Evidence against hepatitis C virus trapping in dialysis membranes

Claudio Angelini, Salvatore Badalamenti, Giovanna Lunghi1, Maurizio Sampietro1, Silvia Finazzi2, Claudio Ponticelli2 and Giorgio Graziani

Unità Operativa di Nefrologia e Dialisi, Istituto Clinico Humanitas, 1 Istituto di Igiene, Medicina Interna, 2 Divisione di Nefrologia, IRCCS Ospedale Maggiore, Milano, Italy

Sir,

Haemodialysis (HD) patients have a high prevalence of hepatitis C virus (HCV) infection due to blood transfusions or nosocomial transmission [1,2]; however, the clinical course of HCV-associated liver disease in HD patients is usually mild [3,4]. Recent studies have described a consistent reduction of plasma HCV-RNA levels after dialysis [57], independently of the membranes and anticoagulants used, suggesting that HD per se could protect patients from an aggressive course of HCV infection. The mechanism by which HD induces the reduction of viraemia remains largely speculative. Two major mechanisms may be involved: (i) a mechanical or physicochemical phenomenon linked to the dialysis technique and (ii) biological changes induced by the host response.

The possible passage of viral particles across the membrane into the dialysate has been investigated previously in our and other laboratories [7,8]. This probably occurs only exceptionally because of the size difference between viral particles and the membrane pores. Only the presence of microlesions in some hollow fibres may allow the transfer of viral particles from blood to dialysate. However, this should involve only few virions and is therefore probably not the cause of the massive reduction of viraemia observed during the dialysis session. Okuda et al. [5] suggested trapping of HCV on the membrane surface during dialysis.

To address this question, we selected 15 non-uraemic patients with HCV-related chronic hepatitis submitted to therapeutic blood letting and used ~400 ml of blood from each patient for a series of 15 haemodialysis procedures in vitro. Blood was collected in bags with Na2 EDTA and immediately used in an in vitro dialysis session, in a closed loop, with the following schedule: blood flow 300 ml/min; dialysate flow 500 ml/min; temperature 37°C; ionic composition (mmol/l) Na+ 138, K+ 3.0, Cl- 110, NaHCO3 31, CH3COONa 5.0, Ca2+ 2.0 and Mg 0.75, with a theoretical osmolality of 290.25 mOsm/l. No haemofiltration was planned and spontaneously occurring haemofiltration was restored by saline infusion, thereby maintaining a constant blood volume of ~400 ml. Membranes employed were ethylenevinyl alcohol (EVAL) in seven experiments, Gambrane (four experiments), Hemophan (two experiments), polyacrylonitrile (PAN-AN69) and polymethylmetacrylate (PMMA) in one experiment. A 2 ml aliquot of blood was collected at baseline and every 30 min for 300 min for the determination of HCV-RNA. The latter was performed using a commercial assay based on competitive amplification with an internal standard (Amplicor HCV Monitor TM, Roche Diagnostic System, Branchburg, NJ, USA). Table 1Go shows the results of HCV viraemia findings from the 15 experiments performed. Only slight reductions of viraemia were seen (P=NS).


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Table 1. HCV viraemia (viral equivalents/ml) in 15 in vitro HD sessions performed with blood from patients with HCV-related chronic hepatitis

 
In another five experiments, all performed with an EVAL 1.2 m2 membrane, blood was submitted for 300 min to haemofiltration without dialysate (100 ml of ultrafiltrate/h, replaced by an equal amount of saline). Again, no changes in HCV-RNA levels were observed.

On one occasion, we studied a PMMA filter immediately after a haemodialysis session in an HCV-positive patient on intermittent HD treatment. The filter was perfused for 10 min with 500 ml of lysing solution, in a closed loop, in order to release viral RNA [9]. Quantitation of the viral genome concentration in three 1 ml fractions of this solution yielded negative results: only 0.1% of the viral equivalents ‘lost’ from patient plasma could be recovered in the eluate from the filter. In fact, the patient's viraemia was 20000 and 1300 viral eq/ml, respectively, before and after HD, with an estimated loss (for 2.76 l of plasma) of 5x107 viral genomes (Table 2Go). The quantitation of viral genomes of the lysing solution consistently yielded values of 103 genomes/ml, with ~5x105 genomes contained in 500 ml.


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Table 2. HCV viraemia of three HCV-positve patients at baseline conditions and after dialysis, and viral particles recovered from membranes

 
Finally, another two attempts to release HCV-RNA from sections of EVAL filters after dialysis of HCV-positive patients yielded negative results, with undetectable HCV-RNA levels in the lysing solution (Table 2Go).

In conclusion, our data do not support the hypothesis that HCV virus is trapped in HD membranes during dialysis of HCV-positive viraemic HD patients on chronic HD treatment. Therefore, alternative possibilities such as as an indirect host-mediated mechanism must be explored to explain the reduction of HCV viral load during HD, by in vivo studies aimed at examining the relationship between endogenous substances with antiviral properties and HCV load during HD.

References

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  7. Noiri E, Nakao A, Oya A, Fujita T, Kimura S. Hepatitis C virus in blood and dialysate in hemodialysis. Am J Kidney Dis2001; 37: 38–42[ISI][Medline]
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  9. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal Biochem1987; 162: 156–169[ISI][Medline]




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