The aetiology and clinical characteristics of cryptococcal infections in Far North Queensland, tropical Australia.

Cryptococcal infections are an important cause of morbidity and mortality in tropical Australia. This retrospective audit was conducted to characterise the aetiology, temporospatial epidemiology, and clinical course of 49 cryptococcal infections in Far North Queensland between 1 January 1999 and 31 December 2019. Cryptococcus gattii was identified in 15/32 (47%) in whom it was possible to speciate the organism. Among these 15 patients, 13 (87%) had a rural residential address, 10 (67%) were Indigenous Australians and 11 (73%) presented during the May-November dry season. When compared to the 17 patients with Cryptococcus neoformans infection, patients with C. gattii were less likely to be immunocompromised (0/15 versus 8/17 (47%), p = 0.003). Neurosurgery was necessary in 5/15 C. gattii cases and 3/17 (18%) C. neoformans cases (p = 0.42). Outcomes were generally good with 42/49 (86%) cases-and 14/15 (93%) with C. gattii infection-surviving to hospital discharge. These positive outcomes are likely to be explained by the development of standardised treatment guidelines during the study period, low rates of comorbidity in the patients with C. gattii infection and access to liposomal amphotericin and neurosurgical support in the well-resourced Australian healthcare system.

Introduction

Cryptococcus gattii and Cryptococcus neoformans are important fungal pathogens which usually affect the central nervous system (CNS) or lungs [1]. However, there are important differences between the two infections. Globally, C. neoformans is a more common cause of disease, predominantly affecting individuals with impaired cell-mediated immunity, particularly those with human immunodeficiency virus (HIV) or transplant recipients [1, 2]. C. gattii, by contrast, commonly presents with large cryptococcomas in patients with no apparent immunocompromise [3]. In the CNS, these cryptococcomas can necessitate urgent neurosurgical intervention and are associated with a poor prognosis [4, 5]. In the lung, they can be mistaken for malignancy, though presentations with pneumonia and respiratory failure have also been reported [1, 6].

C. gattii was responsible for only 15% of the cryptococcal infections in a prospective, community-based Australasian series performed between 1994 and 1997, although this is likely to be reflective of the HIV epidemic at that time [3]. In a more recent study from regional New South Wales, it accounted for 28/107 (26%) cases between 2003 and 2016, while in a study from an urban centre in sub-tropical Queensland between 2001 and 2015, it accounted for 14/97 (14%) infections [7, 8].

In tropical Australia, the proportion of cryptococcal infections caused by C. gattii is higher. In a series from the tropical Northern Territory, C. gattii was responsible for 12/18 (67%) cases of cryptococcal disease between 1993 and 2000 [9]. The authors identified that C. gattii infections occurred more commonly in Aboriginal Australians living in remote locations, a finding that might be explained by their proximity to the Eucalyptus camaldulensis trees that—with E. tereticornis—have long been recognised as an environmental niche for C. gattii in Australia [10, 11]. In this Northern Territory series 13/18 (72%) patients had pulmonary infections, of whom, only 6 (46%) had concurrent CNS disease [9]. The patients had significant morbidity and mortality: 8/18 (44%) required surgery for associated complications, and 4/18 (22%) died [9].

E. camaldulensis and E. tereticornis are abundant in Far North Queensland (FNQ) in tropical Australia (11) (Fig 1). Anecdotally, there is also a significant local burden of cryptococcal infection, however the microbiology, epidemiology, presentation, and clinical course of these infections has been incompletely described. This study aimed to gain a greater understanding of the characteristics and outcomes of cryptococcal infections in tropical Australia during a period which saw enormous progress in HIV care and the development of novel—and better tolerated—antifungal therapies.

Fig 1

The study region.

The map was created using constructed using mapping software (MapInfo version 15.02, Connecticut, USA) using data provided by the State of Queensland (QSpatial). Queensland Place Names—State of Queensland (Department of Natural Resources, Mines and Energy) 2019, available under Creative Commons Attribution 4.0 International licence https://creativecommons.org/licenses/by/4.0/. ‘Coastline and state border–Queensland—State of Queensland (Department of Natural Resources, Mines and Energy) 2019, available under Creative Commons Attribution 4.0 International licence https://creativecommons.org/licenses/by/4.0/.

Methods

Data collection

This retrospective study was performed at Cairns Hospital, a 531-bed tertiary referral hospital that serves a population of approximately 280,000 people who live across an area of 380,748 km2 (Fig 1); 17% of the general local population identify as Indigenous Australians [12]. Patients admitted to Cairns Hospital with a laboratory-confirmed diagnosis of cryptococcal infection between 1 January 1999 and 31 December 2019 were eligible for inclusion into the study. Laboratory confirmed infection was defined as a compatible clinical syndrome with a positive culture for Cryptococcus, a cryptococcal antigen (CrAg) detected on serum or cerebrospinal fluid, or tissue histology consistent with cryptococcosis. The study period was selected as it coincided with the introduction of a statewide electronic pathology system, AUSLAB.

Patients were identified by performing a search in AUSLAB for all laboratory confirmed cases within the study period. Once identified, the patients’ medical records were reviewed and their demographic, clinical, laboratory, and microbiological results were recorded.

Patients were defined as living in a rural/remote, or urban location using the Australian Standard Geographical Classification–Remoteness Areas (ASGC-RA) classification [13]. All individuals receiving care in Queensland’s public health system, are asked whether they identify as an Aboriginal Australian, a Torres Strait Islander Australian, both, or neither. Comorbidity was quantified using the Charlson comorbidity index and determined using the patients’ medical record [14]. If individual comorbidities were not documented, they were presumed to be absent. Patients were defined as immunocompromised if they were receiving any immunosuppressive or immunomodulatory therapy (including any dose of systemic corticosteroids), were living with HIV or had an active malignancy. Hazardous alcohol use was defined as more than 20 grams of alcohol per day as per 2009 Australian guidelines [15]. A cryptococcoma was defined as a lesion (on either lung or brain imaging) ≥ 10mm in size.

Local flowering times of E. tereticornis and E. camaldulensis were determined using data from the Australasian Virtual Herbarium [16]. Australian Bureau of Statistics population data were used to calculate disease incidence [17].

Statistical analysis

Data was de-identified, entered into an electronic database (Microsoft Excel 2016, Microsoft, Redmond, WA, USA) and analysed using statistical software (Stata version 14.2, StataCorp LLC, College Station, TX, USA). Univariate analysis was performed using the Kruskal-Wallis, Chi-squared, Spearman’s rho or Fisher’s exact tests where appropriate. Trends over time with determined using an extension of the Wilcoxon rank-sum test with year as a continuous variable [18].

Ethics approval

The FNQ Human Research Ethics Committee provided ethical approval for the study (HREC/16/QCH/110–1083 LR). As the data were retrospective and de-identified, the Committee waived the requirement for informed consent.

Results

A total of 49 patients were identified and included in the analysis (S1 Fig). The diagnosis of cryptococcal infection was made by culture in 19/49 (39%), serum CrAg in 18/49 (37%), CSF CrAg in 6/49 (12.2%) and histologically in 6/49 (including 1 at autopsy). Speciation was possible in 32/49 (65%): 17/32 (53%) were C. neoformans and 15/32 (47%) were C. gattii.

The median (interquartile range (IQR)) age of patients in the cohort was 48 (37–58) years; 30 (61%) were male; 16 (33%) were immunocompromised. Patients with C. neoformans infection were more likely to be immunosuppressed (47% vs 0%, p = 0.003) while patients with C. gattii were more likely to be smokers (73% vs 24%, p = 0.01). Differences in the other characteristics of the C. neoformans and C. gattii cases are presented in Table 1.

Table 1

Comparison of selected demographic and clinical characteristics of the patients with confirmed C. gattii and C. neoformans infection.

	Unspeciated n = 17	C. gattii n = 15	C. neoformans n = 17	P a
Age	52 (37–71)	45 (18–82)	47 (38–54)	0.64
Male gender	13 (76%)	8 (53%)	9 (53%)	1.0
Indigenous Australian	9 (53%)	10 (67%)	8 (47%)	0.31
Rural residential address b	6 (38%)	13 (87%)	9/13 (69%)	0.37
Charlson Comorbidity Index = 0	2 (12%)	8 (53%)	5 (29%)	0.28
Charlson Comorbidity Index	0 (0–0)	0 (0–4)	3 (0–6)	0.13
Known immunosuppression	8 (47%)	0 (0%)	8 (47%)	0.003
HIV infection	2 (12%)	0 (0%)	3 (18%)	0.23
Solid organ transplant recipient	1 (6%)	0 (0%)	2 (12%)	0.49
Renal replacement therapy	1 (6%)	1 (7%)	1 (6%)	1.0
Hazardous alcohol use (past or current)	5 (29%)	7 (47%)	6 (35%)	0.72
Smoker (past or current)	8 (47%)	11 (73%)	4 (24%)	0.01

All data are presented as n (%) or median (interquartile range)

a For comparison between patients with confirmed C. neoformans and C. gattii

b 4 patients with confirmed C. neoformans and 1 patient with a non-speciated infection were not local residents of FNQ

HIV: Human immunodeficiency virus

Indigenous Australians

Overall, 27/49 (55%) identified as either an Aboriginal or a Torres Strait Islander Australian compared to 49241/287107 (17.2%) of the general FNQ population at the end of the study period (p<0.0001). Indigenous patients were less likely to be immunocompromised than non-Indigenous patients (4/27 (15%) versus 12/22 (55%), p = 0.005); 10/15 (67%) C. gattii cases and 8/17 (47%) C. neoformans cases occurred in Indigenous Australians (p = 0.31). There was no difference between Indigenous and non-Indigenous Australians in age, gender or residence in a remote location. However, Indigenous Australians were more likely to smoke tobacco (17/27 (63%) versus 6/22 (27%), p = 0.02) or have hazardous alcohol use (16/27 (59%) versus 2/22 (9%), p<0.0001). There was no difference in clinical course and outcome (Table 2).

Table 2

Comparison of selected demographic and clinical characteristics of the patients.

	Indigenous Australian n = 27	non-Indigenous Australian n = 22	p
Age (years)	47 (33–53)	49 (39–71)	0.29
Male gender	17 (63%)	13 (59%)	0.78
Rural/remote residence a	20 (74%)	8/17 (47%)	0.11
C. gattii b	10/18 (56%)	5/14 (36%)	0.31
Brain involvement	19 (70%)	14 (64%)	0.76
Lung involvement	23 (85%)	10 (45%)	0.005
HIV/AIDS	1 (4%)	3 (14%)	0.31
Solid organ transplant	0	3 (14%)	0.08
Immunocompromised	4 (15%)	12 (55%)	0.005
Charlson Comorbidity Index	2 (0–4)	3 (0–5)	0.34
Underlying chronic lung disease	4 (15%)	4 (18%)	1.0
Diabetes mellitus	8 (30%)	3 (14%)	0.30
Receiving dialysis	2 (7%)	1 (5%)	1.0
Smoker (past or current)	17 (63%)	6 (27%)	0.02
Hazardous alcohol use (past or current)	16 (59%)	2 (9%)	<0.0001
Lumbar puncture performed	21 (78%)	19 (86%)	0.49
Lumbar puncture opening pressure c	19 (15–33)	34 (17–38)	0.16
Induction Liposomal Amphotericin d	11/19 (58%)	10/17 (59%)	1.0
Neurosurgery required	5 (19%)	3 (14%)	0.72
Intensive Care Unit admission	2 (7%)	5 (23%)	0.22
Died	5 (19%)	2 (9%)	0.44

a There were 5 individuals who were not local residents

b Only includes the 32 cases in which a species was identified

c Only in the patients with CNS involvement

d Of the 36 patients who had liposomal amphotericin

Temporospatial epidemiology

The absolute number of cases of cryptococcal infection that were diagnosed increased over the study period (p for trend = 0.03), although there was no change in the annual incidence per 1,000,000 population (p for trend = 0.89) (Fig 2). In the final year of the study period, the 4 cases of cryptococcal disease—all C. gattii—represent an annual local incidence of 14.3 cases per 1,000,000 population. However, in this small sample there was no discernible change in proportion of C. gattii cases over time; the mean (95% confidence interval) annual incidence of C. gattii over the entire study period was 2.8 (1.1–4.5) per 1,000,000 population. C. gattii cases were observed most commonly in July, which coincides with the peak local flowering of E. camaldulensis (Fig 3) (16).

Fig 2

The proportion of Cryptococcal infections caused by C. gattii during the study period.

Fig 3

Seasonal variation in the presentation of C. gattii cases related to the flowering times of E. tereticornis and E. camaldulensis.

Among the 44 local residents, 27 (61%) lived in a rural/remote location, compared to 10.4% of the local general population at the end of the study period (p<0.0001). Among the remaining 5 cases, 2 were resident in Papua New Guinea, 2 lived in other states in temperate Australia and 1 was a South African resident. All 15 C. gattii cases occurred in local residents, 13/15 (87%) of C. gattii cases occurred in patients living in a rural or remote area, compared with 9/13 (69%) of C. neoformans cases (p = 0.20), however there was no clear temporospatial clustering (Fig 4).

Fig 4

Temporospatial distribution of cases.

These maps were created using constructed using mapping software (MapInfo version 15.02, Connecticut, USA) using data provided by the State of Queensland (QSpatial). Queensland Place Names—State of Queensland (Department of Natural Resources, Mines and Energy) 2019, available under Creative Commons Attribution 4.0 International licence https://creativecommons.org/licenses/by/4.0/. ‘Coastline and state border–Queensland—State of Queensland (Department of Natural Resources, Mines and Energy) 2019, available under Creative Commons Attribution 4.0 International licence https://creativecommons.org/licenses/by/4.0/.

Site of infection

18/49 (36.7%) patients had concurrent pulmonary and central nervous system CNS infection (S2 Fig). Current or past smoking was more common in patients with lung involvement than without lung involvement (19/33 (58%) versus 4/16 (25%), p = 0.04). Among patients with confirmed C. gattii infection, 4/15 (27%) had an isolated CNS presentation, 2/15 (13%) had an isolated respiratory presentation, while in 9/15 (60%) concurrent CNS and lung involvement was confirmed. Patients with C. neoformans infections presented with isolated CNS presentation in 10/17 (59%), isolated respiratory infection in 1/17 (6%), isolated skin infection in 1/17 (6%) and concurrent CNS and lung infections in 5/17 (29%) (S3 Table).

Imaging

The small number of cases in which speciation was possible precluded meaningful comparison of the imaging findings in the C. gattii and C. neoformans cases, however, the lung imaging findings were similar. It was possible to review the brain imaging in 24/33 cases of the patients with CNS involvement; 4/24 (16.7%) had cryptococcomas; all three of the cases with CNS cryptococcomas in which speciation was possible had C. gattii isolated. No patient had hydrocephalus at presentation (Fig 5, S2 Table).

Fig 5

Imaging findings in patients with C. gattii infection.

Laboratory investigations

There was no significant difference in blood results between the C. neoformans and C. gattii cases apart from a lower lymphocyte count in the C. neoformans group (0.8 (0.5–1.3) x 106/L versus 1.4 (0.9–2.3), p = 0.04) (S4 Table), likely reflecting higher rates of immunocompromise in these patients. A lumbar puncture (LP) was performed in 40/49 (82%); 3 patients died before an LP could be performed, 1 declined the investigation, 1 was transferred to another hospital, in the remaining 4, the attending clinicians elected not to perform the procedure. The median (IQR) opening pressure was 27 (16–35) cm H2O in 10 confirmed C. gattii infections compared to 32 (16–35) cm H2O in 11 confirmed C. neoformans cases (p = 0.70) (S5 Table).

Antimicrobial susceptibility

The sensitivities of the isolates are presented in Fig 6 and Table 3. The minimum inhibitory concentrations (MICs) of both organisms to the induction antifungals, amphotericin and 5-flucytosine, were comparable. The median (IQR) MIC to fluconazole was 8 (4–16) μg/mL in C. gattii isolates compared to 4 (2–8) μg/mL in C. neoformans isolates (p = 0.17).

Fig 6

Minimum Inhibitory Concentration (MIC) of C. gattii and C. neoformans isolates to commonly used antifungal agents.

Table 3

Minimum Inhibitory Concentration (MIC) of C. gattii and C. neoformans isolates to commonly used antifungal agents.

	5-Flucytosine	Amphotericin	Fluconazole	Itraconazole	Ketoconazole	Voriconazole	Posaconazole
C. gattii	2 (0.5–8)	0.5 (0.06–2)	8 (1–64)	0.09 (0.015–0.5)	0.09 (0.016–0.125)	0.06 (0.015–0.25)	0.12 (0.03–0.5)
Number of C. gattii isolates	13	13	13	12	6	10	7
C. neoformans	2 (0.5–8)	0.5 (0.12–1)	4 (1–16)	0.125 (0.015–0.25)	0.06 (0.016–0.25)	0.03 (0.015–0.125)	0.12 (0.03–0.5)
Number of C. neoformans isolates	16	16	16	14	14	14	11

All concentrations are in μg/mL

Antifungal therapy

45/49 patients received anti-fungal therapy; 4 (8%) died before treatment could be initiated. All 45 patients received either amphotericin B or liposomal amphotericin induction therapy. One patient with C. gattii and one patient with C. neoformans received monotherapy, but all others received adjunctive 5-flucytosine. Anti-fungal therapy doses were similar in both species: 13/15 C. gattii patients received induction therapy with a median (IQR) daily dose of 3.5 (2.0–4.7) mg/kg liposomal amphotericin or 0.7 (0.6–1.0) mg/kg conventional amphotericin;15/17 C. neoformans patients received a median (IQR) daily dose of 3.5 (3.2–3.8) mg/kg liposomal amphotericin or 0.7 (0.6–0.9) mg/kg conventional amphotericin. The median (IQR) induction period in the patients who survived to the end of their induction was 4 (2–6) weeks in cases of C. gattii compared to 3 (2–6) weeks in cases of C. neoformans (p = 0.72). Consolidation therapy was fluconazole with a median dose of 400 (200–800) mg/day in both groups (p = 0.54).

There was no correlation between fluconazole MIC and fluconazole dose prescribed to either C. gatii (rs = 0.04, p = 0.90) or C. neoformans (rs = 0.10, p = 0.73) infections (S6 Table). Despite this, C. gattii patients had good outcomes. There was only one death in this group which occurred in a patient whose isolate had a fluconazole MIC was 16 μg/mL. This patient died four months after his initial diagnosis, after 8 weeks of induction amphotericin B and 5-flucytosine and following multiple admissions—and neurosurgery—for increased intracranial pressure while receiving a consolidation dose of 800mg fluconazole daily (S7 Table, Fig 7).

Fig 7

Treatment course and outcomes of the patients included in the study.

Clinical course

There were 7/49 (14%) who were admitted to the intensive care unit (ICU) for neurological sequelae. Neurosurgical intervention was necessary in 8/33 (24.2%) cases with CNS involvement; these 8 cases included 5/13 (39%) C. gattii and 3/15 (20%) C. neoformans cases (p = 0.41). Neurosurgical procedures included lumbar and extraventricular drains and ventriculoperitoneal shunting. Steroids were prescribed in 11/33 (33%) of CNS cases; 2/13 (17%) C. gattii and 7/15 (47%) C. neoformans cases (p = 0.11).

Of the 49 patients, 42 (86%) survived to hospital discharge. Twelve months after discharge, 13/42 (31%) had been lost to follow up, 17/42 (40%) were asymptomatic or improving, 6/42 (14%) had been readmitted with relapse, 4/42 (10%) had been hospitalised with an unrelated problem and 1/42 (2%) had died from the underlying lymphoma that had predisposed them to infection (Fig 7).

In this small sample there was no statistically significant association between age, Indigenous status, comorbidity, and in-hospital death. The median (IQR) age of patients that died was 48 (47–76) versus 48 (34–57) in survivors (p = 0.21); 5/27 (19%) Indigenous patients died compared with 2/22 (9%) non-Indigenous patients (p = 0.44); the median (IQR) Charlson Comorbidity index among patients that died was 2 (1–4) versus 2 (0–5) among survivors (p = 0.87); 3/16 (19%) of immunocompromised patients died versus 4/33 (12%) immunocompetent patients (p = 0.67). There was no difference in the case fatality rate of patients with and without confirmed CNS involvement (5/33 (15%) versus 2/16 (13%), p = 1.0).

It was notable that among the 33 patients who received amphotericin induction therapy, there were no deaths among the 22 patients who received liposomal amphotericin in their induction regimen (21 in combination with 5FC and one as monotherapy), whereas 2/11 (18%) who received conventional amphotericin B died (p = 0.04).

Discussion

C. neoformans is the predominant cause of cryptococcal infections globally, however in tropical Australia a significant proportion are due to C. gattii; almost half of the speciated cryptococcal infections in this series were caused by this organism.

Australian studies from other locations have described a significantly lower prevalence of C. gattii infections, in keeping with their predominantly urban population [7, 8, 19]., although the Northern Territory of Australia is a notable exception [3, 9, 20]. The associations with C. gattii infection described in the Northern Territory—Indigenous status and rural residence—were also seen in this FNQ cohort: two-thirds of the patients with C. gattii in FNQ identified as an Indigenous Australian, despite their representing only 17% of the local general population [17]. Meanwhile, almost 90% of patients with C. gattii in FNQ lived in rural regions compared with only 46% of the local general population.

Patients with C. gattii are frequently immunocompetent with no apparent risk factor for the disease [21]. In this series, every patient with confirmed C. gattii infection was immunocompetent, and half had a Charlson Comorbidity Index of zero. However, C. gattii infections were more common in current or former smokers while almost half had a history of hazardous alcohol use [22-25]. Both of these risk factors—markers of greater socioeconomic disadvantage—were more common in the Indigenous patients in this series and might be responsible for their over-representation in this cohort. Indeed, smoking and hazardous alcohol make a significant contribution to the disproportionate burden of communicable and non-communicable diseases in the local Indigenous population and are an obvious target for more aggressive preventative health strategies in the region [26, 27].

Almost 90% of the C. gattii infections in this cohort had CNS involvement, a similar finding to that seen in Northern Territory data where 56% of C. gattii cases also had CNS involvement [9]. In contrast, a large Canadian series reported that CNS involvement was present in less than 20% of their C. gattii cases [28]. Geographic differences in the predominant genotype may account for this: in Australia, the main genotype is VGI, whereas in Canada, it is VGIIa. However, the pathogenesis of C. gattii infections is incompletely defined with the role of the organism’s genotype and virulence factors—and the host’s genetics and immunity—yet to be fully elucidated [21, 28, 29].

Eucalypt trees act as an environmental niche for C. gattii in Australia and this is likely to account for the higher proportion of cases in residents of rural regions [10, 30]. E. camaldulensis and E. tereticornis flower primarily from May to September in FNQ which coincides with the July peak in C. gattii infections seen in this study (Fig 3) [16]. Previous air-sampling experiments in temperate South Australia identified airborne propagules of C. gattii under the canopy of an E. camaldulensis tree in flower, while all other air-sampling experiments—including those conducted under E. camaldulensis trees not in flower, failed to detect C. gattii—suggesting that dispersal of infectious propagules at the time of flowering might represent one mechanism by which C. gattii can infect the host [10, 31]. While hypothesis-generating and worthy of further study, it is difficult to ascribe causation to this observation given the uncertainty over the infection’s incubation period [8, 32, 33]. Furthermore, in British Columbia, while C. gattii was identified in air samples more commonly during the warm, dry summer months, there was no association between the flowering or pollination times of individual tree species and the detected concentrations of airborne C. gattii [34]. It is also possible that other factors which impact on the incidence of infectious diseases in FNQ—including urban expansion, the impact of the local monsoonal wet season on vegetation growth and human activity—play a role in the temporospatial epidemiology of C. gattii in the region [35-40].

Antimicrobial resistance profiles of the C. gattii isolates in this study were similar to those reported in the literature; C. gattii were reliably sensitive to 5-flucytosine and amphotericin at a comparable minimum inhibitory concentration (MIC) to C. neoformans, while the median MIC to fluconazole was twice as high [41]. However, despite this, the dose of fluconazole prescribed by clinicians for consolidation therapy of both C. gattii and C. neoformans was similar.

Outcomes for patients with C. gattii were excellent with only a single death among the C. gattii cases (overall case fatality rate of 6.7%). In the Northern Territory series, which examined cases between 1993 and 2000, the case-fatality rate was 22%. Advances in local healthcare since the turn of the millennium including improved access to healthcare and enhanced aeromedical retrieval may also have contributed to the good outcomes [42-44]. In addition, all our patients were immunocompetent which may explain their lower case fatality rate; in one large Australian series of C. gattii cases, the case fatality rate was 29.2% in immunocompromised patients compared to 4.8% immunocompetent hosts [45]. The greater use of liposomal amphotericin in this series is likely to have reduced adverse effects of therapy [46, 47]. While the efficacy of a lower dose of liposomal amphotericin has been suggested, the dose used in this cohort was within the conventional range [7].

This study was limited by its size and the significant number of infections that could not be speciated; its retrospective nature precluded the collection of comprehensive data in all patients. The patients’ residential address was recorded, and this would not necessarily represent the location where the infection was contracted. The cohort included a heterogeneous group of patients: about a third had disease limited to the lung or skin, a group which traditionally has better outcomes than those with CNS disease [33], although there was no difference in outcome between patients with and without CNS disease in this small study. Some of the patients diagnosed with cryptococcal infection in the Cairns Hospital laboratory were not managed at the hospital and were therefore not included in the analysis. These factors would all tend to increase the likelihood of type 2 statistical errors. It would also tend to under-estimate the local incidence of disease. Nevertheless, it demonstrates the disproportionate burden of C. gattii infection in this region of tropical Australia and provides an insight into the presentation and clinical course of the infection in contemporary Australia. The association between flowering of E. camaldulensis and E. tereticornis and presentation requires further elucidation and is the focus of ongoing work. Future studies focussing on the optimal dose and duration of induction and eradication therapies for C. gattii would be helpful and might inform optimal clinical management strategies for this potentially life-threatening pathogen.

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Consort diagram of the study.

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Site of infection for C.

gattii and C. neoformans infection.

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Imaging findings in patients with pulmonary disease.

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Imaging findings in patients with CNS disease.

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Symptoms of the patients and their vital signs at presentation, stratified by cryptococcal species.

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Laboratory findings of the patients at presentation stratified by cryptococcal species.

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Lumbar puncture findings stratified by cryptococcal species.

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Antifungal doses and duration stratified by cryptococcal species.

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Characteristics and clinical course of the patients with Cryptococcus gattii infection.

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21 Dec 2021

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Reviewer #2: Yes

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Reviewer #1: This paper reports the retrospective experience with 49 patients hospitalized in Northern Queensland with cryptococcosis. The report centers around 32 cases in which the isolate was identified by species: C neoformans or C. gattii. No novel findings were encountered. The higher prevalence of immunosuppressed patients with C. neoformans than C. gattii in Australia was confirmed. Some of the other difference between the two species in other reports were not found, in part due to the small sample size, such as more infections confined to the lung or more mass lesions in the brain among C. gattii cases.

The paper is burdened with masses of data that cannot be interpreted. The speculated connection between the higher prevalence of C. gattii cases in the last half of the year and flowering in Eucalyptus trees is specious. The lower, though normal, lymphocyte counts in the peripheral blood of C. neoformans patients is an example of applying a p value of 0.05 to more than 20 comparisons and finding one value of 0.04. There are larger and more detailed analyses of MRI and CT images, antifungal susceptibility and clinical descriptions of cryptococcosis in Australia than in this report. The long and speculative Discussion in the paper needs drastic shortening. The authors might consider deleting the data on the 17 cases with unspeciated isolates and minimizing speculation. If further analyses can provide insights into how cryptococcosis might differ in indigenous Australians, that might be instructive. Cairns has an experience with that population. Just because no difference was found doesn’t mean there were no differences.

Reviewer #2: In this manuscript, Sim et al. summarized the etiology, epidemiology and clinical course of cryptococcal infections in Far North Queensland between 1999 and 2019. During this period, infections with both Cryptococcus gattii and C. neoformans were identified. Individuals infected with C. gattii were less likely to be immunocompromised compared to patients with C. neoformans. Interestingly, they found that nearly 90% of the C. gattii infections had CNS involvement. Patients with C. gattii infections were more likely to be current or former smoker than patients with C. neoformans. Furthermore, more than 50% of the patients had a history of hazardous alcohol use. These clinical data are interesting; however, the manuscript will definitively benefit from an expanded mechanistic discussion on these clinical observations.

Comments

1) In this manuscript, 90% of the C. gattii infections had CNS involvement. Recent mouse studies showed that the primary target organ of C. gattii is different from that of C. neoformans. C. gattii grows faster in the lung and is primarily linked to pulmonary diseases (Pubmed PMID: 22570277). The authors should expand the discussion on this point.

2) Patients with C. gattii infections more likely to be smoker. It would be nice to have more discussion about that point.

3) It is also interesting that more than 50% of the patients had a history of hazardous alcohol use. The authors should discuss more on the potential mechanisms behind this observation. Actually, recent studies have shown that Liver cirrhosis is an independent risk factor of cryptococcal meningitis (Pubmed PMID: 25747471) and that liver macrophages play a prominent role to filter disseminating Cryptococcal yeast cells (Pubmed PMID: 31594939).

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: Comments PLOS One 2021.docx

Click here for additional data file.

9 Jan 2022

Response to reviewers

We thank the Editorial staff and the reviewers for the time that they have taken to review our manuscript and the very helpful comments that they have made to improve the work. Please find below our point-by-point responses to their comments.

Editorial staff comments

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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Response: We have checked that the manuscript meets the style requirements - including file naming - and feel that it does. We are very happy to address specific concerns if any remain.

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Response: Requirement for informed consent was waived by the Ethics Committee as we highlight in lines 126-128.

“The FNQ Human Research Ethics Committee provided ethical approval for the study (HREC/16/QCH/110 – 1083 LR). As the data were retrospective and de-identified, the Committee waived the requirement for informed consent”.

3. We note that Figures 1 and 4 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

Response: All the maps in the submission were created using constructed using mapping software (MapInfo version 15.02, Connecticut, USA) using data provided by the State of Queensland (QSpatial) which are not copyrighted. Queensland Place Names —State of Queensland (Department of Natural Resources, Mines and Energy) 2019, available under Creative Commons Attribution 4.0 International licence https://creativecommons.org/licenses/by/4.0/ and ‘Coastline and state border–Queensland - State of Queensland (Department of Natural Resources, Mines and Energy) 2019, available under Creative Commons Attribution 4.0 International licence https://creativecommons.org/licenses/by/4.0/

These data - which are freely available in the public domain - are provided by the State Government of Queensland. This is a similar situation to some of the U.S. government sources that were suggested in the decision email.

All the patients were managed in the Queensland State Government’s Public Health system. All the authors contributed to the manuscript when employed by the State Government of Queensland.

We have used these data to create maps in multiple PLoS publications in the past 4 years without any issue.

1. PLoS Negl Trop Dis. 2021 Jun 21;15(6):e0009544. doi: 10.1371/journal.pntd.0009544. eCollection 2021 Jun.

2. PLoS Negl Trop Dis. 2021 Jan 14;15(1):e0008990. doi: 10.1371/journal.pntd.0008990. eCollection 2021 Jan.

3. PLoS One. 2020 Sep 3;15(9):e0238719. doi: 10.1371/journal.pone.0238719. eCollection 2020.

4. PLoS Negl Trop Dis. 2019 Jul 18;13(7):e0007583. doi: 10.1371/journal.pntd.0007583. eCollection 2019 Jul.

5. PLoS Negl Trop Dis. 2019 Feb 13;13(2):e0007205. doi: 10.1371/journal.pntd.0007205. eCollection 2019 Feb.

6. PLoS Negl Trop Dis. 2017 Mar 6;11(3):e0005411. doi: 10.1371/journal.pntd.0005411. eCollection 2017

We have added a footnote to Figures 1 and 4 - specifically addressing this copyright issue - to make this clearer.

4. Please include a copy of Table 2 which you refer to in your text on page 10.

Response: We thank the Editorial staff for highlighting this typographical error in which we had misnumbered the table in the original submission. This table is table 3 in the revised manuscript (page 13).

Reviewer comments

Reviewer #1: This paper reports the retrospective experience with 49 patients hospitalized in Northern Queensland with cryptococcosis. The report centers around 32 cases in which the isolate was identified by species: C neoformans or C. gattii. No novel findings were encountered. The higher prevalence of immunosuppressed patients with C. neoformans than C. gattii in Australia was confirmed. Some of the other difference between the two species in other reports were not found, in part due to the small sample size, such as more infections confined to the lung or more mass lesions in the brain among C. gattii cases.

The paper is burdened with masses of data that cannot be interpreted. The speculated connection between the higher prevalence of C. gattii cases in the last half of the year and flowering in Eucalyptus trees is specious. The lower, though normal, lymphocyte counts in the peripheral blood of C. neoformans patients is an example of applying a p value of 0.05 to more than 20 comparisons and finding one value of 0.04. There are larger and more detailed analyses of MRI and CT images, antifungal susceptibility and clinical descriptions of cryptococcosis in Australia than in this report. The long and speculative Discussion in the paper needs drastic shortening. The authors might consider deleting the data on the 17 cases with unspeciated isolates and minimizing speculation. If further analyses can provide insights into how cryptococcosis might differ in indigenous Australians, that might be instructive. Cairns has an experience with that population. Just because no difference was found doesn’t mean there were no differences.

Response: We thank the reviewer for their constructive comments. We agree that the discussion was too long and have shortened it significantly, by almost 30% (1217 to 864 words), as suggested.

We also agree with their comments about the limitations of the study which are largely a product of its sample size. Indeed, we highlight this specifically in lines 336-337. The risk of type 2 errors that we highlight in the limitations section (line 340) states precisely what the reviewer is suggesting. “Just because no difference was found doesn’t mean there were no differences” is the definition of a type 2 error.

Certainly, while there are other Australian analyses of larger size, this is the largest ever analysis in a tropical Australian location. The only comparable study - contained only 18 patients, only 12 of which were C. gattii - from the Northern Territory (Jenney et al. 2004, reference 7), was published over 15 years ago and examined patients managed between 1993 and 2000. The management of critical illness and cryptococcal disease has evolved since then. The significant difference in case-fatality rate between the two series highlights this and provides updated and contemporary data for the reader.

The fact that Indigenous Australians were over-represented in our cohort is also significant, and the reviewer’s comment regarding further analysis in this population is insightful. Further analyses have been presented as suggested (lines 147-156, lines 300-305 and table 2).

The reviewer suggests that the paper is “burdened with masses of data” but it is important to note that most of this data is presented as supplementary material (7 supplementary tables, 2 supplementary figures and the dataset) which we present for the interested reader. There are only 3 tables of data in the revised submission which includes an additional table examining the burden of disease in Indigenous Australians as suggested by the reviewer.

The reviewer is correct to note that for every 20 analyses of a normal population, 1 - on average - will, by chance, have a p value of <0.05. However, we disagree with the reviewer when they state that the “lower, though normal, lymphocyte counts in the peripheral blood of C. neoformans patients is an example of applying a p value of 0.05 to more than 20 comparisons and finding one value of 0.04.” The median (IQR) lymphocyte count in patients with C. neoformans was 0.8 (0.5-1.3) x 106/L. The median lymphocyte count is below the normal reference range for the lymphocyte count in our laboratory (1.0-4.0 x 106/L), so the majority of patients with C. neoformans had lymphopenia. This is unlikely to be due to chance as 8/17 patients with confirmed C. neoformans had established immunocompromise (including 3 patients with advanced HIV - with lymphocyte counts of 0.1, 0.3 and 0.4 x 106/L respectively - and 2 patients with solid organ transplants with lymphocyte counts of 0.3 and 0.7 x 106/L respectively. This would accord with the knowledge that C. neoformans is a disease of immunodeficiency/immunocompromise and the lymphopenia is a marker of this, rather than a chance finding from multiple analyses.

We acknowledge the reviewer’s caution in suggesting correlation between observed data – it is important not to overstate our findings. Indeed, we specifically highlight this point in lines 344-345. We agree that it is important not to attribute causation between the flowering of the Eucalypt trees and cryptococcosis. Indeed, we are pains to make this point (lines 312-313).

The association between Eucalyptus trees (specifically E. tereticornis and E. camaldulensis) is very well established (Ellis DH, Pfeiffer TJ J Clin Microbiol. 1990;28(7):1642., Sorrell TC, Chen SC, Ruma P, Meyer W, Pfeiffer TJ, Ellis DH, Brownlee AG J Clin Microbiol. 1996;34(5):1253.) so we would respectfully disagree with the reviewer who suggests that the analysis is “specious”. The data are presented only as hypothesis generating and would be, we believe, to be of interest to the readers of the article who would generally be only those with an interest in cryptococcal infection. In the large rural FNQ region there is an opportunity to look at a potential association between flowering of the trees that have been shown to harbour C. gattii; this is our attempt to convey these data to the readers.

Reviewer #2: In this manuscript, Sim et al. summarized the etiology, epidemiology and clinical course of cryptococcal infections in Far North Queensland between 1999 and 2019. During this period, infections with both Cryptococcus gattii and C. neoformans were identified. Individuals infected with C. gattii were less likely to be immunocompromised compared to patients with C. neoformans. Interestingly, they found that nearly 90% of the C. gattii infections had CNS involvement. Patients with C. gattii infections were more likely to be current or former smoker than patients with C. neoformans. Furthermore, more than 50% of the patients had a history of hazardous alcohol use. These clinical data are interesting; however, the manuscript will definitively benefit from an expanded mechanistic discussion on these clinical observations.

Comments

1) In this manuscript, 90% of the C. gattii infections had CNS involvement. Recent mouse studies showed that the primary target organ of C. gattii is different from that of C. neoformans. C. gattii grows faster in the lung and is primarily linked to pulmonary diseases (Pubmed PMID: 22570277). The authors should expand the discussion on this point.

Response: We thank the reviewer for the time that they have taken to review our paper and agree that there are several points that could be expanded in the discussion. We have done our best to do this while also attempting to shorten the discussion overall as per reviewer 1.

We have expanded our discussion about lung involvement particularly its association with alcohol and tobacco use (lines 297-302). We did not add the mouse model reference as we did not feel we had the space in the discussion to do justice to this point. It is also a little beyond the scope of the paper which focuses on human, clinical cases.

2) Patients with C. gattii infections more likely to be smoker. It would be nice to have more discussion about that point.

Response: We thank the reviewer for highlighting this point - we have expanded the discussion accordingly (lines 297-302).

3) It is also interesting that more than 50% of the patients had a history of hazardous alcohol use. The authors should discuss more on the potential mechanisms behind this observation. Actually, recent studies have shown that Liver cirrhosis is an independent risk factor of cryptococcal meningitis (Pubmed PMID: 25747471) and that liver macrophages play a prominent role to filter disseminating Cryptococcal yeast cells (Pubmed PMID: 31594939).

Response: We agree that this is an interesting discussion point and we have expanded our discussion to include this (lines 297-302). We have added both of the suggested references for the interested reader.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

16 Feb 2022

7 Mar 2022

Response to reviewers

We thank the Editorial staff and the reviewers for the time that they have taken to review our manuscript and the very helpful comments that they have made to improve the work. Reviewer 2 appears happy with the revision, however Reviewer 1 still has some valid concerns. Please find below our point-by-point responses to Reviewer 1’s comments.

Reviewers’ comments

Reviewer 1

This is a report of 49 patients with cryptococcosis, of whom 16 may have had infection confined to the lung (table 2). Most analyses were done on a subset of 32 patients in whom the Cryptococcus was speciated as neoformans or gattii. Of the 32, 4 had isolated pulmonary disease. Though the diagnosis, management and outcome differs for cryptococcosis confined to the lung, that distinction does not appear in analysis of management or outcome. This should be corrected.

Response: We thank the reviewer for highlighting this important point. In fact, one of the patients with C. neoformans infection had disease limited to the skin, an oversight we have now corrected (lines 203-204). The reviewer is right to note that patients without CNS disease have different management and a different prognosis to patients with CNS involvement. We have added this as a limitation of the study (lines 350-353). However, as there were only 4/32 (12.5%) in the cohort with a speciated organism without CNS involvement, this is unlikely to have affected our findings significantly. Furthermore, there was no statistical difference in the case fatality rate in this series in patients with and without CNS involvement in the cohort, data that we now present in lines 277-278.

25.change wording to “clinical course of 49 cryptococcal infections”. Please insert total number of case so number of 32 in next line is in perspective

Response: We thank the reviewer for highlighting this point. The text has been corrected accordingly (lines 25-27)

29-30 . omit speculation about flowering of trees because this new speculation does not have sufficient confirmation in the paper and may mislead readers (see below)

Response: This has been removed from the abstract as requested.

31 and 52-53 wording suggests that cryptococcomas may benefit by neurosurgery, presumably meaning resection. This is not true and is misleading.

Response: The neurosurgical management of cryptococcomas that we were describing was, in fact, management of elevated intracranial pressure using ventriculoperitoneal shunting or external ventricular drainage, although resection is also reported in the literature. We have added 2 references - Chan et al. Neurosurgical aspects of cerebral cryptococcosis. Neurosurgery 1989, and Chastain et al. Cerebral Cryptococcomas: A Systematic Scoping Review of Available Evidence to Facilitate Diagnosis and Treatment. Pathogens 2022 - which examine the neurosurgical management of cryptococcomas (references 4 & 5).

93 a map of Australia is readily available to readers outside this manuscript and is unnecessary

Response: The reviewer is right to note that most readers would be aware of the country of Australia. However, we included the map to highlight the tropical - rather than temperate - location of our study and distinguishing it from other published tropical Northern Australia cohorts e.g., the Northern Territory (reference 9). But we would be happy for the Editors to remove the map if they felt that it added little to the paper.

138. Chart reviews about smoking and alcohol use find either no mention, simple mention or quantitation (pack years of cigarettes, amount of alcohol). Lines 109-110 indicate that “no mention”’ was tallied as no use. Cultural expectations may factor into that question. For example, was an indigenous patient more likely to be asked? The authors should be cautious in chart review interpretations, such as smokers being more common.

Response: We thank the reviewer for highlighting this point. We agree that the data collection was limited by its retrospective collection, a point that we acknowledge in the discussion (lines 348-349).

141 table 1 would benefit by a column with the results of the 17 patients with an unspeciated isolate. Readers don’t know how much bias is introduced by omitting a third of the cohort

Response: We thank the reviewer for highlighting this point. We have added a column as suggested by the reviewer.

171-172 This reference to the season of diagnosis correlating with tree flowering has several important limitations that are not adequately taken into account when the authors put this speculation in the abstract. As pointed out in lines 311-312 the time from exposure to clinical infection with C. gattii is unknown. However, it is thought to be from months to many years. This tentative conclusion is based on finding cases of C. gattii in patients who have left known endemic areas years previously. A long incubation period would help explain why there are no case clusters. Even in the Vancouver Island outbreak, there was no temporal relationship between time on the island and onset of symptoms. C. gattii has been isolated from dozens of tree species around the world but exposure history to such trees has not been correlated with disease, a similar situation with C. neoformans and weathered pigeon droppings. Nor has anyone correlated exposure to flowering of any tree nor flowering to isolation of the fungus from the tree. The authors should not put in the abstract (lines 29-30) their speculation that exposure to a flowering eucalyptus tree might lead to C. gattii infection.

Response: It is clear that the reviewer is sceptical about the validity of the potential association between the flowering of Eucalyptus trees and Cryptococcus gattii disease. However, it is not quite correct to state that there is no-one has correlated flowering of any tree and isolation of Cryptococcus gattii. Australian experts in the field have previously presented the correlation between flowering of the Eucalyptus trees and airborne dispersal of C. gattii basidiospores that have the potential to act as infectious propagules (Ellis DH, Pfeiffer TJ J Clin Microbiol. 1990;28(7):1642-1644). In this paper, Ellis and Pfeiffer reported the results of an 8-month sampling period - during which 2100 collections were made - and describe a correlation between isolation of airborne basidiospores and flowering of the E. camaldulensis in late spring. These basidiospores were present in the environment for only short periods of time. When the air sampling was performed under other trees and under E. camaldulensis trees not in flower, no C. gattii was detected.

On Vancouver Island, C. gattii air concentrations were also noted to be higher in the summer months (Kidd et al. Emerg Infect Dis. 2007;13(1):51), although this was not linked to the pollination or flowering of any specific trees and the airborne C. gattii was identified year-round in this series. However, none of the trees that were examined in this series were Eucalypts and the dominant C. gattii genotypes in the two regions are different (VGI in Australia versus VGII in British Columbia). It may be that the different climates and flora of Australia and North America might lead to differences in the ecology of C. gattii in the two regions.

We agree with the reviewer that multiple factors are almost certainly involved in the pathogenesis of the disease and in this revised manuscript we present the limitations of our hypothesis in more detail and propose other factors that might also contribute to the local incidence of disease (lines 321-330). In the interest of not ascribing too much significance to the observation, we have removed the any reference to flowering from the abstract, as requested by the reviewer.

However, the identification of airborne infectious propagules in the aforementioned Australian and Canadian reports would suggest that this is one way that the organism can reach the patients and establish infection in the lung and beyond. There is an association in this and other Australian series between rural residence and C. gattii disease (Jenney et al. JCM 2004, Chen et al CID 2012), and in the large rural FNQ region there is an opportunity to look at a potential association between flowering of the trees that have been shown to harbour C. gattii. This is our attempt to convey these data to the readers. Hopefully the Editor feels that in this revised version we have presented both sides of the argument in a balanced - and less speculative - manner.

Fig.3 There are many possible reasons for differing seasons at diagnosis but recent exposure is not the most likely.

Response: We agree with the reviewer and have revised the manuscript to present these factors - in, we believe, a balanced manner - for the reader (lines 313-330).

Fig. 4 A similar reservation exists about geographic location. The location of a patient at time of diagnosis depends on population mobility. For example, an indigenous population may be less mobile. Location of C. gattii cases in rural areas may be a function of mobility.

Response: We agree with the reviewer; this is a limitation of this retrospective study that we now specifically highlight in the discussion (lines 349-350).

Fig 6 and table 3 include drugs not recommended for treatment and don’t contribute to any conclusions.

Response: It is true that amphotericin, fluconazole and flucytosine are the agents used most commonly in the management of Cryptococcal disease. However, the 2010 IDSA guidelines for the management of Cryptococcal disease suggest that itraconazole, voriconazole and posaconazole are acceptable second line agents if first line therapies in are not tolerated or contraindicated (Perfect et al. Clin Infect Dis 2010).

Other studies reporting the antifungal susceptibilities of C. gattii present data on susceptibility to a variety of agents. This is interesting from a clinical perspective as second line therapies may be required due to drug intolerance or potential drug-drug interactions. It is also interesting from a microbiological perspective and speaks to the global generalisability of treatment recommendations.

One study from Spain, for instance, reported that fluconazole, voriconazole, and posaconazole MICs were significantly higher in C. gattii than C. neoformans (Torres-Rodríguez et al. J Antimicrob Chemother. 2008 Jul;62(1):205-6). While another from Brazil - which examined the susceptibility of 57 strains of C. gattii to nine antifungal agents - reported significantly higher MICs for fluconazole, voriconazole, amphotericin B, and flucytosine in C. gattii than in C. neoformans (Trilles et al. J Clin Microbiol. 2004;42(10):4815.). A Chinese paper published only last month (Zang et al. PLoS NTD. 2022) reported the susceptibility to 6 antifungal agents (including voriconazole, itraconazole and posaconazole)

We have therefore included extended susceptibilities for the interested reader.

8 Mar 2022

The aetiology and clinical characteristics of cryptococcal infections in Far North Queensland, tropical Australia

PONE-D-21-37969R2

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22 Mar 2022

PONE-D-21-37969R2

The aetiology and clinical characteristics of cryptococcal infections in Far North Queensland, tropical Australia

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