Report on novel environmental niches for Cryptococcus neoformans and Cryptococcus gattii in Colombia: Tabebuia guayacan and Roystonea regia

Abstract

Knowledge of the environmental distribution of C. neoformans/C. gattii is important in the epidemiology and ecology of the etiological agent, which causes cryptococcosis, a deadly disease worldwide. The aim of this report is to describe the presence of C. neoformans/C. gattii in new environmental niches in Colombia. A total of 837 environmental samples were collected from six different species of trees across four cities; molecular type was determined by PCR fingerprinting and RFLP. Molecular type VNI and VGIII were isolated from different species of trees, resulting in two novel niches for this pathogen: Tabebuia guayacan and Roystonea regia.

Cryptococcus neoformans, Cryptococcus gattii, environment, niche, Colombia

The genus Cryptococcus is ubiquitous and may survive in a variety of environmental niches. Survival can be influenced by macroenvironmental factors such as humidity, temperature, sunshine, and wind; these conditions play an important role in the propagation and survival of the fungus in the environment.^1,2 Cryptococcosis is an opportunistic infection ^3,4 caused by two species. The first, Cryptococcus neoformans, has a global distribution and comprises the variety grubii (serotype A, molecular types VNI–VNII), the variety neoformans (serotype D, molecular type VNIV), recovered mostly from bird droppings and soil contaminated with this material, and a hybrid variety (serotype AD, molecular type VNIII).^2,5 The second, Cryptococcus gattii, is found to be distributed in tropical, subtropical, and template regions,^2,6 formed by serotypes B and C (molecular types VGI-VGIV) and some interspecies hybrids reported between serotypes BD and AB, and has been recovered mostly from plant material. ^1,3,6,7–8

Cryptococcus has been reported in different families and genders of trees worldwide, including Brazil, Argentina, Puerto Rico, and Turkey.^2,9–11 In India, C. neoformans/C. gattii have been isolated from the trees Tamarindus, Mangiferaindica, Acacia nilotica, among others.¹² In Vancouver Island, European countries and North-South America, C. gattii has been recovered from species such as Cedrus spp., Pseudotsugamenziesii, Quercus garryana, Eucaliptus, and Terminalia catappa.^{2,6,8,12–14}

The incidence of cryptococcosis in Colombia has increased because of the human immunodeficiency virus (HIV) pandemic; in the period 2006–2012 the average annual incidence was 3.3 × 10³ cases per inhabitants/year in patients with AIDS and in the period 1997–2005 was 3.0 × 10³ cases per inhabitants/year. In the general population remains at 2.4 × 10⁶ inhabitants/year for the above two periods. Cryptococcus in the environment have been recovered from different species of trees and types of organic material.^1,4,8 Recently, Noguera et al. in 2015 reported the distribution of this yeast in humans and in the environment in the department of Atlántico and reported for the first time the isolation of C. neoformans from Tabebuia rosea trees in the urban area of Barranquilla.¹⁵ The aim of the present study was to report for the first time the presence of C. neoformans/C. gattii in species of trees not previously described in Colombia.

The sampling procedure used in this study was according to Granados et al. in 2006.¹ Samples from different sources were collected (soil, leaves, cortex, fruit, flowers) from six types of trees present in recreational parks, residential zones and urban trails in four cities; trees are determined according to representation in cities (common trees in each city), sampling was not continuous, between October 2012 through December 2014 (Table 1).

Table 1.

Distribution of species of trees positive for Cryptococcus neoformans and Cryptococcus gattii in four cities in Colombia.

Tree	Month/			Positivity/	Temperature	Relative	Precipitation	Sunshine
Species	year	City	Species	Total	(°C)	Humidity (%)	(mms)	(hours)
Roystonea regia	November 2013	Cali (tropical weather)	C. neoformans	1 /2	24.3	72.5	6.1	3.4
Pithecellobium dulce	March 2014			1/85	25.1	74	4	50.4
Acacia amarilla	July 2013	Medellin (template and humid weather)	C. neoformans	3/137	23.6	57.5	18.1	1.9
	September 2013		…	0/40	23.2	62	4.7	141.9
	October 2013		C. neoformans	1/112	22.7	65	5	154.3
	February 2014		…-	0/56	22.7	64	3.8	116.8
	May 2014		…-	0/44	22.7	68.5	9.3	131.5
	June 2014		…	0/55	23.2	64.6	6.9	157.6
	July 2014		…	0/60	23.9	55	0.9	200.6
Tabebuia guayacán	October 2012	Popayan (template and warm weather)	C. neoformans	1/21	19.2	76.6	42	3.3
	January 2013		…	0/6	20.4	73	6.6	170.1
	February 2013		…	0/52	19	81	5.66	116
	May 2013		…	0/7	19.9	72	13.5	160.2
Cupressus	August 2013		…	0/16	19.6	75.3	14.2	134.5
	December 2013		C. neoformans	5/12	19.2	78	58	134.5
Pinus sp	July 2013		…	0/3	20.1	67	1.18	134.5
	August 2013		C. neoformans	1/23	19.6	75.3	14.2	134.5
	December 2013			5/15	19.2	78	58	134.5
	February 2014		…	0/22	19.2	81	7.3	138.21
	December 2014	La Cruz (cool weather)	C. gattii	1/69	19	65.9	7.4	2.1

Conventional techniques were used for sample processing and isolate confirmation by phenotypic tests.^1,8,16 DNA was extracted as reported by Casali et al.¹⁷, and molecular type identification was done by polymerase chain reaction (PCR) fingerprinting with the unique primer (GTG)₅ and restriction fragment length polymorphism (RFLP) of the URA5 gene, as reported previously.¹⁸ Molecular types were assigned visually by comparing the patterns of the recovered isolates with the patterns obtained from the standard strains.¹⁸ MacroEnvironmental data (temperature, relative humidity, precipitation, and sunshine) during the study period were obtained from the Institute of Hydrology, Meteorology and Environmental Studies (IDEAM).¹⁹

From a total of 837 samples collected from Cupressus, Acacia amarilla, Phitecellobium dulce, Pinus spp., Roystonea regia, and Tabebuia guayacan in four cities in Colombia between October 2012 and December 2014, Cryptococcus neoformans var. grubii and Cryptococcus gattii molecular type VNI and VGIII, respectively, were isolated; Cryptococcus had been previously found in four of these tree types in other areas of Colombia (Cupressus, A. amarilla, P. dulce, and Pinus), the remaining two types of trees are reported for the first time, R. regia and T. guayacan (Table 1, Fig. 1).

Figure 1.

(A) Species of trees that were positive for Cryptococcus neoformans var. grubii and Cryptococcus gattii. (B) Distribution of Cryptococcus neoformans var. grubii and Cryptococcus gattii in four sampled cities in Colombia. Map was generated using Epi Info ™7 and was adapted via Bing Maps to reflect specific sampling sites.

A total of 19 samples harbored the fungus for a positivity of 2.3%, the cortex being the type of material where most of the isolates were recovered in 52.6% of the samples (n = 10), followed by soil, 26.3% (n = 5) and leaves, 21.1% (n = 4) (Fig. 1).

Regarding the climatic conditions in the four sampled cities, the mean temperature was 22.3°C, incidence of sunshine between dawn and late afternoon was an average of 2.5 hours/day, and each city was found to have a moderate intensity of rain and water present in the air (precipitation and relative humidity; Table 1).

The four cities sampled are located in the northern and western parts of the country, characterized by a high density of trees located in urban parks and residential zones; this is relevant since the fungus is the etiological agent of cryptococcosis, an illness initiated by inhalation of infectious propagules present in the environment.^3,4 Granados et al. ¹ reported a retrospective study of environmental isolates of C. neoformans and C. gattii in four cities in Colombia. We report that four of 35 species of trees reported by Granados et al. were also found in this study and are new environmental niches in the following areas of the country: Pinus spp. (La Cruz), Phitecellobium dulce (Cali), Cupressus spp. (Popayán), and Acacia amarilla (Medellín).¹ These species of trees have also been reported as niches in India, Europe and Argentina.^2,12,14,20

Furthermore, the presence of C. neoformans var. grubii molecular type VNI is reported for the first time in Colombia in Tabebuia guayacan (Popayán) and Roystonea regia (Cali).

Recovery of C. neoformans var. grubii has been reported in the genus Tabebuia, and in the different species T. guayacan⁷ and T. avellanedae in Argentina.¹⁴ Additionally, in the Arecaceae family, the fungus has been reported from species of Palmae (species Phoenix)⁷ and is being reported for the first time in Roystonea regia.

The largest frequency of positive samples was in the second semester of the year 2013 (July–December), in the four cities in which macro-environmental data varied; presence of the fungus was related with rainy months in Medellin and Popayán and with dry months in the cities of Cali and La Cruz, as well as with a low solar intensity in the four cities.

This finding correlates with that reported by Granados et al. in which the authors affirm that C. neoformans/C. gattii are present with a higher frequency in the rainy season.¹ Several studies have documented the isolation of the fungus in the dry season, with heavy winds, humidity and low precipitation, in several parts of the world,⁶ this might be explained by the fact that colonization of the fungus in trees varies according to the geographical zone and environmental conditions.

Recovery of the fungus from the reported trees was generally low, similarly to that previously reported in Colombia by Granados et al. in 2006 (2.6%) and in different countries such as Argentina and Puerto Rico.^10,20 This low positivity may be attributable to the fact that the sampling was not continuously done and to environmental factors that may affect the recovery of the fungus.

The finding of C. neoformans/C. gattii in different species of trees, belonging to six families (gymnosperms and angiosperms), reinforces the hypothesis that the fungus does not have a specific association with a host, nor with a specific species of tree.^1,7,14–15 Its association may be favored by specific conditions given by the host and the biological and physical conditions of the environment. In recent years, there has been a noteworthy increase in published reports documenting the presence of the fungus in different families of trees, which suggests that a wide variety of unexplored ecological niches exist. The ongoing search of the environmental distribution is relevant for understanding the epidemiology, ecology, and etiology of this pathogen.

Acknowledgments

Work was supported by the Departamento Administrativo de Ciencia, Tecnologia e Innovación Colciencias for financial support (code: 2011–3600115683) and Instituto Nacional de Salud. Fabiola González, for the collection of environmental samples in Popayán.

Declaration of interest

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