School of Medical Technology

Evaluation of chemiluminescent assay (CMIA) to the core antigen of hepatitis C virus plasma samples from blood donors and hospital patients with chronic renal Catholic University of Chile Clinical

RUBI ARACELI TRONCOSO CALVIO Advisor: TM. Guillermo Alfonso Jerez Jerez. Santiago, Chile

INTRODUCTION It is estimated that worldwide infection with the Hepatitis C virus (HCV) affects 170 million people (3% of world population) creating a huge burden of chronic diseases to be a progressive liver disease.1 The hepatitis C virus (HCV) is one of the most important etiological agents of chronic liver damage (cirrhosis and liver cancer 15 to 20% of cases). It is also the leading cause of liver transplant indic ation in adults worldwide 2 , 3 In our country, the seroprevalence of anti -HCV in blood donors is low reported between 0.28% to 0.3%
3 , 4.

1.1 Major routes of transmission of HCV:

1.1.1 Transmisin transfusional The hepatitis C virus is mainly transmitted via blood transfusion , and all that is contaminated with blood or blood products; being intravenous drug users the second group found more frequently in HCV infected 5. 1.1.2 Transmisin vertical About 6% risk of infection from mother to child transmission is through the birth canal Error! Reference source not found., Error! Reference source not found., Error! Reference source
not found..

1.1.3 Sexual Transmission The infection via sexual intercourse has been reported, although not as efficient as in other viral diseases such as in the case of HIV or hepatitis B. However, in 50% of cases is not possible to identify a clear risk factor. 1.1.4 Otras vas de transmission Evidence suggests indirectly those surgical procedures, medication management, dental surgeries and other medical procedures in which they produce mucosal

contact with potentially infected fluids such as blood could be a route of infection for HCV, particularly in underdeveloped or developing 8.

1.2 General Features of hepatitis C. 1.2.1 Structural characteristics of HCV The hepatitis C virus is an RNA virus belonging to the family Flaviviridae, genus Hepacivirus
9.-10

. The viral RNA has an open reading frame ( ORF), encoding a

11.-12 .-

single polyprotein of about 3000 amino acids

The coding region consists of

two untranslated regions, 5 'and 3' NTR. These regions are highly conserved and play an important role in translation and viral replication. The 5 'NTR contains an internal site of ribosome entry (IRES) that binds to the 40S ribosomal subunit and is responsible for initiating the translation of the polyprotein.

Figure 1. Genomic organization of HCV. Source: Journal of General Virology (2000) 81, 1631-1648.

Both the core structures, E1 and E2 and non -structural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) have been characterized. Core is a protein that is located in the cytoplasm and has been linked to interactions with cellular proteins, modulating gene transcription and proliferation among others, also plays a role in response inhibition inmune 13
.-

E1 and E2 surface glycoproteins that interact with

the receptor and are important in inducing fusion with the cell membrane husped 14

E2 has a hypervariable region has differences of up to 80% in the amino acid

sequence among different viral genotypes. The function of p7 is not known, some authors sugg est that play a role in mating and maturation viral 15 , NS2 autoprotease and NS3 is a serine protease is a multi site cleaving the viral polyprotein, also has a domain that functions as helicasa 16, NS5A is a phosphorylated protein of unknown function. Recent studies have shown that their sequence would be associated with different patterns of response to interferon therapy, identifying a region ISDR (interferon sensitivity Determining)
17 , 18

region

, NS5B polymerase is an RNA-dependent RNA, involved in viral

replication, being a target for the development of antivirales 19 (Figure 1).

1.2.2 Proliferation of HCV. Humans are the only natural host for HCV. Not yet have an efficient system for cell culture or animal model versatile for studies of viral proliferation, -important pillar experimental- of molecular virology. This limits the study and understanding of viral replication and persistence, as well as the development of new antiviral therapeutic agents and vaccines. However, alternative systems have been developed to study such as infectious cDNA; product expression of NS protein subgenomic (replicon) virus-like particle (VLP), among others, that have enabled a breakthrough in the understanding of the state characteristic of chronic persistent infe ction of HCV. A variety of cellular receptors are involved in binding and entry of virus into the host cell. Among these are included in lymphocytes and CD81 cell surface proteins involved in the internalization of lipoproteins such as the LDL receptor class B type I in hepatocitos 20 . It is proposed that mucopolysaccharide with a high degree of sulfation (heparan sulfate) could serve as the initial site of absorption for HCV from which the virus would be transferred to any of the aforementioned receivers, promoting the entry of the virus to clula 21. The envelope glycoprotein complex E1 E2 is expressed on the surface of the viral particle, constituting the natural candidate "ligand" to interact with viral receptors celulares 22.

The virus would enter the cell by endocytosis mechanism receptor-mediated, followed by membrane fusion events between the viral envelope and endosomal pH dependent tanks. The viral capsid is naked after their release into the cytoplasm. The viral genome is the glass for the synthes is of genomic copies and also a substrate for the translation of the viral polyprotein (PPV), which begins with the interaction of IRES with ribosomes and where the 3 'NC play a regulatory role very importante 23. The PPV is processed co-and post-translation by cellular and viral proteases, from which will generate 10 mature proteins. C, E1 and E2, are released by the action of cellular proteases, NS separate finding of a small membrane polypeptide called p7, postulated as viroporins, or ion channel involved in release and maturation of viral progeny. Association of the core (C) mature with neosintetizadas genomic RNA molecules give rise to nascent nucleocapsids of HCV.

2. PROJECT DESCRIPTION AND RATIONALE

Currently in the Blood Bank of the UC Hospital detection of hepatitis C virus in blood donors and in patients with chronic kidney is performed by microparticle chemiluminescent assay (CMIA) for the qualitative detection of HCV antibodies in serum or plasma. Positive results are confirmed by additional technical as the assay RIBA (recombinant immunoblot assay), whereby individuals are detected HCV antibodies against the different recombinant proteins ensayo. 24 The detection of anti-HCV can be carried out only after seroconversion, which is the major disadvantage of this technique, as there is a serological window period of 45 to 68 days in which the virus can not be detected generating a transfusion risk that should not be ignored. Le antibody detection can not distinguish between an acute infection, past or persistent. Moreover, it is important to mention there is a group of patients who have a deficient immune system, such as VHI positive and hemodialysis patients, which take longer to generate antibodies addition to produce in smaller quantities, so these patients could be classified errneamente. 24

Detection of HCV RNA is considered confirmatory technique in both groups of patients, immunocompetent are anti-HCV-positive and immunocompromised individuals can not mount an adequate immune response. The drawback of this technique of HCV RNA amplification is its high cost and time spent, addition to the requirement of sophisticated equipment that require highly trained personnel. These limitations, however, do not apply to the technique of detection of HCV core antigen, it is easy to carry out an immunoassay format, providing results in a comparatively short time and is theoretically less prone to cross contamination of samples of nucleic acid amplification. 25
2.1 Working hypothesis

The core antigen of hepatitis C virus mediante inmunoanlisis quimioluminiscente de micropartculas (CMIA), is a highly sensitive marker of viral presence in plasma samples, CMIA independent of the outcome of anti -HCV antibody. This research aims to determine the characteri stics of the detection of HCV core antigen using magnetic microparticle chemiluminescent assay y compararlo con la tcnica CMIA para la deteccin de anticuerpos anti -VHC and PCR (Polymerase Chain Reaction), to determine its sensitivity. For which use simple random sampling and representative of the population of blood donors and the total population of patients suffering chronic renal UC Hospital. During the months of March to May 2011 will take place in plasma samples from blood donors and patients with c hronic renal failure following determinations: 1. Detection of HCV core antigen by CMIA test. ARCHITECT HCV Ag *. 2. Detection of anti-HCV by CMIA test. Anti-HCV ACHITECT. The core antigen and anti -HCV antibodies was performed in the ARCHITECT i2000 analyzer.

3. Confirmation of reactive results for anti -HCV by Chiron RIBA HCV 3.0 SIA . 4. Confirming results reagents for HCV core antigen by molecular technique. 5. Simple random sampling will be conducted on samples negative for HCV core antigen to which they were made to rule out false negative PCR.
ARCHITECT HCV Ag*

The ARCHITECT HCV Ag * Abbott is a chemiluminescent microparticle immunoassay (CMIA) for the qualitative determination of the core antigen of hepatitis C virus in human serum and plasma.
2.2 Principles biological ARCHITECT HCV Ag Assay:

The ARCHITECT HCV Ag test is a two-step immunoassay using chemiluminescent microparticle technology (CMIA) with a flexible test protocol called Chemiflex, for the qualitative determination of the core antigen of hepatitis C. In the pre-treatment sample is combined with pre-Reagent 1 Reagent 2 treatment and pretreatment. An aliquot of the pretreated specimen is aspirated and dispensed into a new reaction vessel. The sample pre-treated Specific Diluent test and HCV antibody coated microparticles are combined. In the first step of the HCV core antigen in the sample pretreated binds to the antibody coated microparticles. After washing, the conjugate is added antibodies labeled with acridine, this is the second step. After making a second wash cycle solution is added to the Pre-Trigger and Trigger the reaction mix. The result of the chemiluminescent reaction is measured in relative light units, which are detected by the ARCHITECT i 2000sr optical system.
2.3 Reagents

2.2.1 ARCHITECT HCV Ag Kit (6L47). - Particulates: Contains 1 vial of 6.7 ml of microparticles, comprising: microparticles coated with murine anti-HCV in 400 nM Bicine, 50 mM TRIS buffer with bovine protein as a stabilizer. Preservatives: sodium azide as antimicrobial agents. - Conjugate: Contains 1 vial of 6.1 ml of conjugate, comprising: conjugated anti-HCV antibodylabeled murine acridine in BIS-TRIS buffer with 80 nM bovine protein as a stabilizer. Preservatives: sodium azide as antimic robial agents. - Assay Specific Diluent: 1 bottle contains 30 ml Assay Specific Diluent, consisting of: NaOH 1.46 N. - Reagent 1 Pre-Treatment: 1 bottle contains 14.5 ml of Reagent 1 Pre-treatment HCV Ag, consisting of: HCl 0.83 N. - Reagent 2 Pre-Treatment: 1 bottle contains 11.0 ml of Reagent 2 Pre-treatment HCV Ag, consisting of: HCl 0.83 N. - Sample Diluent: Contains 1 vial of 5.9 ml of Sample Diluent, consisting of: phosphate buffered horse serum protein as a stabilizer. Preservatives: sod ium azide as antimicrobial agents.
2.2.2 Other Reagents:

Pre-Trigger Solution, containing 1.32% hydrogen peroxide (w / v) The pretrigger is a pre-activating solution of hydrogen peroxide. Its function is to generate an acidic environment, preventing pr emature loss of light, prevents agglutination of the microparticles and separates the dy e acridine complex

particulate-conjugate, acridine preparing your next step. Is kept refrigerated and stability on board is 28 days. Trigger Solution, containing 0.35 N sodium hydroxide The Trigger is an activator solution of sodium hydroxide provides the alkaline environment is necessary for carrying out the oxidative action of hydrogen peroxide acridine to form the N-methylacridone that upon returning to their origi nal state generates energy in the form of light, thus causing the chemiluminescent reaction. Stored at room temperature and its stability on board is 28 days . Wash buffer, containing phosphate buffer saline solution. Preservative: antimicrobial agent.
2.3 Data collection and sample preparation

2.3.1 Sample Types The sample collection tubes listed below have been tested for use in this trial. Other sample collection tubes have been tested with this assay. Human serum (including serum collected in seru m separator tubes) Human plasma collected at: - Sodium EDTA - Potassium EDTA - Lithium Heparin - Heparin Sodium - Sodium citrate - CPD The liquid can cause thinning of the sample dilution resulting in lower concentrations of individual samples of patients.

ARCHITECT i2000sr system can not verify the type of sample, this is the responsibility of the operator.

2.3.2 Sample Conditions Do not use samples in the following conditions: - Heat Inactivated - Aggregate - hemolyzed - Microbial contaminati on apparent - Samples of body or other body fluid For optimum results the serum and plasma samples should be free of fibrin, red blood cells and other particles.
3. GENERAL AND SPECIFIC OBJECTIVES 3.1 General Objective

1. Knowing the advantages and disadvantages in the detection of antigen hepatitis C Virus Core in plasma samples obtained from blood donors and patients with chronic renal UC Clinical Hospital.
3.2 Objectives Specific

1. Core antigen detection of hepatitis C virus (HCV) plasma from blood donors and patients with chronic renal failure , by CMIA test. 2. To determine the sensitivity and specificity CMIA assay for detection of HCV core antigen.

3. CMIA trials comparing anti -HCV antibody and CMIA HCV Core Antigen. 4. Comparatively analyze the current methods of screening (CMIA) and confirmed (RIBA-HCV) for the detection of anti-HCV with CMIA method of HCV Core Antigen.

4. GENERAL PLAN WORKING

5. BIOSECURITY CONDITIONS

HCV is the leading cause of hepatitis non A -non B. The prevalence of infection varies from 0.2 to 2.5%, in Chile of 0.27% in a population of donors. 75% of infections are asymptomatic and 50% had persistent disease, whose consequences can be severe cirrhosis or hepatocellular carcinoma. The main mechanism of HCV transmission is the direct percutaneous exposure to contaminated blood, such as intravenous drug users, blood transfusions and organ transplants. Sharps accidents transmission with contaminated items is less efficient, with the exception of hemodialysis u nits. They also described sexual and transplacental transmission, but is minor
26

The risk at 20 years of developing cirrhosis has been estimated between 14 and 45% in untreated patients. Once there is cirrhosis, the possibility of developing decompensation or hepatocellular carcinoma is approximately 20% at 5 years. Life expectancy in these patients is reduced by an average of 7 to 10 years. Unfortunately there are no factors that can predict accurately which patients are at increased risk for developing i t.27 Given the above, blood samples from donors or patients should be handled with biohazard level II, by all means of universal precautions,
28

which are based on the

risk of transmission of a biological agent in the health system is due to accidental inoculation with blood from an infected person. As it is impossible to identify all persons are advised to treat all patients as potentially infectious. Furthermore, the risk of infection will be proportional to the prevalence of the disease in the geographic production and the likelihood of accidents wh ile performing procedures.
29

Personal hygiene:

- Cover cuts and wounds with waterproof dressings. - Cover skin lesions with gloves. - Remove rings and other jewelry. - Wash hands before and after seeing pa tients. Barrier protection elements: - Use gloves when handling blood or body fluids potentially infected objects or invasive procedures. - Use of masks on predicted production of splashes of blood or fluids to the nasal or oral mucosa. - Eye protection when splashes provides for the production of the ocular mucosa. - Use waterproof gowns or aprons, when it is expected to produce large volumes of splashes of blood or body fluids. Handling sharps: - Work with extreme care. - No re encapsulate the needles. - Eliminating security rigid containers. - Do not abandon them anywhere. - Check that there are between clothes that are sent to the laundry. Isolation of the sample if the patient has: - uncontrolled bleeding. - Changes uncontrollable behavior. - Profuse diarrhea. - infectious processes that require insulation.

Current recommendations against occupational exposure to HCV are as follows:

27

Determine the source of the baseline anti -HCV antibodies. If the source is reported reactivity for anti-HCV antibodies by enzyme immunoassay (ELISA), these should be repeated in the same sample (repeatedly reactive) and must be confirmed by a supplementary test for detection of anti -HCV antibodies (immunoassays or recombinant immunoblot recombinant line). Not recommended for detection of liver enzymes. The exposed worker, determine anti-HCV antibodies at baseline at 6 weeks and 3 months. Consider that seroconversion occurs, on average, between 8 to 10 weeks post-exposure. Should there be seroconversion: -Refer immediately to a specialist to assess probable therapy. To inform the potential risk of transmission to others: Inability to donate blood. Inability to donate organs or tissues. -Report on the potential risk of sexual transmission. Efforts should be aimed primarily at reducing the occupational risk of HCV infection. That is, using standard precautions and avoiding unnecessary exposure to risk during the study.
6. BIOETHICS STANDARDS

The determination of competence and autonomy of the subject that will be part of the investigation must be established before the study began. In this context,

informed consent (IC) specifies, for the particular case of research, "... The free and rational subject to a procedure proposed by the health t eam, either with intent diagnostic, prognostic, therapeutic
31

or

experimental,

including

competitio n,

information and freedom

".

From this perspective, the IC can be considered a tool to incorporate the subject and / or their families or representatives to process recognizing their interests and allows them to balance situations and choose accordingly. If we focus on this definition,

we understand or consent of the

32

that any medical patientcan be

act performedwithout prior authorization a crime against freedom of the patient authorization of the patient by a CI

. Therefore it is necessary to have the

to use your blood sample in the various

determinations to be performed in this study.

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