Subclinical hepatitis C virus infection in Egyptian patients with rheumatic diseases: a multi-center study

Hepatitis C virus (HCV) infection is highly prevalent in Egypt. It was found to be 7.0% in a study done in 2015. There are some studies on the prevalence of HCV in rheumatoid arthritis, but to our knowledge, no previous study was done to detect it in other rheumatologic diseases. This study aims at detecting the prevalence of subclinical HCV infection in Egyptian patients with different rheumatic diseases. In the current study, eight hundred and three patients with different rheumatic diseases collected from five—geographically different—Egyptian rheumatology departments were studied. Patients with known current or previous HCV infection were excluded from the study. Screening for the positive anti-HCV antibodies was done for all patients. Screening for the presence of HCV ribonucleic acid (RNA) was done in patients with positive serology by reverse transcriptase-polymerase chain reaction. In the studied population, 675 (84.1%) patients were women. The mean age [± SD] was 44.2 [± 12.9] years. Hepatitis C antibody positivity was found in 73 (9.1%) of the patients, while 67 (8.3%) were having positive HCV-RNA quantitative PCR tests. The highest prevalence of seropositive HCV was found in drug-induced vasculitis (DIV) and cryo-vasculitis (100%), while in RA, HCV antibodies and PCR were found to be positive in 9.1% and 8.3% of patients, respectively. Detection of the presence of HCV infection in 9.1 % of the studied middle-aged Egyptian patients with rheumatologic conditions points to the importance of screening for HCV in such population for early detection and intervention especially for those patients that are planned to start biologic therapy.


Background
Hepatitis C virus (HCV) infection is a worldwide major health problem that has health-related and economic impacts including heavy costs. It was estimated to be the 7th leading cause of mortality worldwide and affects 150-170 million people, including 19 million in Europe [1,2]. Egypt constitutes the country with the highest prevalence of HCV in the world, estimated nationally at 14.7% [3,4].
The spectrum of clinical manifestations of HCV infection can range from asymptomatic hepatitis flares to chronic hepatitis with hepatic decompensation, fulminant hepatic failure, and death in about 70-80% of cases due to liver cirrhosis and liver cancer. Therefore, identifying patients at risk and early diagnosis is imperative to decrease its significant morbidity and mortality [5].
Hepatitis C virus is a hepato-and lymphotropic agent that can trigger and sustain a clonal B cell expansion, and produce different autoantibodies causing a broad spectrum of autoimmune and lympho-proliferative disorders. This can complicate the differential diagnosis between primitive and HCV-related rheumatic disorders [6]. Extrahepatic manifestations of HCV including rheumatologic disorders have been reported in up to two thirds of infected patients [7,8]. These manifestations may include frank autoimmune and rheumatic diseases (such as myalgia, arthralgia, arthritis, vasculitis, and sicca syndrome) which may dominate and complicate the course of the disease; moreover, treatment with biologic agents could reactivate latent HCV infection [9].
Having been known with the highest HCV prevalence, yet the rate of rheumatic diseases associated with subclinical HCV remains poorly studied in Egypt. Therefore, we aimed to unveil the prevalence of subclinical HCV in Egyptian populations with different rheumatic diseases. We hypothesized that the rheumatic diseases may increase the vulnerability of the patients to get infected with HCV more than their peers from the general non-rheumatic population. To our knowledge, this is the first study on the prevalence of subclinical seropositive HCV in Egyptian patients with this number of different rheumatic conditions.

Study population and design
Five university centers participated in this multi-center, observational cross-sectional study. These centers were chosen in a way that ensures pooling of the study subjects from nearly all over the country. The two Delta centers received patients from different northern areas in Egypt, while the remaining 3 Upper Egypt centers received patients from southern areas in Egypt.
Each center obtained approval to this study from its Local Ethics Committee, respecting the ethical standards in the Helsinki Declaration of 1975, as revised in 2000. The clinical trial registration number is NCT03587714. Written consent was obtained from each participating subject prior to enrolment.
The study subjects were recruited from patients attending the outpatient Rheumatology Clinics in the aforementioned centers. Patients aged less than 18, pregnant females, patients with current or past history of viral hepatitis, and patients with secondary rheumatic manifestations or with end-organ failure were excluded.
The study was carried out in a 4 months' duration, starting from the beginning of May to the end of August 2017.
Demographic and clinical data were gathered from all patients using a unified questionnaire (Table 1). Diagnoses were made by a qualified rheumatologist according to the fulfillment of diagnostic criteria of different rheumatic diseases (e.g., ACR-EULAR 2012 criteria for RA, SLICC for lupus).
Routine lab works, e.g., CBC, ESR, CRP and other rheumatic diseases' specific autoantibodies to assist diagnoses of primary rheumatic diseases, were done. Liver function tests and HCV antibodies were also done for all participants. HCV-RNA quantitative PCR test was done for patients with positive HCV antibodies tests.

Anti-HCV antibodies quantification
Serum samples withdrawn from patients were stored at − 20°C. Serum anti-HCV antibodies levels were analyzed on ARCHITECT i1000SR Immunoassay (Abbott Diagnostics, Europe), using chemiluminescence/magnetic particle. The results were represented as serum to cutoff ratio (S/CO). All samples were tested in duplicates.

HCV-RNA quantification
Serum HCV-RNA levels were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) on 7500 Fast Real-Time instrument. The results were represented as international unit per milliliter (IU/mL). All samples were tested in duplicates.

Statistical analysis
The descriptive data were presented as number and percentage (N, %), and mean and standard deviation (mean, SD). Cross tabulation was done by chi-square test. All statistical analyses were carried out using the Statistical Package for Social Science (SPSS) version 24 (SPSS Inc.; Chicago, IL, USA).

Results
A total of 803 rheumatic patients from five different Rheumatology centers in Egypt were recruited in this study. The mean age of our patients was 44.2 ± 12.9 years, most of them were females 675 (84.1%), and the mean disease duration was 7.3 ± 6 years. Hepatitis C positivity was found in 73 (9.1%) of the 3 Over half of the patients 483 (66.1%) were diagnosed to have rheumatoid arthritis, while systemic lupus was  Table 2.
The distribution of cases with positive HCV antibodies and positive PCR among the 5 different centers is demonstrated in Table 3. All patients with positive HCV antibody had positive PCR testing except in 3 centers where 8 patients had positive antibody and negative PCR. In one center, isolated positive PCR was found in a total of 2 patients.
The distribution of seropositive HCV and positive PCR among different rheumatic conditions is presented in Table 4. The highest prevalence of seropositive HCV was found in drug-induced vasculitis (DIV) and cryo-vasculitis (100%), followed by polyarthralgia (66.7%) and gout (28.6). In RA, HCV antibodies and PCR were found to be positive in 8.4% and 8.3% of patients, respectively. Negative HCV antibodies were reported in some cases including Behcet's disease, calcium pyrophosphate dehydrogenate deposition disease (CPPD), Sjogren's, sarcoidosis, mixed connective tissue diseases, juvenile-onset idiopathic arthritis (JIA), Wagner's granulomatosis (WG), palindromic rheumatism, primary fibromyalgia, adult-onset Still's disease, and antiphospholipid syndrome.

Discussion
Egypt was reported previously to be among the areas with the highest prevalence of HCV infection in the world. It accounted for up to 40% in some areas [10][11][12][13]. However, the prevalence of this infection has not been determined in rheumatic diseases where it could have a role in their evolution or management. To our knowledge, this is the first study on the prevalence of subclinical seropositive HCV in Egyptian patients with this number of different rheumatic conditions.
In this study, the prevalence of positive HCV antibodies and positive PCR in the studied population was found to be 9.1% and 8.3%, respectively. In a recent epidemiological study on Egyptian populations, patients with rheumatic conditions were classified as "Special clinical population" among 6 groups of populations at risk of exposure to hepatitis C infection. In this population, the prevalence of positive hepatitis C antibodies ranged from 6.7 to 96.1% with a median of 38.0%. This very wide range can be explained by the fact that they included in this study patients with dermatological diseases and non-hepatic malignancies [14].
The pooled mean prevalence of anti-HCV antibodies among the same population was 35.0% (95% CI = 27.3-43.1%). Furthermore, the incidence of hepatitis C infection was found to be higher in the residents of the villages of the Nile Delta compared with residents of the villages of Upper Egypt (10.2 per 1.000 persons versus 0.8 per 1.000 persons, respectively). Their findings suggest that HCV screening in the "special clinical population" should be given a priority [14]. In agreement with the previous finding, we recorded a high prevalence of positive HCV antibodies and PCR (9.9%) each in the patients recruited from center 2 (of a Delta city), while the lowest prevalence (3.3%, 6.6% respectively) was recorded in patients recruited from center 4 (Upper Egypt).
The association between HCV infection and rheumatic diseases especially RA has been reported in many studies. The prevalence of chronic HCV infection in rheumatic patients varied according to the study site, which could reflect differences in the prevalence of HCV among different countries. Some Brazilian and Spanish studies reported the high prevalence of seropositive HCV infection in patients with rheumatic diseases [15,16].
In the current study, the prevalence of positive HCV antibodies and positive PCR in RA patients was 8.5% and 8.3%, respectively. This is markedly higher than what was reported by Maillefert et al. who found that positive HCV antibodies and positive PCR among RA patients were 0.85% and 0.42%, respectively [17].
This great difference could be explained by the endemicity of HCV in Egypt. There is some evidence of the critical role of the liver in modulating the immune response in chronic inflammatory and autoimmune conditions [18][19][20].
It was denoted in the literature that autoimmune and lympho-proliferative diseases are well-known HCVrelated disorders [21]. However, many studies failed to find a positive association between HCV infection and RA, with no support for the participation of HCV in the pathogenesis of RA [15,17,22]. A nationwide study done in Taiwan reported that patients with HCV infection were at higher risk of developing RA later [23].
Some studies found that arthralgia and arthritis were the most common extrahepatic manifestations of chronic HCV infection [24,25]. Others claim that there is high prevalence of seropositive HCV among rheumatologic patients [15,16].
This goes in line with our finding where seropositive HCV was found in (100%) of patients with drug-induced vasculitis and cryo-vasculitis, followed by polyarthralgia (66.7%) and gout (28.6). A possible explanation could be that the state of chronic viremia may trigger these diseases in genetically predisposed persons. Two out of the 3 (66.7%) patients with polyarthralgia were positive for HCV antibodies and PCR. Several studies had reported the high prevalence of vitamin D deficiency in all chronic hepatic diseases irrespective of their etiology [26][27][28]. Serum vitamin D level < 20 ng/ml was found to be highly prevalent in patients with chronic hepatitis B and C worldwide [29]. Furthermore, vitamin D deficiency was delineated to contribute in the pathogenesis of hepatitis B and C and lead to progression of hepatic inflammation and fibrosis [30].
It was reported that clinically evident vasculitis occurs in less than 5% of HCV infected subjects with typical manifestations of purpura, weakness, and arthralgia [6,31,32].
Although anti-HCV antibodies were found in 15.2% of fibromyalgia patients in a Spanish study [36], the increased prevalence of HCV infection in such condition was not confirmed by other studies [37]. However, HCV infection should be kept in mind as a possible cause of secondary fibromyalgia [7].
Detection of seropositive HCV antibodies denotes either active or past infection while detecting the state of viremia assessed by PCR test is a powerful indicator of chronic HCV infection. In this study, 8 patients with positive HCV antibodies were having negative PCR. In one center, isolated positive PCR was found in a total of 2 patients. The occurrence of positive PCR with negative HCV serology can be found but in very low prevalence [38,39], and this denotes an occult HCV infection, which necessitates testing of both HCV antibodies and PCR in all patients.
This study had some limitations including the smallsized population collected from some centers and the cross-sectional type of the study. The great difference in the number of patients in different disease groups was also a weak point. In this study, PCR testing was performed in seropositive HCV cases only, while it would be ideal to perform it in all the participants to detect occult HCV infection.

Conclusion
In conclusion, considering the endemicity of HCV in Egypt and the detection of positive antibody titer in 9.1% of the study patients, regular screening of Egyptian rheumatic patients for HCV infection may not be necessary for the detection of subclinical cases. Rheumatic diseases seem to have no effect on increasing the susceptibility of the patients to get infected by HCV. However, for patients planned to receive biologic therapy, it is a well-known recommendation to have screening for HCV and HBV before initiation of biological treatment.