Improved detection of HCV Infection in hemodialysis patients using a new HCV RNA qualitative assay: experience of a transplant center

Journal of Clinical Virology 30 (2004) 175–182

Improved detection of HCV Infection in hemodialysis patients using a new HCV RNA qualitative assay: experience of a transplant center Nasreen Khan a , Sali Aswad a , Hamid Shidban a , Mehbobeh Aghajani a , Ralph Mendez a , Robert Mendez a , Lorraine Comanor b,∗ a

National Institute of Transplantation, Los Angeles, CA, USA b 1801 Waverley Street, Palo Alto, CA 94301, USA

Received 14 May 2003; received in revised form 3 October 2003; accepted 8 October 2003

Abstract Background: Hepatitis C virus (HCV) is frequently a silent infection in hemodialysis (HD) patients with a prevalence of 8–10%. Improving HCV detection in this population prior to transplantation is critical both for infection control and optimal patient care. Objectives: To assess the current HCV testing practice of the National Institute for Transplantation (PCR testing of enzyme immunoassay (EIA) positive HD patients) by evaluating a subset of EIA positive and EIA negative samples with the VERSANT® HCV RNA Qualitative Assay based on transcription mediated amplification (HCV Qual (TMA)) (sensitivity ≤9.6 IU/ml) and in-house PCR (HCV Qual (PCR)) (sensitivity ∼149 IU/ml). Study design: 2321 HD patients were screened by Abbott HCV EIA 2.0. A subset of 80/169 E IA positive samples and 100/2152 EIA negative samples were tested by both assays. TMA/PCR discordant samples were genotyped. Results: PCR and TMA gave concordant results in 67/80 (83.8%) of EIA positive samples. 11/80 (14.7%) were reactive by HCV Qual (TMA), but not by HCV Qual (PCR); 2/80 (2.7%) were reactive by HCV Qual (PCR), but not by HCV Qual (TMA). 2/100 (2%) EIA negative samples were reactive and 95/100 (95%) were non-reactive by both assays. Three (3%) were only HCV Qual (TMA) reactive. 11/14 TMA+/PCR—samples with sufficient volume were genotyped. Conclusions: HCV Qual (TMA) identified active HCV infection in more EIA positive and EIA negative patients than HCV Qual (PCR) and should be part of our testing algorithm. © 2003 Elsevier B.V. All rights reserved. Keywords: Hepatitis C virus; Hemodialysis; Transplantation; Enzyme immunoassay (EIA); Transcription mediated amplification (TMA); Polymerase chain reaction (PCR)

1. Introduction Hepatitis C virus (HCV) infection remains a significant problem for the hemodialysis (HD) population. Compared to the general population which has a 0.3–1.5% prevalence of HCV infection, the reported prevalence among HD patients in the United States (US) ranges from 8% to 10% (Saab et al., 2001a,b; Tokars et al., 2000) and is considerably higher in many European and middle eastern countries. Within US dialysis centers with 40 or more patients, the reported prevalence ranges from 0% to 51% (Zacks andy Fried, 2000). Moreover, despite a decreased incidence of transfusions and adoption of universal precautions in most dialysis units, a reported annual HCV seroconversion rate ∗ Corresponding author. Tel.: +1-650-321-8871; fax: +1-650-321-8872. E-mail address: [email protected] (L. Comanor).

1386-6532/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2003.10.004

of 0.2–2.59% in HD centers is still present (Furusyo et al., 2001; Saab et al., 2001a,b). This rate appears to increase with length of time on dialysis, suggesting nosocomial or community -acquired transmission (Almroth et al., 2002; Carneiro et al., 2001; Furusyo et al., 2001; Taskapan et al., 2001; Valtuille et al., 2002). Early identification of HCV infection in HD patients is essential both for optimal patient care and infection control practice, especially in the transplant setting. Just as in the general population, HD patients with newly identified HCV infection require education about the implications of their diagnosis—the course of untreated disease, the probability of successful treatment, the possibility of transmission, the need for alcohol abstinence and immunization against hepatitis A and B, as well as the need for further work-up, including genotyping and possible liver biopsy. Even though treatment of HCV in HD patients is poorly tolerated, it can be associated with sustained virologic response and

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histological improvement. It is therefore now advocated in France prior to renal transplantation (ANAES, 2002). Treatment could at least be considered for early HCV infection, as it appears to respond better than chronic infection in the general population (Jaeckel et al., 2001; ANAES, 2002). Also approximately 50% of patients treated after grafting develop rejection (Chan et al., 1993; Koenig et al., 1994). As HCV-infected HD patients are a reservoir of infection to other patients, the HD staff, and transplant team (Pol et al., 1993; Saab et al., 2001a,b), HD centers must be aware of new HCV infections to review their practices and increase their vigilance. Over the last decade it has become increasingly apparent that routinely recommended serological and biochemical tests may fail to reveal active HCV infection in this population (Saab et al., 2001a,b; Zacks and Fried, 2000, 2001). HCV-infected HD patients may test negative for HCV antibodies either because of early stage of infection or because their compromised immune system is unable to mount an antibody response (Almroth et al., 2002; Hanuka et al., 2002). Serum alanine aminotransferase (ALT) levels are also not a reliable indication of HCV infection, since HD patients often exhibit subnormal, normal or only slightly elevated ALT levels (Fabrizi et al., 2002; Natov and Pereira, 2002; Pol et al., 1993; Saab et al., 2001a,b). Even a newly elevated ALT does not predict infection and has been shown to be specific, but not sensitive for chronic HCV infection in this population (Saab et al., 2001a,b). In addition, some PCR assays may have insufficient sensitivity to detect HCV RNA in some HD patients who have low viral levels or exhibit fluctuating patterns of viremia (Umlauft et al., 1997). PCR assays may also be adversely affected by the presence of heparin, a substance commonly used during dialysis and often present in blood samples from HD patients (Beld et al., 2001; Noiri et al., 2001). At the National Institute for Transplantation (NIT), we evaluate annually for transplantation over 1000 end-stage renal disease (ESRD) patients referred from over 200 dialysis centers. It is important for us to accurately diagnose HCV infection both prior to the patient being placed on the transplant waiting list and during the waiting-list period. Nucleic acid testing is critical to distinguish active from resolved infection; viremia may be present in 66–75% of the seropositive HD population and up to 22% of the seronegative HD population (Carneiro et al., 2001). In the past, we screened all new patients with a FDA-approved enzyme immunoassay (EIA) for the qualitative detection of antibody to hepatitis C virus, Abbott HCV EIA 2.0, and tested all positive specimens with an in-house PCR (HCV Qual (PCR)). EIA 2.0-negative pre-waiting-list patients received no further work-up for HCV infection. Waiting -list patients underwent repeat EIA testing on a yearly basis. As a result of increasing reports of silent infection in this group, we decided to evaluate the VERSANT® HCV Qualitative Assay based on transcription-mediated amplification (HCV Qual (TMA)) (Bayer Diagnostics, Tarrytown, NY)

which has a sensitivity of 5.4 IU/ml and is not affected by the presence of heparin. We studied whether HCV Qual (TMA) could identify active HCV infection in a greater number of both EIA positive and negative HD patients than HCV Qual (PCR). In a selected subset of patients from both the pre-waiting list and waiting list, we determined the number who had previously undiagnosed evidence of active infection. Finally, we proposed a new diagnostic algorithm for our patients on both lists.

2. Materials and methods 2.1. Patient population and screening methods A total of 2321 hemodialysis (HD) patients from 230 dialysis centers were screened at the NIT from January 2001 to January 2002 using Abbott HCV EIA 2.0 (Abbott Laboratories, Abbott Park, Illinois). EIA 2.0-positive samples were routinely tested for HCV RNA by HCV Qual (PCR). All patients at NIT gave consent for HCV antibody and RNA testing. Of the 169 EIA 2.0-positive samples, HCV RNA was detected by HCV Qual (PCR) in 110 (65.0%) samples and was not detected in 59 (35.0%). A subset of 80 EIA positive samples with sufficient volume was selected for additional testing with HCV Qual (TMA). In addition, 100 of the 2152 EIA 2.0-negative samples were selected randomly for testing with both HCV Qual (PCR) and HCV Qual (TMA). Three of the EIA 2.0-negative samples that had been tested by both assays had sufficient volume for further testing with another approved anti-HCV screening test, ORTHO® HCV Version 3.0 ELISA (Ortho-Clinical Diagnostics, Raritan, NJ). Additional clinical information was available on 51 of the EIA 2.0-positive patients and 60 of the EIA 2.0-negative patients. Among the 51 EIA 2.0-positive patients, there were 19 females and 32 males with an average age of 50 years. Time on dialysis ranged from <1 years to 22 years, with an average of 4.2 years. Among the 60 EIA 2.0-negative patients, there were 32 females and 28 males with an average age of 50.2 years. Time on dialysis was known for 54 EIA 1.0 negative patients and ranged from <1 years to 16 years, with an average of 3.3 years. 2.2. HCV RNA detection by PCR The presence of HCV RNA was detected by HCV Qual (PCR) using primers for the 5 UTR of the HCV genome. HCV RNA, 4 ␮l (extracted by using NucliSens automated extraction procedure Biomeriuex, NC (Boom et al., 1990; Jaeckel et al., 2001)), was reverse transcribed into cDNA. Nested PCR amplification was performed in a 50 ␮l volume using outer sense primer (5 -CCC TGT GAG GAA CTA CTG TCT TTC ACG-3 ), outer antisense primer (5 -GCT CAT GGT GCA CGG TCT CCG AGA CCT-3 ), a second set of inner sense (5 -TCT AGC CAT GGC GTT AGT ATG AGT GT-3 ) and inner antisense (5 -CAC TCG CAA

N. Khan et al. / Journal of Clinical Virology 30 (2004) 175–182

GCA CCC TAT CAG GCA GT-3 ) primers, 5 U Taq DNA polymerase (Invitrogen), in PCR buffer, and 1.5 mM MgCl2 at the following conditions: 94 ◦ C for 1 min, followed by 40 cycles of 94 ◦ C for 15 s, 55 ◦ C for 30 s, 72 ◦ C for 30 s, and then incubation of samples at 72 ◦ C for 5 min (Gene Amp PCR system 9600, Perkin-Elmer, Tarzana, CA). Final products were visualized via ethidium bromide stained agarose gel electrophoresis. Amplification of the housekeeping gene ␤-actin served as an internal control. In addition a known HCV negative and known HCV positive external control (Boston Biomedica, Inc., West Bridgewater, MA) were tested in parallel to monitor the entire procedure. The assay’s sensitivity of approximately 313 copies/ml was determined using serial dilutions of a control with a known HCV RNA concentration of 100,000 copies/ml. Using a conversion factor of 2.1 copies/IU (personal communication, Boston Biomedica), the sensitivity of the assay is equivalent to 149 IU/ml. 2.3. HCV RNA detection by TMA HCV Qual (TMA) was performed according to instructions and with kits provided by Bayer Corporation. Briefly, three steps were performed in a single tube, including target capture, target amplification by isothermal TMA, and detection of amplified product by hybridization and dual kinetic assays. In the final step, amplicons were detected using complementary single stranded probes labeled with a chemiluminescent tag. The chemiluminescent signal was read as relative light units (RLU), and data were reported as both calculated RLU and as signal-to-cutoff ratios. The specimen was considered reactive, or having detectable HCV RNA, when the signal-to-cutoff ratio was greater than one. The technology and performance of the assay has been previously well described (Comanor et al., 2001; Gorrin et al., 2003; Hendricks et al., 2003; Sarrazin et al., 2000). 2.4. HCV genotyping PCR reactive samples with sufficient volume were genotyped at NIT using the VERSANT HCV LiPA assay (Bayer Diagnostics, Tarrytown, NY; for research use only, not for use in diagnostic procedures) (Stuyver et al., 1996). Samples with TMA positive/PCR negative results and EIA negative/TMA positive samples were sent to the Bayer Reference Testing Laboratory for genotyping using the TMA–LiPA protocol (Comanor et al., 2003).

3. Results

177

All Samples (n = 80)

TMA

PCR +

-

+

43

11

-

2

24

Pre-wait list (n = 22)

TMA

PCR +

-

+

12

4

-

0

6

Wait list (n = 58)

TMA

PCR +

-

+

31

7

-

2

18

Fig. 1. Comparison of HCV RNA detection by PCR and TMA in EIA 2.0-positive HD patients.

assays and 24 (30.0%) were non-reactive. Of the 13 samples that were discordant, 11 (14.7%) were reactive by HCV Qual (TMA), but not by HCV Qual (PCR), and 2 (2.7%) were reactive by HCV Qual (PCR), but not by HCV Qual (TMA). The reactive rates for both assays (54/80 (67.5%)) for HCV Qual (TMA) and 45/80 (56.3%) for HCV Qual (PCR) were compared using McNamara’s test and found to be significantly different (P = 0.02). These results were further analyzed by subdividing HD patients according to their status on the pre-wait list or wait list. Of the 22 samples from HD patients on the pre-waiting list, 12 (54.5%) were reactive and 6 (27.3%) were non-reactive by both assays. The remaining 4 (18.2%) samples were reactive by HCV Qual (TMA), but not by HCV Qual (PCR). Of the 58 samples from HD patients on the waiting list, 31 (53.4%) were reactive and 18 (31.0%) were non-reactive by both assays. Among the nine discordant samples, seven (13.2%) were reactive by HCV Qual (TMA) but not by HCV Qual (PCR), and two (3.8%) were reactive by HCV Qual (PCR) but not by HCV Qual (TMA). Serum ALT information was available for seven of the EIA 2.0-positive HD patients prior to transplantation. Six of 7 were reactive by both HCV Qual (TMA) and HCV Qual (PCR) and had ALT levels ranging from 13 IU/ml to 118 IU/ml, with an average of 41 IU/ml. One sample was non-reactive by both assays and had an ALT of 27 IU/ml.

3.1. HCV RNA testing of EIA 2.0-positive HD patients 3.2. HCV RNA testing of EIA 2.0-negative HD patients A subset of 80 EIA 2.0-positive samples was tested by both HCV Qual (PCR) and HCV Qual (TMA). As shown in Fig. 1, 43 (53.8%) of the samples were reactive by both

A subset of 100 EIA 2.0-negative samples was tested by both HCV Qual (PCR) and HCV Qual (TMA). As shown in

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All Samples (n = 100)

TMA

+

-

+

2

3

-

0

95

Pre-wait list (n = 55)

TMA

PCR +

-

+

1

0

-

0

54

Wait list (n = 45)

TMA

were reactive by HCV Qual (TMA) and one was also reactive by HCV Qual (PCR).

PCR

PCR +

-

+

1

3

-

0

41

Fig. 2. Comparison of HCV RNA detection by PCR and TMA in EIA 2.0-negative HD patients.

Fig. 2, 2 (2.0%) of the samples were reactive and 95 (95.0%) were non-reactive by both assays. The remaining 3 (3.0%) were reactive by HCV Qual (TMA) but not by HCV Qual (PCR). Unfortunately, the reactive rate in this population is low enough to make it difficult to obtain statistically significant reactive rates. Serum ALT information was available for three EIA 2.0-negative HD patients. Levels ranged from 8 IU/ml to 28 IU/ml, with an average of 16 IU/ml. All three patients

3.3. HCV genotyping and resolution of discordant samples HCV genotyping by TMA–LiPA was performed to further characterize discordant samples which were reactive by HCV Qual (TMA) but not by HCV Qual (PCR). Eleven of 14 discordant samples had sufficient volume for additional testing. Table 1 shows the HCV genotype and patient status for each of these discordant samples. Two discordant samples from EIA 2.0-positive HD patients were reactive by HCV Qual (PCR) but not by HCV Qual (TMA). Unfortunately, genotype information was not available for these samples and there was insufficient sample volume for additional testing by TMA–LiPA. HCV genotype also was determined by TMA–LiPA for 2 EIA 2.0-negative samples that had HCV RNA detectable by both HCV Qual (PCR) and HCV Qual (TMA). Combining all genotyping results, HCV genotypes were determined for 51 patients and were distributed as follows: 2 (3.9%) genotype 1, 23 (45.1%) genotype 1a, 18 (35.3%) genotype 1b, 2 (3.9%), genotype 2a/2c, 2 (3.9%) genotype 2b, 5 (9.8%) genotype 3a. 4. Discussion HCV infection is the leading cause of post transplant chronic liver disease and is the third leading cause of death in long-term kidney transplant recipients (Hanafusa et al., 1998; Pereira et al., 1998). In the post transplant period, HCV infection is also associated with a higher incidence of bacterial infection, glucose intolerance, proteinuria, and chronic allograft nephropathy (Bloom et al., 2002; Hestin

Table 1 Resolution of TMA (+) PCR (−) discordant results Sample no.

HCV genotype

Patient status

EIA 2.0-positive 392 211 1133 230 577 1407 209 2410 643 571 699

1a 1a 1a 1a 1a 1b 1 3a 3a nt∗ nt∗

Pre-wait list Pre-wait list Wait list at time Wait list at time Pre-wait list Pre-wait list Wait list at time Wait list Wait list Wait list at time Wait list at time

EIA 2.0-negative 1464 1361

1b 1b

Wait list, original sample tested positive by ELISA 3.0 Wait list at time of study, subsequently transplanted, sample taken at later time point tested positive by ELISA 3.0 Wait list

860 ∗

Not typed.

nt∗

of study, subsequently transplanted of study, subsequently transplanted

of study, subsequently transplanted

of study, subsequently transplanted of study, subsequently transplanted

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et al., 1998). Although HCV posititvity does not appear to impact immediate post transplantation survival, HCV positive patients have dramatically decreased survival 10 years after transplantation compared to their anti-HCV negative counterparts (Batty et al., 2001; Hanafusa et al., 1998; Mathurin et al., 1999). Nevertheless, HCV-infected patients continue to be transplanted at most centers, including ours (Schweitzer et al., 1993), as they appear to survive longer after renal grafting than with continued dialysis (Pereira et al., 1998). However, their placement on our waiting list depends partially on their hepatic evaluation, and our ESRD patients are not referred to the hepatologist unless liver disease, or its causative agent, is identified. Prompt identification of HCV infection is important, because it affects the survival of both long-term HD patients and renal transplant recipients (Espinosa et al., 2001; Fabrizi et al., 2002). More importantly, it now appears that that both transplant candidates and non-candidates may benefit from antiviral therapy (Degos et al., 2001; Gursoy et al., 2001; Huraib et al., 2001; Izopet et al., 1997). For candidates, viral eradication prior to grafting may decrease post-operative liver deterioration and de novo glomerulonephritis. Also post transplant therapy is associated with an average 40% incidence of acute renal failure accompanied by a 50% loss of graft, even with immediate withdrawal of interferon (Rostaing, 2000). In non-candidates with advanced fibrosis, interferon may reduce fibrosis, even if viral eradication is not achieved (Casanovas-Taltavull et al., 2001; Degos et al., 2001; Pol, 1995; Rostaing, 2000). Regardless of transplant candidacy, acute HCV infection should probably be treated, as it appears to have the greatest chance of response. (Gursoy et al., 2001; Jaeckel et al., 2001). The ability of HCV Qual (TMA) to identify active HCV infection in a greater percentage of both the pre-waiting list and the waiting-list populations than did our in-house PCR is not surprising given the improved sensitivity of the HCV Qual (TMA) assay compared to other PCR assays shown in previous studies (Comanor et al., 2001; Sarrazin et al., 2000). One limitation of our study is that we only compared the HCV Qual (TMA) assay to our in-house PCR. Nonetheless, in-house PCR assays are very much in use and currently accepted by the CDC in the diagnostic setting as supplemental tests (Alter et al., 2003). The discordant TMA reactive/PCR non-reactive samples in our study appeared to be true positives, because all with sufficient volume were successfully genotyped using the TMA–LiPA protocol (Comanor et al., 2003). Also the resulting genotypes were representative of those found in the dialysis population in North America. The two discordant samples that were PCR reactive/TMA non-reactive were tested by PCR some months prior to evaluation by the HCV Qual TMA assay. The handling of these specimens, which is integral to the integrity of the HCV RNA, is not known; they were definitely exposed to more freeze–thaw cycles after the PCR testing and may have been held at room temperature for some time.

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Although few ALT measurements were available on our study patients, we observed the reported trend of normal or near normal ALT values in most EIA positive and negative patients infected with HCV (Chan et al., 1993; Natov and Pereira, 2002; Pol et al., 1995; Saab et al., 2001a,b). This trend supported our impression that ALT measurements are inadequate for monitoring acquisition of HCV infection in this population. In a recently published guideline for HCV antibody testing, the CDC endorsed both the Abbott HCV EIA 2.0 assay and the ORTHO Version 3.0 ELISA, stating both FDA-approved assays were suitable for anti-HCV screening. (Alter et al., 2003). Both employ HCV-encoded recombinant antigens; the Abbott EIA uses c100-3, HC-31 and HC-34 from the core, NS3 and NS4 epitopes, while the ORTHO Version 3.0 ELISA adds the NS5 to the other three epitopes, using the C22-3, c200 and NS5 antigens (Pawlotsky, 2002). Unfortunately, we only had sufficient volume to test 3 EIA 2.0-negative/HCV Qual (TMA) reactive samples with the ELISA 3.0 test; two of three were positive and were successfully genotyped. The third ELISA 3.0-negative sample belonged to a patient who subsequently seroconverted 6 months later. Although some investigators have found only minimal increase in sensitivity between the second and third generation ELISA tests in this population (Courouce et al., 1995), others have found the third generation assays to be more sensitive and specific and to detect seroconversion at earlier time points. In addition, the third generation assays also detect presence of antibodies in individuals who either seroconvert very slowly by HCV 2.0 EIA or who a limited serological response to HCV antigens. (Abdel-Hamid et al., 2002; Barrera et al., 1995; Fabrizi et al., 2002; Soffredini et al., 1996; Tobler et al., 2003). Nevertheless, ELISA 3.0-negative/PCR positive patients are still readily observed in the HD population and contribute to the difficulty of identification of active HCV infection prior to grafting (Hinrichsen et al., 2002). Given the findings of our study and the ease of use of HCV Qual (TMA) assay, we are considering adopting the following algorithm for evaluation of both new pre-waiting list and waiting-list patients (Fig. 3a and b). All new pre-waiting-list patients would be initially screened by an approved EIA or ELISA test. ELISA negative patients would undergo further HCV testing only when and if they achieved waiting-list status. ELISA positive patients would also be tested by HCV Qual (TMA). HCV Qual (TMA) non-reactive patients would receive no HCV further work-up and would be considered for the waiting list. HCV Qual (TMA) reactive patients would undergo further evaluation to determine if their liver function was adequate for waiting-list status. Waiting-list patients who are both ELISA 3.0 and HCV Qual (TMA) reactive would receive an annual, or as needed, evaluation of their liver status. Those who are ELISA 3.0 positive/TMA non-reactive would undergo HCV Qual (TMA) testing semi-annually and would undergo further

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All new patients EIA

+

–

TMA testing

End of HCV work-up consider for wait list

+ Evaluation of liver status

Patient not suitable candidate for transplantation

– End of HCV work-up consider for wait list

Consider for wait list

(a)

Patients on Waiting List EIA + TMA (q 6 mos.)

+

–

Maintain on waiting list

TMA (q 6 mos)

+

–

Maintain on waiting list

Evaluation of liver status (annually or as needed) Maintain on waiting list

(b)

Remove from waiting list Fig. 3. (a) All new patients and (b) patients on waiting list.

evaluation, if the results were reactive, to determine their suitability for treatment and maintenance on the waiting list. ELISA negative patients who have not been tested by HCV Qual (TMA) would undergo repeat ELISA and TMA testing semi-annually. An ELISA negative patient who became positive by HCV Qual (TMA) would have follow-up ELISA testing to see if seroconversion takes place. Any patient who became reactive by either test would undergo prompt further evaluation of their liver status. (In the case of new infections, nucleic acid tests are likely to be positive 4–6 weeks prior to seroconversion as demonstrated by an

ELISA test (Krajden, 2000).) We feel monitoring patients on a 6 months basis is justified, as the average wait time for a cadaver kidney now approaches 5 years (Matas and Delmonico, 2001), and HD patients have continual potential exposure to infection. Removal of a patient from the waiting list because of newly identified HCV infection would be unusual. However, a small percentage of recently diagnosed patients will have advanced liver disease that may preclude their candidacy for a single renal transplant (Sterling et al., 1999), but may suggest a combined renal/liver transplant.

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Our algorithms for the HD population differ slightly from the single one recently proposed by the CDC for HCV antibody testing in the general population. In the CDC algorithm, negative EIA screening results are simply reported and no further action is required. Positive EIAs are confirmed either by RIBA or by a NAT and NAT negative results are followed by RIBA to confirm past infection (Alter et al., 2003). We also require no further HCV evaluation for new patients who test EIA negative prior to their being placed on the waiting list. For those with EIA positive results, we recommend testing directly with the most sensitive commercially available NAT, HCV Qual (TMA) (Krajden, 2000), as RIBA is unable to confirm active infection and gives approximately 10% indeterminate results, requiring further testing. (Pawlotsky et al., 1996). We suggest that EIA negative patients on the waiting list be tested with both EIA and HCV Qual (TMA) every 6 months, as a known percentage of HCV actively infected HD patients remain HCV antibody negative (Almroth et al., 2002; Hanuka et al., 2002; Hinrichsen et al., 2002) and HCV Qual (TMA) is FDA approved as a confirmatory diagnostic test, not a screening test. Patients who have been EIA positive/TMA negative prior to being placed on the waiting list should also undergo HCV Qual (TMA) testing every 6 months to see if they become viremic. Additional NAT testing will only slightly increase the healthcare costs of the HD population and the possibility of identifying active infection in a few more patients prior to transplantation seems worth the small extra expense. In summary, we believe adoption of an HCV screening and diagnostic algorithm that follows EIA or ELISA tests with HCV Qual (TMA) will improve HD patient care at all stages. Early diagnosis should increase successful treatment prior to transplantation and perhaps decrease graft rejection. It may allow the HCV positive candidate to receive an HCV positive graft, reducing the waste of HCV positive kidneys and usually decreasing time on the waiting list. (Mandal et al., 2000; Morales et al., 1995, 2000). Finally, reporting of newly identified infections to the dialysis centers should improve infection control practices. These are all compelling reasons for improving detection of HCV infection in the HD population. Acknowledgements We wish to thank Claudia Elkin and Kimmy Leung for genotyping specimens using the TMA LiPA protocol, Linda Wuestehube for writing and editorial assistance, Kristi Whitfield for graphics, and Michel Friesenhahn for statistical input.

References Abdel-Hamid M, El-Daly M, El-Kafrawy S, Mikhail N, Strickland GT, Fix AD. Comparison of second- and third-generation enzyme immunoas-

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says for detecting antibodies to hepatitis C virus. J Clin Microbiol 2002;40:1656–9. Almroth G, Ekermo B, Mansson AS, Svensson G, Widell A. Detection and prevention of hepatitis C in dialysis patients and renal transplant recipients. A long-term follow up (1989–January 1997). J Intern Med 2002;251:119–28. Alter MJ, Kuhnert WL, Finelli L. Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. Centers for Disease Control and Prevention. MMWR Recomm Rep 2003;52:1–13, 15; quiz CE1-4. ANAES. In: Proceedings of the Consensus Conference. Treatment of hepatitis C; Gastroenterol Clin Biol 2000;26(Spec. No. 2):B303–20. Barrera JM, Francis B, Ercilla G, Nelles M, Achord D, Darner J. Improved detection of anti-HCV in post-transfusion hepatitis by a third-generation ELISA. Vox Sang 1995;68:15–8. Batty Jr DS, Swanson SJ, Kirk AD, Ko CW, Agodoa LY, Abbott KC. Hepatitis C virus seropositivity at the time of renal transplantation in the United States: associated factors and patient survival. Am J Transplant 2001;1:179–84. Beld Robers S, Dijkman-de Haan K, Weel J, Sol C, Boom R. Clinical utility of Bayer VERSANT HCV RNA qualitative (TMA) assay above the Roche COBAS AMPLICOR 2.0 assay for detection of HCV RNA in EDTA plasma from dialysis patients. Hepatology 2001;34(Suppl 4):227A. Bloom RD, Rao V, Weng F, Grossman RA, Cohen D, Mange KC. Association of hepatitis C with posttransplant diabetes in renal transplant patients on tacrolimus. J Am Soc Nephrol 2002;13:1374–80. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28:495–503. Carneiro MA, Martins RM, Teles SA, Silva SA, Lopes CL, Cardoso DD, et al. Hepatitis C prevalence and risk factors in hemodialysis patients in central Brazil: a survey by polymerase chain reaction and serological methods. Membr Inst Oswaldo Cruz 2001;96:765–9. Casanovas-Taltavull T, Baliellas C, Benasco C, Serrano TT, Casanova A, Perez JL, et al. Efficacy of interferon for chronic hepatitis C virus-related hepatitis in kidney transplant candidates on hemodialysis: results after transplantation. Am J Gastroenterol 2001;96:1170–7. Chan TM, Lok AS, Cheng IK, Chan RT. Prevalence of hepatitis C virus infection in hemodialysis patients: a longitudinal study comparing the results of RNA and antibody assays. Hepatology 1993;17:5–8. Comanor L, Anderson F, Ghany M, Perrillo R, Heathcote EJ, Sherlock C, et al. Transcription-mediated amplification is more sensitive than conventional PCR-based assays for detecting residual serum HCV RNA at end of treatment. Am J Gastroenterol 2001;96:2968–72. Comanor L, Elkin C, Leung K, Krajden M, Kronquist K, Nicolas K, et al. Successful HCV genotyping of previously failed and low viral load specimens using an HCV RNA qualitative assay based on transcription-mediated amplification in conjunction with the line probe assay. J Clin Virol 2003;28:14–26. Courouce AM, Bouchardeau F, Chauveau P, Le Marrec N, Girault A, Zins B, et al. Hepatitis C virus (HCV) infection in haemodialysed patients: HCV-RNA and anti-HCV antibodies (third-generation assays). Nephrol Dial Transplant 1995;10:234–9. Degos F, Pol S, Chaix ML, Laffitte V, Buffet C, Bernard PH, et al. The tolerance and efficacy of interferon-alpha in haemodialysis patients with HCV infection: a multicentre, prospective study. Nephrol Dial Transplant 2001;16:1017–23. Espinosa M, Martin-Malo A, Alvarez de Lara MA, Aljama P. Risk of death and liver cirrhosis in anti-HCV-positive long-term haemodialysis patients. Nephrol Dial Transplant 2001;16:1669–74. Fabrizi F, Poordad FF, Martin P. Hepatitis C infection and the patient with end-stage renal disease. Hepatology 2002;36:3–10. Furusyo N, Hayashi J, Kakuda K, Ariyama I, Kanamoto-Tanaka Y, Shimizu C, et al. Acute hepatitis C among Japanese hemodialysis patients: a prospective 9-year study. Am J Gastroenterol 2001;96:1592– 600.

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Gorrin G, Friesenhahn M, Lin P, Sanders M, Pollner R, Eguchi B, et al. Performance evaluation of the VERSANT HCV RNA qualitative assay by using transcription-mediated amplification. J Clin Microbiol 2003;41:310–7. Gursoy M, Gur G, Arslan H, Ozdemir N, Boyacioglu S. Interferon therapy in haemodialysis patients with acute hepatitis C virus infection and factors that predict response to treatment. J Viral Hepat 2001;8:70–7. Hanafusa T, Ichikawa Y, Kishikawa H, Kyo M, Fukunishi T, Kokado Y, et al. Retrospective study on the impact of hepatitis C virus infection on kidney transplant patients over 20 years. Transplantation 1998;66:471– 6. Hanuka N, Sikuler E, Tovbin D, Mostoslavsky M, Hausman M, Orgel M, et al. Hepatitis C virus infection in renal failure patients in the absence of anti-hepatitis C virus antibodies. J Viral Hepat 2002;9:141–5. Hendricks DA, Friesenhahn M, Tanimoto L, Goergen B, Dodge D, Comanor L. Multicenter evaluation of the VERSANT HCV RNA qualitative assay for detection of hepatitis C virus RNA. J Clin Microbiol 2003;41:651–6. Hestin D, Guillemin F, Castin N, Le Faou A, Champigneulles J, Kessler M. Pretransplant hepatitis C virus infection: a predictor of proteinuria after renal transplantation. Transplantation 1998;65:741–4. Hinrichsen H, Leimenstoll G, Stegen G, Schrader H, Folsch UR, Schmidt WE. Prevalence and risk factors of hepatitis C virus infection in haemodialysis patients: a multicentre study in 2796 patients. Gut 2002;51:429–33. Huraib S, Iqbal A, Tanimu D, Abdullah A. Sustained virological and histological response with pretransplant interferon therapy in renal transplant patients with chronic viral hepatitis C. Am J Nephrol 2001;21:435–40. Izopet J, Rostaing L, Moussion F, Alric L, Dubois M, That HT, et al. High rate of hepatitis C virus clearance in hemodialysis patients after interferon-alpha therapy. J Infect Dis 1997;176:1614–7. Jaeckel E, Cornberg M, Wedemeyer H, Santantonio T, Mayer J, Zankel M, et al. Treatment of acute hepatitis C with interferon alfa-2. N Engl J Med 2001;345:1452–7. Koenig P, Vogel W, Umlauft F, Weyrer K, Prommegger R, Lhotta K, et al. Interferon treatment for chronic hepatitis C virus infection in uremic patients. Kidney Int 1994;45:1507–9. Krajden M. Hepatitis C virus diagnosis and testing. Can J Public Health 2000;91(Suppl 1):S34–9. Mandal AK, Kraus ES, Samaniego M, Rai R, Humphreys SL, Ratner LE, et al. Shorter waiting times for hepatitis C virus seropositive recipients of cadaveric renal allografts from hepatitis C virus seropositive donors. Clin Transplant 2000;14:391–6. Matas AJ, Delmonico FL. Transplant kidneys sooner: discard fewer kidneys. Am J Transplant 2001;1:301–4. Mathurin P, Mouquet C, Poynard T, Sylla C, Benalia H, Fretz C. Impact of hepatitis B and C virus on kidney transplantation outcome. Hepatology 1999;29:257–63. Morales JM, Campistol JM, Castellano G, Andres A, Colina F, Fuertes A, et al. Transplantation of kidneys from donors with hepatitis C antibody into recipients with pre-transplantation anti-HCV. Kidney Int 1995;47:236–40. Morales JM, Campistol JM, Andres A, Dominguez-Gil B, Esforzado N, Munoz MA. Policies concerning the use of kidneys from donors infected with hepatitis C virus. Nephrol Dial Transplant 2000;15(Suppl 8):71–3. Natov SN, Pereira BJ. Management of hepatitis C infection in renal transplant recipients. Am J Transplant 2002;2:483–90.

Noiri E, Nakao A, Oya A, Fujita T, Kimura S. Hepatitis C virus in blood and dialysate in hemodialysis. Am J Kidney Dis 2001;37:38–42. Pawlotsky JM, Bastie A, Pellet C, Remire J, Darthuy F, Wolfe L, et al. Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay. J Clin Microbiol 1996;34:80–3. Pereira BJ, Natov SN, Bouthot BA, Murthy BV, Ruthazer R, Schmid CH, et al. Effects of hepatitis C infection and renal transplantation on survival in end-stage renal disease. The New England Organ Bank Hepatitis C Study Group. Kidney Int 1998;53:1374–81. Pol S, Romeo R, Zins B, Driss F, Lebkiri B, Carnot F, et al. Hepatitis C virus RNA in anti-HCV positive hemodialyzed patients: significance and therapeutic implications. Kidney Int 1993;44:1097–100. Pol S, Thiers V, Carnot F, Zins B, Romeo R, Berthelot P. Efficacy and tolerance of alpha-2b interferon therapy on HCV infection of hemodialyzed patients. Kidney Int 1995;47:1412–8. Rostaing L. Treatment of hepatitis C virus infection after renal transplantation: new insights. Nephrol Dial Transplant 2000;15(Suppl 8):74–6. Saab S, Brezina M, Gitnick G, Martin P, Yee Jr HF. Hepatitis C screening strategies in hemodialysis patients. Am J Kidney Dis 2001a;38:91–7. Saab S, Martin P, Brezina M, Gitnick G, Yee Jr HF. Serum alanine aminotransferase in hepatitis C screening of patients on hemodialysis. Am J Kidney Dis 2001b;37:308–15. Sarrazin C, Teuber G, Kokka R, Rabenau H, Zeuzem S. Detection of residual hepatitis C virus RNA by transcription-mediated amplification in patients with complete virologic response according to polymerase chain reaction-based assays. Hepatology 2000;32:818–23. Schweitzer EJ, Bartlett ST, Keay S, Hadley GA, Cregar J, Stockdreher DD. Impact of hepatitis B infection on the practice of kidney transplantation in the United States. Transplant Proc 1993;25:1456–7. Soffredini R, Rumi M, Lampertico P, Aroldi A, Tarantino A, Ponticelli C, et al. Increased detection of antibody to hepatitis C virus in renal transplant patients by third-generation assays. Am J Kidney Dis 1996;28:437–40. Sterling RK, Sanyal AJ, Luketic VA, Stravitz RT, King AL, Post AB, et al. Chronic hepatitis C infection in patients with end stage renal disease: characterization of liver histology and viral load in patients awaiting renal transplantation. Am J Gastroenterol 1999;94:3576– 82. Stuyver L, Wyseur A, van Arnhem W, Hernandez F, Maertens G. Second-generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol 1996;34:2259–66. Taskapan H, Oymak O, Dogukan A, Utas C. Patient to patient transmission of hepatitis C virus in hemodialysis units. Clin Nephrol 2001;55:477– 81. Tobler LH, Stramer SL, Lee SR, Masecar BL, Peterson JE, Davis EA, et al. Impact of HCV 3.0 EIA relative to HCV 2.0 EIA on blood-donor screening. Transfusion 2003;43:1452–9. Tokars JI, Miller ER, Alter MJ, Arduino MJ. National surveillance of dialysis-associated diseases in the United States, 1997. Semin Dial 2000;13:75–85. Umlauft F, Gruenewald K, Weiss G, Kessler H, Urbanek M, Haun M, et al. Patterns of hepatitis C viremia in patients receiving hemodialysism. Am J Gastroenterol 1997;92:73–8. Valtuille R, Moretto H, Lef L, Rendo P, Fernandez JL. Decline of high hepatitis C virus prevalence in a hemodialysis unit with no isolation measures during a 6-year follow-up. Clin Nephrol 2002;57:371–5. Zacks S, Fried M. Hepatitis C in renal disease. In: Liang TJ, Hoofnagle JH, editors. Biomedical research reports. San Diego: Academic Press; 2000. p. 329–49.