False-positive results of galactomannan assays in patients administered glucose-containing solutions

Galactomannan (GM) is a polysaccharide cell wall component released by Aspergillus spp., and an immunoenzymatic GM assay is used for the diagnosis of invasive pulmonary aspergillosis. We evaluated the cause of strong positivity for GM in patients with no typical signs of aspergillosis. Repeat assays were performed using different instruments and reagent lots, but there were no differences in results among the assays. Patients with strongly positive GM results were investigated. Medication histories revealed that 14 of 23 patients had been administered total parenteral nutrition solution from one manufacturer and 4 patients had been administered dextrose solution from a different manufacturer before being tested. The results of GM assays conducted on samples of dextrose solution and the glucose fraction of the total parenteral nutrition solution were strongly positive, confirming the causes of the false-positive reactions. We hypothesize that a trace amount of GM was introduced into the glucose-containing solutions because glucoamylase, which is necessary for the saccharification step of glucose synthesis, was derived from Aspergillus niger. To enhance patient care and prevent unnecessary antifungal prescriptions, healthcare providers and manufacturers of healthcare products need to be aware of the possibility of false-positive reactions for GM.


Discussion
Aspergillus spp.are common in the environment, and can cause IPA in immunocompromised patients such as recipients of hematopoietic stem cell transplants or solid organ transplants, patients undergoing chemotherapy, and those with human immunodeficiency virus infection 11 .Cases with positive GM results in this study were suspected to be false positives because the clinical manifestations of most of the patients did not indicate IPA.
The medication histories of the patients revealed that they had been administered glucose-containing solutions,  TPN-A and dextrose-X, which were identified as the causes of the false-positive results, proven by GM assays using direct samples of these solutions.Among the 23 patients, 13 were strongly false-positive for GM after being administered TPN-A (P1-13 in Fig. 1).Follow-up GM assays were considered unnecessary in most of them, and they were not prescribed sufficiently until the results became negative.In 3 patients followed until GM results changed to negative, time to negativity after discontinuation of TPN-A administration ranged 68-125 days (Fig. 2).However, a previous study reported that the average time to negativity was estimated to be 5.5 days and the half-life of GM was estimated to be 2.4 days 5 .This difference is likely because the patients were administered TPN-A for a prolonged period and their index values of GM assays were high (> 5.0), and the metabolic pathways and structures of GM originating from infectious Aspergillus spp.and glucose-manufacturing may be different.P13 and P16 were weakly positive for GM (index < 2.0) in October due to administration of TPN-A, but strongly positive in November.This was because manufacturer A changed the enzyme used to manufacture the glucose-containing solution in November.For the same reason, the index values of TPN-A differed among lots (Table 2).The index value of P14 was low, because this patient received TPN-A for a central line, which had a lower index value than TPN-A for a peripheral line.Patients who had received 5% dextrose-X (P19-22) were strongly positive for GM and remained positive for more than 1 week after cessation of 5% dextrose-X administration.P23 had a history of inpatient treatment at another hospital before being transferred to our hospital and may have received TPN-A or dextrose-X at that hospital.
The glucose in medical solutions is produced from starch in a multi-step process.The saccharification step uses glucoamylase, an enzyme from A. niger or Bacillus licheniformis.Glucoamylase from A. niger is used in the industrial production of glucose from raw starch or soluble oligosaccharides because it is thermostable and highly active in near-neutral pH environments 12 .If there is a problem extracting or purifying glucoamylase from A. niger, GM from the cell wall of A. niger may be introduced, potentially explaining GM positivity in glucose-containing solutions.Manufacturers A and X recently changed their glucoamylase suppliers, resulting in false-positive GM results in subsequent lots of TPN-A and dextrose-X.However, the glucoamylase supplier reported that the enzyme was not contaminated with viable fungi.Manufacturer A notified customers in South Korea of this problem and recalled the problematic lots of TPN-A.The possibility of false-positive results for GM by TPN-A was relayed to the hospital staff, TPN-A prescription was suspended, and comments were added to the results of GM assays.
Several causes of false-positive GM results were reported previously.Not only glucoamylase, but also citric acids are prepared from A. niger.A variety of anticoagulants and additive solutions used in platelet units contain citric acids, and there was a false-positive results of GM after platelet transfusion 13 .GM from Penicillium spp. is used in the manufacture of antibiotics, such as piperacillin/tazobactam and amoxicillin/clavulanate, and has been reported as a cause of false-positive results 14 .Other causes of false-positive GM results include crystalloid solutions, antibiotics, and dietary factors [4][5][6][7][8][9][10] .False-positive GM results may lead to antifungal prescriptions and additional workup such as chest CT, resulting in unnecessary medical costs and even harm to patients.Even worse is that false-positive GM results in symptomatic patients with pulmonary lesions in CT may mask more serious diseases which have symptomatic overlap with IPA, such as mucormycosis.To enhance patient care, healthcare providers and manufacturers of healthcare products need to be aware of the possibility of falsepositive results for GM.
This study had several limitations.Other diagnostic tests for IPA, such as beta-D-glucan, Aspergillus lateralflow assay, culture, and polymerase chain reaction 15 , were not performed.True or false positivity for Aspergillus spp.can be confirmed using other diagnostic tools.If the GM assay is problematic or not available, other diagnostic methods should be considered.Also, in most patients, GM assay results were not followed until they turned negative.As an observational study, this study was based on the dates of prescription of the medical solution and of the GM assay, and no patient intervention was performed.A systematic trial involving a regular follow-up after administration of several glucose-containing solutions is thus needed but may be controversial because of the benefit-risk balance.
In conclusion, patients strongly positive for GM without typical signs of IPA had been administered glucosecontaining solutions from several manufacturers.These solutions were confirmed to be the causes of the falsepositive results.Healthcare providers and manufacturers of medical products should be aware of causes of false-positive results for GM.If a diagnostic test for Aspergillus is problematic, use of alternative diagnostic methods is recommended.

Repeat GM assays
Repeat GM assays were performed on samples from patients suspected by physicians to have false-positive results and on samples from patients with positive or borderline values from 21 to 24 November, 2022.GM assays (Platelia Aspergillus Ag, Bio-Rad, Hercules, CA, USA) were performed on the GEMINI (Diatron, Budapest, Hungary) and Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA) platforms.To confirm reagent denaturation or contamination, GM assays were performed on old and new lots.An index value of ≥ 0.5 was classified as positive for GM.

Medication records
Patients who had strongly positive (index > 5.0) results or suspicion by physicians of false positivity for GM assays from 11 November to 5 December 2022 were included in the analysis.Medical histories and medication records from 1 October to 8 December were investigated to identify commonly prescribed medications.

GM assays on samples from nutritional solutions
Investigation of medication records revealed that the patients, who were strongly positive for GM, had received TPN-A.GM assays were performed on samples of the TPN-A and TPN-B, and dextrose-X and dextrose-Y.Because TPN solutions were divided into glucose, amino acid and electrolytes, and lipids fractions, GM assays were performed on samples of each.

Follow-up GM results after discontinuing administration of TPN-A
To confirm whether the GM results changed to negative after discontinuation of TPN-A administration, the GM results of patients suspected to have false-positive results were tracked.The time taken for GM results to become negative after TPN-A discontinuation was investigated.

Ethical approval
This study was approved by the Institutional Review Board of Seoul National University Bundang Hospital.The need for informed consent was waived by the Institutional Review Board of Seoul National University Bundang Hospital (study number: B-2301-807-101).All methods were performed in accordance with the Declaration of Helsinki.

Figure 1 .
Figure1.Galactomannan (GM) assay results and history of administration of total parenteral nutrition from manufacturer A (TPN-A) and 5% dextrose solution from manufacturer X (dextrose-X).Gray boxes, administration of TPN-A; white boxes, administration of 5% dextrose-X.Numbers indicate GM assay index values (N, negative).P1-P14, patients with falsepositive results for GM due to administration of TPN-A prior to testing.P15-P18, patients with probable invasive pulmonary aspergillosis and an abrupt increase of index values after administration of TPN-A.P19-P22, false-positive results for GM due to administration of 5% dextrose-X prior to testing.P20 received 5% dextrose-X 16-18 September (not shown).P23, medication history in another hospital (22 October to 25 November) prior to transfer to our hospital was not available.

Figure 2 .
Figure 2.Cases in which galactomannan assay results were confirmed to turn negative after discontinuing administration of total parenteral nutrition from manufacturer A (TPN-A).P2, P9, and P10 were administered TPN-A for more than a week and discontinued on 1 December, 12 November, and 26 October, respectively.It took 125, 68, and 79 days, respectively, for the galactomannan assay results of P2, P9, and P10 to change to negative after cessation of TPN-A.

Table 1 .
Timeline of investigation of the cause of false-positive results for galactomannan assay.GM galactomannan, IMI Division of Infectious Diseases, Department of Internal Medicine, IMH Division of Hematology, Department of Internal Medicine, TPN-A total parenteral nutrition solution from manufacturer A.

Table 2 .
Results of galactomannan assays performed on nutrition fluids and their fractions.TPN total parenteral nutrition, C for central line, P for peripheral line.*A, B, X, and Y are tentative names of manufacturers.**Tests were performed in duplicate.