast isolation, identification and quantitation, yeast DNA was extracted and purified according to the protocol recommended for the FTA Classic Card (Whatman PLC, Maidstone, Kent, UK). The DNA fragment covering the nuclear 26S rDNA D1/D2 domain was then amplified with primers 28SF1 and 635, as described previously [13,14]. Thereafter, DNA sequencing was performed using the Applied Biosystems model 310 sequencer and the sequences compared to those in the NCBI Blast database (http://www.ncbi.nlm.nih.gov/blast/) for species identification. Finally, the number of colonies identified as C. neoformans or C. gattii on CHROMagar Candida plates was determined.

For the treatment of cryptococcosis, koalas from which Cryptococcus species were recovered from the nasal cavities were treated with fluconazole (Diflucan; Pfizer Japan Inc., Tokyo, Japan) at 3–10 mg/kg/d PO during the period 2002–2006 and itraconazole (Toracona; Maruko Pharmaceutical Co., Aiti, Japan) at 10–40 mg/kg/d PO from 2007–2009. Amphotericin B (Fungizone; Bristol-Myers K.K., Tokyo, Japan) at 1 mg/d mixed with tyloxapol (Alevaire; Alfresa Pharma Co., Osaka, Japan) at 4 ml/d was vaporized in front of the koala's nose for 5 min and administered as combination therapy from 2006–2010.

For cryptococcal antigen level measurement, blood samples were collected from the cephalic vein of 13 (two koalas, Nos. 14 and 15, were not tested) koalas under manual restrain. Sera were immediately separated and frozen samples were sent to Showa Medical Science Co., Tokyo for measurements using the latex agglutination method.

Eleven koalas that died from age-related diseases were necropsied. Pathologic samples from different organs were assessed at necropsy and by histopathology. Whole internal organs and the brain, as well as serial head sections to assess the nasal cavity and paranasal sinuses were examined. Tissue samples of lesions with suspected cryptococcal etiology and of various organs including the heart, lung, liver, kidney, spleen, stomach, and intestine were placed in 10% buffered formalin and fixed at room temperature (approximately 20°C). The samples were histopathologically examined through the use of the Zelkova Examination Center (Kawasaki, Kanagawa, Japan).

C. neoformans was recovered in culture from the nasal swab samples of nine koalas and C. gattii from one of the 15 koalas (total n = 10; 66.7%), with the remaining five animals showing no cryptococcus colonization. Overall, the koalas could be divided into three groups, i.e., group 1 in which cryptococcus colonization was ascertained and koalas were treated (koalas Nos. 1–6; Table 1), group 2 in which cryptococcus colonization was found but the koalas were not treated (koalas Nos. 7–10; Table 2) and group 3 in which there was no evidence of cryptococcus colonization (koalas Nos. 11–15; Table 3). Cryptococcus species were able to be recovered apart from other fungi through the use of CHROMagar Candida containing micafungin.

Table 1.

Group 1: Nasal colonization, and dosage and period of treatment of Cryptococcus neoformans or C. gattii in koalas treated with antifungal medicine at Kanazawa Zoological Gardens, Yokohama, Japan.

 
 
 

 

 

 

 

 
Koala No. Yeast isolated C. neoformans Treatment C. neoformans Treatment C. neoformans Treatment C. neoformans Treatment C. gattii Treatment C. neoformans Treatment 
2002             
   November Fluconazole (3mg for 2wks)        
   December   Fluconazole (3mg for 2 wks)      
2003             
   May Death            
   July          
2004             
   March          
2005             
   March          
   May        Transferred from a zoo    
   June   97 Fluconazole (6mg for 2 wks)  Fluconazole (6mg for 2 wks) Fluconazole (6mg for 2 wks)   
   July   Fluconazole (10mg for 2 mos)      
   August          Fluconazole (6mg for 2 wks)   
   September      283    
   October          Fluconazole (6mg for 2 wks)   
   December      15  Transferred from a zoo  
2006             
   June    103   19  40  
   July   Death          
   August      Fluconazole (10mg for 2 mos)    Fluconazole (10 mg) Amphotericin B (1 mg for 2 mos)  Fluconazole (10mg for 2 mos) 
   October         
2007             
   March         
   August        28  
   April     Itraconazole (20 mg) Amphotericin B (1 mg for 1 wk)    22  
   May           
   July      Itraconazole (20 mg) Amphotericin B (1 mg for 2 wks)      Itraconazle (20 mg for 2 mos) 
   August      Itraconazole (40 mg) Amphotericin B (1 mg for 1.5 mos)       
   September     Death        
   October       Death    58  
   December            Itraconazle (20 mg for 2 mos) 
2009             
   February           103  
   March            Itraconazole (10 mg) Amphotericin B (1 mg for 2 mos) 
   November           22  
2010             
   January           16 Amphotericin B (1mg for 1 wk) 
 
 
 

 

 

 

 

 
Koala No. Yeast isolated C. neoformans Treatment C. neoformans Treatment C. neoformans Treatment C. neoformans Treatment C. gattii Treatment C. neoformans Treatment 
2002             
   November Fluconazole (3mg for 2wks)        
   December   Fluconazole (3mg for 2 wks)      
2003             
   May Death            
   July          
2004             
   March          
2005             
   March          
   May        Transferred from a zoo    
   June   97 Fluconazole (6mg for 2 wks)  Fluconazole (6mg for 2 wks) Fluconazole (6mg for 2 wks)   
   July   Fluconazole (10mg for 2 mos)      
   August          Fluconazole (6mg for 2 wks)   
   September      283    
   October          Fluconazole (6mg for 2 wks)   
   December      15  Transferred from a zoo  
2006             
   June    103   19  40  
   July   Death          
   August      Fluconazole (10mg for 2 mos)    Fluconazole (10 mg) Amphotericin B (1 mg for 2 mos)  Fluconazole (10mg for 2 mos) 
   October         
2007             
   March         
   August        28  
   April     Itraconazole (20 mg) Amphotericin B (1 mg for 1 wk)    22  
   May           
   July      Itraconazole (20 mg) Amphotericin B (1 mg for 2 wks)      Itraconazle (20 mg for 2 mos) 
   August      Itraconazole (40 mg) Amphotericin B (1 mg for 1.5 mos)       
   September     Death        
   October       Death    58  
   December            Itraconazle (20 mg for 2 mos) 
2009             
   February           103  
   March            Itraconazole (10 mg) Amphotericin B (1 mg for 2 mos) 
   November           22  
2010             
   January           16 Amphotericin B (1mg for 1 wk) 

Nasal colonization (0 = no colonies; figure = number of colonies per plate; + = unnumbered colonies per plate). Treatment (Dosage of fluconazole and itraconazole: mg = mg/kg/d; Dosage of amphotericin B: mg = mg/d).

Table 2.

Group 2: Nasal colonization of Cryptococcus neoformans in koalas not treated with antifungal medicine at Kanazawa Zoological gardens, Yokohama Japan

 10 
Koala no. Yeast isolated C. neoformans C. neoformans C. neoformans C. neoformans 
2002     
   November    
   December    
2003     
   July    
   November  Transferred from a zoo Birth  
2004     
   March  
   September Death    
2005     
   March   
   May    Transferred from a zoo 
   June  
   July  
   September  
   December  
2006     
   June  
   October  
2007     
   March  
   August  20 
2008     
April  
   May  
   July   Transferred to a zoo  
   October   
   November   Transferred from a zoo Death 
2009     
   February   
   July   Transferred to a zoo  
   November  Transferred from a zoo  
   December  Death   
2010     
   January   45  
 10 
Koala no. Yeast isolated C. neoformans C. neoformans C. neoformans C. neoformans 
2002     
   November    
   December    
2003     
   July    
   November  Transferred from a zoo Birth  
2004     
   March  
   September Death    
2005     
   March   
   May    Transferred from a zoo 
   June  
   July  
   September  
   December  
2006     
   June  
   October  
2007     
   March  
   August  20 
2008     
April  
   May  
   July   Transferred to a zoo  
   October   
   November   Transferred from a zoo Death 
2009     
   February   
   July   Transferred to a zoo  
   November  Transferred from a zoo  
   December  Death   
2010     
   January   45  

Nasal colonization: 0 = no colonies; figure = number of colonies per plate; + = unnumbered colonies per plate.

Table 3.

Group 3: Nasal colonization (unfounded) of Cryptococcus neoformans or C. gattii in koalas at Kanazawa Zoological Gardens, Yokohama, Japan.

 11 12 13 14 15 
Koala no. Yeast isolated – – – – – 
2002      
   November   
   December    
2003      
   July Death   
2004      
   March    
   August  Death    
2005      
   March     
   June     
   July     
   September     
   December     
2006      
   March   Death   
2007      
2008      
   January       Birth  
   October     
2009      
   February     
   June     Birth 
   November    
   December    Transferred to a zoo  
 11 12 13 14 15 
Koala no. Yeast isolated – – – – – 
2002      
   November   
   December    
2003      
   July Death   
2004      
   March    
   August  Death    
2005      
   March     
   June     
   July     
   September     
   December     
2006      
   March   Death   
2007      
2008      
   January       Birth  
   October     
2009      
   February     
   June     Birth 
   November    
   December    Transferred to a zoo  

Nasal colonization: 0 = no colonies.

Six of the koalas were treated with antifungals on multiple occasions (Table 1). The animals were not breeding or caring for their young when treated once C. neoformans or C. gattii were recovered on at least two consecutive examinations or when clinical symptoms were found. However, no nasal colonization was found with only three of the treated koalas within 1 year after each treatment.

While subclinical cryptococcosis was found in nine koalas, only one (No. 3) showed clinical symptoms of the disease, i.e., lack of appetite, weight loss, nasal discharge and stertor in 2008. Only koala No. 3 of the 13 tested for antigen titers showed antigenemia in March, May, and June 2008 at the time of the appearance of clinical signs (Table 4).

Table 4

Cryptococcal antigen titer in koalas at Kanazawa Zoological Gardens, Yokohama, Japan.

Koala no. Month/year Cryptococcal antigen titer 
December/2002 – 
December/2002 – 
 August/2005 – 
December/2002 – 
 September/2004 – 
 March/2008 1:1024 
 May/2008 1:4096 
 June/2008 1:32768 
December/2002 – 
 October/2008 – 
October/2005 – 
 August/2007 – 
 February/2008 – 
January/2006 – 
 June/2008 – 
 January/2009 – 
December/2002 – 
May/2008 – 
 November/2009 – 
July/2008 – 
 December/2009 – 
10 June/2008 – 
11 December/2002 – 
 July/2003 – 
12 August/2004 – 
13 December/2002 – 
 December/2005 – 
14  Not tested 
15  Not tested 
Koala no. Month/year Cryptococcal antigen titer 
December/2002 – 
December/2002 – 
 August/2005 – 
December/2002 – 
 September/2004 – 
 March/2008 1:1024 
 May/2008 1:4096 
 June/2008 1:32768 
December/2002 – 
 October/2008 – 
October/2005 – 
 August/2007 – 
 February/2008 – 
January/2006 – 
 June/2008 – 
 January/2009 – 
December/2002 – 
May/2008 – 
 November/2009 – 
July/2008 – 
 December/2009 – 
10 June/2008 – 
11 December/2002 – 
 July/2003 – 
12 August/2004 – 
13 December/2002 – 
 December/2005 – 
14  Not tested 
15  Not tested 

− = Negative for cryptococcal antigen titer.

Pathologic examination of the 11 koalas that died from age-related diseases (Table 5), revealed no gross or histologic lesions consistent with systemic cryptococcosis in whole internal organs, particularly in the brain, nasal cavity, and paranasal sinuses. The fungus was not observed through histopathological examinations of any tissues taken at necropsy. Moreover, there was no neutrophilic inflammatory response usually observed in chronic granulomatous lesions, as well as multinucleated giant cells, scattered lymphocytes, and plasma cells in the brain and spinal tissue.

Table 5.

Pathologic diagnosis in koalas that died at Kanazawa Zoological Gardens, Yokohama, Japan (November 2002–January 2010).

Koala no. Pathologic diagnosis 
Cardiac insufficiency 
Hepatitis 
Cardiac insufficiency 
Hepatitis 
Lymphoma 
Pulmonary fibrosis 
Lymphoma 
10 Tongue ulcer causing inappetence 
11 Lymphoma 
12 Lymphoma 
13 Myocardium fibrosis 
Koala no. Pathologic diagnosis 
Cardiac insufficiency 
Hepatitis 
Cardiac insufficiency 
Hepatitis 
Lymphoma 
Pulmonary fibrosis 
Lymphoma 
10 Tongue ulcer causing inappetence 
11 Lymphoma 
12 Lymphoma 
13 Myocardium fibrosis 

Discussion

In this study, subclinical cryptococcosis and nasal colonization of captive koalas at Kanazawa Zoological Gardens were found through the long-term surveillance. C. neoformans and C. gattii were recovered in culture from the nasal swabs obtained from 10 of 15 koalas (66.7%). Moreover, in the present study, C. neoformans was the predominant species associated with the animals, in contrast to C. gattii, which is principal species in Australia [8,12]. Although the detection rate of nasal colonization in long-term surveillance has not been previously reported, the rate in the present study was midway between 19 and 35% colonization of C. neoformans and/or C. gattii found in Western Sydney Wildlife Park or Port Macquarie Region and 94–100% colonization by C. gattii in Caffs Harbour Wildlife Park or Eastern Sydney Taronga Zoo [12,15]. These findings indicate that C. neoformans, which is considered to be less virulent that C. gattii, may be isolated in relatively high numbers from nasal cavities of captive koalas if nasal swabs are examined throughout koalas' lifetime.

Seven koalas suffered relapses or continuous colonization in the present study. Moreover, the nasal colonization in koala Nos. 7–10 disappeared without treatment. These findings suggest that recurrence might be due to colonization by C. neoformans from the environment or the koalas may have residual cryptococcal infection, particularly in the respiratory tract, which was reactivated due to a lowering in the immune function as a result of independent causes [8]. C. neoformans may have naturally spread through the koala population at the Kanazawa Zoological Gardens [2,3]. The presence of high levels of fungal elements in the environment is believed to be the key factor in etiology of cryptococcosis in koala [8,13]. On the other hand, C. gattii was cultured from koala No. 5 following nasal swab examination performed immediately after it was transferred from a zoo in Japan, suggesting that it was infected at the zoo. The presence of the fungus in the zoo might have been the result of the transfer of animals from Australia which were carriers of C. gatti and its subsequent spread through the zoo population. Most koalas, except for koala No. 3, might have had subclinical cryptococcosis as it has been shown that in cases involving persistent cryptococcal colonization that the animals show no signs of the disease [7,12]. The specific factors underlying the progression of cryptococcosis from subclinical to clinical disease remain unclear. Some possible factors include an extremely heavy environmental cryptococcocal load [12,15], concurrent infection of the respiratory tract with another pathogen such as Bordetella bronchiseptica [16], immune suppression due to stress or concurrent disease [7], or unidentified failure of mucosal defense [17]. The progress from the subclinical stage to the clinical stage of cryptococcosis in the koala should be further clarified.

Although six koalas were treated with fluconazole, itraconazole, or amphotericin B, the treatment was not definitively effective. Treatment of koalas 1 and 2 did seem to prevent recurrence until the death of the animals. However, the period in which the fungus was not detected might have been too short to indicate a therapeutic effect. Although antifungal treatment was given to four koalas (Nos. 3–6), treatments failed to eradicate the cryptococcal colonization. The treatment in koala No. 3 on December 2002 might be effective because Cryptococcus was not recovered in culture for 3 years and 6 months post therapy. However, the animal did show clinical symptoms of crytococcosis in 2008, and 6 months of treatment did not result in any positive effect until its death. The required treatment period for cryptococcosis is 6–12 months in humans [6,18] and 2–3 months to 18 months in the koalas [7], although no successful therapy has been reported in the koala. The treatment period in the present study was shorter than that indicated in prior studies. Moreover, the difference of the absorption of azole antifungals should be considered, as for example, itraconazole has shown unstable absorption, in contrast to fluconazole, in cases of low gastric acid secretion, [19,20]. Although no report have described the koala's stomach pH, the animal has a unique digestive system that is known to be a monogastric hindgut fermenter, and the stomach is always filled with fairly dry, finely chewed leaf material [21]. Therefore, the absorption of itraconazole might have been unstable which could have been a factor in the uncertain efficacy of this antifungal in the treatment of the animals noted since 2007.

Only koala No. 3 showed antigenemia and clinical signs of chronic upper respiratory tract disease. The presence of cryptococcal antigen in serum of koala No. 3 implies tissue invasion [22,23], but unfortunately we could not clearly identify the involved organs or tissues at necropsy and through histopathology. This could be explained by first, the fact that the cryptococcal lesions were missed because of their small size during necropsy while performing systemic examination of the entire respiratory tract [17]. Second, antigenemia might have remained after the resolution of cryptococcal lesions in the host. Antigenemia was shown to persist for 6 months in a rabbit model even after an earlier cryptococcal lesion resolved [24]. Third, laboratory error in the examination center might be considered, but reexamination was impossible because the serum sample was exhausted. Advanced imaging techniques (e.g., computed tomography or magnetic resonance imaging) may be capable of detecting small active or resolving lesions [17].

To the best of our knowledge, this is the first report describing the long-term surveillance and treatment of subclinical cryptococcosis and nasal colonization in captive koalas. In the present cases, the fungus could be recovered in culture from nasal swabs and this was useful in identifying cryptococcal colonies in captive koalas. However, the results also showed that nasal swab alone cannot be used to accurately diagnose subclinical or clinical cryptococcosis in koalas [8,12]. Further research is needed to establish rapid and accurate diagnostic methods to detect the fungal infection in koalas. Moreover, studies on effective treatment regimens for subclinical cryptococcosis and efficient measures for eradicating C. neoformans and C. gattii should also be conducted. The present study probably indicates that treatment may be unnecessary if the koala shows no clinical symptoms of cryptococcosis despite the recovery of the etiologic agent in culture.

Acknowledgements

We thank Dr Edward F. Barroga for the English proofreading of the manuscript.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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This paper was first published online on Early Online on 22 August 2011.