Download Detection of West Nile virus sequences in cerebrospinal fluid

RESEARCH LETTERS
paediatricians. When we compared the computerised notes
with the cases notified to the coordinating centre, the
reporting was complete. We could not assess, however,
how many ADRs were not reported. This system,
therefore, lacks specificity and reported ADRs should be
checked to confirm the relation with drug administration.
In paediatric wards, most drugs are prescribed off-label,4
but there are no data on whether this approach is common
to primary care. Although in our study no drug was
unlicensed or prescribed off-label, our system could
address this issue. Concerns have been raised that with our
reporting system many trivial ADRs may be reported,
which might make detection of important reactions
difficult. Continuation of the study will give us information
to reassess the reporting criteria for all reactions and drugs.
1
2
3
4
Bonati M, Choonara I, Hoppu K, Pons G, Seyberth H. Closing the
gap in drug therapy. Lancet 1999; 353: 1625.
Impicciatore P, Pandolfini C, Bosetti C, Bonati M. Reazioni avverse
da farmaci in età pediatrica. Ital J Clin Pharmacol 1998; 12: 29–35.
Sutcliffe AG. Prescribing medicines for children. BMJ 1999; 319:
70–77.
Conroy S, Choonara I, Impicciatore P, et al. Survey of unlicensed
and off label drug use in paediatric wards in European countries.
BMJ 2000; 320: 79–82.
Department of Epidemiology and Biostatistics, Istituto Superiore
di Sanità, Viele Regina Elena 299, 00161 Rome, Italy
(F Menniti-Ippolito MSc, R Raschetti MSc, R Da Cas MSc); Department
of Paediatrics, University of Padova, Padova, Italy (C Giaquinto MD);
and Family Paediatrician, “Pedianet project”, Padova, Italy
(L Cantarutti MD)
Correspondence to: Ms Francesca Menniti-Ippolito
(e-mail: [email protected])
Detection of West Nile virus
sequences in cerebrospinal fluid
Thomas Briese, William G Glass, W Ian Lipkin
We have established a sensitive and specific real-time PCR
method for detection of West Nile virus. Analysis of
specimens obtained during the 1999 New York outbreak
indicated the presence of viral sequences in cerebrospinal
fluid of all of four individuals with fatal outcomes, and in only
one of four who survived.
Empirical antiherpesvirus therapy has reduced the
morbidity and mortality of encephalitis; however, the
advent of potent antiviral drugs with different targets, such
as enteroviruses, mandates a new focus on rapid pathogen
identification. PCR methods are widely used for detection
of herpesviruses and enteroviruses in cerebrospinal fluid
(CSF).1 At present, diagnosis of infection with West Nile
virus (WNV), an emerging flavivirus recently implicated in
outbreaks of fatal encephalitis in Europe, Asia, and North
America,2 is based on serology, virus isolation and
identification, or molecular analysis of brain materials in a
few reference laboratories. Thus, there is no opportunity
for early intervention with antiviral therapy.
Real-time PCR is a method whereby the presence of a
target sequence is detected by continuous measurement of
a fluorescent dye label generated during the course of
amplification.3,4 Nested PCR is similar in sensitivity to realtime PCR; however, the latter is quantitative over a wide
dynamic range, readily adapted to high throughput in a
clinical laboratory, and less sensitive to false positive results
because assays are done in a closed system. We established
a reverse transcription real-time 5⬘ nuclease PCR assay for
WNV NY1999 using an ABI Prism 7700 Sequence
Detector (PE-Biosystems, Foster City, CA, USA) and two
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Sample
code
Patient number Days after
(outcome)
onset
Serology
(IgM-EIA)*
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
1 (F)
1 (F)
1 (F)
2 (F)
3 (F)
4 (F)
5 (NF)
6 (NF)
7 (NF)
8 (NF)
Control
Control
Control
Control
Control
Control
Control
Control
16·68†
nd
nd
15·36
12·66
12·15
15·42
5·11
16·89
7·06
nd
nd
nd
nd
nd
nd
nd
nd
20
nk
nk
17
29
24
6
14
2
3
nk
nk
nk
nk
nk
nk
nk
nk
Molecules/reaction
prNS3
prNS5
5⫻103
3⫻104
2⫻104
<100
1·5⫻103
4⫻102
<100
<100
<100
<100
<100
<100
<100
<100
<100
<100
<100
<100
1⫻104
1⫻105
5⫻104
1⫻103
1·5⫻104
7⫻102
6⫻102
<100
<100
<100
<100
<100
<100
<100
<100
<100
<100
<100
F=fatal outcome. NF=non-fatal. nk=not known. nd=not done. *IgM-capture enzyme
immunoassay with WNV-Eg101 antigen. †Serological data was obtained for
corresponding serum specimen from same date and individual instead of CSF.
Reverse transcription 5⬘ nuclease PCR analysis of CSF from
New York encephalitis patients
primer/fluorescent-probe sets (TIB MOLBIOL, Freehold,
NJ, USA). One set was selected in the NS3 gene (set
prNS3: fwd, 5⬘-GCA CTG AGA GGA CTG CCC AT;
probe, 5⬘-6FAM-TAC CAG ACA TCC GCA GTG CCC
AGA-T-TAMRA; rev, 5⬘-TGG GTG AGG GTA GCA
TGA CA); the other in the NS5 gene (set prNS5:fwd, 5⬘GCT CCG CTG TCC CTG TGA; probe, 5⬘-6FAMTGG GTC CCT ACC GGA AGA ACC ACG T-TTAMRA; rev, 5-CAC TCT CCT CCT GCA TGG ATG).
Sensitivity of the assays for detection of NS3 or NS5 was
50–100 molecules with synthetic RNA transcripts
corresponding to nucleotides 4966–5438 and 9915–10082
of the WNV-NY1999 genome (hNY1999; GenBank Acc
No AF202541), respectively. Analysis of RNA extracted
from serially diluted virus-tissue culture seed showed a
linear dynamic range of more than five orders of magnitude
with a threshold below ten plaque-forming units.
Specificity for WNV-NY1999 was confirmed by NS3 and
NS5 sequences of St Louis encephalitis virus, the other
flavivirus of the Japanese encephalitis antigenic complex
associated with human disease in North America.
18 coded CSF specimens were obtained from the New
York State Department of Health for blinded analyses. Ten
samples were from individuals with confirmed WNVNY1999 infection;5 eight had neurological disease not due
to WNV. One third of the RNA extracted from individual
250 ␮L samples was reverse transcribed and analysed in
duplicate by real-time 5⬘ nuclease PCR. Seven of ten
samples from individuals with confirmed WNV-NY1999
infection (including three samples obtained at different
times from one individual) were positive for NS5
sequences; five of these were also positive for NS3
sequences (table). Three of the serologically confirmed
cases were negative by PCR for either gene sequence.
Whereas four of five individuals positive for WNV
sequences had fatal outcomes; the three negative by PCR
survived. The one surviving individual with detectable
WNV sequences in CSF (patient 5, table) was a 16-yearold male, the youngest patient with severe disease reported
during the outbreak (age of the other cases ranged from 54
to 87 years). No WNV sequences were detected in
controls. No correlation was found between PCR results
and either the duration of illness at the time of lumbar
puncture or the presence of antibody in CSF by IgMcapture enzyme immunoassay (table) or in sera by plaquereduction neutralising antibody assay (data not shown).
Serum samples from 24 confirmed cases and ten controls
THE LANCET • Vol 355 • May 6, 2000
RESEARCH LETTERS
obtained from the New York State Department of Health
were extracted and tested for the presence of WNVNY1999 sequences by real-time 5⬘ nuclease PCR. Five of
the serologically confirmed cases and none of the controls
were positive for WNV-NY1999 sequences (NS3, 3/5
patients; NS5, 5/5 patients; samples obtained 9–16 days
after onset of illness). Although it was not possible to
analyse matched sets of CSF and sera for all patients, one
of the individuals negative by real-time PCR of CSF was
positive by PCR for NS5 sequences in serum.
The establishment of a real-time PCR method for
detection of WNV sequences in human CSF improves
diagnosis of viral encephalitides. Although CSF containing
WNVs other than WNV-NY1999 were not available for
analysis, the primer/probe sets described here are predicted
to detect lineage I WNVs, viruses associated with outbreaks
of acute illness. Our results suggest that the detection of
WNV-NY1999 sequences in CSF correlates with a poor
prognosis particularly in older individuals. Further
investigation is needed to find whether this correlation can
be extended to other flavivirus encephalitides. As antiviral
research identifies drugs with activity against WNV, the
ability to rapidly implicate this virus is anticipated to
achieve clinical importance similar to that associated with
herpesviral or enteroviral diseases.
We are grateful to Leo Grady, Cinnia Huang, and Susan Wong of the
New York State Department of Health for providing CSF and serum
samples, Rob Lanciotti and John Roehrig from the Division of VectorBorne Infectious Diseases, CDC, for WNV extracts, and clinical and
serological results, and Charles Calisher and Ingo Jordan for helpful
discussions.
1
2
3
4
5
Jeffery KJM, Read SJ, Peto TEA, Mayon-White RT, Bangham RM.
Diagnosis of viral infections of the central nervous system: clinical
interpretation of PCR results. Lancet 1997; 349: 313–17.
Asnis D, Conetta R, Waldmon G, et al. Outbreak of West Nile-like
viral encephalitis: New York 1999. MMWR Morb Mortal Wkly Rep
1999; 48: 845–49.
Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR
analysis: real-time monitoring of DNA amplification reactions.
Bio/Technology 1993; 11: 1026–30.
Lee LG, Connell CR, Bloch W. Allelic discrimination by nicktranslation PCR with fluorogenic probes. Nucleic Acids Res 1993; 21:
3761–66.
CDC. Case definitions for infectious conditions under public health
surveillance. MMWR Morb Mortal Wkly Rep 1997; 46: 1–55.
Emerging Diseases Laboratory, Departments of Neurology,
Microbiology and Molecular Genetics, Anatomy, and
Neurobiology, University of California, Irvine, CA 92697-4292,
USA (T Briese PhD, W G Glass BS, Prof W I Lipkin MD)
Correspondence to: Dr T Briese
(e-mail: [email protected])
Pharmacogenetic prediction of
clozapine response
M J Arranz, J Munro, J Birkett, A Bolonna, D Mancama, M Sodhi,
K P Lesch, J F W Meyer, P Sham, D A Collier, R M Murray,
R W Kerwin
We did association studies in multiple candidate genes to find
the combination of polymorphisms that give the best predictive
value of response to clozapine in schizophrenic patients. A
combination of six polymorphisms in neurotransmitter-receptorrelated genes resulted in 76·7% success in the prediction of
clozapine response (p=0·0001) and a sensitivity of 95% (±0·04)
for satisfactory response. These results will form the basis for a
simple test to enhance the usefulness of clozapine in psychiatric
treatment.
The atypical antipsychotic clozapine was reintroduced into the
UK and USA in 1990, following the demonstration of
THE LANCET • Vol 355 • May 6, 2000
Gene
Polymorphism
Detection method
(restriction enzymes/
electrophoresis conditions)
Adrenergic receptors
␣2A
␣2A
␣1A
⫺1291–C/G
⫺261–G/A
Arg492Cys
MspI/10% PAGE
HhaI/10% PAGE
PstI/3% agarose
Dopamine receptor
D3
Ser9Gly
MscI/3% agarose
Serotonin receptors
5–HT2A
5–HT2A
5–HT2A
5–HT2A
5–HT2A
5–HT2C
5–HT2C
5–HT3A
5–HT3A
5–HT5A
5–HT5A
⫺1438–G/A
102–T/C
516–C/T
His452Tyr
Thr25Asp
Cys23Ser
⫺330–GT/⫺244–CT repeat
178–C/T
1596–G/A
12–A/T
⫺19–G/C
MspI/2% agarose
MspI/2% agarose
Sau96I/2% agarose
BbvI/3% agarose
BstNI/2% agarose
HinfI/4% agarose
10% PAGE
10% PAGE/5% glycerol
NheI/3% agarose
BsrI/2% agarose
BsaJI/10% PAGE
Serotonin transporters
Transporter 5–HTT
VNTR
Transporter promoter 5–HTT 5–HTTLPR
4% agarose
4% agarose
Histamine
H1
H2
BsmI/10% PAGE
HaeIII/10% PAGE
Leu449Ser
⫺1018–G/A
PAGE=Polyacrylamidegel electrophoresis.
Table 1: List of polymorphisms studied and detection methods
for the identification of the alleles
superior efficacy and tolerability in severely treatment-resistant
patients.1 Since then a range of well-tolerated atypical
antipsychotics have been introduced.2 Although the evidence
base for the use of these drugs is compelling, various reasons
seem to prevent use in greater numbers of eligible patients.
The treatment costs are greater than with classic
antipsychotics and the response to these drugs is
heterogeneous, with between 30% and 60% responding to
clozapine, the archetypal atypical antipsychotic.
We have attempted to explore new targets by studying
pharmacogenetic associations in a large number of patients
treated with clozapine. We have previously shown that allelic
variation in the serotonin neurotransmitter receptor 2A gene
(5–HT2A) is a factor in determining clinical response to
clozapine.3,4 However, 5–HT2A polymorphisms on their own
cannot fully explain the variability seen in treatment response,
and it has been postulated that there are contributions from
other mutations in neurotransmitter-receptor-related genes.
We did association studies in multiple candidate genes to
find the combination of polymorphisms that give the best
Polymorphism
p for genotype
p for allele
⫺1291–C/G (ADR2A)
⫺261–G/A (ADR2A)
Arg492Cys (ADR1A)
Ser9Gly (D3)
Leu449Ser (H1)
⫺1010–G/A (H2)
His452Tyr (5–HT2A)
Thr25ASP (5–HT2A)
⫺1438–G/A (5–HT2A)
102–T/C (5–HT2A)
516–C/T (5–HT2A)
⫺330–GT/⫺244–CT (5–HT2C)
Cys23Ser (5–HT2C)
178–C/T (5–HT3A)
1596–G/A (5–HT3A)
⫺12–A/T (5–HT5A)
⫺19–G/C (5–HT5A)
5–HTTLPR
VNTR (5–HTT)
0·85
0·54
0·22
0·89
0·30
0·08
0·01*
0·57
<0·001
<0·001
0·65
0·04†
0·08‡
0·92
0·98
0·20
0·55
0·04
0·89
0·61
0·40
0·10
0·63
0·35
0·43
0·02
0·78
0·001
0·001
0·82
0·31
0·17
0·79
0·85
0·09
0·35
0·36
0·70
*Try recessive: 0·004. †Short allele dominant. ‡Ser23 dominant.
Table 2: Comparison of responders vs non-responders for the
19 polymorphisms genotyped
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