Evaluation of a vanA-Specific PCR Assay for Detection of Vancomycin-Resistant Enterococcus faecium during a Hospital Outbreak

Abstract

We investigated the use of PCR as an alternative to culture of fecal samples for detection of vanA-containing Enterococcus faecium during a recent hospital outbreak. Rectal swabs collected consecutively from 223 patients were analyzed by culture with and without enrichment broth and by vanA-specific PCR of enrichment broth samples. Fifty-five specimens were positive for vanA-containing E. faecium by at least one method. The sensitivities of the vanA-specific PCR assay and agar culture with and without enrichment broth were 94.5, 98, and 89%, respectively. All three methods were 100% specific. Final results were obtained much more rapidly by PCR (within 24 to 30 h of specimen submission) than by the culture methods (4 to 5 days). Thus, PCR is an accurate and rapid alternative to culture for detection of vancomycin-resistant enterococci during hospital outbreaks.

Vancomycin-resistant enterococci (VRE) have emerged as important nosocomial pathogens within the last 10 years (2, 9, 12). These organisms are of great clinical concern because they are often resistant to multiple antimicrobial agents (7, 10, 11). Several recent nosocomial outbreaks attest to the importance of early identification of VRE in colonized patients to prevent person-to-person transmission (10, 11, 15). Surveillance cultures for the detection of VRE with selective media for enhancing the recovery of VRE from stool or rectal samples have been common practice and proven to be reliable (4, 8, 12, 16). However, completion of this method requires at least 4 days, which delays implementation of important preventive measures such as isolation of colonized patients.

Two phenotypes of Enterococcus faecium-acquired vancomycin resistance, VanA and VanB, encoded by gene clusters carried on transposons that include the vanA and vanB genes, respectively, have been described (1). The VanA phenotype confers high-level resistance to both vancomycin and teicoplanin, while the VanB phenotype confers moderate to high-level resistance to vancomycin only. Recently, a PCR assay based on specific amplification of internal fragments of ligase genes was developed for VRE detection (3). This PCR assay was investigated as an alternative to culture for detection of VRE in fecal samples (14). Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. vanA-specific PCR analysis of enrichment broth samples after 15 to 18 h of incubation detected 74 (85.1%) of 87 vanA-containing E. faecium culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. In the present study, we evaluated the use of this vanA-specific PCR assay for detection of vanA-containing enterococci in fecal samples during a recent hospital outbreak. We compared the sensitivity and specificity of PCR with those of culture with and without broth enrichment for the detection of VRE.

The first VRE isolate at the Hôpital Notre-Dame du Centre Hospitalier de l’Université de Montréal—Campus Notre-Dame was isolated from a patient hospitalized in the hematology ward in November 1998. Since then, over 2,400 fecal specimens have been screened on selective media for the presence of VRE. One hundred eighty patients have been found to harbor vancomycin-resistant E. faecium. All isolates expressed the VanA phenotype, and a predominant strain (pulsovar S2) related to the first isolate was recognized by SmaI pulsed-field gel electrophoresis with the GenePath strain typing system (Bio-Rad, Mississauga, Ontario, Canada).

In the present study, rectal swabs collected consecutively from 223 patients between 23 February 1999 and 24 March 1999 were analyzed for the presence of VRE by culture with and without enrichment broth and by vanA-specific PCR assay of enrichment broth samples. Collected samples were placed in Amies transport medium and processed within 8 h of collection. The fecal material from the swabs was first inoculated directly onto Enterococcosel agar (Becton Dickinson Microbiology Systems, Cockeysville, Md.) containing 6 μg of vancomycin per ml (direct plating), and then the swab was agitated in 10 ml of Trypticase soy broth (Becton Dickinson) containing 2 μg of gentamicin per ml, 1 μg of amphotericin B per ml, and 1 μg of vancomycin per ml (enrichment broth). The agar plates and enrichment broths were incubated at 35°C. Ten-microliter samples from the enrichment broths were subcultured onto Enterococcosel agar plates after 15 to 18 h of incubation. The agar plates were examined after 24 h, 48 h, and 5 days of incubation. Colonies resembling enteroccoci by colony morphology were identified by standard laboratory methods (5). The MICs of vancomycin and teicoplanin were determined at the provincial reference laboratory by the NCCLS (National Committee for Clinical Laboratory Standards) broth microdilution method with cation-adjusted Mueller-Hinton broth (Difco) (13). Two microliters of uncentrifuged enrichment broth inoculated and incubated for 15 to 18 h was used for the PCR assay. PCR detection of the vanA resistance gene was carried out with oligonucleotide primers A₁ and A₂ (3) and PCR conditions as previously described (14). Ten-microliter samples of the PCR products were electrophoresed through a 1.5% agarose gel for 45 min at 150 V. The gels were stained with ethidium bromide for visualization on a standard UV box. The vanA-specific PCR product size was 732 bp.

VRE were detected in 55 of the 223 fecal samples by at least one method (Table 1). All 55 isolates were identified biochemically as E. faecium, were found to be highly resistant to vancomycin (MICs, >64 μg/ml) and teicoplanin (MICs, >32 μg/ml), and were shown by PCR to contain the vanA gene. Of the 55 positive samples, 5 specimens (9.1%) were negative by direct plating on agar but positive after broth enrichment by agar culture and by PCR. One specimen (1.8%) was positive by PCR of the enrichment broth sample only. This specimen came from a patient who had tested VRE positive by culture a week earlier. Three specimens (5.5%) were negative by PCR; however, two of these were positive by agar culture with and without broth enrichment and the third was positive by agar culture only after the enrichment step. These three PCR-negative broth specimens were subsequently confirmed to contain the vanA resistance gene by PCR assay directly on isolated colonies with a sample preparation described previously (14). The sensitivities of the PCR assay and agar culture with and without enrichment broth for detection of vanA-containing enterococci were 94.5 (95% confidence interval, 88.5 to 100%), 98, and 89%, respectively. All three methods were 100% specific. Overall, the results of direct plating on agar and PCR assay of enrichment broth samples were identical for 216 of 223 tests (agreement, 96.8%; kappa statistic [6], 0.91).

TABLE 1.

Comparative detection of vanA-containing enterococci in 223 fecal samples by agar culture following direct plating or broth enrichment and by vanA-specific PCR of enrichment broth samples

Method	No. of specimens		Sensitivity (%)	Specificity (%)
	Positive (n = 55)a	Negative (n = 168)b
Agar culture following:
Direct plating
Positive	49	0	89	100
Negative	6	168
Broth enrichment
Positive	54	0	98	100
Negative	1	168
PCR assay
Positive	52	0	94.5	100
Negative	3	168

^aTotal number of vanA-positive samples detected by culture and/or PCR. 

^bTotal number of vanA-negative samples detected by culture and PCR. 

This study validates the use of PCR for detection of VRE in an outbreak setting and confirms the greater sensitivity and rapidity of PCR over traditional culture methods for detection of VRE in colonized patients. Culture of rectal or perirectal samples with Enterococcosel agar containing vancomycin at 35°C followed by biochemical species identification and broth microdilution susceptibility testing is currently used by many clinical laboratories for surveillance of VRE. However, this method required 4 days for completion and was the least sensitive method in the present study and in a previous study conducted by Satake et al. (14). Several groups have advocated the use of broth enrichment to enhance the sensitivities of surveillance culture for VRE (8, 16). We and others (14) have demonstrated that culture on agar after the enrichment step is the most sensitive method for detecting VRE. However, the PCR assay targeted for detection of the vanA resistance gene showed almost equivalent sensitivity to the culture method after the enrichment step, and final results were obtained much more rapidly by PCR (within 24 to 30 h of specimen submission) than by the culture method (5 days). One limitation of this vanA-specific PCR assay for VRE surveillance is that it does not detect all VRE genotypes (vanB, vanC1, and vanC2). A multiplex PCR assay with all four primer sets has been developed but appears to suffer from a lack of sensitivity (14). Nevertheless, the vanA-specific PCR assay is highly sensitive and has proven to be useful for managing vanA-containing enterococcal outbreaks by reducing both the time required for VRE detection and the delay before implementation of preventive measures. For now, our surveillance strategy is to use the vanA-specific PCR assay and to restrict agar cultures to newly hospitalized patients coming from institutions that might harbor other types of VRE.