Reactivity of (1→3)-β-d-Glucan Assay with Commonly Used Intravenous Antimicrobials

Abstract

Forty-four intravenous antimicrobials were tested for the presence of (1→3)-β-d-glucan (BG). Colistin, ertapenem, cefazolin, trimethoprim-sulfamethoxazole, cefotaxime, cefepime, and ampicillin-sulbactam tested positive for BG at reconstituted-vial concentrations but not when diluted to usual maximum plasma concentrations. False-positive BG assays may occur when some antimicrobials are administered; however, this needs to be confirmed.

The Food and Drug Administration (FDA) approved the Glucatell assay (Associates of Cape Cod, Falmouth, MA) in 2004 as an aid in the diagnosis of deep-seated mycoses and fungemia (3, 10, 15). (1→3)-β-d-Glucan (BG) is present in the cell walls of many pathogenic fungi, including Candida sp., Aspergillus sp., and Fusarium sp. (11).

Galactomannan detection by sandwich enzyme-linked immunosorbent assay (Platelia Aspergillus enzyme-linked immunosorbent assay; Bio-Rad Laboratories, Hercules, CA) was approved by the FDA as a diagnostic aid for the diagnosis of invasive aspergillosis in 2003 (2). Although the in vitro cross-reactivity of galactomannan with piperacillin-tazobactam had been reported (1, 18), it was only after marketing the assay that clinical false-positive results were observed (14, 17). This diminished the diagnostic utility of galactomannan detection in centers where piperacillin-tazobactam had become the empirical antibacterial treatment of febrile neutropenic patients.

False-positive BG results are known to occur in patients undergoing hemodialysis with cellulose membranes (5); patients treated with immunoglobulin, albumin (4, 12), or other blood products filtered through cellulose depth filters containing BG (9, 16); and patients with serosal exposure to glucan-containing gauze (6).

Given the potential for fungal cell wall elements or leachates from cellulosic materials to be present in antimicrobial preparations, we sought to determine the reactivity of the BG assay among intravenous antimicrobials available in the United States.

(This work was presented in part previously [F. M. Marty, C. M. Lowry, S. J. Lempitski, D. W. Kubiak, M. A. Findelman, and L. R. Baden, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. M-163, 2005].)

Forty-four commercially available intravenous antimicrobial agents were tested for the presence of BG using the Glucatell assay (Table 1). An initial lot was tested for each agent, with the exception of piperacillin-tazobactam, for which three lots were tested. All drug samples were diluted or solubilized, as specified in the product's package insert, to yield reconstituted-vial concentrations (RVC). All diluents used in the reconstitution process were also tested. Once prepared, samples were coded, placed in BG-free vials, frozen to −80°C, and shipped for analysis to Beacon Diagnostics Laboratory (East Falmouth, MA), where all samples were tested in a blind fashion.

TABLE 1.

Antimicrobials, manufacturers, and lots tested

Antimicrobial	Manufacturer	Lot(s)	RVC (mg/ml)	DIC (mg/ml)	MPC (μg.ml)
Amikacin	Novaplus	617208A	250	NTa	NT
Amphotericin B deoxycholate	Novaplus	677431	5	NT	NT
Amphotericin B, liposomal	Gilead	042458AA	4	2	118
Ampicillin	Abraxis	4A04AH	250	NT	NT
Ampicillin-sulbactam	Baxter	2040114	250	30	150
		R004A
		R684A
Azithromycin	Pfizer	615247	100	2	3.63
		728836
		731654
Aztreonam	Dura	4E80157	100	40	204
Caspofungin	Merck	0103R	5	NT	NT
Cefazolin (premixed bag)	Baxter	LD095678	20	20	185
		LD096487
		LD096636
Cefazolin (vial)	Sandoz	C4274	330	20	185
		C4711
		C4761
Cefepime	Bristol-Myers Squibb	4K89752	100	40	164
		5K09940
		6B17578
Cefotaxime	Novaplus	014640	95	40	214
		075792
		085466
Cefoxitin	Abraxis	400080	100	NT	NT
Ceftazidime	GlaxoSmithKline	4532	100	NT	NT
Ceftriaxone	Roche	U6555	100	NT	NT
Cefuroxime	Novaplus	C3730	94	30	100
Chloramphenicol	Abraxis	131086	100	40	11
Ciprofloxacin	Bayer	2500L75	2	1.6	4.6
Clindamycin	Abbott	16356DK	150	NT	NT
Colistin	X-Gen	YR4CM	50	3.5	5
		5T5CM
		6U5CM
Daptomycin	Cubist	180503A	50	10	133
Doxycycline	Bedford Laboratories	712192	10	1	2.6
Ertapenem	Merck	3741R	100	20	155
		3885P
		3962R
Erythromycin	Abbott	22742Z7	50	NT	NT
Fluconazole	Abbott	20098JT	2	2	8.1
Gentamicin	Abraxis	141293	40	NT	NT
Imipenem	Merck	3829P	50	NT	NT
Itraconazole	Ortho Biotech	04A01A27	10	3.3	3.7
Levofloxacin	Ortho McNeil	25195JT	5	5	12
Linezolid	Pharmacia	04K12Z97	2	2	21
Meropenem	Astra Zeneca	KP0021	50	NT	NT
Metronidazole	Abbott	24073JT	5	5	25
Nafcillin	Sandoz	129903	250	40	30
Oxacillin	Apothecon	2L62896	167	10	43
Penicillin G	Baxter	LN044859	25	25	400
Pentamidine	Abraxis	141387	60	2.5	0.612
		200357
		200407
Piperacillin-tazobactam	Pfizer	A56123	125	68	298
		A91661
		A91796
Quinupristin-dalfopristin	Monarch	2L1356	500	5	3.9
Rifampin	Bedford	575667	60	6	23
Ticarcillin-clavulanate	Novaplus	63787A	200	100	388
Tigecycline	Wyeth	B19951	10	NT	NT
Tobramycin	Abbott	11186DK	40	3.2	12
Trimethoprim-sulfamethoxazole	Sicor	04P114	80	1	9
		05P101
		06A124
Vancomycin	Novaplus	2074327	50	10	40
Voriconazole	Pfizer	A04192	10	5	4

^aNT, not tested.

Briefly, 25 μl of drug sample at the RVC was added to wells in duplicate. One hundred microliters of Glucatell reagent was added to the wells. The sample and reagent were incubated at 37°C, using a time-of-onset kinetic assay (as described in the product insert). Samples that demonstrated inhibition of the reaction at RVC were serially diluted in glucan-free water. Analysis was performed using a log-log plot of time to onset versus standard concentration (pachyman). Duplicate samples were spiked with a positive control reagent. A spike recovery of 50 to 200% was required for a valid test result.

RVC drug solutions that tested positive or inhibited the BG reaction when diluted to concentrations above the drug infusate concentration (DIC) were retested at the DIC and maximum plasma concentrations (MPC) as directed in the package insert or scientific literature (Table 1). Further lots of those antimicrobials that tested positive or inhibited the BG reaction were analyzed for reproducibility. All these samples were prepared and tested in a blind fashion.

Seven antimicrobial agents tested positive for BG at the RVC: colistin, ertapenem, cefazolin (in vials), trimethoprim-sulfamethoxazole, cefotaxime, cefepime, and ampicillin-sulbactam in decreasing order (Table 2). BG was detected in additional lots of these antimicrobials at the RVC, with the exception of ampicillin-sulbactam, of which two additional lots were nonreactive. Colistin, ertapenem, cefotaxime, and cefepime had detectable BG at concentrations greater than 80 pg/ml when diluted to the DIC; two lots of colistin inhibited the reaction and needed to be diluted 500-fold in order to obtain a valid result at the DIC. Although all lyophilized cefazolin vials had detectable BG at the RVC, no premixed cefazolin bag lots from a different manufacturer had detectable BG. None of the seven agents tested positive for BG when diluted to concentrations representing the MPC. Diluents and products used in the transfer of drug did not have detectable BG levels. Several antimicrobials inhibited the BG assay at the RVC, but only azithromycin, pentamidine, and colistin remained highly inhibitory at the DIC (Table 2).

TABLE 2.

BG content of intravenous antimicrobials^a

Medication	Concn of BG (pg/ml) at the:
	RVC	DIC	MPC
Colistin	4,348	627b	<4
Ertapenem	3,472	166	<32
Cefazolin (vials)	2,054	67	<4
Trimethoprim-sulfamethoxazole	1,187	<32	<8
Cefotaxime	560	153	<8
Cefepime	425	106	<8
Ampicillin-sulbactam	519c	<4	<8
Azithromycin	<62,500	<1,250	<8
Pentamidine	<5,000	<2,187	<4
Tobramycin	<1,600	<4	<4
Liposomal amphotericin B	<1,600	<16	4
Vancomycin	<800	<32	<4
Oxacillin	<800	4	<4
Itraconazole	<600	23	<4
Doxycycline	<600	<16	<4
Quinupristin-dalfopristin	<600	5	<4
Nafcillin	<400	22	<4
Rifampin	<400	23	<4
Chloramphenicol	<200	39	5
Amphotericin B deoxycholate	<200	NT	NT
Daptomycin	<200	Optical artifact	4
Voriconazole	<200	14	<4
Amikacin	<80	NT	NT
Aztreonam	<80	14	<4
Caspofungin	<80	NT	NT
Cefuroxime	<80	21	<4
Erythromycin	<80	NT	NT
Gentamicin	<80	NT	NT
Imipenem	<80	NT	NT
Piperacillin-tazobactam	<80	<16	<8
Ticarcillin-clavulanate	<80	13	8
Ampicillin	<40	NT	NT
Cefazolin (bag)	<32	<32	<4
Cefoxitin	<40	NT	NT
Ceftazidime	<40	NT	NT
Ceftriaxone	<40	NT	NT
Ciprofloxacin	<40	<4	<4
Clindamycin	<40	NT	NT
Fluconazole	<40	<4	<4
Levofloxacin	<40	<4	<4
Linezolid	<40	<16	<4
Meropenem	<40	NT	NT
Metronidazole	<40	6	<4
Penicillin G	<40	<16	<4
Tigecycline	<16	NT	NT

^aDrugs were ordered according to their BG reactivity. Antimicrobials that tested positive are listed first, followed by antimicrobials that inhibited the BG assay at the RVC. For positive or highly inhibitory assay results, the value provided is the average of three determinations. NT, not tested.

^bOnly one vial was reactive; the other two lots inhibited the reaction at dilutions up to 1:500 (<1,563 pg/ml of BG).

^cOnly one of three vials was reactive; the other two lots inhibited the reaction at dilutions up to 1:100 (<313 pg/ml of BG).

The availability of noninvasive diagnostic tests for the detection of invasive fungal infections (IFI) is an important advance in the management of such infections (13). As therapeutic decisions may be made based on the results of these tests, it is important to understand their potential limitations.

BG was detected in 7 of the 44 antimicrobial agents at RVC. BG was still detected in four of seven of these antimicrobials at DICs that are above the current positivity threshold for the assay. BG positivity was consistent among different antimicrobial lots, except with ampicillin-sulbactam. Some antimicrobials caused optical artifacts or inhibited the BG assay at high concentrations. Although not available in the United States, intravenous amoxicillin-clavulanic acid was found to contain high BG levels and to cause false-positive results after clinical administration (8).

There was no obvious antimicrobial class effect in terms of the BG reactivity. No antimicrobial solution had detectable BG at the usual drug MPC, but this should be interpreted with caution. As learned from the galactomannan enzyme-linked immunosorbent assay cross-reactivity experience with piperacillin-tazobactam administration (7, 17, 18), the false-positive results were not due to detection of the antimicrobial itself but to the introduction of galactofuran in the manufacturing process, which has a pharmacokinetic behavior different than that of the antimicrobial itself (18).

The findings of BG content cannot be generalized to products produced by manufacturers other than those tested (Table 1). There can be lot-to-lot variability in BG content, as in the case of galactomannan (7), so additional periodic testing of antimicrobial lots commonly used in patients at risk for IFI may be warranted, especially if unexplained BG results are encountered. Interestingly, no piperacillin-tazobactam lots tested positive for BG. This may allow centers that use piperacillin-tazobactam for empirical febrile neutropenia therapy the possibility of using BG for noninvasive surveillance and diagnosis of IFI.

There is limited reactivity between the BG assay and most commonly used intravenous antimicrobial agents. Caution should be used in interpreting BG assay results for drug preparations that do contain BG, as the kinetics of BG are not yet defined. Validation of these findings and correlation with clinical samples are warranted.