- Email: [email protected]
Dopamine-transporter density in patients with ADHD
CORRESPONDENCE
medications until 1 month or more before the SPECT scanning means the patients were not drug-naïve, and that the scan differences between the patients and controls were likely to have been drug-induced. Certainly, it cannot be concluded that the study defines an ADHD phenotype, pathology, pathological physiology, or pathological chemistry. At the National Institutes of Health, Consensus Conference on ADHD, Nov 16–18, 1998, Swanson and Castellanos3 presented a review of the neuroimaging literature that showed that the brains of patients with ADHD were, on average, 10% more atrophic compared with controls without ADHD. Swanson and Castellanos stated there were no such studies in which the ADHD patients were drugnaïve—ie, virtually all ADHD patients had been on stimulant therapy. This being the case, stimulant therapy, not ADHD, is the likely cause of the brain atrophy. There can be no contention that taking study participants off stimulants, or off any psychotropic medication, for 1 month, a few months, or for any period at all, entirely rules out the possibility of drug effect. Also, ADHD is yet to be validated as a disease (with a confirmatory physical or chemical abnormality), a syndrome, or a phenotype (with a confirmatory physical or chemical marker). Fred A Baughman Jr 1303 Hidden Mountain Drive, El Cajon, CA 92019, USA 1
2
3
Dougherty DD, Bonals AA, Spencer TJ, Rauch SL, Madras BK, Fischman AJ. Dopamine transporter density in patients with attention deficit hyperactivity disorder. Lancet 1999; 354: 2132–33 Brown WD, Taylor MD, Roberts AD, et al. FluoroDOPA PET shows the nondopaminergic as well as dopamingergic destinations of levodopa. Neurology 1999; 53: 1212–18. Swanson J, Castellanos FX. Biological Bases of ADHD: neuroanatomy, genetics, and pathophysiology. Programme and abstracts, NIH Consensus Conference on the Diagnosis and Treatment of Attention Deficit Hyperactivity Disorder, Nov 16–18, 1998: 37–42.
Sir—Darin Doughterty and colleagues1 report that the binding potential for the dopamine transporter is extraordinarily high in adults with ADHD compared with controls. If true, this is a major finding and points the way for new investigations of the primary pharmacological treatment for ADHD (with the stimulant drugs —eg, methyphenidate), for which the dopamine transporter is the THE LANCET • Vol 355 • April 22, 2000
primary site of action.2,3 The potential importance of this finding demands special scrutiny of the report. There are two questions that need answers to clarify the reported results: are the error bars shown in figure 2 estimates of the standard error, as stated in the caption? If so then it does not appear that the mean values of the control groups were exceeded by at least 2 SD, as stated in the report. Perhaps the error bars in the figure should be multiplied by 2·4 (for age 21–30 years) to 3·0 (for 31–40 years), since SD=SE⫻sqrt(N); and what was the sex ratio for the controls? The patients were predominantly female, but comparisons were made to controls matched for age and presumably not for sex. What is the magnitude of the effect (in SD units) for the cases compared with controls matched for age and sex? James M Swanson University of California, Child Development Center, Clinic and Clinical Trials, Irvine, CA 92612, USA 1
2
3
Doughtery DD, Bonab AA, Spencer TJ, Raich SL, Madras BK, Fischman AJ. Dopamine transporter density in patients with attention deficit hyperactivity disorder. Lancet 1999; 354: 2132–33. Volkow ND, Ding YS, Fowler JS, et al. Is methylphenidate like cocaine? Studies on their pharmacokinetics and distribution in the human brain. Arch Gen Psychiatry 1995; 52: 456–63. Volkow ND, Wang GJ, Fowler JS, et al. Dopamine transporter occupancies in the human brain induced by therapeutic doses of oral methylphenidate. Am J Psychiatry 1998; 155: 1325–31.
Authors’ reply Sir—None of the six patients with ADHD who we studied took psychotropic drugs for at least 1 month before the study. Four participants had been treated with psychostimulants previously and two had never been exposed to any psychotropic drug. The effects of psychostimulants are probably the result of immediate interactions of the drug with dopamine transporter—ie, alterations in neuronal gene expression do not appear to be necessary. Lastly, several lines of evidence support the contention that these findings are not drug-induced, 1 including previous findings of dopaminergic abnormalities in patients with ADHD.2 The statement made by Fred Baughman that the observed effects in our imaging study are a result of past stimulant use is grossly premature and devoid of any scientific basis. Nonetheless, although the differences we describe in our study may represent a phenotypic difference,
Group
Age-range years
Binding potential
ADHD patients
34 45 24 53 51 41
2·98 2·34 2·71 2·33 2·16 3·51
ADHD group (n=6)
24–53
3·00 (0·51)
Control group (n=30)
19–60 18–30 (n=6) 31–40 (n=9) 41–50 (n=8) 51–60 (n=7)
1·77 (0·53) 2·12 (0·25) 1·90 (0·54) 1·58 (0·51) 1·50 (0·57)
123
I-altropane binding potential in patients with ADHD and age-matched control group
this was a preliminary report of six patients and Baughman has addressed an important point that needs to be examined. Further studies are needed to clarify these issues and such investigations are underway. Lastly, Baughman states that “ADHD is yet to be validated as a disease”. Despite some controversy, an emerging body of scientific data3 consisting of clinical, genetic, neuropsychological, imaging, and treatment studies strongly support not only the validity of the diagnosis of ADHD in adults, 4 but also syndromatic continuity with a paediatric condition. James Swanson raises two important points. First, he requests details regarding the differences in average binding potential between the two groups (table). The six patients with ADHD had a mean agecorrected binding potential of 3·00 (SD 0·51), whereas the 30 control participants had a mean age-corrected binding potential of 1·77 (0·53). An unpaired t test revealed a highly significant difference in age-corrected binding potential between the two study groups (t=5·25; p<0·001). As indicated by the graph in the original report, the binding potential of each patient with ADHD differed from the mean-age-corrected binding potential for age-matched control participants by at least 2 SDs. These data are presented in greater detail in the table shown here. Even for the single patient in the youngest age group, age-corrected binding potential was more than 2 SDs greater than the agecorrected mean for age-matched control participants (2·71 vs 2·12 [0·25]). Second, Swanson asks about the sex ratio in the control group. This group of 30 participants comprised 14 women and 16 men. There was no significant difference in binding potential in the control participant data based on sex. The six patients
1461
CORRESPONDENCE
with ADHD included four women and two men. Given the small sample size, we do not believe any generalisations can be made regarding effects of sex. However, Swanson has broached an important issue and as we expand our database, we plan to test for effects of sex. We thank Scott L Rauch for his help preparing this reply.
Darin D Dougherty, Ali A Bonab, Thomas J Spencer, Bertha K Madras, Alan J Fischman Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114, USA (e-mail: [email protected]) 1
2
3
4
Madras BK. Imaging and dopamine transporter: a window on dopamine neurons. In: Marwah J, Teitelbaum H, eds. Advances in neurodegenerative disorders, volume 1. Parkinson’s disease. Scottsdale: Prominent Press, 1998: 229–53. Ernst M, Zametkin AJ, Matochik JA, et al. High midbrain [18F]DOPA accumulation in children with attention deficit hyperactivity disorder. Am J Psychiatry 1999; 156: 1209–15. Faraone SV, Biederman J. Neurobiology of attention-deficit hyperactivity disorder. Biol Psychiatry 1998; 44: 951–58. Spencer T, Biederman J, Wilens T, Faraone SV. Adults with attentiondeficit/hyperactivity disorder: a controversial diagnosis. J Clin Psychiatry 1998; 59: 59–68.
Human bornaviruses and laboratory strains Sir—Martin Schwemmle and colleagues (Dec 4, p 1973)1 claim that borna disease viruses (BDV) of human origin do not exist. They base their statement on sequences of RW982 and sequencing of their own rat BDV and comparing that information with only seven of 19 available human sequences (N-gene; p 40). Based on nucleotides 277–717 of this gene, we compared RW98 (accession number AF 158629) and ten of the sequences ignored by Schwemmle and colleagues,3,4 with laboratory strain V. Apart from a few individual mutations, eight silent mutations commonly occur. They were present in RW98 (eight of 12 mutations), Berlin-patient III (eight of 15),3 and the Homburg patients (eight of 14–19 mutations).4 Those common mutations (versus strain V) were also found in laboratory strain He/80 and natural horse strain WT-1. Consequently, the divergence of natural isolates to either laboratory strain depends upon the proportion of common versus rare individual mutations—eg, Berlin-patient III, differing by 3·4% from strain V (Ngene-fragment),3 but only 1·6% from He/80 (never handled). The 1·8%
1462
difference is a result of the frequently occurring mutations also present in He/80. Despite various causes for a low degree of genetic divergence, Schwemmle and colleagues gave the impression that contamination is the most likely cause. Isolation of BDV from psychiatric patients 5 was questioned by Schwemmle and colleagues because of only about 1% divergence to strain V. They disregarded the fact that this percentage consists of unique mutations, which are also valid when compared with He/80. Almost all the mutations are individual for each isolate and cause relevant aminoacid changes. No other commonly seen silent mutations were found, which explains the low level of divergence. Moreover, sequence identity of individual isolates with original patients’ samples, as well as biological strain differences, exclude contamination. 5 Schwemmle and colleagues did not explore these arguments and we believe their generalised conclusions are unjustified and wrong. Liv Bode, Roman Stoyloff, *Hanns Ludwig Project Bornavirus infections, Robert Koch-Institut, Berlin, Germany, and *Institute of Virology, Free University of Berlin, 14195 Berlin, Germany (e-mail: [email protected]) 1
2
3
4
5
Schwemmle M, Jehle C, Formella S, Stacheli P. Sequence similarities between human bornavirus isolates and laboratory strains question human origin. Lancet 1999; 354: 1973–74. Planz O, Rentzsch C, Batra A, et al. Pathogenesis of Borna disease virus: granulocyte fractions of psychiatric patients harbour infectious virus in the absence of antiviral antibodies. J Virol 1999; 73: 6251–56. Bode L, Zimmermann W, Ferszt R, Steinbach F, Ludwig H. Borna disease virus genome transcribed and expressed in psychiatric patients. Nat Med 1995; 1: 232–36. Saunder C, Müller A, Cubitt B, et al. Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA. J Virol 1996; 70: 7713–24. Bode L, Dürrwald R, Rantam FA, Ferszt R, Ludwig H. First isolates of infectious human Borna disease virus from patients with mood disorders. Mol Psychiatry 1996; 1: 200–12.
Sir—Schwemmle and colleagues1 give convincing evidence that nearly all published human borna-disease-virus (BDV) isolates and PCR amplification products seem to be contaminations of laboratory strains. We agree that the isolation of BDV from human blood or evidence that BDV nucleic acid has been found in human blood is highly controversial.
BDV is a neurotropic virus, and it is not clear as yet, even in BDV-infected animals, whether there is a period of viraemia during the course of infection, during which it would be possible to detect BDV or BDV nucleic acid in blood. There is the possibility that following nasal uptake of BDV, the virus does not only infect nerve cells, and proceeds via the olfactory bulb to the brain, but also gets into a few immune cells in the nasal mucosa. Thus, during the early stage of infection, and maybe during further acute periods, BDV may be present in blood. There is clear serological evidence for the existence of BDV (or a closely related virus) in human beings. Nearly all laboratories investigating BDV worldwide have found antibodies to BDV or BDV proteins in psychiatric patients and also—at a lower prevalence—in healthy individuals. There is a fundamental difference between BDV infection in naturally infected animals and human beings. BDV infection in animals leads to a severe and often lethal meningoencephalomyelitis, but such inflammation has never been seen in human beings. In human beings only subtle changes are suspected and these may lead to psychiatric disorders. Also, in animals that succumb to BDV infection, BDV, BDV antigen, and nucleic acid can easily be detected using standard techniques including non-nested PCR. In human blood, BDV nucleic acid is only detectable with nested PCR, which has a high chance of becoming contaminated. We have detected BDV nucleic acid sequences, amplified from peripheral blood mononuclear cells, of a patient with chronic fatigue syndrome (CFS).2 The nucleotide sequences from this patient are unique, showing several nucleotide changes that have not been seen in any other BDV. Also, the sequences found are not related to the sequences of any laboratory strain or BDV isolate handled in our laboratory. We constructed a phylogenetic tree (see http://www. thelancet.com) of the nucleotide sequences of the N and P proteins of BDV and included all the sequences that Schwemmle and colleagues used and added the sequences of the patients with CFS (GenBank accession numbers AF094477 [N protein] and AFO94478 [P protein], respectively). The sequences do not group together with any laboratory strain in the N protein region, and show a significant distance from strain He/80 in the P protein gene region. Schwemmle and colleagues
THE LANCET • Vol 355 • April 22, 2000
medications until 1 month or more before the SPECT scanning means the patients were not drug-naïve, and that the scan differences between the patients and controls were likely to have been drug-induced. Certainly, it cannot be concluded that the study defines an ADHD phenotype, pathology, pathological physiology, or pathological chemistry. At the National Institutes of Health, Consensus Conference on ADHD, Nov 16–18, 1998, Swanson and Castellanos3 presented a review of the neuroimaging literature that showed that the brains of patients with ADHD were, on average, 10% more atrophic compared with controls without ADHD. Swanson and Castellanos stated there were no such studies in which the ADHD patients were drugnaïve—ie, virtually all ADHD patients had been on stimulant therapy. This being the case, stimulant therapy, not ADHD, is the likely cause of the brain atrophy. There can be no contention that taking study participants off stimulants, or off any psychotropic medication, for 1 month, a few months, or for any period at all, entirely rules out the possibility of drug effect. Also, ADHD is yet to be validated as a disease (with a confirmatory physical or chemical abnormality), a syndrome, or a phenotype (with a confirmatory physical or chemical marker). Fred A Baughman Jr 1303 Hidden Mountain Drive, El Cajon, CA 92019, USA 1
2
3
Dougherty DD, Bonals AA, Spencer TJ, Rauch SL, Madras BK, Fischman AJ. Dopamine transporter density in patients with attention deficit hyperactivity disorder. Lancet 1999; 354: 2132–33 Brown WD, Taylor MD, Roberts AD, et al. FluoroDOPA PET shows the nondopaminergic as well as dopamingergic destinations of levodopa. Neurology 1999; 53: 1212–18. Swanson J, Castellanos FX. Biological Bases of ADHD: neuroanatomy, genetics, and pathophysiology. Programme and abstracts, NIH Consensus Conference on the Diagnosis and Treatment of Attention Deficit Hyperactivity Disorder, Nov 16–18, 1998: 37–42.
Sir—Darin Doughterty and colleagues1 report that the binding potential for the dopamine transporter is extraordinarily high in adults with ADHD compared with controls. If true, this is a major finding and points the way for new investigations of the primary pharmacological treatment for ADHD (with the stimulant drugs —eg, methyphenidate), for which the dopamine transporter is the THE LANCET • Vol 355 • April 22, 2000
primary site of action.2,3 The potential importance of this finding demands special scrutiny of the report. There are two questions that need answers to clarify the reported results: are the error bars shown in figure 2 estimates of the standard error, as stated in the caption? If so then it does not appear that the mean values of the control groups were exceeded by at least 2 SD, as stated in the report. Perhaps the error bars in the figure should be multiplied by 2·4 (for age 21–30 years) to 3·0 (for 31–40 years), since SD=SE⫻sqrt(N); and what was the sex ratio for the controls? The patients were predominantly female, but comparisons were made to controls matched for age and presumably not for sex. What is the magnitude of the effect (in SD units) for the cases compared with controls matched for age and sex? James M Swanson University of California, Child Development Center, Clinic and Clinical Trials, Irvine, CA 92612, USA 1
2
3
Doughtery DD, Bonab AA, Spencer TJ, Raich SL, Madras BK, Fischman AJ. Dopamine transporter density in patients with attention deficit hyperactivity disorder. Lancet 1999; 354: 2132–33. Volkow ND, Ding YS, Fowler JS, et al. Is methylphenidate like cocaine? Studies on their pharmacokinetics and distribution in the human brain. Arch Gen Psychiatry 1995; 52: 456–63. Volkow ND, Wang GJ, Fowler JS, et al. Dopamine transporter occupancies in the human brain induced by therapeutic doses of oral methylphenidate. Am J Psychiatry 1998; 155: 1325–31.
Authors’ reply Sir—None of the six patients with ADHD who we studied took psychotropic drugs for at least 1 month before the study. Four participants had been treated with psychostimulants previously and two had never been exposed to any psychotropic drug. The effects of psychostimulants are probably the result of immediate interactions of the drug with dopamine transporter—ie, alterations in neuronal gene expression do not appear to be necessary. Lastly, several lines of evidence support the contention that these findings are not drug-induced, 1 including previous findings of dopaminergic abnormalities in patients with ADHD.2 The statement made by Fred Baughman that the observed effects in our imaging study are a result of past stimulant use is grossly premature and devoid of any scientific basis. Nonetheless, although the differences we describe in our study may represent a phenotypic difference,
Group
Age-range years
Binding potential
ADHD patients
34 45 24 53 51 41
2·98 2·34 2·71 2·33 2·16 3·51
ADHD group (n=6)
24–53
3·00 (0·51)
Control group (n=30)
19–60 18–30 (n=6) 31–40 (n=9) 41–50 (n=8) 51–60 (n=7)
1·77 (0·53) 2·12 (0·25) 1·90 (0·54) 1·58 (0·51) 1·50 (0·57)
123
I-altropane binding potential in patients with ADHD and age-matched control group
this was a preliminary report of six patients and Baughman has addressed an important point that needs to be examined. Further studies are needed to clarify these issues and such investigations are underway. Lastly, Baughman states that “ADHD is yet to be validated as a disease”. Despite some controversy, an emerging body of scientific data3 consisting of clinical, genetic, neuropsychological, imaging, and treatment studies strongly support not only the validity of the diagnosis of ADHD in adults, 4 but also syndromatic continuity with a paediatric condition. James Swanson raises two important points. First, he requests details regarding the differences in average binding potential between the two groups (table). The six patients with ADHD had a mean agecorrected binding potential of 3·00 (SD 0·51), whereas the 30 control participants had a mean age-corrected binding potential of 1·77 (0·53). An unpaired t test revealed a highly significant difference in age-corrected binding potential between the two study groups (t=5·25; p<0·001). As indicated by the graph in the original report, the binding potential of each patient with ADHD differed from the mean-age-corrected binding potential for age-matched control participants by at least 2 SDs. These data are presented in greater detail in the table shown here. Even for the single patient in the youngest age group, age-corrected binding potential was more than 2 SDs greater than the agecorrected mean for age-matched control participants (2·71 vs 2·12 [0·25]). Second, Swanson asks about the sex ratio in the control group. This group of 30 participants comprised 14 women and 16 men. There was no significant difference in binding potential in the control participant data based on sex. The six patients
1461
CORRESPONDENCE
with ADHD included four women and two men. Given the small sample size, we do not believe any generalisations can be made regarding effects of sex. However, Swanson has broached an important issue and as we expand our database, we plan to test for effects of sex. We thank Scott L Rauch for his help preparing this reply.
Darin D Dougherty, Ali A Bonab, Thomas J Spencer, Bertha K Madras, Alan J Fischman Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114, USA (e-mail: [email protected]) 1
2
3
4
Madras BK. Imaging and dopamine transporter: a window on dopamine neurons. In: Marwah J, Teitelbaum H, eds. Advances in neurodegenerative disorders, volume 1. Parkinson’s disease. Scottsdale: Prominent Press, 1998: 229–53. Ernst M, Zametkin AJ, Matochik JA, et al. High midbrain [18F]DOPA accumulation in children with attention deficit hyperactivity disorder. Am J Psychiatry 1999; 156: 1209–15. Faraone SV, Biederman J. Neurobiology of attention-deficit hyperactivity disorder. Biol Psychiatry 1998; 44: 951–58. Spencer T, Biederman J, Wilens T, Faraone SV. Adults with attentiondeficit/hyperactivity disorder: a controversial diagnosis. J Clin Psychiatry 1998; 59: 59–68.
Human bornaviruses and laboratory strains Sir—Martin Schwemmle and colleagues (Dec 4, p 1973)1 claim that borna disease viruses (BDV) of human origin do not exist. They base their statement on sequences of RW982 and sequencing of their own rat BDV and comparing that information with only seven of 19 available human sequences (N-gene; p 40). Based on nucleotides 277–717 of this gene, we compared RW98 (accession number AF 158629) and ten of the sequences ignored by Schwemmle and colleagues,3,4 with laboratory strain V. Apart from a few individual mutations, eight silent mutations commonly occur. They were present in RW98 (eight of 12 mutations), Berlin-patient III (eight of 15),3 and the Homburg patients (eight of 14–19 mutations).4 Those common mutations (versus strain V) were also found in laboratory strain He/80 and natural horse strain WT-1. Consequently, the divergence of natural isolates to either laboratory strain depends upon the proportion of common versus rare individual mutations—eg, Berlin-patient III, differing by 3·4% from strain V (Ngene-fragment),3 but only 1·6% from He/80 (never handled). The 1·8%
1462
difference is a result of the frequently occurring mutations also present in He/80. Despite various causes for a low degree of genetic divergence, Schwemmle and colleagues gave the impression that contamination is the most likely cause. Isolation of BDV from psychiatric patients 5 was questioned by Schwemmle and colleagues because of only about 1% divergence to strain V. They disregarded the fact that this percentage consists of unique mutations, which are also valid when compared with He/80. Almost all the mutations are individual for each isolate and cause relevant aminoacid changes. No other commonly seen silent mutations were found, which explains the low level of divergence. Moreover, sequence identity of individual isolates with original patients’ samples, as well as biological strain differences, exclude contamination. 5 Schwemmle and colleagues did not explore these arguments and we believe their generalised conclusions are unjustified and wrong. Liv Bode, Roman Stoyloff, *Hanns Ludwig Project Bornavirus infections, Robert Koch-Institut, Berlin, Germany, and *Institute of Virology, Free University of Berlin, 14195 Berlin, Germany (e-mail: [email protected]) 1
2
3
4
5
Schwemmle M, Jehle C, Formella S, Stacheli P. Sequence similarities between human bornavirus isolates and laboratory strains question human origin. Lancet 1999; 354: 1973–74. Planz O, Rentzsch C, Batra A, et al. Pathogenesis of Borna disease virus: granulocyte fractions of psychiatric patients harbour infectious virus in the absence of antiviral antibodies. J Virol 1999; 73: 6251–56. Bode L, Zimmermann W, Ferszt R, Steinbach F, Ludwig H. Borna disease virus genome transcribed and expressed in psychiatric patients. Nat Med 1995; 1: 232–36. Saunder C, Müller A, Cubitt B, et al. Detection of Borna disease virus (BDV) antibodies and BDV RNA in psychiatric patients: evidence for high sequence conservation of human blood-derived BDV RNA. J Virol 1996; 70: 7713–24. Bode L, Dürrwald R, Rantam FA, Ferszt R, Ludwig H. First isolates of infectious human Borna disease virus from patients with mood disorders. Mol Psychiatry 1996; 1: 200–12.
Sir—Schwemmle and colleagues1 give convincing evidence that nearly all published human borna-disease-virus (BDV) isolates and PCR amplification products seem to be contaminations of laboratory strains. We agree that the isolation of BDV from human blood or evidence that BDV nucleic acid has been found in human blood is highly controversial.
BDV is a neurotropic virus, and it is not clear as yet, even in BDV-infected animals, whether there is a period of viraemia during the course of infection, during which it would be possible to detect BDV or BDV nucleic acid in blood. There is the possibility that following nasal uptake of BDV, the virus does not only infect nerve cells, and proceeds via the olfactory bulb to the brain, but also gets into a few immune cells in the nasal mucosa. Thus, during the early stage of infection, and maybe during further acute periods, BDV may be present in blood. There is clear serological evidence for the existence of BDV (or a closely related virus) in human beings. Nearly all laboratories investigating BDV worldwide have found antibodies to BDV or BDV proteins in psychiatric patients and also—at a lower prevalence—in healthy individuals. There is a fundamental difference between BDV infection in naturally infected animals and human beings. BDV infection in animals leads to a severe and often lethal meningoencephalomyelitis, but such inflammation has never been seen in human beings. In human beings only subtle changes are suspected and these may lead to psychiatric disorders. Also, in animals that succumb to BDV infection, BDV, BDV antigen, and nucleic acid can easily be detected using standard techniques including non-nested PCR. In human blood, BDV nucleic acid is only detectable with nested PCR, which has a high chance of becoming contaminated. We have detected BDV nucleic acid sequences, amplified from peripheral blood mononuclear cells, of a patient with chronic fatigue syndrome (CFS).2 The nucleotide sequences from this patient are unique, showing several nucleotide changes that have not been seen in any other BDV. Also, the sequences found are not related to the sequences of any laboratory strain or BDV isolate handled in our laboratory. We constructed a phylogenetic tree (see http://www. thelancet.com) of the nucleotide sequences of the N and P proteins of BDV and included all the sequences that Schwemmle and colleagues used and added the sequences of the patients with CFS (GenBank accession numbers AF094477 [N protein] and AFO94478 [P protein], respectively). The sequences do not group together with any laboratory strain in the N protein region, and show a significant distance from strain He/80 in the P protein gene region. Schwemmle and colleagues
THE LANCET • Vol 355 • April 22, 2000