Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology
Eun Jeong Won
Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
Corresponding to Eun Jeong Won, E-mail: ejwon@amc.seoul.kr
Ann Clin Microbiol 2024;27(4):245-255. https://doi.org/10.5145/ACM.2024.27.4.4
Received on 1 September 2024, Revised on 4 November 2024, Accepted on 14 November 2024, Published on 16 December 2024.
Copyright © Korean Society of Clinical Microbiology.
This is an Open Access article which is freely available under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/).
Candidemia is the most common healthcare-associated invasive fungal infection with high crude mortality rates. It primarily affects critically ill or severely immunocompromised patients, complicating early diagnosis and prompting the initiation of appropriate antifungal therapy. The gold standard for diagnosing candidemia is blood culture; however, the sensitivity of this test is low and requires at least two days for species identification. These limitations have led to the development of alternative diagnostic methods that are more sensitive and have shorter turnaround times. Here, we review the currently available methods for the nonculturebased diagnosis of candidemia, including (i) immunological diagnostics targeting Candida-related antigens, (ii) human antibodies to Candida-related antigens, and (iii) molecular diagnostics. The strengths, uses, and limitations of each methodology are discussed. Immunological diagnostics targeting Candida-related antigens and human antibodies to these antigens provide supportive evidence for diagnosing candidemia. Advances in molecular diagnostics have shown promising results in facilitating early candidemia diagnosis, potentially improving patient outcomes.
Beta-glucans, Candidemia, Galactomannan, Immunological tests, Molecular diagnostic techniques
Candidemia is the most common healthcare-associated invasive fungal infection [1] and is associated with high morbidity and mortality rates [2,3]. Its increasing incidence has been observed in intensive care units (ICUs), particularly among immunocompromised patients, those treated with broad-spectrum antibiotics, and those requiring invasive procedures and devices [4]. Candidemia is the fifth leading cause of healthcareassociated bloodstream infections (BSIs) in ICUs in European countries. Furthermore, Candida species have been reported as the second most prevalent pathogens causing healthcare-associated BSIs in the United States and South Korea [5,6]. Blood culture is the gold standard for diagnosing candidemia [7]. However, the definitive treatment of candidemia is often delayed because of the limited sensitivity of this method. While blood cultures are essential for performing antifungal susceptibility testing, their overall sensitivity for detecting invasive candidiasis is only approximately 50%, and results may take up to five days to obtain [8]. These unmet requirements have prompted efforts to develop alternative diagnostics with higher sensitivities and faster turnaround times, including the development and validation of nonculture diagnostic tests for candidemia [9]. Over the last 10 years, various nonculture-based methods have been devised to overcome the limitations of conventional culture-based diagnostics. These include (i) immunological diagnostics targeting Candida-related antigens; (ii) the use of human antibodies against Candida-related antigens, such as C. albicans germ tube antibodies and anti-mannan antibodies; and (iii) molecular diagnostics (Table 1). This review aims to provide comprehensive and up-to-date information on the currently available methods for nonculture-based diagnostics of candidemia.
Table 1. Characteristics of nonculture-based diagnostics for candidemia
| Method | Target or principle of assay | Advantage | Shortcoming |
|---|---|---|---|
| Antigen detection-based immunological assay | Candida (1,3)-β-D-glucan | Rapid, high sensitivity, high negative predictive value | Low specificity, high false-positive |
| Candida mannan | Rapid, earlier diagnosis of candidemia | Low sensitivity | |
| Antibody-based immunological assay | Anti-mannan Ab; C. albicans germ tube antibody | Rapid, enhancing performance in combination with antigen testing | Low sensitivity, low specificity |
| Nucleic acid amplification-based molecular assay | Conventional PCR; nested PCR; multiplex PCR; real-time PCR; T2 magnetic resonance | Highly sensitivity, high specificity | Expensive, multiple step, potential false-negative out of specific targets within the panel |
BDG is a cell wall polysaccharide in various medically necessary fungi, including Candida species, but not in Cryptococcus and Mucorales. Several commercial assays capable of detecting circulating
BDG have been developed. These include the Fungitell® assay (Associates of Cape Cod), Fungitec-G assay (Seikagaku Kogyo Corporation), Wako test (Wako Pure Chemical Industries), and Dynamiker Fungus assay (Dynamiker Biotechnology Ltd.) (Table 2). These assays are based on the ability of BDG to activate the horseshoe crab proteolytic coagulation cascade [10]. Each test has different interpretive cutoff values: > 80 pg/mL for the Fungitell and Glucatell assays and > 20 pg/mL for the Fungitec-G assay; values > 20 pg/ mL are considered positive. For the Wako assay and Dynamiker Fungus test, the cutoff values are 11 and > 95 pg/mL, respectively. This may have resulted from the reagents being sourced from different horseshoe crab species, with Fungitell reagents derived from Limulus polyphemus, and Fungitec and Wako reagents obtained from Tachypleus tridentatus. A meta-analysis reported pooled sensitivity and specificity values of BDG of 0.81 (0.75–0.86) and 0.64 (0.55–0.71), respectively (Table 2) [11]. These widely dispersed values can mainly be attributed to the heterogeneity within and between evaluations, which differs for the various BDG assays, the positive cutoff criteria used, the patient and control populations tested, and the number of BDG tests performed per individual. Several studies have explored diagnosis-driven therapies based on BDG detection. However, the diagnostic accuracy of BDG testing appears to be insufficient to inform treatment decisions [12–14] reliably. Nevertheless, the guidelines of the 3rd European Conference on Infections in Leukemia held in 2009 indicate that repeated positive BDG results may be used as supportive evidence for the presence of invasive fungal infections among patients with prolonged neutropenia who present with symptoms consistent with the infection [15]. Several factors should be considered when interpreting the BDG results. The existing assays can produce false-positive results in high-risk populations due to various potential sources of contamination, including human blood products (coagulation factors, immunoglobulins, albumin, and plasma protein fractions), surgical gauzes or other glucan-containing materials, hemodialysis, high levels of triglycerides, certain antibiotics that include intravenous amoxicillin-clavulanic acid or piperacillin-tazobactam, and systemic bacterial infections [16–20]. BDG test performance aspects include high sensitivity, high false-positive rate, low specificity, and high negative predictive value (Table 1). Thus, it is recommended that this test be used to rule out the disease and discontinue empirical antifungal treatment in ICU patients with suspected invasive candidiasis.
Table 2. Characteristics of several immunological diagnostics for candidemia
| General target | Function | Available commercial kit | Overall sensitivity (95% CI) | Overall specificity (95% CI) | Reference |
|---|---|---|---|---|---|
| Candida (1,3)-β-D-glucan | Candida Cell wall component | Fungitell; Wako; Fungitec-G; Dynamiker Fungus | 0.81 (0.75-0.86) | 0.64 (0.55-0.71) | [11] |
| Candida mannan | Candida Cell wall component | Platelia Candida Ag-Plus and Ab-Plus; Serion Mannan kit | 0.58 (0.53-0.62) | 0.93 (0.91-0.94) | [22] |
| Anti-mannan antibody | Antibodies to Candida polysaccharides | Platelia Candida Ab-Plus | 0.59 (0.54-0.65) | 0.83 (0.79-0.87) | [22] |
| C. albicans germ tube antibody (CAGTA) | Antibodies to Candida protein extract | Invasive Candidasis (CAGTA) IFA IgG assays; VirClia IgG Monotest | 0.66 (0.59-0.73) | 0.76 (0.58-0.88) | [32] |
Abbreviation: CI, confidence interval.
Mannans are key cell wall components of Candida spp. They participate in innate and acquired immunity, and are used as biomarkers [21]. Candida mannan can be measured in the serum or plasma of patients with candidemia using a latex agglutination test or sandwich enzyme-linked immunosorbent assay. Commercial assays, such as Platelia Candida Ag-Plus and Ab-Plus (Bio-Rad) and Serion Mannan kits (Serio GmbH) are available. The pooled sensitivity and specificity of mannan tests are reportedly 0.58 (0.53–0.62) and 0.93 (0.91–0.94), respectively (Table 2) [22]. Similar to protein antigen detection assays, mannan assays have low diagnostic sensitivity for candidemia (Table 1) [23]. This is mainly because of the high immunogenicity of mannan, which is rapidly cleared from circulation. They may also form immune complexes with circulating anti-mannan antibodies, which complicates their assessment [24]. The sensitivity and specificity of the mannan assay vary [22]. However, almost all the relevant studies have reported mannan-positive blood cultures, thus allowing for an earlier diagnosis of candidemia. The sensitivity of the mannan assay varies according to different Candida spp. and is highest for C. albicans, followed by C. glabrata and C. tropicalis [25,26]. It was lower in cases of C. parapsilosis and Pichia kudriavzevii, probably because of the lower amount of the mannan produced and released by these species [27,28]. Current guidelines of the European Society of Clinical Microbiology and Infectious Diseases recommend the combined use of mannan antigen and anti-mannan antibody quantification assays and serial determinations for both assays for diagnosing candidemia and chronic disseminated candidiasis [29]. In a meta-analysis of 14 studies [22], the sensitivity and specificity of anti-mannan IgG antibody for invasive candidiasis were 0.59 (0.54–0.65) and 0.83 (0.790.87), respectively (Table 2). The sensitivity and specificity for a combined mannan/anti-mannan assay were 0.83 and 0.86, respectively, with the best performance in patients with C. albicans, C. glabrata, or C. tropicalis infections. Several studies have shown that combining these assays with BDG or mannan detection assays may enhance diagnostic accuracy (Table 1) [30]. Because BDG is nonspecific, additional positive results for mannan or anti-mannan antibodies may indicate fungal disease due to Candida, whereas additional negative results for mannan or anti-mannan antibodies may indicate infection caused by other fungi.
The response to the hyphal wall protein 1 mycelial phase antigen of C. albicans, which is essential for biofilm development and tissue invasion, has been studied for the serodiagnosis of invasive candidiasis [31]. Originally termed CAGTA, reflecting the original design for C. albicans, subsequent research has demonstrated that other Candida species, including C. tropicalis, C. parapsilosis, C. glabrata, C. dubliniensis, C. guilliermondii, and P. kudriavzevii, can also produce CAGTA to a greater or lesser degree [32]. There are two commercial CAGTA assays: an indirect immunofluorescence test [IC (CAGTA) IFA IgG, Vircell Microbiologists] and an indirect chemiluminescent immunoassay [IC (CAGTA) VirClia®, Vircell Microbiologists]. The overall sensitivity and specificity of CAGTA has been reported as 0.66 (0.59–0.73) and 0.76 (0.58–0.88) (Table 2) [32]. Wei et al. [32] suggested that the accuracy of CAGTA is marginal and that the results should be used as part of a full assessment of the clinical features, imaging findings, and other laboratory results for diagnosing candidemia. Furthermore, CAGTA detection does not specify the fungal genus, limiting the use of targeted treatments in practice.
Molecular amplification techniques enable rapid and sensitive detection and identification by directly analyzing small amounts of fungal DNA in clinical samples, thereby eliminating the need for prior cultivation (Table 1). This direct analysis feature makes these tests particularly advantageous for early diagnosis of candidemia, especially in cases often missed by culture methods. Various polymerase chain reaction (PCR) assays have been developed to target different genetic sequences, including 18S, 28S, and 5.8S ribosomal DNA (rDNA), internal transcribed spacer regions, and mitochondrial DNA, to detect various fungi across different specimens. Numerous commercial PCR-based assays have been designed and evaluated to detect Candida DNA in patients at risk of candidemia. The multiplex platforms have been applied, such as the eplex® BCID FP Panel (GenMark DX), CandID (Olm Diagnostics), Fungiplex Candida (Bruker Daltonik), LightCycler® SeptiFast (Roche Diagnostics), Magicplex Sepsis (Seegene), FilmArray BCID2 Panel (bioMérieux), and T2Candida (T2 Biosystems) (Table 3).
Table 3. Characteristics of several molecular diagnostics for candidemia approved by Conformité Européenne/ In Vitro Diagnostic or Food and Drug Administration
| Commercial kit | Manufacturer | Detectable Candida species | Needs of nucleic acid extraction | Principle | Available specimen | Assay time | Reference |
|---|---|---|---|---|---|---|---|
| eplex® BCID FP Panela) | GenMark Dx | C. albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, P. kudriavzevii, C. lusitaniae, C. parapsilosis, C. tropicalis, C. auris | yes | Multiplex PCR | Blood culture positive sample | 90 min | [33] |
| CandID | OLM Diagnostics | C. albicans, C. dubliniensis, C. glabrata, P. kudriavzevii, C. parapsilosis, C. tropicalis | yes | Multiplex qPCR | Blood, synthetic bronchial aspirate lavage | 4 h | [36] |
| Fungiplex Candida | Bruker Daltonics | C. glabrata, Candida species (C. albicans, C. dubliniensis, C. parapsilosis, C. tropicalis), P. kudriavzevii | yes | Multiplex real-time PCR | Blood | < 2 h | [37] |
| SeptiFast Test kit | Roche Diagnostics | C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, P. kudriavzevii | yes | Multiplex real-time PCR | Blood, sterile fluid, tissue, swab | 6 h | [37] |
| Magicplex Sepsis | Seegene | C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, P. kudriavzevii | yes | Multiplex PCR | Blood | 6 h | [47] |
| FilmArray BCID Panel | bioMérieux | C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, P. kudriavzevii | no | Multiplex PCR | Blood culture positive sample | 60 min | [43] |
| FilmArray BCID2 Panel | bioMérieux | C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, P. kudriavzevii, C. auris, Cryptococcus neoformans/C. gattii | no | Multiplex PCR | Blood culture positive sample | 60 min | [44] |
| T2 Candida Panel | T2 Biosystems | C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, P. kudriavzevii | no | Multiplex PCR followed by automated T2MR-based detection | Blood | < 5 h | [45,46] |
a)The ePlex® BCID assay was rebranded as Cobas® eplex Blood Culture Identification Panels (Roche Diagnostics, Basel, Switzerland) in 2021.
Abbreviations: qPCR, quantitative PCR; T2MR, T2 magnetic resonance.
The ePlex® BCID (GenMark DX) assay, now rebranded as Cobas® eplex Blood Culture Identification Panels (Roche Diagnostics), consists of gram-positive (BCID-GP), gram-negative (BCID-GN), and fungal (BCID FP) panels. These panels detected 56 unique bacterial and fungal targets (11 Candida spp., Cryptococcus gattii, C. neoformans, Fusarium, and Rhodotorula) within 90 min [33]. Zhang et al. [34] tested 866 clinical samples and found that the sensitivity and specificity for detecting Candida species ranged from 0.97 to 1.0 and 0.99 to 1.0, respectively. Furthermore, this panel detects C. auris, which has emerged globally as a multidrug-resistant yeast that causes infections and outbreaks in healthcare facilities [35]. The CandID® (Olm Diagnostics) detects C. albicans, C. glabrata, C. parapsilosis, P. kudriavzevii, C. dubliniensis, and C. tropicalis. Rapid time-to-result with nucleic acid extraction through PCR amplification and result interpretation were completed within < 4 h [36]. Price et al. [36] reported that prospective testing generated an overall sensitivity and specificity of 0.88 and 0.82, respectively. The Fungiplex® Candida IVD PCR Kit (Bruker Daltonik, Billerica) detects C. glabrata, P. kudriavzevii, and Candida spp. (including C. albicans, C. parapsilosis, C. tropicalis, and C. dubliniensis) in whole blood, plasma, and serum within 2 h. Kits from Qiagen and bioMérieux are recommended for DNA extraction. The assay manuals provide instrument settings for the different thermocyclers. In a small prospective study on ICU patients, the Fungiplex® Candida revealed a sensitivity of 1.0, a specificity of 0.94, and a diagnostic accuracy of 0.94 [37]. The LightCycler® SeptiFast (Roche Diagnostics) was the first real-time PCR-based system to receive a Conformité Européenne mark for simultaneous pathogen detection and identification in suspected BSIs [38]. A meta-analysis of 54 studies comparing SeptiFast with blood culture found that this assay had an estimated summary sensitivity of 0.65 and specificity of 0.86 [39]. This suggests that a positive blood test at the genus/species level returned by SeptiFast may provide a higher diagnostic value (rule-in) than a negative test result (rule-out) when compared to blood culture [40]. The MagicplexTM Sepsis Real-time Test (Seegene Inc.) can detect 90 pathogens at the genus level and 27 pathogens, including five Candida spp. (C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, P. kudriavzevii), at the species level within 6 h of whole blood collection. In this analysis, DNA pretreatment and extraction are followed by conventional PCR for amplicon generation. If amplicons are detected, conventional PCR is followed by two real-time PCRs for screening and species level identification. Denina et al. [41,42] compared the MagicplexTM test to blood cultures of 150 samples from 89 patients. Candida spp. were detected by the MagicplexTM in four samples, of which only one was accompanied by a positive blood culture. Further evaluation of the clinical performance of this kit is warranted. The FilmArray BCID Panel (bioMérieux) is a nested multiplex PCR system that detects 24 pathogens, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavzevii, within 1 h. In a study involving clinical and spiked samples, this panel demonstrated a sensitivity of 0.99 and a specificity of 0.99 for Candida species compared to conventional cultures [43]. In a study involving clinical and spiked samples, this panel demonstrated a sensitivity and specificity of 0.99 for Candida species compared to conventional cultures [43]. The recently updated FilmArray® BCID2 Panel (bioMérieux) has 43 targets, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, P. kudriavzevii, C. auris, and C. neoformans/ C. gattii. For Candida species, the BCID2 Panel demonstrated a sensitivity of 0.92 and specificity of 0.995 [44]. The T2Candida® assay (T2 Biosystems) is a miniaturized molecular method recently cleared by the Food and Drug Administration for rapid diagnosis of candidemia. It combines PCR with T2 magnetic resonance imaging. This assay reportedly enables the detection of amplified DNA from C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavzevii at concentrations of 1–3 colony forming units/mL in whole blood specimens within 5 h [45]. This new method is highly sensitive and specific for diagnosing candidemia and does not require viable Candida cells or sample purification and preparation. The results of a multicenter study indicated that the T2Candida® assay performed in patients with proven candidemia might be a better marker of complicated infection than follow-up blood cultures or detection of BDG, which may influence the length and type of antifungal therapy in this population as antimicrobial stewardship [46]. In the study, the T2Candida® assay demonstrated limited sensitivity (36%) and negative predictive value (80%) under empirical antifungal therapy, while its specificity/positive predictive value was excellent (100%). These findings indicate that the assay is better suited for confirming the diagnosis of persistent infections [46]. However, this promising molecular diagnostic method has several limitations. It is relatively expensive, its reagents have a short expiration date, and its sensitivity may decrease if intact Candida cells are absent from the whole blood samples. Recently, the detection of microbial cell-free DNA (cfDNA) using next-generation sequencing has been utilized as an accurate and precise method to identify and quantify pathogens [47]. The Karius® Test was developed based on sequencing of microbial cfDNA circulating in plasma to identify over 1,000 pathogens, including bacteria, viruses, and fungi, from 5 mL blood samples. However, this method has not yet been approved [47]. This novel diagnostic tool was validated in a study showing that it could identify 94% of the microbes detected using conventional blood culture in patients with sepsis. Overall, although several molecular diagnostic methods offer rapid identification of medically important Candida species, it is important to be aware of the potential false-negative results that may occur in the absence of specific targets within the diagnostic panel (Table 1).
Candidemia remains a critical clinical challenge due to the limitations of traditional blood culture diagnostics. Among nonculture diagnostics, tests targeting BDG and mannan provide supportive evidence for the diagnosis of candidemia despite some limitations in sensitivity and specificity. Advances in molecular techniques, including PCR and next-generation sequencing, have demonstrated promising sensitivity and specificity for detecting Candida DNA, facilitating early diagnosis and potentially improving patient outcomes. Understanding the advantages and disadvantages of various diagnostic methods is becoming increasingly important to select the most appropriate diagnostic method for candidemia. Combining several nonculture diagnostic methods may help address their performance limitations.
Because this was not a human population study, Institutional Review Board approval and informed consent were not required.
Eun Jeong Won has been an associate editor of the Annals of Clinical Microbiology since January 2024. However, she was not involved in the review of this article. No potential conflicts of interest relevant to this article have been reported.
This research was supported by the Basic Science Research Program through the National Research Foundation of South Korea, funded by the Ministry of Education (Grant No. 2022R1C1C1002741), and by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI23C0319).
None.
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43. Salimnia H, Fairfax MR, Lephart PR, Schreckenberger P, DesJarlais SM, Johnson JK, et al. Evaluation of the FilmArray blood culture identification panel: results of a multicenter controlled trial. J Clin Microbiol 2016;54:687-98.
44. Peri AM, Ling W, Furuya-Kanamori L, Harris PNA, Paterson DL. Performance of BioFire Blood Culture Identification 2 Panel (BCID2) for the detection of bloodstream pathogens and their associated resistance markers: a systematic review and meta-analysis of diagnostic test accuracy studies. BMC Infect Dis 2022;22:794.
45. Park K, Huh JW, Choi SH, Sung H, Kim MN. Clinical evaluation of the T2Candida assay for the rapid diagnosis of candidemia. Diagn Microbiol Infect Dis 2024;110:116406.
46. Clancy CJ and Nguyen MH. T2 magnetic resonance for the diagnosis of bloodstream infections: charting a path forward. J Antimicrob Chemother 2018;73(suppl 4):iv2-5.
47. Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol 2019;4:663-74.
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43. Salimnia H, Fairfax MR, Lephart PR, Schreckenberger P, DesJarlais SM, Johnson JK, et al. Evaluation of the FilmArray blood culture identification panel: results of a multicenter controlled trial. J Clin Microbiol 2016;54:687-98.
44. Peri AM, Ling W, Furuya-Kanamori L, Harris PNA, Paterson DL. Performance of BioFire Blood Culture Identification 2 Panel (BCID2) for the detection of bloodstream pathogens and their associated resistance markers: a systematic review and meta-analysis of diagnostic test accuracy studies. BMC Infect Dis 2022;22:794.
45. Park K, Huh JW, Choi SH, Sung H, Kim MN. Clinical evaluation of the T2Candida assay for the rapid diagnosis of candidemia. Diagn Microbiol Infect Dis 2024;110:116406.
46. Clancy CJ and Nguyen MH. T2 magnetic resonance for the diagnosis of bloodstream infections: charting a path forward. J Antimicrob Chemother 2018;73(suppl 4):iv2-5.
47. Blauwkamp TA, Thair S, Rosen MJ, Blair L, Lindner MS, Vilfan ID, et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol 2019;4:663-74.