BB&T Contributing Editor

WASHINGTON — Infectious disease testing represents one of the largest segments of the in-vitro diagnostics market, second in size only to diabetes testing as a percentage of the total clinical diagnostics market according to supplier estimates. As shown in Table 1, the global market for infectious disease testing products used in diagnosis and screening is estimated at more than $7.6 billion for 2007, growing at 8% annually.

The market includes immunoassay products for infectious disease diagnosis and blood screening as well as molecular diagnostic testing products; microbiology reagents, media and equipment; and rapid infectious disease test kits employing qualitative immunoassay technologies.

Although infectious disease testing has always been an integral part of clinical diagnostics, it continues to be a dynamic segment of the market. New diagnostic technologies are enabling more sensitive and reliable detection of infectious agents, and reducing the time required to provide a result. At the late-July annual meeting of the American Association of Clinical Chemistry (AACC, Washington), new developments in infectious disease testing were discussed by a number of speakers, and multiple new products were introduced.

Molecular diagnostics is playing an increasingly important role in infectious disease testing, as indicated by the figures in Table 1. In the near future, molecular testing may even move to the point of care, providing hospitals as well as remote sites with significantly expanded capability to rapidly diagnose infections and implement appropriate responses when they will be most effective.

Molecular testing is also expanding its role in cancer diagnosis and monitoring as well as in genetic disease testing, and is poised to play a key role in therapy guidance as new pharmacogenomic tests come onto the market as companion tests for targeted drugs.

Demand in infectious disease management

Infectious disease diagnostics is becoming an increasingly important aspect of patient management in the hospital setting. Hospitals are implementing programs to reduce hospital-acquired infections including the growing problem of antibiotic-resistant organisms that rely upon infectious disease testing to identify affected patients, track the source of infections, and guide eradication and treatment.

Sepsis detection is another area of unmet need, where more sensitive tests allowing earlier detection could have a significant impact in reducing in-hospital mortality. As an example of an innovation in the latter area, Seegene (Rockville, Maryland/Seoul, South Korea) described a new molecular sepsis test now under development that could provide hospitals with a new tool to more effectively identify patients with sepsis at an earlier stage and provide guidance for treatment.

As discussed by Seegene CEO Jonh Yoon Chun, PhD, at an AACC press conference, severe sepsis has an incidence rate in the U.S. (300 per 100,000 inhabitants or 750,000 cases annually) which is higher than that for congestive heart failure (196 cases per 100,000), breast cancer (112 cases per 100,000) or AIDS (17 cases per 100,000). Deaths due to sepsis total 215,000 per year in the U.S., compared to 218,000 for acute myocardial infarction.

Often, patients die or suffer shock or coma due to sepsis contracted in the hospital while being treated for a completely unrelated condition. Seegene has developed a research-use sepsis panel, the Seeplex Sepsis multi-pathogen screening test, which analyzes 68 sepsis indicators using molecular diagnostic (PCR-based) technology and a microchip detection array.

The nucleic acid amplification technology employed in the Seeplex assay is based on Seegene's Dual Priming Oligonucleotide (DPO) primers, which consist of a long 5' sequence plus a short 3' sequence. The DPO primer technology allows hybridization to be performed at a higher temperature, preventing primer dimerization and primer competition which are drawbacks of conventional multiplex PCR, and enabling the high level of multiplexing achieved in the Seeplex assay while preserving sensitivity and specificity.

Seegene quotes 10-copy sensitivity for the sepsis assay, and the sample volume requirement is 400 L. In a clinical study conducted in Korea involving 370 hospital patients suspected of having sepsis, the Seeplex assay showed a 17.3% positivity rate compared to 10.7% for blood culture. Turnaround time for the test is five hours.

In addition to screening for 64 sepsis-related pathogens, including 48 Gram-positive and 10 Gram-negative bacteria and six fungi, the Seeplex test analyzes for four drug resistance genes (vanA, vanB, mecA and blaSHV), providing not only detection of sepsis but also information to guide therapy. Importantly, the drug sensitivity test is unaffected by antibiotics given to the patient prior to a blood draw, which is not the case for culture-based assays which are the current gold standard for diagnosis of sepsis. Furthermore, culture tests require two to five days to produce a result, so initial therapy is usually empirical rather than guided by information on the drug sensitivity characteristics of the infection.

Another advantage of the Seegene test is low cost. The company projects that the 68-marker multiplex test will cost about the same as an existing single-analyte molecular test. The product will be introduced first in Europe, and the company plans to initiate clinical trials to generate data for a U.S. regulatory submission.

In addition to the Seeplex sepsis test, Seegene is developing a multiplex test for respiratory infections that will simultaneously detect 13 viruses and 8 bacteria at a cost of $30 per test, as well as multiplex tests for human papilloma virus, sexually transmitted disease, antibiotic resistance, mycobacterium tuberculosis discrimination, and drug response genes.

Detection of antibiotic resistant organisms such as methicillin-resistant staphoccocus aureus (MRSA), vancomycin-resistant staph aureus (VRSA), vancomycin-resistant enterococcus (VRE) and multidrug-resistant TB is another high priority for hospitals. The market for MRSA testing products is between $60 million and $70 million worldwide, with 65% of sales in the U.S. and 35% outside the U.S. according to suppliers, and growing rapidly.

As discussed by Joseph Campos, PhD, of Children's National Medical Center (Washington) at the AACC conference, methicillin-resistant infections in some hospitals now outnumber methicillin-sensitive infections.

The leader in the molecular MRSA testing market is Cepheid (Sunnyvale, California), with its GeneXpert automated rapid molecular testing system. Cepheid is projecting MRSA sales of $52 million to $55 million for 2008. As shown in Table 2, Cepheid's sales in the clinical market have grown rapidly, and a major and growing proportion of clinical sales are attributable to MRSA testing products.

BD (Becton Dickinson; Franklin Lakes, New Jersey) also markets a rapid molecular test for MRSA, the BD GeneOhm MRSA assay, which was introduced to the market before the Cepheid MRSA test and uses the Cepheid SmartCycler for front-end amplification. The test was developed by GeneOhm, a molecular diagnostics company acquired by BD for $230 million in 2006. GeneOhm had revenues of $5 million in 2005. More than 250 hospitals in the U.S., Canada, Europe and Asia-Pacific are now using the GeneOhm MRSA test, and BD plans to expand its menu of hospital-acquired infection (HAI) tests and to introduce a new automated molecular diagnostic platform later this year.

The Cepheid MRSA assay has achieved significantly greater market acceptance than the BD test, however, due to its shorter turnaround time of one hour compared to two hours for the GeneOhm assay, reduced hands-on time (sample preparation for a nasal swab requires only about two minutes vs. 20 to 30 minutes hands-on time for the GeneOhm test), and greater ease of use. Cost is one disincentive for use of the Cepheid test, however. The Cepheid test costs $60 per test, according to Campos (other quotes range from $36 to $50 per test), whereas cost for the GeneOhm test is $25.50. The higher cost has not been a major deterrent to adoption of the Cepheid MRSA test, based on the trend in product sales.

The molecular MRSA testing market is expected to become much more competitive in the future as new products are introduced by a wider array of suppliers, including some of the leaders in the overall molecular diagnostics market. Roche Diagnostics (Indianapolis) is developing a MRSA test that will run on its LightCycler 20, an instrument having a large worldwide installed base. The test will be introduced in both the U.S. and Europe in 2009.

BioHelix (Beverly, Massachusetts) plans to introduce a MRSA nucleic acid test that uses its IsoAmp rapid test format as its first IVD product, with clinical trials scheduled to start in March 2009. IsoAmp is a qualitative, instrument-free test format that uses isothermal Helicase Dependent Amplification (HDA) technology and the BESt detection cassette for visual readout. Turnaround time for the IsoAmp MRSA test is 1.5 hours.

The BioHelix MRSA test is expected to be less expensive than the Cepheid MRSA test, at $20 per test. The test is performed directly from blood culture medium specimens, and is simple to implement since no thermocycling is needed and all reagents and amplicons remain sequestered in the BESt cassette, avoiding issues with cross-contamination.

An initial evaluation of the IsoAmp MRSA test performed at Vanderbilt University Medical Center (Nashville, Tennessee) and the Lahey Clinic (Burlington, Massachusetts) found a sensitivity of 50 colony-forming units, clinical sensitivity and specificity for Staph aureus detection of 100%, and 100% and 98% respectively for detection of methicillin-resistant Staph aureus.

Ibis Biosciences, a subsidiary of Isis Pharmaceuticals (Carlsbad, California), has developed a MRSA test on the Ibis T5000 biosensor platform, a system that employs broad-range PCR amplification followed by mass spectrometric analysis for rapid microbial detection. No culture step is required. The assay turnaround time is five hours. The MRSA test is currently available for research use only.

Iquum (Marlborough, Massachusetts) is developing a MRSA assay for its Liat lab-in-a-tube molecular testing platform that will provide a turnaround time of one hour. The Liat analyzer is designed for point-of-care use, and will provide users with six test channels and a wide range of infectious disease molecular tests including influenza, Chlamydia and gonorrhea, HIV viral load, and a 21-marker sepsis panel as well as a MRSA test. Initial submissions for FDA clearance are planned in early 2009.

The use of MRSA testing has proven effective for reducing the rate of hospital-acquired infections. An analysis conducted by one hospital which implemented Active Surveillance Testing for MRSA using the Cepheid Xpert test found that its infection rate was reduced by 55% (from 11.12 to 5.06 cases per year), saving $30,409. The reduction was attributed primarily to the faster turnaround time for molecular testing compared to culture.

As larger hospitals have begun adopting the Cepheid test, demand has grown for a larger automated testing platform than the 16-channel GeneXpert system. Cepheid has responded by developing the Infiniti-48, a 16- to 48-channel system that employs the same cartridges as the smaller GeneXpert, but enables higher-volume testing to be performed on a single instrument. The Infiniti-48 will be launched in December, and will be priced at $220,000 for the base configuration.

Other high-growth applications for molecular diagnostics in infectious disease testing include respiratory virus assays and tests for sexually transmitted diseases, particularly Human Papilloma Virus (HPV). As those applications expand along with increasing adoption of tests such as MRSA, HIV viral load, and nucleic acid blood screening, the market for molecular infectious disease testing is expected to continue to grow.

As shown in Table 3, the worldwide market is projected to expand from $2.3 billion in 2007 to $3.3 billion by 2012. As an example of new molecular tests that will have a positive impact on the market, Luminex (Austin, Texas) introduced the xTAG respiratory virus panel at the AACC conference, a product which received 510(k) market clearance in January. The xTAG panel detects 12 viruses that collectively are responsible for 85% of all respiratory viral infections.

The xTAG panel is the first multiplex nucleic acid test to be cleared by the FDA, according to Luminex. The product was developed by the Luminex Molecular Diagnostics division, formerly Tm Bioscience, and is targeted for use in hospital and reference laboratories. It is expected to enable hospitals to reduce patient length of stay by allowing rapid determination of the most effective drug or drugs to treat respiratory viral infections, and will also help reduce over-use of antibiotics.

Luminex also has introduced a new analyzer, the Flexmap 3D, that will generate 150,000 data points per hour and provide up to 500 results on a single sample. The Flexmap 3D also has an enhanced lower limit of detection compared to previous Luminex analyzers. The xTAG test will be sold to the clinical diagnostics market in the U.S. through Thermo Fisher Scientific (Waltham, Massachusetts) and internationally by Abbott Diagnostics (Abbott Park, Illinois).

Osmetech Molecular Diagnostics (Pasadena, California) also is developing a molecular respiratory virus panel test that will run on its newly cleared XT-8 analyzer. The XT-8 system uses microfluidic test cartridges using Osmetech's eSensor electronic nucleic acid detection technology.

A number of suppliers have entered the market for HPV testing products as it has expanded. According to Daniel O'Day, head of molecular diagnostics at Roche Diagnostics, the U.S. market for HPV testing products now exceeds $200 million, and is growing at 32% annually. At present, HPV testing is used primarily in ASCUS triage, which addresses only 4% of women in the U.S., equating to 1.1 million HPV tests annually. In the future, Roche believes use will expand to include adjunct HPV molecular testing along with Pap testing, expanding the market to 28 million tests.

Further in the future, Roche expects the market to expand to 50 million tests as HPV molecular testing becomes the preferred method for primary screening for cervical cancer, with PAP testing used in triage of HPV-positive women. Roche is in the process of adding an HPV test to its molecular diagnostics menu in the U.S., and is developing an integrated detection/genotyping platform for HPV. According to O'Day, genotyping is now a requirement for the HPV testing market.

Some leading suppliers in the molecular diagnostics market, such as Gen-Probe (San Diego), Siemens Healthcare Diagnostics (Deerfield, Illinois) and Abbott Diagnostics must overcome a market entry barrier related to the strong patent position of Digene (Gaithersburg, Maryland), now a unit of Qiagen (Hilden, Germany), for HPV diagnostics.

Options for molecular diagnostics multiply

Molecular diagnostics is not limited in its clinical applications to infectious disease testing. In fact, other application segments such as oncology, genetic testing, and pharmacogenomic testing are growing much more rapidly than the molecular infectious disease segment, contributing to overall market growth projected at almost 10% per year over the next five years. A number of new molecular diagnostic systems were exhibited at the AACC conference that will address those other segments of the market.

Sysmex (Kobe, Japan) exhibited the RD-110, a new automated molecular analyzer currently for investigational use in the U.S. but sold in Japan and in Europe under a CE mark. The RD-110 uses RT-LAMP (real-time loop-mediated isothermal amplification), a proprietary nucleic acid amplification technology developed by Eiken Chemical (Tokyo) that eliminates the need for nucleic acid (RNA) extraction. The initial applications under development for the RD-110 are measurement of cell cycle markers, and in particular the CK19 marker which is associated with breast and colon cancer cells.

Sysmex is developing a molecular test for intra-operative use that will measure CK19 levels in lymph node tissue specimens. A special microtome device has been developed that slices the excised node into thin sections, and alternating sections are homogenized on-site in about 90 seconds and then analyzed on the RD-110. Surgeons can obtain an indication of node positivity in essentially real time during a procedure, helping to guide subsequent node dissections. The test is believed applicable to 80% of the roughly 185,000 new cases of breast cancer occurring annually in the U.S.

Sysmex is now completing a U.S. clinical trial of the breast cancer test in preparation for a pre-market approval submission.

AutoGenomics (Carlsbad, California) exhibited new molecular cancer diagnostic tests for its Infiniti analyzer, including the CHEK-2 test for familial breast cancer predisposition, as well as pharmacogenomic tests for NAT2, EGFR, and 5-fluorourocil with applications in prediction of response to chemotherapeutic drugs. The CHEK-2 assay detects a genetic alteration that is present in 5% of all breast cancers, while the NAT2 assay measures a marker of acetylation that helps predict drug activity in a variety of cancer types. All tests are for research use only in the U.S.

AutoGenomics also exhibited FDA-cleared molecular tests for warfarin resistance and for coagulation disorders at the AACC conference.

HandyLab (Ann Arbor, Michigan) introduced the Jaguar molecular diagnostic analyzer at the AACC exhibition. The Jaguar is an open platform for RT-PCR molecular testing, employing microfluidic technology to eliminate the risk of PCR contamination and fluorescence detection for high sensitivity. The system is configured as a random access analyzer, and can generate from one to 24 results in 45 to 90 minutes, including a 15-minute PCR amplification. The Jaguar is well-suited for use in hospital and reference labs that are developing home-brew molecular tests.

In addition, other companies with unique molecular probes are partnering with HandyLab for development of automated assays. For example, Nanogen (San Diego) is developing influenza assays that will run on the Jaguar platform. Other assays being developed for the clinical market include molecular tests for Group B streptococcus, Chlamydia trachomatis, and Neisseria gonorrhea.

Fluidigm (South San Francisco, California) also is planning to collaborate with clinical labs interested in developing in-house molecular RT-PCR tests using its BioMark Digital PCR integrated fluid circuit arrays. Digital PCR is a technique to quantify the amount of DNA in a sample by counting amplifications from single molecules. The DNA sample is diluted and aliquotted via microfluidics into wells such that on average there is less than one copy per well.

The number of positive wells after a PCR reaction corresponds to the number of DNA molecules in the original sample. The method is capable of single-copy sensitivity, and can be used to reduce the amount of DNA needed for analysis. The Biomark system includes an analyzer with a microfluidic chip loader and real-time PCR instrument along with the BioMark Dynamic Array cartridges.

Continued expansion of POC testing

Point-of-care (POC) testing continues to be a high-growth segment of the clinical diagnostics market, and has now expanded to include all types of testing including molecular diagnostics. For example, as discussed by Mark Perkins, PhD, of the Foundation for Innovative New Diagnostics (FIND; Cointrin, Switzerland) at an AACC plenary session, the Cepheid GeneXpert system is being used by FIND in sites in developing countries to perform tuberculosis testing due to its ease of use and culture-equivalent accuracy, and other molecular tests such as a line probe assay from Hain Lifescience (Nehren, Germany) are being implemented in South Africa for multidrug-resistant tuberculosis that reduces turnaround time from two to three months to two days or less.

Genefluidics (Monterey Park, California) exhibited a new molecular diagnostics system at the AACC conference that performs both nucleic acid and immunoassay tests on the same platform, with femtomolar sensitivity for nucleic acid tests and picogram/ml sensitivity for immunoassays. Electrochemical sensing is used, and the system has the capability to perform multiplex analysis. The company first developed a benchtop analyzer, the Athena, for central lab testing, but is developing the Asklepios analyzer for POC use, which will employ microfluidics technology and has a target cost per test of $5.

New technologies for POC immunoassay also were described at the AACC conference. Magnisense (Rosny-sous-Bois, France) discussed a new advance in its magnetic particle sensing technology which for the first time allows multiplex detection of different analytes in a single tube. President Luc Lenglet said at an AACC press conference that the company initially developed the MIAtek technology, which uses nano-sized superparamagnetic particles as labels in immunoassays for proteins, viruses or bacteria, with read-out performed in a flow-through mode using a portable magnetic reader.

Now, Magnisense has demonstrated the capability to simultaneously detect two different labels made of different magnetic materials in a single tube. Three other magnetic materials have been identified which have suitable characteristics for use as labels, in principle enabling a five-plex assay to be performed.

The multiplex assay technology, dubbed MIAplex, is well-suited for use in POC immunoassay testing due to the small size of the reader and the simplicity of the assay configuration. Assays for influenza A and B, salmonella, Legionella and tetanus antibody have been demonstrated using the Magnisense technology.

The company said it plans to partner with established IVD suppliers to commercialize the MIAplex technology for applications in clinical diagnostics, focusing on POC testing where the Magnisense technology has an advantage compared to other multiplex assay methods such as the Luminex xMAP technology.

NanoEnTek (Seoul, South Korea) also is developing a new POC immunoassay system. The Frend analyzer from NanoEnTek performs immunoassays using a chip-based lateral flow technology, enabling analysis of multiple analytes using a 10 uL to 20 uL whole blood sample with a five- to 10-minute turnaround time. The product is not yet on the market, but a CE mark is expected by this month, enabling introduction in the European market, and the company plans to initiate a 510(k) submission to allow entry in the U.S. market.

The initial test menu will focus on cardiac markers, including Troponin I, CK-MB and myoglobin, with NT-proBNP to follow by the end of 2008. Thyroid tests will be introduced by the end of 2008, followed by hormone assays by 2Q09 and immunology assays by Q4 2009. The company is also developing a compact CD4 analyzer, the Adam-CD4, as well as a chip-based CBC test (iCBC), due for introduction in 2010.

Another new POC immunoassay system is under development by e2v Biosensors (Essex, UK). The e2v system uses an integrated microfluidic chip with surface-enhanced resonance Raman spectroscopy (SERRS) detection. The SERRS technology provides high sensitivity, according to the company, with a detection limit of 5 pg/mL demonstrated in a model assay for CRP that requires 10 minutes to perform.

e2v also is developing a Troponin I assay which is expected to have a similar level of sensitivity. A market introduction date has not yet been set.

Chempaq (Farum, Denmark) exhibited the XBC point-of-care hematology analyzer, a compact system using disposable PAQ test cartridges to perform white blood cell counts and hemoglobin measurement using a single drop of blood in about 2.5 minutes. In Europe, the test menu also includes a red cell count, hematocrit and three corpuscular parameters (MCV, MCH and MCHC). The additional parameters are scheduled for launch in the U.S. at year-end.

The XBC has been on the market in Europe since 2005, and is now used in 20% of the physicians' office labs in Denmark, and in labs in other countries throughout Europe. Full-scale launch in the U.S. has been delayed pending receipt of CLIA-waived status, which will open up a large segment of the market comprised of the more than 126,000 labs operating under a CLIA waiver certificate in the U.S. That figure, which includes non-exempt labs only, represents 63% of all non-exempt labs in the U.S. (there are an additional 6,265 labs that are CLIA-exempt).

Chempaq also is developing the XDM analyzer, which adds data management and data transfer capabilities, due out in mid-2009, as well as an advanced PAQ cartridge that will perform a complete blood count. At least 86 million CBCs are performed in the ambulatory care setting in the U.S. each year, according to the most recent data from the National Center for Health Statistics.

Instrumentation Laboratory (IL; Lexington, Massachusetts) introduced a new critical-care POC analyzer, the GEM4000, at the AACC conference. The GEM4000 is one of a number of POC blood gas/electrolyte/metabolite analyzers on the market worldwide that enable hospitals to perform testing in near-patient locations, typically in critical care settings. IL has had considerable success with the GEM POC critical care analyzer product line, with 10,000 instruments installed worldwide.

Decentralized blood gas/electrolyte testing is continuing to expand, and has already penetrated a high percentage of the market in Europe and the medium-to-large hospital market in the U.S., as shown in Table 4.

The newest IL GEM system, GEM4000, integrates co-oximetry along with the parameters offered on the predecessor GEM3000 (blood gases, electrolytes, glucose, lactate and hematocrit). Sensors for BUN and creatinine are to be added to the menu. The GEM4000 also links to the GEM WEB Plus information system, a web-based IT system that allows remote access to critical care test data via the Internet and also links to the laboratory information system.

The new analyzer incorporates IL's IQM automated quality control/quality assurance system which automatically manages running of controls and calibrators, and performs automated error detection and error resolution. The FDA has cleared the IQM system for replacement of traditional quality control procedures.

i-SENS (Seoul, South Korea) previewed its new POC critical care analyzer, scheduled for launch in Korea this month and in Europe in early 2009, with a possible U.S. launch also in 2009. The i-SENS system employs thin-film sensor technology, and is configured to use a multi-test cartridge providing 200 tests with a test turnaround time of 50 seconds.

The system employs two-point calibration, providing a potential accuracy advantage compared to existing POC critical care analyzers that employ single-point calibration. The initial test menu will include sodium, potassium, chloride and hematocrit, with ionized calcium, blood gases and metabolites in development.

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