All patients admitted to the three hospitalization wards of our Liver Unit (Hepatology, Liver Surgery and Transplantation, and Daycare Unit) from August 2000 to October 2002 were screened for anti-HCV. Anti-HCV–negative patients were included in the study. The study was approved by the Local Ethics Committee; all patients received a written synopsis of the aims and methodology of the study and gave their consent for participation.
The Hepatology ward cares essentially for patients with decompensated cirrhosis, whereas the Liver Surgery and Transplantation ward cares for liver transplant recipients and patients with primary or metastasic liver cancer. The Daycare Unit is shared by the Liver and the Gastroenterology units, and patients with liver or gastrointestinal diseases are admitted on a 1-day basis to undergo diagnostic or therapeutic procedures. Medical and nursing staff working at the three hospitalization wards were aware of the aims and methodology of the study. In addition, the Epidemiology Unit of our institution performs regular surveys to ensure full observance of universal precaution measures. Among the common precaution measures used in the units are the routine labeling of HCV infection in the nursing flow sheets, hand washing and change of gloves before and after each patient manipulation, disinfections of nondisposable material (e.g., tourniquets) if used for venous blood sampling, and the nonuse of multidose vials. Capillary blood glucose levels were monitored using an Accu-Chek device (Roche Diagnostics, Barcelona, Spain); patients' fingers were never placed directly on the blood glucose meter. Patients requiring special attention—such as those with major gastrointestinal bleeding or sepsis—are moved into individual rooms and, when necessary, an extra fully dedicated nurse takes care of the patient until the patient is transferred to the intensive care unit or is stabilized.
For anti-HCV–negative patients and during the time of admission, all known risk factors associated with nosocomial transmission of hepatitis C were carefully recorded, including: (1) transfusion of blood products; (2) invasive procedures such as diagnostic endoscopy (gastroscopy, colonoscopy, and endoscopic retrograde cholangiopancreratography), therapeutic endoscopy (variceal sclerosis and banding, polypectomy), angiography and transcutaneous arterial embolization, other radiological procedures requiring intravenous contrast, liver biopsy, percutaneous ethanol injection, radiofrequency ablation, transhepatic cholangiography, hepatic hemodynamics studies and transcutaneous intrahepatic portosystemic shunt, and large volume paracentesis; and (3) minor and major surgical interventions.
Duration of hospitalization was registered for each patient; in individuals admitted more than one instance, the total number of days of hospitalization was considered for analysis. Patient allocation in the different wards was registered every day to identify anti-HCV–positive roommates and the nurse team who was in charge of each patient. A nurse team was defined as all nurses from different shifts (morning, afternoon, night, and weekends) in charge of the same beds (and therefore caring for the same patients). This figure includes registered nurses, licensed practical nurses, and aides.
Anti-HCV was reexamined 6 months after patients' discharge. In patients with more than one hospital admission, anti-HCV was examined at each admission and 6 months after each discharge. If the patient lived outside the Barcelona area, we addressed a letter to the primary care physician asking for a follow-up anti-HCV test. In patients who seroconverted to anti-HCV, infection was confirmed by determination of HCV RNA by a sensitive qualitative assay (Amplicor HCV 2.0; Roche Diagnostics, Branchburg, NJ). In immunocompromised patients, such as patients who underwent liver transplantation or who were under chemotherapy, serum aminotransferases were determined at 3, 6, and 12 months after hospital discharge, and, if elevated, HCV RNA was determined even in the absence of anti-HCV seroconversion.
In case of confirmed HCV infection, a complete epidemiological study based on our records was carried out. Patients were carefully interviewed for lifestyle practices and anti-HCV status of family members. If the patient received blood products, the blood bank was contacted to identify all implicated donors. HCV infection was excluded by serological follow-up of donors and/or by repeating anti-HCV and HCV RNA in stored plasma samples. All anti-HCV–positive (or HCV RNA–positive) individuals who shared a room or nurse with patients who acquired HCV during the study were identified, and a serum sample was obtained when necessary. In patients submitted to invasive procedures, we identified those individuals who had undergone the same procedure on the same day and were anti-HCV–positive; a serum sample was obtained and, if necessary, analyzed. If the source of HCV infection could not be identified, the anti-HCV status of health care staff involved in patients' care was investigated.
HCV sequences from patients who acquired the infection during the study and from individuals with the highest probability to be the source of infection (anti-HCV–positive roommates or individuals who shared the same nurse team) were included in a phylogenetic analysis. If this analysis failed to identify the source of infection, molecular analysis was expanded to other likely sources, such as anti-HCV–positive patients who were simultaneously admitted to the same ward or individuals who underwent the same invasive procedure at the same session.
HCV RNA, Genotype, and Serotype Determination.
HCV RNA was determined with a sensitive qualitative assay (Amplicor HCV 2.0). HCV genotype was determined by restriction fragment length polymorphism after amplification of the 5′ noncoding region of the HCV genome, as described previously.20
If HCV RNA was undetectable, HCV genotype was determined with a serotyping assay that detects type-specific antibodies directed to epitopes encoded by the NS4 region of the genome (Murex HCV Serotyping; Abbot Cientifica S.A., Madrid, Spain). The assay identifies the HCV type (1-6) with high accuracy but does not provide information on the HCV subtype.21
A phylogenetic analysis of HCV sequences was performed to determine if different patients were infected with closely related strains. Partial amplification of the E1 and E2 regions (392 nucleotides encompassing a fragment of the E1 and E2, including the hypervariable region 1) was performed as described elsewhere.5 Amplified fragments were purified, and bidirectional sequence analysis was performed using a commercial kit (Perkin-Elmer Applied Biosystem, Warrington, United Kingdom). Sequence alignment and phylogenetic analysis were performed using the Neighbor Joining program in the PHYLIP package as described previously (Bootstrap support 1000 random resamplings of the sequences K2p (Ti/Tv=2), unrooted).5
We analyzed the data with SPSS version 10 software (SPSS Inc., Chicago, IL). Quantitative variables are expressed as the median (range). For quantitative variables, differences between groups were analyzed using a nonparametric test (Mann-Whitney). For categorical variables, differences between groups were calculated using the Fisher exact test. To identify risk factors of nosocomial HCV acquisition, odds ratios and their respective 95% CIs were calculated (OR ± 1.96 SE).