Fluctuating antibody response in a cohort of hepatitis patients 33 Fluctuating antibody response in a cohort of hepatitis C patients *Said H S Al Dhahry1, Shahina Daar2, Jameel C Nograles1, Situsekara M W W B Rajapakse1 Fadhila S S Al Toqi1, Geraldine Z Kaminski1 ABSTRAC T. Objective: This project was designed to longitudinally study persons who had antibodies to hepatitis C virus (HCV) to characterise the serologic course of infection. Methods: The subjects were 149 multitransfused patients (141 with thalassaemia major, 3 with thalassaemia intermedia, and 5 with sickle cell anaemia) who had been regularly followed up for 3 to 7 years. Sequential serum samples obtained semi-annually between January 1994 and January 2001 were tested, prospectively, by second or third generation HCV enzyme- linked immunosorbent assay (ELISA), followed by confirmatory recombinant immunoblot assay (RIBA-2 or RIBA-3). Results: Of the 149 patients, 90 did not seroconvert to HCV, whereas 59 had detectable antibodies. On the basis of RIBA results in these 59 patients, 24 (41%) had persistent high antibody levels to structural and non structural HCV antigens, 11 (19%) had persistent low antibody levels, 17 (29%) showed fluctuating antibody levels, and in 5 patients (8%) there was a total or a partial disappearance of specific antibodies (seroreversion), mainly anti-core antibodies. Two patients (3%) had antibody responses that did not fit into any of these four categories. In patients with fluctuating antibody levels, there were periods ranging from 6 months to 2 years when anti-HCV antibodies could not be detected. Conclu- sion: This study shows that the antibody response to HCV in patients who receive frequent blood transfusions is very variable. Individuals who exhibit intermittent seropositivity are a challenge to diagnosis. Key words: thalassaemia, antibody response, hepatitis C virus Departments of 1Microbiology and Immunology, and 2Haematology, Sultan Qaboos University Hospital, P O Box 38 , Al Khodh, Muscat 123, Sultanate of Oman. *To whom correspondence should be addressed. Email: dhahrys@omantel.net.om HEPATITIS C VIRUS (HCV), THE MAJOR CAUSE of parenterally-transmitted and community-acquired non-A, non-B hepatitis, was cloned in 989.1 The medical importance of HCV resides in its propensity to persist. In the general population, per- sistent infection leads to chronic hepatitis in 50–80% of patients, often accompanied by an increase in serum transaminase levels.2–4 Chronic HCV infection affects an estimated 70 million individuals worldwide with the majority of countries reporting a prevalence of .0– 5.5%.5,6 However, in patients with haemoglobinopathies such as thalassaemia major, the prevalence of hepatitis C is high. Before the introduction of routine screening of donated blood for HCV antibody, these patients were at high risk of exposure to the virus as a result of frequent transfusions with packed red blood cells. The reported prevalence in thalassaemics varied geographically from 23% to 72%.7 Following the discovery of HCV genome, a variety of diagnostic tests based on detection of anti-HCV anti- bodies have been developed and refined. Three genera- tions of enzyme-linked immunosorbent assay (ELISA) have been developed using recombinant and peptide antigens derived from different regions of HCV genome SQU JOURNAL FOR SCIENTIFIC RESEARCH: MEDICAL SCIENCES 2002, VOL. 4, NO. –2, 33–38 ©SULTAN QABOOS UNIVERSIT Y Arabic abstract Said Al Dhahry Hepatitis C (new �� �� ����) ������� ����� ������ ������ ������� �������� ��������)�( ������ �� ���� ��� ������ ������� �������� �������� ����� ����������� ,���������� ,���������� ,����������������� ,����������� ,�������� �������� �������� :��� ������� ����� ������ ������ ����� ����� ����� ������� �������� �������� �����)� .(�������:���� ������������� ���������� ������ ����������� �������� �������� ����������� ������������ ���� ���� ���� .���� ������ �� ��� ��� ������ ����� �������–������ .�� ������ ����� ������� �������� �������� ���� ���� ��� �� ����� ��� ������� ��� �� ������ ������������������ ��� �������� ����� ����� �� ��� ������ ��� �������� �.�������:��� ����������������� ������� ����������� ����������� �������������� ����� ������ .����� ����� ������ ����������)�� (%������� ������ �������� ������� ������ �� ����� ������� ����� .����������)�� (%������� ����������� ���� �������������������������)�� (% ������ ����� ��� ���� ����� ������ ������� .���������)� (%������ ����� ��������������� ������� �������� �������� . ������)�(%�� ����������������� �������� ������ �� �� ������ � ���� ����� ������.������:������������ �������� �� ����� ��� ������� ����� ������ ������ �������)� (������ �� ���� ��� ����� ��� ���� �������. 34 (Figure ). Each new generation provided incremental improvements in sensitivity to anti-HCV. In high prev- alence populations, such as patients with chronic liver disease, third generation ELISAs have high sensitiv- ity and specificity. Unfortunately, they have suboptimal sensitivity and specificity in low prevalence populations, such as blood donors.8 Thus, to confirm the presence of HCV antibodies, immunoblot assays were developed in parallel with ELISAs. The current (third) generation of recombinant immunoblot assay (RIBA) detects antibod- ies to four HCV antigens (Figure ). Although ELISA and RIBA are useful in the diagnosis of HCV infection, the presence of antibodies which they detect does not neces- sarily mean that the virus is present. Detection of HCV RNA by polymerase chain reaction (PCR) can differen- tiate between ongoing and resolved infection. Further, PCR is useful in assessing the antiviral response to ther- apy, and can help resolve weakly positive or negative anti- body test results when clinical signs and/or risk factors are compatible with HCV infection. The table compares two generations of ELISA and RIBA with HCV PCR in subjects at low- and high-risk for HCV infection Since HCV has been identified only recently, little is known either about the natural history of infection, or about the mechanisms associated with the immuno- logic responses to it. The number of long-term sequen- tial evaluations of viraemia and of anti-HCV immune response has been limited. Thus we undertook a pro- spective, seven-year study of serological markers of HCV infection in multitransfused patients treated at Sultan Qaboos University Hospital (SQUH) to establish anti- body patterns in these subjects. P A T I E N T S A N D M E T H O D S Patients. From January 994 to January 200, a total of 236 patients (27M, 09F) with haemoglobinopa- thies were treated in the Department of Haematology of Sultan Qaboos University Hospital. Their ages at entry into the study ranged from 6 months to 40 years (median 6.5). Among these patients, 29 had thalassaemia major, 9 had thalassaemia intermedia, and 8 had sickle cell dis- ease. The diagnosis of haemoglobinopathy had been made on the basis of family histories, cell counts, and haemoglobin electrophoresis. All the patients were rou- tinely transfused with packed red blood cells (generally every 4 weeks in patients with thalassaemia major) and were followed up at the day care centre of SQUH. At approximately 6-month intervals, blood samples were collected at the time of routine visit before transfu- sion. Serum samples were separated from whole blood within 3 hours after venipuncture, and were kept at +4º C if testing was carried out within 24 hours of collection, or at –20º C if they were to be tested at a later date. Detection of serum HCV antibodies. Serum samples Structural Non-structural Core Envelope Helicase & protease RNA polymerase 5’— C E1 E2 NS2 NS3 NS4 NS5 — 3’ 5-5-1 C22 C33 C100 NS5 Figure 1. Organization of hepatitis C virus genome and location of antigens licensed for diagnostic use. — A L D H A H R Y E T A L Table 1. RIBA and PCR testing in anti-HCV (ELISA) positive blood donors and patients with suspected viral hepatitis C population Study population Generation ELISA /RIBA Number ELISA positive RIBA %PCR positive in RIBA % Posα %Indβ %Negδ Pos Ind Neg Blood donors9 2/2 340 23 38 39 84 6 0 Blood donors10 2/2 1105 17 38 45 75 2 NDφ Blood donors11 2/3 750 37 29 35 80 1 0 Patients10 2/2 283 79 11 10 90 68 ND Patients12 3/3 114 46 34 20 100 46 0 Patients13 3/3 54 94 6 0 94 0 0 αPositive, βIndeterminate, δNegative, φNo data 35 were screened by an enzyme-linked immunosorbent assay (ELISA). To assess the specificity of the ELISA, a confirmation recombinant immunoblot assay (RIBA) was performed on positive sera. Between 994 and 996, second generation ELISAs (Abbott HCV EIA, Abbott IMx HCV, or Abbott Axsym HCV, Abbott Diagnostics Division, Germany) and RIBA (Chiron Corp. Emeryville, California) were used. These assays contain antigens Figure 3. Persistent low levels of antibodies to hepatitis C virus antigens in a patient who was followed over a six-year period Figure 2. Six and half years’ follow-up in a thalassaemic patient, showing persistently high levels of antibodies to hepatitis C virus antigens A N T I B O D Y R E S P O N S E I N H E PAT I T I S C PAT I E N T S 36 coded by the structural gene (core) and non-structural genes (NS3 and NS4) of HCV (Figure ). Third genera- tion ELISA (Abbott Axysm HCV 3.0, Abbott Diagnostics Division, Germany) and RIBA (Chiron Corp., Emeryville or Genelabs Diagnostics, Singapore) contain an extra antigen from the NS5 region of HCV genome (Figure ); they were used from 997 to 200. As instructed by the manufacturer, the intensity of each band in the RIBA was rated from 0 to 4+. Results were interpreted as positive, indeterminate or negative based on the intensity and distribution of the bands. The RIBA score of each band was a measure of the level of antigen-specific antibody in the sample. Detection of human immunodeficiency virus (HIV): Testing for HIV antibodies was performed by ELISA (Abbott Diagnostics, Germany) with confirmatory Western blot assays (Novopath HIV- from Biorad, or New LavBlot I from Diagnostics Pasteur, for HIV- and New LavBlot II, from Diagnostics Pasteur for HIV-2). R E S U L T S When tested by second or third generation ELISA, 39 (59%) patients had no detectable anti-HCV antibodies at any time during the study period, whereas 97 (4%) patients were seropositive. Of this total of 236 patients, 49 had been followed up for 3 to 7 years (mean 6.5), and had serum samples collected and tested every six months. Data of these patients were analysed further to establish antibody responses to HCV. Of the 49 patients, 90 (89 with thalassaemia major,  with thalassaemia interme- dia) did not seroconvert to anti-HCV during the study period. Fifty nine patients (52 thalassaemia major, 2 tha- lassaemia intermedia, 5 sickle cell disease) had detect- able anti-HCV antibodies when tested by second or third generation ELISA and RIBA. On the basis of RIBA results, four serological patterns were observed. Twenty four out of 59 patients (4%) had persistently elevated antibody levels, with RIBA scores of 3+ or 4+ to structural and non structural HCV antigens. Figure 2 shows data of one such patient. Eleven patients (9%) had low antibody levels, with RIBA scores of 2+ for nearly all HCV antigens throughout the study period. Samples from these patients were weakly positive or indetermi- nate (Figure 3). In 7 patients (29%), there were wide fluctuations in antibody levels (Figure 4). They showed transitions between RIBA-indeterminate and –positive status interspersed with RIBA-negative status. Some of Figure 4. Fluctuating levels of antibodies to hepatitis C virus antigens in a atient over a six-year period of follow-up A L D H A H R Y E T A L 37 the patients had no detectable antibodies for periods of 6 months to 2 years. There was progressive loss of HCV antibodies (seror- eversion) in 5 patients (8%), one of whom lost all HCV antibodies (Figure 5). None of these patients was on anti- viral therapy for HCV, and none was HIV-infected. Serological response in 2 patients did not fit into any of these 4 categories. One patient had low antibody lev- els for 5 consecutive years followed by a strong antibody response. The second patient, who had been RIBA-nega- tive for 2 consecutive years, seroconverted in 999, lost all antibodies in 2000, and seroconverted again in 200. D I S C U S S I O N Approximately two thirds of our multitransfused patients who were regularly tested for a mean period of 6.5 years did not seroconvert to anti-HCV, strongly suggesting that they did not acquire HCV infection dur- ing the study period. In the one third that were serop- ositive, our data indicate that the antibody response to HCV infection is variable. Four serological patterns were observed. Most patients (4%) had high antibody levels that were sustained throughout the study period, and 9% had persistent, but low antibody levels. In a similar study in homosexual men, persistent anti-HCV serop- ositivity was reported in 6 of 26 subjects.14 Intermittent seropositivity which we observed rela- tively frequently could be attributed to reinfection. The possibility of reinfection with HCV arises because of at least two reasons. First, chimpanzee studies have shown that recovery from HCV infection does not confer immunity to the individual.15 Second, although screen- ing of blood donations for HCV antibodies has drasti- cally reduced the risk of post-transfusion hepatitis C, a residual risk, mainly linked to viraemic bloods donated by seronegative individuals, persists.5,16 Fluctuating HCV serology may be common in the general population.14 It poses a challenge to diagnosis and contributes to the continuing problem of transfusion-associated hepatitis. Partial or full seroreversion may be observed in three circumstances: spontaneously, induced by therapy, and in conjunction with HIV infection. In our patients, sero- reversion was spontaneous and was probably associated with clearence of infection.17, 18 Our findings indicate that spontaneous seroreversion is relatively rare and are in agreement with observations by others. In a longitudinal study lasting 8 years, only 2 of 30 patients with thalassae- mia major seroreverted.17.An assessment of 03 transfu- sion-associated HCV cases 20 years after disease onset revealed that 0% had lost HCV serologic markers18. A N T I B O D Y R E S P O N S E I N H E PAT I T I S C PAT I E N T S Figure 5. Seroreversion in a patient infected by hepatitis C virus 38 The presence of detectable anti-HCV antibody may reflect a past resolved infection or chronic persistent infection. Usually, the majority (≥80%) of RIBA positives are viraemic.9–13 Samples from persons with persistent and intermittent HCV ELISA seropositivity are positive by polymerase chain reaction 76% and 50% of the time respectively.14 Based on these data, we infer that the anti- body responses we observed reflect different dynamics of HCV infection. We have recently started prospective evaluations of viraemia by PCR in parallel with antibody studies to have a better understanding of the natural his- tory of HCV infection and the antibody response to it. C O N C L U S I O N This study clearly shows that the antibody response to HCV infection in patients who receive frequent blood transfusions, such as those with thalassaemia major, is very variable. HCV infection in these patients is not always characterised by a persistent antibody response. A significant proportion may either show intermit- tent seropositivity, or they may progressively lose anti- HCV antibodies. This variability in antibody levels poses difficulties in the serological diagnosis of viral hepatitis C. R E F E R E N C E S . Choo Q-L, Kuo G, Weiner A, Overby L, Bradley D, Houghton M. Isolation of a cDNA clone derived from a blood-borne viral hepatitis genome. Science, 989, 244, 359–362. 2. van der Poel CL, Cuypers H T, Reesink HW. Hepatitis C virus six years on. 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