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ISSN-2155-9929
Journal of Molecular Biomarkers & Diagnosis
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The Utility of Automated HCV Core Antigen Assay as an Alternative to PCR in Chronic HCV Egyptian Patients

Ehab M Abd El-reheem1*, Magdy Fouad1, Hanaa Kh Fath El-bab1 and Waleed M Abd El-Hmeed2
1Tropical medicine Department, Faculty of Medicine, El-Mania University, Egypt
2Clinical pathology Department, Faculty of Medicine, El-Mania University, Egypt
*Corresponding Author : Francis Ayuka
University of Canterbury
Kirichwa Road, Off Argwings Kodhed Road
Kirichwa Apartments, Nairobi, Nairobi 00100, Kenya
Tel: +254722381401
Mobile: +25473708320
E-mail: [email protected]
Received October 12, 2015; Accepted December 23, 2015; Published December 25, 2015
Citation: Abd El-reheem EM, Fouad M, Fath El-bab HKH, Abd El-Hmeed WM (2016) The Utility of Automated HCV Core Antigen Assay as an Alternative to PCR in Chronic HCV Egyptian Patients. J Mol Biomark Diagn 7:275. doi:10.4172/2155-9929.1000275
Copyright: © 2016 Abd El-reheem EM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Hepatitis C is a disease with a significant global impact. Although the detection and quantitation of HCV RNA have been the standard method for the diagnosis and assessing individual patient response to the antiviral drug regimen, it is very sensitive testing, expensive, labor intensive, and requires technical skill.

Since the HCV core Ag assay is easy to perform in an immunoassay format, cheap, and less prone to sample carryover contamination compared to nucleic acid tests, There is an increasing interest to use HCV core antigen as a reflex test for seropositive individual to identify the active HCV infection also can be used to detect the early HCV infection.

Aim of the work: To compare the diagnostic accuracy of HCV core antigen with HCV-PCR in chronic HCV Egyptian patients.

Patients and methods: HCV core Ag was measured by a fully automated chemiluminescent immunoassay (CLIA) ABBOTT diagnostics in 57 patients proven to be chronic hepatitis C from those 32 were treated with pegylated interferon and ribavirin therpay and achieved HCV RNA negative six monthes after stoppage of treatment ( successful SVR) and 25 chronic HCV patients not considered for treatment . Viral load was quantified with branched DNA (bDNA, Versant, Siemens) also sera were tested with the Architect HCV Ag test (Abbott Laboratories). Statistical analysis was performed on logarithmically transformed values.

Results: HCV core antigen was detectable in 19/25 and grey zone in 4/25 HCV RNA positive sera while 2 sera were negative for HCV Ag. HCV-Ag was undetectable in all 32 HCV RNA negative samples. The sample with the lowest viral load that tested positive for HCV-Ag contained 1200 IU/mL HCV RNA. The levels of HCV core antigen showed a good correlation with those from the HCV RNA quantification (r = 0.907) HCV core Ag levels were correlated significantly with ALT levels (r = 0.516; P < 0.0001)

Conclusion: In conclusion, the Architect HCV antigen assay is highly sensitive, (>90%) reliable, easy to perform, cost-effective, and applicable as a screening, supplemental, and preconfirmatory test for anti-HCV assays used in laboratory procedures for the diagnosis of hepatitis C virus infection.

Keywords
HCV infection; Immunoassay; Hepatitis C; Diagnosis; Virological marker
Introduction
HCV usually suspected by the detection of HCV antibodies. Anti-HCV assays have several disadvantages as the lack of sensitivity of detection in the early window period after infection, the inability to distinguish between different stages of infection. Recombinant immunoblot assays, also have several disadvantages, such as being difficult to perform and having a high percentage of indeterminate results and a high cost [1]. The HCV RNA assay is a reliable method but needs technical skill and may also result in false positivity because of contamination; it is time intensive and more expensive [2].
HCV like other memberes of flavivirdiae family produce an excess of protiens that from the diagnostic point of view may provide usefull marker of infection HCV core antigen is another direct virological marker which has been investigated in last year’s [3]. HCV core antigen assay is as simple as the HCV antibodies assay and can detect HCV infection only 1 day delay compared to the HCV RNA assay. HCV core antigen (Ag) tests have been introduced to supplement anti-HCV tests over the last decade and these quantitative HCV Ag assays could be used for monitoring of antiviral therapy as well as for diagnosis of HCV infection [2]. However, most of the past studies detecting HCV Ag utilized enzyme-linked immunosorbent assays (ELISA) or enzyme immunoassays (EIA) which may need considerable time and skills [3]. In previous years, manual HCV-Ag ELISA tests were available, but they demonstrated significant limitations, mainly in terms of sensitivity [3]. Recently, An HCV core Ag automated chemiluminescent immunoassay (CLIA) has been recently developed with higher sensitivity and throughput was developed to overcome shortcomings of the conventional core Ag assays [4,5].
Thus, we evaluated the newly introduced HCV Ag assay and compared it with a quantitative RNA assay to verify the utility of this automated Ag assay as an alternative to HCV RNA-PCR.
Patients and Methods
This study was carried out on 57 patients who were chosen from 100 chronic HCV patients attending tropical medicine outpatients clinic, EL-Minia university hospital.
They diagnosed as chronic hepatitis C by the presence of HCV Abs by third generation ELISA and detectable HCV RNA in addition to histological evidence of chronic hepatitis C in liver biopsy specimen obtained within the preceding year. Patients who previously received immune-modulatory agents within the last 6 monthes, showed evidence of other liver diseases, had decompansated liver cirrhosis, co infected with shistosomiasis or had pulmonary or cardiac diseases were excluded from the study. The included patients were classified into two groups
Group A: included 32 patients (20 males and 12 females with mean age 47.12 ± 6.89) treated with pegylated interferon and ribavirin therapy for 48weeks and obtained SVR
Group B: included 25 HCV RNA positive patients (14 males and 11 females with mean age 48.23 ± 5.6) not considered for treatment All the patients were subjected to
1. Medical history
2. Stressing upon: possible risk factors of HCV transmission-
3. History of bilharziasis and its treatment
4. History of alcohol intake
5. History of drug use
6. Associated medical diseases etc.
7. Clinical examination
8. General examination: particulary for detection of stigmata of chronic liver disease e.g. jaundice, palmar erythema etc.
9. Abdominal examination to assess the condition of liver (e.g. size, consistency), spleen as well as other abdominal organs
10. Ultra sonographic imaging: to assess the liver size, border, echognicity pattern, presence of focal lesion, spleen, gall bladder, portal vein (diameter and thrombosis) and detection of ascites etc.
11. Laboratory investigation including AST, ALT and HCV RNA with a sensitive PCR assay, the Cobas Amplicor v2.0 test HCV RNA PCR (Roche Molecular Systems, Inc., Pleasanton, CA) according to the manufacturer’s recommendations [5]. In HCV RNA positive sera, viral load was quantified by bDNA (Versant 2.0; Siemens Healthcare Diagnostics, Deerfield, IL), an HCV RNA signal amplification method, with detection limit of 15 IU/mL [6-8].
HCV Ag Detection and Quantification
Sera, stored at -70°C, were thawed once and tested with the Architect HCV-Ag assay. Samples were processed automatically, first treated on-board to dissociate antigen-antibody complexes and release the antigen from the viral particles. HCV core Ag is detected with a cutoff value of 3.0 fmol/L, a grey zone of 3-11 fmol/L and an upper limit of 180,000 fmol/L (1.0 fmol/L of HCV Ag equals to 0.02 pg/mL) [9].
Ethical approval of the study
Ethical approval for the study was obtained from EL-Minia university Ethics committee in accordance with the 1975 Declaration of Helsinki.
Statistical analysis
A statistical analysis was performed by a statistical analysis software version 9.1.3(SAS Institute, Inc., NC, USA.) GraphPad PRISM software and MedCalc statistical software.
The correlation coefficients between HCV-Ag and HCV RNA were calculated by Spearman’s rank test and a comparison between the groups was carried out using the Mann-Whitney U test. P value of less than 0.05 was considered statistically significant.
Results
Table 1 shows the demographic, clinical and laboratory data of the studied groups. there are no significant statistical difference was seen apart from the mean level ± SD of AST and ALT 45.44 ± 4.77)and (47.45 + 3.72) p value (<0.1, <0.2) respectively.
While, Table 2 showed that HCV core Ag was undetected in two patients with HCV RNA was 3.06 and 3.29Log IU, four sera were found in grey zone with value between 6 and 10.6 fmol/L or in Logs 0.84 Log 10 their respective HCV RNA level were between 3.07 and 4.9Log HCV core Ag was clearly positive in 19 patients with HCV RNA levels between 3.12 and 6.87 log10 IU/mL (median 5.55) the correlation coefficient (r) of the logarithmic values of HCV RNA and HCV-Ag, in these cases, was 0.89 (y = 0.8465 x -1.5343) and it was statistically significant (P < 0.001). Figure 1 also the correlation coefficient (r) of the logarithmic values of HCV RNA and ALT was statistically significant a (r = 0.205; P < 0.04) as shown in Figure 2.
Discussion
Since 1999, an ELISA assay has been introduced for serum HCVAg detection and quantification however this method showed marked differences in HCV RNA/core Ag ratios and did not demonstrate the required sensitivity in order to be clinically applied as a substitute for HCV RNA measurements that had become widely used [10]. The employment of chemiluminescent magnetic particle- based technology and immune complex-dissociating reaction increases the sensitivity of the studied Architect HCV-Ag test 8 to 21 fold from previous HCV core Ag ELISA assay [11].
In our study we demonstrated sensitivity in clinical specimen’s equivalent to 1200 IU/mL, corresponding to the serum with the lowest HCV RNA that tested positive for HCV-Ag. This is clear evidence of the enhanced ability of the new HCV core Ag test compared to older ELISA, to detect low level viremia and this in accordance with Medici et al. [12], who studied 193 HCV RNA low positive samples (<3.9 log10 IU/mL), HCV-Ag was detected in low levels (median 16 fmol/L) in 81.9% of studied samples and the sensitivity of HCV-Ag corresponded to 1000 IU/mL of HCV RNA and also in agreement with Emilia et al. [13]. Who demonstrated sensitivity in clinical specimen’s equivalent to 1200 IU/mL, corresponding to the serum with the lowest HCV RNA that tested positive for HCV-Ag. These results significantly correlated with HCV RNA measurements by bDNA with a correlation coefficient of 0.89. Higher correlation was reported in a previous study using a more sensitive Cobas-TaqMan RT-PCR assay (r=0.95).
On the other hand Medici et al. [12]. Concluded that the correlation coefficient between HCV-Ag and bDNA was 0.713 and this could be explained by that these results were based on calculations with absolute and not logarithmically transformed values. We used logarithmic transformation of HCV RNA values as wide range of the previous studies used it which is also common practice in the clinical assessment of chronic hepatitis
HCV core Ag levels were correlated also with ALT levels (0.205; P < 0.04) and this in agreement with Durante-Mangoni et al. [14]. who studied in a prospective cohort of 114 patients with chronic hepatitis whoever this study included genotype 1,2 and 3 only not in genotype 4 as in our study.
Data emerging from this study suggest that, HCV Ag positivity is clear proof of viremia and active HCV infection related to liver disease activity as measured by levels of ALT, The Architect HCV Ag assay potentially offers a cost-effective alternative to HCV RNA quantification. It is operationally easier than molecular assays, being available on a fully automated random access platform. Specimen transit time is short with a time of 36 min to the first result, and its low cost per assay.
Suggesting that it is likely to be established in the clinical laboratories as a routine used tool to aid in early diagnosis of HCV infection. More studies, with concomitant consideration of the financial cost of HCV viral load testing, need to be conducted in order to pinpoint the proper applications of HCV Ag testing.
References

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Review summary

  1. Maddison
    Posted on Oct 17 2016 at 6:32 pm
    In the present manuscript authors have demonstrated the use of HCV antigen assay as a screening tool for anti-HCV assay for diagnosis of hepatitis C virus infection. A very nice, well-structured and informative study. The association presentation is quite remarkable.
 

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