Rapid Identification of Microorganisms from Sterile Body Fluids by Use of FilmArray

Abstract

We evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel in the identification of microorganisms from positive blood culture bottles inoculated with sterile body fluids. All organisms included in the FA BCID panel were accurately identified in 84/84 (100%) and 18/24 (75%) samples with mono- and polymicrobial growth, respectively.

TEXT

Early detection and rapid identification of microorganisms are crucial the for appropriate management of infections of normally sterile body fluids (SBF) (1–3). Previously, SBF other than blood were cultured on solid medium (4). Larger volumes were processed through centrifugation and filtration in order to concentrate and improve the detection of microorganisms. Nonetheless, the microorganism recovery from SBF was unsatisfactory until the introduction of blood culture (BC) bottles. The introduction of BC bottles resulted in increased microorganism recovery in addition to decreased time to detection (5–10). However, the identification of microorganisms from positive bottles with conventional methods may still take up to 48 h for many microorganisms and even longer for others, such as slow-growing bacteria and yeasts (11). Several methods have been evaluated for rapid identification of microorganisms directly from positive blood cultures (12–14). In contrast, the data on the performance of rapid microbiological methods in diagnostics of SBF are scarce. The aim of the present study was to evaluate the performance of the FilmArray blood culture identification (BCID) panel in the identification of microorganisms directly from positive blood culture bottles inoculated with clinical SBF specimens.

This study was performed at Karolinska University Hospital, Huddinge, Sweden. SBF specimens were collected in the clinical wards using standard protocols. Specimen volumes of 1 to 2 ml were inoculated in BacT/Alert pediatric/PF plus (bioMérieux, Durham, NC, USA), and volumes of >2 ml in BacT/Alert aerobic/FA plus and anaerobic/FN plus bottles (bioMérieux). All bottles were incubated in the BacT/Alert 3D blood culture system (bioMérieux) until they gave a positive signal or for a maximum incubation time of 5 days.

Positive BC bottles with SBF were processed as follows. Gram staining was performed immediately after bottles signaled positivity. All positive bottles were subcultured onto a Columbia blood agar plate (aerobic and anaerobic), a cystine-lactose-electrolyte-deficient plate (CLED) agar, and a chocolate agar plate. After an incubation time of 48 h, we analyzed the growth morphology on the agar plates. Identification of microorganisms was done by conventional methods, including matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany) and Vitek2 XL (bioMérieux). Susceptibility testing was performed by the disc diffusion method according to EUCAST guidelines (http://www.eucast.org/). Methicillin resistance in Staphylococcus spp. was evaluated by the cefoxitin disc diffusion method. Similarly, imipenem and meropenem disc diffusion methods were used in order to determine/confirm the absence of Klebsiella pneumoniae carbapenemase (KPC). FilmArray (FA; bioFire, Salt Lake City, UT) analyses were performed as previously described (13). Results that were discrepant between the reference method and the FilmArray were evaluated further. According to the discrepant results, additional subcultures were performed on selective agar plates, such as CLED, colistin-nalidixic acid, Sabouraud, and CHROM agar plates. Subsequently, the identification of microorganisms from these plates was performed by conventional methods as described above.

In total, 116 SBF specimens (51 pleural, 38 synovial, 10 abscess, 9 dialysis, 7 cerebrospinal, and 1 bile fluid) from 106 patients were included during the study period. These included 92/116 (79%) specimens with monomicrobial growth and 24/116 (21%) with polymicrobial growth. During the study period, only 1/116 tests resulted in invalid internal run controls, giving a 1% pouch failure rate.

Thirty different species could be isolated during the study period, and 25/30 (83%) were covered by the FA BCID panel. The three Enterococcus spp., E. faecalis, E. faecium, and E. avium, were identified at the genus level.

The FilmArray results were in concordance with those of conventional identification methods in 84/92 (91%) bottles with monomicrobial growth. The sensitivity, specificity, and negative and positive predictive values for organisms included on the FA BCID panel in bottles with monomicrobial growth were 100%, 100%, 100%, and 98%, respectively. It should be noted that 8 of the 92 (9%) bottles with monomicrobial growth contained microorganisms that were not covered by the FilmArray BCID panel and were negative due to noninclusion on the panel. (Table 1). FilmArray identified all microorganisms included in the FA BCID panel in 18/24 (75%) specimens with polymicrobial growth (Table 2). These constituted 10/24 (42%) specimens where the FilmArray results were in concordance with those of the reference method. In 3/24 (13%) specimens, FilmArray detected additional microorganisms compared to the results for conventional culture methods, and these were later confirmed by additional cultures. FilmArray could identify the microorganisms that were included in the FA BCID panel in 5/24 (21%) specimens; however, these specimens also included microorganisms that were not in the panel. Conflicting results for FilmArray and the reference method were observed in 6/24 (25%) specimens (Table 2).

TABLE 1.

Identification of bacteria and yeasts and detection of antibiotic resistance markers from 92 monomicrobial SBF cultures by FilmArray and reference method

Identification	No. of specimens that were:
	Culture positive and FA positive	Culture positive and FA negative	Culture negative and FA positive
Microorganism or marker included in FA BCID panel
Gram-positive bacteria
Staphylococcus aureus	32
Coagulase-negative staphylococcus	13		1a
Streptococcus pneumoniae	3
Enterococcus spp.	7
Streptococcus spp.	6		1b
Streptococcus agalactiae	3
Alpha-hemolytic streptococcus	7
Streptococcus pyogenes	2
Listeria monocytogenes	1
Gram-negative bacteria
Escherichia coli	3
Klebsiella pneumoniae	3
Pseudomonas aeruginosa	1
Fungi
Candida albicans	2
Candida glabrata	1
Antibiotic resistance marker mecA	2
Microorganisms not included in FA BCID panel
Corynebacterium spp.		3
Micrococcus spp.		2
Aeromonas spp.		1
Bacillus cereus		1
Actinomyces spp.		1

^aThe FilmArray identified Staphylococcus spp., while the reference method as well as additional culturing identified B. cereus.

^bThe FilmArray identified Enterococcus spp. and Streptococcus spp., while the reference method and additional cultures only identified Enterococcus faecalis.

TABLE 2.

Results for polymicrobial specimens analyzed by the FilmArray and reference method

Specimen (n = 24)	Microorganisms identified by FilmArray and the reference methodsa
Pleural fluid	Enterococcus faecalis, E. coli, P. aeruginosa
Pleural fluid	E. faecalis, P. aeruginosa, C. albicans, coagulase-negative staphylococcus, Foxrb
Pleural fluid	E. coli, Staphylococcus epidermidis, Foxr
Pleural fluid	Enterococcus avium, E. coli
CSF	S. epidermidis, Foxs, E. coli
Pleural fluid	E. faecalis, P. aeruginosa
Pleural fluid	Enterococcus faecium, Citrobacter farmeri, S. epidermidis, Foxr
Pleural fluid	E. faecium, E. faecalis, E. coli, C. albicans
Pleural fluid	E. faecalis, E. faecium, S. aureus
Pleural fluid	E. faecalis, E. faecium, C. albicans
Pleural fluid	E. faecalis (Staphylococcus capitis, Foxr)
Pleural fluid	E. faecalis, Proteus vulgaris, Citrobacter freundii (S. aureus)
Abscess	E. faecium, E. coli (K. pneumoniae)
CSF	E. coli, Micrococcus spp.
Bile	E. faecalis, Enterobacter cloacae, Klebsiella oxytoca, Prevotella spp.
Pleural fluid	E. faecium, alpha-hemolytic streptococcus, Candida krusei, Candida parapsilosis, Saccharomyces cerevisiae
Pleural fluid	S. pneumoniae, Prevotella spp.
Pleural fluid	C. glabrata, Pediococcus acidilactici, Lactobacillus spp.
Pleural fluid	C. glabrata, C. albicans, S. epidermidis, Foxr, E. faecium, (Streptococcus salivarius)
Abscess	E. faecalis, E. avium, E. coli, K. oxytoca, anaerobic flora (Enterococcus spp., E. coli, K. oxytoca, Streptococcus oralis, P. aeruginosa, H. influenzaec)
Abscess	E. coli (Streptococcus anginosus, K. oxytoca)
Pleural fluid	C. albicans (S. pneumoniaec; culture broth was Wellcogen positive)
Pleural fluid	Alpha-hemolytic streptococcus, E. faecalis (no growth on culture)
Pleural fluid	S. aureus (S. anginosusd)

^aThe reference methods were conventional culture methods. Boldface shows names or markers identified by both FilmArray and the reference method, lightface shows identification only by the reference method, underlined names were not included in the FA BCID panel, and names or markers in parentheses were identified first only by FA BCID and were later confirmed by the reference method after additional subcultures of the bottle culture broth.

^bFilmArray detected mecA, while conventional methods used the disc diffusion method. Fox^r, cefoxitin resistant; Fox^s, cefoxitin susceptible.

^cWe were unable to confirm H. influenzae and S. pneumoniae on repeated cultures.

^dOn repeated culturing, S. anginosus was found instead of the Enterococcus spp. on FilmArray analyses.

The FilmArray and disc diffusion results were concordant for the detection of seven Staphylococcus spp. with mecA. There were no vanA- or vanB-positive Enterococcus spp. and no bla_KPC-positive organisms detected during the study period.

The FilmArray accurately identified all microorganisms included in the FA BCID panel in 84/84 (100%) mono- and 18/24 (75%) polymicrobial growths, respectively. The remaining microorganisms in 8/92 (9%) bottles with monomicrobial growth were not included in the FA BCID panel (Table 1). The clinical relevance of these eight microorganisms was not analyzed. However, Micrococcus species, Corynebacterium species, and Bacillus cereus are usually considered contaminants, as they are part of normal skin microbiota, while Actinomyces species and Aeromonas species may be clinically significant findings in certain patient populations. This is a common limitation with similar rapid methods that are directed toward identifying the most common clinically significant microorganisms (12, 14, 15).

The identification of microorganisms from bottles with polymicrobial growth is demanding for both conventional and rapid identification methods (12, 14, 16). Certain specimens in this study demonstrated that conventional identification methods may initially fail to identify microorganisms in bottles with polymicrobial growth. There are several reasons for this, including the overgrowth of rapidly growing microorganisms in relation to slow-growing organisms in the same bottles and limited selective agar plates used in routine culture. Interestingly, the FilmArray identified all microorganisms included in the FA BCID panel in 18/24 (75%) bottles with polymicrobial growth and affirmed our previous results on blood cultures (13). In contrast, several rapid methods, including MALDI-TOF MS, have performed poorly in blood cultures with polymicrobial growth (12, 17, 18).

FilmArray could identify 3/3 and 9/9 yeasts in mono- and polymicrobial growth, respectively. Our present results show that, in addition to five Candida species, the FA BCID panel is capable of simultaneous identification of bacteria and antibiotic resistance genes. This is a distinct advantage of the FA BCID panel over many other rapid methods, including Yeast Traffic Light PNA FISH (fluorescence in situ hybridization using peptide nucleic acid probes) assay. This is particularly important when the treatment of polymicrobial infections in SBF is challenging due to the presence of multiple microorganisms with different susceptibility profiles against antimicrobial agents.

In conclusion, this study demonstrates that the FA BCID provides reliable, prompt results from positive blood culture bottles with SBF. Larger clinical studies, preferably in combination with antibiotic stewardship programs, are warranted to assess the impact of the FA BCID on hospital costs and patient outcome.