Central Nervous System Infection Due to Cryptococcus Gattii Sensu Lato in India: Analysis of Clinical Features, Molecular Profile and Antifungal Susceptibility

Received: 20 November 2016 | Revised: 21 June 2017 | Accepted: 22 June 2017 DOI: 10.1111/myc.12656

ORIGINAL ARTICLE

Central nervous system infection due to Cryptococcus gattii sensu lato in India: Analysis of clinical features, molecular profile and antifungal susceptibility

1Department of Neuromicrobiology, National Institute of Mental Health and Neuro Summary Sciences, Bangalore, India Cryptococcus gattii species complex has evolved as a pathogen in the last two decades 2 Department of Neurology, National Institute causing infection among both immunocompetent and immunocompromised hosts. We of Mental Health and Neuro Sciences, Bangalore, India aimed to analyse the clinical features of CNS infection caused by C. gattii sensu lato, mo- 3Department of Neuroimaging and lecular and antifungal susceptibility profile of this pathogen. Cases diagnosed to have CNS Interventional Radiology, National Institute of cryptococcosis were included in the study. Cryptococcus recovered from patient’s speci- Mental Health and Neuro Sciences, Bangalore, India men was identified by standard protocol. Species confirmation, mating type and molecular 4Department of Biostatistics, National type determination were performed by PCR based methods. Antifungal susceptibility was Institute of Mental Health and Neuro Sciences, Bangalore, India tested in VITEK2C to amphotericin B, 5-flucytosine, fluconazole and voriconazole. Among 199 cases, 20 (10%) were due to C. gattii, comprising of 75% cryptococcal meningitis and Correspondence Dr Nagarathna Chandrashekar, Department 25% cryptococcoma cases. Young adult males were commonly affected. Headache and of Neuromicrobiology, National Institute vomiting were prominent symptoms and 50% were immunocompromised. Among the of Mental Health and Neuro Sciences (NIMHANS), Bangalore, Karnataka, India. isolates, 75%, 20% and 5% were C. tetragattii, C. gattii sensu stricto and C. bacillisporus re- Email: [email protected] spectively and all had mating type α. Four (20%) isolates of C. tetragattii and the only iso-

Funding information late of C. bacillisporus were resistant to fluconazole. The most common species isolated University Grant Commission, India from south India is C. tetragattii. The study contributes to the epidemiology of C. gattii and reiterates the need for genotyping and antifungal susceptibility testing.

KEYWORDS antifungal susceptibility, CNS infection, Cryptococcus gattii sensu lato, epidemiology, molecular profile

1 | INTRODUCTION (serotype D).1 Cryptococcus gattii s.l. (serotypes B and C) is recognized as a species complex distinct from C. neoformans s.l. due to their dif- Cryptococcus neoformans, a fungus which belongs to phylum ferences in electrophoretic karyotypes, DNA fingerprints, intergenic Basidiomycota, causes life-threatening infection in humans and animals. spacer sequences of the ribosomal DNA, and biochemical character- There are two main pathogenic species within the genus Cryptococcus: istics.1 Presently C. gattii s.l. includes five distinct species based upon namely C. neoformans sensu lato (s.l.) and C. gattii sensu lato (s.l.). numerous characteristics as described by Hagen et al.1; Cryptococcus Currently, C. neoformans s.l. is recognized as a species complex com- gattii sensu stricto (s.s.) AFLP4/VGI, Cryptococcus bacillisporus AFLP5/ prising C. neoformans molecular type VNI/AFLP1, VNII/AFLP1B, VNB/ VGIII, Cryptococcus deuterogatii AFLP6/VGII, Cryptococcus tetragattii AFLP1A, VNIII/AFLP3 (serotype A) and C. deneoformans VNIV/AFLP2 AFLP7/VGIV and Cryptococcus decagattii AFLP10 VGIV/VGIIIc.

Cryptococcus neoformans s.s. has worldwide distribution and typi- kit (Meridian Bio Sciences, Cincinnati, Ohio, United States), and isola- cally causes cryptococcal meningitis (CM) among hosts with impaired tion of Cryptococcus from CSF on sabouraud dextrose agar (SDA) incu- immunity. Previously, Cryptococcus gattii s.l. was believed to be geo- bated at 25 and 37 °C. In one of the cases of cryptococcoma/abscess, graphically restricted to tropical and subtropical climates and associ- Cryptococcus was identified by Gram staining and culture of the pus ated with the immunocompetent hosts.2 In the last decade, reports specimen. Creamy mucoid colonies on SDA medium were seen within of C. gattii s.l. infection from British Columbia, North West America, 48–72 hours and identified as Cryptococcus by observing the char- Brazil, Sub-Saharan Africa, Mediterranean Europe, India, and Australia acteristic micromorphological features. The isolates were confirmed have established the global distribution of the pathogen.2,3 Moreover, as C. neoformans s.l./C. gattii s.l. based on phenol oxidase positivity several studies have also documented that C. tetragattii (AFLP7/VGIV) on Niger seed agar and urease positivity.13 Cryptococcus neoformans and C. bacillisporus (AFLP5/VGIII) are pathogenic to both immuno- s.l. and C. gattii s.l. were primarily differentiated by chemotyping on compromised and immunocompetent hosts.3–5 canavanine-glycine-bromothymol blue media.13 All cryptococcal iso- Isolated cases of C. gattii s.l. have been reported from southern lates were stocked in 20% glycerol for further analysis. and north-eastern part of India,6–8 but reports on large-scale molecular analysis of clinical isolates are lacking. From South India, we have 2.2 | CSF analysis earlier reported four cases of CNS infection due to C. tetragattii.7 The global occurrence of C. gattii s.l. was determined by analysing CSF cell count and analysis of biochemical parameters were per- several studies on clinical, environmental and veterinary isolates and formed for 19 cases (In one case pus sample was analysed). CSF it was documented that AFLP4/VGI is the most frequent molecular glucose and protein levels were evaluated by automated methods type.9 Later, considering the potential sample biases, it has been con- using Hexokinase/Glucose-6-phosphate Dehydrogenase assay and cluded that C. deuterogattii is more common (47%) followed by C. gattii Pyrogallol red assay respectively.14 s.s. (34%), C. bacillisporus (11%) and C. tetragattii (8%).3 Cryptococcus gattii s.l. is believed to cause mass lesions known as 2.3 | Molecular characterization cryptococcoma among immunocompetent individuals10,11 although clinical presentation may vary depending on the severity of the infec- The following C. gattii s.l. reference strains, representing each of the tion and immunological status of the patient.3 four molecular types, were used for the study. Cryptococcus gattii s.s. In general, C. gattii s.l. has been found to be less susceptible than WM276 (serotype B), C. deuterogattii R265 (serotype B) and C. bacil- C. neoformans s.l. to commonly used antifungal agents except ampho- lisporus NIH312 (serotype C, VGIII) was kindly provided by Dr. Kaustav tericin B (AMB) and flucytosine.12 However, there is scarce data avail- Sanyal, Molecular Mycology Division, Jawaharlal Nehru Center able on antifungal susceptibility of C. gattii s.l. from India. for Advance Scientific Research, Bangalore (India) and C. tetragatti With this background, we aimed to study the clinical features of WM779 (serotype C) was a kind donation by Prof. Joseph Heitman, CNS infection caused by C. gattii s.l., molecular epidemiology and anti- Duke University, USA. These reference strains also served as internal fungal susceptibility profile of the pathogen. controls for the reproducibility of typing technique.

2.4 | Genomic DNA extraction 2 | MATERIALS AND METHODS The cryptococcal cultures were grown on SDA plate containing 1 mg/ This prospective study was carried out in the department of mL chloramphenicol for 48 hours at 35 °C. DNA was extracted using Neuromicrobiology, National Institute of Mental Health and Neuro AmPurE fungal genomic DNA extraction kit (Juniper Life Sciences, Sciences (NIMHANS), Bangalore, the largest tertiary neuro-care cen- Bangalore, India) method. Purified DNA was stored at −20 °C. tre of India. Cases diagnosed to have CNS infection due to C. gattii s.l. from January 2012 to December 2015 were included in the study. 2.5 | Differentiation between Cryptococcus gattii The study was approved by the institute ethical committee. Clinico- species complex and Cryptococcus neoformans demographic features were recorded in a semi-structured proforma species complex (Table 1). The variables recorded were age, gender, immunological status, presenting illness, past medical history, comorbid factors and PCR primer pair CNa-70-S and CNa-70-A as described by Casali geographical location of the patient. Imaging features, CSF analysis, et al.15 were used to amplify a specific DNA fragment from C. neofor- and microbiological results were also recorded. mans s.l. and the PCR primer pair CNa-49-S and CNa-49-A was used to amplify a DNA fragment from C. gattii s.l. Amplification reactions were performed in a volume of 25 L in Applied Biosystem Veriti 2.1 | Diagnosis of cryptococcal infection μ Thermal Cycler, as described by Casali et al.15 All amplified products The diagnosis of cryptococcal infection was based on the presence were characterized by electrophoresis on 2% agarose gels in 1X TAE of round capsulated budding yeast cells in India Ink staining, antigen buffer 80 V for 90 minutes and then stained with a solution of eth- detection by Cryptococcal antigen latex agglutination system (CALAS) idium bromide at 0.5 μg/mL. LAHIRI MUKHOPADHYAY et al. | 3 d d d mo d mo mo after 1 after 3 after 20 after 15 2 d after 2 after 10 after 2 Outcome Succumbed Succumbed Succumbed Recovered Recovered Recovered Recovered Succumbed Succumbed Succumbed Succumbed Succumbed Succumbed in Succumbed Succumbed Succumbed Succumbed Succumbed Succumbed Succumbed μ g/mL) I (8) I (8) I (8) I (8) I (8) S (<1) S (1) S (2) S (4) S (2) S (4) S (<1) S (2) S (<1) S (1) R (32) R (16) R (32) R (16) R (16) FLC sensitivity (MIC in a types Geno- VGI VGI VGI VGI VGIV VGIV VGIV VGIV VGIII VGIV VGIV VGIV VGIV VGIV VGIV VGIV VGIV VGIV VGIV VGIV Diagnosis Cryptococcal meningitis Cryptococcoma Cryptococcal meningitis Cryptococcoma Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis Cryptococcoma Cryptococcoma Cryptococcal meningitis Cryptococcal meningitis Cryptococcoma Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis Cryptococcal meningitis HIV/CD4 Positive Negative Negative Negative CD4: 642 Negative Negative Negative CD4:1123 Positive CD4: 90 Positive Negative Positive CD4: 17 Positive. CD4: 23 Positive CD4: 11 Negative Negative Positive Negative Negative Positive Positive meningitis Comorbid factors Tuberculous meningitis No comorbidity No comorbidity No comorbidity No comorbidity No comorbidity No comorbidity Tuberculous meningitis No comorbidity No comorbidity No comorbidity No comorbidity Tuberculous meningitis No comorbidity No comorbidity Diabetes, Tuberculous No comorbidity Lymphocytic leukaemia Tuberculous meningitis Diabetes mellitus FC, and VOR (not shown on the table). Place (State) Tamil Nadu West Bengal Karnataka Bihar Karnataka Karnataka Tamil Nadu Karnataka Karnataka Andhra Pradesh Karnataka Tamil Nadu Karnataka Karnataka Karnataka Karnataka Karnataka Karnataka Karnataka Karnataka Age in years/ Gender 30/Male 72/Male 32/Male 30/Male 17/Female 20/Female 15/Female 26/Male 26/Female 25/Male 29/Female 30/Female 26/Male 37/Male 32/Male 49/Male 63/Male 68/Male 48/Female 41/Male 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sl no All the isolates were sensitive to AMB, 5- TABLE 1 Demographic details, immune status, comorbidity, final diagnosis, outcome of the cases, molecular types isolated and fluconazole sensitivity a 4 | LAHIRI MUKHOPADHYAY et al.

method. Candida krusei ATCC6258 and Candida parapsilosis ATCC22019 2.6 | Molecular genotyping by PCR fingerprinting were used for quality control in this experiment. Minimum inhibitory 18 Primers of the microsatellite-specific sequences (GTG)5 were used as sin- concentration (MIC90) were determined by the VITEK2 system. gle primers in the PCR, in a slightly modified method originally described by Meyer et al.16 The amplifications were performed in a final volume of 2.9 | Analysis of the clinical presentation 50 μL, containing 15 μL sterile deionized PCR grade water, 27.5 μL 1X PCR master mix (New England Biolabs, Inc, Massachusetts, USA) contain- The clinical manifestations of both cryptococcoma and CM cases were ing 20 mmol/L Tris-HCl, 22 mmol/L KCl, 22 mmol/L NH4Cl, 1.8 mmol/L analysed and shown in Table 2. Statistical analysis was performed by

MgCl2, 5% Glycerol, 0.2 mmol/L dNTPs, 1X Xylene Cyanol, 1X Tartrazine, Fischer’s Exact test in SPSS software version 22.0 (IBM Analytics, 25 units/mL Taq DNA Polymerase (pH 8.9) and 50 ng primer. North Castle, New York, United States) to determine the P values. PCR amplification was performed in 40 cycles using denatur- ation at 94 °C for 20 seconds, annealing at 50 °C for 1 minute, extension at 72 °C for 20 seconds and a final extension cycle at 72 °C 3 | RESULTS for 6 minutes. Amplified products were separated by electrophoresis in 1.6% agarose gel containing ethidium bromide (0.3 mg/mL), in 1X A total of 199 cases of CNS cryptococcosis were diagnosed from 2012 Tris-Acetate EDTA (TAE) buffer at 55 V for 4 hours. The bands were to 2015; out of which 179 (89.9%) were Cryptococcus neoformans s.l. visualized under UV light in Syngene Gel Documentation system. The and 20 (10.1%) were C. gattii s.l. Out of these 20 cases 15 (75%) were molecular types (VGI–VGIV) were assigned by comparing with the ref- CM and five (25%) were cryptococcoma; 10 (50%) immunosuppressed erence strains loaded on each gel. (nine HIV sero positive and one case of leukaemia).

2.7 | Determination of mating type 3.1 | Demography

Two PCR primer pairs specific for mating type α and a of C. neoformans/ Age group of the patients ranged from 15 to 72 years. In both cryp- gattii, were used to detect the mating type, as described by Chaturvedi tococcoma and meningitis, there was male preponderance; 13 (65%) 17 et al. The PCR products were separated on 2% agarose gels in 1X were male with mean age of 40.8 years (σ=16.7) and seven (35%) TAE buffer, stained with ethidium bromide at for 90 minutes at 80 V. were females with a mean age of 26.4 years (σ=11.1) (Table 1).

2.8 | Antifungal susceptibility test 3.2 | Clinical findings

Susceptibility of the isolates to AMB, fluconazole (FLC), voriconazole Headache (100%), neck rigidity (73.3%-80%), vomiting (60%-80%), (VOR), and 5-flucytosine (5-FC) was performed by VITEK 2C automated impairment of vision (40%-60%) were the predominant clinical

TABLE 2 Analysis of the clinical No of cases (%) Cryptococcoma (%) Meningitis (%) manifestations of CNS cryptococcosis due Parameters n=20 n=5 n=15 P values to C. gattii (Cryptococcoma and Male 13 (65%) 4 (80%) 9 (60%) .61* cryptococcal meningitis) Female 7 (35%) 1 (20%) 6 (40%) Headache 20 (100%) 5 (100%) 15 (100%) 1.00* Fever 14 (70%) 0 (0%) 14 (93.3%) .0004# Vomiting 13 (65%) 4 (80%) 9 (60%) .61* Neck rigidity 15 (75%) 4 (80%) 11(73.3%) 1.0* Visual 9 (45%) 3 (60%) 6 (40%) .61* disturbances Altered 10 (50%) 0 (0%) 10 (66.7%) .032# behaviour C. tetragattii 15 (75%) 3 (60%) 12 (80%) .27* VGIV C. gattii s.s VGI 4 (20%) 2 (40%) 2 (13.3%) C. bacillisporus 1 (5%) 0 1 (6.7%) – VGIII

P values determined by Fisher’s exact test. #P < .05 is considered as statistically significant difference between cases of cryptococcoma and meningitis; *P > .05 is considered statistically insignificant. LAHIRI MUKHOPADHYAY et al. | 5 manifestation common to both meningitis and cryptococcoma cases. few in supratentorial white matter with no diffusion restriction or con- Fever (93.3%) and behavioural abnormalities (66.7%) were exclusively trast enhancement suggestive of cryptococcoma. These were seen in observed in meningitis cases (Table 2). four (20%) of subjects, out of which two had co-existence of diffused meningitis. Another patient (5%) also had a mass like lesion in the right cerebellar hemisphere with irregular peripheral ring enhancement and 3.3 | Imaging features intracavitary nodular projections. No diffusion restriction was seen Out of the 20 patients who were included in this study, 11 had com- within the lesion to suggest abscess formation. puted tomographic (CT) scan of the brain for evaluation, two had One patient (5%) had multiple small abscesses in bilateral parietal Magnetic Resonance (MR) images and seven had both CT and MR im- regions with perilesional oedema. These lesions were T1 hypointense ages (Figure 1). and had alternating rings of hypo and hyperintensity in T2 with diffu- Diffuse leptomeningeal enhancement was the commonest finding sion restriction and peripheral ring enhancement. in seven (35%) patients. This leptomeningeal enhancement was pre- Two patients had only calcified granulomas. Imaging features in dominantly sulcal in distribution. The basal meninges were relatively the remaining five subjects were normal. spared. Out of these seven patients, one had communicating hydro- cephalus secondary to meningitis induced impairment of CSF flow. 3.4 | CSF analysis Two had non-enhancing hypodensities in the right caudate nucleus head. CSF was analysed in 19 cases (15 cases of meningitis and four cases The next most common finding was multiple small T1 hypointense, of cryptococcoma). The CSF cell count ranged from nil to 320 cells/ 3 3 T2 and fluid attenuation inversion recovery hyperintense lesions along mm with an average of 70 cells/mm (σ=95) among 18 cases of the perivascular spaces predominantly in bilateral basal ganglia and CM, nine to 34 cells/mm3 with an average of 21.5 cells/mm3

(A) (B)

FIGURE 1 Figure shows imaging of cryptococcal meningitis and cryptococcoma cases: (A) Axial CT image of a proven case of cryptococcosis with diffuse meningeal enhancement. (B, C) Axial T2W images at the level of basal ganglia shows multiple small T2 hyperintense foci predominantly in the caudate head and lentiform nucleus bilaterally without contrast enhancement (not shown) suggestive of cryptococcoma. (D) Cryptococcoma formed mass lesion in the cerebellar hemisphere 6 | LAHIRI MUKHOPADHYAY et al.

(σ=10.4) among cryptococcoma and in one case of lymphocytic leu- Notably, all the isolates from cases of cryptococcoma were sen- kaemia the cell count was 1100 cells/mm3. The predominant cell sitive to all antifungal agents and the isolates that were resistant and type was lymphocytes in both meningitis and cryptococcoma cases. intermediate sensitivity to FLC were from CM. CSF analysis was not performed for one case of cryptococcoma, who underwent surgical removal of the lesions and pus sample was 3.7 | Treatment collected. CSF glucose was lower than normal in 10 (66.7%) and two (50%) The patients were started on intravenous therapy of AMB 0.5 mg/kg cases of meningitis and cryptococcoma respectively. CSF protein level body weight/day and gradually increased up to 1 mg/kg body weight/ was higher than normal range (15-45 mg/dL) in 18 (94.7%) cases and day in combination with oral FLC 400 mg/day (or 5-FC 100 mg/kg normal in one case of meningitis (Figure 2). body weight/day) for 2 weeks followed by a maintenance therapy of FLC 400 mg/day for 3-6 months or more.20 Maintenance therapy with 5-FC 100 mg/kg body weight/day was advised to six cases. Two 3.5 | Genotype and mating type analysis patients with cryptococcoma had underwent surgical removal of the Among the total 199 isolates, 20 were confirmed as C. gattii s.l. by the lesion followed by standard antifungal treatment. variety determination PCR. Out of these 20 isolates, 15 (75%) were detected as C. tetragattii and four (20%) were C. gattii s.s. followed by 3.8 | Outcome one (5%) C. bacillisporus by PCR fingerprinting. Cryptococcus tetragattii was predominant molecular type among both the cases of CM (80%) In both cryptococcoma and meningitis cases mortality was 80%. and cryptococcoma (60%). C. gattii s.s. was identified from 13.3% CM Among the four cases who survived, one was cryptococcoma who un- and 40% of cryptococcoma cases. derwent surgery and rest of three cases of meningitis were seen in the All the isolates were mating type α. out-patient department on a regular basis for a period of 1 year and later was lost for follow up. Among the successfully treated cases, one was caused by C. gattii 3.6 | Antifungal susceptibility s.s. and three were due to C. tetragattii.

All the 20 isolates (100%) were sensitive to AMB (MIC90: 0.25≤1 μg/mL), 5-FC (MIC : ≤1 g/mL), and VOR (MIC : 0.12≤0.5 g/mL). Fluconazole 90 μ 90 μ 3.9 | HIV status and C. gattii s.l. infection sensitivity had a varied pattern with 10 (50%) of the isolates being sensitive (MIC90: 1≤4 μg/mL), five (25%) resistant (MIC90: 16≤32 μg/ Seven (46.7%) among 15 cases of CM, and three (60%) out of five cases of mL) and the rest five (25%) with intermediate sensitivity (MIC90: 8 μg/ cryptococcoma were immunocompromised. Among HIV positive cases, mL). Sensitivity was interpreted by the VITEK2C system and the MIC age group ranged from 26-49 years irrespective of CM or cryptococ- values were compared with epidemiological cut-off values (ECV) re- coma. Among HIV negative cases age group ranged from 15-72 years, ported by Espinel-Ingroff et al.19 for all four antifungal agents (Table 3). which include two cases of CM in children aged 15 years and 17 years.

FIGURE 2 Figure showing protein and glucose levels in CSF LAHIRI MUKHOPADHYAY et al. | 7

TABLE 3 In vitro antifungal susceptibility test of C. gattii (n=20); MIC values expressed in μg/mL

MIC90 range for MIC90 range for Geometric mean ECV for C. gattii ECV for a a Antifungals tested VGI VGIV of MIC MIC90 VGI C. tetragattii Amphotericin B 0.25-0.5 0.25-0.5 0.297 0.5 0.5 1 Fluconazole 1-4 1-32 4.6 16 8 16 5-Flucytosine 1 1 1 1 4 4 Voriconazole 0.12 0.12-0.25 0.143 0.25 0.5 0.25 aECV for the C. gattii s.l isolates were acquired from reports published by Espinel-Ingroff et al.19

Vomiting and altered behaviour were prominent in immunocom- Cryptococcus gattii s.l. causes potentially fatal infection both as op- petent whereas visual difficulties were more common in immunocom- portunistic and primary pathogen.3 Several factors such as oral corti- promised hosts. costeroids, chemotherapy, older age may increase the risk of infection Among immunosuppressed cases eight (80%) were C. tetragattii by this species.37 In our study 10 (50%) cases were immunocompro- and one (10%) each of C. gattii s.s. and C. bacillisporus. Among the im- mised which was similar to the report by Morgan et al.,35 who have munocompetent cases seven (70%) were C. tetragattii and three (30%) documented 61% cases of C. gattii s.l. infection to be HIV seropositive, were C. gattii s.s. supporting the fact that C. gattii s.l. is no longer an exclusive etiologic The five FLC resistance cases, one was immunocompromised and agent for immunocompetent individuals.5 Headache, fever, and neck rest four were immunocompetent. stiffness were the most salient features and were common to both Immunosuppressed patients had 100% mortality within 2 days to cryptococcoma and meningitis patients. Fever (P=.004) and altered 6 months’ post- diagnosis. Four (40%) immunocompetent cases sur- behaviour (P=.032) were prominent among meningitis cases. vived after primary and follow-up therapy. The most common imaging finding in these patients was diffuse leptomeningeal enhancement (35%) followed by cryptococcoma and lesion in 25% of the subjects. All the patients, who had visual difficul- 4 | DISCUSSION ties, the radiological findings featured either presence of abscess, ring enhancing/hypodense lesions or meningeal inflammation. Cryptococcus gattii s.l. was not considered as an important pathogen Most of the clinical isolates from Europe, Asia and Australia are till recently because the infection by this organism represented only C. gattii s.s, whereas in North and South America C. deuterogattii and 1% of the cryptococcosis cases worldwide.21 Even in endemic area C. bacillisporus are more prevalent.3 Cryptococcus tetragattii is pre- like Australia, the reported infection rate was only 0.94 cases per mil- dominant in southern part of Africa3,5,31 and also reported from India, lion.21 The outbreak on Vancouver Island of British Columbia (Canada) Mexico, Colombia, Puerto Rico, Spain.5,25,38–40 Although in India, in 1999, has expanded its epidemiology in temperate regions.22 Till C. gattii s.l. has been isolated from the environment,33 there are limited then C. gattii s.l. has been reported from Columbia23 Papua, New reports of clinical isolation, of which only two mention the molecular Guinea,24 Venezuela,25,26 French Guiana,27 Vietnam,28 Hong Kong,29 types of the isolates. Jain et al.34 have reported five isolates of C. deu- Botswana,30 Zimbabwe5 and Malawi.23 On the other hand, the overall terogattii, and Chowdhary et al.33 have mentioned two clinical isolates prevalence was found to be lower in Europe, South Africa, Mexico, of C. gattii s.s. The present finding agrees with our previous report7 as Argentina, and Asia including China (without Hong Kong), southeast majority of our C. gattii s.l. isolates are C. tetragattii and the patients Asia and India.1,23,31 hailed from three states: Karnataka, Tamilnadu and Andhra Pradesh, There are few reports of clinical isolates of C. gattii s.l., which doc- from southern part of India indicating the probable predominance of uments prevalence rate ranging from 2.8% in south India to 8%-8.8% C. tetragattii in this part of the country. C. gattii s.s. isolated from four in north India.7,32–34 The present series records the increased preva- (20%) cases came from different states; Karnataka, Tamil Nadu (south lence of 10.1% cases, as compared to our previous reports, which is India), West Bengal and Bihar (east India). Thus, it appears that C. gattii probably a consequence of alterations in the ecology and biology of s.s. is widely distributed in India.6,33 We had one case of CM due to this pathogen. C. bacillisporus from Karnataka, which is probably the first report from Most of our patients (15; 75%) presented with meningitis. Morgan India, to the best of our knowledge. et al.35 also report C. gattii s.l. meningitis in their study. Among our During the Vancouver Island outbreak, due to C. deuterogattii, the cases young adults (26-45 years) were commonly affected by both most common clinical presentation was meningitis.23 In this study, cryptococcoma and meningitis. There were two (10%) cases below C. tetragattii was more common among both meningitis and cryp- the age of 18 years who were immunocompetent. A high proportion tococcoma patients although there was no statistically significant of CNS cryptococcosis due to C. gattii s.l. among immunocompetent difference between the molecular types involved and disease presen- young individuals have also been documented in Colombia.23 There tation (P=.27) (Table 2). Cryptococcus tetragattii was also predominant was male preponderance probably due to increased exposure of males species among both immunosuppressed (80%) and immunocom- to environmental sources.36 petent (70%) individuals and C. gattii s.s. was more common among 8 | LAHIRI MUKHOPADHYAY et al. immunocompetent patients (75%). Most of the clinical and environ- CONFLICT OF INTEREST 7,41 mental isolates worldwide belong to mating type α, as were all our The authors report no conflicts of interest in this work. isolates. The isolates were tested for the susceptibility to four most commonly used antifungal agents. MIC90 and the sensitivity pattern was ETHICAL STATEMENT generated by VITEK2C system42 which was comparable to ECVs for This study was approved by institutional ethics committee of National all the antifungals reported by Espinel-Ingroff et al.19,43 Thompson Institute of Mental Health and Neuro Sciences, Bangalore, India. et al.44 described in their study that C. gattii s.l. exhibits low susceptibility to FLC, which supports our present study where five (25%) of the isolates were resistant and five (33.3%) showed intermediate sus- REFERENCES ceptibility to FLC. 1. Hagen F, Khayhan K, Theelen B, et al. Recognition of seven species Four (26.7%) of the 15 C. tetragattii and the C. bacillisporus iso- in the Cryptococcus gattii/Cryptococcus neoformans species complex. late were resistant to FLC. An investigation from USA describing Fungal Genet Biol. 2015;78:16‐48. 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