VOLUME 20 | ISSUE 5-6 | SEPTEMBER-NOVEMBER 2000

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Hepatitis C Virus Genotyping by Polymerase Chain Reaction and DNA Enzyme Immunoassay among Saudi Patients in the Western Province, Saudi Arabia

A.O. Osoba, MD, FRCPath; M. Ibrahim, MD, FRCP; M. A. Abdelaal, MD, FRCPath;

From the Departments of Pathology and Medicine, King Khalid National Guard Hospital, Jeddah, Saudi Arabia.

How to cite this article:

AO Osoba, M Ibrahim, MA Abdelaal, A Al-Mowallad, B Al Shareef, B Al Haj Hussein, Hepatitis C Virus Genotyping by Polymerase Chain Reaction and DNA Enzyme Immunoassay among Saudi Patients in the Western Province, Saudi Arabia. 2000; 20(5-6): 394-397

DOI: 10.5144/0256-4947.2000.394

Abstract

Background: The distribution of hepatitis C virus (HCV) genotypes in the Western Province of Saudi Arabia is unknown. The purpose of our study was to determine the prevalent HCV genotypes among HCV seropositive Saudi patients in the Western Province, and to study the relationship between types/subtypes, clinical status and liver histology.

Patients and Methods: Serum samples were collected from 140 consecutive patients attending the Hepatology Clinic with varying grades of liver diseases, high alanine transferase (ALT) for >6 months, positive HCV, qualitative PCR, and who had had liver biopsy. HCV genotyping was determined on patients who had tested positive by both HCV enzyme immunoassay (EIA) and recombinant immunoblot assay (RIBA).

Results: Of the 140 patients, 97 (69.2%) had genotype 4, 18 (12.8%) had genotype la, and 16 (11.4%) had genotype lb. Genotypes 2b and 5 were found in two patients (1.4%) each, while 5 patients (3.6%) had mixed infections with genotypes 4 and 5. Of the 97 patients infected with genotype 4, 84 (86.6%) had chronic active hepatitis (CAH), two (2.1%) had CAH with active cirrhosis, 9 (9.3%) had cirrhosis and two (2.1%) had normal liver histology (NLH).

Conclusion: The most prevalent HCV genotype in the Western Province of Saudi Arabia was genotype 4 (69.2%). Genotype lb was encountered in 16 (11.4%) patients. For the first time, genotype 5 was identified in the Western Province of Saudi Arabia. Genotypes lb and 4 were associated with different histological grades of liver disease.

Ann Saudi Med 2000;20(5-6):394-397.

Key Words: Hepatitis C virus, genotyping, polymerase chain reaction, DNA enzyme immunoassay.

Chronic infection with the hepatitis C virus (HCV) is estimated to affect about 170 million people worldwide, and about 20%-30% of these cases will eventually progress to liver cirrhosis and its sequelae such as hepatocellular carcinoma.¹ In Saudi Arabia, seroprevalence studies suggest that the overall anti-HCV positivity is about 0.9%, while in subjects over the age of 50 years it is about 3.5%-5%.² These figures suggest that a sizeable number of individuals are at risk of developing complications of HCV infection in the future and will require therapy.

Recent studies have indicated that combination of α-interferon plus ribavirin is more effective in the treatment of HCV infection than monotherapy with interferon alone.¹Using logistic regression, Poynard et al.¹ identified five independent factors significantly associated with response to interferon therapy: genotypes 2 or 3; viral load less than 2 million copies/mL; age ≤40 years; minimal liver fibrosis stage; and female gender. It is, therefore, important that the genotype of the HCV be determined prior to therapy, as it has implications for diagnosis, management and response to therapy. Moreover, HCV genotype determination assays can be particularly useful in studying worldwide and local evolutions of the HCV endemics, since the epidemiology of HCV is changing rapidly.^3,4 There are at least 12 genotypes and 90 subtypes of HCV, but the prevalent HCV genotypes have been determined in most developed countries.^3,5 There are only a few publications to date on HCV genotype determination in Saudi Arabia, and these have indicated that the predominant genotypes were either type 1 or 4,² but none of these have correlated the genotypes with liver histology in the patients. The few published studies have been carried out mainly in institutions in the Riyadh area.^6-12 The purpose of our study was to determine the prevalent HCV genotypes among HCV seropositive Saudi patients in the Western Province, and to study the relationship between types/subtypes, clinical status and liver histology.

Patients and Methods

This was a prospective study on 140 consecutive Saudi patients living in the Western Province with varying grades of liver diseases, and attending the Hepatology Clinic in our hospital. The patients were assessed by detailed history, and specifically on previous transfusion of blood or blood products and other risk factors. Physical examination, basic hematological and biochemical profiles, including liver function tests, were carried out on all patients. Liver biopsy was performed on all patients with high alanine transferase (ALT) for more than six months and positive HCV qualitative polymerase chain reaction (PCR). Serum samples were obtained from all patients and stored at –80°C until genotyping assay was carried out, in order to optimize preservation of HCV RNA. The sera were examined by Axsym hepatitis C virus enzyme immunoassay (HCV EIA) (Abbott Laboratories, Abbott Park, IL, 60064) and recombinant immunoblot assay (RIBA) HCV 3.0 SIA (Chiron Corporation, Emeryville, CA 94608). HCV genotyping was carried out on patients' sera which had tested positive by both HCV EIA and RIBA.

Table 1. Correlation of genotypes/subtypes with liver pathology.

Liver histology/diagnosis	Genotype distribution							Mixed genotypes	Total
Liver histology/diagnosis	1a	1b	2b	3	4	5	6	Mixed genotypes	Total
CAH	17	12	2	0	84	0	0	3 (Genotypes 4/5)	118
CAH with cirrhosis	1	1	0	0	2	0	0	1 (Genotypes 4/5)	5
Liver cirrhosis	0	2	0	0	9	1	0	0	12
Normal	0	1	0	0	2	1	0	1 (Genotypes 4/5)	5
Totals*	18	16	2	0	97	2	0	5*	140
(%)	(12.8)	(11.4)	(1.4)		(69.2)	(1.4)		(3.6)	(100)

*Five patients with mixed infections with genotypes 4 and 5; CAH=chronic active hepatitis.

PCR DNA-EIA Genotyping

The assay is based on a combination of two well-established techniques, the PCR and DNA enzyme immunoassay (DNA EIA). In the first step of the method, a cDNA of about 250 bp corresponding to the HCV core region is amplified by nested PCR. The target cDNA is then hybridized to type-specific oligonucleotides fixed to a solid phase through an avidin-biotin bridge. The formed hybrids are detected by a standard ELISA, using monoclonal antibodies reacting with double-stranded DNA.^3,5,13-16 RNA extraction was carried out on 140 μL of patients' sera, according to the manufacturer's instructions and protocol, using the commercial kit of QIAamp (Qiagen, GmbH, 40724 Hilden, Germany). Buffering conditions were then adjusted to provide optimum binding of the RNA to the QIAamp column and the sample was loaded onto the QIAamp spin column. The RNA binds to the column and contaminants are efficiently washed away in two steps using two different wash buffers. The special QIAamp column guarantees extremely high recovery of pure, intact RNA in just 20 minutes. The final volume of RNA which was resuspended in distilled water was 50 μL. 12 μL of the extracted RNA preparation was transcribed to cDNA using primer 1AS and reverse transcriptase according to the manufacturer's instructions (DiaSorin Saluggia, Vercelli, Italy).

The nested PCR, DNA enzyme immunoassay and identification of genotypes were carried out according to the manufacturer's instructions (DiaSorin Saluggia, Vercelli, Italy), and as described by Viazov et al. Included in each run were PCR-negative and PCR-positive controls, as well as DEIA-negative controls, DEIA positive-specific controls and a DEIA blank. The run is valid when the ratio of the DEIA-positive control absorbance value to the DEIA-negative control mean absorbance value is greater than or equal to 10 (CP DEIA/CN DEIA >10).

Results

There were 66 males and 74 females. The age range of the males was 17 to 64 years, with a mean age of 44.7 years, while the age range of the females was 23 to 66 years, with a mean age of 50.5 years. The age and sex distribution of the patients according to their genotypes are as shown in Figure 1. There was no significant difference in the distribution of the genotypes among the sexes. Seventy-three patients (52%) gave a history of blood transfusion or other known risk factors for HCV infection, such as previous operations, dental procedures and hemodialysis. Our results showed that 97 patients (69.2%) had genotype 4, 18 (12.8%) had genotype la, 16 (11.4%) had genotype lb, two each (1.4%) had genotype 2 and 5, while five patients (3.6%) had mixed infections with genotypes 4 and 5. Genotypes 3 and 6 were not encountered (Table 1). All five genotypes encountered were associated with CAH. Of the 97 patients infected with genotype 4, 84 had CAH, nine had liver cirrhosis, two had CAH and active cirrhosis, and another two had positive HCV serology and normal liver histology (NLH) (Table 1).

Discussion

The main areas of usefulness of molecular biology-based genotyping techniques are direct access to viral genome sequence, high sensitivity due to amplification of targets by PCR, and the possibility of determination of subtypes. ^3,4 Presently, three rapid genotyping methods are available, namely: 1) type-specific PCR amplification with various sets of genotype-specific primers; 2) PCR amplification with conserved primers, followed by restriction fragment-length polymorphism (RFLP) analysis of PCR products with various sets of restriction enzymes; and 3) PCR amplification of conserved primers, followed by reverse hybridization to genotype-specific oligonucleotide probes coated on to microtiter plates or nitrocellulose strips.⁴ Based on the third methodology, three commercial kits have been developed, namely: 1) HCV Line Probe Assay (INNO-LIPA HCV, Innogenetics, Belgium); 2) the GenEtiK DEIA assay (Sorin Biomedics, Italy); and 3) the Amplicis HCV assay (CisBio, France).^3,4In this study, we used the GenEtiK DEIA assay kit, which allowed us to classify all our 140 isolates into types and subtypes, as well as detect mixed infections in five patients, all with genotypes 4 and 5. The fact that we were able to determine the viral genotypes of all our isolates suggests that additional genotypes (7 to 12) are unlikely to be present in the Western Province.

The importance of HCV genotyping has considerably increased in the last few years. It has been used to study worldwide and local molecular epidemiology of HCV, and to trace sources of HCV infection in risk groups such as drug users and blood products. Typing has also been used to study relationships between type/subtype and the clinical status, pathogenesis and/or outcome of disease.³ The major area of clinical application of HCV genotyping has been in the study of the significance of types/subtypes, in response to antiviral treatment of HCV infection with interferon and ribavirin, as well as the identification of patients with mixed infections. It has also been a useful application in vaccine research and development.^3,13

The differential prevalence of HCV genotypes appears to be linked to the geographic areas of origin, and the possible relationship between HCV genotypes and clinical expression of disease has recently aroused considerable interest.¹⁴ The genotypes and their subtypes coexist in various geographic locations but show different prevalence and genetic diversity.^3,15 There is substantial evidence that HCV possesses different pathogenic potentials, since different responses to interferon treatment depending on the HCV type/subtype have been reported.¹⁴ Furthermore, association between some HCV types and the severity of liver disease has been noted in some reports.¹⁴

The most prevalent genotype in the Western Province, based on the result of this study, was genotype 4, which has been identified in the Middle East, Egypt and some African countries.³ It has been found most commonly in the age group of 41 to 60 years (Figure 1). Genotype lb, which is usually unresponsive to interferon, and is the genotype highly prevalent in European countries,³ was encountered in only 16 patients (11.4%). Mixed infections occurred in only five patients (3.6%), and were all genotypes 4 and 5, in contrast to 4.2% of mixed infection cases reported in Riyadh, which had genotypes la, lb, 3a, 3b and 4.

A comparison of our data with that in the published literature from the Kingdom shows some interesting differences from the previous studies. The occurrence of genotype 5 in seven patients was an interesting finding. Only one study from Saudi Arabia has recorded the presence of genotype 5 in the Kingdom,¹² apart from a previous study by our group in the Jeddah area which reported that three out of 10 patients were infected with genotype 5.¹¹ Since some of our subjects gave a history of transfusion of blood or blood products, acquisition of the infection through transfusion from sources outside the Kingdom could not be excluded. However, genotype 5 has been identified mainly in South Africa.³ Blood donors in European countries are almost exclusively infected with genotypes 1, 2 and 3, while 3a has been found in a high percentage of intravenous drug users.^3,17 Only one study in the Kingdom found genotype 3a in two (1.6%) of 119 patients with chronic hepatitis infection.⁹ We did not find genotypes 3 and 6 in any of our patients in the Western Province.

Only two genotypes (lb and 4) encountered in this study were associated with varying grades of liver disease, from normal liver histology to liver cirrhosis (Table 1). Correlation of the genotypes distribution with liver pathology shows that genotype 4 was associated with CAH in 86 (61.4%) of 140 patients. The severity of the histological lesions produced by each of the genotypes could not be ascertained as our sample size was small, but it appears that the most common genotype (genotype 4) produced a wide range of liver pathology. Different pathogenic potentials have been suggested for genotypes lb and 2, in view of the biochemically silent clinical course associated with genotype 2,¹⁷ as opposed to the usually unresponsive nature of genotype lb to interferon. The two cases of genotype 2b encountered in this study had CAH. The liver histology in patients with genotype 5 is shown in Table 1.

The issue of the pathogenicity of the different genotypes/subtypes remains controversial, and long-term prospective studies in various population groups are required. Large series of data are required to determine the epidemiology of HCV genotypes and evidence-based strategy for the management of HCV liver diseases in the Kingdom of Saudi Arabia.

References

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