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Merck Significantly Expands its Patient Assistance Program Offerings to Provide Access to More Medicines for People in Need
Survey shows more than one third of uninsured, unemployed Americans have skipped or incorrectly taken their prescribed medications to save money, yet many may qualify for free medicine
WHITEHOUSE STATION, N.J., Sep 22, 2010 (BUSINESS WIRE) -- Today, Merck announced that it has significantly expanded the number of Merck medicines available through its Merck Helps(TM) patient assistance programs, which include the Merck Patient Assistance Program, the Merck Vaccine Patient Assistance Program, the ACT Program for Oncology and Hepatitis C medicines, and the SUPPORT(R) Program for HIV/AIDS medicines. The Merck Helps programs provide Merck medicines and vaccines free of charge to eligible individuals, primarily the uninsured, who earn up to 400 percent of the federal poverty level who, without assistance, cannot afford needed Merck medicines.
To help increase awareness of all patient assistance programs, the Merck Company Foundation has provided a grant to NeedyMeds, a nonprofit organization with a mission to help people who cannot afford medicine or healthcare costs by making information about these programs available at no cost. NeedyMeds will be using the grant to translate its website into Spanish as well as working closely with various healthcare clinics to increase knowledge of and access to patient assistance programs.
"Merck has historically recognized the critical need for people to have access to the prescription medicines and vaccines they require, even if they lose their insurance," said Michael Rosenblatt, M.D., executive vice president and chief medical officer at Merck. "Our patient assistance programs now provide access to even more medicines for chronic conditions like asthma, diabetes and high blood pressure, allowing us to reach more people in need."
A recent telephone survey of more than 2,000 Americans, conducted by Harris Interactive on behalf of Merck(1), found that more than one third (35%) of those who are uninsured and unemployed did not buy or refill medicines they were prescribed, cut their dosage in half, or took expired medicine as a way to save money. Furthermore, while 32 percent of U.S. adults are potentially eligible for patient assistance programs, 79 percent of those who are uninsured and unemployed are somewhat or not at all aware that such programs exist.
"Unfortunately, many patients don't know that there are patient assistance programs available if they can't afford their prescriptions, and the need is great," said Richard Sagall, MD, co-founder of NeedyMeds. "That's why we are pleased to partner with Merck to help further raise awareness of these programs."
"With an uncertain economy and near double-digit unemployment, more patients are struggling to pay for their medicines than ever before," said Emmanuel Durham, director of Community Healthcare Network -- Helen B. Atkinson Health Center, New York, NY. "I have seen firsthand how the Merck Helps programs can keep needed medicines in reach."
About Merck Helps
For more than 50 years, Merck has helped millions of patients gain access to medicines for chronic conditions like asthma, diabetes and high blood pressure through the Merck Patient Assistance Program. As a global healthcare leader working to help the world be well, Merck provides its medicines and adult vaccines for free to people who do not have prescription drug or health insurance coverage and qualify for a Merck Helps program. Merck Helps programs include:
-- The Merck Patient Assistance Program, which helps eligible patients who earn up to 400 percent of the federal poverty level gain access to Merck medicines for chronic conditions like asthma and diabetes.
-- The Merck Vaccine Patient Assistance Program, which provides free vaccines to adults over age 19 who do not have health insurance coverage for vaccines and who earn up to 400 percent of the federal poverty level.
-- The ACT Program for Oncology and Hepatitis C medicines, which provides free reimbursement support services and refers appropriate patients to a patient assistance program for eligible individuals who earn up to 500 percent of the federal poverty level.
-- The SUPPORTProgram for HIV/AIDS, which provides free reimbursement support services and refers appropriate patients to a patient assistance program for eligible individuals who earn up to 500 percent of the federal poverty level.
All Merck Helps programs are confidential and patients may qualify for the Merck Patient Assistance Program and the Merck Vaccine Patient Assistance Program if they have a household income of $43,320 or less for individuals, $58,280 or less for couples, or $88,200 or less for a family of four, even if the financial situation is temporary due to unemployment or other reasons. Patients may qualify for The ACT Program and the SUPPORT Program if they have a household income of $54,150 or less for individuals, $72,800 or less for couples, or $110,250 or less for a family of four. With the Merck Helps programs, there are no application fees, no co-payments and a simple enrollment process. Many medicines can be delivered to a patient's home or doctor's office at no charge. Patients in need of information should visit www.MerckHelps.com or call (800) PAP-5400.
About NeedyMeds
NeedyMeds is a non-profit organization founded in 1997 as a resource for people who need help with the cost of medicine. The mission of NeedyMeds has been, since its inception, to make comprehensive and reliable information about assistance programs available to low-income patients and their advocates at no cost. NeedyMeds' website is visited by over 14,000 people each workday.
About Merck
Today's Merck is a global healthcare leader working to help the world be well. Merck is known as MSD outside the United States and Canada. Through our prescription medicines, vaccines, biologic therapies, and consumer care and animal health products, we work with customers and operate in more than 140 countries to deliver innovative health solutions. We also demonstrate our commitment to increasing access to healthcare through far-reaching policies, programs and partnerships. For more information, visit www.merck.com.
(1) This telephone survey was conducted by Harris Interactive on behalf of Merck from August 20 to 23, 2010 among 2,012 U.S. adults of whom 549 do not have any prescription drug benefits and 131 do not have any prescription drug benefits and are not employed.
SOURCE: Merck
Media Contact:
Merck
Ron Rogers, 908-423-6449
Monday, September 27, 2010
Mistakes in Genotyping 1, which may occur more in IDUs, becomes more important now with resistance a concern associated with new HCV oral drugs
Mistakes in Genotyping 1, which may occur more in IDUs, becomes more important now with resistance a concern associated with new HCV oral drugs, activity & development of resistance appears to perhaps differ between genotypes 1a and 1b, as well mistakes in genotyping 1 vs 2 or 3 appears to occur
Evaluation of Versant Hepatitis C Virus Genotype Assay (LiPA) 2.0 - pdf attached - (09/21/10)
Concerns About HCV Genotyping: Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice - pdf attached - (09/20/10)
Methods based on the sole analysis of the 5'NCR, namely Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively (Table 1)...... The results clearly show that, although they are by far the most widely used techniques in new HCV drug development trials, genotyping techniques based on the sole analysis of the 5'NCR should be avoided, as they mistype approximately 25% and 10% of HCV subtype 1a and 1b strains, respectively......INNO-LiPA HCV 2.0 displays the same 5'NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1)The real-time PCR-based assay targeting both the 5'NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1)......Novel assays have been recently developed that aim at better discriminating among the different HCV genotype 1 subtypes and between genotypes 1 and 6. Abbott RealTime HCV Genotype II assay is a real-time PCR method using several sets of genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region. As shown in Table 1, adding a second target region for analysis led to substantially improving HCV genotype 1 subtype identification compared to methods targeting the sole 5'NCR. However, in contrast with a previous report [33], we found that this assay failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases"
Evaluation of Versant Hepatitis C Virus Genotype Assay (LiPA) 2.0 - pdf attached - (09/21/10)
Concerns About HCV Genotyping: Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice - pdf attached - (09/20/10)
Methods based on the sole analysis of the 5'NCR, namely Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively (Table 1)...... The results clearly show that, although they are by far the most widely used techniques in new HCV drug development trials, genotyping techniques based on the sole analysis of the 5'NCR should be avoided, as they mistype approximately 25% and 10% of HCV subtype 1a and 1b strains, respectively......INNO-LiPA HCV 2.0 displays the same 5'NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1)The real-time PCR-based assay targeting both the 5'NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1)......Novel assays have been recently developed that aim at better discriminating among the different HCV genotype 1 subtypes and between genotypes 1 and 6. Abbott RealTime HCV Genotype II assay is a real-time PCR method using several sets of genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region. As shown in Table 1, adding a second target region for analysis led to substantially improving HCV genotype 1 subtype identification compared to methods targeting the sole 5'NCR. However, in contrast with a previous report [33], we found that this assay failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases"
Santaris Pharma A/S Advances miravirsen, the First microRNA-Targeted Drug to Enter Clinical Trials
Santaris Pharma A/S Advances miravirsen, the First microRNA-Targeted Drug to Enter Clinical Trials, Into Phase 2 to Treat Patients Infected With Hepatitis C Virus
- Santaris Pharma A/S initiates Phase 2a clinical trial with miravirsen (SPC3649) to assess safety and tolerability in treatment-naive patients with chronic Hepatitis C
By: PR Newswire
Sep. 22, 2010 07:15 AM
Therapeutic Silencing of MicroRNA-122 by SPC3649 in Primates with ...
Science Mag, Dec 3, 2009 ... Here, we investigated the potential of miR-122 antagonism by SPC3649 as ... We next assessed the in vivo antagonism of miR-122 in chimpanzee ...
www.natap.org/2009/HCV/011009_01.htm
HOERSHOLM, Denmark and SAN DIEGO, September 22, 2010 /PRNewswire/ -- Santaris Pharma A/S, a clinical-stage biopharmaceutical company focused on the discovery and development of RNA-targeted therapies, today announced that it has advanced miravirsen (SPC3649), the first microRNA-targeted drug to enter clinical trials, into Phase 2 studies to assess the safety and tolerability of the drug in treatment-naive patients infected with the Hepatitis C virus (HCV).
Paving the way to conduct the first clinical trials of a microRNA-targeted drug in the United States, Santaris Pharma A/S also received acceptance of its Investigational New Drug (IND) application from the U.S. Food and Drug Administration (FDA). In addition to the United States, the Phase 2a clinical trials will be conducted in the Netherlands, Germany, Poland, Romania, and Slovakia.
The World Health Organization estimates about 3% of the world's population has been infected with HCV and that some 170 million are chronic carriers at risk of developing liver cirrhosis and/or liver cancer(2). Approximately 3-4 million Americans are chronically infected with an estimated 40,000 new infections per year(1). In Europe, there are about 4 million carriers(2). The current standard of care, pegylated interferon in combination with ribavirin, is effective in only about 50% of those treated(1).
Developed using Santaris Pharma A/S proprietary Locked Nucleic Acid (LNA) Drug Platform, miravirsen is a specific inhibitor of miR-122, a liver specific microRNA that the Hepatitis C virus requires for replication. Miravirsen is designed to recognize and sequester miR-122, making it unavailable to the Hepatitis C virus. As a result, the replication of the virus is effectively inhibited and the level of Hepatitis C virus is reduced.
"Advancing miravirsen, the first microRNA-targeted drug to enter clinical trials, into Phase 2 studies in patients with Hepatitis C demonstrates Santaris Pharma A/S leadership in developing RNA-targeted medicines," said Arthur A. Levin, Ph.D., Vice President, Chief Development Officer and President, US Operations. "Receiving IND acceptance from the FDA to conduct the first clinical trials with a microRNA-targeted drug in the United States brings Santaris Pharma A/S one step closer to potentially providing a growing number of patients chronically infected with HCV with a more effective and better tolerated treatment option."
The LNA Drug Platform is the only technology with both mRNA and microRNA targeted drugs in clinical trials, reinforcing the broad utility of the platform. The unique combination of small size and very high affinity, which is only achievable with LNA-based drugs, allows this new class of drugs to potently and specifically inhibit RNA targets in many different tissues without the need for complex delivery vehicles. LNA-based drugs are a promising new type of therapy that enables scientists to develop drugs to attack previously inaccessible pathways.
"Using our LNA Drug Platform to advance the first microRNA-targeted therapy into human clinical trials was certainly a scientific breakthrough," said Henrik Oerum, Ph.D., Vice President and Chief Scientific Officer of Santaris Pharma A/S. "We are extremely pleased with the results of the Phase I trials and excited to progress miravirsen into Phase 2 clinical trials. Because of its unique mechanism of action and tolerability profile, miravirsen has the potential to be an effective treatment option for patients with HCV."
The randomized, double-blind, placebo-controlled, ascending multiple-dose Phase 2a study will assess the safety and tolerability of miravirsen and is designed to enroll up to 55 treatment-naïve patients with chronic Hepatitis C virus genotype 1 infection. Secondary endpoints include pharmacokinetics of miravirsen and its effect on viral load. Miravirsen will be given as subcutaneous injections weekly or every other week for four weeks.
Data from Phase 1 clinical studies with miravirsen in healthy volunteers show that the drug is well tolerated. A recent study published in Science demonstrated that miravirsen successfully inhibited miR-122 and dramatically reduced Hepatitis C virus in the liver and in the bloodstream in chimpanzees chronically infected with the Hepatitis C virus(3). Miravirsen provided continued efficacy in the animals up to several months after the treatment period with no adverse events and no evidence of viral rebound or resistance.
In addition to miravirsen, Santaris Pharma A/S has a robust product pipeline targeting mRNAs and microRNAs both internally as well as in partnerships and collaborations with miRagen Therapeutics (cardiovascular diseases), Shire plc (rare genetic disorders), Pfizer (undisclosed therapeutic areas), GlaxoSmithKline (viral disease) and Enzon Pharmaceuticals (oncology).
About microRNAs
MicroRNAs have emerged as an important class of small RNAs encoded in the genome. They act to control the expression of sets of genes and entire pathways and are thus thought of as master regulators of gene expression. Recent studies have demonstrated that microRNAs are associated with many disease processes. Because they are single molecular entities that dictate the expression of fundamental regulatory pathways, microRNAs represent potential drug targets for controlling many biologic and disease processes.
About Locked Nucleic Acid (LNA) Drug Platform
The LNA Drug Platform and Drug Discovery Engine developed by Santaris Pharma A/S combines the Company's proprietary LNA chemistry with its highly specialized and targeted drug development capabilities to rapidly deliver potent single-stranded LNA-based drug candidates against RNA targets, both mRNA and microRNA, for a range of diseases including metabolic disorders, infectious and inflammatory diseases, cancer and rare genetic disorders. The LNA Drug Platform overcomes the limitations of earlier antisense and siRNA technologies to deliver potent single-stranded LNA-based drug candidates across a multitude of disease states. The unique combination of small size and very high affinity, which is only achievable with LNA-based drugs, allows this new class of drugs to potently and specifically inhibit RNA targets in many different tissues without the need for complex delivery vehicles. LNA-based drugs are a promising new type of therapy that enables scientists to develop drugs to attack previously inaccessible clinical pathways. The most important features of LNA-based drugs include excellent specificity, providing optimal targeting; increased affinity to targets providing improved potency; and strong pharmacology upon systemic delivery without complicated delivery vehicles.
About Santaris Pharma A/S
Santaris Pharma A/S is a privately held clinical-stage biopharmaceutical company focused on the discovery and development of RNA-targeted therapies. The Locked Nucleic Acid (LNA) Drug Platform and Drug Discovery Engine developed by Santaris Pharma A/S combine the Company's proprietary LNA chemistry with its highly specialized and targeted drug development capabilities to rapidly deliver potent single-stranded LNA-based drug candidates across a multitude of disease states. The Company's research and development activities focus on infectious diseases and metabolic disorders, while partnerships with major pharmaceutical companies include a range of therapeutic areas including cancer, cardiovascular disease, infectious and inflammatory diseases, and rare genetic disorders. The Company has strategic partnerships with miRagen Therapeutics, Shire plc, Pfizer, GlaxoSmithKline, and Enzon Pharmaceuticals. As part of its broad patent estate, the Company holds exclusive worldwide rights to all therapeutic uses of LNA. Santaris Pharma A/S, founded in 2003, is headquartered in Denmark with operations in the United States. Please visit http://www.santaris.com for more information.
(1) American Association for the Study of Liver Diseases -
http://www.aasld.org/patients/Pages/LiverFastFactsHepC.aspx
(2) World Health Organization -
http://www.who.int/csr/disease/hepatitis/Hepc.pdf
(3) Science. 2010 Jan 8; 327(5962):198-201. Epub 2009 Dec 3
- Santaris Pharma A/S initiates Phase 2a clinical trial with miravirsen (SPC3649) to assess safety and tolerability in treatment-naive patients with chronic Hepatitis C
By: PR Newswire
Sep. 22, 2010 07:15 AM
Therapeutic Silencing of MicroRNA-122 by SPC3649 in Primates with ...
Science Mag, Dec 3, 2009 ... Here, we investigated the potential of miR-122 antagonism by SPC3649 as ... We next assessed the in vivo antagonism of miR-122 in chimpanzee ...
www.natap.org/2009/HCV/011009_01.htm
HOERSHOLM, Denmark and SAN DIEGO, September 22, 2010 /PRNewswire/ -- Santaris Pharma A/S, a clinical-stage biopharmaceutical company focused on the discovery and development of RNA-targeted therapies, today announced that it has advanced miravirsen (SPC3649), the first microRNA-targeted drug to enter clinical trials, into Phase 2 studies to assess the safety and tolerability of the drug in treatment-naive patients infected with the Hepatitis C virus (HCV).
Paving the way to conduct the first clinical trials of a microRNA-targeted drug in the United States, Santaris Pharma A/S also received acceptance of its Investigational New Drug (IND) application from the U.S. Food and Drug Administration (FDA). In addition to the United States, the Phase 2a clinical trials will be conducted in the Netherlands, Germany, Poland, Romania, and Slovakia.
The World Health Organization estimates about 3% of the world's population has been infected with HCV and that some 170 million are chronic carriers at risk of developing liver cirrhosis and/or liver cancer(2). Approximately 3-4 million Americans are chronically infected with an estimated 40,000 new infections per year(1). In Europe, there are about 4 million carriers(2). The current standard of care, pegylated interferon in combination with ribavirin, is effective in only about 50% of those treated(1).
Developed using Santaris Pharma A/S proprietary Locked Nucleic Acid (LNA) Drug Platform, miravirsen is a specific inhibitor of miR-122, a liver specific microRNA that the Hepatitis C virus requires for replication. Miravirsen is designed to recognize and sequester miR-122, making it unavailable to the Hepatitis C virus. As a result, the replication of the virus is effectively inhibited and the level of Hepatitis C virus is reduced.
"Advancing miravirsen, the first microRNA-targeted drug to enter clinical trials, into Phase 2 studies in patients with Hepatitis C demonstrates Santaris Pharma A/S leadership in developing RNA-targeted medicines," said Arthur A. Levin, Ph.D., Vice President, Chief Development Officer and President, US Operations. "Receiving IND acceptance from the FDA to conduct the first clinical trials with a microRNA-targeted drug in the United States brings Santaris Pharma A/S one step closer to potentially providing a growing number of patients chronically infected with HCV with a more effective and better tolerated treatment option."
The LNA Drug Platform is the only technology with both mRNA and microRNA targeted drugs in clinical trials, reinforcing the broad utility of the platform. The unique combination of small size and very high affinity, which is only achievable with LNA-based drugs, allows this new class of drugs to potently and specifically inhibit RNA targets in many different tissues without the need for complex delivery vehicles. LNA-based drugs are a promising new type of therapy that enables scientists to develop drugs to attack previously inaccessible pathways.
"Using our LNA Drug Platform to advance the first microRNA-targeted therapy into human clinical trials was certainly a scientific breakthrough," said Henrik Oerum, Ph.D., Vice President and Chief Scientific Officer of Santaris Pharma A/S. "We are extremely pleased with the results of the Phase I trials and excited to progress miravirsen into Phase 2 clinical trials. Because of its unique mechanism of action and tolerability profile, miravirsen has the potential to be an effective treatment option for patients with HCV."
The randomized, double-blind, placebo-controlled, ascending multiple-dose Phase 2a study will assess the safety and tolerability of miravirsen and is designed to enroll up to 55 treatment-naïve patients with chronic Hepatitis C virus genotype 1 infection. Secondary endpoints include pharmacokinetics of miravirsen and its effect on viral load. Miravirsen will be given as subcutaneous injections weekly or every other week for four weeks.
Data from Phase 1 clinical studies with miravirsen in healthy volunteers show that the drug is well tolerated. A recent study published in Science demonstrated that miravirsen successfully inhibited miR-122 and dramatically reduced Hepatitis C virus in the liver and in the bloodstream in chimpanzees chronically infected with the Hepatitis C virus(3). Miravirsen provided continued efficacy in the animals up to several months after the treatment period with no adverse events and no evidence of viral rebound or resistance.
In addition to miravirsen, Santaris Pharma A/S has a robust product pipeline targeting mRNAs and microRNAs both internally as well as in partnerships and collaborations with miRagen Therapeutics (cardiovascular diseases), Shire plc (rare genetic disorders), Pfizer (undisclosed therapeutic areas), GlaxoSmithKline (viral disease) and Enzon Pharmaceuticals (oncology).
About microRNAs
MicroRNAs have emerged as an important class of small RNAs encoded in the genome. They act to control the expression of sets of genes and entire pathways and are thus thought of as master regulators of gene expression. Recent studies have demonstrated that microRNAs are associated with many disease processes. Because they are single molecular entities that dictate the expression of fundamental regulatory pathways, microRNAs represent potential drug targets for controlling many biologic and disease processes.
About Locked Nucleic Acid (LNA) Drug Platform
The LNA Drug Platform and Drug Discovery Engine developed by Santaris Pharma A/S combines the Company's proprietary LNA chemistry with its highly specialized and targeted drug development capabilities to rapidly deliver potent single-stranded LNA-based drug candidates against RNA targets, both mRNA and microRNA, for a range of diseases including metabolic disorders, infectious and inflammatory diseases, cancer and rare genetic disorders. The LNA Drug Platform overcomes the limitations of earlier antisense and siRNA technologies to deliver potent single-stranded LNA-based drug candidates across a multitude of disease states. The unique combination of small size and very high affinity, which is only achievable with LNA-based drugs, allows this new class of drugs to potently and specifically inhibit RNA targets in many different tissues without the need for complex delivery vehicles. LNA-based drugs are a promising new type of therapy that enables scientists to develop drugs to attack previously inaccessible clinical pathways. The most important features of LNA-based drugs include excellent specificity, providing optimal targeting; increased affinity to targets providing improved potency; and strong pharmacology upon systemic delivery without complicated delivery vehicles.
About Santaris Pharma A/S
Santaris Pharma A/S is a privately held clinical-stage biopharmaceutical company focused on the discovery and development of RNA-targeted therapies. The Locked Nucleic Acid (LNA) Drug Platform and Drug Discovery Engine developed by Santaris Pharma A/S combine the Company's proprietary LNA chemistry with its highly specialized and targeted drug development capabilities to rapidly deliver potent single-stranded LNA-based drug candidates across a multitude of disease states. The Company's research and development activities focus on infectious diseases and metabolic disorders, while partnerships with major pharmaceutical companies include a range of therapeutic areas including cancer, cardiovascular disease, infectious and inflammatory diseases, and rare genetic disorders. The Company has strategic partnerships with miRagen Therapeutics, Shire plc, Pfizer, GlaxoSmithKline, and Enzon Pharmaceuticals. As part of its broad patent estate, the Company holds exclusive worldwide rights to all therapeutic uses of LNA. Santaris Pharma A/S, founded in 2003, is headquartered in Denmark with operations in the United States. Please visit http://www.santaris.com for more information.
(1) American Association for the Study of Liver Diseases -
http://www.aasld.org/patients/Pages/LiverFastFactsHepC.aspx
(2) World Health Organization -
http://www.who.int/csr/disease/hepatitis/Hepc.pdf
(3) Science. 2010 Jan 8; 327(5962):198-201. Epub 2009 Dec 3
HCV: Health Danger of Parties Past
HCV: Health Danger of Parties Past
Wall Street Journal
Sept 20 2010
Most people think their wild-child past is just that—in the past. But some former party animals may be carrying a harmful reminder of their youth and not know it.
People who used intravenous drugs, snorted cocaine with a shared straw, or had an unsterile tattoo or body piercing could be infected with hepatitis C and not realize it. The virus, which spreads via blood-to-blood contact, can cause no symptoms for decades while silently destroying the liver.
Some people may have innocently been infected if they had a blood transfusion before 1992, when the blood supply began to be screened for the virus. Others may have contracted the virus simply by sharing a toothbrush or a razor. More than three million Americans have been diagnosed with hep C, and health experts say at least that many more are unaware that they have it.
"There's a huge reservoir of people who made a few bad decisions many years ago. Now they're successful business people, lawyers, doctors, school principals, and they don't know they are carrying this," says Joseph Galati, medical director of the Center for Liver Disease and Transplantation at Houston's Methodist Hospital. In the meantime, he says, "they could be doing things like drinking alcohol that accelerate the disease or transmitting it to other people."
Hep C, first identified in 1989, is today the leading reason for liver transplants and causes about 12,000 deaths in the U.S. each year. In most cases, the infection becomes chronic, inflaming the liver for years, but often with no apparent symptoms unless the inflammation becomes severe. In about 20% of cases, it progresses to cirrhosis, a severe scarring that shuts down liver function. And about 20% of those cirrhosis cases become liver cancer.
About 20,000 people are diagnosed with hepatitis C each year, and some two-thirds of those are middle-aged, having contracted the disease 20 or 30 years ago.
There is no vaccine against hep C, unlike for hepatitis A and B, which are liver diseases caused by different viruses. Hep C can be cured with a year-long course of chemotherapy drugs, but only about 50% of patients respond to them. A host of new medications now in clinical trials could work faster, and raise the cure rate as high as 80%, according to early results.
Hep C can be diagnosed with an inexpensive blood test that checks for antibodies—if doctors think to look for it. If that test is positive, another test can determine if the virus is still active. (In about 15% of hep C cases, the virus goes away on its own, although the antibodies may still be present.)
A regular annual checkup may reveal elevated liver enzymes. But many people with hep C have normal enzyme levels, and only vague symptoms like fatigue or joint pain, until the damage is well advanced.
"I never had any symptoms. I've had major surgery twice and nobody picked up on this," says a Houston nurse who was diagnosed with hep C in December at age 59. She thinks she was exposed to the virus in 1980, when she was accidentally stuck with a needle while caring for a patient. Her hep C was only found because a new job required the test for hep C antibodies. By then, 35% of her liver was damaged from cirrhosis. She is currently undergoing treatment in a clinical trial with Dr. Galati.
Even minute blood drops—from borrowing a toothbrush or piercing several friends' ears with the same needle—can transmit the virus. "Any blood-to-blood transition route can spread it, no matter how microscopic," says Melissa Palmer, medical director at New York University's Hepatology Associates in Plainview, NY.
"People may have done something once and forgotten about it, like share a $1 or a $100 bill to snort cocaine. The blood vessels in the nose are very weak and could bleed a little, and then the blood gets passed to the next person," says Dr. Palmer.
For now, the standard course of treatment for hep C is two chemotherapy drugs—interferon in weekly injections and ribavirin as pills three times a day—for either 24 or 48 weeks, an arduous regime that can cost more than $50,000 a year. Side effects can include fatigue, weakness, muscle and joint pain, hair loss, nausea and depression. Some patients need additional drugs to boost their red and white blood cells, which the chemo drugs deplete. Some have to stop the treatment because it can be so debilitating.
----------------------------------
Hepatitis A Through E
Hepatitis is an inflammation of the liver, generally caused by viruses, with symptoms ranging from slight to severe. Versions A through C are the most common.
* Hep A: Transmitted via contaminated water or food, particularly in countries with poor hygiene. Symptoms include fatigue, fever, abdominal pain, depression and jaundice. Permanent liver damage is rare. Vaccine recommended for all children at 1 year.
* Hep B: Two billion people world-wide have been infected with hep B, mostly through infected blood or body fluids. It can become chronic and lead to cirrhosis and liver cancer, but most adults clear the virus without treatment and are then immune. Vaccination is now required for many college students and healthcare workers.
* Hep C: Spread by blood-to-blood transmission, with few symptoms either in early stages or for decades later. About 20% of chronic cases develop into cirrhosis or liver cancer. Curable in about 50% of cases by chemotherapy.
* Hep D: Caused by a small RNA virus that only propagates in the presence of hep B, greatly increasing the chance of cancer, cirrhosis and death. Hep B vaccine will prevent illness from D.
* Hep E: Transmitted by fecal-oral contamination in unsanitary conditions. Patients are generally very ill for the first few weeks of infection then the virus usually clears on it own. Vaccine is being tested.
Source: WSJ Reporting
---------------------------------------
"I was really, really sick for a while—I had to hide under the wedding gowns so I could nap," says Sidney Merry, 53, who works for a bridal retailer in Houston. Routine blood tests spotted her hep C, which she thinks she got from a blood transfusion in the 1970s, and later started treatment with Dr. Galati. Ms. Merry stuck with the program and is now free of the virus. She helps counsel other patients undergoing treatment.
Ms. Merry says two of her friends died of hep C they declined to treat, and her own mother died of liver cancer at age 53, from what Ms. Merry suspects may also have been hep C. "This touches many lives—but it's so unspoken about and misunderstood," she says.
Two new drugs on the horizon—boceprevir by Merck & Co. and telaprevir by Vertex Pharmaceuticals Inc.—are protease inhibitors similar to those in AIDS drugs. They could win approval by the Food and Drug Administration next year. Other companies are studying different approaches to fight hep C.
In July, the FDA approved a synthetic form of interferon, called Infergen, by Three Rivers Pharmaceuticals LLC, for use in daily injections for patients who don't respond to the first course of treatment.
In some cases, doctors are advising hep C patients to postpone treatment until the new drugs come on the market. But Dr. Galati, who has been the principal investigator for several industry-sponsored clinical trials, notes that the new drugs will be in addition to the current ones, so waiting for the new regimes won't allow patients to avoid the side effects.
Unborn babies can acquire hep C from infected mothers. Kathryn Maloney had complained of fatigue for years before she was diagnosed with hep C in 2005. "Turns out I had it my whole life and didn't know," says Ms. Maloney, 29, an accountant in Houston.
Since there was no obvious source of her infection, Dr. Galati suggested that her mother, Pamela Grant, be tested too. She tested positive as well, though she has no idea when or where she was exposed. She and her daughter underwent treatment together. They also took part in a clinical trial for one of the new medications and are now free of the virus.
Some patients opt to forgo treatment, since only about 20% progress to cirrhosis. But doctors can't tell in advance which cases will progress. Meanwhile, drinking alcohol, smoking cigarettes and carrying excess weight make cirrhosis more likely.
Some health experts are urging that the general public be screened for hep C; the blood test for antibodies costs only about $12. Short of that, liver specialists urge anyone who might have been exposed, no matter how or how long ago or how well they feel now, to tell their doctors and be tested.
Since hep C can carry a lingering stigma of past drug use, even though there are many other ways to contract it, Dr. Galati says some primary-care physicians routinely hand patients a list of risk factors and say, "If you fit into any of these categories, you should get tested. You don't need to tell me which one."
Wall Street Journal
Sept 20 2010
Most people think their wild-child past is just that—in the past. But some former party animals may be carrying a harmful reminder of their youth and not know it.
People who used intravenous drugs, snorted cocaine with a shared straw, or had an unsterile tattoo or body piercing could be infected with hepatitis C and not realize it. The virus, which spreads via blood-to-blood contact, can cause no symptoms for decades while silently destroying the liver.
Some people may have innocently been infected if they had a blood transfusion before 1992, when the blood supply began to be screened for the virus. Others may have contracted the virus simply by sharing a toothbrush or a razor. More than three million Americans have been diagnosed with hep C, and health experts say at least that many more are unaware that they have it.
"There's a huge reservoir of people who made a few bad decisions many years ago. Now they're successful business people, lawyers, doctors, school principals, and they don't know they are carrying this," says Joseph Galati, medical director of the Center for Liver Disease and Transplantation at Houston's Methodist Hospital. In the meantime, he says, "they could be doing things like drinking alcohol that accelerate the disease or transmitting it to other people."
Hep C, first identified in 1989, is today the leading reason for liver transplants and causes about 12,000 deaths in the U.S. each year. In most cases, the infection becomes chronic, inflaming the liver for years, but often with no apparent symptoms unless the inflammation becomes severe. In about 20% of cases, it progresses to cirrhosis, a severe scarring that shuts down liver function. And about 20% of those cirrhosis cases become liver cancer.
About 20,000 people are diagnosed with hepatitis C each year, and some two-thirds of those are middle-aged, having contracted the disease 20 or 30 years ago.
There is no vaccine against hep C, unlike for hepatitis A and B, which are liver diseases caused by different viruses. Hep C can be cured with a year-long course of chemotherapy drugs, but only about 50% of patients respond to them. A host of new medications now in clinical trials could work faster, and raise the cure rate as high as 80%, according to early results.
Hep C can be diagnosed with an inexpensive blood test that checks for antibodies—if doctors think to look for it. If that test is positive, another test can determine if the virus is still active. (In about 15% of hep C cases, the virus goes away on its own, although the antibodies may still be present.)
A regular annual checkup may reveal elevated liver enzymes. But many people with hep C have normal enzyme levels, and only vague symptoms like fatigue or joint pain, until the damage is well advanced.
"I never had any symptoms. I've had major surgery twice and nobody picked up on this," says a Houston nurse who was diagnosed with hep C in December at age 59. She thinks she was exposed to the virus in 1980, when she was accidentally stuck with a needle while caring for a patient. Her hep C was only found because a new job required the test for hep C antibodies. By then, 35% of her liver was damaged from cirrhosis. She is currently undergoing treatment in a clinical trial with Dr. Galati.
Even minute blood drops—from borrowing a toothbrush or piercing several friends' ears with the same needle—can transmit the virus. "Any blood-to-blood transition route can spread it, no matter how microscopic," says Melissa Palmer, medical director at New York University's Hepatology Associates in Plainview, NY.
"People may have done something once and forgotten about it, like share a $1 or a $100 bill to snort cocaine. The blood vessels in the nose are very weak and could bleed a little, and then the blood gets passed to the next person," says Dr. Palmer.
For now, the standard course of treatment for hep C is two chemotherapy drugs—interferon in weekly injections and ribavirin as pills three times a day—for either 24 or 48 weeks, an arduous regime that can cost more than $50,000 a year. Side effects can include fatigue, weakness, muscle and joint pain, hair loss, nausea and depression. Some patients need additional drugs to boost their red and white blood cells, which the chemo drugs deplete. Some have to stop the treatment because it can be so debilitating.
----------------------------------
Hepatitis A Through E
Hepatitis is an inflammation of the liver, generally caused by viruses, with symptoms ranging from slight to severe. Versions A through C are the most common.
* Hep A: Transmitted via contaminated water or food, particularly in countries with poor hygiene. Symptoms include fatigue, fever, abdominal pain, depression and jaundice. Permanent liver damage is rare. Vaccine recommended for all children at 1 year.
* Hep B: Two billion people world-wide have been infected with hep B, mostly through infected blood or body fluids. It can become chronic and lead to cirrhosis and liver cancer, but most adults clear the virus without treatment and are then immune. Vaccination is now required for many college students and healthcare workers.
* Hep C: Spread by blood-to-blood transmission, with few symptoms either in early stages or for decades later. About 20% of chronic cases develop into cirrhosis or liver cancer. Curable in about 50% of cases by chemotherapy.
* Hep D: Caused by a small RNA virus that only propagates in the presence of hep B, greatly increasing the chance of cancer, cirrhosis and death. Hep B vaccine will prevent illness from D.
* Hep E: Transmitted by fecal-oral contamination in unsanitary conditions. Patients are generally very ill for the first few weeks of infection then the virus usually clears on it own. Vaccine is being tested.
Source: WSJ Reporting
---------------------------------------
"I was really, really sick for a while—I had to hide under the wedding gowns so I could nap," says Sidney Merry, 53, who works for a bridal retailer in Houston. Routine blood tests spotted her hep C, which she thinks she got from a blood transfusion in the 1970s, and later started treatment with Dr. Galati. Ms. Merry stuck with the program and is now free of the virus. She helps counsel other patients undergoing treatment.
Ms. Merry says two of her friends died of hep C they declined to treat, and her own mother died of liver cancer at age 53, from what Ms. Merry suspects may also have been hep C. "This touches many lives—but it's so unspoken about and misunderstood," she says.
Two new drugs on the horizon—boceprevir by Merck & Co. and telaprevir by Vertex Pharmaceuticals Inc.—are protease inhibitors similar to those in AIDS drugs. They could win approval by the Food and Drug Administration next year. Other companies are studying different approaches to fight hep C.
In July, the FDA approved a synthetic form of interferon, called Infergen, by Three Rivers Pharmaceuticals LLC, for use in daily injections for patients who don't respond to the first course of treatment.
In some cases, doctors are advising hep C patients to postpone treatment until the new drugs come on the market. But Dr. Galati, who has been the principal investigator for several industry-sponsored clinical trials, notes that the new drugs will be in addition to the current ones, so waiting for the new regimes won't allow patients to avoid the side effects.
Unborn babies can acquire hep C from infected mothers. Kathryn Maloney had complained of fatigue for years before she was diagnosed with hep C in 2005. "Turns out I had it my whole life and didn't know," says Ms. Maloney, 29, an accountant in Houston.
Since there was no obvious source of her infection, Dr. Galati suggested that her mother, Pamela Grant, be tested too. She tested positive as well, though she has no idea when or where she was exposed. She and her daughter underwent treatment together. They also took part in a clinical trial for one of the new medications and are now free of the virus.
Some patients opt to forgo treatment, since only about 20% progress to cirrhosis. But doctors can't tell in advance which cases will progress. Meanwhile, drinking alcohol, smoking cigarettes and carrying excess weight make cirrhosis more likely.
Some health experts are urging that the general public be screened for hep C; the blood test for antibodies costs only about $12. Short of that, liver specialists urge anyone who might have been exposed, no matter how or how long ago or how well they feel now, to tell their doctors and be tested.
Since hep C can carry a lingering stigma of past drug use, even though there are many other ways to contract it, Dr. Galati says some primary-care physicians routinely hand patients a list of risk factors and say, "If you fit into any of these categories, you should get tested. You don't need to tell me which one."
Evaluation of Versant Hepatitis C Virus Genotype Assay
Evaluation of Versant Hepatitis C Virus Genotype Assay (LiPA) 2.0 - pdf attached
Journal of Clinical Microbiology, June 2008, p. 1901-1906, Vol. 46, No. 6
doi:10.1128/JCM.02390-07
"The reliability of genotyping methods highly depends on the amount of information (i.e., the number of informative sites) that is utilized for the discrimination of genetic variants......Our results indicate that Versant HCV genotype assay (LiPA) 2.0 yielded an interpretable genotype result for 96.0% of the samples and that 99.4% of the interpretable results agreed with the reference method, rendering it an accurate and reliable assay suitable for large-scale genotyping. This new assay outperforms the previous version of the line probe assay, since Versant HCV genotype assay (LiPA) 1.0 has an overall accuracy of 74%, taking subtype i nformation into account (8, 23).....In conclusion, Versant HCV genotype assay (LiPA) 2.0 provides a rapid, sensitive, and accurate means of HCV genotyping and can be used as a routine tool to distinguish between the different HCV genotypes and subtypes. Considering the importance of genotype determination in understanding the epidemiology of the virus and in the management of hepatitis C treatment strategies, efficient genotyping tools are indispensable in clinical diagnostic settings."
Jannick Verbeeck,1 Mark J. Stanley,2 Jen Shieh,2 Linda Celis,3 Els Huyck,3 Elke Wollants,1 Judy Morimoto,2 Alice Farrior,2 Erwin Sablon,3 Margaret Jankowski-Hennig,4 Carl Schaper,4 Pamela Johnson,4 Marc Van Ranst,1* and Marianne Van Brussel3
Laboratory of Clinical Virology, Rega Institute for Medical Research, Leuven, Belgium,1 Department of Microbiology, Kaiser Permanente, TPMG Regional Laboratory, Berkeley, California,2 Innogenetics NV, Gent, Belgium,3 Siemens Healthcare Diagnostics, Berkeley, California4
ABSTRACT
Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5' untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturer's instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison stud y, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.
INTRODUCTION
Hepatitis C virus (HCV) is a leading cause of chronic liver disease and has already infected at least 170 million people worldwide. Each year, 3 to 4 million people are newly infected. HCV creates an extensive disease burden, since it accounts for 20 to 30% of cases of acute hepatitis, 70 to 80% of cases of chronic hepatitis, 40% of cases of end-stage cirrhosis, 50 to 76% of cases of hepatocellular carcinoma, and 30 to 40% of liver transplants (15, 33, 34).
HCV belongs to the family of the Flaviviridae and can be divided into different genotypes based on phylogenetic analysis of full-length or partial sequences of HCV strains. The most current consensus proposal distinguishes six genotypes based on phylogenetic cluster analysis of complete genomes. The genotype formerly designated as 10a has been reassigned as genotype 3, subtype k. Genotypes 7, 8, 9, and 11, belonging to clade 6, have been reassigned to genotype 6, subtypes c to l (25, 26, 27). These six HCV genotypes have different geographical distributions (21, 30, 32).
Treatment options for chronic HCV infections are poor. At the moment, the only accepted antiviral therapy with proven effectiveness is a combination therapy of (peg)interferon alpha and ribavirin. The overall success rate of this antiviral treatment ranges from 50% to 90% (11). According to a National Institutes of Health (NIH) 2002 panel, several factors are associated with successful treatment response, including lower baseline HCV RNA levels, lower fibrosis and inflammation scores upon liver biopsy, lower body weight, and lower body surface area, but the most important predicting factor is HCV genotype (24). Patients infected with HCV genotype 1 respond least to therapy, while patients infected with genotypes 2 and 3 show the best responses (14, 17, 22). For HCV genotypes 4, 5, and 6, treatment data are scarce, but it is recommended to treat these individuals using the same regimen as for patients infected with genotype 1 (7, 13, 18, 31). Nearly all patients experience side effects with the antiviral therapy. These side effects can be severe and contribute to discontinuation rates of 10 to 14% and dose reductions for 7 to 42% of patients, depending on the type and length of treatment (16). Therefore, it is important that clinicians have the appropriate information to make individual treatment choices in order to maximize the chance of successful treatment outcome for each individual patient, rendering HCV genotyping assays important and useful tools to optimize treatment type, duration, and dose.
In this paper, we evaluate Versant HCV genotype assay (LiPA) 2.0 (CE marked in Europe; for research use only; not for use in diagnostic procedures in the United States) (manufactured by Innogenetics, distributed by Siemens Healthcare Diagnostics), which uses sequence information from the core region in addition to sequence information from the 5' untranslated region (5'UTR), allowing an improved and more accurate distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1.
RESULTS
Clinical accuracy study.
Table 1 summarizes the results for the 326 specimens that were used for the reference method comparison. Upon initial testing, 93.3% (304/326) of the specimens gave interpretable genotype results, 2.1% (7/326) failed to amplify, and 4.6% (15/326) amplified but gave uninterpretable results. Of the 304 specimens that yielded a genotype result, 99.3% (302/304) gave results that agreed with the reference method. After specimens that yielded no genotype result were retested, 96.0% (313/326) of the specimens gave interpretable genotype results, 3.5% (12/326) amplified but remained uninterpretable, and 0.3% (1/326) failed to amplify. Of the 313 specimens that yielded a genotype result after repeat testing, 99.4% (311/313) gave results that agreed with the reference method. Table 2 shows that the specimens that did not amplify or give interpretable results were distributed across ge notypes. The two specimens that initially gave results that disagreed with those obtained by the reference method were retested, and both gave retest results that agreed with those obtained by the reference method.
In order to determine the core amplification efficiency, 156 genotype 1 and genotype 6 (c to l) samples were analyzed. Two samples showed negative AMPL CTRL 1 lines and were excluded from further analysis. Of the remaining 154 genotype 1 and genotype 6 (c to l) samples, 1 sample had a negative AMPL CTRL 2, r esulting in the amplification of 99.4% (153/154) samples.
The clinical subtype efficiency for HCV genotypes 1a and 1b was determined using 129 samples that were genotype 1a or 1b based on reference sequencing and genotype 1, 1a, or 1b based on LiPA genotyping; this determination was based on initial testing only, excluding repeat testing of initial amplification failures and uninterpretable results. Three out of 129 samples were indeterminate at the subtype level, resulting in a clinical HCV genotype 1 subtype efficiency of 97.7% after initial testing. Upon repeat testing, all samples gave a correct consensus subtype result. All of the 126 samples that were genotype 1a or 1b by LiPA were concordant with sequencing.
In order to check whether Versant HCV genotype assay (LiPA) 2.0 was able to determine the correct genotype for samples with viral loads at the upper limit of detection, 22 samples with viral loads ranging from 4.0 x 106 IU/ml to 8.7 x 106 IU/ml were selected, and the genotype success rate and the percentage of agreement with the reference method for these high-concentration specimens were estimated. For all these samples, Versant HCV genotype assay (LiPA) 2.0 produced the same genotype results as the genotype result determined by NS5b sequencing and phylogenetic analysis, resulting in both a genotype success rate and an agreement with the reference method of 100%.
Reproducibility study.
Table 3 summarizes the valid, indeterminate, correct, and incorrect genotype results for each reproducibility panel member. In total, 3.3% (16/486) of reactions gave indeterminate results (defined as specimens with either an amplification failure or an uninterpretable result) and 96.7% (LCL, 95.0%) yielded an interpretable genotype result. Of the 470 specimens with interpretable results, 100% (LCL, 99.4%) gave the correct genotype. The indeterminate results occurred at all sites, with all three reagent lots, and in multiple assay runs. There were no significant performance differences seen for the Versant HCV genotype assay (LiPA) 2.0 system across sites/operators (P = 0.43) or across reagent lots (P = 0.88). The genotype success rates at the individual sites were 98.1% for site 1, 97.6% for site 2, and 94.4% for site 3. The genotype success rates for the individual lots were 96.9% using lot 1, 97.5% using lot 2, and 95.7% using lot 3.
Comparison study.
Table 4 gives an overview of the results of the comparison study after original testing and after repeat testing. Of the 100 specimens tested, 13 specimens initially produced uninterpretable results by either Versant HCV genotype assay (LiPA) 1.0 or Versant HCV genotype assay (LiPA) 2.0 or both assays. The HCV RNA concentrations of these 13 samples ranged from 14,615 to 2,500,000 IU/ml. These specimens were retested using both ass ays. After repeat testing, three specimens remained uninterpretable by both versions of the assay, five remained uninterpretable by Versant HCV genotype assay (LiPA) 1.0, and one remained uninterpretable by Versant HCV genotype assay (LiPA) 2.0. For all six specimens that gave a genotype result by only one version of the assay, the observed genotype result agreed with that obtained by sequencing the NS5b region of the HCV genome. After repeat testing, 83 specimens were concordant by both assays at the genotype level. Of these, 16 specimens had concordant genotypes by both assays, but one of the assays failed to give a subtype, resulting in a total of 67 concordant specimens when the subtype level is taken into account. Results from eight specimens were discordant between the two assays, and results from nine specimens were uninterpretable by at least one of the assays. The total number of interpretable specimens by both assays was 91, of which 83 had concordant results at the genotype level only (91.2%; LCL, 84.7%). Table 5 shows the number o f genotype and subtype results produced by both assays for the 100 specimens tested after repeat testing.
The eight samples that showed discordant results were sequenced in the NS5b region of the HCV genome (two samples were HCV genotype 6 subtypes c to l, and six samples were HCV genotype 1a). Results indicated that Versant HCV genotype assay (LiPA) 2.0 gave the correct HCV genotype and subtype, as determined by NS5b sequencing. In contrast, Versant HCV genotype assay (LiPA) 1.0 had misclassified all eight samples as HC V genotype 1b. Of the 96 specimens that were interpretable with Versant HCV genotype assay (LiPA) 2.0, 83 showed concordant results with Versant HCV genotype assay (LiPA) 1.0, while 13 showed improved results over Versant HCV genotype assay (LiPA) 1.0, which leads to 100% concordant or improved results (LCL, 96.9%).
DISCUSSION
Phylogenetic analysis of a coding region, or even more, the complete genome, is considered the gold standard for identifying different HCV genotypes (6). However, since this method is expensive and time-consuming, it is impractical for large-scale genotyping projects (8). For this reason, commercial genotyping kits were developed for routine determination of HCV genotypes. Most commercially available HCV genotyping assays, including Versant HCV genotype assay (LiPA) 1.0, use the 5'UTR, since this region is highly conserved and therefore well suited for the development of detection methods. The reliability of genotyping methods highly depends on the amount of information (i.e., the number of informative sites) that is utilized for the discrimination of genetic variants. The 5'UTR is sufficiently variable for discrimination of HCV genotypes 1 to 5 and most subtypes of HCV genotype 6 (12, 28, 29, 32). However, it does not allow discrimination of HCV genotype 6 subtypes c to l from HCV genotype 1 and has only a limited subtyping accuracy (5, 29). To overcome the limitations of the 5'UTR, a new assay which uses additional sequence information from the core region of the HCV genome, Versant HCV genotype assay (LiPA) 2.0, has recently been developed (20). In this study, we evaluated the new assay and compared it with the previous version of the assay.
Our results indicate that Versant HCV genotype assay (LiPA) 2.0 yielded an interpretable genotype result for 96.0% of the samples and that 99.4% of the interpretable results agreed with the reference method, rendering it an accurate and reliable assay suitable for large-scale genotyping. This new assay outperforms the previous version of the line probe assay, since Versant HCV genotype assay (LiPA) 1.0 has an overall accuracy of 74%, taking subtype information into account (8, 23).
In the comparison study, eight specimens showed discordant results when tested with both assays. The NS5b sequencing results for these samples showed that Versant HCV genotype assay (LiPA) 2.0 gave the correct HCV genotype and subtype and thereby showed an improvement in identifying HCV-positive samples which are subtypes c to l of genotype 6 and in identifying the correct subtype of genotype 1. This improvement can be attributed to the additional information available from the core region of the HCV genome, which can better distinguish between genotype 1 and subtypes c to l of genotype 6 and between subtype a and b of genotype 1. This core information is not available in Versant HCV genotype assay (LiPA) 1.0, and this can lead to misinterpretation. For example, in a study by Chinchai et al., this assay could not discriminate HCV genotype 6a variants from HCV genotype 1b, and two samples found to be genotype 1 by the assay contained genotype 3 core sequences (5). Chen and Weck showed that Versant HCV genotype assay (LiPA) 1.0 cannot accurately distinguish HCV genotypes 1a and 1b, since in most cases, the 5'UTR is not heterogeneous enough for use in determining the HCV subtype (4). Several other studies report on moderate distinction at the subtype level (1, 2, 9, 12, 19). This is not surprising, since the 5'UTR is the most highly conserved region of the HCV genome, and only one or two nucleotide changes distinguish unique subtypes. Assigning correct genotypes and subtypes to HCV specimens is important for several research purposes, including epidemiological, phylogenetic, and natural history studies. Some studies even report that there is a slight difference in treatment outcomes between HCV genotype 1a- and HCV genotype 1b-infected patients, showing that correct subtype assignment is indispensable (3, 10, 30).
In conclusion, Versant HCV genotype assay (LiPA) 2.0 provides a rapid, sensitive, and accurate means of HCV genotyping and can be used as a routine tool to distinguish between the different HCV genotypes and subtypes. Considering the importance of genotype determination in understanding the epidemiology of the virus and in the management of hepatitis C treatment strategies, efficient genotyping tools are indispensable in clinical diagnostic settings.
Journal of Clinical Microbiology, June 2008, p. 1901-1906, Vol. 46, No. 6
doi:10.1128/JCM.02390-07
"The reliability of genotyping methods highly depends on the amount of information (i.e., the number of informative sites) that is utilized for the discrimination of genetic variants......Our results indicate that Versant HCV genotype assay (LiPA) 2.0 yielded an interpretable genotype result for 96.0% of the samples and that 99.4% of the interpretable results agreed with the reference method, rendering it an accurate and reliable assay suitable for large-scale genotyping. This new assay outperforms the previous version of the line probe assay, since Versant HCV genotype assay (LiPA) 1.0 has an overall accuracy of 74%, taking subtype i nformation into account (8, 23).....In conclusion, Versant HCV genotype assay (LiPA) 2.0 provides a rapid, sensitive, and accurate means of HCV genotyping and can be used as a routine tool to distinguish between the different HCV genotypes and subtypes. Considering the importance of genotype determination in understanding the epidemiology of the virus and in the management of hepatitis C treatment strategies, efficient genotyping tools are indispensable in clinical diagnostic settings."
Jannick Verbeeck,1 Mark J. Stanley,2 Jen Shieh,2 Linda Celis,3 Els Huyck,3 Elke Wollants,1 Judy Morimoto,2 Alice Farrior,2 Erwin Sablon,3 Margaret Jankowski-Hennig,4 Carl Schaper,4 Pamela Johnson,4 Marc Van Ranst,1* and Marianne Van Brussel3
Laboratory of Clinical Virology, Rega Institute for Medical Research, Leuven, Belgium,1 Department of Microbiology, Kaiser Permanente, TPMG Regional Laboratory, Berkeley, California,2 Innogenetics NV, Gent, Belgium,3 Siemens Healthcare Diagnostics, Berkeley, California4
ABSTRACT
Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5' untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturer's instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison stud y, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.
INTRODUCTION
Hepatitis C virus (HCV) is a leading cause of chronic liver disease and has already infected at least 170 million people worldwide. Each year, 3 to 4 million people are newly infected. HCV creates an extensive disease burden, since it accounts for 20 to 30% of cases of acute hepatitis, 70 to 80% of cases of chronic hepatitis, 40% of cases of end-stage cirrhosis, 50 to 76% of cases of hepatocellular carcinoma, and 30 to 40% of liver transplants (15, 33, 34).
HCV belongs to the family of the Flaviviridae and can be divided into different genotypes based on phylogenetic analysis of full-length or partial sequences of HCV strains. The most current consensus proposal distinguishes six genotypes based on phylogenetic cluster analysis of complete genomes. The genotype formerly designated as 10a has been reassigned as genotype 3, subtype k. Genotypes 7, 8, 9, and 11, belonging to clade 6, have been reassigned to genotype 6, subtypes c to l (25, 26, 27). These six HCV genotypes have different geographical distributions (21, 30, 32).
Treatment options for chronic HCV infections are poor. At the moment, the only accepted antiviral therapy with proven effectiveness is a combination therapy of (peg)interferon alpha and ribavirin. The overall success rate of this antiviral treatment ranges from 50% to 90% (11). According to a National Institutes of Health (NIH) 2002 panel, several factors are associated with successful treatment response, including lower baseline HCV RNA levels, lower fibrosis and inflammation scores upon liver biopsy, lower body weight, and lower body surface area, but the most important predicting factor is HCV genotype (24). Patients infected with HCV genotype 1 respond least to therapy, while patients infected with genotypes 2 and 3 show the best responses (14, 17, 22). For HCV genotypes 4, 5, and 6, treatment data are scarce, but it is recommended to treat these individuals using the same regimen as for patients infected with genotype 1 (7, 13, 18, 31). Nearly all patients experience side effects with the antiviral therapy. These side effects can be severe and contribute to discontinuation rates of 10 to 14% and dose reductions for 7 to 42% of patients, depending on the type and length of treatment (16). Therefore, it is important that clinicians have the appropriate information to make individual treatment choices in order to maximize the chance of successful treatment outcome for each individual patient, rendering HCV genotyping assays important and useful tools to optimize treatment type, duration, and dose.
In this paper, we evaluate Versant HCV genotype assay (LiPA) 2.0 (CE marked in Europe; for research use only; not for use in diagnostic procedures in the United States) (manufactured by Innogenetics, distributed by Siemens Healthcare Diagnostics), which uses sequence information from the core region in addition to sequence information from the 5' untranslated region (5'UTR), allowing an improved and more accurate distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1.
RESULTS
Clinical accuracy study.
Table 1 summarizes the results for the 326 specimens that were used for the reference method comparison. Upon initial testing, 93.3% (304/326) of the specimens gave interpretable genotype results, 2.1% (7/326) failed to amplify, and 4.6% (15/326) amplified but gave uninterpretable results. Of the 304 specimens that yielded a genotype result, 99.3% (302/304) gave results that agreed with the reference method. After specimens that yielded no genotype result were retested, 96.0% (313/326) of the specimens gave interpretable genotype results, 3.5% (12/326) amplified but remained uninterpretable, and 0.3% (1/326) failed to amplify. Of the 313 specimens that yielded a genotype result after repeat testing, 99.4% (311/313) gave results that agreed with the reference method. Table 2 shows that the specimens that did not amplify or give interpretable results were distributed across ge notypes. The two specimens that initially gave results that disagreed with those obtained by the reference method were retested, and both gave retest results that agreed with those obtained by the reference method.
In order to determine the core amplification efficiency, 156 genotype 1 and genotype 6 (c to l) samples were analyzed. Two samples showed negative AMPL CTRL 1 lines and were excluded from further analysis. Of the remaining 154 genotype 1 and genotype 6 (c to l) samples, 1 sample had a negative AMPL CTRL 2, r esulting in the amplification of 99.4% (153/154) samples.
The clinical subtype efficiency for HCV genotypes 1a and 1b was determined using 129 samples that were genotype 1a or 1b based on reference sequencing and genotype 1, 1a, or 1b based on LiPA genotyping; this determination was based on initial testing only, excluding repeat testing of initial amplification failures and uninterpretable results. Three out of 129 samples were indeterminate at the subtype level, resulting in a clinical HCV genotype 1 subtype efficiency of 97.7% after initial testing. Upon repeat testing, all samples gave a correct consensus subtype result. All of the 126 samples that were genotype 1a or 1b by LiPA were concordant with sequencing.
In order to check whether Versant HCV genotype assay (LiPA) 2.0 was able to determine the correct genotype for samples with viral loads at the upper limit of detection, 22 samples with viral loads ranging from 4.0 x 106 IU/ml to 8.7 x 106 IU/ml were selected, and the genotype success rate and the percentage of agreement with the reference method for these high-concentration specimens were estimated. For all these samples, Versant HCV genotype assay (LiPA) 2.0 produced the same genotype results as the genotype result determined by NS5b sequencing and phylogenetic analysis, resulting in both a genotype success rate and an agreement with the reference method of 100%.
Reproducibility study.
Table 3 summarizes the valid, indeterminate, correct, and incorrect genotype results for each reproducibility panel member. In total, 3.3% (16/486) of reactions gave indeterminate results (defined as specimens with either an amplification failure or an uninterpretable result) and 96.7% (LCL, 95.0%) yielded an interpretable genotype result. Of the 470 specimens with interpretable results, 100% (LCL, 99.4%) gave the correct genotype. The indeterminate results occurred at all sites, with all three reagent lots, and in multiple assay runs. There were no significant performance differences seen for the Versant HCV genotype assay (LiPA) 2.0 system across sites/operators (P = 0.43) or across reagent lots (P = 0.88). The genotype success rates at the individual sites were 98.1% for site 1, 97.6% for site 2, and 94.4% for site 3. The genotype success rates for the individual lots were 96.9% using lot 1, 97.5% using lot 2, and 95.7% using lot 3.
Comparison study.
Table 4 gives an overview of the results of the comparison study after original testing and after repeat testing. Of the 100 specimens tested, 13 specimens initially produced uninterpretable results by either Versant HCV genotype assay (LiPA) 1.0 or Versant HCV genotype assay (LiPA) 2.0 or both assays. The HCV RNA concentrations of these 13 samples ranged from 14,615 to 2,500,000 IU/ml. These specimens were retested using both ass ays. After repeat testing, three specimens remained uninterpretable by both versions of the assay, five remained uninterpretable by Versant HCV genotype assay (LiPA) 1.0, and one remained uninterpretable by Versant HCV genotype assay (LiPA) 2.0. For all six specimens that gave a genotype result by only one version of the assay, the observed genotype result agreed with that obtained by sequencing the NS5b region of the HCV genome. After repeat testing, 83 specimens were concordant by both assays at the genotype level. Of these, 16 specimens had concordant genotypes by both assays, but one of the assays failed to give a subtype, resulting in a total of 67 concordant specimens when the subtype level is taken into account. Results from eight specimens were discordant between the two assays, and results from nine specimens were uninterpretable by at least one of the assays. The total number of interpretable specimens by both assays was 91, of which 83 had concordant results at the genotype level only (91.2%; LCL, 84.7%). Table 5 shows the number o f genotype and subtype results produced by both assays for the 100 specimens tested after repeat testing.
The eight samples that showed discordant results were sequenced in the NS5b region of the HCV genome (two samples were HCV genotype 6 subtypes c to l, and six samples were HCV genotype 1a). Results indicated that Versant HCV genotype assay (LiPA) 2.0 gave the correct HCV genotype and subtype, as determined by NS5b sequencing. In contrast, Versant HCV genotype assay (LiPA) 1.0 had misclassified all eight samples as HC V genotype 1b. Of the 96 specimens that were interpretable with Versant HCV genotype assay (LiPA) 2.0, 83 showed concordant results with Versant HCV genotype assay (LiPA) 1.0, while 13 showed improved results over Versant HCV genotype assay (LiPA) 1.0, which leads to 100% concordant or improved results (LCL, 96.9%).
DISCUSSION
Phylogenetic analysis of a coding region, or even more, the complete genome, is considered the gold standard for identifying different HCV genotypes (6). However, since this method is expensive and time-consuming, it is impractical for large-scale genotyping projects (8). For this reason, commercial genotyping kits were developed for routine determination of HCV genotypes. Most commercially available HCV genotyping assays, including Versant HCV genotype assay (LiPA) 1.0, use the 5'UTR, since this region is highly conserved and therefore well suited for the development of detection methods. The reliability of genotyping methods highly depends on the amount of information (i.e., the number of informative sites) that is utilized for the discrimination of genetic variants. The 5'UTR is sufficiently variable for discrimination of HCV genotypes 1 to 5 and most subtypes of HCV genotype 6 (12, 28, 29, 32). However, it does not allow discrimination of HCV genotype 6 subtypes c to l from HCV genotype 1 and has only a limited subtyping accuracy (5, 29). To overcome the limitations of the 5'UTR, a new assay which uses additional sequence information from the core region of the HCV genome, Versant HCV genotype assay (LiPA) 2.0, has recently been developed (20). In this study, we evaluated the new assay and compared it with the previous version of the assay.
Our results indicate that Versant HCV genotype assay (LiPA) 2.0 yielded an interpretable genotype result for 96.0% of the samples and that 99.4% of the interpretable results agreed with the reference method, rendering it an accurate and reliable assay suitable for large-scale genotyping. This new assay outperforms the previous version of the line probe assay, since Versant HCV genotype assay (LiPA) 1.0 has an overall accuracy of 74%, taking subtype information into account (8, 23).
In the comparison study, eight specimens showed discordant results when tested with both assays. The NS5b sequencing results for these samples showed that Versant HCV genotype assay (LiPA) 2.0 gave the correct HCV genotype and subtype and thereby showed an improvement in identifying HCV-positive samples which are subtypes c to l of genotype 6 and in identifying the correct subtype of genotype 1. This improvement can be attributed to the additional information available from the core region of the HCV genome, which can better distinguish between genotype 1 and subtypes c to l of genotype 6 and between subtype a and b of genotype 1. This core information is not available in Versant HCV genotype assay (LiPA) 1.0, and this can lead to misinterpretation. For example, in a study by Chinchai et al., this assay could not discriminate HCV genotype 6a variants from HCV genotype 1b, and two samples found to be genotype 1 by the assay contained genotype 3 core sequences (5). Chen and Weck showed that Versant HCV genotype assay (LiPA) 1.0 cannot accurately distinguish HCV genotypes 1a and 1b, since in most cases, the 5'UTR is not heterogeneous enough for use in determining the HCV subtype (4). Several other studies report on moderate distinction at the subtype level (1, 2, 9, 12, 19). This is not surprising, since the 5'UTR is the most highly conserved region of the HCV genome, and only one or two nucleotide changes distinguish unique subtypes. Assigning correct genotypes and subtypes to HCV specimens is important for several research purposes, including epidemiological, phylogenetic, and natural history studies. Some studies even report that there is a slight difference in treatment outcomes between HCV genotype 1a- and HCV genotype 1b-infected patients, showing that correct subtype assignment is indispensable (3, 10, 30).
In conclusion, Versant HCV genotype assay (LiPA) 2.0 provides a rapid, sensitive, and accurate means of HCV genotyping and can be used as a routine tool to distinguish between the different HCV genotypes and subtypes. Considering the importance of genotype determination in understanding the epidemiology of the virus and in the management of hepatitis C treatment strategies, efficient genotyping tools are indispensable in clinical diagnostic settings.
Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice
Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice - pdf attached
PLoS ONE 4(12): e8209. doi:10.1371/journal.pone.0008209
Published December 8, 2009
"INNO-LiPA HCV 2.0 currently is the best available commercial assay for HCV genotype 1 subtype identification and should be used in clinical trials and practice.....
Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively....
The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b. It can therefore be used locally in clinical trials to identify the HCV subtype and stratify the patients at inclusion, as well as to interpret efficacy and resistance data. When reporting final data, direct sequence analysis of the NS5B region and/or another coding region (for instance the region encoding the antiviral drug target HCV protein) should always be performed as it may identify mistyping or mis-subtyping with commercial assays, especially in the case of rare subtypes."
Stéphane Chevaliez1,2, Magali Bouvier-Alias1,2, Rozenn Brillet2, Jean-Michel Pawlotsky1,2*
1 French National Reference Center for Viral Hepatitis B, C and delta, Department of Virology, Hôpital Henri Mondor, Université Paris 12, Créteil, France, 2 INSERM U955, Créteil, France
Methods based on the sole analysis of the 5′NCR, namely Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively (Table 1)......
The results clearly show that, although they are by far the most widely used techniques in new HCV drug development trials, genotyping techniques based on the sole analysis of the 5′NCR should be avoided, as they mistype approximately 25% and 10% of HCV subtype 1a and 1b strains, respectively......
INNO-LiPA HCV 2.0 displays the same 5′NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1)
The real-time PCR-based assay targeting both the 5′NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1)......
Novel assays have been recently developed that aim at better discriminating among the different HCV genotype 1 subtypes and between genotypes 1 and 6. Abbott RealTime HCV Genotype II assay is a real-time PCR method using several sets of genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region. As shown in Table 1, adding a second target region for analysis led to substantially improving HCV genotype 1 subtype identification compared to methods targeting the sole 5′NCR. However, in contrast with a previous report [33], we found that this assay failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases.
Table 1. Ability of the different molecular methods tested in this study to correctly identify HCV subtypes 1a and 1b in a series of 500 patients infected by one or the other of these subtypes.
Screen shot 2010-09-18 at 7.38.55 AM.png
Abstract
Background
With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.
Methodology/Principal Findings
A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.
Conclusions/Significance
In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.
Funding: The Trugene HCV 5′NC Genotyping kits and the INNO-LiPA HCV kits were kindly provided by Siemens Medical Solutions Diagnostics. The Abbott RealTime HCV Genotype II kits were kindly provided by Abbott Molecular. This work is part of the activity of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a grant (LSHM-CT-2004-503359) from the Priority 1 “Life Sciences, Genomics and Biotechnology for Health” program in the 6th Framework Program of the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
Over 170 million individuals are infected with hepatitis C virus (HCV) worldwide. Phylogenetic analyses have shown that HCV strains can be classified into at least 6 major genotypes (numbered 1 to 6), and a large number of subtypes within each genotype [1]. Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide, with subtypes 1a and 1b representing the vast majority of circulating strains [2], [3], [4].
Current treatment of chronic hepatitis C is based on the combination of pegylated interferon (IFN)-α and ribavirin [5]. This treatment fails to eradicate infection in 50%–60% of patients infected with HCV genotype 1 and approximately 20% of those infected with HCV genotypes 2 and 3 [6], [7], [8]. Thus the need for more efficacious therapies is urgent, especially for patients infected with HCV genotype 1. A number of novel antiviral molecules currently are in preclinical or clinical development [9]. The most advanced ones are specific inhibitors of viral enzymes and functions involved in the HCV life cycle. Molecules that have reached clinical development include inhibitors of the nonstructural (NS) 3/4A serine protease and inhibitors of HCV replication that belong to different categories: nucleoside/nucleotide analogue and non-nucleoside inhibitors of the HCV RNA-dependent RNA polymerase (RdRp), NS5A inhibitors and cyclophilin inhibitors [9]. These agents have shown potent antiviral efficacy when used alone, and encouraging results have been recently published showing that HCV clearance can be achieved in approximately 70% of cases when a potent NS3/4A inhibitor is used in combination with pegylated IFN-α and ribavirin [10], [11], [12].
HCV genotype 1 is generally considered as a homogeneous group. There are however biological differences between the different subtypes of HCV genotype 1, which are related to differences in their nucleotide and amino acid sequences. Importantly, differences between subtype 1a and 1b (by far the most frequently encountered genotype 1 subtypes in clinical practice) include different efficacies of antiviral drugs and different resistance profiles to such drugs. Indeed, several HCV inhibitors appear to have selective activity against different HCV genotype 1 subtypes, both in vitro and in vivo. Differences have been observed in vitro with NS3/4A protease inhibitors, non-nucleoside inhibitors of HCV RdRp and NS5A inhibitors [13], [14], [15], [16], [17]. For instance, BILB 1941, a non-nucleoside inhibitor of HCV RdRp, has been shown to have better antiviral efficacy in patients infected with HCV subtype 1b than in those infected with HCV subtype 1a, a finding reflecting in vitro experiments [13].
A major issue that limits the efficacy of direct acting antiviral therapies for HCV is the selection by these drugs of resistant variants upon administration [18]. Recent studies with NS3/4A protease inhibitors have shown that the genetic barrier and resistance profiles substantially differ between the different genotype 1 subtypes. For instance, the Arg to Lys substitution at position 155 of the NS3 protease (R155K) is usually selected in subtype 1a replicons treated with telaprevir, but not in subtype 1b replicons [19]. The reason is that only one nucleotide substitution is needed relative to the subtype 1a sequence to generate this variant, whereas two substitutions are needed relative to the 1b sequence (codon usage bias). Overall, natural polymorphisms at positions R155 and V36 are frequent in subtype 1a, but rare in subtype 1b where substitutions at position A156 are preferentially selected in vitro [19]. This is reflected in vivo by the different resistance profiles in patients infected by HCV subtypes 1a and 1b. In the former, the V36 and R155 substitutions represent the backbone of resistance, whereas in the latter resistance is less frequent as it is preferentially associated with substitutions at position A156 that are associated with a decreased fitness of the variants [19], [20], [21]. Similarly, important differences in the resistance profiles have been described in vitro with HCV-796, a non-nucleoside inhibitor of HCV RdRp. The C316Y amino acid substitution has been reported to be selected in both subtype 1a and 1b replicon cells. However, in genotype 1a replicons, the C316Y substitution has low replication capacity that must be compensated for by additional “compensatory” substitutions, including L392F or M414T, resulting in an increase in replication levels of at least 10-fold [19]. A higher genetic barrier to resistance to HCV-796 and related compounds is therefore expected in patients infected with HCV subtype 1a than 1b. In vivo, HCV-796 monotherapy was however shown to select subtype 1a variants with a single C316Y substitution, whereas the C316Y substitution was associated with a number of additional substitutions in subtype 1b patients [22].
As a result of these findings, correct identification of HCV subtypes 1a and 1b is crucial in clinical trials assessing new HCV drugs in order to correctly stratify and interpret efficacy and resistance data. It may also become important in future clinical practice, as tailoring treatment schedules with HCV inhibitors to HCV genotype 1 subtype might become necessary. A variety of molecular methods can be used to identify the HCV genotype and subtype both in clinical trials and practice. Commercial assays have been developed, most of them targeting the 5′ noncoding region (5′NCR) of the HCV genome, although this region is the most conserved one. These methods have been shown to differentiate well the different HCV genotypes (1 to 6), except genotype 1 from genotype 6, a rare HCV genotype in the Western world [23], [24]. The goal of our study was to assess the ability of molecular methods targeting the 5′NCR to correctly identify the HCV genotype 1 subtype in patients eligible for clinical trials, and to identify the best method for this purpose.
Results
Hepatitis C Virus Genotype and Subtype Determination by Phylogenetic Analysis of a Portion of the NS5B Gene
Direct sequence analysis of a sufficiently long portion of the NS5B gene followed by phylogenetic analysis is the reference method for identification of HCV genotype and subtype [1], [25]. It was used to identify the HCV genotype and subtype in 516 treatment-naïve patients included in a multicenter clinical trial assessing different schedules of pegylated IFN-α2a and ribavirin [26]. All of these patients were thought to be infected with HCV genotype 1 at inclusion based on local assessment. In fact, 6 patients were infected with genotype 6, including 2 with subtype 6e, one with subtype 6o, one with subtype 6p, one with subtype 6q and one with subtype 6r. These 6 samples were not considered for further analysis in the present study. The remaining 510 patients were confirmed to be infected with HCV genotype 1: 237 of them (46.5%) were infected with HCV subtype 1a and 263 (51.6%) with subtype 1b (Figure 1). As shown in Figure 1, HCV subtype 1a strains segregated into two distinct clades, that were termed 1a clade I (n = 83, 35.0%) and 1a clade II (n = 154, 65.0%). Eight patients (1.6%) were infected with another HCV genotype 1 subtype, including 4 patients with subtype 1d, 2 with subtype 1e, one with subtype 1i, and one with subtype 1l. The remaining 2 patients (0.3%) were infected with genotype 1 but the subtype could not be determined. The ability of the different molecular methods to correctly identify HCV subtypes 1a and 1b was then tested on the 237 and 263 samples containing HCV subtypes 1a and 1b, respectively.
INNO-LiPA HCV 2.0 displays the same 5′NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1).
The real-time PCR-based assay targeting both the 5′NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1).
5′NCR Sequence Analysis in Misclassified Subtype 1a Strains
Among the HCV subtype 1a strains, 47 were misclassified as subtype 1b by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0, including 33 that were misclassified by both assays, 7 that were misclassified by Trugene HCV Genotyping Kit only, and 7 that were misclassified by INNO-LiPA HCV 1.0 only (Table 2). Figure 2 shows an alignment of their 5′NCR sequences relative to the consensus sequences of the correctly classified strains (including subtype 1a clade I, subtype 1a clade II and subtype 1b). As shown in Figure 2, misclassification of subtype 1a strains into subtype 1b in one or both assays was related to the presence of natural polymorphisms at nucleotide positions 204 and 243, both of which are located within the sequence of an INNO-LiPA HCV 1.0 probe. At position 243, A is the most frequent nucleotide in HCV subtype 1a, in both subtype 1a clade I and clade II. Substitution into a G, the most frequent nucleotide at position 243 in subtype 1b, was found in all cases that were misclassified as subtype 1b by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0 (Figure 2). At position 204, A is the most frequent nucleotide for subtype 1a clade I, whereas C is the most frequent nucleotide for subtype 1a clade II, and C or T are the most frequent nucleotides for subtype 1b. In spite of the presence of a G at position 243, the presence of an A at position 204 allowed correct identification of subtype 1a with Trugene HCV Genotyping Kit but not with INNO-LiPA HCV 1.0 (Figure 2). The usual presence of a C at position 204 in subtype 1a clade II explains why misclassifications were far more frequent with this clade than with subtype 1a clade I.
Among the 12 subtype 1a strains that were classified as genotype 1, indeterminate subtype with Trugene HCV Genotyping Kit, one had a G and 5 had mixed A and G populations at position 243. Two additional patients with an A at position 243 had a C at position 248. In the remaining 4 cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown). Among the 25 subtype 1a strains that were classified as genotype 1, indeterminate subtype with INNO-LiPA HCV 1.0 (including 6 with the same profile in Trugene HCV Genotyping Kit), 4 had a G and 4 had mixed A and G populations at position 243. Three additional patients with an A at position 243 had a C at position 248 (C only in two of them, a mixture of C and T in one). In the 14 remaining cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown).
5′NCR Sequence Analysis in Misclassified Subtype 1b Strains
Among HCV subtype 1b strains, 8 were misclassified as subtype 1a by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0, including 3 that were misclassified by both assays, 4 that were misclassified by Trugene HCV Genotyping Kit only, and 1 that was misclassified by INNO-LiPA HCV 1.0 only (Table 2). Figure 3 shows an alignment of their 5′NCR sequences relative to the consensus sequences of the correctly classified subtype 1a and subtype 1b strains. As shown in Figure 3, and as for misclassified subtype 1a strains discussed above, misclassification of subtype 1b strains into subtype 1a was related to the presence of natural polymorphisms at positions 204 and 243. At position 243, G is the most frequent nucleotide in HCV subtype 1b. Substitution into an A, the most frequent nucleotide at position 243 in subtype 1a, was found in all cases that were misclassified as subtype 1a by both Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0 and by INNO-LiPA HCV 1.0 only, but not in those that were misclassified by Trugene HCV Genotyping Kit only (Figure 3). In the latter, it is the presence of an A at position 204 instead of a C or a T that was responsible for misclassification in all but one case (Figure 3).
Among the 11 subtype 1b strains that were classified as genotype 1, indeterminate subtype with Trugene HCV Genotyping Kit, one had an A at position 243. In the remaining cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown). Among the 15 subtype 1b strains that were classified as genotype 1, indeterminate subtype with INNO-LiPA HCV 1.0 (none of which were classified as indeterminate in Trugene HCV Genotyping Kit), one had an A and one harbored mixed A and G populations at position 243. Both of them had a C at position 248 (C only in one of them and a mixture of C and T in the other one). In the remaining 13 cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown).
Incorrect Subtyping with Abbott RealTime HCV Genotype II Assay, that Targets Both the 5′NCR and NS5B Region
Among the HCV subtype 1a strains, 16 were incorrectly classified by Abbott RealTime HCV Genotype II assay (Table 1): 2 were misclassified as subtype 1b, 12 were classified as genotype 1, indeterminate subtype, one was identified as a mixed 1a/1b infection, and one gave an indeterminate result. In one case, PCR amplification failed, and in one case, not enough serum volume was available for testing.
Among the HCV subtype 1b strains, 27 were incorrectly classified by Abbott RealTime HCV Genotype II assay (Table 1): 3 were misclassified as subtype 1a, 18 were classified as genotype 1, indeterminate subtype, 5 were identified as a mixed 1a/1b infection, and one gave an indeterminate result. In 2 cases, PCR amplification failed, and in one case, not enough serum volume was available for testing.
PLoS ONE 4(12): e8209. doi:10.1371/journal.pone.0008209
Published December 8, 2009
"INNO-LiPA HCV 2.0 currently is the best available commercial assay for HCV genotype 1 subtype identification and should be used in clinical trials and practice.....
Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively....
The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b. It can therefore be used locally in clinical trials to identify the HCV subtype and stratify the patients at inclusion, as well as to interpret efficacy and resistance data. When reporting final data, direct sequence analysis of the NS5B region and/or another coding region (for instance the region encoding the antiviral drug target HCV protein) should always be performed as it may identify mistyping or mis-subtyping with commercial assays, especially in the case of rare subtypes."
Stéphane Chevaliez1,2, Magali Bouvier-Alias1,2, Rozenn Brillet2, Jean-Michel Pawlotsky1,2*
1 French National Reference Center for Viral Hepatitis B, C and delta, Department of Virology, Hôpital Henri Mondor, Université Paris 12, Créteil, France, 2 INSERM U955, Créteil, France
Methods based on the sole analysis of the 5′NCR, namely Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively (Table 1)......
The results clearly show that, although they are by far the most widely used techniques in new HCV drug development trials, genotyping techniques based on the sole analysis of the 5′NCR should be avoided, as they mistype approximately 25% and 10% of HCV subtype 1a and 1b strains, respectively......
INNO-LiPA HCV 2.0 displays the same 5′NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1)
The real-time PCR-based assay targeting both the 5′NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1)......
Novel assays have been recently developed that aim at better discriminating among the different HCV genotype 1 subtypes and between genotypes 1 and 6. Abbott RealTime HCV Genotype II assay is a real-time PCR method using several sets of genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region. As shown in Table 1, adding a second target region for analysis led to substantially improving HCV genotype 1 subtype identification compared to methods targeting the sole 5′NCR. However, in contrast with a previous report [33], we found that this assay failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases.
Table 1. Ability of the different molecular methods tested in this study to correctly identify HCV subtypes 1a and 1b in a series of 500 patients infected by one or the other of these subtypes.
Screen shot 2010-09-18 at 7.38.55 AM.png
Abstract
Background
With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.
Methodology/Principal Findings
A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.
Conclusions/Significance
In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.
Funding: The Trugene HCV 5′NC Genotyping kits and the INNO-LiPA HCV kits were kindly provided by Siemens Medical Solutions Diagnostics. The Abbott RealTime HCV Genotype II kits were kindly provided by Abbott Molecular. This work is part of the activity of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a grant (LSHM-CT-2004-503359) from the Priority 1 “Life Sciences, Genomics and Biotechnology for Health” program in the 6th Framework Program of the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
Over 170 million individuals are infected with hepatitis C virus (HCV) worldwide. Phylogenetic analyses have shown that HCV strains can be classified into at least 6 major genotypes (numbered 1 to 6), and a large number of subtypes within each genotype [1]. Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide, with subtypes 1a and 1b representing the vast majority of circulating strains [2], [3], [4].
Current treatment of chronic hepatitis C is based on the combination of pegylated interferon (IFN)-α and ribavirin [5]. This treatment fails to eradicate infection in 50%–60% of patients infected with HCV genotype 1 and approximately 20% of those infected with HCV genotypes 2 and 3 [6], [7], [8]. Thus the need for more efficacious therapies is urgent, especially for patients infected with HCV genotype 1. A number of novel antiviral molecules currently are in preclinical or clinical development [9]. The most advanced ones are specific inhibitors of viral enzymes and functions involved in the HCV life cycle. Molecules that have reached clinical development include inhibitors of the nonstructural (NS) 3/4A serine protease and inhibitors of HCV replication that belong to different categories: nucleoside/nucleotide analogue and non-nucleoside inhibitors of the HCV RNA-dependent RNA polymerase (RdRp), NS5A inhibitors and cyclophilin inhibitors [9]. These agents have shown potent antiviral efficacy when used alone, and encouraging results have been recently published showing that HCV clearance can be achieved in approximately 70% of cases when a potent NS3/4A inhibitor is used in combination with pegylated IFN-α and ribavirin [10], [11], [12].
HCV genotype 1 is generally considered as a homogeneous group. There are however biological differences between the different subtypes of HCV genotype 1, which are related to differences in their nucleotide and amino acid sequences. Importantly, differences between subtype 1a and 1b (by far the most frequently encountered genotype 1 subtypes in clinical practice) include different efficacies of antiviral drugs and different resistance profiles to such drugs. Indeed, several HCV inhibitors appear to have selective activity against different HCV genotype 1 subtypes, both in vitro and in vivo. Differences have been observed in vitro with NS3/4A protease inhibitors, non-nucleoside inhibitors of HCV RdRp and NS5A inhibitors [13], [14], [15], [16], [17]. For instance, BILB 1941, a non-nucleoside inhibitor of HCV RdRp, has been shown to have better antiviral efficacy in patients infected with HCV subtype 1b than in those infected with HCV subtype 1a, a finding reflecting in vitro experiments [13].
A major issue that limits the efficacy of direct acting antiviral therapies for HCV is the selection by these drugs of resistant variants upon administration [18]. Recent studies with NS3/4A protease inhibitors have shown that the genetic barrier and resistance profiles substantially differ between the different genotype 1 subtypes. For instance, the Arg to Lys substitution at position 155 of the NS3 protease (R155K) is usually selected in subtype 1a replicons treated with telaprevir, but not in subtype 1b replicons [19]. The reason is that only one nucleotide substitution is needed relative to the subtype 1a sequence to generate this variant, whereas two substitutions are needed relative to the 1b sequence (codon usage bias). Overall, natural polymorphisms at positions R155 and V36 are frequent in subtype 1a, but rare in subtype 1b where substitutions at position A156 are preferentially selected in vitro [19]. This is reflected in vivo by the different resistance profiles in patients infected by HCV subtypes 1a and 1b. In the former, the V36 and R155 substitutions represent the backbone of resistance, whereas in the latter resistance is less frequent as it is preferentially associated with substitutions at position A156 that are associated with a decreased fitness of the variants [19], [20], [21]. Similarly, important differences in the resistance profiles have been described in vitro with HCV-796, a non-nucleoside inhibitor of HCV RdRp. The C316Y amino acid substitution has been reported to be selected in both subtype 1a and 1b replicon cells. However, in genotype 1a replicons, the C316Y substitution has low replication capacity that must be compensated for by additional “compensatory” substitutions, including L392F or M414T, resulting in an increase in replication levels of at least 10-fold [19]. A higher genetic barrier to resistance to HCV-796 and related compounds is therefore expected in patients infected with HCV subtype 1a than 1b. In vivo, HCV-796 monotherapy was however shown to select subtype 1a variants with a single C316Y substitution, whereas the C316Y substitution was associated with a number of additional substitutions in subtype 1b patients [22].
As a result of these findings, correct identification of HCV subtypes 1a and 1b is crucial in clinical trials assessing new HCV drugs in order to correctly stratify and interpret efficacy and resistance data. It may also become important in future clinical practice, as tailoring treatment schedules with HCV inhibitors to HCV genotype 1 subtype might become necessary. A variety of molecular methods can be used to identify the HCV genotype and subtype both in clinical trials and practice. Commercial assays have been developed, most of them targeting the 5′ noncoding region (5′NCR) of the HCV genome, although this region is the most conserved one. These methods have been shown to differentiate well the different HCV genotypes (1 to 6), except genotype 1 from genotype 6, a rare HCV genotype in the Western world [23], [24]. The goal of our study was to assess the ability of molecular methods targeting the 5′NCR to correctly identify the HCV genotype 1 subtype in patients eligible for clinical trials, and to identify the best method for this purpose.
Results
Hepatitis C Virus Genotype and Subtype Determination by Phylogenetic Analysis of a Portion of the NS5B Gene
Direct sequence analysis of a sufficiently long portion of the NS5B gene followed by phylogenetic analysis is the reference method for identification of HCV genotype and subtype [1], [25]. It was used to identify the HCV genotype and subtype in 516 treatment-naïve patients included in a multicenter clinical trial assessing different schedules of pegylated IFN-α2a and ribavirin [26]. All of these patients were thought to be infected with HCV genotype 1 at inclusion based on local assessment. In fact, 6 patients were infected with genotype 6, including 2 with subtype 6e, one with subtype 6o, one with subtype 6p, one with subtype 6q and one with subtype 6r. These 6 samples were not considered for further analysis in the present study. The remaining 510 patients were confirmed to be infected with HCV genotype 1: 237 of them (46.5%) were infected with HCV subtype 1a and 263 (51.6%) with subtype 1b (Figure 1). As shown in Figure 1, HCV subtype 1a strains segregated into two distinct clades, that were termed 1a clade I (n = 83, 35.0%) and 1a clade II (n = 154, 65.0%). Eight patients (1.6%) were infected with another HCV genotype 1 subtype, including 4 patients with subtype 1d, 2 with subtype 1e, one with subtype 1i, and one with subtype 1l. The remaining 2 patients (0.3%) were infected with genotype 1 but the subtype could not be determined. The ability of the different molecular methods to correctly identify HCV subtypes 1a and 1b was then tested on the 237 and 263 samples containing HCV subtypes 1a and 1b, respectively.
INNO-LiPA HCV 2.0 displays the same 5′NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1).
The real-time PCR-based assay targeting both the 5′NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1).
5′NCR Sequence Analysis in Misclassified Subtype 1a Strains
Among the HCV subtype 1a strains, 47 were misclassified as subtype 1b by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0, including 33 that were misclassified by both assays, 7 that were misclassified by Trugene HCV Genotyping Kit only, and 7 that were misclassified by INNO-LiPA HCV 1.0 only (Table 2). Figure 2 shows an alignment of their 5′NCR sequences relative to the consensus sequences of the correctly classified strains (including subtype 1a clade I, subtype 1a clade II and subtype 1b). As shown in Figure 2, misclassification of subtype 1a strains into subtype 1b in one or both assays was related to the presence of natural polymorphisms at nucleotide positions 204 and 243, both of which are located within the sequence of an INNO-LiPA HCV 1.0 probe. At position 243, A is the most frequent nucleotide in HCV subtype 1a, in both subtype 1a clade I and clade II. Substitution into a G, the most frequent nucleotide at position 243 in subtype 1b, was found in all cases that were misclassified as subtype 1b by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0 (Figure 2). At position 204, A is the most frequent nucleotide for subtype 1a clade I, whereas C is the most frequent nucleotide for subtype 1a clade II, and C or T are the most frequent nucleotides for subtype 1b. In spite of the presence of a G at position 243, the presence of an A at position 204 allowed correct identification of subtype 1a with Trugene HCV Genotyping Kit but not with INNO-LiPA HCV 1.0 (Figure 2). The usual presence of a C at position 204 in subtype 1a clade II explains why misclassifications were far more frequent with this clade than with subtype 1a clade I.
Among the 12 subtype 1a strains that were classified as genotype 1, indeterminate subtype with Trugene HCV Genotyping Kit, one had a G and 5 had mixed A and G populations at position 243. Two additional patients with an A at position 243 had a C at position 248. In the remaining 4 cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown). Among the 25 subtype 1a strains that were classified as genotype 1, indeterminate subtype with INNO-LiPA HCV 1.0 (including 6 with the same profile in Trugene HCV Genotyping Kit), 4 had a G and 4 had mixed A and G populations at position 243. Three additional patients with an A at position 243 had a C at position 248 (C only in two of them, a mixture of C and T in one). In the 14 remaining cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown).
5′NCR Sequence Analysis in Misclassified Subtype 1b Strains
Among HCV subtype 1b strains, 8 were misclassified as subtype 1a by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0, including 3 that were misclassified by both assays, 4 that were misclassified by Trugene HCV Genotyping Kit only, and 1 that was misclassified by INNO-LiPA HCV 1.0 only (Table 2). Figure 3 shows an alignment of their 5′NCR sequences relative to the consensus sequences of the correctly classified subtype 1a and subtype 1b strains. As shown in Figure 3, and as for misclassified subtype 1a strains discussed above, misclassification of subtype 1b strains into subtype 1a was related to the presence of natural polymorphisms at positions 204 and 243. At position 243, G is the most frequent nucleotide in HCV subtype 1b. Substitution into an A, the most frequent nucleotide at position 243 in subtype 1a, was found in all cases that were misclassified as subtype 1a by both Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0 and by INNO-LiPA HCV 1.0 only, but not in those that were misclassified by Trugene HCV Genotyping Kit only (Figure 3). In the latter, it is the presence of an A at position 204 instead of a C or a T that was responsible for misclassification in all but one case (Figure 3).
Among the 11 subtype 1b strains that were classified as genotype 1, indeterminate subtype with Trugene HCV Genotyping Kit, one had an A at position 243. In the remaining cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown). Among the 15 subtype 1b strains that were classified as genotype 1, indeterminate subtype with INNO-LiPA HCV 1.0 (none of which were classified as indeterminate in Trugene HCV Genotyping Kit), one had an A and one harbored mixed A and G populations at position 243. Both of them had a C at position 248 (C only in one of them and a mixture of C and T in the other one). In the remaining 13 cases, no explanation was found in the 5′NCR sequence for the failure to identify the HCV subtype (data not shown).
Incorrect Subtyping with Abbott RealTime HCV Genotype II Assay, that Targets Both the 5′NCR and NS5B Region
Among the HCV subtype 1a strains, 16 were incorrectly classified by Abbott RealTime HCV Genotype II assay (Table 1): 2 were misclassified as subtype 1b, 12 were classified as genotype 1, indeterminate subtype, one was identified as a mixed 1a/1b infection, and one gave an indeterminate result. In one case, PCR amplification failed, and in one case, not enough serum volume was available for testing.
Among the HCV subtype 1b strains, 27 were incorrectly classified by Abbott RealTime HCV Genotype II assay (Table 1): 3 were misclassified as subtype 1a, 18 were classified as genotype 1, indeterminate subtype, 5 were identified as a mixed 1a/1b infection, and one gave an indeterminate result. In 2 cases, PCR amplification failed, and in one case, not enough serum volume was available for testing.
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