Next Article in Journal
Unveiling the Secretome of the Fungal Plant Pathogen Neofusicoccum parvum Induced by In Vitro Host Mimicry
Previous Article in Journal
Alternative CUG Codon Usage in the Halotolerant Yeast Debaryomyces hansenii: Gene Expression Profiles Provide New Insights into Ambiguous Translation
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
JoF
Volume 8
Issue 9
10.3390/jof8090972
jof-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Performance of Multiplex PCR and β-1,3-D-Glucan Testing for the Diagnosis of Candidemia

by
Özlem Koc
1,
Harald H. Kessler
2,
Martin Hoenigl
3,
Johannes Wagener
4,5,
Sebastian Suerbaum
1,
Sören Schubert
1,* and
Karl Dichtl
1,2,*
1
Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Medizinische Fakultät, LMU München, 80336 Munich, Germany
2
Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austria
3
Division of Infectious Diseases, Department of Internal Medicine, Medical University of Graz, 8010 Graz, Austria
4
Microbiology Department, St. James’s Hospital, D08 RX0X Dublin, Ireland
5
Department of Clinical Microbiology, School of Medicine, Trinity College Dublin, The University of Dublin, St. James’s Hospital Campus, D08 RX0X Dublin, Ireland
*
Authors to whom correspondence should be addressed.
J. Fungi 2022, 8(9), 972; https://doi.org/10.3390/jof8090972
Submission received: 26 August 2022 / Revised: 5 September 2022 / Accepted: 15 September 2022 / Published: 17 September 2022
Browse Figure
Versions Notes

Abstract

:
Bloodstream infections caused by Candida yeasts (candidemia) are associated with high morbidity and mortality. Diagnosis remains challenging, with the current gold standard—isolation from blood culture (BC)—being limited by low sensitivity and long turnaround time. This study evaluated the performance of two nonculture methods: PCR and β-1,3-D-glucan (BDG) testing. The sera of 103 patients with BC-proven candidemia and of 46 controls were analyzed with the Fungiplex Candida Real-Time PCR and the Wako β-Glucan Test. The BDG assay demonstrated higher sensitivity than the multiplex PCR (58% vs. 33%). This was particularly evident in ICU patients (60% vs. 28%) and in C. albicans candidemia (57% vs. 37%). The earlier prior to BC sampling the sera were obtained, the more the PCR sensitivity decreased (46% to 18% in the periods of 0–2 and 3–5 days before BC, respectively), while BDG testing was independent of the sampling date. No positive PCR results were obtained in sera sampled more than five days before BC. Specificities were 89% for BDG and 93% for PCR testing. In conclusion, BDG testing demonstrated several advantages over PCR testing for the diagnosis of candidemia, including higher sensitivity and earlier diagnosis. However, BC remains essential, as BDG does not allow for species differentiation.

1. Introduction

Yeasts of the genus Candida are frequently isolated in the microbiology laboratory, representing a common component of the mucosal flora of the gastrointestinal, respiratory, and female genital tracts. However, as an opportunistic pathogen, this fungus can also cause a variety of infections ranging from superficial and local infections, such as thrush or vulvovaginitis, to invasive systemic infections, including Candida bloodstream infection (BSI). Particularly, the most vulnerable groups, such as intensive care patients, critically ill COVID-19 patients, or hemato-oncological patients, are at specific risk of this manifestation of candidiasis [1,2,3,4]. The incidence of Candida BSI, which typically occurs as a nosocomial infection, has increased over time and was previously estimated to be as high as 50/100,000 hospital admissions [1,5]. Even higher candidemia rates have been reported since the beginning of the SARS-CoV-2 pandemic, which can be attributed to the novel disease entity of COVID-19-associated candidemia (CAC) [4]. Candida BSI is characterized by high mortality rates of up to 40% despite targeted antifungal therapy and causes more than 50,000 deaths every year worldwide [3,5,6]. The outcome has hardly improved over the last decades despite the availability of new antifungal agents such as echinocandins [1,7].
Late initiation of targeted therapy, which is a consequence of late diagnosis, is considered a major cause of the high mortality within patients suffering from Candida BSI [2,6]. This is due to an imperfect diagnostic gold standard, i.e., culture, which is associated with a sensitivity of as low as 50% for diagnosing invasive candidiasis [1,2,7,8,9,10]. Even if cultivation is successful, this result often comes too late due to the slow fungal growth in blood culture (BC) with a time result of several days [7,11].
In contrast, the use of nonculture methods promises more rapid results. The use of combined antigen/antibody testing is well-established in Europe and recommended by current guidelines [1,8,10]. However, to date, none of the available assays has U.S. Food and Drug Administration (FDA) clearance [12]. β-1,3-D-glucan (BDG), a fungal cell wall polysaccharide, is the target of another type of serologic assay that is not based on specific antibodies but the activation of an enzyme cascade [13]. U.S. and European guidelines recommend testing for BDG in the setting of candidemia [1,8,10,12]. Considerably, serologic assays do not allow discrimination between different Candida species, which is a major drawback regarding therapy management. Contrarily, molecular tests such as PCR are capable of overcoming this limitation. However, despite the increasing impact and nearly universal availability of PCR tests, the value of this technique for the diagnosis of invasive candidiasis remains uncertain. Current guidelines recognize the potential of molecular diagnostics but are concerned with the lack of comparable data and assay standardization [1,8,10,12].
This study aimed to evaluate and compare the sensitivities and specificities of a commercially available multiplex PCR and a BDG assay for the diagnosis of candidemia.

2. Materials and Methods

In this study, 103 sera derived from the routine diagnostics of 103 patients with BC-positive candidemia that were treated at the hospital of Ludwig Maximilian University of Munich (Munich, Germany) were analyzed. According to the EORTC/MSG criteria, candidemia was defined by the cultivation of Candida species from BC [9]. Of the 103 samples, 69 were also included in a previously published study, in which BDG testing and mannan antigen/anti-mannan-IgG based serology were compared [14]. The mean age of candidemia patients was 57 years and the women:men ratio was 0.58 in the case cohort. The corresponding data in the negative control cohort were 52 and 0.64. The clinical and microbiology characteristics are summarized in Table 1. Sera were obtained with a maximum interval of seven days before the date of sampling of the corresponding positive BC (day 0). Only one episode of candidemia per patient was included in the study. The sera of 46 patients with negative BC or bacterial BSI and without evidence of Candida BSI were included as negative controls.
All analyses were performed at the Max von Pettenkofer Institute (Munich, Germany). BC diagnostics was based on the BD BACTEC BC system (BD, Franklin Lakes, NJ, USA). Species were identified using a MALDI-TOF MS system (Bruker Daltonics, Bremen, Germany).
For molecular diagnostics, DNA extraction was performed from 1 mL of serum using a MagNA Pure Compact Nucleic Acid Isolation Kit I—Large Volume on the MagNA Pure Compact extraction system (Roche, Rotkreuz, Switzerland). An ABI 7500 FAST cycler was used for real-time PCR (Thermo Fisher Scientific, Waltham, USA). Testing for Candida DNA was performed with a CE/IVD-certified Fungiplex Candida Real-Time PCR kit (Bruker Daltonik, Bremen, Germany). This kit included three PCRs for the detection of Candida from blood samples: one Candida species PCR (Csp), one specific PCR for the detection of Candida glabrata (Cgl), and one specific PCR for the detection of Candida krusei (Ckr). Additionally, an extraction control was included. PCR-positive samples of the negative control cohort were reanalyzed for fungal DNA using a Bruker Fungiplex Universal Realtime PCR kit (research use only), which can detect (but not differentiate) >20 fungal genera, including Candida. Forty-five cycles were run for each PCR assay. Antigen testing from serum was conducted using a Wako β-Glucan Test with a cut-off of 7 pg/mL (FUJIFILM Wako Chemicals Europe, Neuss, Germany). All assays were performed according to the manufacturer’s instructions.
Significances were calculated using Fisher’s exact test (two-tailed) and McNemar’s test (two-tailed) provided by the GraphPad QuickCalcs online tool collection (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Performance of PCR Testing

With the Fungiplex Candida PCR, 34 of 103 (33%) patients with BC-proven candidemia were found to be positive, with ct values ranging from 27.5 to 40.7 (Figure 1). In 31 of these 34 patients, the Csp PCR tested positive. The Cgl and Ckr PCRs identified two (ct value of 32.2 and 33.1) and one (ct value of 35.1) of the cases of candidemia that were missed by the Csp PCR, respectively. Another two C. glabrata and one C. krusei BSI tested positive by the Csp PCR and the specific PCRs. However, one of the C. glabrata positive samples was from a patient, whose BCs only grew C. albicans.
Candida DNA was significantly more likely to be detected in sera obtained closer to the sampling date of the subsequently positive BC (Table 1): sensitivity was 46% in the sera sampled in the three-day period of 0–2 days before BC and 18% in the sera sampled in the three-day period of 3–5 days before BC (p = 0.01). No Candida DNA was detected in the sera sampled 6–7 days before BC. Regarding the underlying conditions, the sensitivity ranged from 28% in a mixed ICU population to 41% in hemato-oncologic patients (not significant). For C. glabrata candidemia, positive results were obtained in 3/19 cases (16%). The corresponding data for C. krusei candidemia were 2/8 cases (25%). For the most common pathogen, i.e., C. albicans (52 cases), the sensitivity was determined to be 37%.
Of the 46 samples of the negative control cohort, 43 tested PCR-negative (93% specificity; Table 1). We found that 1 of 13 S. aureus and 2 of 15 E. coli bloodstream infections yielded positive test results with ct values of 37.0 and 38.3 in the Csp PCR and 38.5 in the Cgl PCR. In total, 5/149 samples tested positive with the Cgl PCR. Of these samples, three were obtained from patients BC positive for C. glabrata, one from a patient BC positive for C. albicans and one from the negative control. All three PCR-positive sera of the negative control cohort were reanalyzed with a Fungiplex Universal kit: two tested negative, and one Csp PCR-positive (ct value of 38.3) sample was found to contain fungal DNA (ct value of 39.6).

3.2. Performance of BDG Testing

Of the 104 samples of patients with BC-proven candidemia, 61 tested positive with the BDG assay, with a broad range of antigen concentrations (7–4495 pg/mL; 59%; Figure 1). The highest concentrations were obtained from C. albicans and C. tropicalis BSI samples. A total of 15 sera demonstrated BDG concentrations under the cut-off and 28 sera did not contain BDG at all. BDG sensitivity was independent of sampling date, underlying clinical conditions, and fungal species (Table 1).
Of the 46 negative control cohort samples, 5 yielded positive BDG results (specificity of 89%). While one serum was found to contain >250 pg/mL BDG, the remaining positive sera were characterized by BDG concentrations close to the cut-off of 7 pg/mL (Figure 1). Three of the five samples were obtained from E. coli BSI patients and two from BC-negative patients. All negative control cohort samples positive for BDG tested negative in the PCR (and vice versa).

3.3. Comparison of Assays

When Fungiplex Candida PCR and the Wako β-Glucan Test were compared, PCR testing was less sensitive (33% vs. 58%; p < 0.001). The superiority of BDG analysis was evident in certain patient groups and almost absent in others (Table 1): while ICU patients particularly benefited from serologic testing (28% vs. 60%; p < 0.003), the selection of the test did not matter in the hemato-oncology subgroup (41% vs. 46%). While patients with C. albicans candidemia were better detected by the BDG assay (37% vs. 57%; p < 0.03), diagnosis of non-albicans Candida BSI was only, to a lesser extent, dependent on the test system (Table 1). BDG testing was found to be superior for the early diagnosis of candidemia, whereas PCR sensitivity continued to decline with increasing time from the date of BC sampling (Table 1). There were no significant differences in specificity.

4. Discussion

BC diagnostics of candidemia are impeded by several drawbacks including low sensitivity and long turnaround time [7,11,15]. This is a particular challenge in the management of Candida BSI patients because the outcome depends on early targeted therapy [16,17,18]. For years, there have been calls for non-culture-based methods to overcome these limitations, with high expectations for molecular techniques [7,11,15]. Since the first attempts at molecular candidemia diagnostics in the early 1990s [19,20,21], several in-house and a few commercial molecular tests have been developed, demonstrating sensitivities varying widely from 25% to 100% [19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37]. Probable explanations for these discrepant results include different sample volumes, different DNA extraction protocols, and particularly statistical effects due to the overall low case numbers. Only five studies had more than twenty cases included (maximum = 47), which all applied in-house PCRs with very heterogenous sensitivities of 25%, 40%, 59%, 72%, and 89% [24,25,29,32,35]. The lack of standardization is the major reason current guidelines do not recommend the use of PCR (or only as an adjunct to other methods) for the diagnosis of candidemia [1,2,8,10,12,38]. Only one pilot study investigating the Bruker Fungiplex assay was recently published. In that study, Fuchs et al. evaluated the performance of BC, Fungiplex Candida PCR, and another multiplex PCR (Roche SeptiFast test), which was discontinued in 2019 [37]. Compared with BC, the Fungiplex PCR demonstrated high diagnostic power with a sensitivity and specificity of 100% and 94%, respectively (SeptiFast: 60% and 96%, respectively). All additional SeptiFast-positive cases were also identified by the Fungiplex PCR. This excellent performance is in notable contrast to the findings of our study (sensitivity of 33% in 103 cases) but again relies on a low case number of only five cases according to the EORTC/MSG criteria [9]. The only other study to investigate a standardized, certified, and commercially available multiplex PCR, the RenDx Fungiplex kit, reported results similar to our findings (sensitivity of 44% and specificity of 87%). However, that study was also based on a very small number of only nine cases of candidemia [36]. The high variability in target sequences might be the reason for the low sensitivity of different Candida-specific PCR assays. For the Fungiplex Candida Real-Time PCR kit, no data on the target sequences are available.
Taking a more detailed look at our study data, the performance for detecting C. glabrata candidemia must be considered with some concern: despite the Fungiplex assay’s dual approach to detecting this species (Csp PCR and species-specific PCR), sensitivity was as low as 17%. Furthermore, the five Cgl PCR-positive results were distributed among one negative control, one C. albicans case, and three C. glabrata cases. However, of these three, two had already tested positive with the Csp PCR. Overall, the C. glabrata PCR was beneficial in only 1 of 19 C. glabrata BSI episodes but caused two false-positive results.
Over the last decade, BDG testing has gained an increasingly prominent role in the diagnosis of invasive candidiasis [7,12,39]. Meta-analyses concluded that both BDG sensitivity and specificity are about 80% in the setting of candidemia [7,15]. Contrarily, lower sensitivity but higher specificity, i.e., 59% and 90%, respectively, were demonstrated in our study. Presumably, this can be attributed to the use of the Wako β-Glucan Test, which is known to be more specific but less sensitive than the more widespread Fungitell assay [40]. Compared with other studies relying on the same test, the present results are consistent with expectations [41,42]. While multiplex PCRs are able to identify Candida at the species level, BDG analysis does not even allow differentiation between different fungal genera such as Aspergillus, Pneumocystis, and Candida [7,15]. Despite this disadvantage for serology, current guidelines favor the use of BDG for the diagnosis of candidemia over the use of PCR [1,2,8,9,10]. This is backed not only by our results (BDG and PCR sensitivity of 58% and 33%, respectively; p < 0.001), but also by those of previous studies [32,36]. Only the in-house assay (seminested block cycler PCR) investigated by Alam et al. in 2007 yielded better results for the molecular method than BDG [24]. However, in none of the studies relying on this PCR were data about the clinical specificity presented, which impairs proper assessment of this comparison [22,23,24]. In virtually all subgroups of our study, BDG testing had a higher sensitivity than PCR testing. There were particularly two conditions in which BDG testing significantly outperformed PCR testing: First, ICU patients had a greater benefit from a BDG than from a PCR testing approach (60% vs. 28% sensitivity, respectively). With some skepticism of this finding, however, one might object that ICU patients are particularly likely to be exposed to factors known to be associated with false-positive BDG results, e.g., immune globulin infusions or surgical materials such as sponges and gauze [43]. Second, BDG was found to yield positive results significantly earlier than PCR in sera obtained up to five days before BC sampling. In sera sampled before this date, PCR did not detect any Candida DNA (BDG: sensitivity of 42%). This is of particular interest as early diagnosis is pivotal, because delayed treatment initiation increases mortality [12]. Finally, a crucial factor should not be neglected when deciding between BDG analysis and PCR testing: serology is typically less expensive than molecular diagnostics.
With respect to the literature and our findings, one could question the value of molecular candidemia diagnostics. However, new techniques and noteworthy developments have been recently reported. The use of digital droplet PCR (ddPCR) promises higher sensitivities and specificities [44]. The T2Candida assay, a combination of nucleic acid amplification and magnetic resonance readout, allows skipping nucleic acid extraction, thereby reducing turnaround time. The clinical sensitivity of this novel assay was as high as 91% in a prospective multicenter study [45].
Our study significantly contributes to the overall small database concerning the value of molecular testing, particularly in comparison with BDG testing, as it included the largest cohort of candidemia cases to date applied for an evaluation of PCR performance. However, the work also has certain limitations such as its retrospective design and two constraints regarding the selection of samples. First, the cases were included based on a positive BC culture. Regarding the fact that BC can be expected to detect only about 75% of candidemia episodes [46], it must be considered that there was a selection bias: the results of this study reflect PCR and BDG performance not in the setting of candidemia but the setting of culture-proven candidemia. Second, for our analysis, the positive results of BDG and PCR testing were considered equal, which may be differently handled in clinical practice. While a positive PCR specific to Candida usually leads to therapy initiation, the significance of a single positive BDG result is still a matter of debate [18,47,48]: with regard to cost efficiency and the idea of antifungal stewardship, some experts advise against basing a therapy decision on a single positive BDG measurement alone due to its low positive predictive value [18]. Third, the fact that none of the individuals in the negative control cohort was BC-positive for Candida could not exclude the presence of invasive fungal infection. Therefore, designating positive samples from the negative control cohort as false positives may be precipitous. Notably, the presence of fungal DNA was confirmed in one control sample by the Fungiplex Universal PCR. However, this multiplex PCR cannot prove candidemia, because it does not differentiate between a range of different fungal species. This also applies to the BDG assay, which had a specificity of only 89%. Because no data are available concerning other invasive mycoses, e.g., Pneumocystis pneumonia or invasive aspergillosis, one could argue that the so-called false-positive results might just indicate another fungal infection. However, designating BDG-positive samples from the case group as true positive due to fungal infection might also be precipitous, because there are different conditions causing false-positive BDG results, e.g., bacterial coinfections, which were not excluded in our study [49]. There are also hints for BDG indicating critical illness rather than fungal infection, which presumably relies on the translocation of BDG from the gut to the bloodstream due to disrupted epithelial integrity [50]. Therefore, one could speculate that the observed early onset (several days before sampling of positive BCs) BDGemia in our patients might not have been a symptom of present candidemia but a symptom of poor health status, which predisposes the patient to subsequent fungal infection. However, the BDG and PCR specificities observed in our control group, which also included critically ill patients, are in good agreement with those reported in previous studies [36,37,41,42].

5. Conclusions

Our results indicate that, depending on the local availability of the assays, both tests can be used to confirm the suspicion of candidemia. The respective specificities argue against their use as screening tests. Our study suggests that BDG analysis might offer higher sensitivity than PCR testing for the diagnosis of candidemia. Positive BDG results were obtained early in the course of infection, which might allow for a timelier diagnosis. Only PCR testing offers the opportunity to identify the causative species, which allows targeted treatment. However, further studies evaluating standardized PCR assays are necessary to determine the value of molecular methods in candidemia diagnostics.

Author Contributions

Conceptualization: K.D. and S.S. (Sören Schubert); methodology: Ö.K. and K.D.; formal analysis: K.D.; investigation: Ö.K. and K.D.; resources: S.S. (Sebastian Suerbaum), S.S. (Sören Schubert) and K.D.; writing—original draft: K.D.; writing—review and editing: H.H.K., M.H., J.W., S.S. (Sören Schubert), S.S. (Sebastian Suerbaum) and K.D.; visualization: K.D.; supervision: S.S. (Sören Schubert) and K.D.; project administration: S.S. (Sören Schubert) and K.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee (Ethikkommission der Medizinischen Fakultät der LMU München; protocol code 17-244, 18.05.2020).

Informed Consent Statement

A waiver of informed consent was granted, because all samples used were residual samples, which were identified in the laboratory and assigned a number without reference to patient or clinical details, and because results from this study had no impact on clinical care.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding authors upon reasonable request.

Acknowledgments

We thank Thomas Grube and Birgit Groß for technical support.

Conflicts of Interest

Ö.K., S.S. (Sebastian Suerbaum), S.S. (Sören Schubert), and H.H.K. declare no conflict of interest. J.W. reports grants from the institution and nonfinancial support from Fujifilm Wako Chemicals Europe and Euroimmun Medizinische Labordiagnostika for past studies and speaker honoraria for lectures from Pfizer (all outside this study). M.H. received research funding from Gilead, Astellas, MSD, Scynexis, F2G, and Pfizer. K.D. reports grants from the institution and nonfinancial support from Fujifilm Wako Chemicals Europe and Euroimmun Medizinische Labordiagnostika outside this study. Neither of the two companies whose products were studied had any role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Keighley, C.; Cooley, L.; Morris, A.J.; Ritchie, D.; Clark, J.E.; Boan, P.; Worth, L.J.; Australasian Antifungal Guidelines Steering, C. Consensus guidelines for the diagnosis and management of invasive candidiasis in haematology, oncology and intensive care settings, 2021. Intern. Med. J. 2021, 51 (Suppl. S7), 89–117. [Google Scholar] [CrossRef] [PubMed]
  2. Ruhnke, M.; Behre, G.; Buchheidt, D.; Christopeit, M.; Hamprecht, A.; Heinz, W.; Heussel, C.P.; Horger, M.; Kurzai, O.; Karthaus, M.; et al. Diagnosis of invasive fungal diseases in haematology and oncology: 2018 update of the recommendations of the infectious diseases working party of the German society for hematology and medical oncology (AGIHO). Mycoses 2018, 61, 796–813. [Google Scholar] [CrossRef] [PubMed]
  3. Kullberg, B.J.; Arendrup, M.C. Invasive Candidiasis. N. Engl. J. Med. 2015, 373, 1445–1456. [Google Scholar] [CrossRef] [PubMed]
  4. Segala, F.V.; Bavaro, D.F.; Di Gennaro, F.; Salvati, F.; Marotta, C.; Saracino, A.; Murri, R.; Fantoni, M. Impact of SARS-CoV-2 Epidemic on Antimicrobial Resistance: A Literature Review. Viruses 2021, 13, 2110. [Google Scholar] [CrossRef] [PubMed]
  5. Arendrup, M.C.; Sulim, S.; Holm, A.; Nielsen, L.; Nielsen, S.D.; Knudsen, J.D.; Drenck, N.E.; Christensen, J.J.; Johansen, H.K. Diagnostic issues, clinical characteristics, and outcomes for patients with fungemia. J. Clin. Microbiol. 2011, 49, 3300–3308. [Google Scholar] [CrossRef]
  6. Andes, D.R.; Safdar, N.; Baddley, J.W.; Playford, G.; Reboli, A.C.; Rex, J.H.; Sobel, J.D.; Pappas, P.G.; Kullberg, B.J. Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: A patient-level quantitative review of randomized trials. Clin. Infect. Dis. 2012, 54, 1110–1122. [Google Scholar] [CrossRef]
  7. Clancy, C.J.; Nguyen, M.H. Non-Culture Diagnostics for Invasive Candidiasis: Promise and Unintended Consequences. J. Fungi 2018, 4, 27. [Google Scholar] [CrossRef]
  8. Cuenca-Estrella, M.; Verweij, P.E.; Arendrup, M.C.; Arikan-Akdagli, S.; Bille, J.; Donnelly, J.P.; Jensen, H.E.; Lass-Florl, C.; Richardson, M.D.; Akova, M.; et al. ESCMID* guideline for the diagnosis and management of Candida diseases 2012: Diagnostic procedures. Clin. Microbiol. Infect. 2012, 18 (Suppl. S7), 9–18. [Google Scholar] [CrossRef]
  9. Donnelly, J.P.; Chen, S.C.; Kauffman, C.A.; Steinbach, W.J.; Baddley, J.W.; Verweij, P.E.; Clancy, C.J.; Wingard, J.R.; Lockhart, S.R.; Groll, A.H.; et al. Revision and Update of the Consensus Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium. Clin. Infect. Dis. 2020, 71, 1367–1376. [Google Scholar] [CrossRef]
  10. Groll, A.H.; Buchheidt, D.; Heinz, W.; Bellmann, R.; Cornely, O.A.; Höhl, R.; Hoenigl, M.; Kluge, S.; Kurzai, O.; Lass-Flörl, C.; et al. Diagnosis and therapy of Candida infections: Joint recommendations of the German Speaking Mycological Society and the Paul-Ehrlich-Society for Chemotherapy. Mycoses 2020, 54, 279–310. [Google Scholar] [CrossRef]
  11. Kidd, S.E.; Chen, S.C.; Meyer, W.; Halliday, C.L. A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready? Front. Microbiol. 2019, 10, 2903. [Google Scholar] [CrossRef] [PubMed]
  12. Pappas, P.G.; Kauffman, C.A.; Andes, D.R.; Clancy, C.J.; Marr, K.A.; Ostrosky-Zeichner, L.; Reboli, A.C.; Schuster, M.G.; Vazquez, J.A.; Walsh, T.J.; et al. Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America. Clin. Infect. Dis. 2016, 62, e1–e50. [Google Scholar] [CrossRef] [PubMed]
  13. Obayashi, T.; Yoshida, M.; Mori, T.; Goto, H.; Yasuoka, A.; Iwasaki, H.; Teshima, H.; Kohno, S.; Horiuchi, A.; Ito, A.; et al. Plasma (1-->3)-beta-D-glucan measurement in diagnosis of invasive deep mycosis and fungal febrile episodes. Lancet 1995, 345, 17–20. [Google Scholar] [CrossRef]
  14. Dichtl, K.; Seybold, U.; Wagener, J. Serological biomarkers of candidemia: A retrospective evaluation of three assays. Infection 2019, 47, 217–224. [Google Scholar] [CrossRef]
  15. Clancy, C.J.; Nguyen, M.H. Diagnosing Invasive Candidiasis. J. Clin. Microbiol. 2018, 56, e01909-17. [Google Scholar] [CrossRef]
  16. Chakrabarti, A.; Sood, P.; Rudramurthy, S.M.; Chen, S.; Kaur, H.; Capoor, M.; Chhina, D.; Rao, R.; Eshwara, V.K.; Xess, I.; et al. Incidence, characteristics and outcome of ICU-acquired candidemia in India. Intensive Care Med. 2015, 41, 285–295. [Google Scholar] [CrossRef]
  17. Puig-Asensio, M.; Peman, J.; Zaragoza, R.; Garnacho-Montero, J.; Martin-Mazuelos, E.; Cuenca-Estrella, M.; Almirante, B.; Prospective Population Study on Candidemia in Spain Project; Hospital Infection Study Group; Medical Mycology Study Group of the Spanish Society of Infectious Diseases; et al. Impact of therapeutic strategies on the prognosis of candidemia in the ICU. Crit Care Med. 2014, 42, 1423–1432. [Google Scholar] [CrossRef]
  18. Rouze, A.; Estella, A.; Timsit, J.F. Is (1,3)-beta-D-glucan useless to guide antifungal therapy in ICU? Intensive Care Med. 2022, 48, 930–932. [Google Scholar] [CrossRef]
  19. Rand, K.H.; Houck, H.; Wolff, M. Detection of candidemia by polymerase chain reaction. Mol. Cell Probes 1994, 8, 215–221. [Google Scholar] [CrossRef]
  20. Chryssanthou, E.; Andersson, B.; Petrini, B.; Lofdahl, S.; Tollemar, J. Detection of Candida albicans DNA in serum by polymerase chain reaction. Scand. J. Infect. Dis. 1994, 26, 479–485. [Google Scholar] [CrossRef]
  21. Kan, V.L. Polymerase chain reaction for the diagnosis of candidemia. J. Infect. Dis. 1993, 168, 779–783. [Google Scholar] [CrossRef] [PubMed]
  22. Khan, Z.U.; Mustafa, A.S. Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia. Microbiol. Res. 2001, 156, 95–102. [Google Scholar] [CrossRef] [PubMed]
  23. Ahmad, S.; Khan, Z.; Mustafa, A.S.; Khan, Z.U. Seminested PCR for diagnosis of candidemia: Comparison with culture, antigen detection, and biochemical methods for species identification. J. Clin. Microbiol. 2002, 40, 2483–2489. [Google Scholar] [CrossRef] [PubMed]
  24. Alam, F.F.; Mustafa, A.S.; Khan, Z.U. Comparative evaluation of (1,3)-beta-D-glucan, mannan and anti-mannan antibodies, and Candida species-specific snPCR in patients with candidemia. BMC Infect. Dis. 2007, 7, 103. [Google Scholar] [CrossRef]
  25. Lau, A.; Halliday, C.; Chen, S.C.; Playford, E.G.; Stanley, K.; Sorrell, T.C. Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR. J. Clin. Microbiol. 2010, 48, 811–816. [Google Scholar] [CrossRef]
  26. Grosse-Onnebrink, J.; Stehling, F.; Tschiedel, E.; Olivier, M.; Mellies, U.; Schmidt, R.; Buer, J.; Rath, P.M.; Steinmann, J. Bacteraemia and fungaemia in cystic fibrosis patients with febrile pulmonary exacerbation: A prospective observational study. BMC Pulm. Med. 2017, 17, 96. [Google Scholar] [CrossRef]
  27. Leon, C.; Ruiz-Santana, S.; Saavedra, P.; Castro, C.; Loza, A.; Zakariya, I.; Ubeda, A.; Parra, M.; Macias, D.; Tomas, J.I.; et al. Contribution of Candida biomarkers and DNA detection for the diagnosis of invasive candidiasis in ICU patients with severe abdominal conditions. Crit Care 2016, 20, 149. [Google Scholar] [CrossRef]
  28. Guo, Y.; Yang, J.X.; Liang, G.W. A Real-Time PCR Assay Based on 5.8S rRNA Gene (5.8S rDNA) for Rapid Detection of Candida from Whole Blood Samples. Mycopathologia 2016, 181, 405–413. [Google Scholar] [CrossRef]
  29. Hasseine, L.; Cassaing, S.; Robert-Gangneux, F.; Fillaux, J.; Marty, P.; Gangneux, J.P.; The PF-qPCR Study Group. High negative predictive value diagnostic strategies for the reevaluation of early antifungal treatment: A multicenter prospective trial in patients at risk for invasive fungal infections. J. Infect. 2015, 71, 258–265. [Google Scholar] [CrossRef]
  30. Taira, C.L.; Okay, T.S.; Delgado, A.F.; Ceccon, M.E.; de Almeida, M.T.; Del Negro, G.M. A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients. BMC Infect. Dis. 2014, 14, 406. [Google Scholar] [CrossRef] [Green Version]
  31. Herne, V.; Nelovkov, A.; Kutt, M.; Ivanova, M. Diagnostic performance and therapeutic impact of LightCycler SeptiFast assay in patients with suspected sepsis. Eur. J. Microbiol. Immunol. 2013, 3, 68–76. [Google Scholar] [CrossRef] [PubMed]
  32. Nguyen, M.H.; Wissel, M.C.; Shields, R.K.; Salomoni, M.A.; Hao, B.; Press, E.G.; Shields, R.M.; Cheng, S.; Mitsani, D.; Vadnerkar, A.; et al. Performance of Candida real-time polymerase chain reaction, beta-D-glucan assay, and blood cultures in the diagnosis of invasive candidiasis. Clin. Infect. Dis. 2012, 54, 1240–1248. [Google Scholar] [CrossRef]
  33. Mokaddas, E.; Burhamah, M.H.; Khan, Z.U.; Ahmad, S. Levels of (1-->3)-beta-D-glucan, Candida mannan and Candida DNA in serum samples of pediatric cancer patients colonized with Candida species. BMC Infect. Dis. 2010, 10, 292. [Google Scholar] [CrossRef] [PubMed]
  34. Schell, W.A.; Benton, J.L.; Smith, P.B.; Poore, M.; Rouse, J.L.; Boles, D.J.; Johnson, M.D.; Alexander, B.D.; Pamula, V.K.; Eckhardt, A.E.; et al. Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood. Eur. J. Clin. Microbiol. Infect. Dis. 2012, 31, 2237–2245. [Google Scholar] [CrossRef] [PubMed]
  35. Moreira-Oliveira, M.S.; Mikami, Y.; Miyaji, M.; Imai, T.; Schreiber, A.Z.; Moretti, M.L. Diagnosis of candidemia by polymerase chain reaction and blood culture: Prospective study in a high-risk population and identification of variables associated with development of candidemia. Eur. J. Clin. Microbiol. Infect. Dis. 2005, 24, 721–726. [Google Scholar] [CrossRef] [PubMed]
  36. McKeating, C.; White, P.L.; Posso, R.; Palmer, M.; Johnson, E.; McMullan, R. Diagnostic accuracy of fungal PCR and beta-d-glucan for detection of candidaemia: A preliminary evaluation. J. Clin. Pathol. 2018, 71, 420–424. [Google Scholar] [CrossRef]
  37. Fuchs, S.; Lass-Florl, C.; Posch, W. Diagnostic Performance of a Novel Multiplex PCR Assay for Candidemia among ICU Patients. J. Fungi 2019, 5, 86. [Google Scholar] [CrossRef]
  38. White, P.L.; Alanio, A.; Brown, L.; Cruciani, M.; Hagen, F.; Gorton, R.; Lackner, M.; Millon, L.; Morton, C.O.; Rautemaa-Richardson, R.; et al. An overview of using fungal DNA for the diagnosis of invasive mycoses. Expert Rev. Mol. Diagn. 2022, 22, 169–184. [Google Scholar] [CrossRef]
  39. Clancy, C.J.; Nguyen, M.H. Rapid diagnosis of invasive candidiasis: Ready for prime-time? Curr. Opin. Infect. Dis. 2019, 32, 546–552. [Google Scholar] [CrossRef]
  40. Yoshida, K.; Shoji, H.; Takuma, T.; Niki, Y. Clinical viability of Fungitell, a new (1-->3)-beta-D: -glucan measurement kit, for diagnosis of invasive fungal infection, and comparison with other kits available in Japan. J. Infect. Chemother. 2011, 17, 473–477. [Google Scholar] [CrossRef]
  41. Forster, J.; Dichtl, K.; Wagener, J. Lower beta-1,3-D-glucan testing cut-offs increase sensitivity for non-albicans Candida species blood stream infections. Mycoses 2022, 65, 500–507. [Google Scholar] [CrossRef]
  42. Friedrich, R.; Rappold, E.; Bogdan, C.; Held, J. Comparative Analysis of the Wako beta-Glucan Test and the Fungitell Assay for Diagnosis of Candidemia and Pneumocystis jirovecii Pneumonia. J. Clin. Microbiol. 2018, 56, e00464-18. [Google Scholar] [CrossRef]
  43. Tortorano, A.M.; Prigitano, A.; Morroni, G.; Brescini, L.; Barchiesi, F. Candidemia: Evolution of Drug Resistance and Novel Therapeutic Approaches. Infect. Drug Resist. 2021, 14, 5543–5553. [Google Scholar] [CrossRef]
  44. Chen, B.; Xie, Y.; Zhang, N.; Li, W.; Liu, C.; Li, D.; Bian, S.; Jiang, Y.; Yang, Z.; Li, R.; et al. Evaluation of Droplet Digital PCR Assay for the Diagnosis of Candidemia in Blood Samples. Front. Microbiol. 2021, 12, 700008. [Google Scholar] [CrossRef]
  45. Clancy, C.J.; Pappas, P.G.; Vazquez, J.; Judson, M.A.; Kontoyiannis, D.P.; Thompson, G.R., 3rd; Garey, K.W.; Reboli, A.; Greenberg, R.N.; Apewokin, S.; et al. Detecting Infections Rapidly and Easily for Candidemia Trial, Part 2 (DIRECT2): A Prospective, Multicenter Study of the T2Candida Panel. Clin. Infect. Dis. 2018, 66, 1678–1686. [Google Scholar] [CrossRef]
  46. Clancy, C.J.; Nguyen, M.H. Finding the “missing 50%” of invasive candidiasis: How nonculture diagnostics will improve understanding of disease spectrum and transform patient care. Clin. Infect. Dis. 2013, 56, 1284–1292. [Google Scholar] [CrossRef]
  47. Bloos, F.; Held, J.; Kluge, S.; Simon, P.; Kogelmann, K.; de Heer, G.; Kuhn, S.O.; Jarczak, D.; Motsch, J.; Hempel, G.; et al. (1 --> 3)-beta-D-Glucan-guided antifungal therapy in adults with sepsis: The CandiSep randomized clinical trial. Intensive Care Med. 2022, 48, 865–875. [Google Scholar] [CrossRef]
  48. Martin-Loeches, I.; Antonelli, M.; Cuenca-Estrella, M.; Dimopoulos, G.; Einav, S.; De Waele, J.J.; Garnacho-Montero, J.; Kanj, S.S.; Machado, F.R.; Montravers, P.; et al. ESICM/ESCMID task force on practical management of invasive candidiasis in critically ill patients. Intensive Care Med. 2019, 45, 789–805. [Google Scholar] [CrossRef]
  49. Albert, O.; Toubas, D.; Strady, C.; Cousson, J.; Delmas, C.; Vernet, V.; Villena, I. Reactivity of (1-->3)-beta-d-glucan assay in bacterial bloodstream infections. Eur. J. Clin. Microbiol. Infect. Dis. 2011, 30, 1453–1460. [Google Scholar] [CrossRef]
  50. Kitsios, G.D.; Kotok, D.; Yang, H.; Finkelman, M.A.; Zhang, Y.; Britton, N.; Li, X.; Levochkina, M.S.; Wagner, A.K.; Schaefer, C.; et al. Plasma 1,3-beta-d-glucan levels predict adverse clinical outcomes in critical illness. JCI Insight 2021, 6, e141277. [Google Scholar] [CrossRef]
Jof 08 00972 g001 550
Figure 1. Plot of analytical measuring results of sera of candidemia patients (cand; full circles) and negative controls (nc; empty circles). The dotted line indicates the cut-off of both assays. For samples, which were tested positive in more than one PCR, only one result was considered for this graph. BDG concentrations below the limit of detection and negative PCR results are plotted (not to scale) in the grey shaded area.
Figure 1. Plot of analytical measuring results of sera of candidemia patients (cand; full circles) and negative controls (nc; empty circles). The dotted line indicates the cut-off of both assays. For samples, which were tested positive in more than one PCR, only one result was considered for this graph. BDG concentrations below the limit of detection and negative PCR results are plotted (not to scale) in the grey shaded area.
Jof 08 00972 g001
Table
Table 1. Comparison of sensitivities (a) and specificities (b) of the Fungiplex Candida PCR and the Wako β-Glucan Test.
Table 1. Comparison of sensitivities (a) and specificities (b) of the Fungiplex Candida PCR and the Wako β-Glucan Test.
a
Candidemia Group		All Samples
n (%)		PCR pos.
n (%)		BDG pos.
n (%)		Significance:
p Value
Total cases103 (-)34 (33)60 (58)0.0002
Underlying conditions
  Hemato-oncologic malignancy39 (37)16 (41)18 (46)0.7893
    HSCT20 (19)7 (35)9 (45)0.7518
  Solid organ transplantation14 (14)4 (29)10 (71)0.0771
  ICU47 (46)13 (28)28 (60)0.0023
Sampling date of serum
  0–2 days before BC63 (61)29 (46)39 (62)0.0890
  3–5 days before BC28 (27)5 (18)16 (57)0.0026
  6–7 days before BC12 (12)0 (0)5 (42)0.0736
Species isolated
  Candida albicans49 (48)18 (37)28 (57)0.0244
  Candida glabrata18 (17)3 (17)8 (44)0.1306
  Candida parapsilosis12 (12)3 (25)8 (67)0.1842
  Candida krusei8 (8)3 (38)6 (75)0.2482
  Others41 (16)7 (44)10 (63)0.5050
b
Control Group		All Samples
n (%)		PCR neg.
n (%)		BDG neg.
n (%)		Significance:
p Value
Total controls46 (-)43 (93)41 (89)0.7237
BC result
  Staphylococcus aureus13 (28)12 (92)13 (100)1.0000
  Escherichia coli15 (33)13 (87)12 (80)1.0000
  Sterile18 (39)18 (100)16 (89)0.4795
Two-tailed p values were calculated using McNemar’s test with continuity correction. Results at a significance level < 0.05 are in bold. pos., positive; neg., negative; BDG, β-1,3-D-glucan; HSCT, hematopoietic stem cell transplantation; ICU, intensive care unit; BC, blood culture.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Koc, Ö.; Kessler, H.H.; Hoenigl, M.; Wagener, J.; Suerbaum, S.; Schubert, S.; Dichtl, K. Performance of Multiplex PCR and β-1,3-D-Glucan Testing for the Diagnosis of Candidemia. J. Fungi 2022, 8, 972. https://doi.org/10.3390/jof8090972

AMA Style

Koc Ö, Kessler HH, Hoenigl M, Wagener J, Suerbaum S, Schubert S, Dichtl K. Performance of Multiplex PCR and β-1,3-D-Glucan Testing for the Diagnosis of Candidemia. Journal of Fungi. 2022; 8(9):972. https://doi.org/10.3390/jof8090972

Chicago/Turabian Style

Koc, Özlem, Harald H. Kessler, Martin Hoenigl, Johannes Wagener, Sebastian Suerbaum, Sören Schubert, and Karl Dichtl. 2022. "Performance of Multiplex PCR and β-1,3-D-Glucan Testing for the Diagnosis of Candidemia" Journal of Fungi 8, no. 9: 972. https://doi.org/10.3390/jof8090972

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop