Multicenter Blinded Analysis of RT-PCR Detection Methods for Paramyxoviruses in Relation to Paget's Disease of Bone

Abstract

Conflicting results have been reported on the detection of paramyxovirus transcripts in Paget's disease, and a possible explanation is differences in the sensitivity of RT-PCR methods for detecting virus. In a blinded study, we found no evidence to suggest that laboratories that failed to detect viral transcripts had less sensitive RT-PCR assays, and we did not detect measles or distemper transcripts in Paget's samples using the most sensitive assays evaluated.Introduction: There is conflicting evidence on the possible role of persistent paramyxovirus infection in Paget's disease of bone (PDB). Some workers have detected measles virus (MV) or canine distemper virus (CDV) transcripts in cells and tissues from patients with PDB, but others have failed to confirm this finding. A possible explanation might be differences in the sensitivity of RT-PCR methods for detecting virus. Here we performed a blinded comparison of the sensitivity of different RT-PCR-based techniques for MV and CDV detection in different laboratories and used the most sensitive assays to screen for evidence of viral transcripts in bone and blood samples derived from patients with PDB. Materials and Methods: Participating laboratories analyzed samples spiked with known amounts of MV and CDV transcripts and control samples that did not contain viral nucleic acids. All analyses were performed on a blinded basis. Results: The limit of detection for CDV was 1000 viral transcripts in three laboratories (Aberdeen, Belfast, and Liverpool) and 10,000 transcripts in another laboratory (Manchester). The limit of detection for MV was 16 transcripts in one laboratory (NIBSC), 1000 transcripts in two laboratories (Aberdeen and Belfast), and 10,000 transcripts in two laboratories (Liverpool and Manchester). An assay previously used by a U.S.-based group to detect MV transcripts in PDB had a sensitivity of 1000 transcripts. One laboratory (Manchester) detected CDV transcripts in a negative control and in two samples that had been spiked with MV. None of the other laboratories had false-positive results for MV or CDV, and no evidence of viral transcripts was found on analysis of 12 PDB samples using the most sensitive RT-PCR assays for MV and CDV. Conclusions: We found that RT-PCR assays used by different laboratories differed in their sensitivity to detect CDV and MV transcripts but found no evidence to suggest that laboratories that previously failed to detect viral transcripts had less sensitive RT-PCR assays than those that detected viral transcripts. False-positive results were observed with one laboratory, and we failed to detect paramyxovirus transcripts in PDB samples using the most sensitive assays evaluated. Our results show that failure of some laboratories to detect viral transcripts is unlikely to be caused by problems with assay sensitivity and highlight the fact that contamination can be an issue when searching for pathogens by sensitive RT-PCR-based techniques.

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures

“…As paramyxoviruses interfere with both IFN production and signalling, there is potentially a mechanistic link between these observations. Others have disputed this link (Nuovo et al, 1992;Ooi et al, 2000;Rima et al, 2002;Ralston et al, 2007), but the picture is complicated by the fact that most paramyxoviruses, and many other viruses, block the IFNsignalling pathways, raising the possibility that persistent infections by different viruses may cause the same manifestations of a chronic disease. Indeed, a number of different paramyxoviruses, including MeV, canine distemper virus (CDV), RSV, PIV3 and PIV5, have been reported to infect osteoclasts persistently (Mills et al, 1984;Basle et al, 1985Basle et al, , 1986Basle et al, , 1987Gordon et al, 1991;Mee et al, 1998;Reddy et al, 1999;Friedrichs et al, 2002;Hoyland et al, 2003).…”

Epidemiological features and the neuropathological manifestations of canine distemper virus-induced infections in Brazil: a review

“…Further, CDV N gene was identified by in situ hybridisation and RT-PCR from bone cells of dogs diagnosed with metaphyseal osteopathy , a canine bone disease of uncertain etiology but with some similar histological features of PDB . However, antagonistic results not in support of this theory were observed by PCR (RALSTON et al, 1991), while a comparative blinded study has suggested that cross-contamination due to the excessive usage of various diagnostic techniques, within the same laboratory, to evaluate the participation of CDV in PDB could have been responsible for the results described by MEE and collaborators (RALSTON et al, 2007).…”

Pathogenesis of Paget Disease of Bone

“…The most widely studied environmental exposure is paramyxoviral infection, although evidence in favor of a viral etiology for PDB remains controversial [30][31][32]. The introduction of immunization programs for measles and distemper in the 1960s has been suggested as a possible reason for the reduction in incidence of PDB, but it has recently been highlighted that this occurred too recently to account for a reduction in prevalence and severity of PDB patients born in the 1930s and 1940s, assuming that a slow viral infection picked up in childhood was indeed the cause of the disease [13,14].…”