BB&T Contributing Editor

SINGAPORE – The in vitro diagnostics market in Asia accounts for only about 12% of the global IVD market, but is attracting growing interest from suppliers based in the U.S. and Europe as well as Asian competitors because of the potential for explosive growth in the region. Advanced technologies, such as those for performing molecular diagnostic testing as well as sophisticated point-of-care tests are becoming important factors in the market. Pharmacogenetic testing is an emerging segment of the Asian market, along with diagnostics employing microarrays and lab-on-a-chip microfluidics technology. Infectious disease testing is one of the most important areas of application for diagnostics in Asia, due to the high prevalence of infections with agents such as hepatitis C, HIV, parasites such as Plasmodium falciparum, the causative agent of malaria, and tuberculosis. For example, China and India together account for about 60% of all new cases of tuberculosis worldwide. Cancer diagnostics is also a major focus of diagnostic laboratories in Asia, as aging populations in the developed countries exhibit increased levels of incidence of neoplastic disease.

As shown in Table 1, a large proportion of the diagnostics market in Asia is attributable to Japan, with other important country markets in China, India and Australia. Market growth, however, is strong, particularly in China and India, although competing against domestic suppliers in countries such as China can prove challenging for U.S. and European companies. In particular, companies in China are beginning to incorporate advanced technologies into their products, and moving beyond simple generic test strip technologies. The market environment in Asia is becoming more favorable for emerging companies as investors increase their presence. Governments in countries such as Singapore are supporting the development of the diagnostics industry through policies that foster research into advanced technologies with clinical applications. Trends in the clinical diagnostics market in Asia were discussed at the inaugural Diagnostics Asia 2006 conference, organized by IBC Asia Pte Ltd. (Singapore) and held here in mid-January.

Molecular diagnostics grows in importance

As discussed by Lucy Lu, vice president, technology development for Roche Diagnostics (Indianapolis) at the conference, molecular diagnostics is the most rapidly growing segment of the diagnostics market, expanding at a 15% annual rate and reaching $1.8 billion worldwide in 2005. As shown in Table 2, more than 85% of the market is attributable to sales of infectious disease testing products on a global basis, including instruments and reagents used for infectious disease diagnosis, blood screening tests, and products used for viral load testing and viral genotyping. The remainder of the market, totaling about $255 million in 2004, is attributable primarily to products used in cancer and genetic disease testing.

Molecular diagnostics has progressed from direct nucleic acid probe testing and a market size of less than $1 million in 1983, to a market of $65 million in 1993 consisting both of tests employing amplification methods such as polymerase chain reaction (PCR) and bDNA as well as direct testing, to the current market that includes products based on at least five amplification technologies, dominated by pcr which accounts for half of the global market. The recent expiration of key patents such as the patents covering PCR has opened the market to smaller suppliers who can sell PCR-based tests free of royalties. Suppliers such as Bayer Diagnostics (Tarrytown, New York) and Abbott Diagnostics (Abbott Park, Illinois) now also are developing new systems that employ PCR technology. Roche is developing a fully automated system for molecular diagnostics that will provide a turnaround time from receipt of sample to result delivery of three hours, and that will be capable of testing 100 patient samples per day.

In the future, a convergence of technologies for nucleic acid testing and proteomics is expected to occur, according to Lu, driven by a clinical need for information on both DNA/RNA and protein markers when performing a differential diagnosis, developing a prognosis, or selecting therapy. New platforms will be required that employ microarrays and microfluidics, allowing high throughput, multi-marker testing to be conducted cost-effectively. A long-term goal, according to leading experts in genomics, is to develop technologies that can analyze the complete genome of an individual at a cost of $1,000.

An example of a new platform employing microfluidics with applications in molecular diagnostics was described at the Diagnostics Asia conference by Robert Hodges of STMicroelectronics (Geneva, Switzerland). The company has developed a new lab-on-a-chip microfluidics platform derived from technology used in inkjet cartridges. The initial applications under development are integrated nucleic acid diagnostic devices for sepsis and influenza testing. The In-Check test platform includes a rapid PCR reactor that performs a typical amplification reaction in nine minutes combined with a 100-cell microarray with a sensitivity of 0.1 nanomolar. At present, a fully integrated version of the system is still in development, but the components of the system already exist. STMicroelectronics said it expects to introduce a system for sepsis testing by the end of this year in partnership with a company that has developed a panel of sepsis markers. Such technology represents the next generation of diagnostics technology to meet the need for multi-marker molecular diagnostic testing systems that will enable clinicians to make productive use of emerging genomic and proteomic biomarker discoveries.

SiMEMS Pte Ltd. (Singapore) is another company that has developed microfabricated device technology with applications in clinical diagnostics. As discussed by Uppili Raghavan of SiMEMS at the conference, the company’s microfluidic chips are designed for isolation and purification of genomic DNA, total RNA, bacterial DNA and viral RNA. The chips integrate between 15 and 20 steps that require separate manipulations using conventional technology for nucleic acid extraction into a single device. The device can accept whole blood or other fluid samples having a volume of between 3 microliters and 400 microliters. A pre-concentration module also has been developed that allows larger sample volumes to be analyzed for targets that occur at very low levels such as organisms that cause sepsis when present at concentrations of one colony-forming unit per milliliter. Between 20 and 40 minutes are required to perform an extraction. The company is targeting applications in point-of-care molecular testing, and plans to reduce the cost per chip from the present level of $12 to $17 down to $6 for specialized IVD testing. Raghavan said the company eventually expects to reduce the cost to about $4 per chip.

Attogenix Biosystems Pte Ltd. (also Singapore) has developed a chip-based nucleic acid diagnostic system with applications in infectious disease testing. The system consists of a pcr amplification chip, the AttoCycler, and a reader, the AttoChip. The first test to be introduced is a kit for detection of dengue virus, launched in late 2005. Use of a microfluidic chip, including a novel self-sealing technology to prevent contamination, allows simplification of the testing process, reducing the number of pipetting steps from 20 for existing PCR formats to four, and enabling the test to be performed by technicians who do not have specialized training. A sensitivity of 98% and specificity of 99% have been demonstrated in clinical trials. Cost per test is only SP$25 (about U.S.$15) vs. SP$50 for competing dengue kits employing nucleic acid diagnostic technology. The company is developing a multi-cartridge extraction system to enable automation of sample preparation.

Boost from pharmacogenetic testing

Pharmacogenetic testing is another application for molecular diagnostics that has the potential to drive expansion of the market in Asia. As discussed by Dr. Christoph Petry, PhD, head of diagnostics research in Germany for Bayer HealthCare AG (Leverkusen, Germany), today’s diagnostic assessment methods rely on a relatively small number of markers that have limited capability to determine if a particular patient will respond to a certain drug, or if that patient will have an adverse reaction. Furthermore, for most diseases existing diagnostic tests are not able to detect causative factors at an early stage. Responses to drug treatment vary widely between patients because information on individual biological variability is not readily available that could allow development of more specific, targeted therapies.

Future generations of molecular diagnostic tests will need to analyze a large number of markers for each patient in order to guide individualized therapy, outstripping the capabilities of existing testing platforms. With today’s technologies, analysis of more than ten markers in parallel creates a major challenge, whereas future applications in pharmacogenetic testing may require analysis of as many as 500 markers. Bayer has analyzed a number of platforms for performing multiplex analysis, but has found most to be deficient. Liquid array technologies, employing microscopic beads and flow cytometric detection of fluorescent labels, typically exhibit low sensitivity, where PCR offers extremely high sensitivity but is difficult to multiplex. Microarrays offer a means to efficiently multiplex, but only provide moderate levels of sensitivity. Coupling of PCR with microarray detection provides a possible solution, but that approach is very expensive, costing approximately $100 per sample, and can also be prone to errors.

To address the next generation of diagnostic testing technologies, with particular emphasis on high-sensitivity testing, Bayer is developing a new direct nucleic acid analysis system using planar waveguide technology. The system offers sensitivity that is 1,000-fold higher than confocal microscope scanning, the technology now used for detection in most microarray systems, along with a wide dynamic range, and the ability to perform multiplex analysis of large numbers of markers in parallel. Waveguide technology employs fluorescence detection of molecules that are bound to the waveguide surface, typically using target-specific capture antibodies or nucleic acid probes to capture the analyte on the surface. Evanescent wave technology is used to achieve highly sensitive detection of the bound targets. Nucleic acid targets are detected directly, without amplification, by using antibodies that bind to DNA/RNA hybrids. While the technology requires further development before it is usable clinically, it promises to provide a simple, fast and inexpensive method for performing highly parallel pharmacogenetic testing.

Dr. Israel Stein, CEO of Clinical Data (Newton, Massachusetts), described his company’s programs in pharmacogenetic testing. Clinical Data recently announced a reorganization to focus on its core pharmacogenomics business. The company’s new Clinical Data Molecular division consists of three businesses acquired in 2005, including Genaissance Pharmaceuticals (New Haven, Connecticut), Lark Technologies and Icoria. The division generated annualized revenues of $33 million for the 12 months ended Sept. 30, 2005, excluding revenue related to Icoria’s agricultural genomic services. Clinical Data is one of the largest independent providers of pharmacogenomics and metabolomics services in the U.S.

The company’s second division, Vital Diagnostics, markets small-volume clinical chemistry analyzers. According to Stein, the blockbuster model of drug development is broken. Increasingly, fewer patients will take a given drug, but treatment efficacy will be higher and the rate of adverse events will be lower. Additional benefits of implementing pharmacogenomics may include reduction in the cost of medical care, retrospective analysis of factors involved in adverse drug reactions, and resurrection of drugs that have become stalled in development due to efficacy and/or safety issues.

Clinical Data now offers two pharmacogenetic tests through its diagnostic services laboratory, including the Familion test for long QT syndrome and a test for genetic mutations that result in an inability to metabolize thiopurine methyltransferase drugs. The company also is developing a new drug, Vilazodone, for use in the treatment of depression, along with a companion genetic test that identifies those patients most likely to benefit from therapy.

Another test is under development to identify the 1.6% of patients with schizophrenia who will have adverse reactions to clozapine. Clozapine has been available for 20 years, but is rarely used because of a low rate of major adverse reactions (agranulocytosis). Use of the new test, which is a home brew genetic assay, could allow clozapine to be offered to the majority of patients with schizophrenia as a new and effective treatment option. Clinical Data has identified 74 candidate genes that are involved in clozapine metabolism or promyelocytic differentiation, or for which associations with clozapine-induced agranulocytosis have been discovered.

Pharmacogenetic testing can potentially be used for management of at least 50 million patients worldwide, a number that could expand significantly as advanced drug treatments become more widely employed in developing countries such as China, India and others in the Asian region.

New technologies for cancer diagnosis

Cancer diagnostics was another topic highlighted at the Diagnostics Asia conference. Cancer is a large and growing public health issue in Asia. Cancer types with high incidence in the region include stomach, colorectal, liver and lung cancer. High rates of liver cancer are due in part to the high prevalence of infection with hepatitis C in parts of Asia. Breast cancer is also a common disease in some countries such as Singapore, where the number of new cases is increasing at 3% annually.

A number of research groups are investigating new biomarkers for use in cancer diagnostics, including Dr. Keli Ou of Agenica Research Pte Ltd. (Singapore), who has developed an integrated proteomic/ genomic platform for breast cancer biomarker discovery. The platform employs 2-D gel electrophoresis or HPLC to separate proteins in a clinical sample followed by mass spectrometry analysis to identify proteins that are useful as cancer biomarkers. A novel aspect of the Agenica approach is the use of cell lines as well as human tumor samples in the biomarker discovery process, as well as combined analysis of both sample types using genomics and proteomics. The method results in discovery of markers that are over-expressed both at the mRNA transcript level and at the protein level, providing increased confidence in the relevance of the markers.

A breast cancer biomarker panel developed using the Agenica discovery method has been shown to unambiguously segregate normal vs. tumor tissue samples using a test set of 100 specimens. Tissue microarrays are used for validation testing. One protein identified by the discovery method looks particularly promising as a breast cancer marker, since its expression is completely eliminated in tumor tissue.

Lance Miller, PhD, of the Genome Institute of Singapore, also presented results at the conference from studies conducted by his group on potential new breast cancer markers. Miller is using DNA microarray analysis to assess patterns of p53 oncogene expression in tumor tissue. A p53 expression signature derived from analysis of 32 signature genes is used to classify breast tumors and correlated vs. histologic grade. In addition, the ability of the p53 signature to predict response to chemotherapy has been assessed, and has been shown to be an independent predictor that identifies patients who can be spared systemic adjuvant chemotherapy.

In comparison to the Nottingham Prognostic Index, which is based on tumor grade, nodal stage and tumor size and is the conventional index used to identify patients who do not need adjuvant therapy, an index that includes the genetic signature identifies twice as many patients who do not need added chemotherapy. The signature appears to be strongly related to tumor proliferation, and is superior to existing markers such as Ki-67 for assessing proliferative index.

Other studies conducted by Miller show that the genetic signature also can be used to characterize other types of cancer such as liver cancer, potentially making it useful in the management of therapy for a number of tumor types.

Cervical cancer is another important cancer type in Asia. It is the second-most-prevalent cancer among women in the developing world, with an incidence rate of 500,000 per year worldwide, resulting in 290,000 deaths. The main causative agent of the disease is human papilloma virus (HPV). Only specific subtypes of HPV are associated with malignancy. For example, 50% of all cervical cancer cases are caused by HPV-16. However, the use of HPV testing to detect cervical cancer at an early stage is complicated by the fact that most women are infected with HPV at some point in life, typically at the stage when they become sexually active.

Dr. Vinay Badal of the Institute of Molecular and Cell Biology (Singapore) is investigating the use of DNA methylation analysis as a tool to detect transformation of HPV-16 from the latent state existing in most women to a tumorigenic state. A clinical study conducted by Badal has shown that the methylation status of the HPV-16 promoter region is negatively correlated with tumorigenesis, i.e., a low level of methylation is correlated with cancer progression. Consequently, Badal’s methylation assay could potentially be used to identify the risk of cervical cancer development in women who are infected with one or more of the high-risk types of HPV. Badal speculated that exposure to carcinogens could be one of the mechanisms resulting in demethylation of HPV genes.

The market for products used in cervical cancer testing is one of the most rapidly growing sub-segments of the in vitro diagnostics market. As shown in Table 3, worldwide sales of products used in cervical cancer screening, including HPV test kits sold by Digene (Gaithersburg, Maryland), as well as slide preparation and automated imaging products from Cytyc (Marlborough, Massachusetts) and TriPath Imaging (Burlington, North Carolina), are projected at well over $500 million in 2005, and have grown at an 18.6% compound annual rate over the past four years.

Point-of-care testing expands in Asia

Point-of-care (POC) testing was another focus of the conference. William Cao of NeuPro Technology Co. Ltd. (Taipei, Taiwan) described a new POC test system based on the company’s NeuChip sensor, a low cost biosensor that is being developed for time-critical diagnostic testing applications such as cardiac testing, rapid pathogen identification, and food toxicology. The sensor is fabricated using screen-printed electrode technology, and features electrochemical techniques using enzyme labels to achieve high sensitivity and low cost.

The test system consists of a test cartridge and a reader that connects to a Personal Digital Assistant for data storage and analysis. Total test turnaround time is less than three minutes for cardiac marker tests now under development. A rate-sensing method is used in the analysis, minimizing background effects. Additional tests for infectious disease are in the pipeline. The company is interested in licensing the technology for diagnostic applications, and is searching for strategic partners.

Another company focusing on point-of-care testing for infectious agents, the Foundation for Innovative New Diagnostics (FIND; Geneva, Switzerland), is developing new POC tests for tuberculosis with funding from the World Health Organization (WHO; also Geneva). FIND is a non-profit company founded in 2003 and has been funded through a $30 million initial investment by WHO as a spin-off from that organization. FIND is focused on the public health sector, with a goal of helping to reduce deaths due to infectious disease, which accounts for 45% of all deaths in Asia.

As discussed at the conference by Giorgio Roscigno, CEO of FIND, the initial focus of development efforts is on TB and malaria diagnosis. Existing TB test methods have a low diagnostic yield, with only 19% of all incident TB cases detected and reported as smear-positive in spite of the fact that over $1.2 billion is spent annually on TB diagnostic tests, as shown in Table 4.

Worldwide, 8.7 million new cases of TB occur annually, mostly in East Asia and Africa. One new test that is being developed by Proteome Systems (North Ryde, Australia) is the DiagnostiQ antigen-based assay for use in developing countries. The test is designed to detect a panel of 62 proteins that are expressed in patients with TB infection. The target patient population for the test is the 16 million patients worldwide who are co-infected with HIV and TB. The test uses sputum, blood or plasma samples to detect active TB infections and has an analytical sensitivity of 100 pg/mL. Diagnostic sensitivity, however, for detection of TB infection is only 70%, and efforts are under way to improve sensitivity prior to launching the test. Jenny Harry, PhD, of Proteome Systems, who discussed the new TB test at the conference, said a prototype test is to be available by December of this year.

FIND also is evaluating low cost nucleic acid amplification technology for TB testing, and is working in partnership with BD (Franklin Lakes, New Jersey) to introduce rapid blood culture testing in Asia using the BD BBL MGIT system, which allows a reduction in turnaround time from 45 days to 14 days. Another TB test under evaluation by FIND uses phage technology combined with nucleic acid amplification for three- to four-day direct (non-culture) detection of TB in sputum.

FIND also is targeting development of improved diagnostic tests for malaria, a disease affecting 40% of the global population with a prevalence of 300 million cases, mostly in Africa, Southeast Asia and India, and the northern countries in South America. Although microscopy is the standard method used in malaria diagnosis, it is highly user-dependent and requires skilled personnel in order to obtain reliable results. There is an increased demand for accurate remote diagnostic malaria tests that can be used by non-skilled personnel.

As discussed at the conference by Dr. Mark Fukuda, chief of the department of immunology & medicine at the Armed Forces Research Institute of Medical Sciences (Bangkok, Thailand), dipstick tests for malaria-specific antigens are available at prices ranging from 60 cents to $2.50 (vs. a cost of 12 cents to 40 cents for microscopy), but such tests cannot differentiate lethal vs. non-lethal forms of the parasite that causes malaria. A new immunochromatographic test developed by Binax (Scarborough, Maine), the Malaria P.f./P.v. NOW ICT, provides subtyping of the parasite using a fingerstick blood sample. The test takes 20 minutes to perform, and the required skill level is low. However, sensitivity is considerably less than microscopy at more than 100 parasites per microliter vs. 50 for microscopy.

Nucleic acid amplification tests for malaria have been developed for research use, and look promising, since sensitivity is less than 5 parasites per microliter and all subspecies can be detected, but at present such tests are research tools, not clinical products. Real-time PCR tests have been evaluated by Fukuda that appear promising, and that can potentially allow therapy to be monitored. However, he said he believes that antigen capture or immunochromatographic methods will prove to be the best methods for future malaria diagnostic testing.

Other important infectious disease testing applications in Asia include detection of Dengue infection and tracking transmission of Avian flu viruses (HN51). As discussed by Dr. Bijan Kumar Sil of the National Environment Agency, Environmental Health Institute (Singapore), a number of tests for dengue virus have been developed by his laboratory that use monoclonal-based competitive ELISA, real-time PCR or antigen ELISA to detect dengue infections at various stages of the disease, improving the ability of public health agencies to monitor infection outbreaks without the need for cell culture facilities.

Dr. Sze-Wee Tan, CEO of Rockeby Biomed Ltd. (Singapore), described a new rapid test for Avian flu that was introduced by his company last November. Rockeby has developed additional tests for vaginal infection in partnership with Pacific Biotech (Thailand). The existing Avian flu test is designed for testing of birds, and has been used in Southeast Asia, the Middle East, Eastern Europe and Japan. A human test also has been developed. The market for a human test, however, is very small due to the fact that only a few cases have so far been diagnosed. That situation could change if the virus mutates and begins to generate a human pandemic.

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