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The Real Spin on Global Flu

By Carren Bersch, Editor, and Kateri Clemons, Admin. Asst.

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In an effort to educate MLO readers about the "start-to-finish" activities of an organization engaged in the "flu business," we asked Gregory R. Chiklis, PhD, vice president, Research and Development, ZeptoMetrix Corp. (ZMC) to address the biosafety laboratory and seroconversion panels, as well as the exact nature of the work of a company like ZeptoMetrix and how that work plays a role in finding solutions to influenza threats. Here are his responses:

Q In one of your company newsletters, a section features ZMC’s Biosafety Level 3-plus (BSL3+) laboratories. Can you reveal more of what it would take for a laboratorian wanting to move into your segment of the marketplace to qualify to be one of the people who could actually work in the BSL3+ lab? When this material was published two years ago, the article mentioned changes in this BSL-3+ lab. Have those changes been achieved, and is the lab now handling the viruses and bacteria mentioned in the article (e.g., lethal primate viruses, avian flu, and mumps/measles, and anthrax and pertussis)?

A Our biological containment laboratories were upgraded last year to BSL3+ enhanced status to support both our work with highly pathogenic organisms, including avian flu, and a contract awarded to the company to develop several testing panels of flu and related organisms for the Centers for Disease Control and Prevention (CDC). This upgrade included the addition of shower-in/shower-out stations and incubators for propagating flu viruses in eggs, as well as real-time polymerase chain reaction (PCR) equipment used to follow the growth of the viruses in culture.

To support these upgrades, we also required additional training for our staff; a new set of emergency-response plans; employee security screening; an upgrade to our select-agent license; and the development of a disaster-recovery plan. This was no small task and took about six months to get in place.

The time between exposure to SARS-CoV and the onset of symptoms is called the “incubation period.” The incubation period for SARS is typically two to seven days, although in some cases, it may be as long as 10 days. In a very small proportion of cases, incubation periods of up to 14 days have been reported. —www.cdc.gov/ncidod/sars/faq.htm.

In order to work in our BSL3+ laboratories, an employee must be at least 18 years old, have earned a bachelor’s degree in a science-related field, and have no criminal record. All laboratory personnel also go through a Security Risk Assessment conducted by the FBI, which includes fingerprinting and a complete background check. Additional experience in the areas of cell culture, molecular biology, and Good Manufacturing Practices, or GMP, are also a plus. Regardless of our scientific backgrounds, we enter all new employees into a six-month training program to work in this laboratory. Once this training is complete, there are monthly training and drills that must also be accomplished to maintain the employee status for working in the laboratory.

With our CDC work in this area concluded, we are now using these upgraded facilities to support influenza and respiratory-virus vaccine and diagnostic-test development through the supply of various organisms, testing services, and finished products. Our main work involves the manufacture of multiple strains of flu virus and related respiratory organisms. These are used to check the sensitivity and specificity of tests being developed to detect these pathogens. In addition to this work, we are also providing kit manufacturers with calibrators and controls for both their antigen- and molecular-based diagnostic tests. These controls are formulated into refrigerator-stable non-infectious materials that allow the technician to validate testing while remaining safe when working with these highly pathogenic organisms.

Another area supported by this laboratory is the proficiency-testing industry through its bulk manufacturing of these flu strains into testing panels. Using the same inactivation technologies described above, we have manufactured and direct shipped more then 4,000 non-infectious influenza-based panels worldwide to monitor the performance of clinical-testing laboratories.

Q In Spring 2006, the company was collaborating with the CDC regarding HIV seroconversion panels. The explanation of this process is relatively simple; however, 1) is ZMC still involved in this collaboration; 2) has ZMC launched the hepatitis B and C virus panels mentioned in this article; and 3) has there been a change in the process by which donors are eliminated, thus making seroconversion panels difficult to construct?

A Diagnostic-test developers are always trying to reduce the time between infection and detection, and seroconversion panels are the perfect tool to accomplish this. A seroconversion panel is a longitudinal series of donor-plasma samples that spans the infection process. In the early samples, the donor’s blood tests remain normal. Then, as the infection progresses, samples begin to exhibit the various diagnostic markers related to that particular pathogen, and the immune system’s response against it. Final sample bleeds will contain disease-specific antibodies, completing the seroconversion process. These longitudinally collected samples represent one of the last ways to examine discreet snapshots in time of the immune system’s response to infection. Our company and our reference-laboratory partners run these panels on every available test kit on the market to compare their detection of the various molecular-, antigen-, and antibody-based tests in these early-stage infections. These panels are sold to the diagnostic-testing industry, vaccine manufacturers, and regulatory authorities worldwide.

A year ago in the fall, the CDC sponsored a workshop to establish new testing guidelines to confirm if a person is infected with HIV-1. While this was first established 20 years ago, there are now many new technologies on the market that are more sensitive then the older first-generation assays. The only way to get an accurate assessment of the capabilities of current tests was to run and compare them using seroconversion panels. For this program, we donated about 60 of our HIV panels to the CDC for evaluation of all the new HIV-testing formats. The data analysis is still in process for this project, and we hope to see some new guidelines for qualifying a donor’s HIV status by the end of 2009.

Since blood donations are now screened by molecular-testing technologies that can measure the RNA or DNA of the infecting virus, blood samples that seroconvert completely to Ab positive are no longer found in the plasma-collection centers. This is because they are deferred from the collection centers before they complete the seroconversion process. Using these new molecular-testing techniques, not only can we now detect copies of virus in people well before their own immune system recognizes the infection but also remove those virus copies earlier from the blood supply, making the blood safer than it has ever been.

Q In Spring 2007, ZMC was engaged in another contract with CDC to help combat H5N1 avian influenza. While you have said this contract is no longer in force, can you tell us whether or not you are still growing the 151 different viruses including 36 different strains of H5N1? Can you give us a list of all of the viruses that your company currently handles in one way or another?

A You are correct in that the CDC cancelled our contract before we reached the point of growing avian flu. They are now releasing a new contract that is much more encompassing and that will cover all aspects of flu diagnostics including virus growth, diagnostic development, and a "storefront" for researchers to purchase these reagents for their work.

Currently, there are more then 20 companies collaborating with ZMC towards the development of flu and respiratory diagnostic tests. For these projects, we are growing hundreds of strains to support their test development and formulating various controls, calibrators, and validation panels to be used with their test kits. The Food and Drug Administration (FDA) also released a guidance document on how to validate respiratory-based molecular tests, and we are working to have all of the strains the FDA currently requires available for researchers. [See a list of some strains on page 28 that ZMC currently supplies these developers.]

As of January 2006, the Joint United Nations Programme on HIV/AIDS (UNAIDS) and the World Health Organization (WHO) estimated that AIDS has killed more than 25 million people since it was first recognized on Dec. 1, 1981, making it one of the most destructive pandemics in recorded history. — www.cdc.gov/ncidod/sars/faq.htm.

Q In one of your newsletters, bubonic plague is mentioned; could it or other ancient/obscure viruses or bacteria cause a threat somewhere in the world? What do you perceive as the major pandemic threat today and why? Are you able to tell us what bioterrorism agents ZMC handles? In terms of your biodefense activities, is that a larger portion of your business than working with public-health issues?

A I still think the major pandemic we could see in the coming years will come from avian influenza. Unlike past pandemics, however, we are able to track outbreaks globally, which gives us the time to quickly develop diagnostic tests specific for the strain — hopefully, before it spreads too far. By identifying these pandemic strains early, we can intervene and reduce the damage they cause. This is a similar concept to that of the SARS outbreak several years ago. As soon as the virus was identified, researchers at the CDC developed a test while we at ZMC developed safe, inactivated molecular and serology (antibody-based) controls from virus provided to us from the CDC. These controls and proficiency materials were quickly distributed to prepare the labs for testing for this virus. These tests were then run at various locations across the globe, allowing the infected donors to be identified and isolated from the general population until they could be treated and released. While a pandemic-flu virus will be harder to isolate because it is airborne, we could follow this same scheme for getting the tests prepared and validated, should this ever occur. As for our biodefense work, we are not at liberty to identify any of the organisms or how they are being used except to say that they are proficiency-testing materials that a biodefense laboratory would run to make sure they can accurately test and identify a biowarfare agents.

Are we due for another pandemic?

A flu pandemic occurs when a novel but normal genetic mutation exists in a strain of circulating influenza. With the age of rapid international air travel, a pandemic is more of a threat today than it ever was. Three major influenza pandemics occurred during the 20th century: the "Spanish" flu in 1918-1919; the "Asian" flu in 1957-58; and the "Hong Kong" flu in 1968-69.

Spanish flu, the most deadly of the three major pandemics, caused approximately 500,000 deaths in the United States alone, and 40 to 50 million deaths across the globe. This highest mortality was among persons from 25 to 30 years of age. Deaths may have been higher but may not have been documented due to restrictions during World War I. First discovered in the Far East, the Asian flu caused about 70,000 deaths in the United States and 2 million deaths worldwide. With better diagnostics, the Asian flu was identified earlier on and was contained better than the Spanish flu.

The Hong Kong flu resulted in 34,000 deaths in the United States and about 1 million deaths in total. As a result of its similarity to the Asian strain, many people may have developed an immunity. This flu abated when schools were closed, so it did not spread easily, proving the value of containment. By this time, other medical advances also enabled the outbreak to be handled and treated more effectively.

In 1997 and 1999, influenza pandemic "scares" occurred due to novel strains of avian influenza, a virus that moved directly from chickens to people, bypassing an intermediary host, such as a pig. Slaughtering potentially infected fowl resulted in quick containment of any outbreak.

While the CDC report that the spread of avian influenza (H5N1) from person to person has been rare, this epizootic continues to pose an important public-health threat. If the virus eventually is transmitted from person to person, a potential pandemic could develop.

The threat of the first pandemic in the 21st century means that continual progress in research and development is essential. Today, patented technologies can inactivate viruses so they are safe to handle in a normal laboratory environment. The viral genome remains unmodified and intact, better reflecting the normal state of the viral agent.

This is an important aspect for rapid development of new diagnostic tests and therapeutics for pathogenic organisms, as it gives researchers unparalleled access to viral pathogens without the need for specialized high-level containment laboratories. This technology enables the shipment of inactivated influenza proficiency samples to more than 4,000 diagnostic testing facilities to assess their ability to accurately screen and report influenza infections. —ZeptoMetrix’ZeptoGram Spring 2007

Faster, more sensitive flu tests to combat flu pandemic

By Graham Lidgard, PhD

Faster and more sensitive tests for influenza — particularly the avian-flu subtypes H5N1, H7N1, and H9N1 — are being developed for the CDC. These tests (being developed under contract by private industry) are expected to be commercially available in the next one to two years. The CDC, working with health agencies in the United States and other countries, has adopted a three-pronged Advanced Development Program for combating influenza outbreaks — early detection and containment, combined with ongoing vaccination programs and antiviral treatments — explains Daniel B. Jernigan, MD, MPH, deputy director of the CDC Influenza Division. The new diagnostic tests, both point of care (POC) and molecular, will be integral components of this global effort to forestall or significantly reduce the severity of a flu pandemic. The molecular tests could be performed in a hospital or a commercial laboratory, and would expedite the diagnosis of large numbers of patients. The expanded testing capability enhances the hospital laboratory-based pandemic and seasonal-flu diagnostic capacity in the United States.¹

HIV is a retrovirus that can lead to AIDS. Currently, there are an estimated 1,039,000 to 1, 185,000 people in the United States living with HIV/AIDS, with 24% to 27% undiagnosed and unaware of their HIV infection.—http://en.wikipedia.org/wiki/HIV, and www.cdc.gov/hiv

Early detection and intervention is critical because vaccinations usually are not effective when new strains suddenly arise and begin spreading within the population and, so, are not as effective as health officials would like. The efficacy of a flu vaccine depends on how well it is matched to the types of flu viruses circulating that year. Scientists try to predict which strains of flu viruses are most likely to spread and cause illness each year, and put these new strains into the vaccine. The 2008-2009 influenza vaccine, for example, contains three new influenza virus strains: A/Brisbane/59/2007 (H1N1)-like virus; A/Brisbane/10/2007 (H3N2)-like virus; B/Florida/4/2006-like virus.² But those who contract the disease from a different strain are not protected — although the vaccine may make the illness milder. Moreover, it takes months to develop a new vaccine to combat mutated virus strains. By the time a novel virus is identified, a flu pandemic could be unleashed before a targeted vaccine is available.

While rapid diagnostic tests are available to detect influenza viruses, none of the tests provide any information about influenza A subtypes. Additionally, these rapid tests differ in the types of influenza viruses they can detect and whether they can distinguish between influenza types. Some tests can detect only influenza A viruses; or they detect both influenza A and B viruses but do not distinguish between the two types.³ In addition, current molecular flu tests take hours and, in some instances, days to obtain results, depending on the type of test and medical-laboratory facilities available, particularly in remote regions of the world. The time lost can make the difference between quickly stamping out an exposure and seeing its uncontrolled spread.

New subtypes are responsible for pandemics and can result from the mutation of human- and avian-influenza viruses. Antigenic changes within a type or subtype of A and B viruses, known as antigenic drift, are ongoing processes responsible for frequent epidemics and regional outbreaks, and these changes make the annual reformulation of influenza vaccine necessary.⁴ To date, these formulations do not include the avian-flu subtype.

Avian H5N1-virus infections of humans are rare, and most cases have been associated with direct poultry contact during poultry outbreaks. While the H5N1 virus does not now infect people easily, infection in humans is serious when it occurs — more than half of people reported to be infected have died.⁵

A major concern, however, is that the deadly H5N1-flu strain will mutate into a virus capable of human-to-human transmission and lead to an influenza pandemic. There is little pre-existing natural immunity to H5N1-virus infection in the human population. If the H5N1 virus gains the ability for efficient and sustained transmission among humans, an influenza pandemic could result, with potentially high rates of illness and death worldwide.⁶ Without intervention, health officials expect a mutated flu virus to spread internationally within one month and U.S. cases in one to two months, according to Martin Cetron, MD, director of the CDC’s Division of Global Migration and Quarantine.⁶

To improve the odds on early detection and containment, the CDC has funded the development of more sensitive and faster tests that can be used in the field or small labs to speed up the detection process and begin immediate intervention. In late 2006, the CDC awarded $11.4 million to four companies to develop new rapid diagnostic tests that will detect seasonal human-influenza viruses and differentiate influenza A H5N1 from those within 30 minutes.⁷ These tests should begin coming to market next year. Two of the firms terminated their contracts with the CDC; the two remaining companies include Meso Scale Diagnostics LLC, of Gaithersburg, MD, using multi-array biomarker detection, and Nanogen Inc., of San Diego, CA, using a novel POC immunoassay system developed in collaboration with HX Diagnostics.

Existing POC tests can only determine if the patient is infected with influenza viruses A or B but cannot differentiate seasonal subtypes from avian influenza H5N1. To test for H5N1, patient samples must be sent to one of about 100 designated labs nationwide. The process can take four to 24 hours to complete, depending on the shipping of samples.⁸

New POC assays, such as those developed through the CDC grants, will be designed to show good reactivity across seasonal changes in current subtypes. If a loss of detection for a seasonal subtype is seen, it will serve as an indication that a new subtype has entered the human population, which often brings significant human disease with it. This sentinel approach is intended to provide an additional tool to identify significant changes in viral subtypes soon after they emerge. In addition to these contracts, CDC is providing funding for a repository of influenza reagents and other materials to aid with the advanced development of these POC diagnostics. Buffalo, NY-based ZeptoMetrix received a contract from the CDC to create a secure collection of influenza viruses, including the avian-flu virus.

H5N1 is a subtype of Influenza A virus. It refers to the subtypes of surface antigens present on the virus: hemagglutinin type 5 and neuraminidasetype 1. —www.wikipedia.com.

While POC tests are valuable, sensitivity is limited to about 75%. A more sensitive test requires molecular diagnostics. Earlier this year, the CDC awarded Meso Scale Diagnostics and Nanogen second contracts for the development of low-cost influenza tests that can detect and differentiate seasonal human-influenza viruses from avian influenza within three hours. Currently, the process for testing for avian influenza A (H5N1) can take up to 24 hours.⁸ Meso Scale develops and markets high-throughput diagnostic tests that allow large numbers of tests to be conducted at once. The company bases its products on a proprietary combination of electrochemiluminescence detection and patterned arrays.

Nanogen, using proprietary minor groove binder (MGB) probe technology for real-time PCR, will develop a fast molecular test that simultaneously detects and differentiates Type A, Type B, seasonal flu (H1N1 and H3N2) strains, and respiratory syncytial virus, or RSV. The target is to achieve 90% to 95% sensitivity. The contract also provides for a secondary "reflex" test for three avian-flu strains (H5N1, H7N3, and H9N2) to be available for samples that are determined to be positive for flu A but negative for seasonal flu. 

The test is being developed in partnership with the Medical College of Wisconsin and HandyLab Inc. It will be significantly more sensitive than current rapid flu tests and is expected to be conducted in less than half the time it takes to run current molecular tests. The company anticipates the use of off-the-shelf instrumentation for sample handling and detection. When these new tests emerge from the pipeline, the medical labs on the frontline of early detection will have more powerful tools than ever to battle a flu pandemic.

Graham Lidgard, PhD, is the senior vice president, Research and Development for Nanogen Inc., in San Diego, CA.

References

1. US Department of Health and Human Services. HHS Awards Contracts for the Development of Faster Influenza Diagnostic Tests. 12 June 2008. http://www.hhs.gov/news/press/2008pres/06/20080612a.html. Accessed September 15, 2008.

2. Centers for Disease Control and Prevention. CDC Media Briefing: Influenza Vaccine Effectiveness. Published April 17, 2008. http://www.cdc.gov/media/transcripts/2008/t080417.htm. Accessed September 15, 2008.

3. Centers for Disease Control and Prevention. Role of Laboratory Diagnosis of Influenza. Published September 26, 2006. http://www.cdc.gov/flu/professionals/diagnosis/labrole.htm. Accessed September 15, 2008.

4. Centers for Disease Control and Prevention. Interim CDC-NIH Recommendation for Raising the Biosafety Level for Laboratory Work Involving Noncontemporary Human Influenza Viruses. Published October 6, 2005. http://www.cdc.gov/flu/pdf/h2n2bsl3.pdf. Accessed September 15, 2008.

5. Centers for Disease Control and Prevention. Avian Influenza: Current H5N1 Situation. Published June 15, 2007. http://www.cdc.gov/flu/avian/outbreaks/current.htm. Accessed September 15, 2008.

6. Cetron M, MD. Non-pharmaceutical Strategies to Limit Spread of Pandemic Influenza. http://www.cdc.gov/flu/avian/pdf/non-pharmaceutical_intervention.pdf. Accessed September 15, 2008.

7. Centers for Disease Control and Prevention. CDC Awards $11.4 Million to Develop New Rapid Diagnostic Tests for Avian Influenza. 4 December 2006. http://www.cdc.gov/media/pressrel/r061204.htm?s_cid=mediarel_r061204_x. Accessed September 15, 2008.

8. Centers for Disease Control and Prevention. HHS Awards Contracts for the Development of Faster Influenza Diagnostic Tests. Published June 12, 2008. http://www.hhs.gov/news/press/2008pres/06/20080612a.html. Accessed September 15, 2008.

Better patient management

"As the market demands faster turnaround times with higher sensitivity and specificity, we see the market evolving over time to molecular product platforms. This will be particularly evident in infectious diseases in general and critically important with influenza where diagnosing early and accurately can lead to better patient management."

— Caren L. Mason
President and CEO
Quidel Corp.
Maker of QuickVue Influenza A+B

Automating next-gen rapid flu tests

“Bringing automation to the reading of influenza tests is a key factor for the next generation of rapid flu tests. Automating and storing the objective result of a flu test will help increase laboratory productivity and minimize the potential for human error, which can contribute to improving patient outcomes.”

— Patrick Parks, MD, PhD
Advanced Division Scientist
3M Health Care
Saint Paul, MN
Maker of 3M Rapid Detection Flu A+B Test

Flu + pneumonia = complications

“As we head into the 2008-2009 respiratory season, both flu and pneumonia will be present. Streptococcus pneumoniae is the most common cause of bacterial pneumonia and is most often a serious complication of the flu. So, when patients present with respiratory issues, it is prudent to test for both flu and pneumonia to improve patient outcomes in many cases.”

— Dotty Peterson, Director of Marketing
Rapid Diagnostics, Inverness Medical
Maker of BinaxNOW Influenza and
BinaxNOW Streptococcus pnuemoniae

ID circulating pathogens

“Molecular tests for respiratory viruses are an exciting development in laboratory medicine. We know that viruses cause about 80% of respiratory infections, yet these viruses are rarely tested for or diagnosed. With new molecular tests, we can identify the cause of an infection efficiently and effectively so doctors can prescribe the right treatment quickly. Moreover, these tests allow us to detect virus outbreaks early and better understand what pathogens are circulating in our communities.”

— Jeremy Bridge-Cook, PhD
Vice President
Luminex Molecular Diagnostics
Maker of the xTAG Respiratory ViralPanel

New ‘gold standard’

“Molecular methods are rapidly displacing viral culture as the ‘gold standard’ for respiratory viruses due to the speed and accuracy of molecular methods. Within the next three to five years, molecular testing for respiratory viruses will be considered standard of care. Most of this testing will be based on the real-time PCR platform which offers the advantages of ease-of-use, high sensitivity, and minimal risk of contamination. This platform will become even more automated as fully-integrated systems move into the marketplace.”

— Steve Visuri, PhD, Chief Science Officer
Prodesse Inc.
Maker of ProFlu+, Pro hMPV+, and ProParaflu+

Observations on viruses

ViraCor Laboratories was the first national clinical laboratory in the United States to offer the xTAG Respiratory Viral Panel (RVP), a unique test that, from one patient sample, can simultaneously detect the 12 viruses and subtypes that are responsible for more than 85% of respiratory viral infections.

ViraCor immediately began running the test following the FDA 510K clearance in January of 2008. Analyzing the first six months of data from the xTAG RVP test has yielded interesting observations, since the test provides a much larger clinical picture than previously available. While the company’s observations are anecdotal and, therefore, cannot provide true incidence rates, the findings provide a unique perspective on pathogens circulating nationwide. Some observations include:

• ViraCor observed the presence of all 12 viruses and subtypes.
• Rhinovirus was the most prevalent pathogen detected in patients, followed by Human Metapneumovirus.
• The pathogens were not commonly tested for before the launch of xTAG RVP.
• A larger than anticipated number of dual and triple infections was observed, with 75% of dual infection combinations containing rhinovirus. Studies have shown rhinovirus can cause severe and fatal lower respiratory-tract infections, especially in elderly and immunocompromised patients; it also has been associated with graft dysfunction in lung-transplant recipients.

Human Metapneumovirus prevalence was much higher than expected, and the virus was distributed throughout all age ranges. The newly discovered virus is similar to RSV in clinical presentation, with serious ramifications in the pediatric and immunocompromised-patient population, as it has been associated with allograft rejection in transplant patients.

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