From 61553 donors analyzed during the study period, 515 (0.84%) were anti-HCV reactive. The overall prevalence descended from 0.93% in 2003 to 0.55% in 2006 (Figure 1). The mean prevalence at the more urbanized central (Angelopolis) region was 0.79% which includes the city of Puebla de los Angeles, the capital of the state. In the other regions, either suburban or rural, prevalence values ranged from 0.86 to 1.04% (Figure 2). No significant difference was observed among these regions (Student's t-test, p > 0.05).

Low S/CO values (from 1.0 to 4.0) were found in 79% of reactive donors. The rest of the donors presented S/CO from 4.1 to 168, which can be considered from medium to relatively high (Figure 3).

Of 515 reactive donors, 96 accepted to be included in the subsequent studies ("recovered" donors) and were interrogated and sampled from June 2005 and December 2006. From these 96 donors, 80% presented low S/CO values (from 1.0 to 4.0) and the rest presented S/CO from 4.1 to 168 (Figure 3). These data show that this sample of 96 donors is representative of the total population of donors previously analyzed.

Of the 96 recovered donors, 37 were HCV-RNA positive (38.5%). The anti-HCV S/CO ratio was related with positivity to HCV-RNA detection; 100% of donors with S/CO > 39 were HCV-RNA positive, whereas donors with lower S/CO values presented progressively reducing percentages of viral RNA detection. In donors with S/CO from 1 to 2, HCV-RNA was detected only in 13.7% (Figure 4).

HCV genotypes and subtypes were analyzed in all 37 HCV-RNA positive recovered blood donors. 34 (91.8%) donors had HCV genotype 1. Of these donors, 2 (5.4%), 15 (40.5%), 10 (27.0%), and 7 (18.9%) had subtypes 1 undetermined, 1a, 1b, and 1a/1b, respectively. One donor had genotype 2a (2.7%) and another had subtype 2b (2.7%). One donor (2.7%) presented the mixed 1a/2a genotypes (Table 1).

The main risk factors recognized in recovered donors were histories of surgery (29.1%) and of blood transfusion (6.2%), with 46 and 50% of HCV-RNA positive, respectively. Other important risk factors identified were history of migration (15.6%), dental treatments (14.5%), multiple sex partners (5.2%), and living with infected family members (4.16%). In 40.6% of the recovered donors the risk factor could not be identified.

Of 37 HCV-RNA-positive donors, 11 (29.7%) presented significantly high levels of ALT (68.9 ± 7.1 IU/l). All donors HCV-RNA-negative presented normal levels of ALT (30.6 ± 5.6 IU/l), which included individuals with anti-HCV S/CO from 1 to 16.

The mean age of the 515 reactive donors was 34.5 years, while that of the 96 recovered donors was 34.6. Among recovered donors, positive and negative HCV-RNA, there was no significant difference (34.7 and 38.1 years, respectively; Student's t-test, p = 0.234). On the other hand, the mean age of recovered HCV-RNA-positive donors was different between the individuals with anti-HCV S/CO ratios ranging from 1 to 16, and those with S/CO > 39 (35.5; 95% CI, 31.1-39.8 years and 43.3; 95% CI, 38.4-48.3 years, respectively; Student's t-test, p = 0.044).

The study population consisted of individuals who donated blood between January 2003 and December 2006 from the State of Puebla, Mexico, at National Health Centre "Manuel Avila Camacho". During this period, 61553 first time donors were analyzed, and were subjected to routine studies by the Blood Bank, according to Mexican Official Standard, NOM-003-SSA2-1993, including a third generation EIA anti-HCV test. After that, we invited the positive anti-HCV donors to participate in this study that included HCV-RNA detection and quantification of alanine-aminotransferase (ALT) activity. Individuals who agreed to enter the study were classified as "recovered" donors. A questionnaire, requesting information on transfusion, surgical or dialysis history, piercing and tattooing, or sexual practices and other data, was applied to recovered donors.

Screening of blood donors was performed using the AxSYM HCV 3.0 assay (Abbott Diagnostics, Wiesbaden, Germany). The AxSYM HCV 3.0 is an indirect microparticle enzyme immunoassay (MEIA) that detects human antibodies against recombinant HCV proteins core, NS3, NS4, and NS5. The values of antibody detection are expressed as the ratio between the signals detected in the sample and the cutoff value (S/CO); samples with S/CO values ≥ 1.0 were considered reactive 22. All reactive samples were submitted to a second anti-HCV detection test.

RNA in serum samples was extracted using QIAamp RNA Viral Mini Kit (Qiagen, Valencia, CA). HCV-RNA was amplified using a nested RT-PCR with the two sets of primers corresponding to the 5' UTR, either in the first or second round, as reported previously 232425. Reverse transcription and the first round of PCR (25 μl reactions) were performed using One-Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, CA). Program parameters were 55°C for 30 min (RT), 94°C for 2 min, and 40 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 30 sec. The second round of PCR (25 μl reactions) was performed using BioMix (Bioline, London, UK) and 2 μl of first-round PCR product. Thermal cycler conditions were 94°C for 2 min and 40 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec. Nested RT-PCR was performed by duplicate in all samples and reaction products were analyzed by 1% agarose gel electrophoresis. This home-made RT-PCR has a detection limit of 50 IU per ml (unpublished results), which is comparable with commercial test used for qualitative detection of HCV genome 4.

Viral genotypes were identified by restriction fragment length polymorphism (RFLP) according to published protocols 25. Briefly, the 251-bp amplicon obtained in nested RT-PCR was digested with enzymes MvaI (Fermentas, Glen Burnie, MD) and HinfI (Promega, Madison, WI) in order to discriminate among viral genotypes 1-6. After that, the 251-bp amplicon was digested with either BstUI or ScrFI enzymes (New England Biolabs, Ipswich, MA) to identify viral subtypes. Restriction fragments were analyzed by 15% polyacrylamide gel electrophoresis.

ALT levels were measured with the Advia 1200 Chemistry System (Siemens healthcare Diagnostics Inc, Tarrytown, NY) and the normal reference interval were 4-36 IU/l.