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fmicb-11-01837 July 27, 2020 Time: 18:12 # 1
ORIGINAL RESEARCH
published: 29 July 2020
doi: 10.3389/fmicb.2020.01837
Edited by:
Miklos Fuzi,
Semmelweis University, Hungary
Reviewed by:
Marcia S. C. Melhem,
Adolfo Lutz Institute, Brazil
Luciana Trilles,
Oswaldo Cruz Foundation (Fiocruz),
Brazil
Kennio Ferreira-Paim,
Universidade Federal do Triângulo
Mineiro, Brazil
Popchai Ngamskulrungroj,
Siriraj Hospital, Mahidol University,
Thailand
*Correspondence:
Binghuai Lu
zs25041@126.com
Specialty section:
This article was submitted to
Fungi and Their Interactions,
a section of the journal
Frontiers in Microbiology
Received: 14 March 2020
Accepted: 14 July 2020
Published: 29 July 2020
Citation:
Li Y, Zou M, Yin J, Liu Z and Lu B
(2020) Microbiological,
Epidemiological, and Clinical
Characteristics of Patients With
Cryptococcal Meningitis at a Tertiary
Hospital in China: A 6-Year
Retrospective Analysis.
Front. Microbiol. 11:1837.
doi: 10.3389/fmicb.2020.01837
Microbiological, Epidemiological,
and Clinical Characteristics of
Patients With Cryptococcal
Meningitis at a Tertiary Hospital in
China: A 6-Year Retrospective
Analysis
Yanbing Li1, Mingxiang Zou1, Jun Yin2, Ziqing Liu1and Binghuai Lu3,4*
1Department of Laboratory Medicine, Xiangya Hospital, Central South University, Changsha, China, 2Department
of Neurology, Xiangya Hospital, Central South University, Changsha, China, 3Laboratory of Clinical Microbiology
and Infectious Diseases, Department of Pulmonary and Critical Care Medicine, National Clinical Research Center
for Respiratory Diseases, China-Japan Friendship Hospital, Beijing, China, 4Guangdong Key Laboratory for Emerging
Infectious Diseases, National Clinical Research Center for Infectious Diseases, Shenzhen Third People’s Hospital, Southern
University of Science and Technology, Shenzhen, China
Cryptococcal meningitis, mainly caused by Cryptococcus neoformans/gattii species
complexes, is a lethal infection in both immunosuppressive and immunocompetent
populations. We characterized 110 Cryptococcus strains collected from Xiangya
Hospital of Central South University in China during the 6-year study period between
2013 and 2018, and performed their antifungal susceptibility testing. Furthermore, the
clinical features, laboratory and imaging data, treatment strategies and outcomes of the
subjects were retrospectively analyzed. Of 110 Cryptococcus strains, C. neoformans
species complexes accounted for 96.4% (106/110), including C. neoformans sensu
stricto (VNI molecular type, 95.5%, 105/110) and Cryptococcus deneoformans (VNIV
molecular type, 0.9%, 1/110), and Cryptococcus deuterogattii (VGII molecular type)
accounted for 3.6% (4/110). The strains were further classified into 17 individual
sequence types (STs) by using multilocus sequence typing (MLST). 89.1% (98/110)
were represented by ST5; seven C. deuterogattii strains and one Cryptococcus
deneoformans strain were assigned as ST7 and ST260, respectively. Antifungal minimal
inhibitory concentrations above the epidemiological cutoff values (ECVs) were found
mainly in C. neoformans species complexes strains (nine for amphotericin B, nine
for fluconazole and seven for 5-fluorocytosine). Furthermore, 60.9% (67/110) of the
subjects were male, and 40.0% (44/110) did not have underlying diseases. Hepatic
diseases (hepatitis/HBV carrier status and cirrhosis) were the most common underlying
health conditions (11.8%, 13/110), followed by autoimmune disorders (10.9%, 12/110)
and chronic kidney disease (6.36%, 7/110). Only 4.5% (5/110) of the patients were
HIV/AIDS positives. For clinical presentation, headache (77.3%, 85/110), fever (47.3%,
52/110), and stiff neck (40.9%, 45/110) were commonly observed. The mortality rate
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Li et al. Cryptococcal Meningitis in China
was 35.0% (36/103). In conclusion, our data were characterized by a high prevalence
of the Cryptococcal meningitis patients without HIV/AIDS and other underlying health
conditions, a relatively high non-wild-type rate of fluconazole and amphotericin B
resistance, and low genetic diversity in Cryptococcus strains. The present study
will provide evidence for further improvement of the diagnosis and treatment of
cryptococcosis in China.
Keywords: cryptococcal meningitis, C. neoformans species complexes, C. gattii species complexes, antifungal
susceptibility testing, cryptococcosis
INTRODUCTION
Cryptococcosis is an opportunistic and potentially
life-threatening infection, not only occurring in
immunocompromised patients, including those with HIV/AIDS
and autoimmune diseases, and transplant recipients, but also
posing threat to apparently immunocompetent subjects (Bratton
et al., 2012;Kwon-Chung et al., 2017;Beardsley et al., 2019;
Ellis et al., 2019). The current genus Cryptococcus contains
10 species, of which seven are pathogenic to humans and
animals: the two members of the Cryptococcus neoformans
species complex that are C. neoformans sensu stricto (serotype A;
AFLP1/VNI, AFLP1A/VNB/VNII, AFLP1B/VNII), Cryptococcus
deneoformans (serotype D; AFLP2/VNIV); and the six species
in the Cryptococcus gattii species complex: Cryptococcus
gattii sensu stricto (serotype B; AFLP4/VGI), Cryptococcus
bacillisporus (serotype B&C; AFLP5/VGIII), Cryptococcus
deuterogattii (serotype B; AFLP6/VGII), Cryptococcus tetragattii
(serotype C; AFLP7/VGIV) and Cryptococcus decagattii
(AFLP10/VGIV/VGIIIc) (Hagen et al., 2015, 2017;Kwon-Chung
et al., 2017;Ashton et al., 2019). Furthermore, a new lineage
of Cryptococcus gattii species complex (serotype B, VGV) was
discovered by Farrer et al. in 2019, while cryptococcosis by
other species has rarely been documented till date (Farrer
et al., 2019). Multilocus sequence typing (MLST) based on
seven housekeeping genes allows for the classification of most
clinical Cryptococcus strains into varied sequence types (STs),
among which ST5 is mainly reported from mainland China
(Fan et al., 2016).
The genus Cryptococcus usually invades the central nervous
system (CNS) and results in cryptococcal meningitis and then a
high mortality rate (Lahiri et al., 2019). In addition, meningitis
by C. neoformans species complex occurred more frequently
than that by C. gattii species complex (Yuchong et al., 2012;
Hagen et al., 2015;Smith et al., 2015;Ferreira-Paim et al., 2017;
Thanh et al., 2018;Rakotoarivelo et al., 2020), and this might
be explained by that the former is globally distributed, while the
latter seems to be geographically restricted (Kidd et al., 2007;
Chen et al., 2014;Smith et al., 2015;Firacative et al., 2016;May
et al., 2016;Souto et al., 2016). Furthermore, both amphotericin
B and fluconazole remain the mainstay treatment in cryptococcal
meningitis (Yao et al., 2014;Beardsley et al., 2019). The resistance
patterns to them are documented worldwide, and geographic
variability is noted (Fan et al., 2016;Nyazika et al., 2016).
Understanding the epidemiological characteristics of local
Cryptococcus strains and clinical features of cryptococcal
meningitis is essential for the development of efficient diagnosis
and treatment strategy. Studies on that are rare in China (Fan
et al., 2016;Guo et al., 2016;Liu et al., 2017;Cao et al.,
2019), and they focused on pediatric patients (Guo et al.,
2016), clinical features (Liu et al., 2017;Cao et al., 2019), or
molecular and antifungal resistance characteristics (Fan et al.,
2016), respectively. Continuous and comprehensive monitoring
of the epidemical changes is crucial for the treatment and
prevention of cryptococcosis. The present study involved 110
cryptococcal meningitis cases from 2013 to 2018 in Xiangya
Hospital of Central South University (XHCSU), Hunan, China.
The molecular characteristics and antifungal agent susceptibility
data of Cryptococcus strains, and the clinical, demographic
features and therapeutic outcomes were documented to help
improve timely diagnosis and reduce the mortality rate.
MATERIALS AND METHODS
Ethical Approval
The institutional review boards at the XHCSU approved the study
protocol. The written informed consent from participants was
waived and the data were analyzed anonymously.
Biosafety Procedures
The procedures, including the incubation, nucleic acid
extraction, susceptibility testing of Cryptococcus strains,
were performed in a Class II, Type B2 biological safety cabinet
(LB2-5B1, ESCO, Singapore) in a biosafety level 2 laboratory.
Furthermore, additional biosafety precautions, including masks
and gloves, were taken.
Case Definition
A case of culture-confirmed cryptococcal meningitis was defined
as the isolation of Cryptococcus strains from cerebrospinal fluid
(CSF). Immunocompromised status was defined as the following:
HIV/AIDS, transplant recipient, diabetes mellitus, malignancy,
glucocorticosteroid treatment, hepatic diseases (hepatitis B virus-
carrier, cirrhosis, and chronic liver failure), etc.
Demographic and Clinical Features of
Cryptococcal Meningitis Cases
Cryptococcus strains were obtained from CSF in 110 patients at
XHCSU, China, between January 2013 and December 2018.
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Li et al. Cryptococcal Meningitis in China
The clinical manifestations, laboratory variables and
demographic characteristics of these above patients were
retrospectively reviewed, including the following variables:
demographic characteristics (age, sex, and place of residence),
symptoms (headaches, altered mental status, fever, speech
difficulties, and others), and laboratory and imaging examination
results, underlying diseases, suspected exposure to pigeon
excrements, main antifungal regimen, laboratory results, and
surgical treatment regimen (use of ventriculoperitoneal shunt).
All 110 patients, except for seven lost to follow-up cases, were
categorized as survival or death during a 1-year follow-up.
Strains Collection and Primary
Identification by Using Matrix Assisted
Laser Desorption Ionization-Time of
Flight Mass Spectrometry (MALDI-TOF
MS)
All Cryptococcus strains were streaked onto Sabouraud medium,
and incubated at 35◦C for 24 h or more if necessary. The
fresh colonies were collected and identified on the basis of
colony morphology and MALDI-TOF MS (Bruker Daltonik,
Bremen, Germany) according to the manufacturer’s suggested
recommendations. The identification was matched with the
Bruker spectra library program (version 4.0.0.1, 5,627 entries),
preinstalled in the Bruker Biotyper device (version 3.1; Bruker.1).
Manufacturer-recommended identification score criteria were
used: a score of =2.000 indicated an identification to species-
level, a score of 1.700 to 1.999 indicated to the genus level, and
a score of <1.700 was interpreted as no identification.
The isolates were identified firstly by using a direct transfer
method. Briefly, fresh colonies were picked up with an
inoculation loop, smeared on an MTP 384 steel target plate,
coated with a matrix solution of α-cyano-4-hydroxycinnamic
acid (HCCA) in 50% acetonitrile with 2.5% trifluoroacetic acid
(TFA), and dried at room temperature. If no reliable result
was obtained, an ethanol/formic acid extraction method was
then applied. One loop of fungal mass was suspended in de-
ionized water (300 µl), and pure ethanol (900 µl) was added.
The suspension was mixed for 1 min using a vortex mixer.
The cell suspension was centrifuged (13,000 rpm for 2 min).
The supernatant was discarded. Then, the pellet was dried and
resuspended with 70% formic acid (50 µl) with thorough mixing,
and then 50-µl acetonitrile was added. After centrifugation
(13,000 rpm for 2 min), the 1-µl pellet was applied on a
steel target plate, dried at room temperature, and coated with
HCCA (1 µl).
DNA Extraction
Genomic DNA was extracted from each Cryptococcus strain
following the protocol described by Chen et al. (2018), with
some modifications. Protoplasts were prepared by incubating the
above-mentioned fresh Cryptococcus strains in a microcentrifuge
tube with 1-ml saline, and the solution was prepared to a
concentration of 2 McFarland, and centrifuged at 12,000 rpm for
1 min. The supernatant was discarded. We added 600-µl PBS
buffer and 6-µl (10 U/µl) cell wall breaking enzyme (Tiangen
biochemical technology co., Ltd., China) into microcentrifuge
tube, and thoroughly mixed and incubated it at 37◦C for 120 min.
After vortexing, 400 µl of 2-µm acid-washed glass beads were
added and further vortexed. Extracted DNAs were dissolved in
TE buffer and stored at -20◦C until used as PCR templates.
Internal Transcribed Spacer (ITS)
Sequencing
Identification of Cryptococcus species through the
amplification of the specific ITS region was performed
using two universal primers ITS1 and ITS4 (ITS1:
50-TCCGTAGGTGAACCTGCGG-30and ITS4: 50-
TCCTCCGCTTATTGATATGC-30), as described previously
(Nascimento et al., 2016). The PCR products were sequenced
in both directions and were compared against those contained
in the Centraalbureau voor Schimmelcultures (CBS) hosted
at the Westerdijk Fungal Biodiversity Institute1. Furthermore,
the sequences were aligned in line with reference sequences
of Cryptococcus type strain of H99 (C. neoformans s.s.,
VNI, GenBank accession number KY102799), CBS 8710
(C. neoformans s.s., VNI, NR130682), WM 148 (C. neoformans
s.s., VNI, KY102824), WM 626 (C. neoformans s.s., VNII,
KY102823), WM 628 (C. neoformans s.s., VNIII, FJ914893),
JEC20 (C. deneoformans, VNIV, KY102637), JEC21
(C. deneoformans serotype D, VNIV, AE017342), CBS 8273
(C. gattii, VGI, NR144805), WM 178 (C. deuterogattii, VGII,
KY102659), WM 161 (C. bacillisporus, VGIII, KY102615), CBS
11249 (C. tetragattii, VGIV, KY102969), downloaded from the
GenBank database to infer species boundaries and identify the
Cryptococcus strains to species level.
Multi-Locus Sequence Typing (MLST)
MLST was performed to identify the molecular type of
Cryptococcus strains using the consensus scheme established by
the Cryptococcal Working Group of the International Society
for Human and Animal Mycology (ISHAM) via amplifying and
sequencing the internal fragments within seven housekeeping
gene loci (namely, CAP59,GPD1, IGS1, LAC1,PLB1,SOD1, and
URA5), as described previously (Meyer et al., 2009). The PCR
products were sequenced in both directions. The allelic numbers
and sequence types (STs) were further identified by querying the
online MLST database2. Molecular types (i.e., VNI to VNIV for
C. neoformans species complex and VGI to VGIV for C. gattii
species complex) were assigned according to their allelic numbers
and STs. The ST was used to infer phylogeny. Briefly, jModelTest
software was used to select the algorithm that best fit our data. Of
88 models, TIM1 +G algorithm demonstrated the lowest Akaike
information criteria (AIC) value (Guindon and Gascuel, 2003;
Darriba et al., 2012). Then, the phylogeny tree was constructed
with IQ-TREE software and iTOL v43by using the TIM1 +G
model (Nguyen et al., 2015;Kumar et al., 2018). The bootstrap
value was set to 1,000.
1http://www.cbs.knaw.nl/collections/BioloMICSSequences.aspx
2http://mlst.mycologylab.org
3https://itol.embl.de
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Li et al. Cryptococcal Meningitis in China
Antifungal Susceptibility Testing
The micro-broth dilution method (Sensititre YeastOne
colorimetric plate, Thermo Fisher Scientific, MA, United States)
was used to determine the susceptibility of all Cryptococcus
strains to the six antifungal drugs, namely, fluconazole, 5-
fluorocytosine, amphotericin B, itraconazole, posaconazole,
and voriconazole. The procedures followed the manufacturer’s
instructions. Two well-trained microbiologists read plates
and interpreted the endpoints for the antifungals. The
results were reported as wild-type (WT) or non-wild-type
(non-WT) in accordance with the epidemiological cutoff
value (ECV) set for Cryptococcus spp. by the Clinical
and Laboratory Standards Institute (Espinel-Ingroff et al.,
2012a,b;CLSI, 2018). Given no recommended ECV for
C. deneoformans, except for voriconazole (ECV = 0.12 µg/ml)
recommended by Espinel-Ingroff et al., the minimum inhibitory
concentration (MIC) value was used for the following analysis
(Espinel-Ingroff et al., 2012a).
Statistical Analysis
We evaluated the differences among groups via the Mann-
Whitney U test or t-test for continuous variables (expressed as
the median [IQR]) or mean value ±standard deviation (Std.)
and χ2 tests for categorical variables, as appropriate. Statistical
analyses were conducted using GraphPad Prism version 8.0.1.
Apvalue of less than 0.05 was considered statistically significant.
MIC data were recorded and analyzed by WHONET 5.6 software,
and MIC50 and MIC90 were also calculated.
RESULTS
Demographic Features of 110
Cryptococcal Meningitis Patients
The demographic features of the subjects were shown in Table 1
and Figures 1–3.
The 110 subjects were from Hunan (102, 92.7%), Jiangxi (6
cases, 5.5%), Fujian (1, 0.9%), and Guizhou (1, 0.9%) in China.
The detailed residence places and numbers of the cryptococcal
meningitis cases were illustrated in Figure 1.
On average, we received 18 strains each year (2013, 6;
2014, 18; 2015, 12; 2016, 28; 2017, 18; 2018, 28). Our
patients aged 47.7 ±16.5 years, ranging from 7 to 78 years,
60.9% (67/110) were males, and 16.4% (18/110) were classified
as being elderly (=65 years, 13 males and 5 females).
Furthermore, in 106 cases with C. neoformans species complex
infection, 60.4% (64/106) were male. Four C. gattii species
complex strains were isolated from three male patients (75%),
aged 20, 58 and 66 years, respectively, and one female
(25%), aged 28 years.
Distribution of Cryptococcus Species by
ITS Sequencing
Of 110 Cryptococcus strains, in line with ITS sequence and MLST
results, C. neoformans species complex was the predominant
species (96.4%, 106/110, including 105 C. neoformans s.s. (VNI
TABLE 1 | Epidemiological characteristics of 110 patients with cryptococcal
meningitis.
Demographic features No. % C. neoformans C. gattii p
Total 110 106 4
Gender 1.000
Male 67 60.9 64 3
Female 43 39.1 42 1
Ages (years) 0.230
≤14 5 4.6 5 0
15–24 5 4.6 4 1
25–34 12 10.9 11 1
35–44 18 16.4 18 0
45–54 30 27.3 30 0
55–64 22 20.0 21 1
=65 18 16.4 17 1
Duration from the
onset of symptoms
to diagnosis
0.624
<2 w 22 20.0 22 0
2 w–6 m 85 77.2 81 4
>6 m 3 2.7 3 0
Underlying status 1.0
No underlying conditions 44 40.0 40 4
Hepatitis and liver cirrhosis 13 11.8 13 0
Autoimmune disorders 12 10.9 12 0
CKD 7 6.4 7 0
HIV/AIDS 5 4.6 5 0
Diabetes 5 4.6 5 0
Immunosuppressants 3 2.7 3 0
Malignancy 2 1.8 2 0
Pregnancy 2 1.8 2 0
Transplant recipient 1 0.9 1 0
Other underlying conditions* 16 14.6 16 0
Contact with pigeon
droppings
9 8.2 9 0 0.296
*Includes hypertension, coronary heart disease, tuberculosis, chronic obstructive
pulmonary disease; CKD, Chronic kidney disease.
molecular type) and one C. deneoformans (VNIV molecular
type), and four C. deuterogattii (VGII molecular type) were
rarely identified (3.6%). This is illustrated in Figure 3 and
Supplementary Figure S1. The ITS sequences and seven MLST
loci-sequences of these 110 Cryptococcus strains are deposited at
GenBank under the accession no. MT437078 to MT437187, and
MT474939 to MT475708, respectively.
Low Genetic Diversity as Shown by
MLST Analysis
Only seven sequence types (STs) were distinguished among
110 Cryptococcus strains. As illustrated in Figure 3, four
C. deuterogattii strains and one C. deneoformans strain were
assigned as ST7 and ST260, respectively. Moreover, 89.1% were
represented by ST5 that has been shown to be widely distributed
in mainland China, and other four STs were ST79 (3, 2.7%),
ST359 (2, 1.8%), ST31 (1, 0.9%), and ST366 (1, 0.9%).
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Li et al. Cryptococcal Meningitis in China
FIGURE 1 | Geographical locations of Cryptococcus strains from patients diagnosed with cryptococcal meningitis in Xiangya Hospital, Hunan, China, between 2013
and 2018. The color-highlighted provinces, cities and counties represent those where Cryptococcus strains were recovered, with the number of strains shown in
brackets. (A) China; (B) Provinces where patients came from, including Hunan, Jiangxi, Fujian, and Guizhou provinces. Purple dot: where one C. deneoformans
strain was detected. Red dot: where four C. deuterogattii strains were detected.
FIGURE 2 | Distribution of the sex and ages in 110 cryptococcal meningitis cases in a tertiary hospital in China.
Clinical Presentation
Of 110 cases, 60.0% had underlying conditions, in which hepatitis
and cirrhosis were the most common (13, 11.8%), followed by
autoimmune disorders (12, 10.9%), chronic kidney disease (CKD,
7, 6.4%), HIV/AIDS (5, 4.5%), and diabetes mellitus (5, 4.5%). No
patients with underlying status were infected by C. gattii species
complex strains. Nine cases (9/110, 8.2%) have pigeon droppings
contact history. These were shown in Table 1.
With regards to the clinical presentations, the headache was
the most common (85, 77.3%), followed by fever (52, 47.3%), stiff
neck (45, 40.9%), and nausea/vomiting (32, 29.1%), as shown in
Table 2 and Figure 3.
In line with the data reviewed, in 43 culture-confirmed
cryptococcal meningitis cases in whom cryptococcal antigen
tests have been conducted in both CSF and blood samples, 43
(100%) and 42 (97.7%) were blood and CSF positive, respectively.
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FIGURE 3 | The phylogenetic tree diagram of 110 clinical unique strains of Cryptococcus and various type strains based on MLST sequences: C. neoformans s.s.
(105 strains) and C. deneoformans (1 strain), and C. deuterogattii (4 strains) clustered into different groups. Reference sequences of VNI, VNII, VNIII, VNIV, and VGII
as outgroups are included. From left to right: 1 Sex; 2–15, clinical features: 2 Speech difficulties; 3 Visual disturbance; 4 Brinell sign; 5 Altered mental status; 6
Dizziness; 7 Fever/chill; 8 Hemiplegia; 9 Nausea/vomiting; 10 Palsies; 11 Unstable walking; 12 Headache; 13 Klinefelter sign; 14 Nick stiffness; 15 Sepsis shock;
16∼18 Laboratory data:16 CSF cryptococcal antigen; 17 Blood cryptococcal antigen; 18 India ink staining; 19 Outcomes; 20 Sequence type (ST); 21 Molecular
type; 22 Year of isolation of Cryptococcus strains; 23 Cryptococcus species.
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TABLE 2 | Clinical presentations of 110 cryptococcal meningitis subjects with or without underlying diseases.
Presentations Total (n= 110) Percent Subjects with underlying
diseases (n= 66), No. %
Previously healthy
subjects (n= 44), No. %
p
Headache 85 77.3 49 72.2 36 81.8 0.353
Fever/chill 52 47.3 32 48.5 20 45.5 0.755
Stiff neck 45 40.9 22 33.3 23 52.3 0.048#
Nausea/vomiting 32 29.1 16 24.2 16 36.4 0.17
Klinefelter 23 20.9 10 15.2 13 29.6 0.069
Dizziness 10 9.1 5 7.6 5 11.4 0.498
Altered mental status 10 9.1 6 9.1 4 9.1 1
Visual disturbance 10 9.1 3 4.6 7 15.9 0.042#
Brinell sign 8 7.3 3 4.6 5 11.4 0.177
Palsies 6 5.5 3 4.6 3 6.8 0.607
Speech difficulties 5 4.6 3 4.6 2 4.6 1
Unstable walking 5 4.6 2 3.0 3 6.8 0.35
Hemiplegia 2 1.8 2 3.0 0 0 0.244
Seizures 1 0.9 0 0 1 2.3 0.412
Septic shock 1 0.9 1 1.5 0 0 0.412
#p<0.05.
However, India ink staining of CSF has been conducted in all 110
cases, and only 53 (48.2%) CSF specimens were positive.
Chest CT and Cranial Magnetic
Resonance Imaging (MRI) Examination
The chest CT examination revealed that there were no obvious
abnormalities in 37 cases (33.6%). Ground-glass opacity and
patchy shadow were the most common pulmonary lesions (33
cases, 30.0%). Single-shot nodules in the right lobe (14 cases,
12.7%) were mostly commonly in those with nodules, followed
by double lung nodule (12, 10.9%) and left lung nodule (5,
4.6%). Stripe-like opacities were found only in 6 (5.5%) cases.
The coexistence of the nodule and cavity was detected in
three (2.7%) cases.
Furthermore, of 110 cases, abnormal changes in head MRI
were observed in 91 cases (82.7%). The mostly observed
abnormalities were multi-site lesions. The details are shown in
Table 3.
Antifungal Susceptibility Results
The antifungal susceptibility and MIC results for six agents tested
against all 110 Cryptococcus strains, including MIC50, MIC90 and
MIC range, are presented in Table 4 and Figure 4. Our results
demonstrate that all four C. deuterogattii strains were uniformly
WT to the studied antifungal agents. In contrast, 8.6% (9/105),
6.6% (7/105), 8.6% (9/105), 3.8% (4/105), and 1.0% (1/105)
of C. neoformans s.s. strains were non-WT to fluconazole, 5-
fluorocytosine, amphotericin B, posaconazole, and itraconazole,
respectively, but all strains were WT to voriconazole. The MICs
of C. deneoformans (VNIV) strain (201416) for fluconazole, 5-
fluorocytosine, voriconazole and amphotericin B were 8, 4, 0.12,
and 0.5 µg/ml, respectively. There is no ECV for C. deneoformans
against antifungal agents recommended by CLSI-M59 (CLSI,
2018), however, in line with the study results on ECV of C.
neoformans/gattii species complex by Espinel-Ingroff et al., the
ECV ranges were recommended as follows, fluconazole (8–32
µg/ml), 5-flucytosine (4–16 µg/ml), and amphotericin B (0.5–1
µg/ml), and the EVC for voriconazole was 0.12 µg/ml (Espinel-
Ingroff et al., 2012a,b). Therefore, the C. deneoformans (201416)
strain in the present study was designated as WT to fluconazole,
5-fluorocytosine, voriconazole, and amphotericin B.
Treatment and Outcomes
During the study period, preferred induction antifungal therapy
in 110 cryptococcal meningitis patients is an amphotericin B plus
5-flucytosine for 2–6 weeks depending on patients’ conditions,
followed by consolidation/maintenance therapy with fluconazole
for 12 months or longer. Of them, the median duration of disease
TABLE 3 | Characteristics of magnetic resonance imaging (MRI) examination
results in 110 patients with cryptococcal meningitis.
MRI characteristics No. %
Local lesions
Frontal lobe 15 13.6
Brain stem 4 3.6
Basal ganglion 5 4.6
Cerebral ventricle 2 1.8
Occipital region 1 0.9
Multi-site lesions 33 30.0
Abnormal meningeal enhancement 2 1.8
Hydrocephalus 7 6.4
Focal lesions +hydrocephalus 6 5.5
Focal lesions +meningeal enhancement 6 5.5
Hydrocephalus +meningeal enhancement 1 0.9
Focal lesions +ventricular dilatation 4 3.6
Focal lesions +ventricular dilatation +meningeal enhancement 4 3.6
Ventricular dilatation +hydrocephalus +meningeal enhancement 1 0.9
No lesions 19 17.3
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TABLE 4 | Antifungal susceptibility and MIC distribution of 110 Cryptococcus strains in a tertiary hospital in mainland China.
VNI (105) VGII (4) VNIV (1)
ECV WT Non-WT MIC50
(mg/L)
MIC90
(mg/L)
Range
(mg/L)
ECV WT Non-WT MIC50
(mg/L)
MIC90
(mg/L)
Range
(mg/L)
MIC
(mg/L)
Fluconazole 8 96 (91.4%) 9 (8.6%) 4 8 1.0–64.0 32 4 (100%) 0 8 16 4.0–16.0 8
5-Fluorocytosine 8 98 (93.3%) 7 (6.6%) 4 8 1.0–64.0 32 4 (100%) 0 4 4 2.0–4.0 4
Amphotericin B 0.5 96 (91.4%) 9 (8.6%) 0.5 0.5 0.12–2.0 1 4 (100%) 0 0.25 0.5 0.25–0.5 0.5
Posaconazole 0.25 101 (96.2%) 4 (3.8%) 0.12 0.25 0.03–0.5 NA NA NA 0.12 0.25 0.12–0.25 0.5
Itraconazole 0.25 105 (99.0%) 1 (1.0%) 0.06 0.12 0.015–0.5 1 4 (100%) 0 0.06 0.25 0.06–0.25 0.25
Voriconazole 0.25 105 (100%) 0 0.06 0.12 0.015–0.25 0.5 4 (100%) 0 0.06 0.25 0.03–0.25 0.12
MIC, minimum inhibitory concentration; WT, wild-type; Non-WT, non-wild-type; ECV, the epidemiologic cutoff value; NA, not available; MIC50, the minimum inhibitory
concentration at which 50% of isolates were inhibited; MIC90, the minimum inhibitory concentration at which 90% of isolates were inhibited; MIC range, range of minimum
inhibitory concentration.
FIGURE 4 | The distribution of the minimum inhibitory concentration (MIC). 110 clinical unique strains of C. neoformans s.s. (VNI molecular type) (105 strains),
C. deneoformans (VNIV) (1 strain), C. deuterogattii (VGII) (4 strains) for six antifungal agents. Purple line: the epidemiological cutoff value (ECV) for C. neoformans s.s.
(VNI molecular type); Red line: ECV for C. deuterogattii (VGII).
was 36.6 days. There are three cases who were hospitalized
after over 6-month recurrent headaches, and the other107
patients admitted within 6 months after the onset of symptoms.
Moreover, 12.7% (14/110) died within 3 days of admission
before the establishment of the diagnosis of cryptococcosis; 6.4%
(7/110) received less than 7 days of treatment, transferred to
other health-care centers, and lost to follow-up; 80.9% (89/110)
patients received more than 7 days of antifungal treatment
and were discharged home with maintenance treatment. In
all 89 cases administrated with antifungal agents, 19 (21.3%)
have received ventriculoperitoneal shunts. All these 89 patients
were followed up to 1-year after hospitalization, and 24.7%
(22/89) died. Table 5 shows the comparison of the detailed
clinical features between the survivors (67 cases) and non-
survivors (36 cases).
Comparison of Clinical and Laboratory
Data in 103 Cryptococcal Meningitis
Cases Caused by C. neoformans
Species Complex and C. gattii Species
Complex, and by ST5 and Non-ST5
C. neoformans Species Complex
There were seven cryptococcal meningitis cases lost to follow-
up. Then, the clinical and laboratory features between 99 cases
by C. neoformans species complex and four cases by C. gattii
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TABLE 5 | Characteristics of 103 survivors and non-survivors with cryptococcal
meningitis.
Total Survival Dead p
No. of cryptococcal
meningitis cases
103a67 36
Cryptococcus species (%) 0.625
C. neoformans s.s. 98 (95.1) 64 (95.5) 34 (94.4)
C. deneoformans 1 (1.0) 1 (1.5) 0
C. deuterogattii 4 (3.9) 2 (3.0) 2 (5.6)
Demographic features
Gender
Male (%) 60 (58.3) 39 (58.2) 21 (58.3) 1
Female (%) 43 (41.7) 28 (41.8) 15 (41.7) 1
Age [mean (SD)] 47.7 (16.5) 45.5 (17.1) 49.9 (14.7) 0.195
Contact to pigeon
droppings = Yes (%)
8 (7.8) 5 (7.5) 3 (8.3) 1
Underlying status
Hepatitis and liver cirrhosis
(%)
13 (12.6) 10 (14.9) 3 (8.3) 0.516
Autoimmune disorders
(including 7 SLE cases) (%)
12 (11.7) 4 (6.0) 8 (22.2) 0.033#
CKD (%) 6 (5.8) 2 (3.0) 4 (11.1) 0.216
HIV/AIDS (%) 4 (3.9) 1 (1.5) 3 (8.3) 0.239
Diabetes (%) 5 (4.9) 4 (6.0) 1 (2.8) 0.812
Long-term use of
immunosuppressants (%)
3 (2.9) 3 (4.5) 0 0.5
Malignancy (%) 2 (1.9) 1 (1.55) 1 (2.8) 1
Pregnancy (%) 2 (1.9) 2 (3.0) 0 0.766
Transplant recipient (%) 1 (1.0) 0 1 (2.8) 0.751
No underlying diseases (%) 40 (38.7) 29 (43.3) 11 (30.6) 0.206
Clinical presentations
Altered mental status (%) 9 (8.7) 4 (6.0) 5 (13.9) 0.322
Fever/chill (%) 49 (47.6) 31 (46.3) 18 (50.0) 0.877
Septic shock (%) 1 (1.0) 0 1 (2.8) 0.751
Seizures (%) 1 (1.0) 1 (1.5) 0 1
Headache (%) 80 (77.7) 53 (79.1) 27 (75.0) 0.819
Stiff neck (%) 43 (41.7) 27 (40.3) 16 (44.4) 0.844
Nausea/vomiting (%) 31 (30.1) 19 (28.4) 12 (33.3) 0.764
Visual disturbance (%) 9 (8.7) 7 (10.4) 2 (5.6) 0.637
Speech difficulties (%) 2 (1.9) 1 (1.5) 1 (2.8) 1
Palsies (%) 4 (3.9) 2 (3.0) 2 (5.6) 0.913
Dizziness (%) 8 (7.8) 5 (7.5) 3 (8.3) 1
Hemiplegia (%) 2 (1.9) 0 2 (5.6) 0.23
Unstable walking (%) 4 (3.9) 3 (4.5) 1 (2.8) 1
Klinefelter sign (%) 23 (22.3) 12 (17.9) 11 (30.6) 0.222
Brinell sign (%) 8 (7.8) 4 (6.0) 4 (11.1) 0.587
Laboratory tests
India ink staining (%) 47 (45.6) 27 (40.3) 20 (55.6) 0.202
Course of the disease
before hospitalization
(days, IQR)
30 (20–60) 22 (15–30) 0.179
Treatment strategies
Shunt-containing regimen
(%)b
20 (19.4) 16 (23.9) 4 (11.1) 0.193
Mortality (hospitalization
to death) (%)
30 days-mortality 29 (28.2) 29 (80.6)
(Continued)
TABLE 5 | Continued
Total Survival Dead p
90 days-mortality 33 (32.0) 33 (91.7)
1 year-mortality 36 (35.0) 36 (100)
Not received treatment
due to death within three
days of admission (%)
14 (13.6) 14 (38.9)
#p<0.05. aThere are seven subjects of loss to follow-up; bshunt: extraventricular
drainage and/or ventriculoperitoneal shunts; CKD, Chronic kidney disease; SLE,
systemic lupus erythematosus; IQR, interquartile range.
species complex were compared, the results were shown in
Supplementary Table S1. All four patients infected by C. gattii
species complex had no underlying diseases, while 36.4% (36/99)
patients by C. neoformans species complex showed underlying
conditions, which is significantly different (p<0.05).
Furthermore, ST5 accounted for 91.9% (91/99) of the 99
C. neoformans strains causing meningitis. The differences
between ST5 and non-ST5 groups were compared, as detailed
in Supplementary Table S2. There were two HIV/AIDS cases in
the non-ST5 C. neoformans cases (25%), significantly more than
those in the ST5 group (2.2%, 2/91) (p<0.05).
Relationship Between Antifungal
Susceptibility Results and Prognosis
As shown in Table 6, for fluconazole-containing regimen, the
mortality rate in those infected with non-WT Cryptococcus
for fluconazole was significantly higher than those with WT
Cryptococcus (p= 0.034, <0.05); however, compared with those
in the survival group, the rate of non-WT to 5-flucytosine and
non-WT to amphotericin B increased, but statically insignificant.
Furthermore, no patient was treated in the present study with
voriconazole, itraconazole, and posaconazole.
DISCUSSION
Cryptococcus, usually acquired by inhalation, causes pneumonia
and cryptococcemia, and exhibits a propensity to disseminate
to the brain and presents as meningitis (Fan et al., 2016;
O’Halloran et al., 2017;Cao et al., 2019;Tsai et al., 2019).
Cryptococcal meningitis is the most severe and common form of
cryptococcosis (Yuchong et al., 2012;Rajasingham et al., 2017).
Of 204 cases of cryptococcosis in a US hospital from 1996
to 2009, 62% (126/204) were cryptococcal meningitis (Bratton
et al., 2013). In the current retrospective study, we analyzed the
clinical features of patients with culture-confirmed meningitis.
Meanwhile, we also provided information about the microbial
characteristics of C. neoformans/gattii species complex isolated.
In this study, of 110 Cryptococcus strains from meningitis
subjects, C. neoformans s.s. predominated (95.5%, 105/110), while
C. deuterogattii (4, 3.6%) and C. deneoformans (1, 0.9%) were
rarely identified. By comparison, Chen et al. also showed that
93.0% (120/129) C. neoformans s.s. and 7.0% (9/129) C. gattii
(VGI) were isolated from 1980 through 2006 from cryptococcosis
patients in 16 provinces of China (Chen et al., 2008). In
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TABLE 6 | Relationship between antifungal susceptibility results and prognosis in
110 cryptococcal meningitis subjects.
Treatment regimen Total Survival (No. %) Dead (No. %) p-value
Amphotericin B
containing regimen
84 64 (76.2) 20 (23.8) 0.588
WT 79 61 (77.2) 18 (22.8)
Non-WT 5 3 (60.0) 2 (40.0)
5-Flucytosine containing
regimen
52 41 (78.8) 11 (21.2) 0.193
WT 48 39 (81.2) 9 (18.8)
Non-WT 4 2 (50) 2 (50)
Fluconazole containing
regimen
57 44 (77.2) 13 (22.8) 0.034#
WT 53 43 (81.1) 10 (18.9)
Non-WT 4 1 (25.0) 3 (75.0)
Amphotericin B,
fluconazole combined
with 5-flucytosine
29 25 (86.2) 4 (13.8) 0.553
WT 24 21 (87.5) 3 (22.5)
Non-WT to any of
amphotericin B,
fluconazole, and
5-flucytosine
5 4 (80.0) 1 (20.0)
Amphotericin B
combined with
fluconazole
23 16 (69.6) 7 (30.4) 0.067
WT 19 15 (78.9) 4 (21.1)
Non-WT to amphotericin B
or/and fluconazole
4 1 (25.0) 3 (75.0)
Amphotericin B
combined with
5-flucytosine
21 16 (76.2) 5 (23.8) 0.429
WT 19 15 (78.9) 4 (21.1)
Non-WT to amphotericin B
or/and 5-flucytosine
2 1 (50.0) 1 (50.0)
Fluconazole combined
with 5-flucytosine
2 0 2 (100) /
WT 1 0 1 (100)
Non-WT to fluconazole
or/and 5-flucytosine
1 0 1 (100)
WT, wild-type; Non-WT, non-wild-type. #p<0.05.
Taiwan during 1997–2010, C. neoformans and C. gattii species
complexes accounted for 95.9% (210/219) and 4.1% (9/219),
respectively, and the predominant molecular type was also VNI
(94.1%, 206/219) (Tseng et al., 2013). However, the species and
molecular types distribution of C. neoformans and C. gattii
species complex might be geographically varied, presenting a
peculiar epidemiological profile (Chen et al., 2014;Smith et al.,
2015;Alves Soares et al., 2019). For example, in 62 Cryptococcus
strains collected from meningitis cases from 2006 to 2010 in
Brazil, C. gattii species complex accounted for 21%, tremendously
higher than that in mainland China (Matos et al., 2012). In
addition, there are several studies in Brazil also showing a high
prevalence of C. neoformans species complex (Andrade-Silva
et al., 2018;Ponzio et al., 2019). Infection due to C. gattii
species complex is more prevalent in the Northeast of Brazil
(Matos et al., 2012), despite has been described in the Southeast
(Vilas-Boas et al., 2020). Furthermore, in the current study, all
four C. gattii species complex strains belonged to VGII molecular
type; however, in Colombia, VGII molecular type was identified
only in 54.3% C. gattii species complex strains (Escandon et al.,
2018). Also, Cryptococcus deuterogattii molecular type is the most
prevalent molecular type in Brazil and it seems to be transmitted
from South America to North America (Souto et al., 2016;David
and Casadevall, 2019).
ITS and MLST are often applied in the evaluation of the
genetic relationship among Cryptococcus strains (Fan et al.,
2016). Differences in ST distribution have previously been
noted across varied populations (Beale et al., 2015;Desnos-
Ollivier et al., 2015;Fan et al., 2016). In the current study,
Cryptococcus strains showed a low degree of genetic diversity,
and only seven STs were detected in 110 Cryptococcus strains,
in which 89.1% were represented by ST5. The ST5 lineage
is the predominant ST reported from China. For example,
in a study in China, in 303 C. neoformans strains from 10
hospitals over 5 years, only 12 STs were identified, and ST5
accounted for 89.2% (272/305) (Fan et al., 2016). In another
Chinese study, MLST analysis assigned 41 C. neoformans strains
into 5 STs, and ST5 accounted for 82.9% (34/41) (Wu et al.,
2015). There were two HIV/AIDS patients among the non-
ST5 C. neoformans cases (25%), significantly more than in
the ST5 group (2.2%, 2/91). In addition, in 183 clinical and
environmental isolates of Cryptococcus strains from Thailand,
Southeast Asia, population genetic analyses showed that Thailand
isolates from 11 provinces were highly homogenous, consisting
of the same genetic background (globally known as VNI)
and exhibiting only 10 nearly identical sequence types (STs),
with three (STs 44, 45 and 46) dominating their strains
(Simwami et al., 2011). In an article in Laos, the strains were
dominated (83%) by STs 4 and 6, while in Vietnam, the
strains were dominated by the ST4/ST6 (35%) and ST5 (48%)
lineages (Thanh et al., 2018). Taken together, the molecular
type VNI C. neoformans species complex strains with low
diversity of STs predominate in China and around Asia,
and the predominant STs of Cryptococcus strains might differ
geographically. The molecular diversity across Southeast Asia
has been explained by ecological rather than human host factors
(Thanh et al., 2018).
A sex bias is observed in cryptococcal studies. The prevalence
of cryptococcosis is consistently common in males in both HIV-
positive and -negative patients (Guess et al., 2018). In our study,
male patients accounted for 60.9% (67/110). Similarly, male
predominance was also shown in Wuhan, China (75.6%, 68/90
cryptococcal meningitis cases) (Cao et al., 2019), in Beijing, China
(73%, 38/52 pediatric patients with disseminated cryptococcosis)
(Gao et al., 2017), in Colombia (79.6% in the general population
and 84.4% in HIV/AIDS patients) (Escandon et al., 2018), and in
Brazil (69% in 5,755 recorded deaths related to cryptococcosis)
(Alves Soares et al., 2019). Furthermore, in our study, 16.4%
(18/110) were elderly patients (=65 years, 13 males and 5
females). In a study in Taiwan, elderly patients were more
vulnerable to cryptococcal meningitis than those aged <65 years,
and fewer males were affected in the elderly group (57.9%, 22/38)
than in non-elderly group (78.7%, 48/61) (Tsai et al., 2019). The
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Li et al. Cryptococcal Meningitis in China
sex bias has been explained by the increased incidence of the
HIV epidemic in males (Liu et al., 2017;Guess et al., 2018).
Nevertheless, in our study, only 4.55% (5/110) were HIV-positive.
Furthermore, Cryptococcus species are commonly distributed in
the environment and associated with bird excreta (mostly pigeon
droppings), soil and wood debris (Alves Soares et al., 2019), and
males are more likely to work outside. This could also partially
explain the sex disparity (Guess et al., 2018). In the present study,
only 9 (8.2%) had been in contact with pigeon droppings, and
similarly, only 10 (10/52, 19.2%) cases had a history of exposure to
pigeon droppings in pediatric patients in China (Gao et al., 2017).
Therefore, the reasonable explanation of male predominance
remained elusive.
Cryptococcosis presents in both immunocompromised
and immunocompetent subjects (Lahiri et al., 2019). Some
cryptococcal meningitis patients might have predisposing
factors, which change over time and are geographically varied
(O’Halloran et al., 2017;Ellis et al., 2019). The common risk
factors for cryptococcosis generally involve HIV/AIDS, organ
transplant, corticosteroid use, and malignancy (Hong et al., 2017;
Kashef Hamadani et al., 2018;Ellis et al., 2019). In our study, 60%
(66/110) of the strains were isolated from patients with apparent
risk factors, and the underlying status included hepatitis and
cirrhosis (13, 11.8%), autoimmune disorders (12, 10.9%) and
diabetes mellitus (5, 4.5%). Comparatively, in Taiwan during
1997–2010, HIV infection was the most common underlying
condition (54/219, 24.6%), and among HIV-negative patients,
liver diseases (HBV carrier or cirrhosis) were common (30.2%)
and 15.4% did not have any underlying condition (Tseng et al.,
2013). The current study was conducted in XHCSU, China. It
is not a reference service for HIV. HIV-affected patients will be
transferred to the specific infectious disease hospital. In another
study in China, 71% (91/129) of cryptococcosis cases during
1985–2006 had no apparent risk factor and only 8.5% (11/129)
were HIV/AIDS patients (Chen et al., 2008). In addition, in a
study in the Southwest of China, among 85 patients with CM
were identified, 32 (37.6%) were HIV-uninfected patients (Liu
et al., 2017). The less HIV-infected patients might be explained
by the bias in the population studied. As previously reviewed,
cryptococcosis in non–HIV-infected patients, compared to those
HIV-infected, the substantial differences in terms of natural
history, clinical course, diagnosis, and outcome should be noted
in China, especially the transplant recipients with cryptococcosis
(Pappas, 2013).
Different from C. neoformans species complex, C. gattii
species complex-caused cryptococcosis occurs mainly in non-
elderly and immunocompetent hosts (Chen et al., 2008;
Alves Soares et al., 2019). We compared the clinical and
laboratory data of meningitis cases caused by C. neoformans
and C. deuterogattii, and found that four C. deuterogattii-
infected patients had no underlying diseases, significantly fewer
than among C. neoformans cases (100 vs. 36.4%). Similarly, in
Colombia between 1997 and 2011, 91.1% (41/45) C. gattii species
complex-caused cases had no predisposing factors and only 6.7%
(3/45) were HIV-positive (Lizarazo et al., 2014). It is documented
in a previous study that dual tubercular/cryptococcal meningitis
was the most frequent (54.0%) and most easily misdiagnosed
(95.2%, 40/42) co-infection (Fang et al., 2017). However, in
our study, only three tuberculosis patients (2.73%, 3/110) were
detected, which could be explained by geographical differences
(Fang et al., 2017).
Cryptococcal meningitis is highly lethal without early
diagnosis and proper treatment. Its clinical presentation is
often not specific (Liu et al., 2017;Alves Soares et al.,
2019). In the current study, headache was the most common
presentation (85, 77.3%), followed by fever (52, 47.3%), and
nausea/vomiting (32, 29.09%), in accordance with another
study involving 90 cryptococcal meningitis patients (Cao
et al., 2019). In 45 cryptococcosis cases caused by C. gattii
species complex, their clinical features also included headache
(80.5%) and nausea/vomiting (56.1%) (Lizarazo et al., 2014).
Additionally, in our study, in 103 patients, the mortality rate
was 35.0% (36/103), lower than that in Colombia (47.5%)
(Escandon et al., 2018). In Taiwan, patients infected with
C. gattii species complex, compared to those with C. neoformans
species complex, were more likely to have a higher 10-week
mortality rate (44.4 vs. 22.2%) (Tseng et al., 2013). As is
the case with our study, in four C. deuterogattii meningitis
subjects, two died.
The emergence of Cryptococcus strains with resistance or
elevated MIC above ECVs is of concern as well. Antifungal
susceptibility revealed species-specific differential susceptibility,
but the acquired resistance was still an infrequent phenomenon
(Hagen et al., 2016). Fluconazole and amphotericin B are the
most commonly used antifungal agents for the treatment of
cryptococcal meningitis. As shown in Table 4 and Figure 4,
8.6% (9/105), 6.6% (7/105), and 8.6% (9/105) of C. neoformans
s.s. strains had higher MIC values than the recommended
ECVs of fluconazole, 5-fluorocytosine, and amphotericin B,
respectively. All four C. deuterogattii strains were uniformly
designated as WT to the above antifungal agents. In a study
from Denmark, all 108 clinical C. neoformans and C. gattii
species complex strains were amphotericin B susceptible (Hagen
et al., 2016). In a Chinese study from 10 hospitals over 5 years,
among 303 C. neoformans species complex strains, 7.6% (23/303)
were non-WT to fluconazole, however, seven C. gattii species
complex strains had WT MICs to all drugs tested except for
one C. gattii species complex strain with a fluconazole MIC of
16 µg/ml (Fan et al., 2016). By comparison, the susceptibilities
of the 52 Cryptococcus spp. strains in Zhejiang, mainland
China, only one C. neoformans s.s. (1/51, 2.0%) was non-WT
to amphotericin B (1.0 mg/L) and one (2.0%) non-WT to
fluconazole (16 µg/ml) (Fu et al., 2019). Taken together, the
elevated MIC values for antifungal agents are rarely observed and
partially related to the use of unstandardized regimens, which
should attract more attention.
It is recommended that amphotericin B and 5-flucytosine
are the preferred agents for the initial or induction therapy,
whereas the azoles (especially fluconazole) are generally used
in the consolidation and maintenance phases of therapy (Beale
et al., 2015;Fan et al., 2016). In 204 adults with cryptococcosis
from 1996 to 2009 in the United States of America, 5-flucytosine
exposure was demonstrated to be associated with a lower
mortality rate (Bratton et al., 2013). In a previous study,
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Li et al. Cryptococcal Meningitis in China
the proportion of patients receiving amphotericin B-containing
regimen was 70.6% (48/68), and had a lower 30-day mortality
rate than those treated with other regimens, but the difference
was not statistically significant (16.7%, 8/48 vs. 25.0%, 5/20) (Fu
et al., 2019). In our study, the regimen containing amphotericin
B, fluconazole and 5-flucytosine demonstrated a lower mortality
rate. Furthermore, as revealed in Table 6, the elevated MIC
value for fluconazole was statically related to a higher mortality
rate (22.8 vs. 18.9%, P= 0.034). Nevertheless, the conclusion
should be interpreted with caution due to the small size of
the present study.
In summary, the present study demonstrated that more
C. neoformans species complex isolates (mainly C. neoformans
s.s.) were observed than C. gattii species complex (mainly
C. deuterogattii). Low prevalence of HIV patients with
cryptococcal meningitis and relatively high non-WT rates to
Amphotericin B and fluconazole in Cryptococcus strains in China
were also noted. The study will be helpful for understanding
the genetic diversity of Cryptococcus strains and for decision
making in the context of the diagnosis, treatment and prevention
strategies in cryptococcosis.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation, to any
qualified researcher.
ETHICS STATEMENT
The institutional review boards at the XHCSU approved the study
protocol. The written informed consent from participants was
waived and the data were analyzed anonymously.
AUTHOR CONTRIBUTIONS
YL and MZ isolated the Cryptococcus spp. and performed the
tests. JY collected the clinical data. ZL collected the laboratory
data. YL and BL made substantial contributions to conception
and design, and drafted the manuscript. All authors contributed
to the article and approved the submitted version.
FUNDING
We gratefully acknowledge funding from the National Key
Research and Development Program of China (Grant Nos.
2018YFC1200100 and 2018YFC1200102), and the Sanming
Project of Medicine in Shenzhen (No. SZSM201911009).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2020.01837/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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