Molecular epidemiology of Cryptococcus neoformans ... - Springer Link

Front. Med. China 2010, 4(1): 117–126 DOI 10.1007/s11684-010-0011-z

RESEARCH ARTICLE

Molecular epidemiology of Cryptococcus neoformans species complex isolates from HIV-positive and HIV-negative patients in southeast China *

2*

, BS, Shao-Xi WU3, MD, Xiao-Ping TANG4, MD, Bi-Wei FENG , BS, Zhi-Rong YAO5, MD, Wei-Hua PAN1, MD, Wan-Qing LIAO (✉)1, MM, Zhe-Xue QUAN (✉)2, PhD

Min CHEN1 , MM, Xiao-Ran LI 2

1 National Laboratory of Cryptococcus Neoformans, Department of Dermatology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China 2 Department of Microbiology & Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China 3 Chinese Cultural Collection Commission for Microbiology, Institute of Dermatology, Chinese Academy of Medical Science, Nanjing 210042, China 4 Guangzhou No.8 People’s Hospital, Guangzhou 510060, China 5 Department of Dermatology, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai 200092, China

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010

Abstract This study investigated the molecular types of the Cryptococcus neoformans species complex isolates and their clinical manifestations among human immunodeficiency virus (HIV)-positive and HIV-negative patients in southeast China in the past 15 years. The molecular types of 109 isolates from 108 patients were analyzed by the PCR fingerprinting method, sequences of internal transcribed spacers of rDNA (ITS region), and sequences of the capsule-associated gene (CAP59). In HIV-positive patients, clinical isolates were grouped into molecular types VNI (75%, 15/20), VNII (15%, 3/20), and VNIII (10%, 2/20). In HIV-negative patients, the majority of the clinical isolates were grouped into molecular types VNI (72%, 64/89), VNII (13%, 12/89), VGI (12%, 11/89), VNIII (1%, 1/89), and VGII (1%, 1/89). In reference to the mating type of the isolates, 97% (106/109) were of the MATα, 2% (2/109) were of the MATα/- and 1% (1/109) were of the MATα/a. As for the clinical manifestations of the molecular types among the patients, the average cerebrospinal fluid (CSF) pressure of the patients infected by the C. gattii was higher than that of the patients infected by the C. neoformans. These results suggest that both HIVpositive and HIV-negative cryptococcal patients in the southeast of China are mostly infected by the C. neoformans strains. No C. gattii strains were found in HIVpositive patients. Received November 24, 2009; accepted December 8, 2009 E-mail: [email protected], [email protected] *

Both authors contributed equally to this work.

Keywords Cryptococcus neoformans; Cryptococcus gattii; cryptococcosis; molecular epidemiology; molecular type; mating type

1

Introduction

Cryptococcus neoformans, a fatal fungal pathogen, was previously divided into two varieties comprising C. neoformans var. neoformans, which is the opportunistic agent of cryptococcosis in immunodepressed hosts, and C. neoformans var. gattii, which is a probable cause of cryptococcosis in immunocompetent hosts [1,2]. Recently, C. neoformans var. gattii has been defined as a separate species, C. gattii, due to the divergence of ecological, biochemical, and molecular characteristics [3]. Today, these C. neoformans species complex contains C. neoformans var. neoformans (serotype D), the hybrid isolates (serotype AD), C. neoformans var. grubii (serotype A) [4] and C.gattii (serotypes B and C) [3]. In addition, C. neoformans species complex is a heterothallic yeast with two alternative mating types, α or a, which can multiply by budding or sexual reproduction, respectively [5]. Several molecular typing methods have been widely used in epidemiological molecular analyses of the C. neoformans species complex including electrophoretic karyotyping [6], random amplified polymorphic DNA (RAPD) [7], restriction fragment length polymorphism (RFLP) [8], amplified fragment length polymorphism

118

Front. Med. China 2010, 4(1): 117–126

(AFLP) [9], internal transcribed spacer rDNA (ITS region) [10] and multilocus sequence typing (MLST) analysis [11]. For example, polymerase chain reaction (PCR) fingerprint patterns based on M13 microsatellite DNA identified eight major molecular types among the C. neoformans species complex isolates [7]. To summarize the molecular types and serotypes within C. neoformans, the molecular types VNI and VNII isolates belong to serotype A (C. neoformans var. grubii) and VNIII or VNIV isolates belong to serotype AD or serotype D (C. neoformans var. neoformans). Although the majority of molecular types VGI and VGII isolates belong to serotype B (C. gattii), molecular types VGI, VGII, VGIII and VGIV have no homologous relationship with serotype B and C in C. gattii isolates [12]. Despite recent reports for the molecular types of clinical isolates from China, the cryptococcal infections have been mostly reported from human immunodeficiency virus (HIV)-negative patients [13]. The isolates and clinical manifestation of cryptococcosis from Chinese HIVpositive or other immunocompromised patients have rarely been included. Interestingly, China has also been confronted with a significant increase in acquired immune deficiency syndrome (AIDS) cases with cryptococcosis, which continues to be associated with HIV infections in China in recent years [14,15]. According to the most recent data of The Ministry of Health of the People's Republic of China, southeast China was also an important region where the incidence of HIV infections was increased (http://www. gov.cn/gzdt/2009-02/17/content_1233236.htm). To fill in the gap of available data, this study was performed to analyze the different molecular types of C. neoformans species complex isolates and the clinical manifestations of cryptococcosis between HIV-positive and HIV-negative patients in southeast China.

(CBS10081, VGIII, serotype B), and WM779 (CBS10101, VGIV, serotype C). Ethical approval was granted by the Second Military Medical University Ethics Committee, Shanghai, China. 2.2

DNA extraction

Genomic DNA was extracted with benzyl chloride following the procedures described previously [17] and purified using the Wizard DNA Clean-Up system according to the manufacturer’s protocol. Concentration and quality of the purified DNA was evaluated by 1.4% agarose gel electrophoresis and fluorescence spectrophotometry (Nanodrop 3300, Rockland, DE, USA). 2.3 Internal transcribed spacer (ITS) region and CAP59 gene sequencing

The ITS region including 5.8S rDNA cluster was amplified using the ITS5 and ITS4 primer set [10]. The CAP59 gene was amplified using the primer set CH-CAP59F (5'-CCT YGC CGA AGT YCG AAA CG-3') and CH-CAP59R (5'AAT CGG TGG TTG GAT TCA GTG T-3') designed by Enache-Angoulvant et al. [18] with slight modifications. All amplification reactions were performed with Taq DNA MasterMix (TIANGEN, China) with a total volume of 50 μL and 20 ng of DNA as template. PCR products were directly sequenced on an ABI 3730 automated DNA sequencer twice in opposite directions. 2.4

Phylogenetic analysis

2

Materials and methods

The sequences were aligned with ClustalX version 1.83 [19]. A phylogenetic tree was constructed by MEGA software version 3.0 [20] with the neighbour joining method based on the nucleic acid sequences. Bootstrap analysis with 1000 replications was done to test the robustness of the internal branches.

2.1

Cryptococcal isolates

2.5

A total of 109 clinical strains from 108 patients were obtained from ten Chinese university hospitals, and the patients came from 26 cities of ten provinces in southeast China where the subtropical climate dominates. Each strain submitted for analysis was subcultured and a single colony was selected. After identification was confirmed by caffeic acid agar, positive urease test, and its ability to grow at 37°C, species were differentiated by culturing the isolates on L-canavanine-glycine-bromothymol blue medium [16]. The reference strains of each PCR fingerprint pattern were: WM148 (CBS10085, VNI, serotype A), WM626 (CBS10084, VNII, serotype A), WM628 (CBS10080, VNIII, serotype AD), WM629 (CBS10079, VNIV, serotype D), WM179 (CBS10078, VGI, serotype B), WM178 (CBS10082, VGII, serotype B), WM161

PCR fingerprinting

A primer of the minisatellite-specific core sequence of the wild-type phage M13 was used as a single primer, and the PCR condition was in accordance with that originally described by Meyer et al. [7]. Amplification reactions were performed with Taq DNA MasterMix (TIANGEN, China) with a total volume of 50 μL and 20 ng of DNA as template. Amplification products were separated by gel electrophoresis on 1.4% agarose gels in 1 TAE buffer at 110 V for 1 h. DNA bands of each fingerprinting pattern were defined manually by comparison to the reference strains of the eight major molecular types. 2.6

Mating type

Determination of mating type was established by PCR

Min CHEN et al. C. neoformans isolates in southeast China

using two specific primer sets in accordance with Chaturvedi et al. [21]. Amplification reactions were performed with Taq DNA MasterMix (TIANGEN, China) with a total volume of 50 μL and 20 ng of DNA as template. Products were separated by gel electrophoresis on 3.5% agarose gels in 1 TAE buffer at 110 V for 1.5 h. 2.7

Statistical analysis

The data collected during this study period were analyzed using Minitab 15.0. Percentages were compared using χ2 or Fisher’s exact test, and mean values were compared using Student’s t-test when the data followed a normal distribution. A P value less than 0.05 was considered statistically significant.

3

Results

3.1 Molecular types determined by culturing and molecular methods

The results of species identification by culturing showed that most of the isolates were C. neoformans (89%, 97/109), and only 11% of the isolates were C. gattii (11%, 12/109). Several molecular typing methods were used to analyze C. neoformans species complex isolates from the HIVpositive and HIV-negative patients. However, 99 samples can yield results through ITS sequencing while only 59 samples can yield results through CAP59 gene sequencing. Analysis of M13-PCR fingerprint pattern and sequences of the ITS region and CAP59 gene showed that molecular type VNI was the most represented (75%, 15/20), followed by VNII (15%, 3/20) and VNIII (10%, 2/20) among HIVpositive patients. Molecular type VNI was also the most highly represented (72%, 64/89), followed by VNII (13%, 12/89), VGI (12%, 11/89), VNIII (1%, 1/89) and VGII (1%, 1/89) in HIV-negative patients. The geographical distribution of the molecular types is shown in Fig. 1. The details can be seen in Tables 1 and 2. 3.2

Mating type determination

Ninety-seven percent of strains (106/109) were MATα, followed by 2% (2/109) MATα/- and 1% (1/109) MATα/a (Tables 1 and 2).

4

Discussion

The incidence of cryptococcal infection has risen markedly as a result of HIV epidemic and increasing use of immunosuppressive therapies [1]. However, with the wide use of highly active antiretroviral therapy (HAART) since 1996, the incidence of cryptococcosis

119

among HIV-infected persons in America and Europe has decreased in recent years [22,23]. Almost all C. neoformans species complex strains analyzed in China in previous studies were isolated from HIV-negative patients [13,24]; however, a recent clinical survey of 85 HIVpositive patients in China showed that 7% of the patients were associated with this deadly infection [15]. In a recent similar study in China, the difference in the male-to-female gender ratio of immunocompetent patients was not significant (54 ∶ 50) [24]; however, the results of this study show that among both HIV-positive and HIVnegative patients, the infected C. neoformans species complex was more frequently observed in males (male: female ratio = 4∶ 1 vs 1.5∶ 1). These findings were in agreement with data reported from a recent European survey ( 5∶ 1 vs 2∶ 1) [25]. Moreover, our results show that the most represented age groups of HIV-positive and HIVnegative patients were at 21–30 years (35%, 7/20) and 31– 40 years (25%, 22/88), respectively. These findings are somewhat different from data reported from an European study, which states that the most represented age groups were at 31–40 years (53.8%) of HIV-positive patients and > 60 years (39%) of HIV-negative patients [25]. Over 95% of all clinical and environmental isolates of C. neoformans species complex strains are MATα serotype A isolates with worldwide distribution. These wild-type haploid MATα cells could develop a hyphal phase under appropriate conditions, producing basidia with viable basidiospores [26]. In this study, three molecular methods grouped 109 clinical cryptococcal strains into five of the eight major molecular types described previously [7]. Among the 97 clinical C. neoformans investigated, 81% (79/97) were of molecular type VNI, while the remaining clinical isolates were of VNII (15%, 15/97) and VNIII (3%, 3/97). Within the 12 clinical C. gattii isolates, 11 were of VGI type, and one was of VGII. These data are similar to the results of previous data from China [13,24] and Malaysia [27], where molecular type VNI accounted for the vast majority in C. neoformans while molecular type VGI accounted for the majority in C. gattii. Remarkably, there were 20 strains of C. neoformans species complex isolated from HIV-positive patients in Shanghai and Guangdong Province, among which 75% (15/20) were of molecular type VNI/MATα strain. The remaining strains were of molecular type VNII/MATα (3/ 20) and VNIII (MATα/a and MATα/ – ) (2/20), which represent the first hybrid (VNIII) strains isolated from HIVpositive Chinese patients. In this study, the molecular type and mating type characteristics of clinical isolates from HIV-positive Chinese patients were similar to reports from other Asian countries such as Thailand [28] and Malaysia [27]. C. neoformans and C. gattii differ from each other not only in host range and geographic distribution, but also in clinical manifestations [1]. Although both species infect the central nervous system, patients infected with C. gattii

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Front. Med. China 2010, 4(1): 117–126

Fig. 1 Geographical distribution of molecular types of 109 Cryptococcus neoformans species complex isolates from southeast China

seem to maintain symptoms longer before presentation, and therapy is often required for a longer period of time [29]. Among HIV-negative patients, the median cerebrospinal fluid (CSF) pressure of C. gattii-infected patients (34947 mmH2O) was higher than the median CSF pressure of C. neoformans-infected patients (300 56 mmH2O, P = 0.007). In addition, abnormal neurology was also more frequently observed among C. gattiiinfected patients than C. neoformans-infected patients (P = 0.009). This observation is consistent with the previous results discussed above [29,30]. Moreover, among the C. neoformans-infected patients, the clinical manifestations of body temperature > 39°C, death attributed to cryptococcosis, serum antigen detection and positive CSF antigen were observed less frequently (P < 0.05) in the HIV-positive patients than in the HIV-negative patients, which is also in agreement with the aforementioned results [29,30]. This phenomenon may be associated with a lower immune response of the AIDS patients. Furthermore, none of the C. gattii strains were isolated from HIV-positive Chinese patients. This is not surprising since there have been many reports showing that C. gattii strains tend to infect immunocompetent patients [1,31]. Dromer et al. proposed that male sex, HIV status, and serotype A are the major determinants of presentation and outcome during cryptococcosis [32]. In this study, a total of nine patients died during the anti-fungal treatment among 88 HIV-negative patients. As for the molecular type of the isolates, seven out of nine were of VNI, followed by VNII (1/9) and VGI (1/9). Among the HIV-negative patients who died, there were six patients without other immunocompromised diseases such as alvine tuberculosis

or pulmonary tuberculosis. Interestingly, four of these six patients were infected by molecular type VNI (serotype A) strains. Due to the small number of the samples, we could not comparatively analyze the toxicity diversity of C. neoformans and C. gattii isolates among different molecular type isolates. However, this phenomenon may suggest that patients who are infected by VNI strains would be more likely to have poor outcomes. In addition, since C. gattii was first isolated from Eucalyptus camaldulensis in 1989 [33], it was soon reported to be isolated from other trees such as E. tereticornis, E. microcorys and S. glomulifera [34]. In this study, all the 12 C. gattii clinical strains were from Shanghai city, Jiangsu, Zhejiang and Guangdong provinces where the subtropical climate dominates and many E. camaldulensis trees were once transplanted. In recent literature, C. gattii has not been isolated from environmental samples in China. Whether or not these clinical C. gattii strains actually descended from E. camaldulensis trees in China, further research is necessary. The PCR fingerprinting method has been widely used in studies of genetic diversity within the C. neoformans species complex isolates. However, PCR fingerprinting patterns are not highly reproducible in different laboratories. Furthermore, numbers detected with faintly stained bands and the intensity varies somewhat with the staining process, making allocation to subtypes of the major groups difficult [7]. In this study, it was difficult to discriminate between fingerprinting patterns of VNI and VGI. Thus, the ITS region and CAP59 gene sequencing methods were chosen to assist in the determination of the genetic diversity within the C. neoformans species complex

2006.01

2006.05 Guangdong CSF

2006.05

2006.08 Guangdong blood














SHCZ78

SHCZ79

SHCZ80

SHCZ84

SHCZ85

SHCZ86

SHCZ89

SHCZ90

SHCZ91

SHCZ92

SHCZ97

SHCZ100

SHCZ101

SHCZ103

SHCZ105

SHCZ110

SHCZ114

Shanghai

Shanghai

Shanghai

CSF

CSF

CSF

CSF

F

F

M

M

M

M

M

M

M

M

M

F

M

F

M

M

M

M

M

M

34

30

27

49

29

55

35

48

62

56

35

44

34

50

25

27

24

22

48

40

age

no

no

yes

no

yes

no

yes

nd

no

no

yes

yes

no

no

yes

no

yes

yes

no

yes

no

no

no

no

no

no

no

nd

no

yes

no

no

no

no

yes

no

no

yes

no

no

no

no

yes

no

yes

no

no

nd

yes

yes

no

no

no

yes

yes

yes

yes

yes

no

yes

b

no

no

yes

no

yes

no

yes

nd

yes

yes

yes

no

yes

no

yes

yes

yes

yes

no

no

body nervous death attrib- serum temperature system uted to antigen > 39°C a abnormali- cryptococ- detection cosis tiesb

310

260

275

220

340

340

320

ND

300

ND

320

295

265

270

350

320

210

255

280

240

ND

6

680

40

90

38

2180

ND

2

10

12

74

34

400

132

21

26

280

210

12

maximal maximum CSF CSF cell pressure /count /mmH2Oc /mm3

no

no

yes

yes

yes

yes

yes

nd

yes

no

yes

yes

yes

no

yes

yes

yes

yes

no

yes

positive CSF antigen

yes

yes

yes

yes

yes

no

yes

nd

yes

yes

yes

yes

yes

yes

no

yes

yes

yes

yes

yes

VNI VNI

α α

VNI VNIII VNIII VNI VNI VNI VNI VNI VNI VNI VNI VNI

α α&α&a α α α α α α α α α

VNI

VNI

α

VNII

VNII

α

α

VNI

α

α

VNII

α

CAP59 accession number

GQ850168

GQ850157 c

GQ850156 GQ850253

GQ850141 GQ850258

GQ850189 GQ850232

GQ850212 GQ850271

GQ850206 GQ850270

GQ850208 GQ850255

GQ850182 GQ850278

GQ850137 GQ850279

GQ850138 GQ850280

GQ850152

GQ850214 GQ850262

GQ850223 GQ850268

GQ850213 GQ850231

GQ850186 GQ850229

GQ850179

GQ850201

positive mating type molecular ITS India ink type accession test number

: body temperature>39°C: body temperature upon arrival at or above 39°C is “yes”, otherwise is “no”. : Nervous system abnormalities: eye, ear and other nervous systems being implicated and function impaired. : Maximal CSF pressure: the highest CSF pressure during the CSF test. ND: not determined; CSF: cerebrospinal fluid; F: female; M: male; ITS: internal transcribed spacer.

a

2006.01

SHCZ74

Shanghai

CSF

2004.10

Shanghai

2003.08

SHCZ63

source sex

SHCZ50

locality

collecting date

isolate

Table 1 Mating type, molecular types and clinical features of clinical Cryptococcus neoformans species complex isolates from the HIV-positive patients in southeastern China

Min CHEN et al. C. neoformans isolates in southeast China 121

Jan 1994

May 1994

Mar 1995

SHCZ4

SHCZ5

SHCZ6

CSF CSF CSF CSF CSF CSF CSF CSF

Jiangsu Henan Shanghai jiangsu Anhui Shanghai Jiangsu Shanghai

Oct 2000

Dec 2000

Dec 2000

Dec 2000

Dec 2000

SHCZ25

SHCZ26

SHCZ27

SHCZ28

SHCZ29＊

SHCZ30＊

Apr 2001

May 2001

Jun 2001

SHCZ33

SHCZ34

SHCZ35

Feb 2001

Sept 2000

SHCZ24

Mar 2001

Sept 2000

SHCZ23

SHCZ32

Sept 2000

SHCZ22

SHCZ31

Jun 1999

Dec 1999

SHCZ21

CSF CSF CSF

Guangdong

CSF

Jiangxi Shanghai

CSF

Zhejiang Shanghai

sputum

Shanghai

CSF

CSF

Shanghai

CSF

Shanghai

Feb 1999

SHCZ20

CSF

Shanghai

Jan 1999

Feb 1999

Zhejiang

SHCZ18

CSF

Shanghai

SHCZ19

sputum

Shanghai

Dec 1998

CSF

Jiangsu

Nov 1998

CSF

Fujian

SHCZ17

CSF

Jiangsu

SHCZ16

CSF

Shanghai

May 1998

CSF

Jiangsu

SHCZ15

CSF

Zhejiang

Feb 1998

CSF

Shanghai

SHCZ14

CSF

Shanghai

Jan 1998

CSF

Shanghai

Sept 1997

CSF

Shanghai

SHCZ13

CSF

Jiangsu

SHCZ12

CSF

Zhejiang

Dec 1996

CSF

Shanghai

Mar 1997

CSF

Shanghai

SHCZ10

source

locality

SHCZ9

Jan 1996

Dec 1993

SHCZ3

Oct 1996

May 1993

SHCZ2

SHCZ8

May 1993

SHCZ1

SHCZ7

collecting date

M

F

M

M

F

F

F

F

M

M

F

M

M

M

F

M

M

F

M

F

M

M

F

F

F

F

M

M

F

M

M

F

F

M

sex

30

51

15

14

50

40

40

12

21

61

53

36

17

4

22

54

8

41

40

37

60

21

41

14

37

23

27

27

42

22

43

38

32

54

age

VNI VNI VNI VNI VGI VNI VNI VGI VGI VNI VNI VNI VNI VNI VNI VGI VNI VNII VNI VNI

α α α α α α α α α α α α α α α α α α

VNI

α α

VNI

α

VNI

VNI

α

α

VNIII

α& –

α

VGI

α

VNII

VGI

α

α

VNI

α

VNI

VNI

α

VNII

VNI

α

α

VNI

α

α

genotype

mating type

GQ850181

GQ850200

GQ850184

GQ850160

GQ850126

GQ850195

GQ850150

GQ850146

GQ850199

GQ850163

GQ850176

GQ850129

GQ850125

GQ850154

GQ850170

GQ850128

GQ850140

GQ850173

GQ850194

GQ850147

GQ850190

GQ850222

GQ850183

GQ850166

GQ850202

GQ850177

GQ850162

GQ850204

GQ850134

GQ850135

GQ850167

GQ850218

GQ850198

GQ850191

ITS accession number

Mating type, molecular types and clinical features of Cryptococcus neoformans species complex isolates from the HIV-negative patients in southeastern China

isolate

Table 2

GQ850249

GQ850227

GQ850225

GQ850282

GQ850254

GQ850276

GQ850246

GQ850273

GQ850283

GQ850238

GQ850243

GQ850257

GQ850251

GQ850226

GQ850266

GQ850224

GQ850284

GQ850248

GQ850244

GQ850228


122 Front. Med. China 2010, 4(1): 117–126

Oct 2004

Dec 2004

SHCZ62

SHCZ64

Aug 2005

Sept 2005

Nov 2005

Dec 2005

Jan 2006

Jan 2006

SHCZ70

SHCZ71

SHCZ72

SHCZ73

SHCZ75

SHCZ76

Aug 2005

Sept 2004

SHCZ61

SHCZ69

Aug 2004

SHCZ60

Aug 2005

Jul 2004

SHCZ59

SHCZ68

May 2004

SHCZ58

Mar 2005

May 2004

SHCZ57

Mar 2005

Apr 2004

SHCZ56

SHCZ67

Apr 2004

SHCZ55

SHCZ66

Oct 2003

Feb 2004

SHCZ53

Oct 2003

SHCZ52

SHCZ54

Jul 2003

Aug 2003

SHCZ49

May 2003

SHCZ47

SHCZ48

Feb 2003

Dec 2002

SHCZ43

Mar 2003

Dec 2002

SHCZ42

SHCZ44

Dec 2002

SHCZ41

SHCZ45

Dec 2001

May 2002

Nov 2001

SHCZ37

SHCZ40

Jul 2001

SHCZ36

SHCZ38

collecting date

isolate

sputum CSF CSF CSF CSF

Shanghai Guangdong Zhejiang Shanghai Zhejiang

CSF CSF CSF CSF CSF CSF CSF CSF CSF CSF

Guangdong Shanghai Shanghai Shanghai Shanghai Jiangsu Guangdong Guangdong Shanghai Shanghai

CSF

CSF

Shanghai

Guangdong

CSF

CSF

CSF

Shanghai Guangdong

CSF

CSF

Shanghai

Guangdong

CSF

Guangdong

CSF

Henan

CSF

Zhejiang

Henan

CSF

Jiangsu

CSF

CSF

Guangdong

Guangdong

CSF

Shanghai

CSF

sputum

Shanghai

CSF

skin

Hubei

CSF

Anhui Hunan

Guangdong

source

locality

M

F

M

M

F

M

F

M

M

F

M

M

M

F

M

F

F

M

F

M

M

F

M

F

F

M

M

M

F

M

F

M

M

F

sex

49

38

43

58

38

67

31

31

46

46

31

27

29

41

40

43

41

32

41

22

5

34

3

25

26

30

8

59

38

39

74

41

5

8

age

VNI VNII VNII VGI

α α α α

VNII VGI

α

α

α

VNII

α

VNI

VNI

α

VGII

VNI

α

α

VNI

α

α

VNI VNII

α

VNI

VNI

α

α

VGI

α

VNI

VNII

α

VNII

VNII

α

α

VNI

α

α

VNI VNI

α α

VNI

VNI

α

α

VNI

α VNI

VNI

α

VNII

VGI

α

α

VNI

α

α

VNI VNI

α α

genotype

mating type

GQ850132

GQ850136

GQ850196

GQ850197

GQ850142

GQ850215

GQ850153

GQ850207

GQ850130

GQ850192

GQ850161

GQ850169

GQ850188

GQ850133

GQ850209

GQ850205

GQ850217

GQ850158

GQ850139

GQ850155

GQ850180

GQ850216

GQ850171

GQ850210

GQ850127

GQ850149

GQ850178

GQ850193


(Continued)

GQ850236

GQ850234

GQ850233

GQ850252

GQ850285

GQ850245

GQ850237

GQ850235

GQ850277

GQ850272

GQ850263

GQ850230

GQ850247

GQ850240

GQ850241

GQ850260


Min CHEN et al. C. neoformans isolates in southeast China 123

Dec 2007

Guangdong

Jiangsu

Guangdong

Guangdong

Guangdong

CSF

CSF

blood

CSF

blood

CSF

CSF

CSF

CSF

＊ SHCZ29 and SHCZ30 were from the same patient. Abbreviations are the same as in Table 1.

Dec 2007

SHCZ113

SHCZ115

Zhejiang

Nov 2007

Nov 2007

Oct 2007

SHCZ109

SHCZ111

Sept 2007

SHCZ112

Guangdong

Sept 2007

SHCZ108

Guangdong

Zhejiang

SHCZ107

blood

Guangdong

Aug 2007

CSF

SHCZ106

CSF

Shanghai Guangdong CSF

CSF

Guangdong

CSF

Anhui Shanghai

Jul 2007

CSF

Guangdong

Jul 2007

CSF

Guangdong

SHCZ102

CSF Lung

CSF

Shanghai Shanghai

CSF

Shanghai Shanghai

source

locality

SHCZ104

Apr 2007

SHCZ99

Jan 2007

SHCZ95

Feb 2007

Jan 2007

SHCZ93

Apr 2007

Dec 2006

SHCZ88

SHCZ98

Oct 2006

SHCZ87

SHCZ96

Jul 2006

Jun 2006

SHCZ83

SHCZ81

SHCZ82

Jan 2006

May 2006

SHCZ77

collecting date

isolate

F

M

M

F

M

M

M

M

F

M

M

M

M

F

M

M

M

M

M

F

F

sex

24

44

58

10

40

44

26

24

45

36

20

56

67

18

35

36

42

37

47

53

70

age

VNI VNI VNI VNI VNI

α α α α

VNI

α

α

VNI

α

VNI

VNI

α

VNI

VNI

α

α

VNI

α

α

VNI

α

VNI

VNI

α

α

VNI

α

VNI

VGI

α

VNI

VNI

α

α

VNI

α

α

genotype

mating type

GQ850219

GQ850145

GQ850220

GQ850185

GQ850211

GQ850203

GQ850174

GQ850159

GQ850151

GQ850221

GQ850175

GQ850165

GQ850164

GQ850143

GQ850172

GQ850144

GQ850131

GQ850187

GQ850148


(Continued)

GQ850269

GQ850250

GQ850264

GQ850275

GQ850274

GQ850256

GQ850267

GQ850239

GQ850265

GQ850261


124 Front. Med. China 2010, 4(1): 117–126

Min CHEN et al. C. neoformans isolates in southeast China

isolates. Sequences retrieved from the CAP59 gene were analyzed using a phylogenetic tree. The amplified fragment contains an intron region and two regions of exons. The intron region was found to have a large divergence within eight molecular types. Hence, the nucleic acid sequences were used for analysis. However, nearly half of the samples could not be amplified by the current primer set of the CAP59 gene, although the primer set used in this study contains two sites of degenerate bases according to the related sequences from GenBank. Meanwhile, another primer set [35] used to amplify samples in this study (data not shown) also indicates that the current primer sets of the CAP59 gene may lack coverage. Therefore, more effective primers need to be designed for the CAP59 gene.

9.

10.

11.

Acknowledgements The study was supported by the National Natural Science Foundation of China (Grant No. 30771927). We thank Dr. Teun BOEKHOUT and Ferry HAGEN (Centraalbureau voor Schimmelcultures, The Netherlands) for the reference strains and their professional advice. We also thank Qiang-Qiang ZHANG (Huashan Hospital, Fudan University, Shanghai), Yin-Zhong SHENG and Hong-Zhou LU (Public Heath Clinical Center, Fudan University, Shanghai), Qi-Qiang XIE (The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou), Li-Yan XI (The Second Affiliated of Sun Yat-sen University, Guangzhou) and Wan-Shan CHEN (No.8 People’s Hospital, Guangzhou) for contribution of clinical isolates from southeast China.

14.

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