Serum Galactomannan: A Predictor of Poor Outcomes in Peritoneal Dialysis Patients With Fungal Peritonitis

Abstract

Introduction

The potential value of serum galactomannan index (GMI) in monitoring treatment response in patients with fungal peritonitis who are receiving peritoneal dialysis (PD) was assessed in the present study.

Methods

The study included all Thailand fungal PD-related infectious complications surveillance (MycoPDICS) DATA study participants who had timely PD catheter removal and availability of both baseline and ≥2 subsequent serum GMI measurements after starting antifungal therapy (if available). Serum GMI was assessed by direct double-sandwich enzyme-linked immunosorbent assay with reference to positive and negative control samples. Comparisons of categorical variables among groups were analyzed by Fisher’s exact test for categorical data and the Wilcoxon rank-sum test for continuous variables. Mortality outcomes were analyzed by survival analyses using Kaplan-Meier curves with Log-rank test.

Results

Seventy-six (46%) of 166 participants from 21 PD centers between 2018 and 2022 were included. The median age was 58 (50–65) years, and a half of the patients (50%) had type II diabetes. Nineteen (25%) and 57 (75%) episodes were caused by yeast and mold, respectively. Death occurred in 11 (14%) patients at 3 months, and no differences were observed in demographics, laboratories, treatment characteristics, or in baseline serum GMI between those who died and those who survived. Serum GMI progressively declined over the follow-up period after the completion of treatment. Patients who died had significantly higher posttreatment serum GMI levels and were more likely to have positive GMI after treatment.

Conclusion

Serum GMI is an excellent biomarker for risk stratification and treatment response monitoring in patients on PD with fungal peritonitis.

Graphical abstract

Fungal peritonitis constitutes 1% to 23% of peritonitis cases.¹ It leads to serious complications of PD, high rates of transfer to hemodialysis (40%–55%) and mortality (5%–53%).^1,2 PD Catheter removal within 24 hours after the diagnosis of fungal peritonitis markedly reduces the mortality rate compared to those with delayed catheter removal (13% vs. 32%, respectively).³ Therefore, the 2022 International Society for Peritoneal Dialysis Peritonitis Guidelines recommend immediate catheter removal when the diagnosis of fungal peritonitis is made. Although the guideline recommended a continuation of antifungal agent for a minimum of 2 weeks after PD catheter removal, the level of evidence is weak due to insufficient data.⁴ Follow-up posttreatment biomarkers could provide benefits in risk-stratifying patients and determining the duration of treatment.

Serum levels of the fungal cell wall component, galactomannan (GM), which is released from fungi during the hyphal growth,⁵ have been validated as an accurate marker for the diagnosis of invasive aspergillosis in patients with hematologic malignancy or hematopoietic stem cell transplantation.⁶ The study in patients on PD confirmed that serum GMI was also useful in the diagnosis of fungal peritonitis.⁷ In this study, we evaluated the clinical utility of serum GMI as a biomarker for risk stratification and treatment response monitoring.

Methods

The study included the Thailand fungal PD-related infectious complications surveillance (MycoPDICS) DATA study participants who had timely PD catheter removal (within 3 days after the onset of fungal peritonitis) and availability of both baselines (before antifungal or antibiotic agent administration) and ≥2 subsequent serum GMI measurements after starting antifungal therapy. The MycoPDICS database is a national registry designed to survey the incidence of PD-related infections with fungus or environmental organisms under the Nephrology Society of Thailand, which has been launched since 2015 and aims to monitor incidence, disease trends and risk factors of fungal and environmental infections in patients on PD.¹ All reporting was performed under the Strengthening the Reporting Diagnostic Accuracy Studies guidelines (Supplementary Figure S1).

Sample Collection

GMI sera were sequentially collected at 5 periods, including baseline, during treatment, immediate posttreatment (within 1 week after the completion of antifungal course), 1-month posttreatment (2–6 weeks), and 3 to 6 months posttreatment (if available). The recommended regimens of antifungal agents used in this study were either of the following: (i) voriconazole 200 mg tablet at a dose of 6 mg/kg/dose twice daily on the first day, followed by 4 mg/kg/dose twice daily for 14 days and (ii) amphotericin B at 1 mg/kg/day in combination with 5-fluorocytosine 500 mg tablet twice daily for 14 days. To mitigate the risk of artifactual serum GM results during blood collection and handling, a protocol collection⁷ was emphasized to charge nurses as follows: applying the tourniquet during venous puncture for no longer than 1 minute and avoiding fist-clenching, completing air dry after cleaning the puncture area with 70% alcohol (avoiding iodine-containing or chlorhexidine-containing antiseptic agents), using a 20 to 22 gauge needle, placing the bevel of the needle face-up, collecting blood only from the antecubital region of the arm, pulling the syringe plunger gently, drawing the blood into sterile sodium citrate tube and mixing thoroughly by inverting the tube slowly 3 to 4 times, separating the serum by centrifugation at 150 g for 5 minutes, and shipping the separated serum to the local laboratory. Serum samples were stored at −70 °C until analyzed.

For PD effluent (PDE) collection, the drained bags with a dwell period of more than 4 hours were collected. PDE was aliquoted from the bag at the injection port using an aseptic technique for local microbiologic culture according to the standard operating procedure of each study site. The bag with the remaining solution was then placed in an ice-shield box and shipped directly to the central laboratory as soon as possible. Any bag with delayed shipping (longer than 24 hours from the onset of bag collection) was discarded.

Sample Preparation and Microbiology Analysis at the Central Laboratory

At the central laboratory, 50 ml of PDE was centrifuged at 3500g for 15 minutes, discarding the supernatants. The remaining solution (about 5 ml) was mixed up with pellet and injected into Bactec Plus Aerobic/F vial (Dun Laoghaire, Ireland) and was also spread onto several agar plates, including blood agar, MacConkey agar (Oxoid, Basingstoke, United Kingdom), and Chocolate agar (Oxoid, Basingstoke, United Kingdom) for 7 days at 37 °C for bacterial culture. For fungal culture, the pellet from another 50 ml of centrifuged PDE was streaked on Sabouraud dextrose agar, blood agar (Oxoid), and specific agar plates (as needed) then incubated at 25 °C and 37 °C for 15 to 30 days. Bacterial pathogens were identified by Gram stain and Vitek MS system (bioMérieux, USA),⁷ whereas fungi were identified by API20c AUX kit (bioMérieux, Marcy l’Etoile, France) based on biochemical reactions and stained by Lactophenol Cotton Blue technique to classify mold-form fungi based on the morphology of their sexual spores and conidia. To exclude coincidental infection with Mycobacterium organisms, the pellet from an additional 50 ml PDE was inoculated in Ogawa medium slants and BACTEC MGIT 960 media for 2 months.⁸

GM Testing

The Platelia Aspergillus EIA test kit (Plateria Aspergillus, Bio-Rad Laboratories, Marnes La Coquette, France), which utilizes a direct double-sandwich enzyme-linked immunosorbent assay technique, was used to measure GM in the prepared serum samples as well as the control samples (1 positive control with GM >4 ng/ml, 1 negative control, and 1 cut-off control with 1 ng/ml GM), as described previously.⁹ The results were reported quantitatively as optical density determined by a spectrophotometer set at 450 and 620/630 nm wavelengths.⁹ The presence or absence of GM is determined by its index or GMI, which is calculated by dividing the optical density of samples by the mean optical density of the controls’ cut-off values.¹⁰ The positive cut-off value of serum GMI was defined as ≥0.56 because this been found by previous investigators to provide diagnostic value in fungal peritonitis.⁷

Statistical Analysis

The descriptive statistical analyses were described as frequency with percentage for categorical variables and median with interquartile range (IQR) for continuous variables. Group comparisons were performed by Fisher’s exact test for categorical data and Wilcoxon rank-sum test for continuous variables. The primary outcome was overall survival at 3 months after the completion of antifungal treatment. Mortality outcomes were assessed by survival analyses using Kaplan-Meier curves with Log-rank test. All included patients had complete follow-up and there was no censoring for loss of follow-up. The association between serum GMI and candidate predictors of mortality using linear and logistic regression. In addition, we explored the association between initial antifungal treatment and other baseline characteristics. Data were analyzed using R 4.0.5 (R Core Team, Vienna). P-values less than 0.05 were considered statistically significant.

Results

Patient Demographics and Characteristics

Participant flow is shown in Figure 1. During the observation period, there were 166 patients on PD with fungal peritonitis having serum GMI, of whom only 115 (69%) patients had timely PD catheter removal (within 3 days) and received an appropriate dosage of antifungal medication according to the 2022 International Society for Peritoneal Dialysis Peritonitis Guidelines.⁴ After excluding those with fungal episodes with serum GMI less than the cut-off level (33 patients) and those with no follow-up serum GM (6 patients), 76 (46%) consented participants from 21 PD centers between 2018 and 2022 were included.

Figure 1.

Patient flow diagram. GMI, galactomannan index; PD, peritoneal dialysis.

The median age of included participants was 58 (50–65) years with female predominance (53%) and half of them (50%) having type II diabetes mellitus. There were no significant differences in demographics, laboratory or treatment characteristics, or in baseline serum GMI between those who died and those who survived, except that patients who died were more likely to have diabetes and had lower body mass index and serum albumin (Table 1). None had clinical gut perforation or required abdominal exploratory laparotomy.

Table 1.

Patient characteristics comparing baseline characteristics between patients who were deceased within 3 months postfungal peritonitis and survivors

Characteristics	Total (N = 76)	Deceased (n = 11)	Survivors (n = 65)	P-value
Age, yrs	58 (50–65)	65 (55–69)	58 (48–64)	0.06
Male, n (%)	36 (47%)	4 (36%)	32 (49%)	0.5
BMI, kg/m2	22 (20–26)	20 (19–21)	23 (20–27)	0.02a
Diabetes, n (%)	38 (50%)	9 (82%)	29 (45%)	0.047a
SIRS, n (%)	13 (17%)	4 (36%)	9 (14%)	0.2
Hemoglobin, g/dl	9.4 (8.4–10.5)	9.2 (8.6–9.6)	9.4 (8.2–10.6)	0.7
Serum BUN, mg/dl	39 (27–55)	36 (32–41)	41 (27–58)	0.7
Serum potassium, mmol/l	3.6 (3.3–4.1)	3.8 (3.4–4.2)	3.6 (3.3–4.1)	0.8
Serum chloride, mmol/l	93 (89–97)	91 (88–93)	93 (90–97)	0.2
Serum bicarbonate, mmol/l	27 (25–29)	27 (25–28)	27 (26–29)	0.7
Serum total bilirubin, mg/dl	0.3 (0.2–0.4)	0.4 (0.2–0.4)	0.3 (0.2–0.5)	0.8
Serum ALT, units/l	9 (5–15)	11 (6–15)	9 (5–15)	0.9
Serum ALP, IU/l	95 (73–129)	86 (76–93)	99 (70–132)	0.3
Serum albumin, g/dl	2.6 (2.3–3.1)	2.2 (1.7–2.7)	2.6 (2.3–3.1)	0.047a
Effluent leukocyte count, ×1000 cells/μl	1.0 (0.4–3.1)	1.3 (0.7–1.7)	1.0 (0.3–3.5)	0.8
Effluent neutrophil, %	74 (54–88)	86 (76–91)	72 (53–88)	0.2
Baseline serum GMI	0.87 (0.64–1.54)	0.89 (0.61–1.58)	0.85 (0.65–1.52)	0.7
Fungal characteristics				0.7
Yeast	19 (25%)	2 (18%)	17 (26%)
Mold	57 (75%)	9 (82%)	48 (74%)
Initial antifungal therapy				0.2
Amphotericin B	47 (62%)	9 (82%)	38 (58%)
Voriconazole	29 (38%)	2 (18%)	27 (42%)

ALP, alkaline phosphatase; ALT, alanine transaminase; BID, twice daily; BMI, body mass index; BUN, blood urea nitrogen; GMI, galactomannan index; IU, international units, SIRS, systemic inflammatory response syndrome.

Continuous variables are presented as medians and interquartile ranges, whereas categorical variables are presented as frequencies and percentages. The recommended doses of antifungal agents used in this study were as follows: (i) voriconazole 200 mg tablet at a dose of 6 mg/kg/dose BID on the first day, followed by 4 mg/kg/dose BID for 14 days and (ii) amphotericin B at a dose of 1 mg/kg/day in combination with 5-fluorocytosine 500 mg tablet BID for 14 days.

^aIndicates significant value at P < 0.05.

Patients with low body mass index, diabetes, and hypoalbuminemia had worse outcomes. In the sensitivity analysis, there were no associations between serum GMI measurements (at baseline, during, and posttreatment) and the poor outcome indicators (Supplementary Tables S1 and S2). Furthermore, there were no differences observed in baseline characteristics and posttreatment serum GMI measurements between patients who initially received amphotericin B versus voriconazole.

Nineteen (25%) and 57 (75%) fungal peritonitis episodes were caused by yeasts and molds, respectively. The most common fungal pathogen was Aspergillus spp. followed by Candida spp., Fusarium spp., and Paecilomyces spp. Of note, serum GMI sera gradually decreased after treatment (Figure 2).

Figure 2.

Serial serum GMI during the study period. IQR, interquartile range.

Association of Serum GMI With Patient Survival

Death occurred in 11 (14%) participants; 8 died from fungal peritonitis (2 with fungemia), 2 from withdrawn dialysis, and 1 from an unknown cause. All deaths occurred after completion of the antifungal course. Nine and 2 died within 1 and 3 months, respectively. Compared with survivors, deceased participants had higher serum GMIs during treatment (IQR: 0.91 [0.63–1.21] vs. IQR: 0.48 [0.34–0.71]; P = 0.04) and postcomplete treatment (IQR: 0.91 [0.66–1.39] vs. IQR: 0.37 [0.29–0.59]; P < 0.001) and were less likely to have serum GMI return to normal levels after treatment completion (82% vs. 29%, P = 0.001) (Table 2). Of note, the serum GMI prior to death in the deceased participants was higher than immediately postcomplete treatment of the survivors (0.86 [0.66–1.28], P < 0.001) with a median interval between the collection and the death dates of 5 (2–14) days. Using the Kruskal-Wallis rank test, the survival probability of patients with GMI below cut-off after treatment was significantly higher than that of patients with GMI above cut-off after treatment (P = 0.0007) (Figure 3). There were no differences in baseline characteristics and GMI measurements (baseline, during, and posttreatment) between patients who initially received amphotericin B and those who received voriconazole (Supplementary Table S3).

Table 2.

Serum galactomannan index measurements and patient outcome

Characteristics	Deceased (n = 11)	Survivors (n = 65)	P-value
During-treatment serum GMI	0.91 (0.63–1.21)	0.48 [0.34–0.71]	0.04a
Posttreatment serum GMI	0.91 (0.66–1.39)	0.37 [0.29–0.59]	< 0.001a
% Posttreatment serum GMI reduction	7 (−50 to 43)	59 [40–78]	0.002a
Positive serum GMI after antifungal treatment, n (%)	9 (82%)	19 (29%)	0.001a

GMI, galactomannan index.

Continuous variables are presented as medians and interquartile ranges, whereas categorical variables are presented as frequencies and percentages.

^aIndicates significant value at P < 0.05.

Figure 3.

The survival probability between patients with GMIs below vs. above cut-off after the completion of antifungal treatment.

Remark: One death occurred at 3 months after submitting serum GM.

Discussion

This is the first proof-of-concept study describing the utility of serum GM as a prognostic marker and for monitoring treatment response in patients on PD with fungal peritonitis. Serum GMI gradually decreased after treatment of fungal peritonitis. Following treatment completion, serum GMI was inversely associated with patient survival.

Fungal peritonitis has worse outcomes (death hazard ratio 9.4, 95% confidence interval: 2.4–36.4 and hemodialysis transfer death hazard ratio 19. 6, 95% confidence interval: 8.1–47.0) and a lower rate of cure (adjusted odd ratio 0.01, 95% confidence interval 0.00–0.09) compared with Gram-positive bacterial peritonitis. In other words, fungal peritonitis actually has the worst outcomes of all peritonitis.¹¹ The mortality rate ranges from 9% to 60%,^1,3,12, 13, 14, 15 and is 2 to 3 folds higher among those who do not undergo catheter removal.⁴ Our study demonstrates a consistent mortality rate of 15%. The 2022 International Society for Peritoneal Dialysis Peritonitis Guidelines recommend that after the catheter is removed, the antifungal treatment should be continued for at least 2 weeks.⁴ Generally, follow-up markers should be used to guide the management after initiation of antimicrobial therapy; for example, blood cultures among septicemic patients^16,17 and PDE leukocyte counts and cultures among PD peritonitis patients.⁴ Nevertheless, among patients with fungal peritonitis, it is mandatory to remove the PD catheter; thus, PDE leukocyte monitoring is unfeasible. Although abdominal paracentesis could theoretically be used to sample PDE, it is invasive, inconvenient, and may be complicated by bleeding or bowel perforation, particularly in the setting of ileus or localized fluid collections. In this study, we found that posttreatment serum GMI provided prognostic value in predicting and monitoring treatment responsiveness.

In patients with hematologic malignancy or those who have received hematopoietic stem cell transplantation, the diagnostic performance of serum GMI values ≥0.5 for invasive aspergillosis is comparable to that of blood polymerase chain reaction. Therefore, serial monitoring of serum GMI could be used for monitoring therapeutic response and predicting outcomes.⁶ A study of patients with hematologic disorders who experienced invasive aspergillosis demonstrated that persistently negative serum GMI values (<0.5) were strongly correlated with increased survival and response rates. Eighty-three percent of nonsurvivors had a positive serum GMI at death,¹⁸ which was similar to our study in which 9 (82%) of nonsurvivors had a positive serum GMI after treatment. A GMI reduction of >35% between baseline and week 1 can also predict the clinical response.¹⁹ Besides Aspergillus spp., a positive GM enzyme-linked immunosorbent assay test can also be elicited by other mold and dimorphic fungal infections; for example, Cladosporium spp., Fusarium spp. and Paecilomyces spp., Penicillium spp., and Trichophyton spp.⁶ Thus, serum GMI could have benefits in the detection of these fungal infections.

In patients on PD with fungal peritonitis, GM can be detected in the circulation due to the translocation of GM from the peritoneal fluid to the bloodstream. A cut-off value of serum GMI of ≥0.56 had a sensitivity and specificity of 65% and 85%, respectively for diagnosing fungal peritonitis.⁷ However, posttreatment serum GMI among fungal peritonitis had never been evaluated. We found that serum GMI reduction after treatment can predict clinical response. Following completion of treatment, serum GMI among patients who subsequently died was higher than that of the survivors (0.91 [IQR 0.66–1.39] vs. 0.37 [IQR 0.29–0.59], respectively). Of interest, most patients with serum GMI measurements above the cut-off level of ≥0.56 following treatment had poor outcomes, whereas most cases with GMI levels below the cut-off value had excellent outcomes.

Using serum GMI as a fungal peritonitis marker must be interpreted with caution. The potential for false positive and negative results should be borne in mind. Patients with Pseudomonas aeruginosa septicemia have been reported on occasions to have a positive serum GMI.²⁰ False positive GMI has also been reported in rhodococcal peritonitis.²¹ Some generic formulations of piperacillin/tazobactam, colistin inhalation, and massive hemolysis may also result in false positive serum GMI values.22, 23, 24, 25 Yeasts have minimal GM in their cell walls, such that serum GMI has limitations for detection of yeast infection.²⁶ Minimal peripheral blood translocation of GM may also lead to undetectable serum GMI.⁷ Previous studies have revealed negative serum GMI in patients with infections due to Alternaria spp., Aspergillus flavus, Candida spp., Exophiala spp., Exserohilum rostratum, Fusarium solani, and Trichosporon spp. peritonitis.⁷

The study has certain strengths to be mentioned. First, it is the first proof-of-concept study evaluating the prognostic value of serum GMI for peritonitis outcomes. Serum GMI is practical and needs no invasive procedure. We propose a practical measure for monitoring the treatment response in patients on PD with fungal peritonitis after PD catheter removal. Second, the study was conducted in multiple centers, including community-based hospitals, reflecting real-life practices. Lastly, all recruited participants received timely PD catheter removal and antifungal medications, which are consistent with the 2022 International Society for Peritoneal Dialysis Peritonitis Guidelines recommendation.⁴ Balanced against these strengths, this study has some limitations. First, only 11 patients died, which might have decreased the power of the study. Despite this, a statistically significant difference in serum GMI following completion of treatment was observed between those who did and did not survive. Second, only half of the participants with fungal peritonitis had serum specimens collected at 6 months following completion of antifungal treatment, thereby limiting the ability to assess long-term clinical usefulness and predict repeat episodes. A further study including more cases and extended follow-up should be conducted. Third, 33 (29%) episodes had normal serum GMI values (20 Candida, 4 Fusarium, 2 Acremonium, 2 Cladosporium, and 5 other molds); therefore, the results of this study may not generalize to PD populations in which Candida is the dominant fungal infection.

In conclusion, serum GMI is a practical and valuable biomarker for predicting prognosis and monitoring response to antifungal treatment in patients with fungal PD peritonitis who have undergone PD catheter removal.

Disclosure

DWJ has previously received consultancy fees, research grants, speaker's honoraria, travel sponsorships from Baxter Healthcare and Fresenius Medical Care, consultancy fees from AstraZeneca, Bayer, and AWAK, and speaker's honoraria from Ono and BI & Lilly, and travel sponsorships from Amgen. He is also supported by an Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grant. JP has received speaking honoraria from Astra Zeneca, Baxter Healthcare, DaVita Healthcare Partners, Fresenius Medical Care, Dialysis Clinics Incorporated, Satellite Healthcare, and has served as a consultant for Baxter Healthcare, DaVita Healthcare Partners, Fresenius Medical Care, and LiberDi. TK has received consultancy fees from VISTERRA, ELEDON, Otsuka OLE, and Otsuka VISIONARY as a country investigator and current recipient of the National Research Council of Thailand and received speaking honoraria from Astra Zeneca and Baxter Healthcare. All the other authors declared no competing interests.

Supplementary Material

Figure S1. STARD Statement.

Table S1. Association between baseline characteristics and serum GMI measurements using univariable linear regression.

Table S2. Association between baseline characteristics and serum GMI measurements using univariable logistic regression.

Table S3. Baseline characteristics and serum GMI measurements between patients who initially received amphotericin B and those who received voriconazole.