Virus detection and semiquantitation in explanted heart tissues of idiopathic dilated cardiomyopathy adult patients by use of PCR coupled with mass spectrometry analysis

Abstract

Viral detection in heart tissues has become a central issue for the diagnosis and exploration of the pathogenesis of idiopathic dilated cardiomyopathy (IDCM). In the present study, common cardiotropic viruses in 67 explanted heart samples of 31 IDCM adult patients were detected and semiquantified by using for the first time a new technology based on PCR assay coupled to electrospray ionization-time of flight mass spectrometry analysis (PCR-MS), with comparison to reference quantitative real-time PCR (RT-qPCR) assay. PCR-MS identified single or mixed enterovirus (EV) and parvovirus B19 (PVB19) infections in 27 (40.2%) of 67 samples, corresponding to 15 (48.3%) of the 31 patients, whereas RT-qPCR identified viral infections in 26 (38.8%) samples, corresponding to 16 (51.6%) of the patients. The PCR-MS results correlated well with EV and PVB19 detection by RT-qPCR (kappa = 0.85 [95% confidence interval {CI}, 0.72 to 1.00] and kappa = 0.82 [95% CI, 0.66 to 0.99], respectively). The levels of EV RNA (median, 550 [range, 178 to 3,200] copies/μg of total extracted nucleic acids) and of PVB19 DNA (median, 486 [range, 80 to 1,157] copies/μg of total extracted nucleic acids) were measured using PCR-MS and correlated with those obtained by RT-qPCR (r(2) = 0.57, P = 0.002 and r(2) = 0.64, P < 0.001 for EV and PVB19, respectively). No viruses other than EV and PVB19 strains were detected using the new PCR-MS technology, which is capable of simultaneously identifying 84 known human viruses in one assay. In conclusion, we identified single or mixed EV and PVB19 cardiac infections as potential causes of IDCM. The PCR-MS analysis appeared to be a valuable tool to rapidly detect and semiquantify common viruses in cardiac tissues and may be of major interest to better understand the role of viruses in unexplained cardiomyopathies.

Fig 1

Viral findings obtained in 67 heart samples taken from 31 patients with dilated cardiomyopathy using classical quantitative real-time PCR (RT-qPCR) and PCR followed by electrospray ionization-mass spectrometry analysis (PCR-MS). (A) Number of samples positive (+) or negative (−) by indicated assay. (B) Number and percentage of cases positive for virus(es) by indicated assay. EV, enteroviruses; PVB19, parvovirus B19; *, P > 0.05 according to Fischer's exact test.

Fig 2

Viral loads in cardiac samples obtained by the new PCR-MS system in comparison to those obtained by classical RT-qPCR. (A and B) Box plots demonstrating the distribution (median, first and third quartiles, and range values) of the viral loads. Enteroviruses (EV) and parvovirus B19 (PVB19) were measured by RT-qPCR and by the new PCR-MS system. (C and D) Correlation curves obtained by comparison of the estimated viral loads obtained by RT-qPCR and the new PCR-MS system for EV and PVB19 strains in heart tissue samples.

Fig 3

Genotyping identification of viruses by use of the new PCR-MS system in heart tissue samples from two study patients. (A and B) Examples of mass spectra obtained from the PCR-MS for one enterovirus-positive heart sample (A) and one parvovirus B19-positive heart sample (B). For each identified viral amplicon, two peaks appeared in the mass spectra (plus and minus strands), as well as two peaks for the internal calibrant, allowing a semiquantitative detection approach. The x axis represents the exact molecular mass of the single-stranded amplicon, while the y axis represents the number of molecules detected.