Study population and questionnaire
The recruitment of patients with symptomatic acute hepatitis C has already been described in our previous report of a pilot study performed in 2002 in Cairo 
. Adjustments to the protocol were made at the end of the pilot study, and the main components of the study are presented here. Patients were recruited from two “fever hospitals” (Abbassia and Imbaba Fever Hospitals), which are public hospitals specialized in infectious diseases offering care at moderate cost to the disadvantaged populations of Cairo. Inclusion criteria were age above 18 years, symptoms (fever or jaundice) lasting less than 21 days, and elevated serum alanine aminotransferase (ALT) ≥3 times the upper limit of normal (ULN
All patients were interviewed by trained medical doctors, using a questionnaire on socio-demographic characteristics and risk factors for HCV infection in the past six months. Risk factors were categorized as high risk and low risk exposures based on the magnitude of the associations with HCV transmission documented in previous studies 
. High risk exposures included surgery, blood transfusion, hemodialysis, biopsy, endoscopy, Caesarean section, episiotomy, uterine curettage, injection, infusion, catheter, sclerotherapy of varicose veins, and dental care. Low risk exposures included acupuncture, shaving at barber, tattooing, pedicure, manicure, and circumcision. Also assessed was exposure to other potential causes of hepatitis, including drugs, pesticides, and other chemicals known for their hepatotoxicity.
A 10 ml venous blood sample was collected for liver functions [ALT, aspartate aminotransferase (AST), total and indirect bilirubin, alkaline phosphatase], and exclusion of acute hepatitis A and B by serological analysis [IgM anti-hepatitis A virus (HAV) (HAVAB®, M EIA, Abbott Laboratories, Diagnostics Division, Abbott Park, IL); IgM anti-hepatitis B virus (HBV) core (CORZYME®, M rDNA, Abbott Laboratories, Diagnostics Division); hepatitis B surface antigen (AUSZYME MONOCLONAL®, third generation EIA, Abbott laboratories, Diagnostics Division, Abbott Park, IL)]. In patients with non-A non-B hepatitis, anti-HCV antibodies were assessed serologically (INNOTEST® HCV Ab IV, Innogenetics, Ghent, Belgium), and HCV RNA Extraction was performed using QIAmp Viral RNA Kit (QIAGEN, Santa Clarita, U.S.A.) according to manufacturer's recommendations. The Reverse Transcription and Polymerase Chain Reaction (RT-PCR) was done according to Abdel-Hamid et al, 1997 
with some modifications to increase the sensitivity of the assay (50 IU/mL). The RT and first round of PCR amplification were performed in a single step as follows: the RT-PCR master mix consisted of 1X Taq buffer with 1.5 mM MgCl2 (Roche Molecular Biochemicals), 0.2 mM dNTP (Promega), 20 pmol of each primer (P1 and P2), 20 U of Human Placental Ribonuclease Inhibitor (RNasin, Promega), 10 U of AMV reverse transcriptase (Promega), and 2.5 U of Taq DNA polymerase (Roche Molecular Biochemicals). The master mix was added to 55 µl of RNA from each sample, and the tubes were subjected to the following thermal cycles: 42°C for 30 minutes (one cycle) for RT, 35 cycles at 94°C for denaturation for 1 minute, 50°C annealing for 1 minute, and 72°C extension for 1 minute and a final extension step at 72°C for 10 minutes (one cycle). Nested PCR was conducted with 10 µl of the first PCR reaction added to 90 µl of the master mix, consisting of the same reagents used in the first PCR expect for using P-3 and P-4 and without adding RT or RNasin. Thermo cycling for 35 cycles was performed as in the first PCR but without the initial RT step. The outer (P-1, P-2) and inner primers (P-3, P-4) for the nested PCR reaction were derived from the highly conserved 5′ untranslated region (5′UTR) of the HCV genome. PCR results were visualized by electrophoresis using ethidium bromide in a 3% agarose gel.
In patients with positive HCV antibodies and RNA, exacerbation of chronic hepatitis C by other infectious agents was ruled out using reverse transcriptase PCR for HEV-RNA (in house assay using ORF1 and ORF2 primers) and serological testing [anti Epstein-Barr virus (EBV) IgM (ETI-EBV-M reverse P001605, Dia Sorin, Vercelle, Italy), anti-cytomegalovirus (CMV) IgM (AXSYM® system-CMV-IgM, Abbott Laboratories, Wiesbaden, Delknheim, Germany), and anti-Toxoplasma IgM (AXSYM® system-Toxo-IgM, Abbott Laboratories, Wiesbaden, Delknheim, Germany)]. All tests were performed at the Viral Reference Hepatology Laboratory (VHRL).
Definitions of acute hepatitis C
The diagnosis of acute hepatitis C was based on epidemiological findings, clinical examination, and laboratory test results. Accordingly, patients were categorized as 1) Definite acute hepatitis C if negative anti-HCV antibody and positive HCV RNA; 2) Probable acute hepatitis C if positive anti-HCV antibody and HCV RNA associated with ALT≥10 times the ULN, history of high risk exposure 1 to 3 months prior to diagnosis, negative serology/PCR (see above) for other infectious agents (HAV, HBV, HEV, EBV, CMV, toxoplasmosis), no history of drugs or pesticides exposure, and no other cause of hepatitis (e.g., autoimmune hepatitis, ischemic liver injury, Wilson disease).
All patients with acute hepatitis C were offered follow-up at the National Hepatology and Tropical Medicine Research Institute (NHTMRI) in Cairo. Visits were scheduled for clinical and biochemical assessment at 2, 3, 6, 12, 18 and 24 months, calculated from onset of symptoms. Antiviral treatment was not offered immediately in order to allow for spontaneous clearance of the virus; however, non-specific symptomatic treatment, such as antiemetics for nausea and vomiting, antipyretics for fever and analgesics for headache, were offered to all patients. Patients who were still HCV RNA positive three months after the onset of symptoms were screened for treatment eligibility. Exclusion criteria for treatment were age <18 years or >65 years; poorly controlled diabetes; thyroid disease (TSH outside the normal range); autoimmune diseases; alcoholism; liver cirrhosis; hepatocellular carcinoma; psychiatric disease such as history of severe depression, psychosis, suicidal ideas; epilepsy; non-stabilized medical or surgical condition; hemoglobin<11 g/dl, leucocytes <3000/µL, polynuclear neutrophils <1500/µL, platelets <100 000/µL, and blood creatinin >150 µmol/L. For females: pregnancy or breastfeeding were contra-indications (an effective contraception was requested during the treatment period).
Treatment with free pegylated interferon [PEG-IFNα-2a (PEGASYS®, Roche)] (180 µg/week) for 12 weeks was started 4–6 months after onset of symptoms in those still positive at that date, provided there were no contraindications. Injections were provided at the NHTMRI clinic under medical supervision. In patients still HCV RNA positive after 12 weeks of treatment, treatment was extended for another 12 weeks. Patients were seen weekly during the treatment period, and every six weeks afterwards until week 36 (end of follow-up for those treated for 12 weeks), or week 48 (end of follow-up for those treated for 24 weeks). The dosage of PEG-IFN α-2a was reduced for two weeks to 90 µg/week if neutrophil or platelet counts fell below 750/µL and 50,000/µL, respectively. After the 2 weeks, dose was raised back to 180 µg/week if neutrophil and platelet counts went above 750/µL and 50,000/µL, respectively. Treatment was discontinued if neutrophil or platelet counts fell below 500/µL and 25000/µL, respectively, or when serum hemoglobin concentrations decreased below 8.5 g/dL. Doctors' judgment was the basis for dose reduction and discontinuation of PEG-IFN α-2a if other commonly reported adverse events (such as flu-like symptoms and emotional side effects) were reported. The primary endpoint was sustained virological response (SVR), defined as negative serum HCV RNA 24 weeks after the end of treatment. Secondary endpoints were the absence of detectable levels of serum HCV RNA at the end of treatment and the normalization of ALT levels. Viral load was determined using the Cobas Amplicor HCV Monitor test, v 2.0 (Roche Diagnostic Systems).
All patients with acute hepatitis consulting or hospitalized in the two participating hospitals were included in the study after providing a written informed consent. A separate informed consent was required for the patients who were treated with pegylated interferon. The entire study protocol has obtained clearance from the Ministry of Health and Population in Egypt, and the Institutional Review Board for Human Subject Research at NHTMRI. The 12-week pegylated interferon trial protocol for patients with acute hepatitis C has been registered under the following number: NCT00158522 (ANRS1213).
Spontaneous viral clearance (SVC) was defined as no HCV RNA in serum, in the absence of treatment, for two consecutive HCV PCR tests during follow-up. Patients treated with pegylated interferon were censored at the time of treatment initiation. Date of clearance was chosen as the midpoint between the last positive PCR and the first negative PCR defining clearance. The SVC rate was estimated using Kaplan-Meier estimates, with time 0 equivalent to onset of symptoms. A Cox proportional hazards model was used to identify factors associated with SVC. Factors with p values less than 0.25 in univariate analysis were tested in the multivariate model, and p values less than 0.05 were considered significant. Statistical analyses were performed using Stata 8 statistical software (Stata Corporation, College Station, Texas, USA).