Genetic and serological evidence for multiple instances of unrecognized transmission of hepatitis C virus in hemodialysis units

Abstract

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.

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.

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.

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.

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).