Evaluation of the QIAstat-Dx BCID GN and GPF kits for direct identification and antimicrobial resistance prediction from blood culture bottles

ABSTRACT

Rapid multiplex PCR kits have been used for rapid identification of blood culture isolates and prediction of antimicrobial resistance. We performed an evaluation of the QIAstat-Dx BCID GN and GPF research use only (RUO) kits on positive blood culture bottles using routine laboratory testing as the reference standard. Positive blood culture bottles between November 2023 and January 2024 were tested with QIAstat-Dx BCID GN and GPF kits based on initial Gram stain results and compared against routine identification and phenotypic susceptibility testing. A total of 174 monomicrobial blood cultures were included in the final analysis. The 174 monomicrobial blood cultures composed of 129 BCID GN tests and 45 BCID GPF tests. The majority of on-target Gram-negative organisms in monomicrobial cultures were identified. One Escherichia coli isolate was not identified as such, although the pan-Enterobacterales target was positive. All on-target Gram-positive organisms in monomicrobial cultures were identified. Overall sensitivity and specificity of tem/shv for detection of aminopenicillin resistance in E. coli was 94.7% (18/19) and 95.8% (23/24). The presence/absence of ctx-m and ampC had 100% sensitivity and specificity for identification of third-generation cephalosporin resistance in E. coli and Klebsiella pneumoniae. The combination of blaZ and mecA gene detection was fully predictive of phenotypic susceptibility results to penicillin and cloxacillin for Staphylococcus aureus. Overall, the QIAstat-Dx BCID GN and GPF kits were able to identify on-target pathogens. Detected resistance mechanisms were highly predictive of β-lactam resistance. Prediction of resistance for non-β-lactam antimicrobial was more variable.

IMPORTANCE

This is one of the first evaluations of the QIAstat BCID kit and demonstrates high levels of correlation for both identification and antimicrobial resistance prediction.

INTRODUCTION

Patients with bacteremia are identified by growth of microorganisms in blood culture bottles. Bacteremia represents invasive infection with these pathogens and requires prompt initiation of effective antimicrobials to optimize outcomes. In the age of widespread antimicrobial resistance, clinicians are often faced with a clinical dilemma of selecting optimal antibiotics. If broad-spectrum antibiotics are prescribed empirically, a risk of selecting drug resistance occurs. But this has to be balanced against concerns that individual patient outcomes may be compromised if an ineffective antibiotic is prescribed instead. However as with traditional culture techniques, the final identification and susceptibility testing require time (1). Routine identification and susceptibility testing results may not be immediately available. This has prompted efforts to improve turnaround time for the availability of actionable susceptibility testing results.

Rapid diagnostic tests from blood cultures are increasingly available in the market (2). Testing is performed on positive blood culture bottles and reduces the turnaround time for the availability of identification and/or susceptibility testing results. These include modifications of existing methods to be performed directly such as MALDI-TOF or phenotypic susceptibility testing (1, 3). These are often labor-intensive and alternative options such as multiplex PCR kits that are also increasingly available. These can rapidly identify pathogens, as well as detect key antimicrobial resistance (AMR) genes for prediction of resistance. The targets in terms of organisms (for identification) and AMR genes vary between manufacturers and may also vary with time (4, 5). These rapid results are then used for escalating and de-escalating antibiotics depending on the identified pathogen(s) and corresponding AMR genes (6, 7). Ideally, these should also be coupled with direct antimicrobial stewardship efforts to maximize impact on management and outcomes (2).

We performed an evaluation of the QIAstat-Dx BCID GN and GPF research use only (RUO) kits on positive blood culture bottles using routine laboratory testing as the reference standard.

MATERIALS AND METHODS

Positive BacTAlert FAN Plus bottles incubated in BacT/Alert Virtuo machines between November 2023 and January 2024 were included. Blood culture bottles that flagged positive were processed per routine with Gram stain performed and subcultured onto agar plates. In addition, based on the Gram stain result, bottles with Gram-negative organisms were tested with the QIAstat-Dx BCID GN Plus AMR RUO kit (BCID GN), and bottles with Gram-positive organisms or yeast were tested with the QIAstat-Dx BCID GPF Plus AMR RUO kit (BCID GPF). To conduct this study, an RUO version of the QIAstat-Dx kits was used.

Testing was performed on the QIAstat-Dx analyzer 1.0 and performed as per manufacturer’s recommendations. In brief, 300 µL of blood culture broth was diluted in 1:1 ratio, and 100 µL of sample was loaded into the cartridge and run on the analyzer. The analyzer performs automated DNA extraction, polymerase chain reaction, and interpretation of result targets. Successful amplification of an internal control is required for each test run for results to be valid.

The full range of target pathogens and antimicrobial resistance (AMR) genes targeted is in the supplemental material. Of note, BCID GN kits also have a pan-Enterobacterales target, and the BCID GPF kits have a pan Gram-negative (GN) target. Also, Candida spp. are not identified to species level and are grouped as either group 1 (Candida albicans, Candida tropicalis, Candida dubliniensis, Candida famata, Candida guilliermondii, and Candida kefyr) or group 2 (Candida glabrata, Candida krusei, Candida parapsilosis, Candida auris, and Candida lusitaniae). The antimicrobial resistance (AMR) gene results provided in each test run is dependent on the identified pathogen(s) and is also summarized in the supplemental material.

Identification results were compared against routine identification performed using Bruker MALDI Biotyper (Bruker, MA, USA). AMR gene results were compared against routine susceptibility testing as summarized in Table 1. Detected AMR genes were considered concordant if they corresponded with phenotypic resistance identified as per Table 1, accounting for intrinsic resistance mechanisms.

TABLE 1.

Detected AMR genes in this study and expected phenotypic results^a

BCID kit	AMR genes	Expected resistant antimicrobials	Evaluated organisms	Phenotypic testing method
BCID GN	tem, shv	Ampicillin and/or amoxicillin-clavulanic acid	Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii	Vitek 2
	ctx-m	Third- and/or fourth-generation cephalosporins
	ampC	Third-generation cephalosporins
	oxa-23	Meropenem, imipenem
	ges	Meropenem, imipenem
	imp	Meropenem, imipenem
	aac(6')-lb, armA	Gentamicin, amikacin
BCID GPF	blaZ	Penicillin	Staphylococcus aureus Staphylococcus lugdunensis	Vitek 2
		Ampicillin	Enterococcus faecalis
	mecA	Cloxacillin	All Staphylococcus spp.
	ermC	Erythromycin, clindamycin	All Staphylococcus spp. All Streptococcus spp.	Vitek 2b (Streptococcus pyogenes and S. pneumoniae tested with disk diffusion including Dtest)
	tetM	Minocycline	All Staphylococcus spp. Streptococcus agalactiae	Vitek 2
	aac(6’)/aph(2”)	Gentamicin	All Staphylococcus spp. Enterococcus faecalis	Vitek 2

^a Only AMR genes which were detected in this study are listed.

^b Isolates which are erythromycin resistant and clindamycin susceptible are further tested with disk and Dtest for confirmation of susceptibility.

Susceptibility testing was performed with Vitek 2 (bioMérieux, Marcy-l'Étoile, France) for Enterobacterales, Pseudomonas spp., Acinetobacter spp., Staphylococcus spp., Enterococcus spp., and Streptococcus agalactiae. Routinely tested and reported antimicrobials for Gram-negatives include ampicillin, amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem, gentamicin, and amikacin. Susceptibility results to other antimicrobials such as co-trimoxazole and ciprofloxacin were also available but not evaluated in this study as there are no specific AMR gene targets. Routinely tested antimicrobials are as follows: Staphylococcus spp. (penicillin, cloxacillin and cefoxitin screen, vancomycin, erythromycin, clindamycin, and minocycline), Streptococcus agalactiae (penicillin, erythromycin, clindamycin, and minocycline), and Enterococcus spp. (ampicillin and vancomycin). Susceptibility testing of penicillin, erythromycin, and clindamycin for other Streptococcus spp. was performed using disk diffusion and Etest (bioMérieux). All results were interpreted based on EUCAST break points.

RESULTS

A total of 174 monomicrobial blood cultures and 14 polymicrobial blood cultures were included in the final analysis. The 174 monomicrobial blood cultures composed of 129 BCID GN tests and 45 BCID GPF tests and 80 aerobic, 90 anaerobic, and four paediatric bottles. The BCID identification results as well as AMR gene results for resistance to β-lactams are summarized in Table 2 (BCID GN) and Table 3 (BCID GPF) for monomicrobial cultures. Additional results for correlation of AMR genes with phenotypic results are provided in Table 4 and the supplemental material. Results for polymicrobial cultures are listed in Table 5.

TABLE 2.

Correlation between BCID GN kit with final culture and phenotypic susceptibility testing results for β-lactams for blood cultures with monomicrobial Gram-negative organisms

Organism identification by routine methods	BCID identification and number of isolates		Detected AMR genes and number of isolates		Number of isolates with discordant phenotype
Escherichia coli	Escherichia coli, pan-Enterobacterales	57	tem	17	1
	Pan-Enterobacterales only	1	shv	1
			ctx-m	8
			ctx-m, tem	6
			ampC	1
			ampC, tem	1
			Nil detected	24	1c
Klebsiella pneumoniae	Klebsiella spp., pan-Enterobacterales	31	shv	21
			shv, tem	5
			ctx-m, tem	1
			ampC, shv	1
			ctx-m, shv, tem	2
			ampC, shv, tem	1
Klebsiella variicola	Klebsiella spp., pan-Enterobacterales	1	Nil detected	1
Enterobacter cloacae complex	Enterobacter spp., pan-Enterobacterales	1	Nil detected	1
Proteus mirabilis	Proteus spp., pan-Enterobacterales	3	Nil detected	3
Salmonella Typhi	Salmonella spp., pan-Enterobacterales	1	tem	1
Serratia spp.	Serratia spp., pan-Enterobacterales	5	Nil detected	5
Morganella morganii a	Pan-Enterobacterales only	1	Nil detected	1
Citrobacter koseri a	Pan-Enterobacterales only	2	Nil detected	2
Citrobacter freundii complexa	Pan-Enterobacterales only	1	ampC	1
Acinetobacter baumannii complex	Acinetobacter baumannii complex	4	Nil detected	3
			oxa-23	1
Pseudomonas aeruginosa	Pseudomonas aeruginosa	9	Nil detected	9	1d
Stenotrophomonas maltophilia	Stenotrophomonas maltophilia	3	Nil detected	–e	N/Af
Bacteroides thetaiotaomicronb	No pathogen detected	1	N/A		N/A
Elizabethkingia anophelisb	No pathogen detected	1	N/A		N/A
Fusobacterium spp.b	No pathogen detected	1	N/A		N/A
Parabacteroides distasonisb	No pathogen detected	1	N/A		N/A
Pseudomonas otitidisb	No pathogen detected	1	N/A		N/A
Roseomonas mucosab	No pathogen detected	1	N/A		N/A
Burkholderia cepacia complexb	No pathogen detected	1	N/A		N/A
Gabonibacter massiliensisb	No pathogen detected	1	N/A		N/A
Porphyromonas speciesb	No pathogen detected	1	N/A		N/A

^a No species targets available.

^b Not targeted in BCID GN kit.

^c One E. coli isolate with ampicillin and amoxicillin-clavulanic acid resistance.

^d One P. aeruginosa isolate was resistant to all β-lactams.

^{e –} , not tested due to intrinsic resistance.

^f N/A, not applicable.

TABLE 3.

Correlation between BCID GPF kit with final cultures and phenotypic susceptibility testing results for β-lactams for blood cultures with monomicrobial Gram-negative organisms

Organism identification by routine methods	BCID identification and number of isolates		Detected AMR genes and number of isolates		Number of isolates with discordant phenotype
Staphylococcus aureus	Staphylococcus aureus	11	blaZ	3
			mecA	1
			blaZ, mecA	6
			Nil detected	1
Staphylococcus epidermidis	Staphylococcus epidermidis	7	mecA	2
			blaZ, mecA	2
			Nil detected	3
Staphylococcus capitis	Staphylococcus capitis/hominis	4	mecA	3
			Nil detected	1
Staphylococcus lugdunensis	Staphylococcus lugdunensis	1	Nil detected	1
Streptococcus pneumoniae	Streptococcus pneumoniae	1	N/Ab		N/A
Streptococcus pyogenes	Streptococcus pyogenes	3	N/A		N/A
Streptococcus agalactiae	Streptococcus agalactiae	1	N/A		N/A
Enterococcus faecalis	Enterococcus faecalis	3	Nil detected
Staphylococcus argenteusa	No pathogen detected	1	N/A		N/A
Bacillus cereus group	Bacillus cereus group	1	N/A		N/A
Candida albicans	Candida spp. group 1	1	N/A		N/A
Candida glabrata	Candida spp. group 2	2	N/A		N/A
Clostridium perfringensa	No pathogen detected	1	N/A		N/A
Cutibacterium acnesa	No pathogen detected	2	N/A		N/A
Dermacoccus spp.a	No pathogen detected	1	N/A		N/A
Paraclostridium spp.a	No pathogen detected	1	N/A		N/A
Streptococcus anginosusa	No pathogen detected	1	N/A		N/A
Streptococcus equinusa	No pathogen detected	1	N/A		N/A
Streptococcus canisa	No pathogen detected	1	N/A		N/A
Streptococcus mitisa	No pathogen detected	1	N/A		N/A

^a Not targeted in BCID GPF kit.

^b N/A, not applicable.

TABLE 4.

Summary of sensitivity and specificity of AMR genes for prediction of phenotypic resistance^c

Organism	Aminopenicillin	shv and/or tem (in isolates without ctx-m/ampC)						Third-generation cephalosporins	ctx-m and/or ampC
		Det	ND	Sens.	Spec.	PPV	NPV		Det.	ND	Sens.	Spec.	PPV	NPV
Escherichia coli (n = 58)	Resistant	18	1	94.7%	95.8%	94.7%	95.8%	Resistant	16	0	100.0%	100.0%	100.0%	100.0%
	Susceptible	1	23					Susceptible	0	42
Klebsiella pneumoniae groupa (n = 32)	Resistant	N/A – intrinsic resistance to aminopenicillins						Resistant	5	0	100.0%	100.0%	100.0%	100.0%
	Susceptible							Susceptible	0	26
Organism	Gentamicin	aac(6’)-lb						Amikacin	aac(6’)-lb
		Det	ND	Sens.	Spec.	PPV	NPV		Det.	ND	Sens.	Spec.	PPV	NPV
Escherichia coli (n = 58)	Resistant	2	5	28.6%	98.0%	66.7%	90.9%	Resistant	1	0	100.0%	96.5%	33.3%	100.0%
	Susceptible	1	50					Susceptible	2	55
Klebsiella pneumoniae groupa (n = 32)	Resistant	3	1	75.0%	100.0%	100.0%	96.6%	Resistant	0	0	-	90.6%	0.0%	100.0%
	Susceptible	0	28					Susceptible	3	29
Organism	Penicillin	blaZ (in isolates without mecA)						Cloxacillin	mecA
		Det	ND	Sens.	Spec.	PPV	NPV		Det.	ND	Sens.	Spec.	PPV	NPV
Staphylococcus aureus (n = 11)	Resistant	3	0	100.0%	100.0%	100.0%	100.0%	Resistant	7	0	100.0%	100.0%	100.0%	100.0%
	Susceptible	0	1					Susceptible	0	4
Coagulase-negative Staphylococcusb (n = 11)	Resistant	N/A – No break points for penicillin						Resistant	7	0	100.0%	100.0%	100.0%	100.0%
	Susceptible							Susceptible	0	8
Organism	Gentamicin	aac(6’)/aph(2”)						Minocycline Sens.	tetM
		Det	ND	Sens.	Spec.	PPV	NPV		Det	ND	Sens.	Spec.	PPV	NPV
All Staphylococcus spp.b (n = 22)	Resistant	8	0	100.0%	100.0%	100.0%	100.0%	Resistant	0	0	-	100.0%	-	100.0%
	Susceptible	0	15					Susceptible	0	22
Organism	Erythromycin	ermC						Clindamycin	ermC
		Det	ND	Sens.	Spec.	PPV	NPV		Det.	ND	Sens.	Spec.	PPV	NPV
All Staphylococcus spp.b (n = 22)	Resistant	6	1	85.7%	87.5%	75.0%	93.3%	Resistant	6	0	100.0%	100.0%	100.0%	100.0%
	Susceptible	2	14					Susceptible	0	17

^a Includes 31 K. pneumoniae and one K. variicola isolate.

^b Detailed breakdown is available in the supplemental material.

^c Det, detected; ND, not detected: sens., sensitivity; spec., specificity; PPV, positive predictive value; NPV, negative predictive value; N/A, not applicable.

TABLE 5.

Comparison of BCID results with routine testing for polymicrobial cultures

	BCID GP results		BCID GN results		Organism identification by routine methods	Resistant antibiotics
	Identification results	AMR genes detected	Identification results	AMR genes detected
Polymicrobial cultures with Gram-positive organisms only
1	Staphylococcus capitis/hominis, Staphylococcus epidermidis	aac(6’)/aph(2”), blaZ, ermC, mecA, tetK	N/Aa	N/A	Staphylococcus epidermidis	Penicillin, cloxacillin, erythromycin, clindamycin
					Staphylococcus capitis	Penicillin, cloxacillin
2	No pathogen detected	Nil detected	N/A	N/A	Staphylococcus haemolyticus
					Corynebacterium striatumb
3	Staphylococcus aureus	mecA, ermC	N/A	N/A	Staphylococcus aureus	Penicillin, cloxacillin, erythromycin, clindamycin
					Staphylococcus capitisb	Not performed
4	No pathogen detected	Nil detected	N/A	N/A	Staphylococcus haemolyticus	N/A
					Dermabacter hominis	N/A
Polymicrobial cultures with Gram-negative organisms only
1	N/A	N/A	Pseudomonas aeruginosa	ges, imp, aac(6’)-lb	Pseudomonas aeruginosa	β-lactams, gentamicin
					Pseudomonas putida	Ceftazidime, piperacillin-tazobactam, gentamicin
2	N/A	N/A	Klebsiella spp., Escherichia coli, pan-Enterobacterales	shv, ampC	Escherichia coli	AmpC phenotype
					Klebsiella pneumoniae	AmpC phenotype
3	N/A	N/A	Klebsiella spp., Escherichia coli, pan-Enterobacterales	Nil	Escherichia coli	Wild type
					Klebsiella oxytoca	Ampicillin resistance
4	N/A	N/A	Klebsiella spp., pan-Enterobacterales	Nil	Klebsiella aerogenes	AmpC phenotype
					Pseudomonas aeruginosab	Wild type
Polymicrobial cultures with Gram-positive and Gram-negative organisms
1	Staphylococcus epidermidis, pan Gram-negative	mecA	Escherichia coli, pan-Enterobacterales	TEM	Escherichia coli	Ampicillin
					Staphylococcus epidermidis	Not performed
2	Enterococcus faecium, Staphylococcus epidermidisc	meCA, aac(6’)/aph(2”), ermC, vanA	Pseudomonas aeruginosa	Nil	Pseudomonas aeruginosa	Ceftazidime, piperacillin-tazobactam, meropenem, imipenem
					Enterococcus faecium	Vancomycin, gentamicin
3	Enterococcus faecalis, pan Gram-negative	Nil detected	Klebsiella spp., Escherichia coli, pan-Enterobacterales	shv, ctx-m, tem	Escherichia coli	Ampicillin, cephalosporins
					Klebsiella pneumoniae	Wild type
					Enterococcus faecalis	Wild type
					Enterococcus casseliflavus	Wild type
4	Pan Gram-negative	Nil detected	Escherichia coli pan-Enterobacterales	ctx-m	Escherichia coli	Ampicillin, cephalosporins
					Streptococcus bovis	N/A
5	Enterococcus faecium	aac(6’)/aph(2”)	No pathogen detected		Enterococcus faecium	Wild type
					Elizabethkingia spp.	N/A
6	Enterococcus faecium, pan Gram-negative	aac(6’)/aph(2”)	Not performed as initial Gram-stain was negative for Gram-negative organisms		Enterococcus faecium	Gentamicin
					Escherichia coli	N/A

^a N/A, not applicable.

^b Target organisms which were not identified by BCID kits (false negative).

^c S. epidermidis identified on BCID testing but not culture. This patient had other cultures that were positive with S. epidermidis. AmpC phenotype: resistant to third-generation cephalosporins and piperacillin-tazobactam but susceptible to cefepime.

Identification of pathogens with BCID panels

The majority of on-target Gram-negative organisms in monomicrobial cultures were identified. One E. coli isolate was not identified as such, although the pan-Enterobacterales target was positive. As expected, all non-target Enterobacterales were positive on the pan-Enterobacterales target only (Morganella morganii, Citrobacter koseri, and Citrobacter freundii complex did not have genus or species targets).

All on-target Gram-positive organisms in monomicrobial cultures were identified. Of note, one isolate identified as Staphylococcus argenteus on routine testing by MALDI-TOF had no pathogens detected on the BCID panel. Candida albicans (n = 1) and Candida glabrata (n = 2) were also detected by the GPF BCID kit as belonging to Candida spp. group 1 and Candida spp. group 2, respectively.

No pathogens were detected as expected for all non-target pathogens (Tables 2 and 3).

For polymicrobial cultures, three false-negatives were observed (S. capitis, Corynebacterium striatum, and Pseudomonas aeruginosa), while all other on-target pathogens were identified (Table 5). Staphylococcus epidermidis was detected on BCID GPF kit in one polymicrobial culture but was not isolated on culture. However, S. epidermidis was cultured from other blood culture bottles from the same patient, supporting the BCID result.

Prediction of antimicrobial resistance in Gram-negative organisms

For Enterobacterales, the presence of a β-lactamase generally correlated with phenotypic resistance. Results were considered concordant if there were at least one listed antimicrobial with acquired resistance, taking into account intrinsic resistance as per EUCAST (8).

As Klebsiella spp. are expected to be intrinsically resistant to ampicillin due to chromosomal β-lactamases, the correlation between shv/tem and aminopenicillin resistance is not evaluated.

One TEM-positive Escherichia coli isolate remained susceptible to aminopenicillins, and one E. coli without tem or shv detected was resistant to ampicillin and amoxicillin-clavulanic acid. The presence of tem/shv (in isolates without ctx-m/ampC) otherwise correlated with ampicillin/amoxicillin-clavulanic acid resistance in E. coli. Overall sensitivity and specificity of tem/shv for detection of aminopenicillin resistance was 94.7% (18/19) and 95.8% (23/24).

Fourteen ctx-m and two ampC-positive E. coli and three ctx-m and two ampC-positive K. pneumoniae were detected. One ampC-E. coli was resistant to ceftazidime only, while all other isolates were resistant to ceftriaxone and ceftazidime. All other E. coli (n = 42) and K. pneumoniae (n = 26) isolates did not have ctx-m or ampC detected and were susceptible to ceftriaxone, ceftazidime, and cefepime. The presence/absence of ctx-m and ampC had 100% sensitivity and specificity for identification of third-generation cephalosporin resistance in E. coli and K. pneumoniae. oxa-23 was detected in one A. baumannii complex isolate and correlated with resistance to carbapenems. ges and imp was detected in a polymicrobial culture with Pseudomonas aeruginosa and Pseudomonas putida identified. No other carbapenemase genes were detected in this study.

The armA gene was detected in only one A. baumannii complex isolated and correlated with resistance to gentamicin and amikacin. The aac(6’)-lb gene was detected in E. coli and K. pneumoniae isolates only and negative in all other bacterial species. aac(6’)-lb had a sensitivity and specificity of 28.6% and 98.0% for gentamicin resistance in E. coli, 75.0% and 100.0% for gentamicin resistance in K. pneumoniae, and 100.0% and 96.% for amikacin resistance in E. coli (Table 4). Specificity for amikacin resistance was 90.6% in K. pneumoniae. There were no K. pneumoniae isolates with phenotypic amikacin resistance detected. All other isolates (Enterobacterales and P. aeruginosa) were negative for both genes and remained susceptible to gentamicin and amikacin.

Prediction of antimicrobial resistance in Gram-positive organisms

In this evaluation, only eleven S. aureus isolates were included, of which one was penicillin susceptible, three were methicillin susceptible (penicillin resistant), and seven were methicillin-resistant S. aureus. The presence of blaZ and mecA genes was concordant with the phenotypic susceptibility results to penicillin and cloxacillin for Staphylococcus aureus. The detection of mecA genes in other Staphylococcus spp. was also fully concordant with phenotypic methicillin resistance. No mecC genes were detected in our isolates. Taken together, mecA gene detection had 100% (14/14) sensitivity and 100% (9/9) specificity for methicillin resistance.

No ermA genes were detected in this cohort. ermC detection had 100% (6/6) sensitivity and 100% (17/17) specificity for clindamycin resistance for Staphylococcus spp. but not Streptococcus spp. Correlation with erythromycin resistance was more variable, with sensitivity of 85.7% (6/7) and specificity of 87.5% (14/16) in Staphylococcus spp. None of the erythromycin and clindamycin-resistant isolates in Streptococcus spp. had erm genes detected (Table S2).

Detection of aac(6’)/aph(2”) had 100% (8/8) sensitivity and 100% specificity (15/15) for gentamicin resistance in Staphylococcus spp. Two E. faecalis isolates without high-level gentamicin resistance did not have aac(6’)/aph(2”) detected, but there were no gentamicin-resistant E. faecalis isolates.

Only one isolate had phenotypic minocycline resistance (S. agalactiae), which corresponded with tetM gene detection. All Staphylococcus spp. were minocycline susceptible and negative for tetM gene.

Only three monomicrobial E. faecalis isolates were included of which all were vancomycin susceptible and negative for vanA/vanB genes. vanA was detected in a vancomycin-resistant E. faecium isolate in a polymicrobial culture (Table 4).

Additional details are provided in the supplemental material.

DISCUSSION

A recent meta-analysis reported a potential mortality benefit with the use of a rapid diagnostic tests for blood cultures in combination with antimicrobial stewardship (2). The benefits are largely due to reducing turnaround time to microbiological results for optimizing effective antimicrobial therapy. The results between different studies are however heterogeneous, and patient selection and methods used may be key factors that impact outcomes. The methods available include the use of multiplex PCR kits on positive blood culture bottles. These kits are designed to rapidly identify pathogens and to detect key antimicrobial resistance genes. The clinical utility and impact likely depend on multiple factors, one of which includes the predictive accuracy of tests in identifying optimal or effective antimicrobial therapy.

Overall concordance between identified AMR genes on the QIAstat-Dx BCID RUO kit and phenotypic susceptibility results was high, particularly for β-lactam antibiotics. β-lactams are key antimicrobials used for treatment of invasive infections such as bacteremia (9). Rapid pathogen identification could be used for guiding treatment based on the presence of known intrinsic resistance mechanism (e.g., Stenotrophomonas maltophilia are resistant to β-lactams) or based on local antibiogram data (e.g., rates of carbapenem resistance among different bacterial species).

The presence of AMR genes predicts phenotypic resistance and could be used for escalation to appropriate antimicrobials. All mecA-positive isolates were methicillin-resistant Staphylococcus aureus, and blaZ-positive isolates were penicillin-resistant methicillin-sensitive Staphylococcus aureus. blaZ and mecA targets together could be used to rapidly determine effective first-line therapy for S. aureus infections.

All E. coli and K. pneumoniae isolates that were resistant to a third-generation cephalosporin had either ctx-m or ampC detected and vice versa. Harris et al. (10) demonstrated that although ctx-m is the predominant β-lactamase in E. coli and K. pneumoniae that are resistant to third-generation cephalosporins (11), ampC β-lactamases were found in 17.1% of isolates. Although much less common, other non-ctx-m extended-spectrum β-lactamases will not be detected by this assay. In our study, 4/21 (19.1%) of third-generation cephalosporin-resistant E. coli and K. pneumoniae were ampC positive. At the time of writing, other multiplex PCR panels included ctx-m as a target but not ampC. This presents as an advantage for this kit in some clinical settings where acquired ampC genes are a significant contributor to phenotypic third-generation cephalosporin resistance. The presence/absence of ctx-m or ampC may be useful in determining the need for antibiotic escalation to carbapenems (11) or whether cephalosporins remain a suitable treatment option.

Enterobacter spp., K. aerogenes, and C. freundii are organisms known to carry chromosomal AmpC β-lactamases, although ampC was only positive in a C. freundii isolate. This may be due to differences in target genes as ampC is a representation of a group of different β-lactamases. Moreover, the antimicrobial susceptibility phenotype may not always correlate with the presence of AMR genes due to low levels of expression, but may result in inducible resistance. Thus, isolate identification (e.g., Enterobacter cloacae complex) must always be taken into consideration despite the absence of detected AMR genes. A limitation of this study is that the number of positive cases with AMR genes was limited for some targets. Of note, the number of detected carbapenemases was limited, which is reflective of local epidemiology. Where positive however, the carbapenemase genes were predictive of phenotypic carbapenem resistance and could be used to prompt escalation of targeted antimicrobials based on the class of carbapenemase present (6). Carbapenem-resistant Pseudomonas aeruginosa may remain negative for carbapenemases as they often have an array of non-β-lactamase resistance mechanisms such as efflux pump and porin loss. Similarly, only one vancomycin-resistant Enterococcus isolate was detected and was positive for vanA (from a polymicrobial culture). Ideally, a larger collection of isolates/samples should be tested to confirm the sensitivity/specificity of the test kits and correlation with phenotypic resistance. Local diagnostic laboratories should consider their local antibiogram data and prevalence of particular AMR genes when evaluating multiplex PCR tests as the positive and negative predictive values of tests will vary with local epidemiology.

Candida spp. are reported as a group in the BCID GPF kit and does not identify yeast to species level. This a potential limitation depending on local antifungal resistance rates. Empiric treatment for the common Candida spp. is with echinocandins, and in our setting, acquired echinocandin resistance is largely sporadic (12). Acquired fluconazole resistance rates vary with species and occur particularly in C. tropicalis and C. parapsilosis. Rapid identification to species level may be useful, particularly with the emergence of other species with higher potential for acquired echinocandin resistance such as C. auris (13).

Future studies could focus on effecting antimicrobial interventions and assessing the impact on clinical outcomes. In general, the use of rapid molecular testing should be coupled with antimicrobial stewardship efforts (14). Direct communication of results in real time with treating teams would result in earliest possible intervention and change to effective antimicrobials. However, this needs to be balanced against costs. The reported direct impact on mortality of these rapid diagnostics on outcome has been variable (2). Targeting rapid diagnostic tests to certain groups such as patients with carbapenemase-producing Enterobacterales infections may improve cost-effectiveness (6). Depending on local antibiograms and populations, the highest benefit or yield of performing rapid BCID multiplex PCRs may be in immunocompromised patients, vulnerable patients in intensive care units, or patients with other risk factors for infection with multi-drug-resistant organisms.

ETHICS APPROVAL

This project was reviewed and approved by the National Healthcare Group Domain Specific Review Board (NHG DSRB reference number: 2023/00213).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.01169-24.

Supplemental material. jcm.01169-24-s0001.docx.

Supplemental text; Tables S1 to S3.

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.