- Email: [email protected]
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii inhabiting Eucalyptus terreticornis and Eucalyptus camaldulensis trees in Jabalpur City of Madhya Pradesh, Central India
Journal de Mycologie Médicale (2012) 22, 341—347
Available online at
www.sciencedirect.com
SHORT COMMUNICATION/COURTE COMMUNICATION
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii inhabiting Eucalyptus terreticornis and Eucalyptus camaldulensis trees in Jabalpur City of Madhya Pradesh, Central India ´ valence saisonnie ` re de Cryptococcus neoformans var. grubii et de Pre Cryptococcus gattii colonisant Eucalyptus terreticornis et Eucalyptus calmadulensis dans la ville de Jabalpur Madhya Pradesh, Inde centrale N.G. Bedi a,b, S.R. Nawange a,c, S.M. Singh a,c,*, J. Naidu a, A. Kavishwar d a
Medical Mycology Research Laboratory, Department of Biological Science, Rani Durgavati Vishwavidyalaya, Jabalpur, 482001 Madhya Pradesh, India b Amity Institute of Biotechnology, Amity University, Noida, 201303 Uttar Pradesh, India c Center for Medical Mycology, Fungal Disease Diagnostic and Research Center (Society for Research Diagnosis and Treatment of Human Fungal Diseases), Jabalpur, 482002 Madhya Pradesh, India d Regional Medical Research Centre for Tribal, RMRCT Complex, Jabalpur, 482002 Madhya Pradesh, India Received 1st June 2012; received in revised form 29 August 2012; accepted 31 August 2012 Available online 2nd November 2012
KEYWORDS Cryptococcus neoformans var. grubii; Cryptococcus gattii; Seasonal prevalence; Eucalyptus sp.; Jabalpur; Madhya Pradesh; Central India
Summary This study presents a two-year retrospective analysis of the work done during 2003— 2005 on the distribution population density and isolation frequency of Cryptococcus neoformans var. grubii and Cryptococcus gattii recovered from different parts of Eucalyptus tree spp., at Jabalpur Madhya Pradesh, Central India. Of the 1000 samples collected from bark, flowers, fruits, buds and leaves of Eucalyptus trees E. terreticornis and E. camaldulensis, 32 (3.2%) were found to be positive for C.n var. grubii and 28 (2.8%) for C. gattii respectively. While both the pathogens were isolated through all the seasons, no significant difference was found in prevalence of the two species (P > 0.05) from different Eucalyptus tree samples. For C. neoformans var. grubii the highest isolation frequency of the pathogen was in spring followed by autumn, summer, winter and rainy season. For C. gattii, highest isolation frequency of the pathogen was in summer, followed by autumn, spring, winter and rainy season. Significant difference was seen in the isolation frequency of C. neoformans var. grubii and C. gattii during autumn and rainy season (P < 0.01), spring and rainy season (P < 0.001) and summer and rainy season (P < 0.001). Population density and isolation
* Corresponding author. SRDTHFD Flat 2C, Pavitra Apartment, South Civil lines, Jabalpur 482001, Madhya Pradesh, India. E-mail addresses: [email protected] (S.R. Nawange), [email protected] (S.M. Singh). 1156-5233/$ — see front matter # 2012 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.mycmed.2012.09.001
342
N.G. Bedi et al. frequency of the both pathogens were significantly lower in rainy season. Bark of the Eucalyptus tree yielded the highest frequency of C. neoformans var. grubii followed by flower, fruits, buds and debris. Trees located in the densely populated area of the city yielded highest frequency of the pathogens followed by trees located in sparsely populated area on the outskirt of the city and areas near the river Narmada. Further comprehensive study is suggested to assess the overall impact of seasonal prevalence in the isolation frequency and population density of both the pathogens and their clinical significance across climatically divergent region of India. # 2012 Published by Elsevier Masson SAS.
MOTS CLÉS Cryptococcus neoformans var. grubii ; Cryptococcus gattii ; Prévalence saisonnière ; Eucalyptus spp. ; Jabalpur ; Madhya Pradesh ; Inde Centrale
Re ´sume ´ Cette étude présente une analyse rétrospective des travaux effectués de 2003 à 2005 sur la densité de population et la fréquence d’isolement de Cryptococcus neoformans var. grubii et de Cryptococcus gattii isolés sur différentes parties d’Eucalyptus sp. à Jabalpur, Madhya Pradesh, Inde Centrale. Sur 1000 échantillons recueillis sur les écorces, les fleurs, les fruits, les bourgeons et les feuilles d’Eucalyptus terreticornis et d’Eucalyptus camaldulensis, 32 (3,2 %) ont été trouvés positifs pour C. neoformans var. grubii et 28 (2,8 %) pour C. gattii. Alors que ces deux espèces ont été isolées en toutes saisons, il n’a pas été trouvé de différence significative dans leur prévalence ( p > 0,05) sur les différentes espèces d’Eucalyptus. Pour C. neoformans var. grubii, la plus grande fréquence d’isolement se situe au printemps, suivie par l’automne, l’été, l’hiver et la saison des pluies. Pour C. gattii, la plus grande fréquence d’isolement se situe en été, suivie par l’automne, le printemps, l’hiver et la saison des pluies. Une différence significative a été observée dans la fréquence d’isolement de C. neoformans var. grubii et de C. gattii pendant l’automne et la saison des pluies ( p < 0,01), le printemps et la saison des pluies ( p < 0,001) et l’été et la saison des pluies ( p < 0,001). La densité de population et la fréquence d’isolement sont significativement plus faibles pendant la saison des pluies. Les écorces d’Eucalyptus donnent la plus grande fréquence d’isolement de C. neoformans var. grubii, suivie par les fleurs, les fruits, les bourgeons et les débris. Les arbres situés dans les endroits peu peuplés de la ville montrent une grande fréquence de ces deux pathogènes suivie par les endroits peu peuplés des banlieues et par les endroits situés près de la rivière Narmada. Plusieurs études complémentaires sont nécessaires pour déterminer l’impact global de la prévalence saisonnière dans la fréquence d’isolement et dans la densité de population de ces deux pathogènes et leur signification clinique à travers les régions très différentes au plan climatique de l’Inde. # 2012 Publié par Elsevier Masson SAS.
Introduction Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous yeasts causing respiratory and disseminated infection in man and animals [19]. C. neoformans and C. gattii were isolated from Eucalyptus tree species worldwide [3,14]. In our earlier publications [6,15,16], we reported that decayed wood inside tree hollows of a wide spectrum of tree species and pigeon excreta from Central India served as an important natural reservoir for C. neoformans and C. gattii. In Central India, the climate varies widely from season to season. In Central India, Grover et al. [6] reported for the first time the effect of seasonal variation on the prevalence of these pathogens in decayed tree trunk hollows. In northwest India, Randhawa et al. [17] also reported the impact of climatic factors on the prevalence of C.n. var. grubii and C. gattii. In the present retrospective study, the effect of various climatic conditions on the prevalence of C. neoformans species complex inhabiting Eucalyptus trees had been determined and results presented.
Materials and methods Study area The Jabalpur city is situated in surroundings of rocky basin at a height of 402 m above sea level and lies at 238100 North
latitude and 798580 East longitude. The climate of Jabalpur is humid subtropical. On the basis of temperature, relative humidity and rainfall, the year has been divided into the following five seasons: summer (21st April—21st June), rainy (22nd June—22nd September), autumn (23rd September— 21st November), winter (22nd November—19th February) and spring (22nd February—22nd April) based on temperature, relative humidity and rainfall (Table 1).
Sample collection A total of 1000 samples of Eucalyptus trees parts that of flowers (n = 225), buds (n = 100), fruits (n = 150), leaves (n = 110), bark (n = 240) and debris (n = 175) were collected over a period of 2 years (2003—2005) from three species of Eucalyptus and these species were E. terreticornis, E. camaldulensis and E. citriodora.
Sample processing and identification of Cryptococcus spp. Approximately 5 g of each sample was suspended in 45 ml of sterilized distilled water containing 20 mg/ml penicillin and 40 mg/ml streptomycin. The suspension was shaken vigorously for few minutes and then was allowed to settle at 37 8C for 1 hour. Serial dilution i.e. 1:10, 1:100, 1:1000 of the suspension was prepared and 0.1 ml of each dilution was
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii
343
Table 1 Data of mean temperature, rainfall and relative humidity during the study period. ´ trie et humidite ´ relative pendant la pe ´ riode d’e ´ tude. ´ rature moyenne, pluviome Tempe Factors
Seasons
Jan.
Feb.
Mar.
Apr.
May
Mean Temp. range (8C)
Min. Max.
8.46 24.1
11.5 27.3
15.45 33.8
22.16 39.1
26.8 41.6
27 37.6
25.3 32.3
23.8 28.8
20.3
27.8
1.9
0.9
11.6
141.3
278.3
589.2
40.3 92
39.3 88.6
21 69.5
13.6 44.3
16.3 40
39.6 61.3
63.6 82.3
80.3 92
Rainfall (MM) Relative humidity (%)
Eve. Mor.
Jun.
Jul.
Aug.
Sept. 23.3 30.36 288.3 68.3 87.66
Oct.
Nov.
Dec.
18.43 30.6
13.06 29.3
6.86 6.36
42.7
10.5
2.9
45.6 85.6
34.6 89.3
36 92.5
plated on Staib’s Guizotia abyssinica creatinine agar medium [22] of the following composition (70 g pulverized niger seed, 10 g dextrose, 1 g KH2PO4, 1 g creatinine and 20 g agar, and pH of medium was set at 5.5). The medium was autoclaved at 15 lbs pressure at 121 8C for 15—20 min. The medium was cooled to approximately 50 8C, and streptomycin (40 mg/ml), penicillin (20 mg/ml) and 10 ml of biphenyl solution (1 g/ 10 ml 95% alcohol) were added. The inoculated plates were incubated at 28 8C and examined daily for 8 days. Yeast like brown colonies were examined microscopically and isolated in pure culture. All brown yeast isolates were subjected to physiological and morphological identification tests in order to identify Cryptococcus species [12]. Canavine glycine bromothymol medium [12] was used to screen the varieties. Serotyping of the isolates identified as C. neoformans was done by the slide agglutination test based on monoclonal antibody [9] (CRYPTO CHECK 25 test kit, Iatron Laboratories Inc. Chiyoda, Japan).
and detritus. Similarly for C. gattii flower was the best source of isolation followed by buds, fruits, debris and bark. No isolate of C. neoformans species complex was obtained from leaves (Table 4). Though both the Cryptococcus species were isolated throughout the year but the frequency of isolation of C.n. var. grubii was higher in spring followed by autumn, summer, winter and rainy season. In case of C. gattii, the highest frequency was obtained during summer, followed by autumn, winter, spring and rainy season. Highest frequency of C. gattii was in summer and that of C. neoformans var. grubii was in spring and least isolation frequency of the both species was in rainy season. Both pathogens were isolated throughout all the seasons, but no significant difference was found in the prevalence of the two species (P > 0.05). However, statistically significant difference was found in the isolation of C. neoformans var. grubii and C. gattii during autumn and rainy season (P < 0.01), spring and rainy season (P < 0.001) and summer and rainy season (P < 0.001) (Table 5).
Statistical analysis
Discussion
The data collected on the seasonal prevalence of C.n. var. grubii and C. gattii was analyzed by using t-test.
Results In the present study, population density and isolation frequency of C. gattii and C.n. var. grubii were investigated from various parts of E. terreticornis (hybrid) and E. camaldulensis trees from Jabalpur city. The highest population density (2.1 104—2.2 105 cfu/g) of C.n. var. grubii was obtained from buds of E. terreticornis (Tree no E150), located at a vehicle factory on the outskirts of the city. The highest population density (1.2 105—2 105 cfu/g) of C. gattii was obtained from flower of E. camaldulensis (Tree no. E165) located at Medical college campus of the city. There was no remarkable difference in association of C.n. var. grubii and C. gattii observed with any of the Eucalyptus trees (P > 0.05). The observation span of this study ranges between 231 days to 640 days (Table 2). Maximum positive samples found for the pathogens were from Eucalyptus trees located in city area followed by trees located on the outskirts and river site. Though no statistically significant difference (P > 0.05) was found in the isolation of the both species from any particular site. However, more isolates of the pathogens were obtained from Eucalyptus trees located near the river Narmada of Jabalpur City (Table 3). For C. neoformans var. grubii, bark of Eucalyptus trees was found to be the best source followed by flower, bud, fruit
Contrary to the report of Randhawa et al. [17] seasonal variation in the prevalence of C. neoformans var. grubii and C. gattii from decayed wood inside trunk hollows of diverse tree species from Jabalpur city of Madhya Pradesh, Central India was highlighted first time by Grover et al. [6] In the present study, out of 1000 samples studied from different tree parts of Eucalyptus trees investigated, 3.2% yielded C. neoformans var. grubii and 2.8% yielded C. gattii all through the seasons in a year. However, overall prevalence of C. neoformans var. grubii over C. gattii was not statistically significant. On the contrary, Randhawa et al. [17] reported much higher prevalence of C. neoformans var. grubii (17.2%) over that of C. gattii (11.9%) which was statistically significant in decayed wood inside trunk hollows of diverse tree species excluding Eucalyptus trees. Similarly Nawange et al. [15] had reported the prevalence of C. neoformans var. grubii (5.56%) and C. gattii (9.26%) from decayed wood inside trunk hollow of diverse tree species which is found to be higher than present study from the same area but no statistical significance was mentioned in paper. It suggests that decayed wood inside trunk hollows of living trees serve as a good natural habitat for C. neoformans species complex in the environment because the pathogens inside woody debris in trunk hollows is well protected from lethal effects of sunlight and drying. Moreover, these pathogens can utilize the woody material rich in polyphenol compounds and lignin as substrate for growth due to its phenoloxidase activity [20]. On the contrary, these pathogens
344
N.G. Bedi et al.
Table 2 Distribution and population density of C. neoformans var. grubii and C. gattii from various parts of E. terreticornis (hybrid) and E. camaldulensis trees located at different parts of Jabalpur city of Madhya Pradesh, Central India. ´ de population de C. neoformans var. grubii et C. gattii dans diffe ´ rentes parties de E. terreticornis (hybride) Distribution et densite ´ s dans diffe ´ rents endroits de la ville de Jabalpur, Madhya Pradesh, Inde Centrale. et d’E. camaldulensis situe Tree species
Tree Type of Site for number samples isolation
Sample Positive sampling positive dates
Observation Variety/ CFU/g span (days) Serotype (lowest and highest range)
E. terreticornis
E15
Bark
Polypathar
4
Cnvg/A
Bark
Hattital colony
4
490
Cnvg/A
E4
Flower
Polypathar
4
640
C.g/B
E216
Flower
Civic centre
3
360
C.g/B
E178
Buds
3
503
C.g/B
E150
Buds
Medical college Road Vehicle factory
3
410
Cnvg/A
E28
Fruit
Polypathar
4
545
C.g/B
E28
Debris
Polypathar
4
2.5 104— 2.9 10 4 2.4 104— 2.6 10 4 0.7 104— 0.8 10 4 1.4 104— 1.8 10 4 2.0 104— 2.5 10 4 2.1 104— 2.2 10 5 0.7 104— 0.8 10 4 0.7 104— 1.1 10 4
450
E114
6 Mar., 10 June 2004; 20 Apr., 15 June 2005 6 Mar., 9 Apr. 2004; 16 June, 1st Nov. 2005 6 Mar., 10 June 2004; 15 June, 6 Dec. 2005 6 Dec. 2004; 23 Mar., 25th May 2005 6 Mar., 25 Sept., 26 Oct. 2004 26 Oct, 16 Nov. 2004; 21 Dec. 2005 21 Apr., 10 June 2004; 15 June, 22 Oct. 2005 10 June, 19 Sept. 2004; 15 June, 18 Oct. 2005
495
C.g/B
E. Camaldulensis E75
Bark
RDVV campus
4
C.g/B
Bark
RDVV campus
4
1.2 104— 1.1 10 5 1.4 104— 1.5 10 5
611
E80
503
Cnvg/A
E137
Flower
Bargi hills
4
Cnvg/A
Flower
Bargi hills
3
385
C.g/B
E165
Flower
3
503
C.g/B
E248
Flower
465
Cnvg/A
E30
Fruit
Medical college campus Boys hostel Krishinagar Adhartal Polypathar
1.6 104— 1.8 10 4 2.2 104— 2.0 10 5 1.2 105— 2 10 5 1 104— 1.2 10 4
446
E147
15 Feb, 9 Apr. 2004; 16 June, 18 Oct. 2005 6 Mar., 9 June, 16 June 2004; 15 Apr. 2005 30 Jan., 25 May 2005; 5 Mar., 22 Apr. 2004 30 Jan. 2005; 5 Mar., 26 Oct. 2004 14 Oct., 6 Dec. 2004; 21 Feb. 2005 14 Oct., 14 Dec. 2004; 16 Aug. 2005
Cnvg/A
Debris
Behraghat thane 3
1.1 104— 1.3 10 4 1.0 104— 1.0 10 5
559
E119
10 Apr., 28 June 2004; 24 Sep., 21 Oct. 2005 28 Oct. 2004; 23 Mar., 16 June 2005
231
Cnvg/A
3
4
Cnvg/A denotes Cryptococcus neoformans var. grubii. C.g./B denotes Cryptococcus gattii.
inhabiting different parts of Eucalyptus trees bark, flower, bud, leaves are exposed to much more hostile environment in terms of sunlight, temperature, wind, relative humidity, substrate which might be inducing and inhibitory effect on their growth as evidenced by the low percentage of prevalence of these pathogens in Eucalyptus trees [1]. The result of the present study has revealed that tree parts of E. camaldulensis and E. terreticornis serves as natural habitat for C. neoformans var. grubii and C. gattii. For C. neoformans var. grubii bark was the best natural habitat and for C. gattii flowers were the best ecological niche. Both pathogens were also isolated from fruits and buds of these Eucalyptus trees. To our knowledge,
this is the first report of the isolation of C. neoformans var. grubii and C. gattii from Eucalyptus trees at Jabalpur, Central India. Montenergo and Paula [14] showed a close association between the flowering season from November to February of Eucalyptus tree and occurrence of C. gattii because they could not isolate the pathogens from Eucalyptus trees beyond March. Montagna et al. [13] on the contrary made three isolations of the pathogen during the month of June from E. camaldulensis. Similarly, in the present study we could isolate C. gattii from various samples of Eucalyptus trees during its non-flowering season. We therefore, support the hypothesis that there is non-flowering seasonal
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii
345
Table 3 Distribution of Cryptococcus neoformans species complex in positive samples from trees located at three test sites in the different parts of Jabalpur city of Madhya Pradesh, Central India. ` ces de C. neoformans dans les ´echantillons positifs des Eucalyptus situe ´ s dans trios endroits diffe ´ rents Distribution des deux espe de la ville de Jabalpur, Madhya Pradesh, Inde Centrale. Location of positive sites
Total number of samples
Number of C.n. var. grubii/A
Percentage (%)
Number of C. gattii/B
Percentage (%)
River site City area Outskirts
220 453 327
7 12 10
3.18 2.6 3.05
3 15 10
1.36 3.3 3.05
Table 4 Isolation frequency of C. neoformans var. grubii and C. gattii in different parts of Eucalyptus tree. ´ quence d’isolement de C. neoformans var. grubii et de C. gattii au niveau de diffe ´ rents sites sur les Eucalyptus. Fre Type of Samples
Total number of samples
Number of C.n var. grubii positive
Frequency and percentage (%)
Number of C. gattii positive
Frequency and percentage (%)
Bark Flower Fruit Buds Leaves Debris
240 225 150 100 110 175
12 10 4 3 0 3
5 4.4 2.7 3 0 1.71
4 13 4 3 0 4
1.6 5.7 2.7 3 0 2.28
dispersion as well of the pathogen in Jabalpur city. In the present study though, both pathogens were isolated from Eucalyptus trees through all the seasons. However, no significant difference was found in their distribution and isolation frequency (P > 0.05). For C. neoformans the highest isolation frequency was seen in spring followed by autumn, summer, winter and least in rainy season. Significant difference was seen in the isolation frequency of C. neoformans and C. gattii during autumn and rainy season (P < 0.001) and spring and rainy season (P < 0.001) respectively. Colom et al. [2] also reported low rate of isolation of each pathogenic Cryptococcus species from Eucalyptus trees. This is also in accordance with previous finding from Mediterranean area [7,8,18]. The present study revealed that the prevalence of C. neoformans var. grubii was higher in spring (5.2%) than during autumn (4.11%) and summer (4%). On the contrary, prevalence of C. gattii was higher in summer (4.5%) than during winter (4.11%). This difference was however not statistically significant. Randhawa et al. [17] reported low prevalence of C. gattii in decayed wood during winter
season. We also found low prevalence of C. neoformans var. grubii and C. gattii during winter months. Grandos and Casteneda [4] did not find any C. gattii in winter in the decayed wood or tree trunk hollows. Moreover, they also found low population density of the pathogens in the bark of the trees investigated during winter. However, their population was higher during rainy season. Later, Granados and Castaneda [5] analyzed the relationship between the occurrence of various serotypes of C. neoformans species complex in tree samples and climatic conditions recorded during sampling in four cities of Colombia between 1992 and 2004. Their investigations using logistic regression and lagged Pearson correlation indicated that environmental conditions mainly humidity, temperature and solar radiation variably affected the occurrence of serotypes A, B and C. It was also noted that serotypes A was more thermotolerant and hygrophobic than serotype B. Our data on the prevalence of C. neoformans var. grubii and C. gattii during all seasons provide further support for their hypothesis. Grover et al. [6] also reported highest isolation frequency of C. neoformans var. grubii in spring in Jabalpur, when minimum and
Table 5 Seasonal variation in isolation frequency of Cryptococcus neoformans var. grubii and C. gattii in various samples of Eucalyptus spp. ` re de la fre ´ quence d’isolement de Cryptococcus neoformans var. grubii et C. gattii dans les diffe ´ rents Variation saisonnie ´echantillons d’Eucalyptus. Seasons
Number of samples tested
Number of positive C.n. var. grubii
Isolation of frequency Percentage (%)
Number of positive C. gattii
Isolation of frequency Percentage (%)
Autumn Winter Spring Summer Rainy
170 200 170 200 260
7 2 9 9 2
4.11 1.5 5.2 4 0.77
7 6 5 9 1
4.11 2.5 3.5 4.5 0.38
346 maximum temperature varied between 15 8C and 33 8C and less rainfall were observed and this was found to be the favorable condition for the growth of pathogenic yeast. Grover et al. [6], for the first time, reported seasonal variation in the isolation frequency and population density of C. neoformans var. grubii and C. gattii from decaying wood of trunk hollows of living trees at Jabalpur Central India. They reported that isolation frequency of C. neoformans var. grubii was highest in March (Spring) followed by October (Autumn) and June (late summer and early rainy season). On the contrary, isolation frequency of C. gattii was highest in June followed by October (autumn) and March (spring). Randhawa et al. [17] from Delhi, India reported that both the pathogens exhibited seasonal variation in their prevalence with the highest variation, found during the autumn followed by that in summer. In the present study we also report the seasonal variation in the isolation frequency of C. neoformans var. grubii and C. gattii from various parts of Eucalyptus tree. Isolation frequency of C. neoformans var. grubii was highest in spring followed by autumn and summer. Similarly, highest isolation frequency of C. gattii was seen in summer followed by autumn and spring. In rainy season the isolation frequency of the pathogen was low. Interestingly, similar results of a low frequency of isolation during the rainy season as against a highest prevalence of C. neoformans var. grubii during rainy season have been reported in an investigation of chicken faeces in Northern Thailand [11]. Kidd et al. [10] in an environmental survey in British Columbia Canada and Pacific Northwest of the USA reported significantly higher concentration of C. gattii in the air during warm, dry and low humidity summer months than during cold wet and high relative humidity months. Interestingly, Grover et al. [6] and Randhawa et al. [17] also found similar observation regarding seasonal prevalence of C. gattii in their study during their wood sampling at Jabalpur and Delhi respectively. In the present study we also found similar pattern of seasonal prevalence of C. gattii in Eucalyptus trees. The present study as well as those of other authors [6,15,16] have shown the presence of C. neoformans var. grubii and C. gattii in the environment of Jabalpur city in all seasons throughout the year. Despite the widespread presence of the pathogens in the environment there is paucity of clinical data related to Cryptococcosis in this region. It would be however interesting to correlate such environmental data with the prevalence of Cryptococcosis in the same region as has been reported by Nawange et al. [16] in Jabalpur. In India we do not have such linked data available. However, such comparisons have been done in USA [21]. In contrast, the USA study found a significantly higher prevalence of Cryptococcosis in the fall and winter month than in spring and summer. We suggest a comprehensive study to determine the potential health impact of seasonal prevalence of C. neoformans. var. grubii and C. gattii in decayed wood and Eucalyptus trees in climatically divergent region of India.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
N.G. Bedi et al.
Acknowledgements The authors wish to thank the Vice-Chancellor R.D. University, for providing laboratory facilities and to The DirectorGeneral, Indian Council of Medical Research (ICMR), New Delhi for awarding Post Doctoral Research Associateship and Government of India, Ministry of Science and Technology, Department of Science and Technology SERC FAST Track Scheme for Young Scientist to one of the author (S.R.N).
References [1] Colom MF, Fras´es S, Ferrer. et al. First case of human cryptococcosis due to Cryptococcus neoformans var. gattii in Spain. J Clin Microbiol 2005;43:3548—50. [2] Colom MF, Hagen F, Gonzalez A, et al. Ceratonia siliqua trees as natural habitat and source of infection by Cryptococcus gattii in the Mediterranean environment. Med Mycol 2012;50:67—73. [3] Ergin C, Ilkit M, Kaftanoglu O. Detection of Cryptococcus neoformans var. grubii in honeybee (Apis mellifera) colonies. Mycoses 2004;47:431—4. [4] Granados DP, Castaneda E. Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogota, Colombia, and study of ecological conditions in the area. Microb Ecol 2005;49:282—90. [5] Granados DP, Castaneda E. Influence of climatic conditions on the isolation of members of the Cryptococcus neoformans species complex fro trees in Colombia from 1992—2004. FEMS Yeast Res 2006;6:636—44. [6] Grover N, Nawange SR, Naidu J, Singh SM, Sharma A. Ecological niche of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decaying wood of trunk hollows of living trees in Jabalpur City of Central India. Mycopathologia 2007;164: 159—70. [7] Hagen F, Boekhout T. The search for the natural habitat of Cryptococcus gattii. Mycopathologia 2010;170:209—11. [8] Hamasha AMS, Yildiran ST, Gonulum A, Saracli MA, Doganci L. Cryptococcus neoformans varieties from material under the canopies of Eucalyptus trees and pigeon dropping samples from four major cities in Jordan. Mycopathologia 2004;158:195—9. [9] Ikeda R, Shinoda T, Fukazawa Y, Kaufman L. Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol 1982;16:22—9. [10] Kidd SE, Chow Y, Mak S, et al. Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States. Appl Environ Microbiol 2007;73:1433—43. [11] Kuroki M, Phichaichumpon C, Yasuoka A, et al. Environmental isolation of Cryptococcus neoformans from an endemic region of HIV-associated cryptococcosis in Thailand. Yeast 2004;21:809—12. [12] Kwon-Chung KJ, Bennett JE. Cryptococcus and Cryptococcosis. In: Kwon-Chung KJ, Bennett JE, editors. Medical mycology. Philadelphia: Lea and Febiger; 1992. p. 397—446. [13] Montagna MT, Viviani MA, Pulito A. Cryptococcus neoformans var. gattii in Italy. Note II. Environmental investigations related to an autochthonous clinical case in Apulia. Mycol Med 1997;7:93—6. [14] Montenegro H, Paula CR. Environmental isolation of Cryptococcus neoformans var. gattii and C. neoformans var. neoformans in the city of Sao Paulo, Brazil. Med Mycol 2000;38: 385—90. [15] Nawange SR, Shakya K, Naidu J, Singh SM, Jharia N, Garg S. Decayed wood inside trunk hollows of living trees of T. indica S. cumini and M. indica as natural habitat of
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii Cryptococcus neoformans and their serotypes in Jabalpur city of Central India. J Mycol Med 2006;16:63—71. [16] Nawange SR, Singh SM, Naidu J, et al. Serotype distribution of Cryptococcus neoformans and Cryptococcus gattii in patients and in the environment of Jabalpur, a city of Madhya Pradesh India. Asian J Microbiol Biotechnol Environ Sci 2011;13:735—42. [17] Randhawa HS, Kowshik T, Chowdhary A, et al. Seasonal variations in the prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decayed wood inside trunk hollows of diverse tree species in north-western India: a retrospective study. Med Mycol 2010;49:320—3. [18] Rodriguez TJM, Hermoso de Mendoza M, Alvarado-Ramirez E, Segura-Roca G. Cryptococcosis by Cryptococcus gattii in
[19] [20] [21]
[22]
347
immunocompetent goats in Spain and review of the literature. Acta Sci Ve 2006;34:345—53. Sorrell TC. Cryptococcus neoformans var gattii. Med Mycol 2001;39:155—68. Sorrell TC, Ellis DH. Ecology of Cryptococcus neoformans. Rev Iberoam Micol 1997;14:42—3. Sorvillo F, Beall G, Turner PA, et al. Incidence and factors associated with extra pulmonary cryptococcosis among persons with HIV infection in Los Angeles County. AIDS 1997;11: 673—9. Staib F. Cryptococcus neoformans und Guizotia abyssinica (Syn. G. oleifera D.C.). Z Hyg 1962;148:466—75.
Available online at
www.sciencedirect.com
SHORT COMMUNICATION/COURTE COMMUNICATION
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii inhabiting Eucalyptus terreticornis and Eucalyptus camaldulensis trees in Jabalpur City of Madhya Pradesh, Central India ´ valence saisonnie ` re de Cryptococcus neoformans var. grubii et de Pre Cryptococcus gattii colonisant Eucalyptus terreticornis et Eucalyptus calmadulensis dans la ville de Jabalpur Madhya Pradesh, Inde centrale N.G. Bedi a,b, S.R. Nawange a,c, S.M. Singh a,c,*, J. Naidu a, A. Kavishwar d a
Medical Mycology Research Laboratory, Department of Biological Science, Rani Durgavati Vishwavidyalaya, Jabalpur, 482001 Madhya Pradesh, India b Amity Institute of Biotechnology, Amity University, Noida, 201303 Uttar Pradesh, India c Center for Medical Mycology, Fungal Disease Diagnostic and Research Center (Society for Research Diagnosis and Treatment of Human Fungal Diseases), Jabalpur, 482002 Madhya Pradesh, India d Regional Medical Research Centre for Tribal, RMRCT Complex, Jabalpur, 482002 Madhya Pradesh, India Received 1st June 2012; received in revised form 29 August 2012; accepted 31 August 2012 Available online 2nd November 2012
KEYWORDS Cryptococcus neoformans var. grubii; Cryptococcus gattii; Seasonal prevalence; Eucalyptus sp.; Jabalpur; Madhya Pradesh; Central India
Summary This study presents a two-year retrospective analysis of the work done during 2003— 2005 on the distribution population density and isolation frequency of Cryptococcus neoformans var. grubii and Cryptococcus gattii recovered from different parts of Eucalyptus tree spp., at Jabalpur Madhya Pradesh, Central India. Of the 1000 samples collected from bark, flowers, fruits, buds and leaves of Eucalyptus trees E. terreticornis and E. camaldulensis, 32 (3.2%) were found to be positive for C.n var. grubii and 28 (2.8%) for C. gattii respectively. While both the pathogens were isolated through all the seasons, no significant difference was found in prevalence of the two species (P > 0.05) from different Eucalyptus tree samples. For C. neoformans var. grubii the highest isolation frequency of the pathogen was in spring followed by autumn, summer, winter and rainy season. For C. gattii, highest isolation frequency of the pathogen was in summer, followed by autumn, spring, winter and rainy season. Significant difference was seen in the isolation frequency of C. neoformans var. grubii and C. gattii during autumn and rainy season (P < 0.01), spring and rainy season (P < 0.001) and summer and rainy season (P < 0.001). Population density and isolation
* Corresponding author. SRDTHFD Flat 2C, Pavitra Apartment, South Civil lines, Jabalpur 482001, Madhya Pradesh, India. E-mail addresses: [email protected] (S.R. Nawange), [email protected] (S.M. Singh). 1156-5233/$ — see front matter # 2012 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.mycmed.2012.09.001
342
N.G. Bedi et al. frequency of the both pathogens were significantly lower in rainy season. Bark of the Eucalyptus tree yielded the highest frequency of C. neoformans var. grubii followed by flower, fruits, buds and debris. Trees located in the densely populated area of the city yielded highest frequency of the pathogens followed by trees located in sparsely populated area on the outskirt of the city and areas near the river Narmada. Further comprehensive study is suggested to assess the overall impact of seasonal prevalence in the isolation frequency and population density of both the pathogens and their clinical significance across climatically divergent region of India. # 2012 Published by Elsevier Masson SAS.
MOTS CLÉS Cryptococcus neoformans var. grubii ; Cryptococcus gattii ; Prévalence saisonnière ; Eucalyptus spp. ; Jabalpur ; Madhya Pradesh ; Inde Centrale
Re ´sume ´ Cette étude présente une analyse rétrospective des travaux effectués de 2003 à 2005 sur la densité de population et la fréquence d’isolement de Cryptococcus neoformans var. grubii et de Cryptococcus gattii isolés sur différentes parties d’Eucalyptus sp. à Jabalpur, Madhya Pradesh, Inde Centrale. Sur 1000 échantillons recueillis sur les écorces, les fleurs, les fruits, les bourgeons et les feuilles d’Eucalyptus terreticornis et d’Eucalyptus camaldulensis, 32 (3,2 %) ont été trouvés positifs pour C. neoformans var. grubii et 28 (2,8 %) pour C. gattii. Alors que ces deux espèces ont été isolées en toutes saisons, il n’a pas été trouvé de différence significative dans leur prévalence ( p > 0,05) sur les différentes espèces d’Eucalyptus. Pour C. neoformans var. grubii, la plus grande fréquence d’isolement se situe au printemps, suivie par l’automne, l’été, l’hiver et la saison des pluies. Pour C. gattii, la plus grande fréquence d’isolement se situe en été, suivie par l’automne, le printemps, l’hiver et la saison des pluies. Une différence significative a été observée dans la fréquence d’isolement de C. neoformans var. grubii et de C. gattii pendant l’automne et la saison des pluies ( p < 0,01), le printemps et la saison des pluies ( p < 0,001) et l’été et la saison des pluies ( p < 0,001). La densité de population et la fréquence d’isolement sont significativement plus faibles pendant la saison des pluies. Les écorces d’Eucalyptus donnent la plus grande fréquence d’isolement de C. neoformans var. grubii, suivie par les fleurs, les fruits, les bourgeons et les débris. Les arbres situés dans les endroits peu peuplés de la ville montrent une grande fréquence de ces deux pathogènes suivie par les endroits peu peuplés des banlieues et par les endroits situés près de la rivière Narmada. Plusieurs études complémentaires sont nécessaires pour déterminer l’impact global de la prévalence saisonnière dans la fréquence d’isolement et dans la densité de population de ces deux pathogènes et leur signification clinique à travers les régions très différentes au plan climatique de l’Inde. # 2012 Publié par Elsevier Masson SAS.
Introduction Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous yeasts causing respiratory and disseminated infection in man and animals [19]. C. neoformans and C. gattii were isolated from Eucalyptus tree species worldwide [3,14]. In our earlier publications [6,15,16], we reported that decayed wood inside tree hollows of a wide spectrum of tree species and pigeon excreta from Central India served as an important natural reservoir for C. neoformans and C. gattii. In Central India, the climate varies widely from season to season. In Central India, Grover et al. [6] reported for the first time the effect of seasonal variation on the prevalence of these pathogens in decayed tree trunk hollows. In northwest India, Randhawa et al. [17] also reported the impact of climatic factors on the prevalence of C.n. var. grubii and C. gattii. In the present retrospective study, the effect of various climatic conditions on the prevalence of C. neoformans species complex inhabiting Eucalyptus trees had been determined and results presented.
Materials and methods Study area The Jabalpur city is situated in surroundings of rocky basin at a height of 402 m above sea level and lies at 238100 North
latitude and 798580 East longitude. The climate of Jabalpur is humid subtropical. On the basis of temperature, relative humidity and rainfall, the year has been divided into the following five seasons: summer (21st April—21st June), rainy (22nd June—22nd September), autumn (23rd September— 21st November), winter (22nd November—19th February) and spring (22nd February—22nd April) based on temperature, relative humidity and rainfall (Table 1).
Sample collection A total of 1000 samples of Eucalyptus trees parts that of flowers (n = 225), buds (n = 100), fruits (n = 150), leaves (n = 110), bark (n = 240) and debris (n = 175) were collected over a period of 2 years (2003—2005) from three species of Eucalyptus and these species were E. terreticornis, E. camaldulensis and E. citriodora.
Sample processing and identification of Cryptococcus spp. Approximately 5 g of each sample was suspended in 45 ml of sterilized distilled water containing 20 mg/ml penicillin and 40 mg/ml streptomycin. The suspension was shaken vigorously for few minutes and then was allowed to settle at 37 8C for 1 hour. Serial dilution i.e. 1:10, 1:100, 1:1000 of the suspension was prepared and 0.1 ml of each dilution was
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii
343
Table 1 Data of mean temperature, rainfall and relative humidity during the study period. ´ trie et humidite ´ relative pendant la pe ´ riode d’e ´ tude. ´ rature moyenne, pluviome Tempe Factors
Seasons
Jan.
Feb.
Mar.
Apr.
May
Mean Temp. range (8C)
Min. Max.
8.46 24.1
11.5 27.3
15.45 33.8
22.16 39.1
26.8 41.6
27 37.6
25.3 32.3
23.8 28.8
20.3
27.8
1.9
0.9
11.6
141.3
278.3
589.2
40.3 92
39.3 88.6
21 69.5
13.6 44.3
16.3 40
39.6 61.3
63.6 82.3
80.3 92
Rainfall (MM) Relative humidity (%)
Eve. Mor.
Jun.
Jul.
Aug.
Sept. 23.3 30.36 288.3 68.3 87.66
Oct.
Nov.
Dec.
18.43 30.6
13.06 29.3
6.86 6.36
42.7
10.5
2.9
45.6 85.6
34.6 89.3
36 92.5
plated on Staib’s Guizotia abyssinica creatinine agar medium [22] of the following composition (70 g pulverized niger seed, 10 g dextrose, 1 g KH2PO4, 1 g creatinine and 20 g agar, and pH of medium was set at 5.5). The medium was autoclaved at 15 lbs pressure at 121 8C for 15—20 min. The medium was cooled to approximately 50 8C, and streptomycin (40 mg/ml), penicillin (20 mg/ml) and 10 ml of biphenyl solution (1 g/ 10 ml 95% alcohol) were added. The inoculated plates were incubated at 28 8C and examined daily for 8 days. Yeast like brown colonies were examined microscopically and isolated in pure culture. All brown yeast isolates were subjected to physiological and morphological identification tests in order to identify Cryptococcus species [12]. Canavine glycine bromothymol medium [12] was used to screen the varieties. Serotyping of the isolates identified as C. neoformans was done by the slide agglutination test based on monoclonal antibody [9] (CRYPTO CHECK 25 test kit, Iatron Laboratories Inc. Chiyoda, Japan).
and detritus. Similarly for C. gattii flower was the best source of isolation followed by buds, fruits, debris and bark. No isolate of C. neoformans species complex was obtained from leaves (Table 4). Though both the Cryptococcus species were isolated throughout the year but the frequency of isolation of C.n. var. grubii was higher in spring followed by autumn, summer, winter and rainy season. In case of C. gattii, the highest frequency was obtained during summer, followed by autumn, winter, spring and rainy season. Highest frequency of C. gattii was in summer and that of C. neoformans var. grubii was in spring and least isolation frequency of the both species was in rainy season. Both pathogens were isolated throughout all the seasons, but no significant difference was found in the prevalence of the two species (P > 0.05). However, statistically significant difference was found in the isolation of C. neoformans var. grubii and C. gattii during autumn and rainy season (P < 0.01), spring and rainy season (P < 0.001) and summer and rainy season (P < 0.001) (Table 5).
Statistical analysis
Discussion
The data collected on the seasonal prevalence of C.n. var. grubii and C. gattii was analyzed by using t-test.
Results In the present study, population density and isolation frequency of C. gattii and C.n. var. grubii were investigated from various parts of E. terreticornis (hybrid) and E. camaldulensis trees from Jabalpur city. The highest population density (2.1 104—2.2 105 cfu/g) of C.n. var. grubii was obtained from buds of E. terreticornis (Tree no E150), located at a vehicle factory on the outskirts of the city. The highest population density (1.2 105—2 105 cfu/g) of C. gattii was obtained from flower of E. camaldulensis (Tree no. E165) located at Medical college campus of the city. There was no remarkable difference in association of C.n. var. grubii and C. gattii observed with any of the Eucalyptus trees (P > 0.05). The observation span of this study ranges between 231 days to 640 days (Table 2). Maximum positive samples found for the pathogens were from Eucalyptus trees located in city area followed by trees located on the outskirts and river site. Though no statistically significant difference (P > 0.05) was found in the isolation of the both species from any particular site. However, more isolates of the pathogens were obtained from Eucalyptus trees located near the river Narmada of Jabalpur City (Table 3). For C. neoformans var. grubii, bark of Eucalyptus trees was found to be the best source followed by flower, bud, fruit
Contrary to the report of Randhawa et al. [17] seasonal variation in the prevalence of C. neoformans var. grubii and C. gattii from decayed wood inside trunk hollows of diverse tree species from Jabalpur city of Madhya Pradesh, Central India was highlighted first time by Grover et al. [6] In the present study, out of 1000 samples studied from different tree parts of Eucalyptus trees investigated, 3.2% yielded C. neoformans var. grubii and 2.8% yielded C. gattii all through the seasons in a year. However, overall prevalence of C. neoformans var. grubii over C. gattii was not statistically significant. On the contrary, Randhawa et al. [17] reported much higher prevalence of C. neoformans var. grubii (17.2%) over that of C. gattii (11.9%) which was statistically significant in decayed wood inside trunk hollows of diverse tree species excluding Eucalyptus trees. Similarly Nawange et al. [15] had reported the prevalence of C. neoformans var. grubii (5.56%) and C. gattii (9.26%) from decayed wood inside trunk hollow of diverse tree species which is found to be higher than present study from the same area but no statistical significance was mentioned in paper. It suggests that decayed wood inside trunk hollows of living trees serve as a good natural habitat for C. neoformans species complex in the environment because the pathogens inside woody debris in trunk hollows is well protected from lethal effects of sunlight and drying. Moreover, these pathogens can utilize the woody material rich in polyphenol compounds and lignin as substrate for growth due to its phenoloxidase activity [20]. On the contrary, these pathogens
344
N.G. Bedi et al.
Table 2 Distribution and population density of C. neoformans var. grubii and C. gattii from various parts of E. terreticornis (hybrid) and E. camaldulensis trees located at different parts of Jabalpur city of Madhya Pradesh, Central India. ´ de population de C. neoformans var. grubii et C. gattii dans diffe ´ rentes parties de E. terreticornis (hybride) Distribution et densite ´ s dans diffe ´ rents endroits de la ville de Jabalpur, Madhya Pradesh, Inde Centrale. et d’E. camaldulensis situe Tree species
Tree Type of Site for number samples isolation
Sample Positive sampling positive dates
Observation Variety/ CFU/g span (days) Serotype (lowest and highest range)
E. terreticornis
E15
Bark
Polypathar
4
Cnvg/A
Bark
Hattital colony
4
490
Cnvg/A
E4
Flower
Polypathar
4
640
C.g/B
E216
Flower
Civic centre
3
360
C.g/B
E178
Buds
3
503
C.g/B
E150
Buds
Medical college Road Vehicle factory
3
410
Cnvg/A
E28
Fruit
Polypathar
4
545
C.g/B
E28
Debris
Polypathar
4
2.5 104— 2.9 10 4 2.4 104— 2.6 10 4 0.7 104— 0.8 10 4 1.4 104— 1.8 10 4 2.0 104— 2.5 10 4 2.1 104— 2.2 10 5 0.7 104— 0.8 10 4 0.7 104— 1.1 10 4
450
E114
6 Mar., 10 June 2004; 20 Apr., 15 June 2005 6 Mar., 9 Apr. 2004; 16 June, 1st Nov. 2005 6 Mar., 10 June 2004; 15 June, 6 Dec. 2005 6 Dec. 2004; 23 Mar., 25th May 2005 6 Mar., 25 Sept., 26 Oct. 2004 26 Oct, 16 Nov. 2004; 21 Dec. 2005 21 Apr., 10 June 2004; 15 June, 22 Oct. 2005 10 June, 19 Sept. 2004; 15 June, 18 Oct. 2005
495
C.g/B
E. Camaldulensis E75
Bark
RDVV campus
4
C.g/B
Bark
RDVV campus
4
1.2 104— 1.1 10 5 1.4 104— 1.5 10 5
611
E80
503
Cnvg/A
E137
Flower
Bargi hills
4
Cnvg/A
Flower
Bargi hills
3
385
C.g/B
E165
Flower
3
503
C.g/B
E248
Flower
465
Cnvg/A
E30
Fruit
Medical college campus Boys hostel Krishinagar Adhartal Polypathar
1.6 104— 1.8 10 4 2.2 104— 2.0 10 5 1.2 105— 2 10 5 1 104— 1.2 10 4
446
E147
15 Feb, 9 Apr. 2004; 16 June, 18 Oct. 2005 6 Mar., 9 June, 16 June 2004; 15 Apr. 2005 30 Jan., 25 May 2005; 5 Mar., 22 Apr. 2004 30 Jan. 2005; 5 Mar., 26 Oct. 2004 14 Oct., 6 Dec. 2004; 21 Feb. 2005 14 Oct., 14 Dec. 2004; 16 Aug. 2005
Cnvg/A
Debris
Behraghat thane 3
1.1 104— 1.3 10 4 1.0 104— 1.0 10 5
559
E119
10 Apr., 28 June 2004; 24 Sep., 21 Oct. 2005 28 Oct. 2004; 23 Mar., 16 June 2005
231
Cnvg/A
3
4
Cnvg/A denotes Cryptococcus neoformans var. grubii. C.g./B denotes Cryptococcus gattii.
inhabiting different parts of Eucalyptus trees bark, flower, bud, leaves are exposed to much more hostile environment in terms of sunlight, temperature, wind, relative humidity, substrate which might be inducing and inhibitory effect on their growth as evidenced by the low percentage of prevalence of these pathogens in Eucalyptus trees [1]. The result of the present study has revealed that tree parts of E. camaldulensis and E. terreticornis serves as natural habitat for C. neoformans var. grubii and C. gattii. For C. neoformans var. grubii bark was the best natural habitat and for C. gattii flowers were the best ecological niche. Both pathogens were also isolated from fruits and buds of these Eucalyptus trees. To our knowledge,
this is the first report of the isolation of C. neoformans var. grubii and C. gattii from Eucalyptus trees at Jabalpur, Central India. Montenergo and Paula [14] showed a close association between the flowering season from November to February of Eucalyptus tree and occurrence of C. gattii because they could not isolate the pathogens from Eucalyptus trees beyond March. Montagna et al. [13] on the contrary made three isolations of the pathogen during the month of June from E. camaldulensis. Similarly, in the present study we could isolate C. gattii from various samples of Eucalyptus trees during its non-flowering season. We therefore, support the hypothesis that there is non-flowering seasonal
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii
345
Table 3 Distribution of Cryptococcus neoformans species complex in positive samples from trees located at three test sites in the different parts of Jabalpur city of Madhya Pradesh, Central India. ` ces de C. neoformans dans les ´echantillons positifs des Eucalyptus situe ´ s dans trios endroits diffe ´ rents Distribution des deux espe de la ville de Jabalpur, Madhya Pradesh, Inde Centrale. Location of positive sites
Total number of samples
Number of C.n. var. grubii/A
Percentage (%)
Number of C. gattii/B
Percentage (%)
River site City area Outskirts
220 453 327
7 12 10
3.18 2.6 3.05
3 15 10
1.36 3.3 3.05
Table 4 Isolation frequency of C. neoformans var. grubii and C. gattii in different parts of Eucalyptus tree. ´ quence d’isolement de C. neoformans var. grubii et de C. gattii au niveau de diffe ´ rents sites sur les Eucalyptus. Fre Type of Samples
Total number of samples
Number of C.n var. grubii positive
Frequency and percentage (%)
Number of C. gattii positive
Frequency and percentage (%)
Bark Flower Fruit Buds Leaves Debris
240 225 150 100 110 175
12 10 4 3 0 3
5 4.4 2.7 3 0 1.71
4 13 4 3 0 4
1.6 5.7 2.7 3 0 2.28
dispersion as well of the pathogen in Jabalpur city. In the present study though, both pathogens were isolated from Eucalyptus trees through all the seasons. However, no significant difference was found in their distribution and isolation frequency (P > 0.05). For C. neoformans the highest isolation frequency was seen in spring followed by autumn, summer, winter and least in rainy season. Significant difference was seen in the isolation frequency of C. neoformans and C. gattii during autumn and rainy season (P < 0.001) and spring and rainy season (P < 0.001) respectively. Colom et al. [2] also reported low rate of isolation of each pathogenic Cryptococcus species from Eucalyptus trees. This is also in accordance with previous finding from Mediterranean area [7,8,18]. The present study revealed that the prevalence of C. neoformans var. grubii was higher in spring (5.2%) than during autumn (4.11%) and summer (4%). On the contrary, prevalence of C. gattii was higher in summer (4.5%) than during winter (4.11%). This difference was however not statistically significant. Randhawa et al. [17] reported low prevalence of C. gattii in decayed wood during winter
season. We also found low prevalence of C. neoformans var. grubii and C. gattii during winter months. Grandos and Casteneda [4] did not find any C. gattii in winter in the decayed wood or tree trunk hollows. Moreover, they also found low population density of the pathogens in the bark of the trees investigated during winter. However, their population was higher during rainy season. Later, Granados and Castaneda [5] analyzed the relationship between the occurrence of various serotypes of C. neoformans species complex in tree samples and climatic conditions recorded during sampling in four cities of Colombia between 1992 and 2004. Their investigations using logistic regression and lagged Pearson correlation indicated that environmental conditions mainly humidity, temperature and solar radiation variably affected the occurrence of serotypes A, B and C. It was also noted that serotypes A was more thermotolerant and hygrophobic than serotype B. Our data on the prevalence of C. neoformans var. grubii and C. gattii during all seasons provide further support for their hypothesis. Grover et al. [6] also reported highest isolation frequency of C. neoformans var. grubii in spring in Jabalpur, when minimum and
Table 5 Seasonal variation in isolation frequency of Cryptococcus neoformans var. grubii and C. gattii in various samples of Eucalyptus spp. ` re de la fre ´ quence d’isolement de Cryptococcus neoformans var. grubii et C. gattii dans les diffe ´ rents Variation saisonnie ´echantillons d’Eucalyptus. Seasons
Number of samples tested
Number of positive C.n. var. grubii
Isolation of frequency Percentage (%)
Number of positive C. gattii
Isolation of frequency Percentage (%)
Autumn Winter Spring Summer Rainy
170 200 170 200 260
7 2 9 9 2
4.11 1.5 5.2 4 0.77
7 6 5 9 1
4.11 2.5 3.5 4.5 0.38
346 maximum temperature varied between 15 8C and 33 8C and less rainfall were observed and this was found to be the favorable condition for the growth of pathogenic yeast. Grover et al. [6], for the first time, reported seasonal variation in the isolation frequency and population density of C. neoformans var. grubii and C. gattii from decaying wood of trunk hollows of living trees at Jabalpur Central India. They reported that isolation frequency of C. neoformans var. grubii was highest in March (Spring) followed by October (Autumn) and June (late summer and early rainy season). On the contrary, isolation frequency of C. gattii was highest in June followed by October (autumn) and March (spring). Randhawa et al. [17] from Delhi, India reported that both the pathogens exhibited seasonal variation in their prevalence with the highest variation, found during the autumn followed by that in summer. In the present study we also report the seasonal variation in the isolation frequency of C. neoformans var. grubii and C. gattii from various parts of Eucalyptus tree. Isolation frequency of C. neoformans var. grubii was highest in spring followed by autumn and summer. Similarly, highest isolation frequency of C. gattii was seen in summer followed by autumn and spring. In rainy season the isolation frequency of the pathogen was low. Interestingly, similar results of a low frequency of isolation during the rainy season as against a highest prevalence of C. neoformans var. grubii during rainy season have been reported in an investigation of chicken faeces in Northern Thailand [11]. Kidd et al. [10] in an environmental survey in British Columbia Canada and Pacific Northwest of the USA reported significantly higher concentration of C. gattii in the air during warm, dry and low humidity summer months than during cold wet and high relative humidity months. Interestingly, Grover et al. [6] and Randhawa et al. [17] also found similar observation regarding seasonal prevalence of C. gattii in their study during their wood sampling at Jabalpur and Delhi respectively. In the present study we also found similar pattern of seasonal prevalence of C. gattii in Eucalyptus trees. The present study as well as those of other authors [6,15,16] have shown the presence of C. neoformans var. grubii and C. gattii in the environment of Jabalpur city in all seasons throughout the year. Despite the widespread presence of the pathogens in the environment there is paucity of clinical data related to Cryptococcosis in this region. It would be however interesting to correlate such environmental data with the prevalence of Cryptococcosis in the same region as has been reported by Nawange et al. [16] in Jabalpur. In India we do not have such linked data available. However, such comparisons have been done in USA [21]. In contrast, the USA study found a significantly higher prevalence of Cryptococcosis in the fall and winter month than in spring and summer. We suggest a comprehensive study to determine the potential health impact of seasonal prevalence of C. neoformans. var. grubii and C. gattii in decayed wood and Eucalyptus trees in climatically divergent region of India.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
N.G. Bedi et al.
Acknowledgements The authors wish to thank the Vice-Chancellor R.D. University, for providing laboratory facilities and to The DirectorGeneral, Indian Council of Medical Research (ICMR), New Delhi for awarding Post Doctoral Research Associateship and Government of India, Ministry of Science and Technology, Department of Science and Technology SERC FAST Track Scheme for Young Scientist to one of the author (S.R.N).
References [1] Colom MF, Fras´es S, Ferrer. et al. First case of human cryptococcosis due to Cryptococcus neoformans var. gattii in Spain. J Clin Microbiol 2005;43:3548—50. [2] Colom MF, Hagen F, Gonzalez A, et al. Ceratonia siliqua trees as natural habitat and source of infection by Cryptococcus gattii in the Mediterranean environment. Med Mycol 2012;50:67—73. [3] Ergin C, Ilkit M, Kaftanoglu O. Detection of Cryptococcus neoformans var. grubii in honeybee (Apis mellifera) colonies. Mycoses 2004;47:431—4. [4] Granados DP, Castaneda E. Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources in Bogota, Colombia, and study of ecological conditions in the area. Microb Ecol 2005;49:282—90. [5] Granados DP, Castaneda E. Influence of climatic conditions on the isolation of members of the Cryptococcus neoformans species complex fro trees in Colombia from 1992—2004. FEMS Yeast Res 2006;6:636—44. [6] Grover N, Nawange SR, Naidu J, Singh SM, Sharma A. Ecological niche of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decaying wood of trunk hollows of living trees in Jabalpur City of Central India. Mycopathologia 2007;164: 159—70. [7] Hagen F, Boekhout T. The search for the natural habitat of Cryptococcus gattii. Mycopathologia 2010;170:209—11. [8] Hamasha AMS, Yildiran ST, Gonulum A, Saracli MA, Doganci L. Cryptococcus neoformans varieties from material under the canopies of Eucalyptus trees and pigeon dropping samples from four major cities in Jordan. Mycopathologia 2004;158:195—9. [9] Ikeda R, Shinoda T, Fukazawa Y, Kaufman L. Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol 1982;16:22—9. [10] Kidd SE, Chow Y, Mak S, et al. Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States. Appl Environ Microbiol 2007;73:1433—43. [11] Kuroki M, Phichaichumpon C, Yasuoka A, et al. Environmental isolation of Cryptococcus neoformans from an endemic region of HIV-associated cryptococcosis in Thailand. Yeast 2004;21:809—12. [12] Kwon-Chung KJ, Bennett JE. Cryptococcus and Cryptococcosis. In: Kwon-Chung KJ, Bennett JE, editors. Medical mycology. Philadelphia: Lea and Febiger; 1992. p. 397—446. [13] Montagna MT, Viviani MA, Pulito A. Cryptococcus neoformans var. gattii in Italy. Note II. Environmental investigations related to an autochthonous clinical case in Apulia. Mycol Med 1997;7:93—6. [14] Montenegro H, Paula CR. Environmental isolation of Cryptococcus neoformans var. gattii and C. neoformans var. neoformans in the city of Sao Paulo, Brazil. Med Mycol 2000;38: 385—90. [15] Nawange SR, Shakya K, Naidu J, Singh SM, Jharia N, Garg S. Decayed wood inside trunk hollows of living trees of T. indica S. cumini and M. indica as natural habitat of
Seasonal prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii Cryptococcus neoformans and their serotypes in Jabalpur city of Central India. J Mycol Med 2006;16:63—71. [16] Nawange SR, Singh SM, Naidu J, et al. Serotype distribution of Cryptococcus neoformans and Cryptococcus gattii in patients and in the environment of Jabalpur, a city of Madhya Pradesh India. Asian J Microbiol Biotechnol Environ Sci 2011;13:735—42. [17] Randhawa HS, Kowshik T, Chowdhary A, et al. Seasonal variations in the prevalence of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decayed wood inside trunk hollows of diverse tree species in north-western India: a retrospective study. Med Mycol 2010;49:320—3. [18] Rodriguez TJM, Hermoso de Mendoza M, Alvarado-Ramirez E, Segura-Roca G. Cryptococcosis by Cryptococcus gattii in
[19] [20] [21]
[22]
347
immunocompetent goats in Spain and review of the literature. Acta Sci Ve 2006;34:345—53. Sorrell TC. Cryptococcus neoformans var gattii. Med Mycol 2001;39:155—68. Sorrell TC, Ellis DH. Ecology of Cryptococcus neoformans. Rev Iberoam Micol 1997;14:42—3. Sorvillo F, Beall G, Turner PA, et al. Incidence and factors associated with extra pulmonary cryptococcosis among persons with HIV infection in Los Angeles County. AIDS 1997;11: 673—9. Staib F. Cryptococcus neoformans und Guizotia abyssinica (Syn. G. oleifera D.C.). Z Hyg 1962;148:466—75.