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71―123（ 23 ）〈Original Article〉

Significant 1, 3-β-D-glucan content
in a penicillin G potassium productJun Suzuki1, Shuji Hatakeyama1,2,*,
Yuji Morisawa1 and Toshiyuki Yamada3
1

Division of Infectious Diseases, Jichi Medical University Hospital,
3311–1 Yakushiji, Shimotsuke-shi, Tochigi 329–0498, Japan
2
Division of General Internal Medicine, Jichi Medical University Hospital,
3311–1 Yakushiji, Shimotsuke-shi, Tochigi 329–0498, Japan3
Department of Clinical Laboratory Medicine, Jichi Medical University,
3311–1 Yakushiji, Shimotsuke-shi, Tochigi 329–0498, Japan(Received for publication December 4, 2017)

β-D-Glucan (BDG) is a fungal cell wall component and a useful marker indiagnosing invasive fungal infections. However, false-positive BDG test results
occasionally limit its utility. We recently treated a patient whose plasma BDG levels
were elevated during administration of penicillin G potassium despite the absence offungal infections. We therefore investigated the BDG contents of different penicillin
G potassium products available in Japan. BDG levels were measured using twodifferent assays. An injectable penicillin G potassium product, the only productapproved for clinical use in Japan, was found to contain signiﬁcant amounts of BDG
in all batches and lots tested. According to the Fungitec G test MK II “Nissui” (cutoff value, 20 pg/mL) and β-glucan test Wako (cut-off value, 11 pg/mL) assays, BDG
levels in 10,000 units/mL solutions of this agent (5 lots) ranged from 79.4 to
192.9 pg/mL and 16.9 to 34.5 pg/mL, respectively. The two other penicillin G
potassium products tested, which are used for cell culture, did not contain signiﬁcantamounts of BDG. The amounts of BDG contained in the injectable product areprobably sufﬁcient to cause false-positive test results in patients receiving clinicaldoses (e.g., 24 million units per day). Further clinical studies are needed to conﬁrm
this result.

*Corresponding author, Tel: ＋81–285–58–7498, Fax: ＋81–285–40–5160, E-mail: shatake-tky@umin.ac.jp

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Introduction
The fungal cell wall component 1,3-β-D-glucan (BDG) is frequently used in clinical settings
to diagnose invasive fungal infections. However, positive BDG test results associated with nonfungal etiology (clinical false-positive results) occasionally limit the utility of the test. False-positive BDG results have been reported with several causes, including hemodialysis with cellulose
membrane, transfusion of blood products, and the use of wound gauze1,2,3). An antibiotic, intravenous amoxicillin-clavulanic acid (not available in Japan), has also been recognized as a causative
agent of false-positive BDG test results4). Previous studies have demonstrated that several otherantibiotics are reactive to the BDG assay in vitro, but practical administration of the contaminateddrugs is thought to have a small or unknown inﬂuence on plasma BDG concentrations5,6).
Regarding penicillin G potassium, it has been reported in two studies that no signiﬁcant levels of BDG were detected5,6). However, we recently treated a patient whose plasma BDG levels
were elevated during administration of intravenous penicillin G potassium despite not having any
fungal infections or other known causes of false-positive results. The patientʼs plasma BDG levels (20 pg/mL is the cut-off to be considered positive) were elevated from 8.5 pg/mL before starting penicillin G potassium at 24 million units (MU) per day to above 75.0 pg/mL 14 days afteradministration of the drug (a case report is in preparation).
Therefore, we hypothesized that the penicillin G potassium product used in Japan might beassociated with false-positive BDG results. In this study, we measured the BDG levels of threepenicillin G potassium products available in Japan.

Materials and Methods
Penicillin G potassium products tested
We purchased three different penicillin G potassium products: penicillin G potassium 1 MUfor injection (Meiji Seika Pharma Co., Ltd., Tokyo, Japan), penicillin G potassium salt 5 g (1,530units/mg) (Nacalai Tesque, Inc., Kyoto, Japan), and benzylpenicillin potassium 5 g (1,430 units/mg) (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Penicillin G potassium 1 MU for injection (Meiji Seika Pharma) is the only penicillin G potassium product licensed for use in humansin Japan, and ﬁve different lots (lot numbers 840, 930, 932, 934, and 942) were examined in thisstudy. Single lots of penicillin G potassium manufactured by Nacalai Tesque (lot numberM6N1379) and Wako Pure Chemical Industries (lot number LKK2659), both of which are used
for cell culture, were also tested for comparison. Each penicillin G potassium product was diluted
with 0.9% saline, and solutions with a ﬁnal concentration of 10,000 units/mL were used for BDG
measurements.

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71―125（ 25 ）BDG assay
We measured BDG in the penicillin products using two different assays, the Fungitec G test
MK II “Nissui” (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and the β-glucan test Wako
(Wako Pure Chemical Industries, Ltd.), according to the manufacturersʼ instructions. Glucan-freeequipment and saline were used. The detection ranges and the cut-off values for positivity were4.0–500.0 pg/mL and 20.0 pg/mL, respectively, for the Fungitec G test MK II “Nissui” assay and6.0–600.0 pg/mL and 11.0 pg/mL, respectively, for the β-glucan test Wako assay. All samples
were measured in triplicate using the Fungitec G test MK II “Nissui” and measured singly using
the β-glucan test Wako. For the former assay, the means of the triplicate measurements are shownin the results. When the test results were negative according to the Fungitec G-test MK II “Nissui”, the β-glucan test Wako was not performed.

Results
Regarding the penicillin G potassium 1 MU for injection (Meiji Seika Pharma Co., Ltd.), themean (±standard deviation) BDG levels of triplicate 10,000 units/mL solutions of lot numbers
840, 930, 932, 934, and 942 were 79.4 (±4.0), 192.9 (±12.0), 187.0 (±26.0), 156.6 (±1.9), and146.3 (±9.8) pg/mL, respectively, according to the Fungitec G test MK II “Nissui” assay. Highlevels of BDG were also detected using the β-glucan test Wako assay, with values of 16.9, 34.5,30.1, 24.5, and 20.3 pg/mL, respectively (Table 1). In contrast, BDG concentrations in the penicilTable 1. β-D-Glucan levels measured by two assays in penicillin G potassium productsNT: not tested.
1
Products with lot numbers 900–999 are from different batches as those with lot numbers 800–899.
2
Values above the positive cut-off for each assay are shown in bold. Cut-off values for positivity were 20.0 pg/mL and 11.0 pg/
mL for the Fungitec G test MK II “Nissui” and β-glucan test Wako, respectively.

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lin G potassium products for cell culture manufactured by Nacalai Tesque and Wako Pure Chemical Industries were below the cut-off value (Table 1).

Discussion
The penicillin G potassium for injection that is available in Japan contained signiﬁcantamounts of BDG, regardless of the batch or lot numbers of the product. Amounts of BDG in
1 MU (1 vial) penicillin G potassium were estimated to be 7,940–19,290 pg and 1,690–3,450 pg
as measured by Fungitec G test MK II “Nissui” and β-glucan test Wako, respectively, indicatingsome variation in BDG concentrations across batches and lots. For adult patients, penicillin G potassium is generally administered intravenously as 18–24 MU per day. To date, there are few reports on the kinetics of plasma BDG. With that in mind, based on the estimated half-life of BDG
in humans (median, 20 h; range, 3–181 h)1) and the estimated volume of distribution in rabbits7), it
is postulated that the clinical use of this drug may lead to signiﬁcant elevations (false positives) inplasma BDG levels in humans. However, further kinetic studies of BDG in humans are needed toconﬁrm this estimate.
To the best of our knowledge, there are no other studies showing signiﬁcant BDG contamination in penicillin G potassium products. Marty et al. measured BDG levels in 44 intravenous
antibiotics and demonstrated that BDG was not detected in a penicillin G product (manufacturedby Baxter, USA; lot number, LN044859) using the Glucatell assay5). In a study conducted by Lisset al. that investigated BDG contents in 35 antimicrobial agents, another penicillin G product(Infectopharm, Germany; batch number, B011401,1) did not contain signiﬁcant levels of BDG
according to two different BDG tests6).
The BDG-positive penicillin G potassium product (Meiji Seika Pharma) in this study is produced by fed-batch fermentation of Penicillium species, the same technique adopted in the products manufactured by Nacalai Tesque (tested in this study) and Baxter (tested in the study byMarty et al.5)). Because signiﬁcant amounts of BDG were not contained in the latter two penicillin G products, BDG contamination may depend on the manufacturing processes of speciﬁc manufacturers. Contamination with Aspergillus galactomannan seems to have been eliminated from
non-generic piperacillin-tazobactam8), which had been described as a cause of false-positive results in patients tested using the Aspergillus galactomannan assay. Likewise, as Liss et al. havealso pointed out6), it is probably important for manufacturers to take measures to reduce the riskof BDG contamination in their products.
The penicillin G potassium for injection used in Japan was found to contain a signiﬁcantamount of BDG. Although this amount is probably enough to cause false-positive test results inpatients receiving clinical doses of this agent, further clinical (in vivo) studies are needed to conﬁrm these results.

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71―127（ 27 ）Acknowledgments
We would like to thank Tomoyuki Yamazaki (Nissui Pharmaceutical Co., Ltd.) for BDG testing support. We would like to thank Dr. Takehito Yamamoto (The Education Center for ClinicalPharmacy, Graduate School of Pharmaceutical Sciences, The University of Tokyo) for estimating
the kinetics of BDG in humans.
Conﬂict of Interest
The authors state that they have no conﬂict of interest (COI).

References
1) Kanda H, Kubo K, Hamasaki K, et al.: Inﬂuence of various hemodialysis membranes on theplasma (1→3)-β-D-glucan level. Kidney Int. 2001; 60: 319–23.
2) Kanamori H, Kanemitsu K, Miyasaka T, et al.: Measurement of (1–3)-beta-D-glucan derived fromdifferent gauze types. Tohoku J Exp Med. 2009; 217: 117–21.
3) Barton C, Vigor K, Scott R, et al.: Beta-glucan contamination of pharmaceutical products: Howmuch should we accept? Cancer Immunol Immunother. 2016; 65: 1289–301.
4) Mennink-Kersten MA, Warris A, Verweij PE: 1,3-beta-D-glucan in patients receiving intravenous
amoxicillin-clavulanic acid. N Engl J Med. 2006; 354: 2834–5.
5) Marty FM, Lowry CM, Lempitski SJ, Kubiak DW, Finkelman MA, Baden LR: Reactivity of(1→3)-β-D-glucan assay with commonly used intravenous antimicrobials. Antimicrob Agents
Chemother. 2006; 50: 3450–3.
6) Liss B, Cornely OA, Hoffmann D, Dimitriou V, Wisplinghoff H: 1,3-β-D-Glucan contamination ofcommon antimicrobials. J Antimicrob Chemother. 2016; 71: 913–5.
7) Yoshida M, Roth RI, Grunfeld C, Feingold KR, Levin J: Soluble (1→3)-beta-D-glucan puriﬁed
from Candida albicans: Biologic effects and distribution in blood and organs in rabbits. J Lab
Clin Med. 1996; 128: 103–14.
8) Mikulska M, Furfaro E, Del Bono V, et al.: Piperacillin/tazobactam (Tazocin) seems to be no longer responsible for false-positive results of the galactomannan assay. J Antimicrob Chemother.
2016; 67: 1746–8.