Department of Immunology and Rheumatology, Hôpital E. Herriot and
1 Department of Virology, Domaine Rockefeller, Lyon, France
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Abstract |
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KEY WORDS: Dermatomyositis, Autoimmune disease, Parvovirus B19, Muscle biopsy, Polymerase chain reaction
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Introduction |
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Case report |
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In February 1997, treatment with prednisone (20 mg/day) and i.m. methotrexate (MTX) (12.5 mg/week) was started. The clinical outcome was good and 3 months later the patient did not complain of any pain but had some proximal lower limb muscle weakness. A biopsy of the right quadriceps femoris muscle was performed in June 1997. In this biopsy, the initial lesions were less severe. In June 1997, serum creatinine phosphokinase was normal but aldolase was 9 IU/l. However, doses of prednisone and MTX were decreased gradually from June to September 1997 without relapse. The patient was maintained on MTX alone for a total of 2 yr and has been disease-free since MTX was stopped.
Because of the association of arthralgia and a rash, serum and muscle biopsies were analysed for the presence of parvovirus B19 antibodies and DNA.
Serological analysis of parvovirus B19 antibodies was performed on four sequential serum samples (4 January, 24 February, 17 June and 9 September 1997), using different commercial kits for enzyme immunoassay (EIA). The presence of specific IgG was tested with a synthetic peptide derived from two capsid proteins, viral proteins 1 (VP1) and 2 (VP2) (Parvoscreen-B19TM IgG; Euro-Diagnostica, Gentilly, France). A µ-antibody-capture assay based on a recombinant VP2 antigen (Parvovirus B19 IgM EIA; Biotrin, Dublin, Ireland) was used for detection of specific IgM. Serological analysis for other viruses [coxsackie virus, echo virus, influenza virus, adenovirus, human immunodeficiency virus (HIV), human T-cell leukaemialymphoma virus type I (HTLV-1)] were performed on the same samples.
Detection of parvovirus B19 DNA in the four serum samples and two muscle biopsies from the patient was performed after amplification by the polymerase chain reaction (PCR). Vastus lateralis muscle biopsies of three patients undergoing hip replacement for osteoarthritis were used as controls. Briefly, muscle biopsies were mechanically disrupted in RBS buffer (10 mM TrisHCl, pH 7.4, with 10 mM NaCl and 25 mM EDTA) then digested by proteinase K (1 mg/ml in sodium dodecyl sulphate 10%; Sigma, St Louis, MO, USA) at 37°C overnight. After phenol extraction and ethanol precipitation, air-dried DNA pellets were resuspended in 0.1 M TrisEDTA. The same procedure was applied to two parvovirus B19-positive sera used as a positive PCR control and a negative control (200 µl H2O instead of serum). Serum samples were treated with proteinase K. Serum samples (200 µl) were mixed with 100 µl 10 x lysis buffer (10 mM TrisHCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2 and 0.45% Tween 20) and 100 µg/ml proteinase K, then incubated at 56°C for 1 h, then at 95°C for 10 min. The total volume of PCR mixture was 100 µl, and included either 1 or 10 µl of muscle and either 0.1 or 1 µl of serum DNA extracts.
Two sets of primers were selected to amplify two genomic sequences, the first coding for VP1 and the second for non-structural protein 1 (NS1). Primers A (5' GTG CTT ACG TGT CTG GAT TGC 3', sense nucleotides 24082422) and B (5' GCT AAC TTG CCC AGG CTT GT 3', antisense nucleotides 28092790) amplified (34 cycles at an annealing temperature of 48°C) a 402-base pair (bp) fragment located within the VP1 coding sequence [3]. Amplified products were detected by ethidium bromide staining after agarose gel electrophoresis. Specific identification was performed with a biotin-labelled probe (5' AAT ATT AAA AGA TCA TTA TAA TAT TTC TTT AGA TAA TCC CC 3', nucleotides 25602600) according to the hybridization procedures of GEN-ETI-KTMDEIA; DiaSorin, Saluggia, Italy.
A second set of primers, C (5' AAT ACA CTG TGG TTT TAT GGG CCG 3', sense nucleotides 13991422) and D (5' CCA TTG CTG GTT ATA ACC ACA GGT 3', antisense nucleotides 16821659) [4], was used for the amplification (35 cycles at an annealing temperature of 55°C) of a 284 bp fragment corresponding to the NS1 coding sequence. PCR products were visualized by gel staining.
Specific parvovirus B19 IgG antibodies were present at a stable level in the different samples. No specific IgM antibodies were detected. The samples were also negative for antibodies against other viruses (coxsackie virus, echo virus, influenza virus, adenovirus, HIV and HTLV-1).
None of the four serum samples was positive for parvovirus B19 DNA. Conversely, the two muscle biopsies (27 February and 17 June) contained parvovirus B19 VP1 sequences (Fig. 1). The second one was also positive for NS1. Parvovirus B19 DNA was not detected in muscle biopsies of three control individuals.
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Parvovirus B19 is a single-stranded DNA virus that causes erythema infectiosum, arthralgia, aplastic crisis (particularly in patients with red cell defects) and chronic anaemia in immunocompromised patients [6]. More recently, parvovirus B19 has been associated with autoimmune connective tissue diseases [7]. In particular, a large proportion of rheumatoid arthritis synovial biopsies was found to contain parvovirus B19 DNA [2] but the significance of this remains controversial [8].
Regarding muscle diseases, several cases of myositis with the possible involvement of parvovirus B19 have been described. In two patients with a clinical picture highly suggestive of systemic lupus erythematosus, increased creatinine phosphokinase and aldolase levels were associated with acute parvovirus B19 infection, as indicated by elevated IgM and IgG antibodies, but no viral examination was done in the muscle biopsy [9]. One child with recent onset of juvenile dermatomyositis showed serological evidence of acute parvovirus B19 infection. Amplification of viral DNA by PCR was positive in an early serum sample but was negative in muscle biopsy and peripheral blood leukocytes drawn at the time of biopsy [10]. A 7-yr-old girl exposed to parvovirus B19 developed acute hepatitis and myositis followed by a life-threatening interstitial lung disease [11]. Parvovirus B19 DNA in bone marrow, lung and serum and elevated IgM and IgG antibodies were detected. PCR-amplified viral DNA remained positive in serum until the end of the survey. Muscle biopsy showed type II fibre atrophy without significant inflammation, but parvovirus B19 DNA detection was not performed.
The absence of IgM in our patient could be explained by the recurrence of parvovirus B19 infection in December 1996. However, the presence of specific IgM is not sufficient to link a parvovirus B19 infection to the diagnosis of DM. Viral detection directly at the site of the disease by PCR or Southern hybridization was therefore necessary. The presence of parvovirus B19 DNA in muscle biopsy was confirmed twice in our patient, using two different sets of primers. However, the extent of the contribution of a latent parvovirus B19 infection to myositis remains to be determined. Parvovirus B19 DNA has been shown to persist in synovial membrane, bone marrow or skin, not only in patients with autoimmune diseases or chronic urticaria but also in healthy immunocompetent individuals [1214]. Our control muscle samples were negative.
Since the cause and pathogenesis of DM remain unclear, the contribution of an infectious agent is also difficult to establish. Many factors are probably involved, including genetic and hormonal components. Conversely, many viruses have been studied for their possible contribution to DM [1, 5]. Regarding this case, it is likely that the virus itself was not the cause of muscle damage, but that an undefined aberrant host immune response was triggered by the parvovirus, leading to muscle destruction. The presence of parvovirus B19 DNA in our two muscle biopsies was established using primers for VP1 and NS1. At a 5-month interval, the two samples were positive for VP1, which may be active in the formation of infectious viral progeny. Only the second sample was positive for NS1. In this context, it is of interest to note that NS1 has been associated with persistent infection and may therefore be involved in the pathogenesis [15]. Similarly, recent data suggest a link between the apoptotic pathways activated by the combination of tumour necrosis factor and NS1 in human erythroid cells [16].
This is the first report of viral parvovirus B19 DNA in muscle of a patient with all the classical features of DM. The demonstration of a direct link will need a prospective study with large numbers of patients with such a rare disease.
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Notes |
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References |
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