Abstract

Single-strand conformation polymorphisms (SSCP) of Candida albicans’ microsatellite CAI were characterized. Among the 76 clinical isolates recovered from different patients (independent strains), 60 distinct CAI SSCP patterns were recognized, resulting in a discriminatory power of 0.993. The multiple isolates recovered sequentially from the same or different body locations of the same patient showed exactly the same CAI SSCP pattern. The reliability of the SSCP analysis was confirmed by GeneScan and sequence analyses. From the same set of independent strains, 59 distinct CAI genotypes were identified by GeneScan analysis. Sequence comparison showed the advantage of SSCP over GeneSan analysis in the detection of point mutations in the microsatellite. The results indicated that PCR SSCP analysis of CAI microsatellite is a powerful and economical approach for rapid strain typing of C. albicans in clinical laboratories, especially in the detection of microevolutionary changes in microsatellites and in large-scale epidemiological investigation.

Introduction

Candida albicans is the most common opportunistic fungal pathogen of humans. It causes from benign infections such as oral and vaginal candidiasis to fatal, systematic disease in immunocompromised or critically ill patients. Nearly three-quarters of all healthy women experience at least one episode of candida vulvovaginitis during their lifetime and about 5% endure recurrent bouts of the disease 1, 2. Due to the growing population over the last two to three decades of transiently or permanently immunocompromised patients, invasive infections caused by C. albicans have become an increasingly important clinical problem, i.e., it is recognized as the fourth leading cause of nosocomial infections 3, 4. As a result of the difficulties in the early accurate diagnosis of systemic candidiasis, a limited number of suitable and effective antifungal drugs and the increasing drug resistance of the etiologic agents, mortality rates from systemic candidiasis remain high 5. In addition to improved therapy, the rapid and accurate identification of the disease-causing strains is crucial for diagnosis, clinical treatment and epidemiological studies.

A variety of methods for typing strains of C. albicans have been described, including electrophoretic karyotyping 6, restriction fragment length polymorphism analysis 7, 8, southern blot hybridization with discriminating probes 4, 9–12, randomly amplified polymorphic DNA (RAPD) analysis 13, and multilocus sequence typing 14, 15. In recent years, short tandem repeats (STRs) or microsatellites have been increasingly used as molecular markers for population genetics and genotyping of different organisms. Several polymorphic microsatellite loci have been identified in the genome of C. albicans16–20. Among them, the locus called CAI located in a noncoding region appeared to be the most polymorphic one exhibiting a discriminatory power of 0.97 19, 20.

The polymorphisms of microsatellite loci were usually designated by either the total lengths of the alleles 16, 17 or the total number of repeat units in the alleles 19, 20. The advantage of the quantitative designation is essential for reproducible, inter-laboratory comparisons and database construction. However, since the quantitative data can only be accurately determined by GeneScan analysis or sequencing with an automatic DNA sequencer, the use of the method is limited by the unavailability of the equipment in routine laboratories and by the high cost of the studies. Furthermore, the GeneScan analysis can not reveal the other two different levels of polymorphism that usually exist in microsatellite loci, i.e., the structure of the repeated region and point mutations in the sequence.

We have investigated an alternative approach to reveal the polymorphisms of microsatellites by utilizing the technique of single-strand conformation polymorphism (SSCP). This method was initially developed for point mutation detection in human DNA 21, 22. It has subsequently been demonstrated to be a powerful tool for gene mutation and variation analysis 23, 24 and has been used in species identification and strain typing of human and plant pathogenic fungi 25–29. The molecular markers of the previous studies were mainly rRNA gene fragments. The SSCP profiles of microsatellites remain to be characterized. The present study shows that SSCP technique is a powerful tool for rapid detection of microsatellite polymorphisms in C. albicans.

Materials and methods

Collection and identification of test isolates

A total of 123 clinical isolates of C. albicans, recovered from nine hospitals located in Beijing and Tianjing (northern China) and Guangdong (southern China), were employed in this study. Among them, 76 were isolated from different patients (independent strains) and the remaining 47 were sequentially collected from the same or different locations of 13 patients (Tables 1 and 2). C. albicans strain ATCC 90028 was used as a reference and species identification of all the clinical isolates was confirmed by 26S rRNA gene D1/D2 domain sequence analysis 30.

Table 1

CAI genotypes of independent Candida albicans strains.

Observed Genotype No. of strains Specimen Frequency 
11–11 Urine 0.013 
11–17 Sputum 0.025 
11–18* Sputum 0.013 
11–21 Sputum 0.013 
12–12 Feces 0.013 
12–19 Feces 0.013 
12–21 Sputum, blood 0.025 
12–22 Sputum 0.025 
16–16 Sputum 0.038 
16–18 Choler 0.013 
16–19 Peritoneal fluid 0.013 
16–23 Sputum 0.013 
16–24 Sputum 0.013 
16–28 Sputum 0.013 
16–30 Cancer tissue 0.013 
16–37 Feces 0.013 
17–17* Sputum 0.025 
17–20 Sputum 0.013 
17–21* Sputum 0.038 
17–23* Sputum 0.013 
17–26 Sputum 0.013 
17–34 Sputum 0.013 
18–18* Sputum, feces 0.025 
18–19 Feces 0.013 
18–25* Sputum 0.025 
18–26 Sputum 0.025 
18–27* Sputum 0.025 
18–31 Feces 0.013 
18–40 Perineum 0.013 
19–19 Sputum 0.013 
19–28 Sputum 0.013 
20–20* Sputum 0.013 
20–25 Sputum 0.013 
20–31 Wound 0.013 
21–21* Sputum 0.013 
21–34 Sputum, skin 0.025 
22–26 Sputum 0.013 
23–23 Sputum 0.013 
23–26 Sputum 0.025 
23–28 Sputum 0.013 
23–30 Sputum 0.013 
25–25* Urine 0.013 
25–28 Oral mucosa 0.013 
25–32 Sputum 0.013 
25–33 Sputum, pleural fluid 0.025 
25–34 Sputum, skin, perineum 0.038 
26–28 Sputum 0.013 
26–33 Feces 0.013 
26–34 Sputum 0.013 
26–45 Sputum 0.013 
27–27* Feces 0.013 
27–34 Sputum 0.013 
27–45 Perineum 0.013 
28–28* Choler 0.013 
30–45 Perineum 0.013 
30–46 Sputum 0.013 
31–45 Sputum 0.013 
32–44 Sputum 0.013 
34–34 Sputum 0.013 
Observed Genotype No. of strains Specimen Frequency 
11–11 Urine 0.013 
11–17 Sputum 0.025 
11–18* Sputum 0.013 
11–21 Sputum 0.013 
12–12 Feces 0.013 
12–19 Feces 0.013 
12–21 Sputum, blood 0.025 
12–22 Sputum 0.025 
16–16 Sputum 0.038 
16–18 Choler 0.013 
16–19 Peritoneal fluid 0.013 
16–23 Sputum 0.013 
16–24 Sputum 0.013 
16–28 Sputum 0.013 
16–30 Cancer tissue 0.013 
16–37 Feces 0.013 
17–17* Sputum 0.025 
17–20 Sputum 0.013 
17–21* Sputum 0.038 
17–23* Sputum 0.013 
17–26 Sputum 0.013 
17–34 Sputum 0.013 
18–18* Sputum, feces 0.025 
18–19 Feces 0.013 
18–25* Sputum 0.025 
18–26 Sputum 0.025 
18–27* Sputum 0.025 
18–31 Feces 0.013 
18–40 Perineum 0.013 
19–19 Sputum 0.013 
19–28 Sputum 0.013 
20–20* Sputum 0.013 
20–25 Sputum 0.013 
20–31 Wound 0.013 
21–21* Sputum 0.013 
21–34 Sputum, skin 0.025 
22–26 Sputum 0.013 
23–23 Sputum 0.013 
23–26 Sputum 0.025 
23–28 Sputum 0.013 
23–30 Sputum 0.013 
25–25* Urine 0.013 
25–28 Oral mucosa 0.013 
25–32 Sputum 0.013 
25–33 Sputum, pleural fluid 0.025 
25–34 Sputum, skin, perineum 0.038 
26–28 Sputum 0.013 
26–33 Feces 0.013 
26–34 Sputum 0.013 
26–45 Sputum 0.013 
27–27* Feces 0.013 
27–34 Sputum 0.013 
27–45 Perineum 0.013 
28–28* Choler 0.013 
30–45 Perineum 0.013 
30–46 Sputum 0.013 
31–45 Sputum 0.013 
32–44 Sputum 0.013 
34–34 Sputum 0.013 
*

These genotypes are shared with those observed in the Portuguese C. albicans strains 19.

Table 2

CAI genotypes of multiple strains isolated sequentially from the same patient.

Patient Specimen No. of isolates CAI genotype 
Sputum 21–21 
Sputum 11–21 
Sputum 26–28 
Vaginal exudate 18–26 
 Sputum 18–26 
Scrotum 30–46 
 Hand 30–46 
Sputum 26–34 
Pleural fluid 17–21 
 Urethral exudate 17–21 
 Blood 17–21 
 Urine 17–21 
Sputum 21–23 
Sputum 18–18 
Sputum 10 11–11 
Sputum 11–18 
Sputum 23–23 
Sputum 11–17 
Patient Specimen No. of isolates CAI genotype 
Sputum 21–21 
Sputum 11–21 
Sputum 26–28 
Vaginal exudate 18–26 
 Sputum 18–26 
Scrotum 30–46 
 Hand 30–46 
Sputum 26–34 
Pleural fluid 17–21 
 Urethral exudate 17–21 
 Blood 17–21 
 Urine 17–21 
Sputum 21–23 
Sputum 18–18 
Sputum 10 11–11 
Sputum 11–18 
Sputum 23–23 
Sputum 11–17 

DNA extraction and PCR

Nuclear DNA was extracted in general accord with the method described by Kaiser et al. 31. The microsatellite locus CAI was amplified by PCR using a pair of primers (forward, 5′ -ATG CCA TTG AGT GGA ATT GG -3′; reverse, 5′ -AGT GGC TTG TGT TGG GTT TT -3′) according to Sampaio et al. 19. For GeneScan analysis, the forward primer was 5′ fluorescently labeled with 6-carboxyfluorescein. PCR amplification was performed in a thermocycler (ICycler, Bio-Rad, Hercules, CA) with a program consisting of an initial denaturing step at 95°C for 4 min; 33 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and extension at 72°C for 1 min; and a final extension step of 7 min at 72°C.

SSCP analysis

In order to obtain an equal amount of DNA loading for SSCP electrophoresis, the PCR product was first electrophoresed on agarose gel and the amount of DNA was estimated by its band intensity. Three to five microliters of PCR products (approximately 100 ng DNA) were mixed with the same volume of denaturing loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05% bromophenol blue). Mixtures were heated at 95°C for 10 min and then chilled on ice. Denatured PCR products were loaded on an 8% acrylamide:bis (29:1) nondenaturing gel (200×200×0.75 mm) cast using the set provided in the DCode Universal Mutation Detection System (Bio-Rad, Hercules, CA). Electrophoresis was performed in the same system in pre-chilled 1×TBE buffer (89 mM Tris-borate, 2 mM EDTA, pH 8.0) at 240 V for 10 h at 10°C.

After electrophoresis, silver staining of the gel was carried out, with minor modifications, using the procedure reported by Beidler et al. 32. Specifically, the polyacrylamide gel was peeled off from the glass plate, soaked in 300 ml of 10% ethanol for 5 min and then in the same volume of 0.05% acetic acid solution for 5 min. After one brief wash with 300 ml dH2O, the gel was soaked in 300 ml of 0.1% (w/v) silver nitrate for 15 min and then washed two times with 300 ml dH2O. The gel was developed by rinsing in 300 ml of 1 ppm formaldehyde in 1.5% sodium hydroxide solution. When the desired intensity was reached the gel was washed with 300 ml of dH2O and fixed in 0.75% sodium carbonate.

GeneScan analysis

The fluorescently labeled PCR products were run in an ABI 370 genetic analyzer (Applied Biosystem, Foster City, CA). Fragment sizes were determined automatically using the GeneScan 3.7 analysis software.

Cloning and sequencing

The purified PCR products of CAI alleles from three strains (H1, J1 and K1) were cloned into the PUCm-T vector (Bio Basic Inc., Ontario, Canada) and transformed in Escherichia coli DH5α cells according to the manufacturer's instructions. For each strain studied, two to three positive clones containing the target fragments confirmed by colony PCR were selected for plasmid proliferation, extraction and sequencing. Plasmids were sequenced with the universal primers M13F and M13R using the ABI BigDye terminator cycle sequencing kit (Applied Biosystem, Foster City, CA).

Results

SSCP analysis

The fragments of CAI alleles from all the C. albicans strains in this study were successfully amplified by PCR. Due to the diploid nature of C. albicans, double CAI fragments were usually amplified from heterozygous isolates which were dominant (77.6%) in the test population. When a single fragment was obtained, the strain was regarded as being homozygous.

Most of the independent C. albicans strains were clearly distinguished from each other by SSCP analysis of their PCR products of the CAI microsatellite. Three to six major bands (those with heavier relative intensity) and a few paler, minor bands were usually found in the lanes of the SSCP analysis gels (Fig. 1). The SSCP patterns of CAI fragments amplified from 19 randomly selected strains and reference strain ATCC 90028 are shown in Fig. 1A. Among these strains as compared in this gel, 16 distinct genotypes were identified, with lanes 2, 11 and 12 having identical SSCP patterns with those in lanes 16, 19 and 14, respectively.

Fig. 1

PCR SSCP patterns of CAI microsatellite of Candida albicans strains: (A) 19 randomly selected independent clinical strains showing 16 distinct genotypes (lanes 2, 11 and 12 share the same genotype with lanes 16, 19 and 14, respectively; lanes 9, 13 and 15 are homozygous strains and the others are heterozygous strains); (B) multiple strains from the same patient (the sources of the strains are listed in Table 2); and (C) strains with the same CAI genotype 17–21 determined by GeneScan analysis. M, reference strain ATCC 90028.

Fig. 1

PCR SSCP patterns of CAI microsatellite of Candida albicans strains: (A) 19 randomly selected independent clinical strains showing 16 distinct genotypes (lanes 2, 11 and 12 share the same genotype with lanes 16, 19 and 14, respectively; lanes 9, 13 and 15 are homozygous strains and the others are heterozygous strains); (B) multiple strains from the same patient (the sources of the strains are listed in Table 2); and (C) strains with the same CAI genotype 17–21 determined by GeneScan analysis. M, reference strain ATCC 90028.

In the SSCP profiles, heterozygous strains usually showed six major bands, corresponding to two double-stranded and four single-stranded bands. In contrast, homozygous strains generally exhibited three major bands corresponding to one double-stranded and two single-stranded bands (e.g. Fig. 1A lanes 9, 13 and 15). The SSCP patterns of different isolates were mainly distinguished by the number and relative positions of the major bands. The minor bands were probably formed by the denatured CAI fragments that folded differently in the nondenaturing gel. They may contribute to further differentiation of C. albicans strains with similar patterns of the major bands (data not shown). Although the lanes near both edges of the gel were usually slightly curved, the banding patterns were comparable to the straight lanes near the center of the gel without artificial correction (e.g., lanes 11 and 19 in Fig. 1A). Using the CAI SSCP pattern of strain ATCC 90028 as the reference in comparisons among different gels, 60 distinct genotypes were recognized in the 76 independent clinical strains. The discriminatory power calculated according to the method of Hunter and Gaston 33 was 0.993.

The multiple isolates recovered sequentially at different intervals (from 3–90 days) from sputum samples of the same patient showed exactly the same CAI SSCP patterns (e.g. isolates from patients A, B, C and F in Fig. 1B). These results suggest that only one strain of C. albicans is present and maintained in the sampling body site of the same patient. This is in agreement with a previous study that found that multiple isolates from the same body location of the same patient possessed the same CAI genotypes 19. In addition, multiple strains isolated from different body locations of a single patient also exhibited the same CAI SSCP patterns (e.g. isolates from patients D, E and G in Fig. 1B), indicating that the infecting population may have dispersed to different sites of a host.

GeneScan analysis

In order to investigate the reliability and specificity of SSCP analysis, the fragment sizes of the amplified CAI alleles from all the isolates compared in this investigation were determined by GeneScan analysis (Tables 1 and 2). The CAI genotypes of the isolates were designated by the number of trinucleotide repeat units according to the method described by Sampaio et al. 19. Among the 76 independent clinical strains, a total of 24 different alleles varying in sizes between 189 bp (11 repeats) to 294 bp (46 repeats) and 59 distinct genotypes were recognized. Except for K1, strains exhibiting different SSCP patterns had different CAI genotypes, while strains with identical SSCP patterns, including unrelated strains and multiple isolates recovered from the same patients, had exactly the same genotypes.

Among the 59 CAI genotypes identified from 76 non-related clinical C. albicans strains from China and the 44 CAI genotypes observed from 73 non-related strains of the species from north Portugal 19, only 12 genotypes were shared among the isolates in these two countries (Table 1). This result implies that the genotype distribution of C. albicans strains may not be the same in different countries or regions.

Sequence comparison

GeneScan analysis showed that strain K1 possessed the same CAI genotype 17–21 with strains H1 and J1 and the same size of their corresponding CAI alleles. However, PCR SSCP analysis revealed that the CAI SSCP pattern of strain K1 was clearly different from those of strains H1 and J1 (Fig. 1C). The reason for this inconsistency was investigated by sequence comparison and the result showed that the lengths of the corresponding CAI alleles of the three strains were identical. However, in CAI-21 allele, strain K1 differed from strains H1 and J1 by 3 nucleotide substitutions located inside and outside the repeated regions (Fig. 2).

Fig. 2

The sequence alignment of CAI-21 allele of three Candida albicans strains. At a given position, a nucleotide identical to that in the top line is indicated by a dot.

Fig. 2

The sequence alignment of CAI-21 allele of three Candida albicans strains. At a given position, a nucleotide identical to that in the top line is indicated by a dot.

Discussion

The present study demonstrated that PCR-SSCP analysis of CAI microsatellite provides a powerful approach for strain typing of C. albicans. The SSCP analysis could distinguish not only the CAI microsatellite alleles with different sizes (Fig. 1A), but also the alleles with point mutations (Fig. 1C). The microsatellites differing in structure of the repeated region have different sequences and theoretically can readily be detected by SSCP analysis 21, 22. Therefore, SSCP technique can reveal three different levels of polymorphism in CAI microsatellite simultaneously, i.e., (i) the total lengths; (ii) the structure of the repeated regions, and (iii) point mutations inside or outside the repeat regions. Point mutations have been observed in several CAI alleles 19. CAI-21a (AY693676) and CAI-21b (AY693677) differ by two point mutations whereas CAI-32a (AY693687) differs from CAI-32b (AY693688) by four point mutations. Two new subtypes of CAI-21 allele, CAI-21c (EF203895) and CAI-21d (EF203896) were identified in the present study. These differed from each other and from the other two previously reported subtypes by 3–6 point mutations. A new subtype of CAI-25 allele (EF203897) was also recognized, which differed by 4 nucleotide substitutions from the other subtype of the same allele (AY693681) reported by Sampaio et al. 19. Since GeneScan analysis is unable to detect the point mutations occurring in the same alleles, a higher discriminatory power can be achieved by using SSCP analysis in C. albicans strain typing as shown in the present study. In addition to strain typing, point mutation detection is valuable for the identification of fine microevolutionary changes in C. albicans which have occurred in response to environmental stress conditions.

The high discriminatory power and simplicity of use make the CAI SSCP analysis a rather competitive method among the image-based techniques that have been commonly used in C. albicans genotyping, e.g. RAPD analysis, multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization 10, 11, 13, 34. The main disadvantages of the image-based strain typing methods are the limitations in making comparisons among different gels and in the establishment of databases which would allow for easy data sharing among investigators in different laboratories. Comparison of SSCP patterns between different gels can be achieved by employing a reference strain (as in this study) or a DNA ladder 26 and by standardizing the parameters of the SSCP analysis procedure, including PCR amplification, denaturation, electrophoretic conditions (including buffer composition, gel matrix, gel temperature during electrophoresis and DNA concentration) and detection.

SSCP analysis can be performed in the same apparatus as used for polyacrylamide gel electrophoresis 26 and radioactive labeling is not necessary. Moreover, the PCR products of CAI microsatellite alleles are amplified from a specific locus using a pair of highly specific primers. As a result, the SSCP profiles are more specific, reproducible and predictive. The stability of the CAI marker has been demonstrated by Sampaio et al. 19, 20 and reconfirmed in our study. The identical SSCP profiles of multiple strains from the same patients showed the stability of the microsatellite in vivo (Fig. 1B). In vitro experiment have shown that after daily subculturing for one month (over 400 generations), multiple randomly selected colonies of strain ATCC 90028 exhibited the same CAI SSCP pattern as the original strain (data not shown). Though GeneScan analysis is necessary for CAI genotype comparison of C. albicans among different laboratories in different regions, PCR SSCP analysis of CAI microsatellite can be used as an economically valuable alternative method for rapid strain typing of C. albicans in routine clinical laboratories, especially in the detection of point mutation and microevolutionary changes in microsatellites, and in large-scale epidemiological investigation.

Acknowledgements

We thank Dr Yingchun Xu, Peking Union Medical College Hospital, Beijing, Dr Fang Li, Chaoyang Hospital, Beijing, Dr Hao Wu, Youan Hospital, Beijing, Dr Dongming Li, the Third Hospital of Peking University, Beijing, Dr Hongqi Wang, the PLA 302 Hospital, Dr Jiangang Jin, PLA 307 Hospital, Dr Hong Lei, PLA 309 Hospital, Dr Zhenhua Nie, Changzheng Hospital, Tianjin, and Dr Wenming Huang, the Applied Hospital of Guangdong Medical College, Zanjiang, Guangdong, for their kind supply of clinical strains and related clinical records. This study was supported by grants 2004BA720A05-02 from the Key Technologies R&D Program of the Ministry of Science and Technology, China, and 30570098 from the National Natural Science Foundation of China (NSFC).

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