Received 13 November 1997/Returned for modification 22 December
1997/Accepted 29 June 1998
We investigated the unrecognized patient-to-patient transmission of
hepatitis C virus (HCV) in hemodialysis units by performing phylogenetic and serological analyses of hypervariable region 1 (HVR1)
of HCV. Of the 62 patients in one center, 11 were positive for HCV RNA.
A total of 24 HVR1 sequences, including the minor population of
sequences of HCV isolates, from each patient were closely related and
classified into five clusters by phylogenetic analysis. Of the 11 patients, 5 were infected with multiple clusters of HCV. Two patients
were infected with HCV during an 18-month interval between
examinations, and these HVR1 sequences fell into one of the five
clusters. In another hemodialysis center, 5 of the 20 patients were HCV
RNA positive, and two HVR1 sequences were found to be closely related
and phylogenetically derived from the same cluster. The antibody
responses of these patients to the HVR1 peptides representative of the
genetic clusters revealed exactly the same clustering as that shown by
phylogenetic analysis. These findings suggest that phylogenetic and
serological analyses of HVR1 sensitively detect unrecognized and
multiple transmission of HCV occurring within the same room in
hemodialysis centers. Fingerprinting analyses using hypervariable
regions of infectious agents are useful in identifying the precise
route of transmission of infection.
 |
INTRODUCTION |
Hepatitis C virus (HCV) is a major
causative agent of posttransfusion non-A, non-B hepatitis. Screening
and confirmatory assays for circulating antibodies to HCV became
available (10, 27) after the molecular cloning of the HCV
genome in 1989 (3). The second-generation enzyme immunoassay
for detection of anti-HCV antibody has revealed a high prevalence of
antibodies to HCV in hemodialysis (HD) patients (5). Most
cases of HCV infection in HD patients are thought to be related to
blood transfusions, but several reports from different parts of the
world have also shown the presence of HCV infection in nontransfused HD
patients as well (8, 22, 30), and the anti-HCV
antibody-positive rates have been found to increase with the duration
of dialysis (8, 22, 30). This suggests that nosocomial
transmission of HCV occurs in HD units. Recently, patient-to-patient
transmission of HCV in HD units has been demonstrated by molecular
biology techniques, but the frequency of transmission was low (2,
20, 25). In these studies the sequences of HCV dominantly
propagating in patients were determined and compared. Allander et al.
(2) were the first to detect nucleotide sequence similarity
in variable parts of the HCV genome in several HD patients. Sampietro
et al. (20) and Stuyver et al. (25) found a rare
HCV variant in several patients treated in the same HD unit by
sequencing the relatively conserved region of the HCV genome, the 5'
untranslated region (5'-UTR), and the core region, respectively.
However, no evidence of transmission had ever been demonstrated by
sequence analysis of a minor population of HCV isolates.
The HCV genome exhibits different variability of nucleotide sequences
in different regions. There is a hypervariable region, hypervariable
region 1 (HVR1), in the putative second envelope glycoprotein (E2) of
the HCV genome (7). HVR1 consists of 27 amino acid residues
located in the N-terminal region of E2 and is the most variable region
in the HCV genome. Accordingly, it appeared that HVR1 might be useful
for discriminating HCVs the same as a polymorphic marker for genetic
fingerprinting and for detecting nosocomial transmission of HCV. It was
thought that not only rare but also common genotypes of HCV might be
found to be transmitted. Furthermore, most patients with chronic
hepatitis C possess antibody against the HVR1 of their own isolates
(9, 29), and anti-HVR1 antibody was also thought to be
useful for demonstrating nosocomial transmission. In this study, we
examined HCV transmission in HD units by performing phylogenetic
analysis of HCV HVR1 sequences and testing for antibodies to HVR1
peptides. We also analyzed multiple sequences of HVR1 from each
individual and investigated nosocomial transmission of HCV, including a
minor population of HCV isolates transmitted in HD patients.
| |
MATERIALS AND METHODS |
Patients.
We studied patients attending two dialysis
centers. In one dialysis center, 62 patients were examined for HCV RNA
twice, once in October 1994 and again in April 1996, by a two-step PCR
amplifying the 5'-UTR. Nine patients were positive for HCV RNA the
first time they were examined and two additional patients were found to
be positive the second time. A total of 20 samples of patient serum
were used for the sequence analysis of HCV HVR1 and the assay for
anti-HVR1 antibody. Serial samples of serum from the patients were
tested for alanine aminotransferase (ALT) and anti-HCV antibodies with
a second-generation enzyme-linked immunosorbent assay (ELISA) (ELISA
II; Ortho Diagnostic Systems, Tokyo, Japan). In the other center, 5 of
the 20 patients were found to be positive for HCV, and their HCV HVR1
sequences were then examined. The HCV isolates from all patients were
genotype 1b as determined by the SMI TEST HCV-Genotype (Sumitomo Metal
Industries, Tokyo, Japan). All dialysis machines in each center were
located in a single room. All patients were hemodialyzed against
standard bicarbonate dialysate three times weekly, for 4 h each
time. Dialysate was delivered from the central station, and the types
of dialysis membranes used were regenerated cellulose membrane
(AM-FP-15; Asahi Medical Co., Tokyo, Japan), polysulfone membrane
(PS-1.3UW; Fresenius, Bad Homburg, Germany), and acrylonitrile-sodium
methallyl membrane (Filtral-12; Hospal, Lyon, France). No dialyzers
were reused. Informed consent was obtained from all patients examined in this study.
Amplification of HCV cDNA by reverse transcription-PCR.
RNA
was extracted from 100 µl of serum with TRIzol (Life Technologies
Inc., Gaithersburg, Md.) according to the manufacturer's instructions.
To synthesize cDNA, the RNA was reverse transcribed in 20 µl of
reaction mixture containing 100 pmol of random primer, 20 U of
ribonuclease inhibitor (Toyobo, Co., Osaka, Japan), and 100 U of
Moloney murine leukemia virus reverse transcriptase (Life Technologies
Inc.). Half of the synthesized cDNA was used for the PCR. The PCR was
performed in 50 µl of solution containing 0.5 U of Taq DNA
polymerase (Life Technologies Inc.), 50 µM concentrations of four
deoxynucleoside triphosphates, and 0.3 µM concentrations of each
primer by 35 cycles of 30 s at 94°C, 1 min at 60°C, and 1 min
at 72°C, followed by 10 min of extension at 72°C. Five microliters of the product was used for the second-round PCR using nested primers
under the same conditions as the first PCR. The primers used for the
first PCR of HCV HVR1 were 5'-GCGGGCCTTGCCTACTATTC-3' (sense; positions 1069 to 1088 [numbered from the ATG initiation codon]) and 5'-GGGCACCCGGACGAGTTGAA-3' (antisense;
positions 1339 to 1358), and those for the second were
5'-CATGGCGGGGAACTGGGCTAAGGT-3' (sense; positions 1089 to
1112) and 5'-CAGGGCAGTCCTGTTGATGTGCCA-3' (antisense;
positions 1258 to 1281). The primer pairs for the first and second PCR
of HCV 5'-UTR were 5'-CACTCCCCTGTGAGGAACTA-3' (sense;
positions 
304 to 285) with 5'-GGTCTACGAGACCTCCCGGG-3' (antisense; positions 20 to 1) and
5'-TTCACGCAGAAAGCGTCTCTAG-3' (sense; positions 279 to
260) with 5'-CCCTATCAGGCAGTACCACA-3' (antisense; positions
60 to 41), respectively.
Subcloning, sequencing, and phylogenetic analysis.
The PCR
product was purified with phenol-chloroform, and then subcloned into
T-tailed plasmid vector of pGEM-4Z by the T-A overhang cloning method
(12). Ten recombinant clones were isolated from each sample
and sequenced by the cycle sequencing method (16) using
Taq DNA polymerase and 32P-labeled primer. The
PCR product was also sequenced directly after the removal of excess
primers by three cycles of centrifugation in a Microcon 100 centrifuge
(Amicon, Inc., Beverly, Mass.).
The nucleotide sequences were translated by the DNASIS program (version
3.6; Hitachi Software Engineering Co., Ltd., Yokohama, Japan), and the
amino acid sequences and the nucleotide sequences of HVR1, including
unrelated HVR1 sequences from Japanese patients and blood donors, were
aligned by using the CLUSTAL W program, version 1.5 (26).
Phylogenetic trees were constructed by the neighbor-joining method
(19), and 1,000 trials of bootstrap analysis were performed.
Peptide ELISA.
Six peptides of HVR1 sequences derived from
five clusters were synthesized by the solid-phase method using a
simultaneous multiple solid-phase peptide synthesizer (PSSM-8; Shimadzu
Co., Kyoto, Japan). After the peptidyl resin was cleaved, crude
peptides were purified by a preparatory reverse-phase high-performance liquid chromatography and the purified peptides were characterized by
sequence analysis using a protein sequencer (PPSQ-10; Shimadzu Co.) in
addition to analytical high-performance liquid chromatography. Using
the synthesized peptides, indirect ELISA was performed as described
previously (1). Briefly, microtiter wells (Nunc Immuno Plates, Maxisorp; Nunc A/S, Roskilde, Denmark) were coated with peptide
(1 µg/ml), followed by blocking. Patient serum diluted 20 times was
added to the wells, and reacting antibody was detected by incubation
with peroxidase-conjugated goat F(ab)2 fragment to human
immunoglobulin (Cappel Inc., Durham, N.C.) and colorization with
tetramethylbenzidine (Sigma Co., St. Louis, Mo.) and
H2O2. After stopping the reaction with
H2SO4, absorbance at 450 nm
(A450) was measured in a Multiskan Bichromatic
System (Labsystems Inc., Helsinki, Finland). The data were estimated as
an average of three independent experiments.
Nucleotide sequence accession numbers.
The nucleotide
sequences described in this work have been deposited in the DDBJ,
GenBank, and EMBL databases under accession no. AB001390 to AB001416
and no. AB015321 to AB015326.
| |
RESULTS |
Phylogenetic analysis of HCV HVR1 sequences.
To examine the
relationship among HCV isolates infecting HD patients in a dialysis
center, HCV HVR1 sequences were amplified from the RNAs of patients'
sera and determined. The amplified cDNA was subcloned into plasmid DNA
to determine the sequences of both major and minor HCV isolates in each
patient.
Nine patients were positive for HCV RNA among 62 patients examined at
the center the first time. Unfortunately, 18 months after the first
examination, two additional patients in the same unit were found to be
positive. Figure 1A shows 24 sequences of 27 amino acids of HCV HVR1 isolated from these 11 patients. Each patient had a major clone of HCV individually accounting for more than
70% of the population. Patients F and I had minor clones related to
their major clones, respectively, so-called quasispecies of HCV,
whereas patients A, C, D, E, and H had clones unrelated to their own
major species. A phylogenetic tree was constructed to determine the
genetic relationship among these isolates (Fig. 1B). All 24 sequences,
which were genotype 1b, were classified into five clusters. They were
clearly separated from 25 unrelated sequences of HCV genotypes 1b, 2a,
and 2b from Japanese patients. Major isolates A and B; C and D; E, F,
and G; and I, J, and K were related and formed clusters I, II, III, and
V, respectively. Since two isolates, J and K, from two additional
patients were related to isolate I, HCV seemed to have been transmitted
from patient I to patients J and K within 18 months. Minor isolates F1
and F2 and I1, I2, and I3, were quasispecies of their major isolates, F
and I, respectively, and each grouped into the same cluster as its
major isolate. On the other hand, minor isolates from the other
patients were grouped into one of the three other clusters. As a
control cluster, three related isolates which were major sequences from
a single patient isolated at three different times, 0, 10, and 21 months (11), were included in this tree, and these actually
formed an independent cluster. These results suggest that
patient-to-patient transmission of HCV occurs in HD units, and the
minor isolates from individuals in particular suggest that simultaneous
transmission of mixed populations of HCV or multiple transmission may
occur, since five patients, A, C, D, E, and H, were infected with one
or two more isolates from different clusters. The phylogenetic tree of
19 HCV HVR1 sequences isolated from nine patients, A to I, during the
first examination was also constructed. It revealed the same five
genetic clusters of the sample sequences shown in Fig. 1B, with higher
bootstrap values: 1,000, 887, 576, and 1,000 for clusters I, II, III,
and V, respectively (data not shown). The phylogenetic tree of
nucleotide sequences also confirmed these five clusters. In addition,
phylogenetic analysis of all 43 sequences identified both times in the
HD patients revealed that the classification into five clusters was
maintained (data not shown). Amplifying the more conserved
nonstructural protein 5a region for comparison with the results for
HVR1, we found all nine sequences of the nonstructural protein 5a
region from nine patients, A to I, to be within a single cluster (data not shown). Thus, HVR1 is sensitive for phylogenetic analysis of HCV
transmission.
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FIG. 1.
Comparison of deduced amino acid sequences of the HCV
HVR1 isolated from 11 patients, A to K, 18 months after the first
examination. Minor isolates from each patient are shown as patient
letters with numbers. (A) Alignment of the amino acid sequences. Only
amino acid residues differing from the reference sequences (A, C, E, H,
and I) of each cluster are shown. The actual number of plasmid clones
obtained from each patient is shown on the right. (B) Phylogenetic tree
of HVR1 nucleotide sequences constructed by the neighbor-joining method
using 24 isolates from 11 HD patients and 25 unrelated isolates. The
Roman numerals on the right indicate the genetic clusters of 24 isolates from HD patients. Three related isolates ( ) obtained from a
single patient at different months, indicated by numbers, were included
as a control cluster (11). Bootstrap analysis was performed
for 1,000 trials, and calculated values are shown at each branch.
|
|
Figure 2 shows the epidemiological data
for these 11 patients: serum ALT levels, seroconversion of anti-HCV
antibody and HCV RNA, and history of blood transfusion. All patients
seroconverted to HCV positivity after admission to the dialysis center.
Since both patients J and K seroconverted for HCV RNA between July and October 1995, HCV transmission from patient I probably occurred at the
same time. However, the other transmissions were not epidemiologically clear, since the time point of seroconversion for HCV RNA was not
determined in each patient. In view of the pattern of anti-HCV antibody
detection, HCV transmission probably occurred from patient A to B, from
patient C to D, and from patient G to E and F, resulting in the
generation of clusters I, II, and III, respectively. The second (and
third) transmission of HCV from a different cluster, at least in
patient A, was presumed to have occurred on the basis of results of the
phylogenetic analysis of multiple isolates from each patient alone,
with no substantiation by the epidemiological data. Thus, it is
difficult to conclude that there was patient-to-patient transmission of
HCV based on the epidemiological data alone. The phylogenetic tree is
very informative in determining detailed transmission, including cases
of multiple and mixed infection.
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FIG. 2.
Serum ALT levels and HCV infection markers of 11 patients, A to K. Open bars, negative for anti-HCV antibody; shaded
bars, positive for anti-HCV antibody; open circles, negative for HCV
RNA; solid circles, positive for HCV RNA; arrows, blood transfusion;
triangles, sequence examination.
|
|
Figure 3 shows the HCV HVR1 sequences of
five HD patients in another dialysis center. The phylogenetic tree
indicates that minor isolate X1 from patient X was unrelated to major
isolate X but was related to isolate Y. Four control clusters included in the phylogenetic tree revealed that isolates X1 and Y constructed a
single genetic cluster. This transmission was not detected by sequence
analysis of major isolates, e.g., direct sequencing of the amplified
products. Sequence analyses following subcloning revealed unrecognized
transmission of HCV in HD units.
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FIG. 3.
Comparison of deduced amino acid sequences of the HCV
HVR1 isolated from five patients, V to Z. Only residues differing from
the reference sequence (V) are shown in the alignment. Isolate X1 was
obtained from patient X as a minor clone. The actual number of plasmid
clones obtained from each patient is shown to the right of the
sequence. The phylogenetic tree of nucleotide sequences was constructed
by the neighbor-joining method using 6 isolates from HD patients and 25 unrelated isolates, including a control cluster (), as shown in Fig.
1B.
|
|
Serological analysis of anti-HVR1 antibody.
Most patients with
chronic hepatitis C possess specific antibody against HVR1 of their own
isolates, which is thought to be neutralizing antibody (9,
29). However, emergence of an escape mutant not selected by the
host immune response seems to be involved in the development of
chronicity of infection (9, 29). To assess whether anti-HVR1
antibody proves the footprinting of HCV transmission, the
representative HVR1 peptides from five clusters were synthesized and
used to detect anti-HVR1 antibody in patients' sera. As shown in Fig.
4, the serum of all patients was
specifically immunoreactive to HVR1 peptides related to their own major
isolates individually. Furthermore, patients with mixed populations of HCV were also reactive to the HVR1 peptides derived from the cluster of
minor isolates. For example, the antibody response of patient A was
positive for not only peptide I but also peptides II and III. Patients
C, D, E, and H were also specifically reactive to one more cluster
peptide. Peptides V(I) and V(K) were derived from the same cluster,
cluster V, but 5 amino acids were changed and phylogenetically distant,
even within a cluster (Fig. 1). However, patients I and J were both
immunoreactive to these two peptides. Patient K was still negative for
the third generation of anti-HCV antibody and therefore had not
produced antibody against his own HVR1 sequence.
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FIG. 4.
Antibody responses of 11 patients, A to K, to six HVR1
peptides of different clusters, I to V. Patients' sera were obtained
at month 18. The amino acid sequences of the six peptides used are as
follows: I, FTRVTGGAQAVPTHGLTSLFTFGAQQN; II,
RTLVMGGAATLTTRGRVSLFTFINSQR; III, ETKVMGGAQAPTTSGFTSLFALGSRQN; IV,
NTYVTGGQAGYTTMALSSLFAPGAQQN; V(I), NTYTTGGQAGKTVSTFTSLFTLGASQN; V(K),
NTYTSGGTAGKTVSTLTSLFTPGQSQN. The small Roman numerals
under A, C, D, E, and H indicate the cluster numbers of minor isolates
from each patient, and the large Roman numerals at the bottom indicate
those of predominant isolates from each patient. The
A450 was estimated as the average ± standard deviation (error bar) of three independent experiments.
Negative control values of the patients' sera were 0.48 ± 0.13 (patient A), 0.46 ± 0.11 (patient B), 0.51 ± 0.13 (patient
C), 0.53 ± 0.02 (patient D), 0.49 ± 0.09 (patient E),
0.47 ± 0.01 (patient F), 0.56 ± 0.07 (patient G), 0.50 ± 0.05 (patient H), 0.40 ± 0.16 (patient I), 0.48 ± 0.19 (patient J), and 0.52 ± 0.04 (patient K).
|
|
These results confirm the fidelity of phylogenetic clustering of these
HCV isolates and suggest that the anti-HVR1 antibody assay is simple
and useful for detecting HCV transmission in a unit only if the
patients are immunocompetent.
| |
DISCUSSION |
In this study we demonstrated that multiple instances of
unrecognized transmission of HCV occurred in HD units by constructing the phylogenetic tree of multiple HVR1 sequences isolated from each
patient. In addition to several reports of molecular evidence of
nosocomial transmission of HCV in HD units (2, 4, 14, 15, 18, 20,
25, 31), we found that patient-to-patient transmission of HCV
occurred more frequently than expected in HD units, as shown by our
amplifying the HVR1 sequences as individual markers of HCV isolates and
comparing multiple clones isolated from each sample. However, there is
some concern, as indicated by Munro et al. (15), that the
high and inconstant mutation rate of HVR1 sequences causes spurious
clustering or unclustering in the phylogenetic analysis. To dispel such
concerns in this study, the phylogenetic analysis was performed in the
presence of numerous unrelated sequences together with bootstrap
analysis (Fig. 1B) and was performed by using sequences from different time points and constructing a mixed tree to confirm the genetic clustering. Recently, phylogenetic analysis using the HVR1 sequences clearly proved that a cardiac surgeon with chronic hepatitis C transmitted HCV to patients during open-heart surgery (6). Thus, HVR1 sequences may be a useful probe for fingerprinting of HCV
isolates, provided that patients have been infected with HCV for less
than 5 years, as in this study, because of differences in the selective
forces and the mutation rate of HVR1.
The second point in this study is that the sequence analysis of
multiple clones from each patient revealed complicated HCV infection;
one possibility is that mixed populations of HCV (clusters II and III)
were simultaneously transmitted in patients A, C, D, and E. Another is
that transmission occurred more than once in these patients, especially
in patient A. These infections are not detected by the epidemiological
data or direct sequence analysis of the amplified products. Multiple
sequence analysis in each patient suggests that patient-to-patient
transmission of HCV occurs more frequently than expected in HD units.
As shown in Fig. 4, we also found that the antibody response to HVR1
peptides monitored the historical transmission of HCV, including
unrecognized and multiple infection. For analysis of multiple plasmid
clones, however, we have to carefully consider artificial variants
which were produced by high error rates of reverse transcriptase and
Taq DNA polymerase during reverse transcription-PCR of the
virus genome, as indicated by Smith et al. (24).
The transmission mechanism in HD units has not yet been identified. A
possible cause of transmission is thought to be a lack of careful
infection control by HD personnel. Okuda et al. (17) found
that transmission of HCV was associated with the location of the HD
console and suggested strict aseptic precautions by the staff to
prevent nosocomial spread of HCV infection. The location of the
consoles occupied by the 11 patients in this study indicates that
transmission occurred more frequently between patients treated during
the same shift than between those occupying the same dialysis machine.
The consoles of patients J and K were located side by side and near
that of patient I in the same shift; thus, transmission probably
occurred as a result of the practices of a staff member common to these
three patients. It is important to educate HD personnel to be aware of
the risk of unexpected HCV transmission from HCV-positive patients. In
order to precisely identify HCV-positive patients, regular testing for
HCV RNA should be performed, because there are some populations of HD
patients who are negative for anti-HCV antibody and do not show ALT
elevation but who carry HCV RNA (23).
Patient-to-patient transmission of human immunodeficiency virus
(28) and hepatitis GB virus C (13, 21) has also
recently been suspected in HD units. Thus, there is a high possibility that not only HCV but also other infectious viruses in blood are transmitted in HD units. The phylogenetic analysis of the variable region of these infectious agent clones may be useful for sensitive detection of the agents and identification of the transmission route.
We thank M. Yanai, Department of Clinical Pathology, Nihon
University School of Medicine, and M. Mizokami, Nagoya City University Medical School, for helpful discussion, and C. Yamamoto for technical assistance.
This work was supported by Health Science Research Grants (Non-A, Non-B
Hepatitis Research Grants), the Kurozumi Medical Foundation, and an
Interdisciplinary General Joint Research Grant for Nihon University (DA
97-003).
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Abstract
Introduction
