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Journal of Clinical Microbiology, July 1998, p. 1895-1901, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Algorithmic Approach to High-Throughput Molecular Screening for
Alpha Interferon-Resistant Genotypes in Hepatitis C Patients
Srinand
Sreevatsan,1
Jack B.
Bookout,1
Fidel M.
Ringpis,1
Mridula R.
Pottathil,1
David J.
Marshall,2
Monika
De
Arruda,2
Christopher
Murvine,2
Lance
Fors,2
Raveendran M.
Pottathil,3 and
Raj R.
Barathur1,*
Center for Innovative Technologies, ClinCyte,
San Diego, California 921211;
Third Wave
Technologies, Madison, Wisconsin 537192; and
AccuDx, San Diego, California 921263
Received 17 September 1997/Returned for modification 28 November
1997/Accepted 2 April 1998
 |
ABSTRACT |
This study was designed to analyze the feasibility and validity of
using Cleavase Fragment Length Polymorphism (CFLP) analysis as an
alternative to DNA sequencing for high-throughput screening of
hepatitis C virus (HCV) genotypes in a high-volume molecular pathology
laboratory setting. By using a 244-bp amplicon from the 5' untranslated
region of the HCV genome, 61 clinical samples received for HCV reverse
transcription-PCR (RT-PCR) were genotyped by this method. The genotype
frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2%
for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by
nucleotide sequence analysis provided 100% concordance with
those obtained by CFLP analysis at the major genotype level, with
resolvable differences as to subtype designations for five samples.
CFLP analysis-derived HCV genotype frequencies also concurred with
the national estimates (N. N. Zein et al., Ann. Intern. Med.
125:634-639, 1996). Reanalysis of 42 of these samples in parallel in a
different research laboratory reproduced the CFLP fingerprints for
100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation
temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay,
CFLP analysis, and automated DNA sequencing indicated that the average
cost per amplicon was lowest for CFLP analysis, at $20 (direct costs).
On the basis of these findings we propose that CFLP analysis
is a robust, sensitive, specific, and an economical method for
large-scale screening of HCV-infected patients for alpha
interferon-resistant HCV genotypes. The paper describes an algorithm
that uses as a reflex test the
RT-PCR-based qualitative screening of samples for HCV
detection and also addresses genotypes that are ambiguous.
| |
INTRODUCTION |
Characterization of hepatitis
C virus (HCV), the primary cause of transfusion-associated and
community-acquired non-A, non-B hepatitis, has been refined by the
development of a battery of successful molecular biology-based methods
that probe for viral traits at the genotype level (1, 5, 7, 8,
12-14, 21-24, 26). Correlations have also been found between
HCV genotyping and severity of disease, rate of disease progression,
and response to therapy (21).
Hepatitis C is caused by a positive-strand RNA virus which has a high
degree of sequence homology to members of the families Pestiviridae and Flaviviridae. The viral genome
is 9,425 bp in length and appears to code for a single polyprotein that
is subsequently cleaved into a series of structural proteins and
nonstructural peptides with presumed enzymatic roles in virus
replication (11, 20, 21). HCV becomes chronically
established in 70 to 90% of the patients and 20 to 30% of patients
progress on to cirrhosis (2, 21, 26). The most effective
therapeutic approach against HCV infection is long-term treatment with
alpha interferon (IFN-
). The success rate with interferon is only
about 25% (21). IFN- therapy is very expensive and
lengthy (6 to 18 months of treatment time costing between $12,000 and
$15,000 per patient). Also, infection caused by type 1b is more
resistant to IFN- therapy (3, 12, 18, 21, 24, 28) than
infection caused by other genotypes, genotype 1a, 2b, or 2c.
Furthermore, there is evidence that associates certain genotypes of HCV
with more severe hepatic pathology or quicker progression to
chronicity. These data underscore the need to identify HCV strains at
the genotype level to institute potentially beneficial therapeutic or
other intervention strategies.
Various molecular and serological methods (26) for
classifying HCV samples into one of the known genotypes (genotypes 1a, 1b, 2b, 2c, 3a, 4a, 5b, and 6 to 8) are available. The results of
serological genotyping methods based on the NS-4 protein have shown a
high degree of concordance with those of sequencing but are limited to
the identification of only major types (13, 24). Among the
molecular or nucleic acid-based genotyping assays of virus are the commercially available line probe assay (LiPA;
Innogenetics, Ghent, Belgium) (3, 6, 12-14, 21-25) and
automated DNA sequencing of various regions of the HCV genome. Many
approaches to genotyping target the 5' untranslated region (5' UTR) of
the genome because it is highly conserved (6% sequence divergence
between genotypes), and consequently, use of this target has the
advantage of allowing a high success rate in an amplification reaction
(21), thereby simplifying meaningful phylogenetic analysis.
Recently, a structure-specific endonuclease (Cleavase I; Third
Wave Technologies, Inc., Madison, Wis.) has been identified (4). This method, termed Cleavase Fragment Length
Polymorphism (CFLP) analysis, relies on the ability of the enzyme
Cleavase I to recognize and cleave DNA based on structure rather than
sequence. Specifically, the Cleavase I enzyme recognizes the junctions
between single-stranded and duplexed regions of DNA, such as
those that occur at the base of a hairpin, and cleaves on the 5'
side of the first paired base. These secondary structures can be
formed by brief thermal denaturation of double-stranded DNA, followed by rapid cooling. Since these structures are a reflection of the sequence composition, the cleavage patterns produced from Cleavase I
enzyme digestion can identify, with high degrees of sensitivity and
specificity, changes in the DNA sequence (4, 14, 19). In
this study, we compare this new structural fingerprinting technology with DNA sequencing for analysis of HCV-positive clinical samples. Through examination of the 5' UTR of the HCV genome by both methods, we
show that the CFLP screening and fingerprinting technique is simple, rapid, reliable, and cost-effective for routine use in a
high-throughput molecular pathology laboratory.
| |
MATERIALS AND METHODS |
Clinical specimens.
Seventy-two serum samples which were
defined by reverse transcription-PCR (RT-PCR) as having detectable
levels of HCV RNA were obtained from LabCorp (Center for Molecular
Biology and Pathology, Research Triangle Park, N.C.). The source,
patient information, and other demographic attributes were blinded for
the purposes of this study and to maintain confidentiality.
RNA extraction and RT-PCR.
RNA was extracted from
approximately 100 µl of serum with a commercially available
extraction kit (Puregene; RNA extraction systems; Gentra Systems,
Minneapolis, Minn.) and was resuspended in 30 µl of RNase-free
distilled water. Approximately, 1 to 3 µl of RNA extracts was used to
amplify a 244-bp fragment of the 5' UTR with the Amplicor HCV
amplification kit (Roche Molecular Systems, Branchburg, N.J.).
Amplicons generated by this method contain a biotin label on the 5' end
of the antisense strand. All protocols were performed as per the
manufacturer's recommendations.
CFLP structural fingerprint analysis.
The Cleavase I enzyme
(Third Wave Technologies), a structure-specific endonuclease, was used
to generate cleavage patterns for all amplicons as described previously
(4), with a few modifications. Briefly, amplicons were
purified with spin columns (High Pure PCR product purification kit;
Boehringer Mannheim, Indianapolis, Ind.) to remove residual
uracil-N-glycosylase and primers present in the
amplification master mixture. Subsequently, the recovered amplicons
were treated with exonuclease I (United States Biochemical, Cleveland,
Ohio) in the presence of 10× PCR buffer I with MgCl2 (Perkin-Elmer, Branchburg, N.J.) to remove residual primers and truncated single-stranded amplification products. The excess
exonuclease and salts were removed with a second spin column
purification step, and DNA was eluted in 70 µl of autoclaved
distilled (DI) H2O. Purified amplification products were
treated with the Cleavase I enzyme as described previously
(4).
CFLP patterns are the result of partial digestion with the Cleavase I
enzyme. Therefore, the optimal conditions that provide the most
representative distribution of partial digestion bands while still
maintaining an adequate amount of undigested (uncut) amplicon must be
defined. All conditions for the Cleavase I reaction were optimized by
using amplicons from two different patients in a checkerboard format of
various temperatures and incubation times. Approximately 25 to 30 ng of
the amplicons was reconstituted in DI H2O to a final volume
of 10 µl and heated to 95°C for 15 s, after which the
temperature was dropped to 45, 50, or 55°C as the digestion condition
to be tested required. Ten microliters of a master mixture (containing
2 mM MnCl2 [2 µl], 2 µl of 10× CFLP buffer (10 mM
morpholinepropanesulfonic acid {MOPS; pH 7.5}; 0.5% Tween 20, 0.5% Nonidet P-40], and 25 U of Cleavase I [1 µl] and DI
H2O [5 µl]) was added to each tube, and the tubes were incubated at the respective temperatures for either 4 or 6 min. Optimal
cleavage occurred when amplicons were incubated with the master mixture
at 55°C for 4 min. All samples in this study were analyzed at this
incubation temperature and time.
Cleaved products were electrophoresed on a 10% denaturing acrylamide
gel (20 by 20 by 0.5 cm) containing 7 M urea and were
transferred onto
a 0.2-µm-pore-size nylon membrane (Nytran Plus;
Schleicher & Schuell,
Keene, N.H.) by dry blotting overnight.
The membranes were baked at
120°C for 15 min and blocked in 1%
blocking agent (Boehringer
Mannheim) for 90 min. Streptavidin-
alkaline phosphatase conjugate
(Sigma Chemical Co., St. Louis,
Mo.) at a 1:20,000 dilution in 1%
blocking buffer was used as
a secondary detection agent. The membranes
were washed three times
(for 10 min each time) with 0.1% sodium
dodecyl sulfate in Genius
Buffer I (0.3 M NaCl, 0.2 M Tris-HCl [pH
7.5]) followed by three
washes (for 5 min each time) with Genius
buffer III (Boehringer
Mannheim). Five milliliters of CDP star (Tropix
Inc., Bedford,
Mass.) was applied to each membrane as a
chemiluminiscent substrate
in the detection system. The membranes were
carefully wrapped
in clean Saran wrap without drying and were exposed
to an X-OMAT
(Kodak, Rochester, N.Y.) radiographic film for 1 to
2 h and developed
in an automatic film developer (Konika QX-130A).
All cleavage patterns analyzed were compared to the fingerprints
generated from samples that were infected with known genotypes
and that
were available in our laboratory. Genotypes were identified
for each
sample independently by three researchers (D.J.M., S.S.,
and J.B.B.).
The cleavage patterns were also generated with the
same set of samples
in another laboratory independently, and the
genotype fingerprint
patterns were correlated. Additionally, two
different lots of the
Cleavase I enzyme and reagents were evaluated
to assess lot-to-lot
variance.
Automated DNA sequencing.
Forty-two amplicons were DNA
sequenced by the dideoxy terminator cycle sequencing method with
Taq FS (Applied Biosystems Inc., Foster City, Calif.) on an
automated 377A sequencing instrument (Applied Biosystems Inc.).
Sequences from both strands were assembled with the SEQMAN and MEGALIGN
software (DNASTAR, Madison, Wis.). The data generated were compared
with the CFLP fingerprints of each isolate. DNA sequence data were used
as a "gold standard" for comparisons.
Comparative cost analysis.
Direct costs for performing CFLP,
LiPA (Innogenetics), and automated DNA sequencing of the amplicons were
calculated on the basis of a full load analysis (24 samples) for each
assay with 2.5 technicians (Table 1). Assumptions and the
costs that were itemized are described in Table 1.
| |
RESULTS |
Genotyping by CFLP analysis.
Of the 72 samples evaluated in
this study, 11 (15.3%) could not be genotyped by either CFLP analysis
or sequencing because of the availability of suboptimal
amounts of RNA for adequate generation of cDNA and subsequent
amplification. The remaining 61 amplicons, when analyzed by CFLP
analysis (Fig. 1; Table
2), generated patterns consistent with
genotype 1 in 49 (80.3%) samples compared to the patterns from
samples with known genotypes. Twenty-seven of 61 (44.3%) samples were
typed as 1a. Sixteen samples (26.2%) were typed as 1b. Five samples
had banding patterns which were shifted in size from type 1 only in the
50- and 100-bp region and were identified as 1ab variants (Fig. 1). In
addition, one sample resulted in a poor (or marginal) yield of DNA,
precluding analysis of discriminator bands for subtype determination
but yielding enough of a structural fingerprint to determine that it
belonged to genotype 1. Eight (13.1%) samples were found to be type 2, while three (5%) samples were typed as type 3. One sample (with <500
copies/ml) gave a pattern that was not represented among reference
samples.
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FIG. 1.
HCV genotyping with Cleavase fragment polymorphisms. A
representative chemiluminescent blot of HCV genotyping by CFLP analysis
shows molecular mass markers (lane 1), CFLP analysis of 5' UTRs for
clinical isolates of HCV (unnumbered lanes 2 to 27; the numbers above
each lane denote isolate identifications), and an uncut amplicon
electrophoresed on a 10% denaturing acrylamide gel. Also shown at the
bottom of the gel are the genotype for each fingerprint (1, mixed
infection by sequencing; v, variant within each subtype identified by
sequencing and CFLP analysis). For comparisons, previously established
genotype fingerprints (14) were used. Samples D2, F3, and X
had established genotype fingerprints prior to this study and had been
sequenced and were included as representative of types 2b, 3a, and 1b
patterns, respectively.
|
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TABLE 1.
Comparative cost analysis (direct costs) of
LiPA,a CFLP analysis, and automated DNA
sequencing for genotyping of HCV isolatesb
|
|
Reliability and precision of CFLP analysis.
CFLP reanalysis of
19 amplicons in different batches reproduced identical fingerprints.
Samples analyzed as many as six times produced identical fingerprints
on each analysis. Cross matching of data generated in two different
laboratories for 42 samples also produced identical CFLP fingerprints
for each isolate. Specimens for this correlation study also
included samples exchanged between the two laboratories as sera
(n = 5), RNA preparations from live specimens
(n = 42), and amplified products (n = 10). Additionally, analysis of a set of 19 amplicons cleaved under
different conditions (45°C for 4 min or 55°C for 4 min) yielded
identical patterns.
Correlation of genotype fingerprints with DNA sequences.
The
5' UTR regions of 42 amplicons from the set described above were
sequenced. Sequence information for all samples analyzed was identical
to previously reported 5' UTR nucleotide sequences (23),
with five exceptions (Fig. 2). Complete
agreement between CFLP analysis and sequence analysis was defined for
92.5% specimens at the subtype level.
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FIG. 2.
5' UTR nucleotide sequence alignments of common HCV
subtypes identified (19) (M, A or C; R, A or G; W, A or T;
Y, C or T). The periods between nucleotides  137 and 138 in most
sequences correspond to the insertion in some type 1 and 4 samples
(19; this study). Also shown are the sequences of
clinical samples from this study with variations in nucleotides 94
and 99 that resulted in changes in their CFLP banding patterns.
|
|
Samples 1, 22, 23, 41, and 63 were clearly defined as type 1 strains by
either CFLP analysis or DNA sequencing. However, there
were
interpretive differences between the two techniques regarding
subtype
designations for these samples due to the presence of
mixed signals at
key positions indicative of type 1a or 1b. This
ambiguity centers
around two possible base substitutions: an A-to-G
change at nucleotide
99 and a T-to-C polymorphism at nucleotide
94. The
94
substitution may exist alone or in combination with
the
99 change.
While a change to a G at position
99 is indicative
of type 1b for
>90% of strains, the subtype association dictated
by the
94
polymorphism can be used only to establish type 1a
or b (1ab)
(
18). Sample 41 (Fig.
2) is one such variant; it
contains a
C at position
94, suggesting that it can be classified
only as
type 1ab.
Analysis of the sequence chromatograms indicated that samples 1 and 22 both exhibited mixed peaks at position
99, consisting
of G and A
signals, indicating the presence of viruses of types
1a and 1b. In
addition, sample 22 had a mixed peak at position
94 (C and T
signals). Inspection of relevant sample lanes demonstrates
that the
CFLP structural fingerprints generated from these samples
are
consistent with the presence of these mixed types. Similarly,
samples
23 and 63 all had an A at position
99, indicative of
type 1a (Fig.
2). However, these two samples had mixed peaks at
position
94 that
suggest the presence of an additional viral
population that is of
indeterminate subtype 1ab. In addition,
sample 63 had an A at position
138. Again, the structural fingerprints
(Fig.
1) reflect these
composite sequence changes.
While subtyping of these mixed samples by either sequencing or CFLP
analysis is complicated by the presence of indeterminate
alleles at
positions
94 and/or
99, these base changes are reflected
in the
CFLP structural fingerprints generated by these samples.
Therefore,
CFLP analysis identified simple base substitutions
or mixtures of bases
at positions
94 and
99 accurately. When
analyzed for major types
alone, there was a 100% concordance between
sequence information and
the results of CFLP analysis.
| |
DISCUSSION |
In this investigation we examined the feasibility and
validity of a new HCV genotyping method using a structure-specific
endonuclease. The method has been used to type various genes including
analysis of bacterial 16S rRNA for epidemiological analysis and
katG of Mycobacterium tuberculosis to identify
isoniazid resistance (4) as well as mutations in
CFTR, COL4A5, and FGFR3 genes
(19).
Analysis of a set of clinically positive HCV samples with the CFLP
technology identified that the method was accurate on the basis of the
good concordance of CFLP fingerprinting results with those of automated
DNA sequencing. Additionally, the method was repeatable, with no
variations in CFLP fingerprints between assays, with different lots of
Cleavase I enzyme or associated reagents, or by execution by two
different laboratories. Minor variations in the banding patterns
identified for samples from five patients could not be correlated with
a single reference fingerprint. Careful inspection of the
sequence chromatograms confirmed the presence of double peaks at
key bases, indicating the presence of a mixed sample, although only the
major peak was identified by the base-calling software. It has been
suggested that in the event of the concurrent presence of a mixture of
genotypes in the patient, minor genotypes (representing <10% of
the circulating viral population) are missed by automated DNA
sequencing (13, 21). Similarly, CFLP analysis may not be
able to differentiate infection with a variant from a true mixed
infection. However, CFLP analysis may be equivalent or superior to
automated DNA sequencing (14a) because of its ability to
identify single-base-pair substitutions with a high degree of
sensitivity as reflected by a shift in the banding pattern.
Clinical and biological attributes of HCV genotyping.
The
importance of HCV genotyping is to provide understanding
of the association of particular genotypes with a higher
rate of progression of HCV infection to cirrhosis or hepatocellular carcinoma and in delineating differences in response to IFN- therapy. Although HCV genotyping is still considered a research tool
(9), its use in defining type and subtype relationships with
liver disease may eventually aid in establishing the clinical basis for
meaningful, successful therapeutic or other intervention strategies.
Various studies have documented a correlation between genotype
and viral load as well as the response to IFN- therapy (3,
12-14, 21, 24, 28). In particular, a study involving 139 chronic
HCV patients identified that HCV type 2 infection was associated with
greater histologic activity in the liver but had what appeared to
be lower serum HCV RNA levels, whereas type 3 was associated with lower
serum alanine aminotransferase levels (24). These
findings corroborate those of other studies which have demonstrated
that type 2 was associated with more aggressive liver disease
(3, 18). Another study on viral load and response to
IFN- therapy demonstrated that patients infected with type 1 had a higher viral load than patients infected with type 2 and were
less responsive to IFN- therapy (24).
However, some investigators have recently noted biases in
commercially available amplification assays which result in
reduced
quantitative accuracy for various subtypes demonstrating
89 to
92% reduced sensitivity for the detection of
genotypes 2 and 3
compared to genotype 1 by three different
quantitative assays
(
9,
10). Such biases may result in
misinterpretations of
viral loads and even compromise genotype
identification in mixed
subtype infections. Given this limitation of
viral quantitation,
we believe that HCV genotyping by a method such
as CFLP analysis
would identify heterogeneous populations of viral
subtypes (as
in mixed infections identified in this study for sample 63 and
other 1ab variants). The CFLP assay may also have potential as
a
screening tool for investigating genotype associations with
disease
correlates (e.g., severity) and possibly even in determining
genetic
complexity and diversity in infections as demonstrated
by Polyak et al.
(
17) by the analysis of the HVR1 region by
gel shift
analysis.
Comparisons with other HCV detection and/or genotyping assays.
Numerous nucleic acid-based assays are available for HCV genotyping.
Among these are restriction fragment length polymorphism analysis of
the 5' UTR (7) that is able to discriminate types 1a, 1b,
2a, 3a, 3b, 4, 5, and 6, the allele-specific oligonucleotide analysis
such as the LiPA (Innogenetics) that can identify these genotypes, in
addition to types 2b, 2a/2c, 2d, and 3c-f (11, 12, 19, 20),
and a group-specific PCR of the sequence within the core region
(15, 27). The results of these assays and other serotyping
methods (based on the NS-4 region) have shown a high concordance
to each other and to those of DNA sequencing (13, 24). The
group-specific PCR has been demonstrated to have a sensitivity of
88.8% (15). On the other hand, an analysis of serological
responses as determined by three different immunoassays (RIBA-4 [Ortho
Diagnostics, Raritan, N.J.], enzyme immunoassay [Abbott Laboratories,
Chicago, Ill.], and Inno-EIA [Innogenetics]) and LiPA (Innogenetics)
failed to demonstrate any strict correlation between genotypes and
anti-HCV responses (25). Although LiPA is adapted to a
convenient plate-tray format and can be performed on an
instrument, some deficiencies have been identified. First, the
probe-based assays are limited in terms of the number of types and
subtypes that they can identify and would be difficult to apply in
areas where different genotypes predominate, such as in the Middle East
and south and southeast Asia (8). Second, due to the high
level of stringency required to perform these assays, they are
laborious and limiting for adaptation in a high-throughput laboratory.
Third, they are more expensive (Table 1)
to perform as either a routine or a reflex genotyping strategy for HCV
patients. An ideal genotyping assay will need to be inexpensive,
reliable (repeatable and valid), and easy to use. CFLP analysis, in our experience, has been found to be simple and applicable to a
high-throughput laboratory. In addition, the assay is robust because it
performs under different conditions (different temperatures and times
of incubation) and can be used in low- and high-throughput
laboratories. The assay can use amplicons generated by the Amplicor HCV
detection kits (Roche Molecular Systems) or other noncommercial
protocols as long as there is basic homogeneity within the amplified
product.
Selection and interpretation of diagnostic tests.
Currently,
diagnosis of HCV infection is based upon the ability to detect specific
antibody by enzyme immunoassay and strip immunoblot assays that use
recombinant HCV antigens. Positive results by both antibody assays
indicate a high likelihood of HCV infection in the patient. Hepatic
function is monitored by determination of liver enzyme levels, and a
liver biopsy is indicated when the clinical state is aggravated.
Qualitative amplification assays for the detection of HCV RNA are
indicated to demonstrate actual infection and for assessment of viral
clearance. The molecular tests (branched-chain DNA [Chiron Corp.] and
RT-PCR [Roche Molecular Systems]) that detect HCV RNA or measure
viral load have been used to predict the response to treatment
with IFN- and to monitor antiviral therapy (11-13, 18, 21, 24,
26). On the basis of these practices and the difficulty in
identifying HCV genotypes (which may be important when placing
patients on expensive IFN- therapy), we propose the use of a
simplified algorithm to identify and genotype HCV (Fig.
3) by a single reliable assay. The
current standard of practice calls for qualitative detection of HCV RNA followed by a separate sequencing protocol. This protocol is expensive and may require two separate amplifications. The proposed CFLP analysis
protocol and the algorithm require a single amplification. However, if
an internal control is used for PCR detection (as is the case with the
Roche Amplicor HCV monitor kit), CFLP analysis will also require a
second amplification without the addition of the internal control. Once
the viral threshold is confirmed, the amplicons from the same sample
are processed for CFLP analysis and fingerprints of the genotypes are
determined. We also recommend, on the basis of the results of our
studies, that those samples with ambiguous banding patterns or those
that are untypeable by CFLP analysis, e.g., due to the occurrence of an
unusual subtype, be reflexed to a sequencing protocol. As seen in this
study, only 1 of 61 amplicons was not typeable through comparison to
our known reference standards. It is expected that in routine use, a
similar number of samples would require additional testing as the
patterns for rare and new genotypes are established.
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FIG. 3.
Broad schematic of a proposed algorithmic approach for
the identification and genotyping of HCV. ALT, alanine
aminotransferase; EIA, enzyme immunoassay.
|
|
In our analysis, the frequency of genotypes identified by CFLP analysis
of the clinical samples concur with the national estimates
(
1,
5,
8,
11-13,
28). The slight underrepresentation
of type 1a in our
data probably reflects a sampling bias. However,
the sampling frame for
this study was a clinical reference laboratory
with acquisitions from
all 50 states of the United States. Alternately,
the underestimate of
type 1a may be due to the fairly small sample
size used in this
investigation. This is evident by the fact that
this investigation did
not find type 4, 5, or 6 in the analysis,
as may be predicted due to
their low prevalence nationwide. However,
CFLP fingerprints for types 4 and 5 from other sources have been
defined by the principal
investigator (data not shown) and formed
a part of the library for
comparisons.
In conclusion, HCV genotyping by CFLP analysis is accurate, rapid,
repeatable, and economical and can be applied in a high-volume
molecular pathology laboratory. This investigation validates and
extends the previous finding on the use of CFLP analysis for various
other molecular genetic analyses for the typing of microorganisms
or
for early disease marker identification in genetic diseases
of humans
(
4,
13,
16).
| |
ACKNOWLEDGMENTS |
We acknowledge the following personnel at Third Wave
Technologies: Mary Oldenburg and Bruce Neri for assistance on technical matters related to CFLP analysis and Laura Heisler and Mary Ann Brow
for advice and critique of the manuscript. The technical assistance
provided by San Duo Xu (ClinCyte, Center for Innovative Technologies)
is also appreciated. We also thank LabCorp, Center for Molecular
Biology and Pathology) for providing us with the HCV samples.
A portion of this project was funded by a grant from the NIH, under the
SBIR program (GM51704-03) awarded to Third Wave Technologies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for
Innovative Technologies, ClinCyte, 5627 Oberlin Dr., Suite 120, San
Diego, CA 92121. Phone: (619) 455-1221, ext. 3626. Fax: (619) 457-1827. E-mail: RajBarathur{at}worldnet.att.net.
| |
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Journal of Clinical Microbiology, July 1998, p. 1895-1901, Vol. 36, No. 7
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
