Keywords
vancomycin - clinical laboratory services - immunoassay synovial fluid - antibacterial
agents
Introduction
Clinical laboratories are required to answer several emerging clinical questions;
however, any request for implementation, in terms of a new biomarker, innovative assay,
or unconventional samples, and alternative matrices, needs to be evaluated according
to best practice and to certification rules.
When instruments or reagents, intended for a specific sample matrix, are used to analyze
a different matrix, laboratory personnel must validate the procedure, according to
the international guideline such as Clinical and Laboratory Standards Institute (CLSI)
EP19.
Clinical laboratories routinely perform serum vancomycin assay for therapeutic drug
monitoring (TDM). Recently, assessment of vancomycin levels in synovial fluid from
patient affected by periprosthetic joint infection (PJI) and undergoing revision surgery
has been suggested.[1]
PJI is a rare yet devastating complication after total joint replacement, ranging
between 0.5 and 3%, with total knee arthroplasty (TKA) having a higher prevalence
than total hip arthroplasty.[2] Two-stage revision surgery with antibiotic-loaded spacer implantation is now considered
the standard of care.[3]
[4]
[5] Vancomycin is widely used and, although the pharmacokinetic properties of aminoglycosides
have been thoroughly investigated in vitro and in vivo studies,[5]
[6]
[7]
[8] only limited data are available on the vancomycin release in vivo from prosthetic
devices after implantation.[1] Accordingly, it appears to be of paramount importance the availability of an affordable
and reliable analytical method to study vancomycin in vivo.
The aim of this study was to verify if vancomycin assay validated for TDM in human
plasma and serum can be used to measure vancomycin in drainage fluid.
Methods
K2EDTA blank drainage fluid samples were obtained from patients (n = 3) who underwent primary TKA. No systemic vancomycin was given before or after
surgery in any patient. The samples collected 24 hours after surgery were pooled,
centrifuged, transferred to Microcon YM10 filters (Merck Millipore, Billerica, Massachusetts,
United States), designed to remove proteins from viscous biological samples. According
to manufacturer instructions, filters were loaded and centrifuged at 15,000 g for
20 minutes, and the ultrafiltrates were then collected (Filtered Synovial Fluid [FSF]).
Vancomycin hydrochloride for injection USP 500 mg was purchased from Mylan N.V. Pharmaceutical
(Amsterdam, The Netherlands) and a stock solution of vancomycin (1 mg/mL) was prepared
in blank FSF. Calibration standards at vancomycin concentrations of 5, 10, 25, 50,
and 100 µg/mL were obtained from appropriate additions of stock solution to blank
FSF.
Three levels of quality control (2.5, 25.0, and 50.0 µg/mL) were analyzed during each
routine analysis, and these were prepared by the appropriate addition of another (independently
prepared and weighed) stock solution of vancomycin (1 mg/mL).
All samples were analyzed on CDx90 (Thermo Scientific, Whaltam, Massachusetts, United
States) instrument by Quantitative Microsphere System (QMS) vancomycin assay (Thermo
Scientific); the vancomycin assay is a homogeneous particle-enhanced turbidimetric
immunoassay, and it is based on competition between drug in the sample and drug coated
onto a microparticle for antibody-binding sites of the vancomycin antibody reagent.
The vancomycin-coated microparticle reagent is rapidly agglutinated in the presence
of the antivancomycin antibody reagent and the absence of any competing drug in the
sample. The rate of absorbance change is measured photometrically.
When a sample containing vancomycin is added, the agglutination reaction is partially
inhibited, slowing down the rate of absorbance change. A concentration-dependent classic
agglutination inhibition curve can be obtained with a maximum rate of agglutination
at the lowest vancomycin concentration and the lowest agglutination rate at the highest
vancomycin concentration.
Results
The method was linear from 0 to 100 µg/mL (r2 = 0.987), selectivity was checked by analyzing 25 blank drainage fluid samples, the
limit of quantification was 2.5 µg/mL and the accuracy ranged from 82 to 93% at three
concentration levels (2.5, 25, and 50 µg/mL).
The precision of the method was determined by calculating the coefficient of variability
at three concentration levels (2.5, 25, and 50 µg/mL) for 10 times in the same day
(intra-assay precision) and once for 10 days (inter-assay precision). Intra-assay
precision ranged from 1.8 to 4.9% at each level and the inter-assay precision from
4.0 to 10.4%.
Discussion
Since the introduction of antibiotic-loaded acrylic bone cement, antibiotic-impregnated
bone cement have been widely used as a delivery vehicle for the local administration
of antibiotics in joint-infected sites, reaching higher concentrations compared with
systemic administration.[5]
The most commonly used antibiotics include tobramycin, gentamicin, vancomycin, and
cephalosporins that show a wide spectrum activity even against the multidrug-resistant
bacteria. Strikingly, a recent systematic review of the use of antibacterial cement
spacers reports that a significantly high infection eradication rate, ranging from
73 to 100%, can be achieved even when the antibacterials placed into the spacers are
not active against the infecting organisms.[5]
Despite these appealing results, several questions still remain to be addressed to
optimize the use of these devices and to define the ideal moment for prosthetic reimplantation.
Indeed, the therapeutic concentration levels of antibiotics present at the implantation
site and the actual inhibitory activity of antibiotics released at the infection site,
in the first postoperative period, are still open questions, along with the duration
of the antimicrobial effect.
The possibility to measure vancomycin levels in drainage fluid is the basis for studying
the antibiotic release in vivo from prosthetic devices after implantation and might
represent a step ahead toward treatment optimization. Nevertheless, significant heterogeneity
caused by differences in methods and experimental conditions have complicated the
attempts to draw definite conclusions from available studies. The QMS method guarantees
enough precision and accuracy at low costs to answer, at least, some of the clinical
questions related to PJIs.
