December 30, 2011

Needles and the damage done

Posted: Fri, Dec. 30, 2011, 3:01 AM

By Christopher Moraff

With a single sentence buried in the 1,200-page omnibus spending package for 2012, signed into law last week by President Obama, social conservatives in the House quietly took a hatchet to one of the most effective strategies cities have for preventing the spread of HIV. The provision ended federal support for needle exchange programs that have been backed by most major health organizations, the U.S. Conference of Mayors, and Obama himself.

The subcommittee responsible for the rider is led by Rep. Denny Rehberg (R., Mont.), who once told a reporter: "The problem with AIDS is, you got it, you die. So why are we spending money on the issue?" AIDS advocates say the change will likely doom countless addicts to contracting HIV as well as hepatitis C.

Needle exchange programs have been a controversial but highly effective barrier to the spread of infectious blood-borne illnesses for nearly three decades. As the AIDS epidemic reached crisis levels in the mid-1980s, cities such as New York and Los Angeles began to copy European programs providing clean needles to addicts. Early studies showed immediate decreases in syringe sharing among drug users, one of the leading causes of AIDS.

In 1988, however, North Carolina Sen. Jesse Helms, a staunch conservative, successfully lobbied to ban all federal funding of syringe exchange programs. The prohibition lasted more than two decades, until a Democratic Congress finally voted to lift it in 2009.

According to the Kaiser Family Foundation, there are 221 needle exchange programs in 33 states and the District of Columbia, many relying on federal funding. Their impact on new HIV infections is nothing less than dramatic, research shows, reducing them by as much as a third.

Moreover, there is no attendant increase in drug use. On the contrary, the National Institutes of Health has found that drug users in areas that offer syringe exchange are more likely to get treatment due to regular contact with outreach workers.

In Philadelphia, where Prevention Point Philly's needle exchange program has been distributing clean needles since 1992, public health officials have seen a marked decrease in HIV infections. According to a study by the Center for Studies of Addiction at the University of Pennsylvania, the years following establishment of a citywide needle exchange saw the annual rate of new HIV infections among intravenous drug users drop from 6.8 percent to less than 0.05 percent.

With the fourth-largest exchange program in the country, Prevention Point Philly exchanged more than one million syringes last year, serving nearly 4,000 drug users. In 1991, a year before the program was sanctioned by then-Mayor Ed Rendell, nearly half of those people would have had HIV. Today, only one in 10 does.

And those are just the public health implications. Consider the financial ramifications: It costs just 8 cents to provide a clean needle to an addict, compared with more than $300,000 in medical costs alone to care for someone with AIDS. Even those without a care for the suffering of HIV-infected addicts can do that math.

According to executive director Jose A. Benitez, Prevention Point is unlikely to be directly affected by the reinstated federal ban, because Pennsylvania already prohibits money passing through its coffers - including federal funds - from supporting needle exchange. That's a dubious oversight in a state whose former governor helped create the program, and it has forced Prevention Point to depend exclusively on the city and a handful of foundations. Benitez expects private funding to become even tighter once federal money dries up.

By prohibiting states from using their federal HIV funding as they see fit, Congress has allowed a handful of zero-tolerance zealots to hijack a public health initiative that saves taxpayers money and prevents unnecessary pain and suffering. And these are the people we hired to look out for us?

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December 29, 2011

FDA Rings in 2012 with HIV Vaccine Trials

By: Jessica Grogan, MDNews.com
Thursday, December 29, 2011

The U.S. Food and Drug Administration has approved Phase I human clinical trials for a Canadian vaccine designed to prevent HIV.

A team of researchers led by Dr. Chil-Yong Kang at the University of Western Ontario developed the SAV001 vaccine, with the support of Sumagen Canada. The team plans to begin Phase I of clinical trials on 40 HIV-positive patients in January 2012. The FDA has given the green light to test the safety, not the effectiveness, of the vaccine.

The SAV001 vaccine is based on a genetically modified killed whole virus and not a live virus. In preliminary toxicology tests, the vaccine has been shown to stimulate a strong immune response. If found safe, two more clinical trials will be conducted. Dr. Kang estimates it will cost $100 million to complete those two trials.

Phase II would look at the immune responses and see whether they make antibodies against the virus. This trial would be conducted on roughly 600 subjects who are HIV-negative but whose lifestyle puts them at high risk of contracting the disease.

Phase III would test the effectiveness of the vaccine in a larger at-risk group. Researchers hope to recruit about 6,000 volunteers for the final phase; half of the individuals would be vaccinated and half would not.

Dr. Kang said participants in the study would be tracked for three years to see how many become infected with the virus.

“FDA approval for human clinical trials is an extremely significant milestone for our vaccine, which has the potential to save the lives of millions of people around the world by preventing HIV infection,” said Dr. Kang in a press release.

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Hepatitis PAP and Co-Pay Programs

Patient Assistance and Co-Pay Programs for Viral Hepatitis Drugs

Provided by Hep Magazine

Introduction

Do you lack health insurance but make too much money to qualify for government assistance? Are your prescription co-payments so high that you can’t afford other basic necessities? Help may be available from pharmaceutical companies in the form of patient assistance programs (PAPs) and co-pay programs.

Over the past two years, the Fair Pricing Coalition (FPC) has been working closely with the pharmaceutical industry to streamline access to co-pay programs and PAPs for people living with viral hepatitis. The FPC has negotiated co-pay programs with virtually every major hepatitis drug manufacturer. Following is a list of co-pay and patient assistance programs for hepatitis B and C, and contact information for these programs. This is a living document that will be updated as program changes are implemented.

Continue Reading Here.....



To hear Maura Shaw tell it, her husband died in the line of duty.

But, as MyFOX Boston reports, Kenneth Shaw's widow has been repeatedly denied accidental death benefits because she can't prove exactly how her husband died.

Shaw worked in the Boston Police Department's identification unit where his wife and two doctors believe he contracted hepatitis C while working in gruesome crime scenes. However, no benefits have been awarded 13 years after Shaw died from the disease.

"It's like a slap in the face. He loved his job. He loved doing what he did," Maura Shaw told MyFOX Boston.

Joseph Connarton, a spokesperson for the Public Employee Retirement Administration Commission, told The Huffington Post that Shaw's situation was an "unfortunate case," but said current laws prevent the commission from awarding benefits.

"It is sadly the fact that Massachusetts law requires a specific criteria to be met prior to either accidental disability or accidental death benefits being granted," Connarton wrote in an email. "In the case the statutory requirements just were not met by either the Boston Retirement Board or the Boston Police Dept."

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Peregrine Provides Update on HCV Clinical Program

Preliminary Data From Phase II Study Shows Antiviral Activity and Positive Safety Profile at Both Bavituximab Doses Evaluated; Supporting Further Dosing and Combination Studies; Company to Seek Collaboration to Advance HCV Program While Continuing to Focus on Its Lead Bavituximab Clinical Program in Multiple Solid Tumor Indications Including Lung Cancer

TUSTIN, CA -- (MARKET WIRE) -- 12/29/11 -- Peregrine Pharmaceuticals, Inc. (NASDAQ: PPHM), a clinical-stage biopharmaceutical company developing first-in-class monoclonal antibodies for the treatment of cancer and viral infections, today provided an update from its randomized Phase II bavituximab study in patients infected with genotype-1 chronic hepatitis C virus (HCV). Patients were randomized in the three-arm study to receive one of two doses of bavituximab (0.3mg/kg or 3mg/kg) or pegylated interferon alpha-2a, in combination with ribavirin. The goals of the study were to determine if bavituximab plus ribavirin has a better safety profile as compared to interferon plus ribavirin, to confirm that the combination of bavituximab and ribavirin has antiviral activity defined as 12 week early virologic response (EVR)(1) and to compare antiviral activity of peg-interferon plus ribavirin versus bavituximab plus ribavirin.

A preliminary data analysis indicates that the combination of bavituximab and ribavirin appeared safe and well tolerated with patients reporting fewer side effects than in the interferon-containing arm. Initial data from the study also indicated that both dose levels of bavituximab with ribavirin demonstrated antiviral activity, however the antiviral effects in patients receiving the 0.3 mg/kg dosing level were more pronounced. A comparison of the viral data indicated that the kinetics of antiviral activity were different between the interferon and bavituximab treatment groups with a high percentage of those patients achieving EVR in the interferon arm of the study doing so between week 4 and 8 and the majority of patients achieving EVR in the bavituximab groups doing so at the week 12 end of study timepoint. More patients had achieved EVR in the interferon-containing group by the end of the study, however based on the nature of late EVR development in the bavituximab containing arms at the very end of the 12 week trial, a longer-term evaluation is needed to adequately compare the effectiveness of bavituximab and interferon. The company plans to present full results from the study at a medical conference in 2012.

"We are pleased with the initial results we have seen from this clinical study evaluating the combination of bavituximab with an established antiviral drug in HCV patients. We see good evidence that the combination of bavituximab with ribavirin has a better safety profile than an interferon containing regimen which was one of the primary objectives of the study," said Joseph S. Shan, vice president of clinical and regulatory affairs at Peregrine Pharmaceuticals. "In addition, we also see that while both dose levels of bavituximab were active, the lower dose level appears more active in HCV patients than the high dose level. Taken together, these early results are very important in validating that the combination of bavituximab with its immunological mechanism of action with an active antiviral agent has a good safety profile and promising antiviral activity. These results suggest that future studies evaluating longer bavituximab treatment durations at or around the lower dose level in combination with ribavirin and potentially direct acting antivirals in certain patient populations may hold promise as interferon-free HCV therapeutic regimens."

"The early data from this trial are promising and suggest that continued development of bavituximab in HCV patients is warranted to explore the full immune-modulating potential of the compound in combination with antiviral agents," said Steven W. King, president and chief executive officer of Peregrine. "With this data in hand, we plan to actively seek development partners interested in working with us to move the PS-targeting antiviral program forward while we continue to focus our resources on the advancement of our bavituximab oncology clinical program in multiple solid tumor indications including non-small cell lung cancer (NSCLC) and pancreatic cancers as well as other indications with high unmet medical need. With as many as six data points coming over the next six months or so from our ongoing phase II trials in front and second line NSCLC and the additional possible data points coming from five additional oncology trials, this is a good time to seek partners for the antiviral program which has shown promise in this study. We look forward to sharing full data from the HCV trial in 2012 and to moving the program forward in the future."

1. EVR is defined as equal or greater than a 2 log reduction in HCV RNA from baseline.

About the Phase II HCV Trial

In this multicenter Phase II randomized trial, 66 patients with previously untreated genotype-1 chronic HCV infection were randomly assigned to one of three treatment arms. Patients received daily oral ribavirin (1000 mg) with either weekly bavituximab (0.3 mg/kg or 3 mg/kg) or pegylated interferon alpha-2a (180 µg) for up to 12 weeks and were tested for safety parameters and antiviral activity.

About Bavituximab's Antiviral Approach

Bavituximab is the first in a new class of patented antibody therapeutics that target and bind to phosphatidylserine (PS), a specific phospholipid component of cell membranes. Bavituximab helps reactivate and direct the body's immune system to destroy infected cells and virus particles that exhibit this specific phospholipid on their surface. Since their target is host-derived rather than pathogen-derived, PS-targeting antibodies have the potential for broad-spectrum antiviral activity and are also expected to be much less susceptible to the viral mutations that often lead to drug resistance.

About Peregrine Pharmaceuticals

Peregrine Pharmaceuticals, Inc. is a biopharmaceutical company with a portfolio of innovative monoclonal antibodies in clinical trials for the treatment of cancer and serious viral infections. The company is pursuing multiple clinical programs in cancer and hepatitis C virus infection with its lead product candidate bavituximab and novel brain cancer agent Cotara®. Peregrine also has in-house cGMP manufacturing capabilities through its wholly-owned subsidiary Avid Bioservices, Inc. (www.avidbio.com), which provides development and biomanufacturing services for both Peregrine and outside customers. Additional information about Peregrine can be found at www.peregrineinc.com.

Safe Harbor Statement: Statements in this press release which are not purely historical, including statements regarding Peregrine Pharmaceuticals' intentions, hopes, beliefs, expectations, representations, projections, plans or predictions of the future are forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. The forward-looking statements involve risks and uncertainties including, but not limited to, the risk that results from the randomized Phase II trial will not be consistent with results experienced in the earlier single-arm Phase I studies, the risk that results from the randomized Phase II trial may not support registration filings with the U.S. Food and Drug Administration, the risk that Peregrine may not have or raise adequate financial resources to complete the planned clinical programs, and the risk that Peregrine will not find a development partner interested in the antiviral applications of its PS-targeting technology. Factors that could cause actual results to differ materially or otherwise adversely impact the company's ability to obtain regulatory approval for its product candidates include, but are not limited to, uncertainties associated with completing preclinical and clinical trials for our technologies; the early stage of product development; the significant costs to develop our products as all of our products are currently in development, preclinical studies or clinical trials; obtaining additional financing to support our operations and the development of our products; obtaining regulatory approval for our technologies; anticipated timing of regulatory filings and the potential success in gaining regulatory approval and complying with governmental regulations applicable to our business. Our business could be affected by a number of other factors, including the risk factors listed from time to time in the company's SEC reports including, but not limited to, the annual report on Form 10-K for the year ended April 30, 2011 and quarterly report on Form 10-Q for the quarter ended October 31, 2011. The company cautions investors not to place undue reliance on the forward-looking statements contained in this press release. Peregrine Pharmaceuticals, Inc. disclaims any obligation, and does not undertake to update or revise any forward-looking statements in this press release.

Peregrine Contact:

Jay Carlson

Peregrine Pharmaceuticals

(800) 987-8256

info@peregrineinc.com

Source: Peregrine Pharmaceuticals

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December 28, 2011

SAMHSA Releases New Working Definition of 'Recovery'

Deborah Brauser

December 28, 2011 — After considering more than 1,400 comments during a public feedback period this summer, the Substance Abuse and Mental Health Services Administration (SAMHSA) has released a new working definition of "recovery" from mental disorders and substance use disorders.

The organization began consulting members of the behavioral healthcare community more than 1 year ago to develop both major guiding principles and a definition that would capture "the essential, common experiences of those recovering," they write.

The new definition, which was published on SAMHSA's Web site on December 22, now states that recovery is a "process of change through which individuals improve their health and wellness, live a self-directed life, and strive to reach their full potential."

"Over the years it has become increasingly apparent that a practical, comprehensive working definition of recovery would enable policy makers, providers, and others to better design, deliver, and measure integrated and holistic services to those in need," Pamela S. Hyde, SAMHSA administrator, said in a news release.

"I believe SAMHSA has achieved a significant milestone in promoting greater public awareness and appreciation for the importance of recovery, and widespread support for the services that can make it a reality for millions of Americans," added Ms. Hyde.

Public Feedback

In August 2010, SAMHSA invited "mental health consumers and individuals in addiction recovery" to a meeting to discuss the need for a new definition of recovery as part of its Recovery Support Strategic Initiative. There, the participants developed the first draft of a definition as well as a draft of guiding principles of recovery.

Over the following 12 months, SAMHSA worked with other participants at meetings and conferences to clarify and revise the drafts. The first official working definition and principles were then posted on a Web site blog in August 2011, along with an invitation for public feedback.

The initial blog post received 287 comments, with more than 1,200 additional comments left on SAMHSA's Feedback Forums.

Altogether, 1,000 participants left comments; the participants included representatives from organizations such as the National Alliance on Mental Illness (NAMI) and the National Association of State Alcohol and Drug Abuse Directors (NASADAD).

NAMI and NASADAD Respond

"SAMHSA's 10 'Guiding Principles of Recovery' are laudable as a vision to aspire to," Michael J. Fitzpatrick, executive director of NAMI, wrote at that time in one of the blog's comments. "Unfortunately, these principles fail to acknowledge that some people living with the most serious mental illnesses are sometimes limited or unable to exercise self-determination."

He added that neglect or lack of treatment can lead to "horrendous consequences," including large numbers of those with mental illnesses being incarcerated in jails and prisons, "boarded" in emergency departments, or becoming homeless.

"The solution to these problems is a system that is individualized, respectful, allows people to exercise personal choice and autonomy whenever possible, but also has the capacity to intervene and provide humane, compassionate assistance to people when they most need it," wrote Mr. Fitzpatrick.

He also suggested that the important role families play in helping adults living with mental illness should be acknowledged and that the guidelines should emphasize that recovery means different things for different people.

Robert I. L. Morrison, executive director of NASADAD, wrote in his comments that more work should be done in understanding current state-funded recovery services and that SAMHSA should draw from states' experiences with the various Access to Recovery models already implemented.

"In addition, we agree there are commonalities between recovery from mental and substance use disorders that are important to understand, particularly in the context of reimbursement strategies," wrote Mr. Morrison.

He added that it should also be acknowledged that recovery experiences may be different for these people.

SAMSHA noted that many of these comments have been incorporated into the current working definition and principles.

"The response to our request for feedback was tremendous and clearly demonstrated the field's interest and concern on this important issue. Over 8,500 votes were also cast in support of the ideas on the forums," they write.

New Guidelines

The new guiding principles now state that recovery:

  • emerges from hope;
  • is person-driven;
  • occurs through many pathways;
  • is holistic;
  • is supported by peers and allies;
  • is supported through relationships and social networks;
  • is culturally based;
  • is supported by addressing trauma;
  • involves the individual, family, and community; and
  • is based on respect.

Along with the definition and principles, SAMHSA has also created "4 major dimensions that support a life in recovery," consisting of health, home, purpose, and community.

Further, they added a preamble emphasizing that "there are many different pathways to recovery, and each individual determines his or her own way."

SAMHSA notes that although several effective models exist for promoting recovery, more work is needed to make sure services and systems are put into place in every state.

"Drawing on research, practice, and personal experience of recovering individuals, within the context of health reform, SAMHSA will lead efforts to advance the understanding of recovery and ensure that vital recovery supports and services are available and accessible to all who need and want them."

The full report was published online December 22 on SAMHSA's Web site.

Source

From Journal of Viral Hepatitis

S.-C. C. Sun; A. Bae; X. Qi; J. Harris; K. A. Wong; M. D. Miller; H. Mo

Posted: 12/27/2011; J Viral Hepat. 2011;18(12):861-870. © 2011 Blackwell Publishing

Abstract and Introduction
Abstract

To assess the natural variation in drug susceptibility among treatment-naïve hepatitis C virus (HCV) patient isolates, the susceptibilities of chimeric replicons carrying the HCV NS5B polymerase from up to 51 patient isolates against a panel of diverse HCV nonnucleoside polymerase inhibitors were evaluated using a replicon-based transient replication assay. Some patient to patient variation in susceptibility to the panel of three HCV nonnucleoside polymerase inhibitors was observed. Linear regression and correlation analyses revealed no correlations among the susceptibilities to the polymerase inhibitors tested. Our results suggest that variable antiviral responses to HCV nonnucleoside polymerase inhibitors may be observed because of the natural variation in baseline susceptibility. In addition, the lack of correlation among the susceptibilities to three classes of HCV polymerase inhibitors evaluated here supports their possible combined use in a combination therapy strategy

Introduction

Hepatitis C virus (HCV) is a positive-strand RNA virus and is estimated to infect over 170 million people worldwide.[1,2] To date pegylated interferon-α plus ribavirin, the current standard of therapy for HCV infection, is associated with incomplete efficacy and various side effects.[3–6] Therefore, intense effort and time have been devoted to the discovery and development of novel and selective small-molecule inhibitors of HCV. Although no direct small-molecule antivirals have yet been approved for therapeutic use, the NS3 protease and NS5B polymerase are considered to be prime targets, and inhibitors of each enzyme have shown strong antiviral activity in early clinical trials.[7–11]

HCV RNA-dependent RNA polymerase is the essential enzyme for replication of HCV RNA. A number of HCV polymerase inhibitors have been discovered; some have advanced to phase I/II clinical trials and have demonstrated antiviral activity in HCV-infected subjects in monotherapy.[9,10,12] Among these HCV polymerase inhibitors, a number of nucleoside analogues (2'-Me-C, R1479, NM283, R1626 and R7128) bind to the active site of the HCV polymerase.[13–15] In vitro resistance selection in the presence of nucleoside analogues identified a S282T resistance mutation for 2'-Me-C, and two (S96T and N142T) for R1479.[16] The S282T mutation has also been reported in virologic breakthrough in patients on an NM283 (Idenix Pharmaceuticals, Cambridge, MA, USA) clinical trial.[17,18] In addition to the active site, there are four allosteric binding sites for nonnucleoside inhibitors (NNIs) within the NS5B polymerase including: palm I (palm domain near the active site), palm II (partially overlapping palm I and towards the active site), thumb I (thumb domain near the fingertips) and thumb II (the outer surface of the thumb domain).[19–21] First, the benzothiadiazine and acylpyrrolidine class of NNIs bind to the palm I domain of the polymerase.[7,20] Resistance selection in the presence of benzothiadiazines revealed amino acid substitutions at residues 411, 414, 448, 451, 553, 554, 555, 556 and 559.[22–25] Second, the benzofuran class of NNIs (HCV-796; Wyeth/Viropharma) binds to the palm II domain. HCV-796 (benzofuran) has been reported to select a major resistance mutation at amino acid residue 316 in an in vitro replicon system, and in early clinical trials.[26,27] Distinct from the above HCV polymerase inhibitors, benzimidazoles and indoles bind to the thumb I site.[28] Reduced susceptibility to this class of compounds was associated with the selection of mutations at residues 495 and 496 of the NS5B polymerase.[28] Lastly, the thiophene carboxylic acids bind to the thumb II site.[20,29,30] The most frequent mutations observed in vitro and in vivo for this class are located at amino acid position 423 (M423T/V/I) followed by positions 419 (L419M) and 482 (I482L).[20,30,31]

HCV is characterized by a high degree of genetic diversity.[32,33] The nucleotide sequences among the six different genotypes differ at 30–35% of the nucleotide sites.[34,35] Each of the six major genotypes of HCV contains a series of more closely related subtypes that typically differ from each other by 20–25% in nucleotide sequences. Furthermore, 5–8% sequence divergence was observed between individual strains (variants) of HCV within a given subtype (GT-1a and GT-1b). It is possible that this high degree of natural sequence variation may have an impact on the susceptibility of patient isolates to HCV NS5B inhibitors, possibly, influencing the clinical response to direct antivirals. Therefore, it is of interest to test drug candidates against circulating genotypes from a variety of clinical isolates. In the present study, the susceptibilities of a panel of patient-derived NS5B polymerase to a number of nonnucleoside polymerase inhibitors were evaluated using a transient replication assay. The correlations in drug susceptibility among the different polymerase inhibitors were analysed.

Materials and Methods
Clinical Isolates

Patient serum samples were obtained from untreated GT-1a and GT-1b HCV-infected patients. All patients gave informed consent and originated from the USA.

Compounds

The NNIs benzofuran (NNI-1), benzothiadiazine (NNI-2) and thiophene carboxylic acid (NNI-3) were synthesized at Gilead Sciences, Inc. (Foster City, CA, USA).

Construction of NS5B Shuttle Vector

The genotype 1b-Con-1 subgenomic replicon construct 1b-PI-luc used to create the shuttle vectors has been described by Friebe et al.[36] The components of the replicon are depicted in Fig. 1. A poliovirus IRES element was added at the 5' end after the HCV 5'NTR to increase firefly luciferase translation and RNA replication. Translation of HCV replicon from NS3 to NS5B is driven by the EMCV IRES. Three adaptive mutations, two in NS3 (E1202G+T1280I) and one in NS4B (K1846T) were introduced for efficient replication.[37] Luciferase activity was used as an end-point readout.

754991-fig1

Figure 1. Design of shuttle vector. Vector used for GT-1a and GT-1b NS5B gene insertion from the clinical isolates. The backbone of the shuttle vector is from 1b-Con-1.

To clone NS5B from patient isolates, a BclI site located at the 9th amino acid residue of NS5B was used as the 5'-end cloning site (Fig. 1). An AseI site was inserted directly after the TGA stop codon of NS5B, giving an insertion of ATTAAT at the 5' end of the 3' NTR. The restriction site was generated by site-directed mutagenesis using a QuickChange XL mutagenesis kit (Agilent, Santa Clara, CA, USA). The 1.0-kb fragment between two BglII sites in NS5B was removed to prevent contamination of recombinant shuttle vectors with the parental NS5B gene in luciferase readouts (Fig. 1).

NS5B Gene Isolations and Subcloning to Shuttle Vector

Viral RNA was isolated from 140 μL of serum from HCV-infected subjects using the QiaAmp Viral RNA isolation kit (Qiagen, Valencia, CA, USA) according to the supplier's instructions. cDNA was synthesized in a 20-μL reaction containing 0.2 mm of each dNTP, 0.125 μm of reverse primers and 50 U/μL MonsterScript reverse transcriptase (Epicentre, Madison, WI, USA) as recommended by the manufacturer. Ten microlitres of the RNA isolated from 140 μL of serum was denatured at 65 °C for 1 min, chilled on ice and added as template for reverse transcription. The reverse transcription reaction was incubated at 50 °C (1a) or 54 °C (1b) for 10 min and then at 60 °C for 40 min. Subsequently, the reverse transcriptase was heat inactivated at 90 °C for 2 min. The cDNA was used as template for PCR amplification of the NS5B gene.

A nested PCR strategy was used with genotype-specific primers to amplify the NS5B gene that was then used as a template for sequencing. GT-1a was amplified using primers 1aNS5B-5'7380-5' GAA TCA ACC CTA TCT ACT GCC TTG GCC GAG C-3' and 1aNS5B-3'9386-5' CTA AGA GGC CGG AGT GTT TAC-3' for the first round of PCR, and primers 1aNS5B-5'-7419-5'- TTT GGC AGC TCC TCA ACT TCC GG-3' and 1aNS5B-3'-9372-5' GAG TGT TTA CCC CAA CCT TCA TCG-3' for nested PCR. GT-1b was amplified using primers 1bNS5B-5'7595 -5' TCG TAC TCC TCC ATG CCC CCC CTT GA-3' and 1bNS5B-3'9459 -5' CCT ATT GGC CTG GAG TGT TTA GCT C-3' for the first round of PCR, and primers 1bNS5B-5'-7637-5'- GAT CTC AGC GAC GGG TCT TGG TC-3' and 1bNS5B-3'-9424-5' TTG GGG AGC AGG TAG ATG CCT AC-3' for nested PCR. PCR parameters were as follows for both genotypes: 94 °C, 2 min, 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2:10 min for first round PCR and 94 °C, 2 min, 35 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2:10 min for nested PCR. All nested PCRs were performed using the High Fidelity Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). PCR products were purified on a QiaQuick PCR clean-up column (Qiagen), according to the supplier's recommendations. The cycle sequencing of purified PCR product was conducted by ABI BigDye Terminator Sequencing technology (Applied Biosystems, Inc. Carlsbad, CA, USA) with 50 ng of purified PCR product. DNA sequencing conditions were 96 °C for 1 min, followed by 25 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 4 min. The dried products were then denatured in Hi-Di formamide and run on an ABI 3100 genetic analyzer. DNA sequences were analysed with the Sequencher 4.0 program. Reference sequences of 1a-H77 and 1b-Con-1 were used as comparators to report any changes in the clinical isolates for GT-1a and GT-1b, respectively. Amino acid (AA) sequence analyses were performed from AA 1-591 and AA 1-582 of the NS5B gene for GT-1a and GT-1b, respectively.

The final PCR products for cloning into the NS5B shuttle vector were amplified from the above PCR products with primers containing 5' BclI and 3' AseI restriction sites. The final PCR was performed in the buffer supplied with the polymerase, plus 1.25 mm MgCl2, 0.3 mm each primer. After digestion with AseI (New England Biolabs, Ipswich, MA, USA) at 37 °C for 3 h and subsequent BclI digestion at 50 °C for 3 h, the PCR product was cleaned with a QiaQuick PCR column (Qiagen) according to the supplier's recommendations. The shuttle vector DNA was similarly digested and gel purified with QIAEX II Gel Extraction kit (Qiagen) according to manufacturer's protocol. Purified shuttle vector DNA was then treated with shrimp alkaline phosphatase (Roche) for 15 min at 37 °C and then 65 °C for 15 min to heat inactivate the enzyme. Ligations were performed with 100 ng each of vector and insert DNA in 20 μL of 50 mM Tris–Cl, pH 7.8, 10 m MgCl2, 10 mM DTT, 1 mm ATP, 25 μg/mL bovine serum albumin and 0.15 Weiss units of T4 DNA ligase (New England Biolabs) at 16 °C, overnight.

Plasmid Purification and RNA Synthesis

The ligation reaction was coprecipitated with Pellet Paint® Co-Precipitant (Novagen, San Diego, CA, USA) according to the supplier's recommendations. The precipitated DNA was then resuspended in 2 μL of water and transfected by electroporation into Escherichia coli ElectroTen-Blue® Electroporation Competent Cells (Agilent) according to the supplier's recommendations. Ten per cent of the transformation mixture was plated on antibiotic selection plates to determine the transformation efficiency and the remaining transformants were expanded in liquid culture to propagate the quasi-species pool. Plasmid DNA from each overnight culture was then purified using two QiaPrep mini spin columns (Qiagen). Plasmid DNA was linearized by digestion with ScaI, and purified by extraction once with an equal volume of phenol/chloroform (1/1). DNA was precipitated with ethanol, and template RNA was synthesized using a T7 Megascript RNA synthesis kit (Ambion, Austin, TX, USA) according to the supplier's instructions. After lithium chloride precipitation, RNA was used for the transient replication assays.

Transient Replication Assay

The Huh-7 cells used in the transient replication assay were derived from a cured replicon cell clone as described previously,[36] referred to as Lunet cells. For all experiments, Lunet cells were grown to a density of (6–10) × 104 cells/cm2 in Dulbecco's minimal essential medium (DMEM; Invitrogen) containing 10% foetal bovine serum (HyClone, Waltham, MA, USA), penicillin (100 U/mL)/streptomycin (100 μg/mL) (Gibco/Invitrogen) and 100 μM MEM Non-Essential Amino Acids Solution (Invitrogen). Cells were harvested by trypsinization and washed twice with ice-cold PBS; cell concentration was adjusted to 1 × 107 cells/mL with ice-cold PBS, and 0.4 mL was transferred to a cold cuvette with a 0.4-cm gap, along with 5–10 μg of template RNA. Electroporation was performed with a Gene Pulser II (BioRad, Hercules, CA, USA) at 960 μF and 270 V using 1 manual pulse. Transfected cells were diluted to 2 × 105 cells/mL and plated in 96-well plates at 2 × 104 cells per well in complete DMEM medium. A duplicate plate was analysed for luciferase activity at 4 h posttransfection for transfection efficiency normalization. Compounds (or DMSO alone as a control) were added 24 h posttransfection in threefold dilutions at a final DMSO concentration of 0.5% (v/v). Firefly luciferase reporter signal was read 72 h after addition of compounds using the Luciferase assay system (Promega, Madison, WI, USA) with a Victor Luminometer (Perkin-Elmer, Waltham, MA, USA).

EC50 Determination and Replication Capacity of HCV NS5B Clinical Isolates

The EC50 values were assessed as the compound concentration at which a 50% reduction in the level of firefly reporter activity was observed when compared with control samples in the absence of compound. Dose response curves and EC50 values were generated by using GraphPad Prism 4.0 (GraphPad Software, La Jolla, CA, USA) with nonlinear regression analysis. The signal-to-noise window was determined as the ratio of luciferase activity from cells treated with 0.5% DMSO vs activity from cells treated with 500 nM of BILN-2061 in 0.5% DMSO.

The replication level of either reference strains (1b-Con-1) or chimera replicons derived transiently from clinical isolates was determined as the ratio of the firefly luciferase signal at day 4 to that at 4 h postelectroporation, to normalize for transfection efficiency. The replication capacity of each chimera replicon derived from clinical isolates was expressed as their normalized replication efficiency when compared with that of the reference strain (1b-Con-1) within the same experiment.

Statistical Regression Analysis of Correlation Between Different HCV Inhibitors

The mean EC50 values derived from each individual treatment-naïve HCV patient were compared in a statistical analysis to determine the correlation between different HCV inhibitors. This was calculated using the Pearson product-moment correlation coefficient (r) and the coefficient of determination (r2) of linear regression analysis. Both the correlation and linear regression analyses were performed using GraphPad Prism 4.0 (GraphPad Software).

Results
Characterization of the Phenotypic Assays

The plasmid used for the construction of the NS5B designated as 1b-PI-luc, includes three adaptive mutations (E1202G, T1280I and K1847T) and a poliovirus IRES element that has been shown to enhance both translation of the luciferase gene and RNA replication.[36] This replicon replicated to high levels in Lunet cells. The NS5B shuttle vector was modified by addition of the 3' restriction site (AseI) for insertion of NS5B genes from clinical isolates. Insertion of the new restriction site did not adversely affect the replication capacity of the replicon (data not shown).

To evaluate the reproducibility of the NS5B phenotypic assay, the susceptibility of reference replicon (1b-Con-1) to the thiophene carboxylic acid, benzofuran and benzothiadiazine NNIs was obtained from between 15 and 16 independent determinants. Table 1 summarizes the mean EC50 values, standard deviations, the 95% confidence intervals, and the maximum fold changes for the NNIs. The assay was highly reproducible with the EC50 determinations from independent assays differing by <2-fold with respect to the mean EC50 for each drug.

The Replication of the Chimeric Replicons Carrying Heterologous NS5B From Clinical Isolates

To test the clinical isolates, the HCV NS5B polymerase gene was amplified from 55 clinical isolates from untreated patients infected with HCV (41 GT-1a and 14 GT-1b) and were ligated into the NS5B shuttle vector. By mimicking the intrinsic HCV heterogeneity present in patients, these pooled populations of molecules were transfected into Lunet cells. The replication capacity of the patient isolates was determined by comparing the luciferase activity in the test sample to the parental 1b-PI-Luc at day 4 in a transient replication assay. As shown in Fig. 2, the replication capacity of the chimeric replicons harboring the NS5B genes from patient sera ranged from 0.01% to 50% of the wild-type 1b-Con-1. In this assay, the luciferase signal-to-noise ratio of the parental 1b-PI-Luc was high (>1000, data not shown). EC50 values can be accurately determined if the clinical sample can replicate at 0.01% of the wild-type replicon (corresponding to signal/noise ratio of >10). Among the 55 patient isolates, 51 of 55 (92.7%) demonstrated sufficient replication levels to allow for drug susceptibility determination while the remaining four clinical isolates for NS5B phenotypic analysis either did not replicate or replicated at a level too low to allow for accurate EC50 determination.

754991-fig2

Figure 2. Relative replication capacity of chimeric NS5B clinical isolates from 52 HCV treatment-naïve patients.

Susceptibility of Treatment-naïve Clinical Isolates to HCV Polymerase Inhibitors

To investigate the natural variation in drug susceptibility of the baseline clinical isolates, the chimeric replicons were tested for their susceptibilities to three HCV NNIs. Susceptibilities to the thumb II inhibitor thiophene carboxylic acid were comparable to the laboratory strain 1b-Con-1 (Fig. 3a and Table 1). Thirty-nine of these samples were GT-1a and eight were GT-1b. Four of the clinical isolates failed to generate an EC50 value because of poor curve fitting. The EC50 values ranged from 100 to 764 nM with a mean EC50 of 322 ± 148 nM for GT-1a and 344 ± 148 nM for GT-1b patients. Thus, no difference in susceptibility to the thiophene carboxylic acid tested was observed between GT-1a and GT-1b.

Against the benzofuran, the majority of the GT-1 isolates (45 of 51) were slightly more sensitive to the benzofuran than the laboratory stain 1b-Con-1 (Fig. 3b and Table 1). The EC50 ranged from 1.3 to 13 nM and the mean EC50 values were 5.9 ± 2.9 nM from 39 GT-1a patients and 5.8 ± 1.7 nM from 12 GT-1b patients, which were slightly lower than that of the laboratory strain 1b-Con-1 (9.74 nM. Similar to the thiophene carboxylic acid, no significant difference in sensitivity between GT-1a and GT-1b was observed for the benzofuran.

754991-fig3

Figure 3. Susceptibility to HCV polymerase inhibitors of treatment-naïve HCV NS5B clinical isolates indicates as EC50 values. (a) A total of 47 NS5B clinical isolates (39 GT-1a and 8 GT-1b) were tested to determine the sensitivity to thiophene. The mean EC50 is 322 ± 148 nM from GT-1a patients and 344 ± 148 nM from GT-1b patients. (b) A total of 51 NS5B clinical isolates (39 GT-1a and 12 GT-1b) were tested to determine the sensitivity to benzofuran. The mean EC50 is 5.9 ± 2.9 nM from GT-1a patients and 5.8 ± 1.7 nM from GT-1b patients. (c) A total of 44 NS5B clinical isolates (33 GT-1a and 11 GT-1b) were tested to determine the sensitivity to benzothiadiazine. The mean EC50 is 302 ± 165 nM from GT-1a patients and 130 ± 44 nM from GT-1b patients.

In contrast, the GT-1a clinical isolates showed a decreased sensitivity to the palm I inhibitor benzothiadiazine compared to GT-1b clinical isolates, and the laboratory strain 1b-Con-1 (Fig. 3c and Table 1). The reduced susceptibility to the compound ranged from 1.1- to 3.4-fold in EC50 values when compared to the mean of GT-1b clinical isolates. We were unable to obtain reportable EC50 values for seven of the clinical isolates because of poor curve fitting. The mean EC50 was 302 ± 165 nM from 33 GT-1a and 130 ± 44 nM from 11 GT-1b patients.

As shown in Fig. 3 and Table 2, some natural variation in susceptibility among these isolates was observed with all three different classes of HCV polymerase inhibitors tested (benzofuran, benzothiadiazine and thiophene carboxylic acid). The EC50 values in the 95th percentile were 4.5-, 6- and 7.5-fold higher than the 5th percentile EC50 values for the thiophene carboxylic acid, benzofuran, and benzothiadiazine compounds, respectively.

Genetic Diversity of Variants Within Clinical Isolates

Population sequence analysis of full-length NS5B was performed for all 55 baseline clinical isolates, and each clinical isolate was aligned against respective subtype reference, GT-1a: 1a H77-AF009606 or GT-1b: 1b-Con-1-AJ238799, to identify differences between patient and reference amino acid sequences. Genotypic analysis revealed intrinsic genetic diversity among the clinical isolates, but did not identify any known resistance mutations that have been reported to be associated with resistance to any of the three classes of compounds tested in 52 of 55 isolates including all 41 GT-1a isolates (Table 3). In contrast, 1 of 14 GT-1b isolates harboured S556S/G as a single mutant/wild-type mixture; S556S/G is known to confer resistance to the benzothiadiazines (Table 3). This isolate had EC50 values of 546.5, 5.42 and 144.6 nM for the thiophene carboxylic acid, benzofuran and benzothiadiazine, respectively (Table 4). The EC50 value against the benzothiadiazine was the third highest among the 11 GT-1b isolates, but was within the natural variation. In addition, 2 of 14 (14%) GT-1b isolates contained both C316N and S556G mutants, known to confer low-level of resistance to the benzofuran and/or benzothiadiazine classes (Table 3). The drug susceptibility data were available from only one of these two isolates because the chimeric replicons from the second replicated poorly. The EC50 values were 9.14 nM for the benzofuran, the highest among the 12 GT-1b isolates tested, and 112.27 nM for the benzothiadiazine, comparable to other GT-1b isolates (Table 4).

Linear Regression Analysis of Correlation

To provide more information regarding susceptibilities of baseline clinical isolates to HCV nonnucleoside polymerase inhibitors, the susceptibilities of the baseline clinical isolates were compared pairwise among the three NNIs. Linear regression analysis was performed with the mean EC50 values of each clinical isolate obtained in the transient phenotypic assays (Fig. 4). The correlations between the susceptibilities of the clinical isolates to any of the three NNIs tested were extremely poor as the coefficients of determination (r2) ranged between 0.0001 and 0.043. The P-values from the Pearson correlation of each analysis are much greater than the preset threshold value alpha of 0.05 (≥0.17). These data indicate there is no significant correlation among the susceptibilities to the HCV inhibitors tested in this study.

754991-fig4

Figure 4. Correlation between different HCV inhibitors. The x-axis and y-axis represent mean EC50 values from the corresponding NS5B chimeric clinical isolates. The line (–) represents the best unconstrained regression through the points. Sample size (n), the coefficient of determination (r2) and the P-value of each correlation plot are indicated.

Discussion

In this study, the sensitivity of up to 51 clinical baseline isolates from untreated HCV GT-1-infected patients to a panel of nonnucleoside HCV polymerase inhibitors was determined to investigate the effect of HCV genetic diversity on the inhibitor's antiviral potency. Some natural variation in susceptibility among these isolates was observed with all three different classes of HCV polymerase inhibitors tested (benzofuran, benzothiadiazine and thiophene carboxylic acid). The degree of the variation in EC50 was slightly different among these three drug classes of nonnucleoside HCV polymerase inhibitors. The EC50 values in the 95th percentile were 4.5-, 6.0- and 7.5-fold higher than the 5th percentile EC50 values for the thiophene carboxylic acid, benzofuran and benzothiadiazine, respectively. Interestingly, no significant difference in sensitivity between GT-1a and GT-1b was observed for the tested benzofuran and thiophene carboxylic acid. In contrast, the benzothiadiazine was significantly more potent against GT-1b than GT-1a. The differential potency against GT-1a vs GT-1b for only the benzothiadiazine, and the high assay reproducibility for the reference standard suggests that the natural variation in drug susceptibility observed among baseline clinical isolates is not caused by assay variation. In addition, the results in the study are consistent with the findings of previous reports that described variable activity for HCV nonnucleoside polymerase inhibitors among clinical baseline isolates using similar phenotypic analysis assays.[38,39]

Given the observation of the natural variation in drug susceptibility to these three classes of nonnucleoside inhibitors, it is possible that this natural variation may cause variable antiviral response among different patients. For example, one dose may be optimal for patients containing more sensitive variants, but this same dose may be suboptimal for patients who have less susceptible variants. Indeed, variable response to some of the HCV nonnucleoside polymerase inhibitors among different patients has been observed in clinical studies, especially in lower-dose groups. Furthermore, consistent with the in vitro findings of less potent activity against GT-1a than GT-1b for the benzothiadiazines in this study, the response in HCV GT-1a-infected patients was poorer than the GT-1b-infected patients during monotherapy with ABT-333 and ANA-598 (both benzothiadiazines).[9,10] In addition to the susceptibility of baseline isolates, the pharmacokinetics of the drug also influences response. The natural variation in drug susceptibility coupled with pharmacokinetic information could be used to predict the response to drug treatment and provide guidance on the drug concentration needed for achieving a maximal response. If sufficiently high levels of drug exposure relative to the cluster of EC50 values is achieved, the natural variation in drug susceptibility would be of less concern. Finally, the natural variation in baseline susceptibility could provide a threshold for defining abnormal reductions in drug susceptibility and serve as an indicator for an increased probability of drug resistance.

Sequence analysis of NS5B revealed C316N and S556G double mutants in 2 of the 14 GT-1b isolates. Phenotypic analysis of the one isolate with sufficient replication capacity to test drug susceptibility had the highest EC50 against the benzofuran. This finding is consistent with previous reports, demonstrating that C316N confers a low-level of resistance to the benzofuran.[26] However, the benzofuran susceptibility of this isolate was well within the natural variation of the GT-1a isolates and it had wild-type susceptibility to the benzothiadiazine tested in this study. Similarly, no significant change in EC50 to the benzothiadiazine was seen in the isolate containing S556S/G. Previous studies showed that both C316N and S556G were associated with reduced susceptibility to other compounds of the benzothiadiazine class.[23,24,26] The discrepancy may be due to the fact that the benzothiadiazine compound tested in this study has subtle differences in chemical structure with the benzothiadiazine compounds in previous studies resulting in a slightly different interaction with the NS5B polymerase.

In contrast to the above three GT-1b isolates, NS5B sequence analysis did not identify any of the mutations that are known to confer resistance to the tested compounds in 52 of 55 patient samples (41 GT-1a and 11 of 14 GT-1b). In addition, no clear pattern of the NS5B sequence was revealed in baseline clinical isolates with higher EC50 values (data not shown). Thus, the natural variation in drug susceptibility observed in this study may be caused by the intrinsic genetic diversity of HCV.

The mean EC50 values derived from each baseline clinical isolate against the benzofuran, benzothiadiazine and thiophene carboxylic acid were compared between drugs using the statistical regression analysis of correlation. Overall, the correlation was poor between any of the three compounds. These results suggest that the HCV variants which are less susceptible to one of these three classes of nonnucleoside polymerase inhibitors may not be less sensitive to the other two different classes of nonnucleoside polymerase inhibitors. Therefore, a combination of two or three different classes of nonnucleoside polymerase inhibitors may be advantageous for maximal antiviral response and reducing the selection of resistance. However, other aspects including synergistic/antagonistic inhibitory effects, drug–drug interactions and overlapping toxicity profiles should be taken into consideration for potential combination therapy strategies. The lack of correlation between two different classes of nonnucleotide polymerase inhibitors agrees with the fact that these three nonnucleoside inhibitors target different sites of the HCV polymerase and also exhibit different resistance profiles.

In summary, the activity of three classes of nonnucleoside polymerase inhibitors (benzofuran, benzothiadiazine and thiophene carboxylic acid) against a panel of baseline clinical isolates was evaluated using a replicon-based transient replication assay. Some variation in drug susceptibility was observed for all three classes of nonnucleoside polymerase inhibitors. However, there was no correlation between susceptibilities from one compound to another among those tested. Our findings suggest that the existing natural variation in baseline susceptibility should be taken into account for the optimal dose selection for the development of future nonnucleoside polymerase inhibitors. The lack of correlation between drug susceptibilities supports the combination of these different classes of HCV nonnucleotide polymerase inhibitors in the clinic.

References

  1. Seeff LB. Natural history of chronic hepatitis C. Hepatology 2002; 36(5 Suppl. 1): 35–S46.
  2. Purcell RH. Hepatitis C virus: historical perspective and current concepts. FEMS Microbiol Rev 1994; 14(3): 181–191.
  3. Fried MW, Shiffman ML, Reddy KR et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Eng J Med 2002; 347(13): 975–982.
  4. Fried MW. Side effects of therapy of hepatitis C and their management. Hepatology 2002; 36(Suppl. 1): S237–S244.
  5. Foster GR. Past, present, and future hepatitis C treatments. Semin LiverDis 2004; 24(Suppl. 2): 97–104.
  6. Foster GR. Review article: pegylated interferons: chemical and clinical differences. Aliment Pharmacol Ther 2004; 20(8): 825–830.
  7. Kwong AD, McNair L, Jacobson I, George S. Recent progress in the development of selected hepatitis C virus NS3.4A protease and NS5B polymerase inhibitors. Current OpinPharmacol 2008; 8(5): 522–531.
  8. Kieffer TL, Sarrazin C, Miller JS et al. Telaprevir and pegylated interferonalpha-2a inhibit wild-type and resistant genotype 1 hepatitis C virus replication in patients. Hepatology 2007; 46(3): 631–639.
  9. Lawitz E, Rodriguez-Torres M, Cohen D, et al. Dose-dependent decrease in HCV viral load following two-day monotherapy with ABT-333 in treatment-naive, HCV genotype 1-infected subjects. 4th International Workshop on Hepatitis C resistance & new compounds, June, 25–26th, Boston, USA 2009; Abstract# 18.
  10. Lawitz E, Rodriguez-Torres M, DeMicco M et al. Antiviral activity of ANA598, a potent non-nucleoside polymerase inhibitors, in chronic Hepatitis C patients, 44th Annual Meeting of the European Association for the Study of the Liver (EASL) in Copenhagen, Denmark, Absract# 1055. J Hepatol 2009; 50: S384.
  11. Sarrazin C, Rouzier R, Wagner F et al. SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders. Gastroenterology 2007; 132(4): 1270–1278.
  12. Klein CE, Cohen D, Menon R et al. Safety, tolerability and antiviral activity of the HCV polymerase inhibitor ABT-072 following single and multiple dosing in healthy adult volunteers and two days of dosing in treatment-naive HCV genotype-1-infected subjects. Dec 6–10, Kohala Coast, Hawaii, Abstract # 56. HepDART 2009; 5(Suppl. 1): 52–53.
  13. Murakami E, Bao H, Ramesh M et al. Mechanism of activation of beta-D-2¢-deoxy-2¢-fluoro-2¢-c-methylcytidine and inhibition of hepatitis C virus NS5B RNA polymerase. AntimicrobAgents Chemother 2007; 51(2): 503–509.
  14. Toniutto P, Fabris C, Bitetto D, Fumolo E, Fornasiere E, Pirisi M. R-1626, a specific oral NS5B polymerase inhibitor of hepatitis C virus. IDrugs 2008; 11(10): 738–749.
  15. Brown NA. Progress towards improving antiviral therapy for hepatitis C with hepatitis C virus polymerase inhibitors. Part I: nucleoside analogues. Expert Opin Investig Drugs 2009; 18(6): 709–725.
  16. Ali S, Leveque V, Le Pogam S et al. Selected replicon variants with low-level in vitro resistance to the hepatitis C virus NS5B polymerase inhibitor PSI-6130 lack cross-resistance with R1479. Antimicrob AgentsChemother 2008; 52(12): 4356–4369.
  17. O_Brien C, Godofsky E, Rodriguez-Torres M, et al. Randomized trial of Valopicitabine (NM283), alone or with Peg-Interferon, vs. retreatment with Peg-Interferon plus Ribavirin (PEGIFN/RBV) in hepatitis C patients with previous non-response to PEGIFN/RBV: first interim results. 56th Annual Meeting of AASLD Hepatology 42 (S1), 234A (Abstract 96). 2005.
  18. Afdhal N, Godofsky E, Dienstag J, et al. Final phase I/II trial results for NM283, a new polymerase inhibitor for hepatitis C: antiviral efficacy and tolerance in patients with HCV-1 infection, including previous interferon failures. 55th Annual Meeting of AASLD Hepatology 40 (S4), 726A (Abstract LB03). 2004.
  19. Love RA, Parge HE, Yu X et al. Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme. J Virol 2003; 77(13): 7575–7581.
  20. Beaulieu PL. Non-nucleoside inhibitors of the HCV NS5B polymerase: progress in the discovery and development of novel agents for the treatment of HCV infections. CurrOpin Investig Drugs 2007; 8(8): 614–634.
  21. Sarisky RT. Non-nucleoside inhibitors of the HCV polymerase. J AntimicrobChemother 2004; 54(1): 14–16.
  22. Wagner R, Maring C, Flentge P, et al. Preclinical characterization of ABT-333 and ABT-072: novel nonnucleoside HCV NS5B polymerase inhibitors. Dec 6–10, Kohala Coast, Hawaii, Abstract # 108. Hep DART 2009; 5(Suppl. 1):100–101.
  23. Lu L, Dekhtyar T, Masse S et al. Identification and characterization of mutations conferring resistance to an HCV RNA-dependent RNA polymerase inhibitor in vitro. AntiviralRes 2007; 76(1): 93–97.
  24. Mo H, Lu L, Pilot-Matias T et al. Mutations conferring resistance to a hepatitis C virus (HCV) RNA-dependent RNA polymerase inhibitor alone or in combination with an HCV serine protease inhibitor in vitro. AntimicrobAgents Chemother 2005; 49(10): 4305–4314.
  25. Showalter RE, Thompson PA, Steffy KR, Appleman JR. ANA598 displays potent in vitro antiviral activity against diverse clinical isolates of genotype 1 HCV in a transient replicon shuttle vector system. American Association for the Study of Liver Diseases (AASLD), Boston, Oct 30–Nov. 3, Abstract# 1586. Hepatology 2009; 50(Suppl. 4): 1037A.
  26. Kneteman NM, Howe AY, Gao T et al. HCV796: a selective nonstructural protein 5B polymerase inhibitor with potent anti-hepatitis C virus activity in vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus. Hepatology 2009; 49(3): 745–752.
  27. Flint M, Mullen S, Deatly AM et al. Selection and characterization of hepatitis C virus replicons dually resistant to the polymerase and protease inhibitors HCV-796 and boceprevir (SCH 503034). AntimicrobAgents Chemother 2009; 53(2): 401–411.
  28. Kukolj G, McGibbon GA, McKercher G et al. Binding site characterization and resistance to a class of nonnucleoside inhibitors of the hepatitis C virus NS5B polymerase. J Biol Chem 2005; 280(47): 39260–39267.
  29. Cooper C, Lawitz EJ, Ghali P et al. Evaluation of VCH-759 monotherapy in hepatitis C infection. J Hepatol 2009; 51(1): 39–46.
  30. Shi ST, Herlihy KJ, Graham JP et al. In vitro resistance study of AG-021541, a novel onnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase. Antimicrob Agents Chemother 2008; 52(2): 675–683.
  31. Shi ST, Herlihy KJ, Graham JP et al. Preclinical characterization of PF-00868554, a potent nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase. Antimicrob Agents Chemother 2009; 53(6): 2544–2552.
  32. Herring BL, Tsui R, Peddada L, Busch M, Delwart EL. Wide range of quasispecies diversity during primary hepatitis C virus infection. J Virol 2005; 79(7): 4340–4346.
  33. Zhang YY, Lok AS, Chan DT, Widell A. Greater diversity of hepatitis C virus genotypes found in Hong Kong than in mainland China. J ClinMicrobiol 1995; 33(11): 2931–2934.
  34. Simmonds P. Viral heterogeneity of the hepatitis C virus. J Hepatol 1999; 31(Suppl. 1): 54–60.
  35. Simmonds P. The origin and evolution of hepatitis viruses in humans. J Gen Virol 2001; 82(Pt 4): 693–712.
  36. Friebe P, Lohmann V, Krieger N, Bartenschlager R. Sequences in the 5¢ nontranslated region of hepatitis C virus required for RNA replication. J Virol 2001; 75(24): 12047–12057.
  37. Lohmann V, Hoffmann S, Herian U, Penin F, Bartenschlager R. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol 2003; 77(5): 3007–3019.
  38. Middleton T, He Y, Pilot-Matias T et al. A replicon-based shuttle vector system for assessing the phenotype of HCV NS5B polymerase genes isolated from patient populations. J VirolMethods 2007; 145(2): 137–145.
  39. Le Pogam S, Seshaadri A, Kosaka A et al. Existence of hepatitis C virus NS5B variants naturally resistant to non-nucleoside, but not to nucleoside, polymerase inhibitors among untreated patients. J Antimicrob Chemother 2008; 61(6): 1205–1216.

Source

Cirrhosis Tied to Increased Risk of Liver Cancer in Diabetics

By David Douglas

NEW YORK (Reuters Health) Dec 26 - Cirrhosis and hepatitis are associated with the occurrence of hepatocellular carcinoma (HCC) in patients with diabetes, and hepatitis C is of particular importance, Taiwanese researchers report in a November 15 online paper in The American Journal of Gastroenterology.

As Dr. Shih-Wei Lai told Reuters Health by email, "In our study, diabetic patients comorbid with liver cirrhosis, hepatitis B, and hepatitis C were at significantly increased risk of developing hepatocellular carcinoma, and the risk associated with hepatitis C was stronger than that with hepatitis B."

Dr. Lai of China Medical University, Taichung and colleagues note that there is accumulating evidence that patients with diabetes mellitus are more prone to cancer in general and liver cancer in particular. An American study indicated that the risk of HCC in diabetic patients was more than twice that in non-diabetics, and there have been some similar findings in Taiwan.

To clarify the effect of diabetes on HCC risk, the team examined a health insurance database covering the years 2000 to 2005 and used the information to compare 19,349 newly diagnosed diabetes patients with 77,396 matched controls. Where possible, they were followed until the end of 2008.

The HCC incidence was doubled in diabetics compared with controls (21.0 versus 10.4 per 10,000 person-years), which translated to an adjusted hazard ratio of 1.73, the team found. Hazard ratios were also significantly and independently increased by being male (2.32), and having cirrhosis (8.65), hepatitis B (2.52), and hepatitis C (5.61).

Stratified analyses showed that subjects with diabetes and cirrhosis along with hepatitis C had the greatest elevation in risk (hazard ratio, 72.4).

The researchers then went on to examine the association between HCC and anti-diabetic medication. After adjustment, metformin use was associated with significant protection (hazard ratio, 0.49). This was also true of thiazolidinediones (hazard ratio, 0.56). Taking insulin, sulfonylureas, and other agents also reduced the risk, but not significantly.

Overall, given the influence of the studied comorbidities, concluded Dr. Lai, "These observations suggest that patients with these disorders may be the high-risk group that deserves to be closely monitored. The importance of hepatitis C should not be overlooked."

SOURCE: http://bit.ly/vHt1Dv

Am J Gastroenterol 2011.

Source

Authors: Itoh, Yoshito1; Nishimura, Takeshi1; Yamaguchi, Kanji1; Yokomizo, Chihiro1; Fujii, Hideki1; Minami, Masahito1; Nagao, Yasuyuki2; Sumida, Yoshio3; Hashimoto, Hiroaki4; Umemura, Atsushi4; Shima, Toshihide4; Okanoue, Takeshi4; Yoshikawa, Toshikazu1

Source: Hepatology Research, Volume 41, Number 12, 1 December 2011 , pp. 1145-1152(8)

Publisher: Wiley-Blackwell

Abstract:

Aim: Hepatic steatosis is one of the factors limiting the virological response to interferon-based antiviral therapy for chronic hepatitis C (CH-C) patients infected with genotype 1, while contradictory results have been reported for genotype 2. We aimed to clarify the effect of hepatic steatosis on therapeutic outcome and cumulative positivity of serum HCV RNA in CH-C patients infected with genotype 2 treated by peginterferon (PEG-IFN)α2b and ribavirin (RBV) combination therapy.

Methods: A total of 74 treatment-naïve non-cirrhotic CH-C patients infected with genotype 2 who received PEG-IFNα2b and RBV according to the standard regimen were divided into hepatic steatosis 0-10% and >10% groups. The clinical backgrounds, sustained virological response (SVR) rates and cumulative positivity of serum HCV RNA were compared between the two groups.

Results: Among the 74 patients, 61 (82.4%) had hepatic steatosis 0-10% and 13 (17.6%) had hepatic steatosis >10%. Scores of homeostasis model assessment-insulin resistance and hepatic fibrosis were higher in patients with hepatic steatosis >10% than hepatic steatosis 0-10% (P = 0.040 and 0.042, respectively). Non-SVR was more frequent in patients with hepatic steatosis >10% than hepatic steatosis 0-10% (P = 0.003). Cumulative positivity of serum HCV RNA was significantly higher in patients with hepatic steatosis >10% than hepatic steatosis 0-10% (P = 0.004).

Conclusions: In CH-C patients infected with genotype 2 treated by PEG-IFNα2b and RBV combination therapy, hepatic steatosis >10% was associated with increased insulin resistance, advanced hepatic fibrosis and higher cumulative positivity of serum HCV RNA, which lead to a higher risk of non-SVR.

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1872-034X.2011.00886.x

Affiliations:1: Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto2: Department of Gastroenterology, Matsushita Memorial Hospital3: Department of Gastroenterology and Hepatology Nara City Hospital, Nara, Japan4: Hepatology Center, Saiseikai Suita Hospital, Osaka

Publication date: 2011-12-01

Source

Authors: Tamori, Akihiro1; Kioka, Kiyohide2; Kurai, Osamu3; Sakaguchi, Hiroki4; Enomoto, Masaru1; Fujii, Hideki1; Kobayashi, Sawako1; Iwai, Shuji1; Morikawa, Hiroyasu1; Yamaguchi, Seiko3; Kawasaki, Yasuko2; Oka, Hiroko3; Tanaka, Yasuhito5; Kawada, Norifumi1

Source: Hepatology Research, Volume 41, Number 12, 1 December 2011 , pp. 1169-1177(9)

Publisher: Wiley-Blackwell

Abstract:

Aim: Effect of re-treatment for pegylated interferon (PEG-IFN) plus ribavirin was not fully evaluated. We examined the effects of re-treatment with PEG-IFN plus ribavirin in patients with high viral loads of genotype 1 hepatitis C virus who failed to achieve a sustained virological response (SVR) with combination therapy.

Methods: We examined 38 patients who were re-treated with PEG-IFN α2a plus ribavirin for more than 60 weeks, among whom 14 were non-responders and 24 were relapsers after previous treatment with PEG-IFN α2b plus ribavirin. IL28B genotyping was done in 21 patients.

Results: The overall SVR rate was 34%. Analysis of baseline characteristics showed that the relapsers had a significantly higher SVR rate than the non-responders (50.0%, 12/24 vs. 7.1%, 1/14, respectively, P = 0.012) The SVR rates of re-treated patients who had turned hepatitis C virus (HCV) RNA-negative at weeks 8, 12, 24, and 48 of the previous therapy were 67% (4/6), 67% (4/6), 29% (2/7), and 25% (1/4), respectively. Re-treatment achieved an SVR in five of 12 patients with IL28B major alleles and three of nine patients with IL28B minor alleles. During the re-treatment, patients with complete viral suppression at week-12 achieved a significantly higher SVR rate (P = 0.001).

Conclusions: Re-treatment with PEG-IFN α2a plus ribavirin therapy is effective in patients who relapse after a course of PEG-IFN α2b plus ribavirin therapy. Re-treatment is a particularly useful option for patients who achieve early viral clearance during previous therapy.

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1872-034X.2011.00887.x

Affiliations:1: Department of Hepatology, Osaka City University Graduate School of Medicine2: Department of Hepatology Osaka City General Hospital3: Department of Gastroenterology and Hepatology, Osaka City Juso Hospital, Osaka4: Internal Medicine, Izumi Municipal Hospital, Izumi5: Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Publication date: 2011-12-01

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Authors: Tamai, Hideyuki; Shingaki, Naoki; Shiraki, Tatsuya; Tukuda, Hiroshi; Mori, Yoshiyuki; Moribata, Kosaku; Enomoto, Shotaro; Deguchi, Hisanobu; Ueda, Kazuki; Inoue, Izumi; Maekita, Takao; Iguchi, Mikitaka; Yanaoka, Kimihiko; Oka, Masashi; Ichinose, Masao

Source: Hepatology Research, Volume 41, Number 12, 1 December 2011 , pp. 1137-1144(8)

Publisher: Wiley-Blackwell

Abstract:

Aim: Continuation of pegylated interferon (PEG-IFN) plus ribavirin at the recommended dose is difficult in elderly patients and/or patients with cytopenia or complications. Whether the therapeutic efficacy of low-dose PEG-IFN plus ribavirin therapy could be predicted based on virological response within 2 weeks of therapy initiation was evaluated.

Methods: A total of 106 patients with a high viral load of genotype-1b hepatitis C virus (HCV) underwent low-dose PEG-IFN plus ribavirin therapy. PEG-IFN alpha 2b (0.75 µg/kg per week) and ribavirin (600-800 mg/day) were administered for 48 weeks.

Results: Sustained virological response (SVR) was achieved in 37%, and treatment was discontinued in 9%. On univariate analysis of SVR-contributing factors, significant differences were noted in the white blood cell count, platelet count, fibrosis markers, and viral reduction within 2 weeks from therapy initiation. On multivariate analysis, the platelet count and the reduction in the HCV core antigen level at week 2 were independent factors. The positive predictive value (PPV) and the negative predictive value (NPV) for SVR based on a 1-log or greater HCV-RNA level reduction at week 2 were 65% and 90%, respectively, and those based on HCV core antigen level at week 2 were 64% and 97%, respectively. PPV and NPV based on a 2-log or greater reduction of the RNA level were 86% and 67%, respectively, and those based on the core antigen level were 93% and 69%, respectively.

Conclusion: Evaluation of viral reduction at week 2 after therapy initiation is useful for predicting SVR to low-dose PEG-IFN plus ribavirin therapy.

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1872-034X.2011.00879.x

Affiliations:1: Second Department of Internal Medicine, Wakayama Medical University, 811-1 Kimiidera, Wakayama city, Wakayama 641-0012, Japan

Publication date: 2011-12-01

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Am J Gastroenterol. 2011 Dec;106(12):2121-2. doi: 10.1038/ajg.2011.343

Lai M, Afdhal NH.

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Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

Abstract

The limitations of and the invasive nature of liver biopsy has spurred extensive interest in the development of non-invasive tests to measure liver fibrosis in patients with chronic hepatitis C. Clinically applicable non-invasive tests, including radiological studies, elastography, and serum markers, all of which perform extremely well in excluding significant disease and diagnosing cirrhosis. FibroScan and acoustic radiation force impulse elastography are two elastography-based tests that show promise. In this new era of increased cure rates with newly Food and Drug Administration-approved drugs and the availability of multiple non-invasive tests of liver fibrosis, we anticipate a decreasing need for liver biopsies in the management of chronic hepatitis C.Am J Gastroenterol 2011; 106:2121-2122; doi:10.1038/ajg.2011.343.

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Gastroenterology. 2011 Nov 30. [Epub ahead of print]

Fletcher NF, Wilson GK, Murray J, Hu K, Lewis A, Reynolds GM, Stamataki Z, Meredith LW, Rowe IA, Luo G, Lopez-Ramirez MA, Baumert TF, Weksler B, Couraud PO, Kim KS, Romero IA, Jopling C, Morgello S, Balfe P, McKeating JA.

Source

Hepatitis C Research Group, Institute for Biomedical Research, University of Birmingham, Birmingham, UK.

Abstract
BACKGROUND & AIMS:

Hepatitis C Virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS.

METHODS:

We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells using quantitative PCR and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication.

RESULTS:

Using quantitative PCR we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor-BI, and Claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis.

CONCLUSIONS:

Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Drug Trials Not Representative, Researchers Charge

By Emily P. Walker, Washington Correspondent, MedPage Today
Published: December 27, 2011

Few major randomized, controlled clinical trials examine the effects of a drug in patients who have multiple chronic conditions, even though more than one-quarter of all Americans are living with at least two chronic health conditions, researchers reported.

The proportion is even greater for older individuals, two out of three of whom are likely to have at least two chronic health conditions, according to Alejandro Jadad, MD, and colleagues from the Centre for Health, Wellness and Cancer Survivorship at the University Health Network in Toronto.

That means that most trials on which the FDA bases its approval of new drugs are not generalizable to the U.S. population, they wrote in a research letter published in the Dec. 28 issue of the Journal of the American Medical Association.

Jadad and his colleagues examined all randomized controlled studies that dealt with an intervention for a long-lasting or chronic disease or condition that were published from January-March in 1995, 2000, 2005, and 2010 in five major peer-reviewed medical journals -- BMJ, CMAJ, JAMA, The Lancet, and the New England Journal of Medicine -- as well as six journals that focus on the most prevalent chronic conditions, including Circulation and Annals of General Psychiatry.

Only six of the 284 published trials analyzed (2.1%) explicitly included patients with multiple chronic conditions, and that percentage didn't change much from 1995 to 2010. In 179 of the randomized controlled trials, patients with multiple medical conditions were explicitly excluded from the trial.

Patients with multiple conditions were mentioned often in trial reports, although not actually included in the trial. About 70% of the published trial reports mentioned multiple coexisting diseases; with general medical journals describing them more often than specialized journals (72% versus 69%; P=0.02).

The letter authors concluded that few randomized controlled clinical trials published in the last 15 years have included patients with multiple chronic conditions.

Although the study was small, the authors said the finding "invites reflection about the risk of unintended harm from inappropriate generalization of trial results conducted in populations with a single disease."

"Given the possible drug-to-drug, drug-to-disease, and disease-to-disease interactions that remain unexamined, most of the evidence gathered to date by [randomized controlled clinical trials] is of limited value to guide decisions," they wrote.

The study authors said it may be useful for the FDA to have drug companies include subgroups of patients with the most common combinations of diseases in their drug development process; to observe safety outcomes of adding a new drug to patients who are already medicated for other conditions; and to have post-marketing studies that include the risk stratification to allow for meta-analyses across populations, such as those with multiple conditions.

The study was supported by funds from the Canada Research Chair in eHealth Innovation, the University of Toronto, and the University Health Network.

One of the letter writers, Alejandro Jadad, reported being a consultant to Foresight Links Corporation and that he receives royalties from Wiley for a book on randomized controlled trials.

None of others reported having any financial disclosures.

Primary source: Journal of the American Medical Association
Source reference:
Jahad A, et al "Consideration of multiple chronic diseases in randomized controlled trial" JAMA 2011; 306(24): 2670-2674.

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Researchers strike gold with nanoparticle virus test

Nehal Lasheen 27 December 2011 | EN

[CAIRO] An Egyptian research team has won a prize for developing a hepatitis C test using gold nanoparticles. The test could become a cheap and rapid way to screen people and blood banks for the virus.

Hassan Azzazy, chair of the chemistry department at The American University in Cairo (AUC), and his team, won third place at the Intel Global Challenge held at the University of California, Berkeley, in the United States, last month (7–10 November) winning US$10,000.

"Nanoparticles are promising tools for developing the next generation of diagnostic assays because of their unique properties," Azzazy told SciDev.Net. He said gold nanoparticles exhibit a phenomenon known as surface plasmon resonance, which causes them to change colour from an intense red to blue when they aggregate. The test works by adding a target agent that binds to viral genetic material, forcing the nanoparticles to aggregate and turning the solution blue.

Azzazy said that "conventional detection of active hepatitis C generally requires two tests that take from three to four days and cost about US$78 per sample, while the nanogold test can generate similar results in less than one hour and for one-tenth of the cost. One gram of gold tetrachloride costs US$260 and is enough for 10,000 samples."

According to the WHO, hepatitis C infects 200 million people worldwide, but is especially prevalent in the Middle East and North Africa (MENA) with about 9.2 million individuals infected. For most MENA countries, the prevalence is between one and two per cent; the exception is Egypt where estimated prevalence is 14 per cent for adults aged 15–60.

"Early identification and treatment of infected patients is critical to reducing transmission of the disease, and a fast and relatively cheap new assay will be of great help," said Hala Gabr, professor of haematology at Cairo University.

Amr Abul-Fotouh, lecturer in tropical medicine and hepatology at Cairo University, added: "Although the nanogold test is a good one, it is not quantitative so it cannot be used to treat and follow up patients, as this needs the quantity of the virus to be detected by a PCR [polymerase chain reaction] test".

He pointed out that the new assay could be used in blood banks to check for hepatitis C. "We have seen that about 20 per cent of Egyptian blood donors test positive for the virus," he said.

Sherif Shawky, a doctoral fellow at AUC, and a member of the research team, told SciDev.Net that the technology can be adapted for detecting other infectious agents such as tuberculosis, and for use as cancer biomarkers. A spin-off company, NanoDiagX, has been set up to develop these bio-nanotechnology platforms.

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