Introduction

Cryptococcus neoformans species complex is the leading cause of fungal attributable mortality in HIV/AIDS [1]. The basidiospores of C. neoformans species complex inhaled from environmental sources such as wood debris and birds’ droppings rarely cause disease in immunocompetent individuals [2]. However, disseminated disease with a predilection for the meninges to cause cryptococcal meningitis (CM) occurs due to abrogation of cell-mediated immunity following depletion of CD4 + T cells (< 100/mL) [3]. CM is usually the first AIDS-defining illness in HIV infection [3]. The annual global burden of CM is estimated at 152,000 incident cases which results in 112, 000 cryptococcal-related of which 70% occur in Sub-Saharan Africa AIDS [1]. CM is so highly fatal that even with ART and antifungal therapy, the 6-month survival remains ≤ 40% in routine care [4,5].

Two closely related opportunistic yeasts; Cryptococcus neoformans and C. gattii species complexes cause CM [6]. The two Cryptococcus species complexes are basidiomycetous encapsulated yeast species which are likely inhaled from the environment However, the two closely related species complexes differ in ecology and geographic distribution [7].

C. neoformans species complex which causes CM in immunocompromised individuals, for example, in patients with cancer, solid organ transplant, and HIV/AIDS is the most prevalent and globally distributed [8,9]. C. neoformans species complex is ubiquitous in the environment, where it is usually associated with avian droppings [10]. The sibling species Cryptococcus gattii species complex, which induces cryptococcal meningitis more often than C. neoformans species complex in immunocompetent patients, has traditionally shown a geographic restriction to tropical and subtropical regions [11,12] and [13–16]. C. gattii species complex is often associated with the red gum trees; Eucalyptus species (Eucalyptus camaldulensis) [17–19]. More than 50 tree species have yielded C. gattii species complex [20], especially, in Egypt and Tunisia [21,22], Cassia tree in Kenya [10] and Almond tree in Tunisia [22]. In Zambia, environmental sampling identified C. neoformans species complex from Zambezi Mopane woodlands and C. gattii species complex primarily recovered from Central Miombo woodlands [23]. Since these fungi are acquired from nature, environmental sampling is key to understanding their ecological niches. However, the source of Cryptococcus in nature has not yet been identified in Uganda. Therefore, we aimed to screen C. neoformans and C. gattii species complexes environmental isolates from Eucalyptus tree barks, Marabou stork bird droppings and domestic chicken droppings in Kampala, Uganda.

Materials and methods

Results

In this study, 150 environmental samples from different sites within Kampala Metropolitan area were analysed for Cryptococcus strains. A positive control for C. neoformans species complex was included; however, we did not have a positive control for C. gattii species complex. The samples included swabs from 24 chicken droppings, 25 Marabou stork droppings and 101 eucalyptus tree bark swabs (Table 1). Of the N = 150 specimens, 4% (6/150) isolates (0 Chicken, 2 Marabou stork and 4 eucalyptus tree barks) were India ink and urease test positive and thus, confirmed as C. neoformans species complex (Tables 2 and 3, Fig 1). However, all the isolates were negative on CGB agar which suggests that no C. gattii species complex was present (Table 3).

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Table 1. Growth of environmental specimens on *SAB agar (n = 150).

https://doi.org/10.1371/journal.pone.0329947.t001

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Table 2. India ink stain of positive culture on SAB agar (n = 150).

https://doi.org/10.1371/journal.pone.0329947.t002

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Table 3. Urease test, blue colonies on CGB agar and species names of India ink positive isolates (n = 6).

https://doi.org/10.1371/journal.pone.0329947.t003

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Fig 1. Kampala Metropolitan area showing where all the 150 samples were collected from.

The GPS locations of Kampala metropolitan areas where samples of eucalyptus tree bark, marabou stork and chicken droppings were collected are shown in the map of Uganda. The inset of Kampala metropolitan areas within the map of Uganda is marked with a square and an arrow used to show the display.

https://doi.org/10.1371/journal.pone.0329947.g001

Discussion

This study reports the environmental screening report for Cryptococcus species in Uganda where 70% of all meningitis cases especially in immunocompromised patients are due to cryptococcal meningitis [2]. We found 4% (150) environmental prevalence of C. neoformans species complex, however, no C. gattii species complex was isolated. This suggests that there was no C. gattii species complex in the sampled specimens in Uganda’s environment. Our finding is in consistence with our previous publication in which we demonstrated C. neoformans species complex infections but no C. gattii species complex infections among Ugandan HIV-infected patients presenting with cryptococcal meningitis [24]. C. neoformans species complex were predominantly identified in marabou stork droppings at 8% (2/25) followed by eucalyptus tree barks; 3.96% (4/101) similar to other environmental studies reported in the nearby Kenya [10]. However, no C. neoformans species complex was isolated from chicken droppings.

C. gattii species complex is traditionally associated with the red gum trees, Eucalyptus species [19,22], and has shown a geographic restriction to tropical and subtropical regions, however, in this study, none was identified from the samples analyzed. Several other studies have reported environmental presence of C. neoformans/C. gattii species complexes globally [11,19,22].

In this study, samples were analyzed phenotypically as described above in the culture and identification section because of the limited resources within the scope of this study. However, recent studies have employed genetic and molecular means in determining genotypic diversity of C. neoformans/C. gattii species complexes [5,18,25] which would give a better accuracy of identification.

This study highlights the isolation of C. neoformans species complex from eucalyptus tree barks and marabou stork droppings. Eucalyptus tree species grow across the whole country in both cooler and hotter areas and are choice for many commercial farmers because it is multipurpose, fast-growing and has a ready market. This followed decimation of natural forests and woodlands across Africa which could no longer keep pace with the required demand for forest cover, energy usage, timber and construction work associated urbanization [26]. Marabou storks are common in urban areas especially around the garbage collection sites and markets due to their vulturous nature of diet. The finding of the urban ecological niches of C. neoformans species complex in this study is in agreement with the previous study which showed that up to 93.5% (n = 200) of the Infectious Diseases Institute’s recruited patients with AIDS-associated cryptococcal meningitis were from Uganda’s central region the capital city; Kampala, is located [24]. Furthermore, the previous clinical study showed that 45% of the patients with cryptococcal meningitis were in the age category 30–39 years old [24], which represents the most physically productive age group that would be involved with urban economic activities such as building using timber from eucalyptus tree. This calls for safety measures when dealing with Marabou stork and farmers/ individuals who work in eucalyptus tree plantations.

However, there could be many more undocumented environmental reservoirs of C. neoformans species complex/C. gattii species close to or within human habitation which might be a significant exposure risk factor for the high prevalence of cryptococcal meningitis especially among the immunocompromised patients in Uganda.

Conclusion

The prevalence of C. neoformans species complex in Uganda’s environment is 4%, predominantly, in marabou stork droppings. However, there seems to be no species belonging to the C. gattii species complex in our pool of samples. We recommend further genetic and molecular studies on a larger number of isolates from increased numbers of different environmental specimens which may unearth the distribution of the of a C. neoformans and C. gattii species complexes in Uganda.

Supporting information

Acknowledgments

We would like to thank the staff and management of Clinical Microbiology laboratory, Department of Medical Microbiology, College of Health Sciences, Makerere University for their support, permission and assistance in samples collection and laboratory work. We thank Luwum Innocent for administrative support.