Seronegative hepatitis C virus infection in Polish blood donors—Virological characteristics of index donations and follow‐up observations

Abstract Nucleic acid testing (NAT) was implemented in Poland in 1999 for screening of plasma for fractionation and for all blood donors in 2002. To analyze seronegative NAT‐positive samples representing hepatitis C virus (HCV) window‐period (WP) in the years 2000 to 2016 and to determine infection outcome. We analyzed results of 17 502 739 donations screened in minipools (6‐48) or individually. Index samples underwent viral load (VL) quantification, genotyping and Ag, and anti‐HCV re‐testing using chemiluminescence (CMIA), electrochemiluminescence (ECLIA), and fourth‐generation enzyme‐linked immunosorbent assay (IV EIA) assays. HCV‐seronegative infections were identified in 126 donations (7.2/mln donations; 95% confidential intervals, 6.0‐8.6). Frequency of NAT yields was decreasing over time. Of the initial 126 seronegative index cases 106 were retested: 32.1% were reactive in IV EIA, 11.3% in ECLIA, and 1.9% in CMIA. The lowest VL correlated with absent anti‐HCV and HCV Ag, while VL was highest when the antigen was detectable and then it decreased when anti‐HCV appeared at a level detectable by sensitive third generation tests while retesting. The proportion of genotype 1 was 38.9% in samples positive only for HCV RNA and 71.4% in samples that were anti‐HCV reactive in re‐testing. In parallel, genotype 3 frequency was 50% in the former group and 21% in the latter. NAT is an effective measure to limit HCV transmission by transfusion and IV EIA seems to have higher clinical sensitivity than ECLIA. Samples representing likely successive phases of early HCV infection were characterized by different genotype distribution probably due to very early elimination of genotype 3.


| BACKGROUND
Poland was one of the first countries to introduce nucleic acid amplification testing (NAT) for blood donor screening. Molecular   Figure S1).

| Confirmatory testing
First step of confirmatory procedure was performed in screening laboratory. If minipool containing 24 to 48 donations was reactive, subpools prepared from 6 to 12 donations were tested with assay used for screening and in further step donations from reactive subpool were tested individually using the same assay.  available index samples 70 were tested for HCV core antigen with Architect HCV Ag assay (ABBOTT), while 36 were tested using Ortho HCV core Ag Assay (Ortho Clinical Diagnostics, Rochester).

| Other testings
All HCV RNA positive samples identified by NAT were analyzed with four different serological HCV screening assays: chemiluminescence assays (CMIA) Vitros aHCV (Ortho-Clinical Diagnostics) and Architect Anti-HCV (ABBOTT); electrochemiluminescence assay (ECLIA)-Elecsys Anti-HCV II (Roche Diagnostics GmbH, Mannheim, Germany) and fourth-generation enzyme-linked immunosorbent assay (ELISA) which was also able to detect HCV core antigen (combo assay)-Monolisa HCV Ag-Ab Ultra V2 (Bio-Rad, Marnes-la-Coquette, France). In case of a reactive result, testing was repeated twice. Re-testing was performed in plasma bags that were not accepted for clinical use due to reactive result in NAT.

| Statistical analysis
Frequency of seronegative HCV infected donations was calculated per one million with 95% confidence intervals (95% CI). Differences between two frequencies were expressed as a relative risk with 95% CI. Differences in VL due to non-normal distribution, were analyzed using the Kruskal-Wallis test.
Significant differences were observed between 31 and 40 years donors and group aged 21 to 30 and <20 years (P < .05).   Table S1.

| Follow-up of seronegative donors
However, four received antiviral treatment. Details on VL in the follow-up samples are presented in Table S1.  The observed decreasing number of seronegative HCV infections over the years may also result from improved awareness of donors as to their responsibility for blood safety as well as from reduction in the number of potential sources of infection in the population due to effective and wide available antiviral treatment. 12,13 In the current analysis, we found that the distribution of genotypes in seronegative donors has not significantly changed over the years as compared to our previous report. 1 [16][17][18] It is also worth noting that out of the third generation tests, ECLIA showed higher sensitivity compared to both CMIAs (P < .05).

| DISCUSSION
Our results allowed for characterization of the early stages of HCV infection. VL differed between donors at different stage of early infection defined by distinct diagnostics markers patterns ( Figure 3A). The lowest VL correlated with absent anti-HCV and HCV Ag, while VL was highest when the antigen was detectable and then it decreased when anti-HCV appeared at a level detectable by sensitive third generation tests while retesting.
Interestingly, changes in VL were accompanied gradual decrease in genotype 3 and increase in genotype 1 frequency.
The most likely explanation is the more frequent spontaneous clearance of genotype 3, and more frequent progression of genotype 1 infection into chronicity 19 that could also explain the domination of genotype 3a in Polish NAT yields and domination of genotype 1b among seropositive donors and patients with chronic hepatitis. 1 Our observations suggest that the process of infection elimination could take place even before the appearance of specific antibodies and thus is likely to be dependent on cellular mechanisms. This is in line with finding of multispecific and sustained cell immune response as a key determinant of HCV clearance. 20 In conclusion, we demonstrated high effectiveness of NAT in prevention of HCV transmission from seronegative donors. We observed a high, but decreasing over time, number of seronegative NAT yields among Polish blood donors. In nearly 7% of followed up donors antibodies appeared later than 50 days after index donation. The highest clinical sensitivity, as assessed by NAT yields testing, was demonstrated by the IV generation tests, while among the third generation assays, ECLIA showed higher sensitivity than CMIA. Samples representing successive stages of early HCV infection were characterized by differences in viral load and genotypes distribution.