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Context
Hepatitis C is a contagious disease caused by the HCV virus. It is one of the main causes of chronic liver disease and cirrhosis worldwide, resulting in a large proportion of all liver transplants in the United States, Australia & most of Europe. Hepatitis C was first isolated from non-A, non-B Hepatitis in 1989 and accounts for 90% of such cases (Chen & Morgan, 2006). An estimated 180 million people are infected with Hepatitis C globally with highest concentrations in Sub-Saharan Africa and East Asia (Strader & Seeff, 2009). In Ireland, around 700-800 new cases are identified each year. However, as the symptoms are similar to that of the flu, many cases go unidentified. Its prevalence rate is estimated at 2.2% globally and between 0.5% and 1.2% in Ireland (Thornton et al, 2011). Hepatitis C is divided into two types depending on duration of illness, Acute Hepatitis C and Chronic Hepatitis C. Acute Hepatitis is generally diagnosed as a short-term illness that develops within the first 6 months of exposure to the virus. Approximately 75%-85% of those with Acute Hepatitis C will develop Chronic Hepatitis C (where the infected persons body cannot get rid of the virus). Depending on the presence of cofactors (such as heavy alcohol consumption and older age when HCV is acquired), between 10% and 40% of patients with chronic Hepatitis C infection will develop cirrhosis (European Association for the Study of the Liver 2011 EASL Clinical Practice Guidelines Management of hepatitis C virus infection, 2011). Unlike Hepatitis A and B, there is no Hepatitis C vaccine.
Hepatitis C can be split into at least six genotypes (with genotypes 1 and 3 being the most prevalent in Ireland) and consequently, in this study, we are dealing with genotypes 1, 2 and 3. Each genotype can be further classified according to its subtype (which is denoted by a lower-case letter). The classification of genotypes and subtypes is important in order to determine the most suitable treatment method for an infected patient.
The Hepatitis C virus is carried in the blood, and most commonly infects a person if blood from someone who is already infected enters their body. The most common way this occurs is via the sharing of needles between a person who is infected and a person who is not, as a means of injecting drugs. Therefore, drug users are more likely to be infected with Hepatitis C.
It is also possible for Hepatitis C to spread as a result of sexual transmission, getting tattooed or being born to someone infected, although this is rare. Only 6% of infants born to mothers infected with Hepatitis C will, themselves, get infected (Centres for Disease Control and Prevention, 2015).
Of those with acute Hepatitis C, approximately 10-15% experience flu-like symptoms including fever, nausea and jaundice. The majority of acute cases dont exhibit symptoms, so many are unaware that they have become infected. Most chronic cases of Hepatitis C are also asymptomatic, hence the virus usually only becomes noticeable after several years after infection, when the liver is sufficiently damaged. These patients can develop serious issues such as cirrhosis, fibrosis and liver cancer in approximately 6-8% of cases infected for over twenty years (Keating, 2003).
Motivation/Rationale
Chronic Hepatitis C is diagnosed using viral load tests and hence, it is important to be able to identify a 95% hit rate and a 95% confidence interval for the LOD for each genotype as we are doing in this study. Viral Load tests are blood tests in which the genetic material of the HCV virus, or its ribonucleic acid (RNA) is measured. Viral load tests have two main categories: qualitative tests, which determine whether or not HCV RNA is present in the blood, and have either a positive or a target not detected test result; and quantitative tests, which measure the amount of HCV RNA in one millilitre of blood. Viral load is typically measured in International Units per mL (IU/mL). A viral load test result is interpreted as low for a measurement of less than 800,000 IU/mL (5.90 log10 IU/mL) and high if a measurement greater than this is obtained, though it is disputed whether or not this is a feasible cut-off point (Franciscus & Highleyman, 2012). Log10 IU/mL units are commonly used to represent the viral load due to a tendency for transcription errors to occur when dealing with large figures of IU/mL.
Viral Load tests are used to track the progression in the treatment of HCV patients. When the virus can no longer be detected (ie. the Viral Load is below the LLOD), a virologic response (VR) is said to have taken place, which gives an indication that this method of treatment is effective on the patient. The patients Viral Load then continues to be monitored over time and if it remains below the LLOD (ie. result of Target Not Obtained, TND), a sustained virological response (SVR) is said to have occurred. However, it is pertinent to mention that due to the high cost of testing, the patient is generally tested at commencement of treatment, after 4 weeks and again usually after 3 months. It is very rare for a patient who achieves a SVR to ever become infected with HCV again. In a study on the Risk of Late Relapse or Reinfection With Hepatitis C Virus After Achieving a Sustained Virological Response carried out B. Simmons in 2016 of low-risk patients (n = 7969),the 5-year recurrence rate was found to be 0.95%. (B. Simmons, 2016)
Safety issues and side effects of the treatment are a major concern for many treatment approaches. For example, a small sample-size study carried out by Ghany, Stradder, Thomas and Seeff in 2009 found that despite 90% of patients who were treated with the drugs peginterferon and ribavirin achieving a SVR, the majority of patients experience adverse events during the course of therapy. Influenza like side effects such as fatigue and headache occurred in greater than half of the patients and Neuropsychiatric side effects (including anxiety, depression, insomnia,, mood disorders,, suicidal ideation, actual suicide, and homicide) occurred in 22% to 31% of patients. (Ghany Strader Thomas & Seeff 2009) These side effects of the drug, coupled with the high rates of adverse events such as severe neutropenia and anemia, means that it is crucial for whoever is administering the test to know the 95% hit rate and a 95% confidence interval for the LLOD, to limit the occurrence of false diagnoses or incorrect interpretation of the treatment progression of a patient.
Research Questions
This study aims to identify the 95% hit rate known as the Lower Limit of Detection (LLOD), a 95% confidence interval for the Lower Limit of Detection samples and finally, relate this to the manufacturing standard. In other words, we are trying the find the values for the Viral Load in International Units per ml (IU/mL) which will result in a greater than 95% probability of a positive hit. The hit rate is the number of positive tests (ie. hits) divided by the total number of replicates performed at each Viral Load. The total number of replicates performed at each Viral Load can be thought of as the sample size at that Viral Load (VL). For example, the COBAS® AmpliPrep/COBAS®TaqMan®HCVTest (diluted in EDTA plasma matrix) has 95% confidence limits for the hit rate of 1119.8 log10 IU/mL, while the same test diluted in serum matrix (instead of plasma matrix) has 95% confidence limits for the hit rate of 8.414.8 log10 IU/mL (Sizmann et al, 2007).
In addition, we want to ascertain whether or not this 95% Confidence Interval can be considered to be the same across the three genotypes which are dealt with in this study, namely Genotypes 1, 2, and 3. Each genotype has a separate set of data, which tells us the Viral Load of the sample, the number of hits and the total total number of replicates performed. We are not given data regarding what sub-genotype each sample belongs to and hence, do not check whether or not this 95% Confidence level is the same across sub-genotypes.
Finally, the study seeks to identify the level of the Lower Limit of Detection and to compare this level to the manufacturing standard. The Lower Limit of Detection (LLOD) is the lowest actual value for the Viral Load in a sample that can be detected with 95% probability. We want to compare these 95% hit rates for the three genotypes to manufacturing standards for the LLOD, given to us as a figure for the LLOD of 1.18 log10 IU/mL.
There are numerous possible reasons for differences between the value of the LLOD indicated by our samples and those given by the manufacturer. Firstly, the sample of blood we are testing might not have a uniform level of viral load. This could lead to us taking a sample that does not reflect the viral load of the blood as a whole. The blood is also frozen multiple times, which could have an effect on the viral load. Other factors such as the Assay Manufacture/Device used and whether the test was carried out on Blood Serum or Plasma also provide potential for our level for the LLOD to different from manufacturing standards. Hence, it is important to calculate this LLOD value (as is the purpose of this study) instead of assuming that the manufacturing standard holds.
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