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Methods
Patients
The municipal hospital in which the HCV outbreak occurred provides general as well as specialty medical and surgical services. Between July 1 and October 13, 1998, HCV infection was diagnosed in four patients (Patients 2, 3, 4, and 6 in this report) on the basis of clinical symptoms, a rise in the serum alanine aminotransferase concentration, or detection of serum HCV antibodies and HCV RNA. All of these patients had undergone orthopedic or general surgery in the same hospital 6 to 18 weeks earlier. A comprehensive investigation was initiated by the public health authorities, and we were asked to determine the circumstances of the suspected nosocomial HCV infections. An institutional review board of Essen University Hospital approved the study protocol, and the patients provided written informed consent.
Epidemiologic Studies
The charts of all patients with HCV infection were reviewed in detail. Interviews were conducted to obtain further information about prior medical interventions, prior hepatitis infections, and risk factors for the acquisition of HCV. To search for other potential cases of HCV transmission, we performed a retrospective seroepidemiologic study of all patients who had undergone surgery in the hospital between January and July 1998. Fifty-eight of these patients had died, and 904 were still alive; serum was obtained for antibody testing from 833 of these 904 patients. Hospital personnel were interviewed with special attention to compliance with infection-control practices and were tested for HCV antibodies. The hospital — in particular, the surgical facilities — was inspected by experts in hygiene and occupational health.
Virologic and Molecular Studies
The presence of HCV antibodies was determined by enzyme-linked immunosorbent assay (ELISA; Sanofi Diagnostics Pasteur, Freiburg, Germany). Reactivity was confirmed by immunoblot assay (Mikrogen, Munich, Germany). HCV RNA was detected qualitatively and was also quantified with polymerase-chain-reaction (PCR) kits (Roche Diagnostics, Mannheim, Germany). HCV isolates were identified by genotyping, and HCV hypervariable region 1 (nucleotides 1491 to 1572, numbered as reported by Choo et al.8) was amplified as described elsewhere.9,10 Products of the second PCR were purified from the agarose gel (QIAquick, Hilden, Germany) and cloned into a plasmid vector (TOPO TA cloning kit, Invitrogen, Groningen, the Netherlands). Four to six clones from each subject were sequenced in both directions (with the Dye Terminator DNA sequencing kit, Perkin–Elmer, Norwalk, Conn.).
As area controls, the PCR products of HCV isolates that were obtained from chronically infected patients (located within a radius of approximately 200 km from the hospital) were subjected to direct sequencing.11 To prevent possible cross-contamination of the samples, stringent procedures were used for nucleic acid extraction and amplification,12 and the analyses were performed several weeks apart. Sequences of HCV hypervariable region 1 that were obtained from the samples have been submitted to GenBank (accession numbers AF227763 through AF227786).
Statistical Analysis
A matrix of nucleotide distances was calculated by Kimura's two-parameter method.13 The statistical significance of the differences was assessed by a two-sided Wilcoxon rank-sum test with the use of SPSS statistical software (SPSS, Chicago). Phylogenetic trees were constructed with use of the neighbor-joining algorithm on the previous sets of pairwise distances (PHYLIP, version 3.514). The significance of the grouping was evaluated by the bootstrap method (1000 replicates).
Results
Epidemiologic Findings
Six patients were found to have hepatitis C viremia. They are listed according to the dates of surgery in Table 1. The HCV infection of Patient 1 was first diagnosed in 1996; she probably contracted the virus through the transfusion of contaminated blood or clotting-factor concentrates at the time of surgery for heart-valve replacement in 1980. Patients 2, 3, and 6 had acute icteric hepatitis C 6 to 18 weeks after orthopedic or general surgery, whereas Patients 4 and 5 were asymptomatic after surgery. Only Patient 1 had ever received blood or blood products. None of the other patients had a history of hepatitis, nor were they aware of any history of hepatitis in their families. All reported no other risk factors for HCV infection, including tattooing or body piercing, intravenous drug use, or high-risk sexual behavior. The operations were performed in two rooms. There was no known contact between the patients either before or during their hospitalizations, which were in different parts of the hospital.
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On questioning, he reported that during the time under investigation he had a wound on the medial side of the third finger of his right hand, sustained in the middle of April 1998 when he opened a box containing infusion solutions. The wound was initially the size of a thumbnail and bled repeatedly. He used a bandage for three or four days but not thereafter, although the wound was still weeping. The assistant admitted that this was negligent behavior, but at the time he already considered the open wound to be an old injury and was not aware that such an attitude might be risky for him as well as for his patients. Between April 28, 1998, the day of surgery in Patient 1, and June 9, 1998, the day of surgery in Patient 6, the assistant participated in 39 operations. Between the time he went on sick leave because of acute hepatitis C and July 1998, another 118 operations were performed in the hospital, and no further HCV infections occurred.
Hygiene and occupational health inspections as well as interviews with staff members indicated that numerous breaches of general infection-control practices had taken place. For instance, needles were frequently recapped after use, and gloves were not always worn in settings in which exposure was likely. Multidose vials for flushing solutions, saline, local anesthetic drugs, and heparin were often used in the operating rooms, although the solutions were changed every second day. As a disinfectant for surfaces, the hospital used a product based on a peroxide compound (Dismozon pur, Bode Chemie, Hamburg, Germany) that is not recommended for areas grossly contaminated with blood. The central sterilization facility worked properly, as indicated by the relevant technical protocols.
Virologic and Molecular Findings
All six patients and the anesthesiology assistant were positive for serum HCV antibodies and HCV RNA. At the time of the investigation, Patient 1 and the assistant had high plasma levels of HCV RNA (2.6x107 copies of HCV RNA per milliliter and at least 1x106 copies of HCV RNA per milliliter, respectively). Genotyping revealed HCV type 1a infection in all cases. The alignment of sequences of HCV hypervariable region 1 demonstrated a very high degree of homology between the isolates obtained from the patients and those obtained from the anesthesiology assistant (Figure 1). Among the subjects, nucleotide distances varied only from 0 to 0.05, whereas a com-parison of the subjects' sequences with those of the 10 area controls with type 1a HCV and the 13 genotype-1a sequences drawn from GenBank showed nucleotide distances that ranged from 0.25 to 0.59 (P<0.001) (data not shown).
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The available epidemiologic and molecular biologic evidence suggested that Patient 1, who had chronic HCV infection, was the index patient in the chain of transmission. The anesthesiology assistant contracted HCV from Patient 1 and subsequently transmitted the virus, during the incubation stage of his disease, to at least five patients.
Discussion
Our findings provide evidence that a nonsurgical staff member infected with HCV transmitted the virus to at least five patients. To our knowledge, such events have been occasionally recorded only for nonsurgical personnel infected with hepatitis B virus (HBV).15,16,17,18 Our conclusion is supported by both epidemiologic and molecular evidence. The five patients had no known risk factors for HCV infection, and there were no evident contacts among them — a fact that excludes the possibility of patient-to-patient transmission. On the other hand, the anesthesiology assistant was the only staff member infected with HCV and could be identified as the sole common denominator in all six cases. He tested negative for serum HCV antibodies approximately eight weeks before Patient 1 underwent surgery on April 28, 1998, and had symptoms of acute hepatitis C six weeks after this operation. Since viral RNA usually appears in the blood within the first week after the transfusion of contaminated blood,19 HCV was probably present in the blood of the anesthesiology assistant and transmissible starting in the first week of May 1998. Furthermore, the incubation periods of HCV recorded in the patients, which ranged from 6 to 18 weeks after surgery, are in agreement with the incubation times reported for HCV after post-transfusional infection (2 to 26 weeks20).
Our molecular investigations were based on sequence analyses of HCV hypervariable region 1, which is commonly used to distinguish between related and unrelated isolates of the same subtype.21 The evolutionary distances between the sequences of hypervariable region 1 obtained from our subjects were similar to those reported previously for epidemiologically linked strains of HCV in infections caused by needle-stick injuries22 or transmitted from mothers to their babies.23,24 Phylogenetic analysis confirmed that the assistant and all six patients were infected with the same HCV isolate.
We could identify an index case as well as the direction of the spread of HCV. However, we were not able to pinpoint the precise mechanisms leading to the infections. The only identifiable condition that might have caused the spread of the virus was the wound on the assistant's right hand. Given the high plasma levels of HCV RNA in both Patient 1 and the assistant, and given that the assistant usually did not wear gloves in the operating room, it is possible that a fraction of a microliter of blood or wound secretions might have transmitted HCV from Patient 1 to the assistant and subsequently from him to the five other patients. Wound secretions due to exudative dermatitis have previously been implicated in provider-to-patient transmission of HBV,15 and the high prevalence of HCV in patients with chronic skin diseases like psoriasis is most likely attributable to infection through minute skin abrasions.25
In our study, patients could have been exposed to minimal and invisible amounts of the assistant's blood or wound secretions directly through mucosal lesions caused by intubation or through indwelling venous and arterial cannulas. Other possible but less likely routes of transmission include inadvertent contamination of instruments or multidose vials with blood or wound secretions from the assistant. Multidose vials have been implicated previously in nosocomial HCV,26 HBV,27,28 and human immunodeficiency virus29 infections.
Whatever the precise mechanisms of HCV transmission in this outbreak, the spread of the virus could probably have been prevented if so-called universal precautions for infection control had been taken.30 Breaches of infection control are therefore associated with a high risk of the transmission of blood-borne pathogens.
Supported in part by a grant to the German National Reference Center for Hepatitis C.
We are indebted to the local authorities for their help in the epidemiologic investigations; to S. Hoffmann, L. Gallina, and S. Sarr for their excellent technical assistance; to P. Galle, M.D., for providing serum and extracted RNA; and to D. Danzglock, M.D., for helpful discussions concerning the mechanisms of viral-pathogen transmission.
Source Information
From the Institute of Virology, National Reference Center for Hepatitis C, University of Essen, Essen (R.S.R., S.V., T.G., M.R.); the Department of Occupational Health, University of Wuppertal, Wuppertal (F.H.); and the Institute of Hygiene, Dr.-Horst-Schmidt-Kliniken, Wiesbaden (H.-M.S.) — all in Germany.
Address reprint requests to Dr. Ross at the Institute of Virology, National Reference Center for Hepatitis C, University of Essen, Hufelandstr. 55, D-45122 Essen, Germany, or at stefan. ross{at}uni-essen.de.
References
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