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Journal of Clinical Microbiology, August 2001, p. 2860-2863, Vol. 39, No. 8
Divisione Malattie Infettive Arcispedale
"Santa Maria Nuova" Azienda Ospedaliera, Reggio
Emilia,1 Istituto Nazionale per le
Malattie Infettive, IRCCS "L. Spallanzani,"
Rome,2 Dipartimento Scienze Biomediche,
University of Trieste, Trieste,4 and
Istituto di Microbiologia, University of Ancona,
Ancona,3 Italy
Received 15 November 2000/Returned for modification 2 February
2001/Accepted 30 May 2001
A cluster of hepatitis C virus (HCV) infections among gynecological
patients who underwent surgical intervention in the same setting is
described. An epidemiological investigation was conducted to identify
the cases, the likely source of infection, and the route of
transmission. Four recent HCV infections were identified. Based on
molecular fingerprinting analysis and epidemiological investigation,
transmission between the putative source patient (an
HCV-positive woman who was the first patient of the surgical session)
and outbreak patients was highly suggestive. All patients, including
the source patient, were infected with HCV type 1b. Molecular
characterization of HCV clones by sequence analysis of both structural
envelope regions (20 clones from the source patient and 58 from the
outbreak patients) and the nonstructural NS5 region of the viral genome
(12 clones from the source patient and 32 from the outbreak patients)
showed close homology between the viral isolates from the source and
those from the outbreak patients that was higher than that observed
between the viral isolates from the source and those from four
unrelated, HCV type 1b-infected patients from the same geographical
area (in the latter case, 33 clones were sequenced for the envelope
regions and 30 were sequenced for the NS5 region). The mean percent
divergence between clones was 4.69 for the envelope and 3.71 for the
NS5 region in the source patient and the outbreak patients compared with 6.76 (P = 0.001) and 5.22 (P = 0.01) in the source patient and control
patients, respectively. Among the risk factors investigated, only that
of having undergone surgery in the morning session of the same day
reached statistical significance (P = 0.003). The investigation showed that the source patient and outbreak patients shared only the administration of propofol in multidose vials. The
study documents the risk of nosocomial transmission of HCV and the
importance of infection control procedures in the operating room and
highlights the crucial role of molecular strategies, especially
sequence-based phylogenetic analysis of cloned viral isolates, in the
investigation of HCV outbreaks.
Hepatitis C virus (HCV) infection is
a major health problem worldwide. Approximately 80% of the individuals
infected with HCV progress to chronic infection (4), and
0.4 to 2.5% of these develop hepatocellular carcinoma
(11). In the past, blood transfusion and administration of
blood products were important sources of HCV transmission, but
currently, high-risk drug and sexual exposures account for most cases
of HCV transmission. However, for approximately 10% of patients the
source of transmission is unknown (2).
Nosocomial HCV infection, which is mostly due to patient-to-patient
transmission, can be identified by genotyping of HCV strains and
through sequence-based molecular fingerprinting (1, 2, 4).
In some hospital settings commonly using intravenous lines (i.e.,
dialysis and hematology wards), blood-borne pathogens are more easily
transmitted. However, owing to the peculiar characteristics of HCV
(high proportion of asymptomatic cases, long incubation period, and the
fact that patients may never return to the same care provider), the
actual risk of nosocomial infection with HCV has rarely been measured.
Risk factors for nosocomial HCV infection include transmission through
blood components (3) (currently very rare), organ transplantation (12), patient-to-patient transmission
through shared dialysis equipment (23) or devices such as
colonoscopes and breathing circuits (8, 9), and multidose
vials (24). Unfortunately, however, in many cases it is
nearly impossible to establish or even surmise the source of infection.
Moreover, since most cases of HCV infection are asymptomatic, the
spread of HCV among hospitalized patients may often go unnoticed.
In March 1998, two women with recent HCV infection who had both
undergone gynecological surgery on 9 January 1998 in the same operating
room were admitted to the Infectious Diseases Unit of Reggio Emilia
Hospital. An investigation was conducted to identify further cases, the
likely source of infection, and the route of transmission. Molecular
characterization of HCV genomes conducted through genotype analysis and
sequencing of the structural envelope regions 1 and 2 (E1 and E2),
including the hypervariable region 1 (HVR-1) and the nonstructural
region NS5 of the viral genome, revealed close homology
between the HCV genome of an HCV-positive woman, who was the first
patient of the day's session, and those of four outbreak patients, who
underwent surgery later in the same morning.
Epidemiological investigation.
At the end of March 1998, the
medical records of the 16 patients who had undergone gynecological
surgery on 8 January (8 patients), 9 January (6 in the morning and 1 in
the afternoon), and 10 January (1 patient) were reviewed. The patients
were traced to obtain information on demographic characteristics, HCV
serological status, hair removal before the operation, preanesthesia
medications, antibiotic prophylaxis, anesthetics administered, whole
blood or other blood component transfusions, length of hospital stay, and clinical condition.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2860-2863.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Transmission of Hepatitis C Virus in a Gynecological
Surgery Setting
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Virological study. Anti-HCV antibodies were detected by a third-generation enzyme-linked immunosorbent assay (Abbott Laboratories, Chicago, Ill.); serological confirmation of antibody reactivity was performed with immunoblot assays (Matrix 2.0; Abbott).
Detection of HCV RNA was performed for all patients and for the sexual partners of patients with a positive anti-HCV test (reverse transcriptase PCR [RT-PCR] Amplicor assay; Roche, Basel, Switzerland). HCV genotyping was performed by nested PCR of the HCV core region according to the method of Okamoto et al. (19), with minor modifications (22). For HCV RNA quantitation, RNA was extracted from 100 µl of plasma using the guanidinium thiocyanate method (10); 10 µl of RNA dissolved in diethyl pyrocarbonate-treated water was quantified using competitive RT-PCR (16). For sequence analysis, a total of 835 nucleotides encompassing partial fragments of the E1 and E2 regions, the entire HVR-1, and the nonstructural NS5 region of the HCV genome were amplified by RT-PCR from plasma samples of all patients and from four control patients infected with HCV type 1b, unrelated to this cluster (admitted to the Hospital of Ancona, Ancona, Italy, for primary HCV infection); cloned into a plasmid vector; and transformed to competent cells. The plasmid DNA extracted from transformant colonies was sequenced in both forward and reverse directions by fluorescence-labeled dideoxynucleotides with an automated sequencer (model 373A; Perkin-Elmer, Norwalk, Conn.), according to methods described previously (15). Nucleotide sequences were edited and assembled using the Sequence Navigator program included in the ABI373 software package. Multiple nucleotide and amino acid sequences were aligned using the Megalign (DNAstar Inc., Madison, Wis.) and the CLUSTAL W 1.7 programs. A pairwise matrix of evolutionary distances of nucleotide sequences was generated using DNADIST (Kimura's two-parameter method) included in version 3.572 of the PHYLIP package (J. Felsenstein, Phylogeny Inference package, version 3.5; Department of Genetics, University of Washington, Seattle). Phylogenetic trees were constructed from the same distance matrices with the NEIGHBOR program (neighbor-joining algorithm). Bootstrap values were calculated using SEQBOOT (100 resamplings), followed by DNADIST, NEIGHBOR, and CONSENSE in order to generate the majority rule consensus tree.Statistical methods. Attack rates were calculated for patients exposed and not exposed to potential risk factors and compared using, when possible, relative risk. Ninety-five-percent confidence intervals of relative risk were computed by the exact method. Fisher's exact test was used to evaluate the association of outcome with potential risk factors. The unpaired t test was used to compare group means of nucleotide sequence divergence. Two-tailed P values of <0.05 were considered statistically significant. Calculations were performed using STATA (STATA statistical software, release 5.0; Stata Corporation, College Station, Tex.).
Nucleotide sequence accession numbers. The sequences described in this study have been submitted to the EMBL data bank and assigned accession no. AJ310571 to AJ310608 and AJ409828 to AJ409866.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cases of HCV infection. Of the 16 patients who underwent surgery on 8, 9, or 10 January 1998, 5 tested anti-HCV and HCV RNA positive in March 1998. The other 11 patients tested anti-HCV and HCV RNA negative then and 6 months later.
All anti-HCV-positive women underwent surgery in the morning of 9 January 1998. One of them, the first patient of the day, was a 51-year-old woman known to have chronic hepatitis C at the time of the intervention. The four cases with acute, type C hepatitis were identified following admission to the hospital in March 1998 (two patients aged 67 and 33 years) or following epidemiological investigation (two patients aged 67 and 47 years). All surgical personnel tested anti-HCV negative. The sexual partners of the anti-HCV-positive women tested anti-HCV and HCV RNA negative. No other risk factor for a blood-borne infection was identified by interviewing the four outbreak patients. The review of infection control procedures adopted in the gynecological surgery unit did not show failures in the infection control routine. Decontamination, disinfection, and sterilization procedures were appropriate. Fresh sucker heads and/or suction catheters were reportedly used on each patient. Breathing circuits were replaced after the operation on the patient with chronic HCV infection. Finally, new high-efficiency filters (Hygroboy; DAR, Mirandola, Italy) were fitted on the patient circuits of anesthesia equipment at each operation. In the operating room, a two-way peripheral venous catheter was inserted before surgery following skin cleaning and preparation. One channel was used for administering fluids, and the other was used for intravenous anesthetics; an antireflux valve in the latter prevents blood reflux during infusion. Occasionally, anesthetics were administered through the other channel (which does not have an antireflux valve). Intraoperative blood pressure was always monitored with a cuff sphygmomanometer placed on the arm where the peripheral venous catheter was inserted. Finally, multidose anesthetic vials (propofol and fentanyl) were used.Virological data.
All samples from the five anti-HCV-positive
patients were HCV RNA positive. The patients were all infected with HCV
type 1b. For each patient, the nucleotide sequences of 10 to 20 clones, corresponding to partial fragments of the E1-E2 and the NS5
regions, were analyzed and aligned. A total of 185 clones were
sequenced: 111 for the E1-E2 region (20 from the putative source
patient, 58 from the outbreak patients, and 33 from the control
patients) and 74 for the NS5 region (12 from the putative source
patient, 32 from the outbreak patients, and 30 from the control
patients). Mean percent divergence values of each pairwise comparison
between the putative source patient and the outbreak patients ranged
from 3.63 to 5.32 for E1-E2 and from 2.70 to 4.48 for NS5,
respectively. The percent divergence among sequences of the same
nucleotide fragments between the putative source patient and the
control patients was significantly higher (t test for E1-E2
region [including HVR-1] = 5.2893; P = 0.001;
t test for NS5 region = 3.2737; P = 0.01). The phylogenetic tree reconstruction of cloned sequences is
shown in Fig. 1.
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Epidemiological data. According to regional records, no case of acute HCV infection was reported in 1997 through 12 January 1998 among women undergoing operations in a gynecological unit.
Outbreak patients were defined as those individuals with previously unknown HCV serostatus who had antibodies to HCV and homology of HCV RNA sequences in serum or plasma samples by RT-PCR and genotyping in March 1998. The source patient was traced among those patients who already were anti-HCV positive at the time of the operation and showed molecular homology in the HCV genome with those of the outbreak patients. Based on genotyping, sequencing, and phylogenetic analysis of HCV RNA-positive samples, the woman with chronic hepatitis C is the putative source patient and the four anti-HCV-positive women are cases of recent HCV infection. On 9 January 1998, the day-specific attack rate of HCV infection was 80% (four of five). Among the risk factors investigated, those common to all the patients who underwent surgery on the morning of 9 January were administration of atropine, fentanyl, and propofol. However, propofol was not administered to the patient who did not become infected. The retrospective cohort study conducted among the patients who underwent surgical intervention on 8, 9, and 10 January showed that having undergone surgery in the morning session of 9 January was the sole significant risk factor (P = 0.003).| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study describes the molecular epidemiology of a cluster of cases of primary HCV infection that involved four gynecological patients who underwent surgery on the same day in the same gynecological surgery unit.
The investigation identified a woman with chronic hepatitis C as the putative source patient. She was the first of the six patients of the operative session of 9 January: all the other women but one became outbreak patients. The phylogenetic analysis of viral sequences from the source and outbreak patients revealed very close homology between viral sequences, consistent with nosocomial transmission; this highlights the potential of molecular strategies to unambiguously trace cases of HCV transmission from a common source of infection.
It is extremely likely that HCV infection resulted from a point source and that it occurred in the operating room. Among the risk factors identified, the source patient and outbreak patients shared only the administration of propofol. This anesthetic was not administered to the woman who did not seroconvert.
Review of infection control procedures showed the possibility of a blood reflux in the peripheral venous line during blood pressure monitoring.
Accordingly, if the syringe used to administer propofol to the source patient was also used to draw propofol from the multidose vials and administer it to the subsequent patients, blood contamination of the syringe is likely to have resulted and to have determined the spread of HCV infection. However, the surgical team members were adamant in stating that the syringe used to draw fluid from the multidose vials was changed after each patient.
Despite the well-known risks of blood-borne infection transmission to both patients and operators, reuse of disposable syringes and multidose drug vials in anesthesia remains a major concern, as microscopic amounts of blood in the intravenous tubing can cause contamination of syringes, needles, and drug vials (18).
Contaminated multidose vials have been implicated in the transmission of hepatitis B (13, 14, 20), hepatitis C (24), and bacterial infections (5). Indeed, as in-use contamination of propofol multidose vials is not uncommon (6, 7), specific precautions have been suggested to prevent propofol-related infection because its lipid base can support bacterial growth in case of contamination (21).
In conclusion, two factors seem to have been decisive in causing this cluster of HCV infection: the operating schedule and a common vehicle of infection. Had the source patient been the last to be operated on, HCV infection might never have occurred. This case emphasizes the risk of nosocomial transmission and the importance of universal infection control procedures in the operating room. Some health care procedures, i.e., surgical and dental treatments, have recently been indicated as risk factors for acute HCV (17), likely due to breaches in infection control measures. The present study highlights the usefulness of molecular epidemiological strategies and particularly of cloning and sequencing techniques for the investigation of outbreaks of HCV infection.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from the Consiglio Nazionale delle Ricerche (target project "Biotechnology") to M.C. The epidemiological investigation was conducted as part of Ricerca Corrente of IRCCS "Spallanzani."
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FOOTNOTES |
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* Corresponding author. Mailing address: Istituto di Microbiologia, University of Ancona, Via Pietro Ranieri, I-60100 Ancona, Italy. Phone: 39 071 596 4849. Fax: 39 071 596 4852. E-mail: manzin{at}popcsi.unian.it.
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Journal of Clinical Microbiology, August 2001, p. 2860-2863, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.2860-2863.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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