Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests

Abstract

Staphylococcus aureus strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which S. aureus had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to S. aureus culture supernatants. The corresponding S. aureus isolates were characterized by PCR for the presence of the PVL locus (lukS-PV and lukF-PV) and the mecA gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185 S. aureus isolates were PVL locus positive (PVL(+)); 28 of these were also mecA positive. PVL was detected in the supernatants of all PVL(+) strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL(+) strains ranged from 0 to 399 microg/ml by ELISA. By the use of 0.015 microg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by S. aureus during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.

FIG. 1.

Variability of PVL production among clinical isolates of S. aureus with a given genetic background. Isolates were grouped according to their multilocus sequence type. ST8 MRSA strains correspond to the USA300 CA-MRSA clone, ST80 strains correspond to the European CA-MRSA clone, and ST93 strains correspond to the Queensland ST93 CA-MRSA clone, while ST121 strains correspond to a predominant methicillin-sensitive S. aureus PVL⁺ clone spreading in Europe (20, 25, 29). The level of PVL production in vitro was measured by ELISA. The results are expressed as the means of three experiments ± standard errors of the means. *, P < 0.05, Mann-Whitney U test.

FIG. 2.

Variability of PVL concentration among clinical specimens containing S. aureus isolates with a given genetic background. Isolates were grouped according to their multilocus sequence types, while the PVL concentrations in samples from which the corresponding strains were isolated were measured by ELISA.