ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2006, p. 3450–3453 0066-4804/06/$08.00⫹0 doi:10.1128/AAC.00658-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 50, No. 10
Reactivity of (133)--D-Glucan Assay with Commonly Used Intravenous Antimicrobials Francisco M. Marty,1,2,3* Colleen M. Lowry,1 Steven J. Lempitski,4 David W. Kubiak,1 Malcolm A. Finkelman,4 and Lindsey R. Baden1,2,3 Brigham & Women’s Hospital,1 Dana-Farber Cancer Institute,2 Harvard Medical School,3 Boston, Massachusetts, and Associates of Cape Cod, Inc., East Falmouth, Massachusetts4 Received 30 May 2006/Returned for modification 15 July 2006/Accepted 31 July 2006
Forty-four intravenous antimicrobials were tested for the presence of (133)--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 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 reconstitutedvial 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. Briefly, 25 l 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), trimethoprimsulfamethoxazole, 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
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). (133)--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 crossreactivity 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 glucancontaining 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
* Corresponding author. Mailing address: Division of Infectious Diseases, Brigham & Women’s Hospital, 75 Francis Street, PBB-A4, Boston, MA 02115. Phone: (617) 732-8881. Fax: (617) 732-6829. E-mail:
[email protected]. 3450
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TABLE 1. Antimicrobials, manufacturers, and lots tested Antimicrobial
Manufacturer
Lot(s)
Amikacin
Novaplus
617208A
Amphotericin B deoxycholate
Novaplus
Amphotericin B, liposomal
RVC (mg/ml)
DIC (mg/ml) a
MPC (g.ml)
250
NT
NT
677431
5
NT
NT
Gilead
042458AA
4
2
Ampicillin
Abraxis
4A04AH
250
NT
Ampicillin-sulbactam
Baxter
2040114 R004A R684A
250
30
Azithromycin
Pfizer
615247 728836 731654
100
2
Aztreonam
Dura
4E80157
100
40
Caspofungin
Merck
0103R
Cefazolin (premixed bag)
Baxter
LD095678 LD096487 LD096636
Cefazolin (vial)
Sandoz
Cefepime
118 NT 150
3.63
204
5
NT
20
20
185
C4274 C4711 C4761
330
20
185
Bristol-Myers Squibb
4K89752 5K09940 6B17578
100
40
164
Cefotaxime
Novaplus
014640 075792 085466
95
40
214
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
Clindamycin
Abbott
16356DK
150
Colistin
X-Gen
YR4CM 5T5CM 6U5CM
50
Daptomycin
Cubist
180503A
50
10
Doxycycline
Bedford Laboratories
712192
10
1
Ertapenem
Merck
3741R 3885P 3962R
100
20
Erythromycin
Abbott
22742Z7
50
NT
NT
Fluconazole
Abbott
20098JT
2
2
8.1
Gentamicin
Abraxis
141293
40
NT
NT
2
1.6 NT 3.5
NT
4.6 NT 5
133 2.6 155
Continued on following page
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NOTES
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1—Continued
Antimicrobial
Manufacturer
Lot(s)
RVC (mg/ml)
DIC (mg/ml)
MPC (g.ml)
NT
NT
Imipenem
Merck
3829P
50
Itraconazole
Ortho Biotech
04A01A27
10
Levofloxacin
Ortho McNeil
25195JT
5
5
12
Linezolid
Pharmacia
04K12Z97
2
2
21
Meropenem
Astra Zeneca
KP0021
50
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 200357 200407
60
Piperacillin-tazobactam
Pfizer
A56123 A91661 A91796
125
68
Quinupristin-dalfopristin
Monarch
2L1356
500
5
Rifampin
Bedford
575667
60
6
23
Ticarcillin-clavulanate
Novaplus
63787A
200
100
388
Tigecycline
Wyeth
B19951
10
Tobramycin
Abbott
11186DK
40
3.2
Trimethoprim-sulfamethoxazole
Sicor
04P114 05P101 06A124
80
1
9
Vancomycin
Novaplus
2074327
50
10
40
Voriconazole
Pfizer
A04192
10
5
4
a
3.3
2.5
NT
3.7
NT
0.612
298
3.9
NT 12
NT, not tested.
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). 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 piperacillintazobactam 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
VOL. 50, 2006
NOTES
TABLE 2. BG content of intravenous antimicrobialsa Concn of BG (pg/ml) at the: Medication RVC
Colistin Ertapenem Cefazolin (vials) Trimethoprim-sulfamethoxazole Cefotaxime Cefepime Ampicillin-sulbactam Azithromycin Pentamidine Tobramycin Liposomal amphotericin B Vancomycin Oxacillin Itraconazole Doxycycline Quinupristin-dalfopristin Nafcillin Rifampin Chloramphenicol Amphotericin B deoxycholate Daptomycin Voriconazole Amikacin Aztreonam Caspofungin Cefuroxime Erythromycin Gentamicin Imipenem Piperacillin-tazobactam Ticarcillin-clavulanate Ampicillin Cefazolin (bag) Cefoxitin Ceftazidime Ceftriaxone Ciprofloxacin Clindamycin Fluconazole Levofloxacin Linezolid Meropenem Metronidazole Penicillin G Tigecycline
4,348 3,472 2,054 1,187 560 425 519 c ⬍62,500 ⬍5,000 ⬍1,600 ⬍1,600 ⬍800 ⬍800 ⬍600 ⬍600 ⬍600 ⬍400 ⬍400 ⬍200 ⬍200 ⬍200 ⬍200 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍40 ⬍32 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍40 ⬍16
DIC
MPC b
627 166 67 ⬍32 153 106 ⬍4 ⬍1,250 ⬍2,187 ⬍4 ⬍16 ⬍32 4 23 ⬍16 5 22 23 39 NT Optical artifact 14 NT 14 NT 21 NT NT NT ⬍16 13 NT ⬍32 NT NT NT ⬍4 NT ⬍4 ⬍4 ⬍16 NT 6 ⬍16 NT
⬍4 ⬍32 ⬍4 ⬍8 ⬍8 ⬍8 ⬍8 ⬍8 ⬍4 ⬍4 4 ⬍4 ⬍4 ⬍4 ⬍4 ⬍4 ⬍4 ⬍4 5 NT 4 ⬍4 NT ⬍4 NT ⬍4 NT NT NT ⬍8 8 NT ⬍4 NT NT NT ⬍4 NT ⬍4 ⬍4 ⬍4 NT ⬍4 ⬍4 NT
a Drugs 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. b Only one vial was reactive; the other two lots inhibited the reaction at dilutions up to 1:500 (⬍1,563 pg/ml of BG). c Only one of three vials was reactive; the other two lots inhibited the reaction at dilutions up to 1:100 (⬍313 pg/ml of BG).
encountered. Interestingly, no piperacillin-tazobactam lots tested positive for BG. This may allow centers that use piperacillintazobactam for empirical febrile neutropenia therapy the possibility of using BG for noninvasive surveillance and diagnosis of IFI.
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