Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort

Abstract

Objective: The presence of the retrovirus xenotropic murine leukaemia virus-related virus (XMRV) has been reported in peripheral blood mononuclear cells of patients with chronic fatigue syndrome. Considering the potentially great medical and social relevance of such a discovery, we investigated whether this finding could be confirmed in an independent European cohort of patients with chronic fatigue syndrome.

Design: Analysis of a well defined cohort of patients and matched neighbourhood controls by polymerase chain reaction.

Setting: Certified (ISO 15189) laboratory of clinical virology in a university hospital in the Netherlands. Population Between December 1991 and April 1992, peripheral blood mononuclear cells were isolated from 76 patients and 69 matched neighbourhood controls. In this study we tested cells from 32 patients and 43 controls from whom original cryopreserved phials were still available.

Main outcome measures: Detection of XMRV in peripheral blood mononuclear cells by real time polymerase chain reaction assay targeting the XMRV integrase gene and/or a nested polymerase chain reaction assay targeting the XMRV gag gene.

Results: We detected no XMRV sequences in any of the patients or controls in either of the assays, in which relevant positive and negative isolation controls and polymerase chain reaction controls were included. Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences per 10(5) peripheral blood mononuclear cells by real time as well as by nested polymerase chain reaction, demonstrating high sensitivity of both assays.

Conclusions: This study failed to show the presence of XMRV in peripheral blood mononuclear cells of patients with chronic fatigue syndrome from a Dutch cohort. These data cast doubt on the claim that XMRV is associated with chronic fatigue syndrome in the majority of patients.

Fig 1 Results of XMRV integrase real time polymerase chain reaction assay. (A) All 32 patients with chronic fatigue syndrome (CFS) compared with positive 22Rv1 control, which yielded a crossing point value of about 23. Results for neighbourhood controls not shown. (B) 22Rv1 total nucleic acid (DNA, solid), reverse transcribed total nucleic acid (cDNA, dashed). The additional reverse transcription step increased the sensitivity of the polymerase chain reaction, decreasing the crossing point (Cp) value by 3.5. One of three independent experiments is shown. (C) Sensitivity of the assay. The inlay shows linear relation between number of spiked molecules and crossing point value from 10¹ to 10⁶ copies per reaction

Fig 2 Results of XMRV gag nested polymerase chain reaction assay. (A) Results for 11 patients with chronic fatigue syndrome and negative controls. Results for neighbourhood controls not shown. (1) The positive 22Rv1 control yielded a product of the expected size of 92 base pairs (arrow); (2) negative polymerase chain reaction control; (3) phocine distemper virus (internal control); (4) negative reverse transcription control; (5)-(11) and (13)-(16) patient samples; (12) negative isolation control; (M) 100 base pairs size marker. (B) Sensitivity of the XMRV gag first polymerase chain reaction. (C) Sensitivity of the nested reaction. White arrows indicate the 613 base pairs (B) and 92 base pairs (C) reaction products. (1) 22Rv1; (2) negative polymerase chain reaction control; (3-9), dilution series of 10⁶ to 10⁰ copies of calibrator per reaction; (10) negative isolation control; (11) negative nested polymerase chain reaction control; (12) positive nested polymerase chain reaction control (22Rv1); (M) 100 base pairs size marker. Black arrows 1-4 in (C) indicate polymerase chain reaction products that are formed in the nested reaction (see D). (D) Positions of the gag primers and the gag polymerase chain reaction products formed in the nested reaction. In addition to the primers of the nested reaction (GAG-I-F, I-F and GAG-I-R2, I-R2), primers from the first reaction (GAG-O-F, O-F and GAG-O-R, O-R) are also present in the nested reaction, yielding reaction products 1-4 that correspond to the black arrows in (C). Numbers represent the positions on the XMRV genome (VP42, accession DQ241302)