Comparison of API20C with molecular identification of Candida spp isolated from bloodstream infections

Abstract

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

Candida species are important fungal pathogens in several patient populations, particularly when causing bloodstream infections. There have been recent reports on the misidentification of several candida species when using phenotypic identification techniques.^1,2 More recently, molecular identification techniques for fungi have been described, which allow for more accurate and rapid identification of Candida spp based on differences within the ribosomal RNA.³ Of these methods, polymerase chain reaction (PCR) amplification of the short internal transcribed spacer 2 (ITS2) region has been described as a rapid and reliable method to characterise yeast infections. At present, most laboratories in the UK use a combination of microscopic tests together with a commercial test system, such as the API20C, API32C, or API candida schema for the identification of clinically important yeasts. Previous work^4,5 showed that the API20C scheme was more reliable at identifying common (97%) and rare (88%) yeast isolates than the API32C scheme, which could reliably identify 92% of common yeasts and 85% of rare isolates, but there have been few data comparing the use of the API schemes with the recently described ITS sequence based identification methods. Therefore, this study aimed to carry out a comparison of the API20C identification scheme with molecular identification using the ITS rRNA regions. In addition, we wanted to evaluate laboratory parameters associated with these respective phenotypic and genotypic techniques, to help provide guidance on their advantages and disadvantages in diagnostic applications.

“Our study aimed to carry out a comparison of the API20C identification scheme with molecular identification using the internal transcribed spacer region rRNA regions”

MATERIALS AND METHODS

Candida spp isolates (n = 58) were obtained from the blood cultures of 58 patients with bloodborne candidiasis attending Belfast City Hospital during the period 1994 to 2000. All isolates were stored at −80°C in accordance with the method of Moore et al.⁶ Isolates were resuscitated from storage on to Sabouraud dextrose agar (Oxoid CM0041; Oxoid Ltd, Basingstoke, UK), and passaged at least twice before phenotypic and genotypic characterisation.

All DNA isolation procedures were carried out in a class II biological safety cabinet in a room geographically separate from that used to set up the reaction mixes and also from the “post-PCR” room to minimise the production of false positive results. Total genomic fungal DNA was extracted from a single colony using the Roche high purity PCR template kit (Roche Diagnostics, East Sussex, UK), in accordance with the manufacturer’s instructions, following pretreatment with lyticase (Boehringer Mannheim, East Sussex, UK), as described previously.⁷ All reaction mixes were set up in a PCR hood in a room separate from that used to extract DNA and the amplification and post-PCR rooms to minimise contamination. Reaction mixes (50 μl) were set up as follows: 10mM Tris/HCl, pH 8.3, 50mM KCl, 2.5mM MgCl₂, 200μM (each) dATP, dCTP, dGTP, and dTTP, 1.25 U of Taq DNA polymerase (Amplitaq; Perkin Elmer, Cheshire, UK), 0.2μM (each) of the ITS2 region primers (ITS3/ITS4; namely: ITS3, 5`-GCA TCG ATG AAG AAC GCA GC-3` and ITS4 (reverse) 5`-TCC TCC GCT TAT TGA TAT GC-3`, as described previously),³ and 4 μl of DNA template. After a “hot start”, the reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 96°C for three minutes, followed by 40 cycles of 96°C for one minute, 55°C for one minute, and 72°C for one minute, with a final extension at 72°C for 10 minutes. During each run molecular grade water (LAL Grade; Biowhittaker Inc, Walkersville, Maryland, USA) samples were included randomly as negative controls. Where there was disagreement between the phenotypic and genotypic result using the ITS2 region, a further PCR reaction was set up, amplifying the ITS1–5.8S–ITS2 region, using the primers ITS1 (forward) 5`-TCC GTA GGT GAA CCT GCG G-3` and ITS4 (reverse), as described above. After amplification, aliquots (15 μl) were removed from each reaction mixture and examined by electrophoresis (80 V, 45 minutes) in gels composed of 2% (wt/vol) agarose (Gibco, Paisley, UK) in TAE buffer (40mM Tris, 20mM acetic acid, 1mM EDTA, pH 8.3), stained with ethidium bromide (5 μg/100ml). Gels were visualised under ultraviolet illumination using a gel image analysis system (UVP Products, UK) and all images archived as digital (*.bmp) graphic files.

All amplicons were purified using a QIAquick PCR purification kit (Qiagen, Crawley, Sussex, UK) eluted in Tris/HCl (10mM, pH 8.5) before sequencing, particularly to remove dNTPS, polymerases, salts, and primers. For the short ITS2 region, the sequencing primer ITS3 was used in the forward direction and for the large ITS1–5.8S–ITS2 region, the ITS1 primer was used in the forward direction. In both cases, the sequencing primer was labelled with Cy-5` and was used in conjunction with the ALF Express II (Amersham-Pharmacia, Little Chalfont, Buckinghamshire, UK) using the Thermo Sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP (catalogue no: RPN 2438; Amersham Pharmacia Biotech) (96°°C for one minute, followed by 25 cycles of 96°C for 10 seconds, 50°C for five seconds, and 60°C for five seconds, followed by a 4°C hold). The resulting sequences obtained were compared with those stored in the GenBank Data system using BLAST alignment software (http://www.blast.genome.ad.jp/) and sequence homology identity determined in accordance with the criteria, as described previously by Goldenberger et al.⁸

Isolates were identified using the API20C scheme (bioMérieux, Les Halles, France), in accordance with the manufacturer’s instructions.

RESULTS AND DISCUSSION

Table 1 shows the phenotypic and genotypic characterisation of the isolates. Overall, there was only one disagreement between the phenotypic and genotypic identifications (isolate 23), where the API scheme identified this isolate as C albicans but the molecular method identified this as C dubliniensis. In this case, a larger section of the ITS1–5.8S–ITS2 region was sequenced to confirm the initial identification of the smaller ITS2 region.

Table 1.

Comparison of phenotypic and genotypic identification within a collection of 58 invasive Candida spp isolated over the seven year period, 1994–2000

			Small ITS region (ITS3/ITS4)
Isolate number	Phenotypic identification (API 20C)	Molecular identification	Base pairs	% Homology
1	C albicans	C albicans	295	100
2	C albicans	C albicans	299	100
3	C albicans	C albicans	290	100
4	C albicans	C albicans	303	99
5	C albicans	C albicans	302	99
6	C albicans	C albicans	293	99
7	C parapsilosis	C parapsilosis	276	99
8	C parapsilosis	C parapsilosis	267	99
9	C tropicalis	C tropicalis	295	100
10	C krusei	Issachenkia orientalis*	289	99
11	C krusei	Issachenkia orientalis*	271	99
12	C parapsilosis	C parapsilosis	263	99
13	C krusei	I orientalis*	271	99
14	C albicans	C albicans	294	100
15	C tropicalis	C tropicalis	289	99
16	C parapsilosis	C parapsilosis	277	99
17	C glabrata	C glabrata	320	99
18	C glabrata	C glabrata	332	100
19	C albicans	C albicans	393	100
20	C glabrata	C glabrata	344	100
21	C albicans	C albicans	264	100
22	C albicans	C albicans	289	99
23	C albicans	C dubliniensis	299	100
24	C albicans	C albicans	259	100
25	C albicans	C albicans	269	98
26	C albicans	C albicans	280	100
27	C glabrata	C glabrata	343	99
28	C albicans	C albicans	271	100
29	C albicans	C albicans	294	100
30	C albicans	C albicans	299	100
31	C albicans	C albicans	287	100
32	C glabrata	C glabrata	357	100
33	C glabrata	C glabrata	333	100
34	C glabrata	C glabrata	376	100
35	C parapsilosis	C parapsilosis	272	99
36	C glabrata	C glabrata	300	100
37	C parapsilosis	C parapsilosis	270	99
38	C parapsilosis	C parapsilosis	271	99
39	C tropicalis	C tropicalis	278	100
40	C dubliniensis	C dubliniensis	285	100
41	C albicans	C albicans	289	100
42	C albicans	C albicans	283	100
43	C albicans	C albicans	282	100
44	C albicans	C albicans	300	100
45	C albicans	C albicans	294	99
46	C albicans	C albicans	294	100
47	C glabrata	C glabrata	380	100
48	C albicans	C albicans	294	100
49	C albicans	C albicans	295	100
50	C glabrata	C glabrata	384	100
51	C albicans	C albicans	295	100
52	C albicans	C albicans	283	100
53	C glabrata	C glabrata	380	100
54	C glabrata	C glabrata	375	99
55	C albicans	C albicans	293	99
56	C glabrata	C glabrata	376	100
57	C parapsilosis	C parapsilosis	262	100
58	C albicans	C albicans	295	99

*I orientalis is the teleomorphic and sexual form of C krusei. All sequences entered in GenBank for C krusei have been renamed as I orientalis. Resulting sequences for I orientalis, C dubliniensis, C tropicalis, C glabrata, C parapsilosis, and C albicans were subsequently deposited in GenBank with the respective accession numbers, AF417255, AF430249, AF441197 AF441198, AF441199, and AF441200. …ITS, internal transribed spacer region.

Over the past few years, molecular techniques have greatly enhanced the diagnosis of causal agents of infectious disease, particularly when the causal agent is difficult to culture and identify.^9–11 The results from our present study suggest that such a molecular identification scheme may be useful in differentiating C dubliniensis from C albicans, but is of little benefit in relation to the other non-albicans Candida spp. In addition, molecular techniques may be of benefit for the rapid identification of yeasts. Table 2 compares various laboratory parameters of the API20C and PCR schema, as experienced in our study, in the identification of Candida spp.

Table 2.

Comparison of laboratory parameters of API20C and PCR sequencing techniques

Laboratory parameter	PCR sequence identification	API20C
Time to result
Culture time	–	72 hours
DNA extraction	1 hour
PCR amplification	3 hours
Agarose gel electrophoresis	15 minutes
Clean up of PCR product	—
Sequencing protocol	16 hours (overnight)
Total time	20 hours 15 minutes	72 hours5
Relative cost/isolate	High	Moderate
Ease of use	Complex	Simple
Repeatability	Excellent	Good
Subjectivity	Low	Moderate
Requirements for complex equipment	High	Low
Applicability to routine diagnostic laboratory	Poor	Good

Because previous studies have shown that C dubliniensis is often present in mixed cultures with C albicans,¹² it is important that routine diagnostic laboratories have the ability to differentiate between these two species. To date, the main problem has been in the selection of representative colonies of C dubliniensis from primary plates, before isolates are further characterised to the species level. There have been numerous reports of various phenotypic tests that have been used in an attempt to differentiate between these two species. These tests, including characteristic appearance on indicator media, chlamydospore formation, growth/restricted growth at raised temperature, carbohydrate assimilation, and intracellular β-D-glucosidase activity, have had varying degrees of success.¹² Initially, it was suggested that the use of CHROMagar was a suitable means of discrimination; however, subsequent studies have shown this phenotypic characteristic to be an unreliable marker.^12,13 Tintelnot and colleagues¹² found that a more reliable phenotypic test was growth at increased temperature (42°C), where 117 of 117 C albicans and 53 of 53 C dubliniensis isolates were able to grow and not grow, respectively. However, Tintelnot and colleagues¹² and other workers¹³ have shown that increased growth temperature cannot be used as the sole marker on all occasions.

“Molecular differentiation may be of increasing importance, given the emerging role of C dubliniensis in the current literature”

Take home messages.

• The API20C technology was compared with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region (ITS2) for the identification of 58 isolates of invasive candida species

• Overall, there was only one disagreement: the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis.

• Thus, the API20C method is useful in the identification of Candida spp isolated from blood culture and molecular methods do not enhance its use

• However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, we recommend that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically

Consequently, because of the variable phenotypic results and evidence of the presence of atypical C albicans organisms, various workers have examined the usefulness of molecular assays to aid in the discrimination of these two species.^14–17 Of these, the method of Elie and colleagues¹⁴ is particularly useful because it does not rely on colony picking from primary plates, and DNA can be extracted from the entire candida flora represented on the plate and differentiated by the use of specific probes. Such molecular differentiation may be of increasing importance, given the emerging role of C dubliniensis in the current literature.¹⁸

In conclusion, our study demonstrated that the API20C is a useful method in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance API20C identification. However, for correct reporting of C dubliniensis, we recommend that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

Abbreviations

• ITS, internal transcribed spacer region

• PCR, polymerase chain reaction