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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 Shayanki Lahiri Mukhopadhyay1
| Veenakumari H. Bahubali1 | Netravathi Manjunath2 |
Aarthi Swaminathan3 | Sayani Maji1 | Marimuthu Palaniappan4 | Satishchandra Parthasarathy2 | Nagarathna Chandrashekar1 1 Department of Neuromicrobiology, National Institute of Mental Health and Neuro Sciences, Bangalore, India 2
Department of Neurology, National Institute of Mental Health and Neuro Sciences, Bangalore, India 3
Department of Neuroimaging and Interventional Radiology, National Institute of Mental Health and Neuro Sciences, Bangalore, India 4
Department of Biostatistics, National Institute of Mental Health and Neuro Sciences, Bangalore, India Correspondence Dr Nagarathna Chandrashekar, Department of Neuromicrobiology, National Institute of Mental Health and Neuro Sciences (NIMHANS), Bangalore, Karnataka, India. Email:
[email protected] Funding information University Grant Commission, India
Summary Cryptococcus gattii species complex has evolved as a pathogen in the last two decades causing infection among both immunocompetent and immunocompromised hosts. We aimed to analyse the clinical features of CNS infection caused by C. gattii sensu lato, molecular and antifungal susceptibility profile of this pathogen. Cases diagnosed to have CNS cryptococcosis were included in the study. Cryptococcus recovered from patient’s specimen was identified by standard protocol. Species confirmation, mating type and molecular type determination were performed by PCR based methods. Antifungal susceptibility was 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 25% cryptococcoma cases. Young adult males were commonly affected. Headache and vomiting were prominent symptoms and 50% were immunocompromised. Among the isolates, 75%, 20% and 5% were C. tetragattii, C. gattii sensu stricto and C. bacillisporus respectively and all had mating type α. Four (20%) isolates of C. tetragattii and the only isolate of C. bacillisporus were resistant to fluconazole. The most common species isolated 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.
Mycoses. 2017;1–9.
wileyonlinelibrary.com/journal/myc © 2017 Blackwell Verlag GmbH | 1
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LAHIRI MUKHOPADHYAY et al.
2
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 earlier reported four cases of CNS infection due to C. tetragattii.7
2.2 | CSF analysis
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 cryptococcoma among immunocompetent individuals10,11 although
2.3 | Molecular characterization
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 | MATERIALS AND METHODS
2.4 | Genomic DNA extraction 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. The study was approved by the institute ethical committee. Clinico- demographic features were recorded in a semi-structured proforma (Table 1). The variables recorded were age, gender, immunological
2.5 | Differentiation between Cryptococcus gattii species complex and Cryptococcus neoformans species complex
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
2.1 | Diagnosis of cryptococcal infection The diagnosis of cryptococcal infection was based on the presence
were performed in a volume of 25 μL in Applied Biosystem Veriti Thermal Cycler, as described by Casali et al.15 All amplified products 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.
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
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Karnataka
Karnataka
Karnataka
Karnataka
Karnataka
Karnataka
Karnataka
Karnataka
Tamil Nadu
Karnataka
Andhra Pradesh
Karnataka
Karnataka
Tamil Nadu
Karnataka
Karnataka
Bihar
Karnataka
West Bengal
Tamil Nadu
Place (State)
Diabetes mellitus
Tuberculous meningitis
Lymphocytic leukaemia
No comorbidity
Diabetes, Tuberculous meningitis
No comorbidity
No comorbidity
Tuberculous meningitis
No comorbidity
No comorbidity
No comorbidity
No comorbidity
Tuberculous meningitis
No comorbidity
No comorbidity
No comorbidity
No comorbidity
No comorbidity
No comorbidity
Tuberculous meningitis
Comorbid factors
All the isolates were sensitive to AMB, 5-FC, and VOR (not shown on the table).
a
30/Male
Age in years/ Gender
1
Sl no
Positive
Positive
Negative
Negative
Positive
Negative
Negative
Positive CD4: 11
Positive. CD4: 23
Positive CD4: 17
Negative
Positive
Positive CD4: 90
Negative CD4:1123
Negative
Negative
Negative CD4: 642
Negative
Negative
Positive
HIV/CD4
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcoma
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcoma
Cryptococcoma
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcal meningitis
Cryptococcoma
Cryptococcal meningitis
Cryptococcoma
Cryptococcal meningitis
Diagnosis
VGIV
VGIV
VGIV
VGIV
VGIV
VGIV
VGIV
VGIV
VGIV
VGIV
VGIV
VGIII
VGIV
VGIV
VGIV
VGIV
VGI
VGI
VGI
VGI
Geno-types
T A B L E 1 Demographic details, immune status, comorbidity, final diagnosis, outcome of the cases, molecular types isolated and fluconazole sensitivitya
I (8)
I (8)
S (1)
R (16)
S (<1)
R (16)
S (2)
S (<1)
S (4)
I (8)
I (8)
R (32)
I (8)
R (16)
R (32)
S (2)
S (4)
S (2)
S (1)
S (<1)
FLC sensitivity (MIC in μg/mL)
Succumbed
Succumbed
Succumbed after 2 mo
Succumbed after 10 d
Succumbed
Succumbed
Succumbed after 2 mo
Succumbed in 2 d
Succumbed
Succumbed after 15 d
Succumbed after 20 d
Succumbed after 3 d
Succumbed
Recovered
Recovered
Recovered
Recovered
Succumbed
Succumbed after 1 mo
Succumbed
Outcome
LAHIRI MUKHOPADHYAY et al. 3
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LAHIRI MUKHOPADHYAY et al.
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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
Primers of the microsatellite-specific sequences (GTG)5 were used as sin-
concentration (MIC90) were determined by the VITEK2 system.18
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 50 μL, containing 15 μL sterile deionized PCR grade water, 27.5 μL 1X
2.9 | Analysis of the clinical presentation
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 denaturation 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%)
et al.17 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
Parameters
No of cases (%) n=20
Cryptococcoma (%) n=5
Male
13 (65%)
4 (80%)
Female
Meningitis (%) n=15 9 (60%)
P values .61*
7 (35%)
1 (20%)
6 (40%)
Headache
20 (100%)
5 (100%)
15 (100%)
Fever
14 (70%)
0 (0%)
14 (93.3%)
Vomiting
13 (65%)
4 (80%)
9 (60%)
Neck rigidity
15 (75%)
4 (80%)
11(73.3%)
Visual disturbances
9 (45%)
3 (60%)
6 (40%)
.61*
Altered behaviour
10 (50%)
0 (0%)
10 (66.7%)
.032#
C. tetragattii VGIV
15 (75%)
3 (60%)
12 (80%)
.27*
C. gattii s.s VGI
4 (20%)
2 (40%)
2 (13.3%)
C. bacillisporus VGIII
1 (5%)
0
1 (6.7%)
1.00* .0004# .61* 1.0*
–
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.
T A B L E 2 Analysis of the clinical manifestations of CNS cryptococcosis due to C. gattii (Cryptococcoma and cryptococcal meningitis)
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LAHIRI MUKHOPADHYAY et al.
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 in seven (35%) patients. This leptomeningeal enhancement was predominantly sulcal in distribution. The basal meninges were relatively
sion restriction and peripheral ring enhancement. Two patients had only calcified granulomas. Imaging features in the remaining five subjects were normal.
spared. Out of these seven patients, one had communicating hydrocephalus secondary to meningitis induced impairment of CSF flow. Two had non-enhancing hypodensities in the right caudate nucleus head.
3.4 | CSF analysis 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/
T2 and fluid attenuation inversion recovery hyperintense lesions along
mm3 with an average of 70 cells/mm3 (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
F I G U R E 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
(A)
(B)
(C)
(D)
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LAHIRI MUKHOPADHYAY et al.
6
(σ=10.4) among cryptococcoma and in one case of lymphocytic leu3
Notably, all the isolates from cases of cryptococcoma were sen-
kaemia the cell count was 1100 cells/mm . 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 collected.
3.7 | Treatment
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
3.5 | Genotype and mating type analysis Among the total 199 isolates, 20 were confirmed as C. gattii s.l. by the
with 5-FC 100 mg/kg body weight/day was advised to six cases. Two patients with cryptococcoma had underwent surgical removal of 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 one (5%) C. bacillisporus by PCR fingerprinting. Cryptococcus tetragattii was predominant molecular type among both the cases of CM (80%)
3.8 | Outcome 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.
3.6 | Antifungal susceptibility
Among the successfully treated cases, one was caused by C. gattii 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 (MIC90: ≤1 μg/mL), and VOR (MIC90: 0.12≤0.5 μg/mL). Fluconazole sensitivity had a varied pattern with 10 (50%) of the isolates being
3.9 | HIV status and C. gattii s.l. infection
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.
F I G U R E 2 Figure showing protein and glucose levels in CSF
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LAHIRI MUKHOPADHYAY et al.
T A B L E 3 In vitro antifungal susceptibility test of C. gattii (n=20); MIC values expressed in μg/mL MIC90 range for VGI
Antifungals tested
MIC90 range for VGIV
Geometric mean of MIC
MIC90
ECV for C. gattii VGIa
ECV for C. tetragattiia
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
a
ECV 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 rest four were immunocompetent.
agent for immunocompetent individuals.5 Headache, fever, and neck stiffness were the most salient features and were common to both
Immunosuppressed patients had 100% mortality within 2 days to 6 months’ post- diagnosis. Four (40%) immunocompetent cases survived after primary and follow-up therapy.
cryptococcoma and meningitis patients. Fever (P=.004) and altered behaviour (P=.032) were prominent among meningitis cases. 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,
21
lion.
The outbreak on Vancouver Island of British Columbia (Canada)
Mexico, Colombia, Puerto Rico, Spain.5,25,38–40 Although in India,
Till
C. gattii s.l. has been isolated from the environment,33 there are limited
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-
in 1999, has expanded its epidemiology in temperate regions. then C. gattii s.l. has been reported from Columbia
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,
Botswana,
30
5
Zimbabwe and Malawi.
23
23
22
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
|
LAHIRI MUKHOPADHYAY et al.
8
immunocompetent patients (75%). Most of the clinical and environmental isolates worldwide belong to mating type α,
7,41
as were all our
isolates.
CO NFL I C T O F I NT ER ES T The authors report no conflicts of interest in this work.
The isolates were tested for the susceptibility to four most commonly used antifungal agents. MIC90 and the sensitivity pattern was 42
generated by VITEK2C system
which was comparable to ECVs for
all the antifungals reported by Espinel-Ingroff et al.19,43 Thompson et al.44 described in their study that C. gattii s.l. exhibits low suscep-
ET HI C AL S TAT EM ENT This study was approved by institutional ethics committee of National Institute of Mental Health and Neuro Sciences, Bangalore, India.
tibility to FLC, which supports our present study where five (25%) of the isolates were resistant and five (33.3%) showed intermediate susceptibility to FLC. Four (26.7%) of the 15 C. tetragattii and the C. bacillisporus isolate were resistant to FLC. An investigation from USA describing FLC resistance of C. gattii, also showed C. tetragattii (Colombia) and C. bacillisporus (Australia, USA) isolates from several sources were resistance to FLC,45 which explains that FLC resistance is present in C. bacillisporus and C. tetragattii in endemic areas. All the four (100%) C. gattii s.s. isolates were sensitive to all the antifungal drugs tested including FLC, which supports the previous study by Chowdhary et al.33 Among the four patients who survived the infection, two were due to C. tetragattii FLC resistant isolates, who had undergone long-term maintenance therapy with 5-FC, rest of the immunocompetent cases could not survive because they visited the hospital in the advanced stage of the disease. A recent review suggests that treatment of C. gattii s.l. infection comprises of induction therapy with AMB and 5-FC for 2-6 weeks followed by maintenance therapy with FLC for 6-12 months can eradicate the infection.3 Thus, FLC resistance remains a factor of concern for both the clinicians and patients.
5 | CONCLUSION Cryptococcus gattii s.l. can cause meningitis or cryptococcoma in both
immunocompromised
and
immunocompetent
individuals.
Cryptococcus tetragattii is probably more prevalent in south India and C. gattii s.s. is well distributed in the country. We report the first clinical case of meningitis caused by C. bacillisporus. Fluconazole resistance was documented among C. tetragattii and C. bacillisporus isolates and C. gattii s.s. isolates were sensitive to all antifungal agents. It is important to understand the emergence and the geographic distribution of this important fungi as the geographic variation in the distribution of this fungus is striking.
ACKNOWLE DG E MEN TS We thank University Grant Commission, India for providing funds required for the study. We convey our sincere thanks to Dr. Rose Dawn Bharath, Additional Professor, Neuroimaging & Interventional Radiology, NIMHANS, Bangalore for her contribution to this study. We are thankful to Dr. Kaustav Sanyal, JNCASR, Bangalore and Dr. Joseph Heitman, Duke University, USA for providing necessary standard strains of cryptococcus for this research.
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How to cite this article: Lahiri Mukhopadhyay S, Bahubali VH, Manjunath N, et al. Central nervous system infection due to Cryptococcus gattii sensu lato in India: Analysis of clinical features, molecular profile and antifungal susceptibility. Mycoses. 2017;00:1–9. https://doi.org/10.1111/myc.12656
