Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 23, Num. 2, 2005, pp. 106-110

Indian Journal of Medical Microbiology, Vol. 23, No. 2, April-June, 2005, pp. 106-110

Original Article

Prevalence of HCV Infection in Patients on Haemodialysis: Survey by Antibody and Core Antigen Detection

Reddy A.K., Murthy K.V.D., Lakshmi V.

Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad

Correspondence Address: Department of Microbiology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad -500 082, lgorthi@hotmail.com

Code Number: mb05028

Abstract

Purpose: The present study was undertaken to determine the prevalence of HCV infection by antibody testing and HCV core antigen (HCVcAg) determination by ELISA in haemodialysis patients and to evaluate the HCV c Ag assay in the detection of HCV infected patients on haemodialysis.
Materials and Methods: A total of 151 chronic renal failure patients on haemodialysis from May 2003 to October 2004 were studied. One hundred patients out of 151 were followed for 2-5 months. All the patients were tested for anti HCV and HCV core antigen once a month. Anti HCV ELISA positive specimens were confirmed by RIBA.®
Results: The overall prevalence of HCV infection was 13.23%. Antibody positivity was observed in 9.93% and HCVcAg alone was detected in 2.64%. One patient (0.66%) was initially positive for core antigen and later seroconverted.
Conclusions: Screening for HCV antibodies alone does not exclude infection with HCV in patients on haemodialysis and HCVcAg may be a useful test for identifying HCV infected patients on haemodialysis in the early phase of infection before seroconversion.

Keywords: Chronic renal failure, HCV c Ag, seroconversion

Chronic renal failure patients on haemodialysis (HD) are at high risk for blood borne infections because of prolonged vascular access and the potential for exposure to infected patients and contaminated equipment.[1] Infection due to hepatitis viruses is one such infection that continues to be a major concern in dialysis setting. Development of diagnostic testing for hepatitis B confirmed a high incidence and prevalence for HBV infection both in patients and staff in HD units.[2] Introduction of vaccination, isolation of hepatitis B virus (HBV) positive patients, dedicated dialysis machines and regular surveillance for HBV infection dramatically reduced the spread of HBV infection.[2],[3] Patients on haemodialysis have a high risk of acquiring HCV infection. Transfusion of unscreened blood, duration of dialysis and nosocomial transmission within haemodialysis unit are implicated as the main transmission routes of HCV infection in haemodialysis patients.[4] HCV infection in patients on HD has been associated with greater morbidity and mortality.[5]

The prevalence of antibodies to HCV (anti HCV) in HD patients ranges world wide from 1% in UK to 62% in Portugal[6] and is highly variable between different countries in the same locality.[7] There is also great variability in HCV testing practices in dialysis centre.[1] Data on prevalence of HCV among Indian haemodialysis patients are scanty. The prevalence of anti HCV in patients on haemodialysis from India is reported to be in the range of 3-45%.[8]

Virological diagnosis and monitoring of HCV infection are based on two categories of laboratory tests, namely serologic assays detecting specific antibody to HCV (indirect test) and assay that can detect, quantify or characterise the component of HCV viral particles (direct tests), such as HCV RNA.[9] Antibody detection tests are considered important tools to assess the magnitude of HCV infection in patients on HD. The window phase in HD patients can be longer as these patients are immunocompromised[10] and the anti HCV ELISA test alone may fail to detect the infected patients in the acute phase of the disease.[11]

HCV RNA detection is widely accepted as a gold standard test in the diagnosis of HCV infection in HD patients. However there have been concerns that wide spread use of PCR may be limited by availability, the need for meticulous specimen handling and concerns regarding reproducibility.[12],[13] Many of these reservations have been addressed by introduction of automated PCR assays shown to be accurate and suitable for the diagnosis and monitoring of viral load in patients infected with HCV.[14]

The latest break through in diagnosing early HCV infection is by detecting the HCV core antigen (HCVcAg) that is present during the early stage of infection when anti HCV antibodies have not been produced.[15] A highly sensitive enzyme immunoassay for HCVcAg has been developed by Ortho Clinical Diagnostics. There is a positive correlation between the concentration of core antigen and HCV RNA in anti HCV negative specimens. This study was under taken to find out the prevalence of HCV infection and utility of the HCVcAg assay in the early diagnosis of HCV infection in patients on HD.

Materials and Methods Study design and patients

The study was performed in the department of microbiology in collaboration with department of nephrology of the Nizam′s Institute of Medical Sciences (NIMS). The study protocol was approved by the ethics committee of NIMS. A total of 151 chronic renal failure patients on haemodialysis from May 2003 to October 2004 were studied. Eleven out of 151 patients were anti HCV positive before enrolling them in to the study and were excluded.

Patients follow up program

Of the remaining 140 / 151 patients, 40 were on HD for a short period i.e., less than one month and could not be continued in the study. The actual study comprised of the remaining 100 patients who were followed for two to five months.

Haemodialysis unit

The haemodialysis unit has one routine haemodialysis area and two isolated areas, one for HBsAg positive patients and other for anti HCV positive patients. The routine haemodialysis area has six haemodialysis machines, HBsAg positive area has one machine and anti HCV positive area has one machine. All the patients underwent serological testing for HBsAg, anti HCV and HIV before initiating the dialysis. Patients who were negative for HBsAg and anti HCV before initiating dialysis were dialyzed in routine haemodialysis area. Patients who were positive for anti HCV and / HBsAg before initiating the dialysis were dialyzed on dedicated machines in the respective isolated areas. Any patient who seroconverted to HCV or HBV during haemodialysis treatment was shifted to the respective isolated area.

All haemodialysis machines were chemically disinfected between each dialysis session. Dialyzer membranes and tubings were reused. Reuse areas and areas for storage of dialyzers are separate for HBsAg and anti HCV positive patients and for patients who are negative for both. In these three different areas (routine haemodialysis area, HBsAg positive area, anti HCV positive area) dedicated nursing staff were posted to look after each patient.

Specimen collection

Blood samples were drawn from the patients before the start of the first haemodialysis and every month there after. Serum was separated within two hours after blood sampling. All the serum samples were divided in to 0.5 mL aliquots and stored at -20oC.

Serology

All the 100 patients were tested once monthly for anti HCV, HCV core antigen with Ortho HCV 3.0 ELISA test system and Ortho antibody to HCV core antigen ELISA test system (Ortho clinical diagnostics) respectively. Anti HCV ELISA positive specimens were confirmed with supplemental anti HCV serological tests by using CHIRON RIBA HCV 3.0 SIA.Ò All core antigen ELISA positive specimens were retested in duplicate and the specimen was considered repeatedly reactive for HCV core antigen if either or both duplicate determination(s) was (were) reactive.

Results

Four other patients were positive for HCVcAg. Two out of four patients were positive for HCVcAg in the initial specimen that was collected before initiating the haemodialysis. They were continued to be followed up for seroconversion. Both these patients were negative for anti HCV in the first month and second month follow up specimens. However, these patients were lost to further follow up beyond two months. In the remaining teo patients, one patient was positive for HCVcAg in the first month follow up specimen and the other patient in the fourth month follow up specimen. The next follow up specimens of these two patients were negative for anti HCV. There was only one patient out of the 100 patients that had both demonstrable HCVcAg and anti HCV. The initial specimen of this patient was positive for HCVcAg and the first month follow up specimen was positive for anti HCV.

Out of 151 patients, 15 patients were anti HCV positive (9.93%). All anti HCV positive specimens were positive in supplemental anti HCV tests (RIBA). In the RIBA most of the anti HCV positive patients showed antibody response to NS3 antigen band compared to core antigen band. The overall prevalence of hepatitis C (HCV antibody positivity and / or HCVcAg Positivity) was 13.23%. The prevalence data are given in [Table - 2].

Discussion

The performance parameters of the testing method used have a direct impact on the detection of hepatitis C and thus can lead to differences in the prevalence data.[20] In the early 1990s, the first generation HCV antibody testing kits were introduced using NS4 antigen repertoire as the solid phase.[21] These tests were further improvised with the addition of NS3 and the core regions of the viral genome. This second generation ELISA assay had a higher sensitivity and specificity over the earlier one.[21] At present, the third generation ELISA assays use highly purified antigens with the addition of NS5 region of HCV genome and have the highest sensitivity and specificity.[20] The antigens are either a purely recombinant (Ortho), or purely synthetic peptides (UBI), or a mixture of recombinant and synthetic peptides (General Biologicals). The sensitivity and specificity of these 3rd generation kits depend on the type of antigen used.[21]

With the advent of the molecular techniques, the circulating virus (viraemia) can now be detected by HCV RNA measurement. This testing is used for early detection (before seroconversion) and also is essential for confirmation of active HCV infection and monitoring of antiviral therapy. Several authors reported high prevalence of HCV infection among haemodialysis patients by using anti HCV and HCV RNA detection.[20],[22] To perform the HCV RNA detection by RT-PCR extreme care and high standards must be maintained to avoid a false positive or negative result.[23] Well trained technologists are required and all runs need negative controls and multiple low and high quality standards to achieve maximum sensitivity and specificity of RNA assays.[23]

More recent studies have shown that the HCV core antigen is detectable throughout viraemic preseroconversion period.[24] Studies in HCV seroconversion series have shown that the average time to detection of core antigen is 1-2 days after the appearance of RNA.[24] Peterson et al reported that HCV core antigen appears substantially earlier than the HCV antibody during early phase of HCV infection and simultaneously with HCV RNA in most seroconversion series.[25] It became undetectable in a large proportion of seroconversion specimens after the development of anti HCV.[25] In our study we detected four HCV infected patients with core antigen ELISA and all these patients were anti HCV negative. Core antigen was negative in the 15 anti HCV positive specimens.

Hinrichsen et al reported that seroconversion to HCV antibodies does not occur in all haemodialysis patients.[20] In our study we observed that four patients were HCVcAg positive. Two of these patients were positive for core antigen in the initial specimens and one patient was positive in first month follow up specimen and the other patient in the fourth month follow up specimen. Two patients were followed for two months, one patient was followed for four months and the other patient was followed for one month. These four patients remained anti HCV negative during their follow up. We observed only in one patient who was core antigen positive in the initial specimen and anti HCV positive in the first month follow up specimen.

In patients 7 and 8 [Table - 1] core antigen was positive once and negative thereafter. Since we do not have enough studies on HCVcAg in patients on haemodialysis, such responses have to be confirmed using RT-PCR for RNA. However, one can probably say that the expression of antigen and antibody in immunocompromised patients is very inconsistent, irregular and may fluctuate.

Lee et al reported that HCV antigen ELISA can identify 94% of viremic blood donations given during the seronegative window phase of infection.[24] Courouse et al studied 135 serial specimens from 28 haemodialysis patients, and found that 88% of viraemic specimens contain HCVcAg.[26] One of our patients (patient 4 in [Table - 1], became anti HCV positive in the fourth month follow up specimen. The initial specimen, first, second and third month follow up specimens were negative for core antigen. We are in agreement with Courouse et al that in all preseroconversion phase specimens we can not detect core antigen. The antibody positivity in the absence of the antigen is probably because the antigen kit may not be sensitive enough to detect low and falling levels of antigen that may be present before the seroconversion. In this patient we started collecting the samples only three months before seroconversion and hence the antigen status is not known in this patient before three months. Hence we do not know the exact stage of infection in this patient.

Devesa et al observed restricted antibody reactivity to Hepatitis C Virus synthetic antigens in patients on haemodialysis.[27] In the RIBA strip NS3 and NS5 antigen bands are recombinant proteins and the core and NS4 bands are of synthetic antigens.This could be one of the reasons for greater antibody response towards NS3 antigen among our haemodialysis patients.

In conclusion, the prevalence of HCV infection in our haemodialysis patients with anti HCV detection alone was 9.93% but when we used HCVcAg along with anti HCV ELISA for diagnosis of HCV infection the prevalence was 13.23%. Screening for HCV antibodies alone does not exclude infection with HCV in patients on haemodialysis and HCVcAg may be useful and an early test for identifying antibody negative HCV infected patients on haemodialysis.

1.	Meyers CM, Seeff LB, Breen CO, Hoofangle JH. Hepatitis C and renal disease: Update. Am J Kidney Dis 2003;42 :631-57.  Back to cited text no. 1
2.	Fabrizi F, Poordad FF, Martin P. Hepatitis C infection and the patients with end stage renal disease. Hepatology 2002;36 :3-10.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.	Froio N, Nicastri E, Comandini UV, Cherubini C, Felicioni F, Solmone M, et al . Contamination by hepatitis B and C viruses in dialysis setting. Am J Kidney Dis 2003;42 :546-50.  Back to cited text no. 3
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6.	Dalekos GN, Boumba DS, Katopodis K, Zervou E, Sferopoulos G, Elisaf M, et al . Absence of HCV viremia in anti-HCV negative hemodialysis patients. Nephrol Dial Transplant 1998;13 :1804-6.  Back to cited text no. 6
7.	Devesa M, Khudyakov YE, Capriles F, Blitz L, Fields HA, Liprandi F, et al . Reduced antibody reactivity to Hepatitis C Virus antigens in Hemodialysis patients co infected with Hepatitis B Virus. Clin Diagn Lab Immunol 1997;4 :639-42.  Back to cited text no. 7
8.	Agarwal SK, Dash SC, Irshad M. Hepatitis C Virus infection during hemodialysis in India. J Assoc Physic Ind 1999;47 :1139-43.  Back to cited text no. 8
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10.	Lok AS, Chien D, Choo QL, Chan TM, Chiu EK, Cheng IK, et al . Antibody response to core, envelope and non-structural Hepatitis C Virus antigens: Comparison of immunocompetent and immunosupressed patients. Hepatology 1993;18 :497-502.  Back to cited text no. 10
11.	Moreira R, Pinho JR, Fares J, Oba IT, Cardoso MR, Saraceni CP, et al . Prospective study of Hepatitis C Virus infection in hemodialysis patients by monthly analysis of HCV RNA and antibodies. Can J Microbiol 2003;49 :503-7.  Back to cited text no. 11
12.	Schiff ER, Medina MD, Khan S. New perspectives in the diagnosis of Hepatitis C. Semin Liver Dis 1999;19 :3-15.  Back to cited text no. 12
13.	Gretch DR. Diagnostic tests for hepatitis C. Hepatology 1997;26:43s-7s.  Back to cited text no. 13
14.	Young KK, Archer JJ, Yokosuka O. Detection of hepatitis C RNA by a combined reverse transcription PCR assay Comparison with nested amplification and antibody testing. J Clin Microbiol 1995;33 :654-7.  Back to cited text no. 14
15.	Saab S, Brezina M, Gitnick G, Martin P, Yee HF. Hepatitis C screening strategies in Hemodialysis patients. Am J Kidney Dis 2001;38 :91-7.  Back to cited text no. 15
16.	Salunkhe PN, Naik SR, Semwal SN, Naik S, Kher V. Prevalence of antibodies to Hepatitis C Virus in HBs Ag negative hemodialysis patients. Indian J Gastroenterol 1992;11 :164-5.  Back to cited text no. 16  [PUBMED]
17.	Chadha MS, Arankalle VA, Jha J, Banerjee K. Prevalence of Hepatitis B and C virus infection among hemodialysis in Pune (Western India). Vox Sang 1993;64 :127-8.  Back to cited text no. 17  [PUBMED]
18.	Sumathi S, Valliammai T, Thyagarajan SP, Malathy S, Madanagopalan N, Sankarnarayan V, et al . Prevalence of hepatitis C virus infection in liver disease, renal disease and voluntary blood donors in south India. Indian J Med Microbio l 1993;11 :291-7.  Back to cited text no. 18
19.	Jaiswal SP, Chitnis DS, Salgia P, Sepaha A, Pandit CS. Prevalence of hepatitis viruses among chronic renal failure patients on hemodialysis in central India. Dialys Transplant 2002;31 :234-8.  Back to cited text no. 19
20.	Hinrichsen H, Leimnstoll G, Stegen G, Shrader H, Folsch UR, Schmidt WE, Prevalence and risk factors of Hepatitis C Virus infection in hemodialysis patient's multi center study in 2796 patients. Gut 2002;51 :429-33.  Back to cited text no. 20
21.	Uyttendale S, Clayes H, Mertens W, Verhaert H, Vermylen. Evaluation of third generation screening and confirmatory assays for HCV antibodies. Vox Sang 1994;66 :122-9.  Back to cited text no. 21
22.	Schneerberger PM, Keur I, Vliet W, Hoek K, Boswijk H, Loon AM, et al . Hepatitis C virus infection in dialysis centers in The Netherlands: national survey by serological and molecular methods. J Clin Microbiol 1998;36 :1711-5.  Back to cited text no. 22
23.	Lai KN. Hepatitis C infection screening in hemodialysis units. Am J Kidney Dis 2001;38 :186-8.  Back to cited text no. 23
24.	Lee SR, Peterson J, Niven P, Bahl C, Page E. Efficacy of Hepatitis C core antigen enzyme linked Immuno Sorbent assay for the identification of window phase blood donations. Vox Sang 2001;80 :19-23.  Back to cited text no. 24
25.	Peterson J, Green G, Lida K, Well CB, Aoyagi K. Detection of Hepatitis C core antigen in antibody negative window phase of Hepatitis C infection. Vox Sang 2000;78 :80-5.  Back to cited text no. 25
26.	Courouce AM, Marrec NL, Bouchardeau F, Razer A, Maniez M, Laperche S, et al . Efficacy of HCV core antigen detection during the preseroconversion period. Transfusion 2000;40 :1198-202.  Back to cited text no. 26
27.	Devesa M, Saez A, Leon G, Sirit F, Cosson C, Bermudez H, et al . Restricted isotypic antibody reactivity to Hepatitis C Virus synthetic peptides in immunocompromised patients. Clin Diagn Lab Immunol 1999;6 :279-81.  Back to cited text no. 27

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