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Letters |
1
MEDICANALYSIS Institute of Molecular Biology Applications, GR-15121 Athens, Greece
2
Nursing Military Academy, Laboratory of Research, GR-16201 Athens, Greece
3
University of Athens, School of Medicine, Laboratory of Histology, and Embryology, GR-11527 Athens, Greece
4
"401" General Army Hospital, Department of Pathology, GR-11525 Athens, Greece
5
"HYGEIA" Therapeutic Center, Unit of Nephrology, GR-15123 Athens, Greece
a Address correspondence to this author at: Erasmus University Rotterdam, Faculty of Medicine, Department of Cell Biology, PO Box 1738, 3000 DR, Rotterdam, The Netherlands. E-mail patrinos{at}ch1.fgg.eur.nl
To the Editor:
The prevalence of anti-hepatitis C virus (HCV) antibody among patients on hemodialysis (HD) is consistently higher than in healthy populations, suggesting that dialysis patients may be at a higher risk of acquiring HCV infection (1).
At present, the diagnosis of HCV infection relies mostly on immunoserological screening assays (ELISA) because they combine a direct evaluation of immune anti-HCV response, simplicity in both handling and performance, and a substantially lower cost (2). However, anti-HCV positivity may indicate past infection, current infection, or even nonspecific reactivity (3). HCV RNA determination through qualitative reverse transcription-PCR (RT-PCR) screening assays allows direct HCV detection before any serological alteration, including generation of antibodies or an increase in aminotransferase (4). In fact, several recent studies have demonstrated that de novo cases of HCV infection occur in HD units in the absence of other parenteral exposure (5). This finding underlines the spread of HCV among HD patients and the need for HCV RNA screening assays in the diagnosis of acute HCV infection in seronegative HD patients (6). Until recently, both qualitative and quantitative RT-PCR techniques had no practical application in diagnostic laboratories. Recently, several commercial diagnostic kits have been developed, offering high reproducibility and reliability (7). However, applying HCV genetic testing by means of an approved HCV RNA detection assay to each HD patient often is not suitable because it is a relatively expensive assay and should be performed frequently if acute infection is to be detected. Recently, transfusion services have applied nucleic acid amplification technology by means of the "minipool" methodology (8). In this screening strategy, samples are pooled together and tested at once. Hence, we investigated whether pooling candidate HCV samples before testing them individually, by means of a minipool RT-PCR methodology, may decrease the cost without markedly decreasing the sensitivity of the assay.
We examined 200 HD patients (age, 46.5 ± 18 years; 96 males and 94 females) three consecutive times over a 1.5-year follow-up period for the presence of anti-HCV antibodies and HCV RNA. The procedures followed were approved by our institution’s responsible committee.
HCV RNA qualitative determination was performed in three well-isolated areas (sample preparation, amplification, and detection) to avoid contamination. Specimen preparation was performed with an optimized isopropanol-based method, and detection of HCV RNA was performed by a nonisotopic method adapted to the microwell format, as instructed by the manufacturer (HCV AmplicorTM, La Roche). Both positive and negative controls were treated as unknown samples and were included in the initial step of sample preparation. For the minipool RT-PCR qualitative determination, we produced 20 master pools of 10 samples each. If a minipool was reported as HCV RNA negative, then no further HCV RNA evaluation was performed. On the other hand, if a minipool was reported as HCV RNA positive, it was divided in two smaller primary pools of five samples each. Subsequently, if a five-sample minipool was reported as positive, each of the five samples was tested separately for the presence of HCV RNA. In the minipool methodology, the RNA extraction step was performed separately for each sample. Samples were pooled together before the cDNA amplification step. Before the minipool testing, positive controls were diluted 10-fold and tested separately to confirm the sensitivity of the assay. The remaining procedure was performed according to the manufacturer’s instructions supplied by the provided protocol.
Serum samples were tested three consecutive times during the study period for the presence of anti-HCV antibodies by the ORTHO ELISA III (ELISA III HCV; Ortho Clinical Systems) and the MONOLISA anti-HCV New Ag (Monolisa HCV; Sanofi Diagnostics Pasteur) assays. The ORTHO ELISA III detects antibodies directed to core, nonstructural 3 (NS3), NS4, and NS5 antigens; the MONOLISA anti-HCV plus detects antibodies directed to core, NS3, and NS4 antigens. Both assays were performed according to the manufacturers’ instructions. Low- and high titer-positive controls were included in each assay, and these were always positive. Detection of anti-HCV antibodies (c33, NS5, C22p, and c100p) by means of the RIBA third-generation assay was also performed three consecutive times according to the manufacturer’s instructions (Chiron). Reverse transcription, PCR amplification, and detection and colorimetric quantification (based on a method adapted to the microwell format) were performed simultaneously, with the incorporation of an internal standardized target sequence control according to the manufacturer’s instructions (HCV-MonitorTM Test; La Roche). Results were expressed as viral copies/mL of human serum.
Results obtained with the minipool RT-PCR methodology were HCV positive for 16 of 200 HD patients (8%). All 16 were found anti-HCV positive by the ORTHO ELISA III, MONOLISA anti-HCV plus, and RIBA assays. Quantitative RT-PCR analysis showed that 3 of 16 PCR-positive samples had viral loads of >500 000 copies/mL and that each patient’s viral load remained relatively stable in three consecutive measurements.
Some of the difficulties in formulating strategies to control the transmission of HCV infection include the high prevalence of HCV in HD patients, the limitations of immunoserological tests in identifying these patients, and the uncertainties regarding the modes of transmission within dialysis units. At this point, detection of acute HCV infection in seronegative HD patients by the highly sensitive RT-PCR screening assay may represent a critical step in preventing patient-to-patient HCV transmission. Pooling candidate HCV samples may have certain limitations in detecting low concentrations of HCV RNA. This inadequacy may be further strengthened by the nonrandom distribution of HCV quasispecies in plasma and peripheral blood mononuclear cell subsets (9). A role for additional refinement of a HCV diagnosis using tests to determine genotype and subtype as well as quasispecies should be explored. We cannot confirm the sensitivity of this methodology in samples with low HCV RNA; the cutoff for the quantitative HCV RNA determination assay for this study was <2000 viral copies/mL. However, quantitative assays are not intended for use in HCV diagnosis. These tests, in conjunction with the clinical presentation and other laboratory markers, may provide an aid in assessing viral response to antiviral treatment as measured by changes in serum or plasma HCV RNA concentrations. In essence, our findings suggest that pooling candidate HCV samples before testing them individually decreases the time and cost of qualitative RT-PCR analysis up to 50% without reducing sensitivity. In seronegative HD patients, this screening approach may be helpful in identifying currently HCV-infected patients, thus allowing a higher flexibility in implementing policies to prevent patient-to-patient HCV transmission.
Acknowledgments
This work was supported by the Department of Research of MEDICANALYSIS Institute of Molecular Biology Applications (MIMBA 99-02).
References
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