Molecular diagnostics is perhaps the most important and prime diagnostic IVD technique. Molecular diagnostics tests are being done in all six elementary areas including infectious diseases, pharmacogenomics, oncology, genetic disease screening, coagulation, and human leukocyte antigen typing. The rising prevalence of diseases, especially among the ageing population is the key driver. They are associated with detection of specific sequences in DNA and RNA that may or may not be associated with the diseases including single nucleotide polymorphism, deletions, rearrangements, insertions, and others.

One prime reason that goes in favor of molecular diagnostics testing other IVD diagnostics testing it its inherent nature of generating a profit margin that increases as the testing volume rises. The doctors and patients are increasingly finding the need to undergo clinical examinations that give results in less than a day. The infectious disease applications among many are the most common areas of clinical interest. Hospital acquired infection including methicillin-resistant Staphylococcus aureus, Clostridium difficile, or Vancomycin-resistant enterococci represent another set of applications, which involve the infectious disease surveillance using molecular diagnostics.

Availability of assays for the molecular diagnostics testing at commercial level has also increased to a high extent. These assays are available for both qualitative (for verifying the presence of the virus) and quantitative (for determination of the number of viruses existing in particular area) applications. Additionally, there are two other categories of assays available in the market, which differ on their uses. The first are the ones that may be reported in the same patient including recurrence of an infectious disease or cancer, and detection of minimal residual disease. The other ones are those conducted only once in any patient's lifetime to confirm the mutations that would have modified genetic makeup. The mutations such as Clopidogrel (Plavix) or the Leiden Mutation or warfarin metabolism are analyzed through these types of assays.

Global Scenario

The molecular diagnostics instruments and reagents market is estimated as the highest growth segment of the global IVDs market, valued at USD 5.3 billion in 2014 and is expected to expand at a CAGR of 6.6 percent to reach USD 7.3 billion by 2019, according to Medtech Insight, higher than the IVD industry, at a CAGR of 4.2 percent for the same time period. Increasing demand for a few conventional diagnostics products t including immunoassay products for infectious disease, cell culture testing, immunoassay for cancer diagnosis, and products available for genetic testing are key drivers.

The infectious disease molecular diagnostics immunoassay segment will witness important developments in field of reagent systems and automation and also the introduction of new products. The rate of market penetration and commercialization of products, however, will depend on innovative marketing approaches and automated systems with amplification methods. Integrating new technology planning with business, marketing, and corporate strategies will be a major challenge for diagnostics companies.

Indian Market

The Indian molecular diagnostics market is estimated at Rs.91 crore (does not include placed systems) in 2014. Reagents contributed Rs.90 crore and the analyzers a mere Rs.1 crore, which came from five numbers of standalone, real-time PCRs. All other systems were placed. These were placements of 4 numbers of extraction analyzers and 12 numbers of standalone PCRs. This is gradually emerging as an acceptable trend in the Indian medical ecosystem.

Installations included two numbers at NACO, 1 system at Metropolis Hospital, Mumbai; 1 unit at Dr Lal Pathlabs, Delhi; and 1 system at Shah Satnam Ji Speciality Hospital, Sirsa, Haryana.

Abbott Diagnostics, Roche Diagnostics India, and Qiagen India Private Limited are the leading players in the Indian market. LabIndia Healthcare, Bio-Rad Laboratories Inc., and some Chinese brands also have aggressive presence.

2014 saw the entry of Cepheid, with its expanded claims on its Xpert MTB/RIF test, which detects Mycobacterium tuberculosis complex DNA and, in positive specimens, provides simultaneous identification of rifampin-resistance associated mutations of the rpoB gene.

Technology Trends

Advancement and inclusion of modern technologies in genetic analysis have revolutionized the practice of medicine, improving the reproductive and prenatal care, enabling earlier and effective disease detection and advancing treatment of inheritable diseases. New and technologically advanced assays have been developed by market players, which are based on next-generation sequencing and microarray technologies assisting us to determine the true power of the genome. In future scenario, when laboratories would begin to take advantage and use these tools, the growth in healthcare would be revolutionary. The development of personalized medicines would be possible, consequently breaking the grounds to improve human health. Cystic fibrosis is one such disease which could be treated appropriately if detected in early stages and testing with molecular diagnostics techniques.

Next-generation Sequencing. NGS technology has become the norm of the day and large number of molecular diagnostics testing is dependent upon this platform for accurate, and comprehensive results. The growing popularity of NGS platform has paralleled the advancements in technology, particularly in the field of high throughput systems and bench-top sequencing, resulting in increased automation, ease to conduct tests and cost efficiency. Next generation sequencing is considered as the answer to all complex genomic research questions, majorly as it allows the user to rapidly sequence whole genomes, zoom in to deeply sequence target regions, utilizing RNA sequencing to determine novel RNA split sites and variants or more precisely for quantification of gene expression analysis, analysis of DNA-protein interaction and for studying microbial diversity in human beings. The efficacy and efficiency of NGS could be determined in a way that the Human Genome Project took more than 10 years and cost approximately USD 3 billion when conducted through capillary electrophoresis based Sanger sequencing. However, NGS makes it possible for an average researcher to make large scale whole scale sequencing accessible and practical.

Considered as the dynamic range for sequence based gene expression profiling, the technology is a digital alternative to analog technique, which let the user quantify RNA expression with the breadth of microarray and the resolution of qPCR. Microarray gene expression measurement could be limited by noise at the low end and single saturation at the higher side. In contrast to which, NGS is capable of quantifying discrete, digital sequencing read counts offering unlimited dynamic range. Moreover, the technology offers tunable resolution to meet specific experimental needs of the user. Targeting sequencing allows the user to focus on particular regions of the genome. For instance, researcher can select whether the analysis needs to be conducted on multiple samples through shallow scan or on fewer samples at greater depth to find rare variants in a given region.

Personalized Medicines. Personalized medicine therapeutics, also known as companion diagnostics is expected to be the future of the healthcare industry.. These tests are carried out once a disease has been diagnosed to analyze the best possible treatment method as per a genetic makeup. This method reduces the severity of adverse effects approach to drug therapies. For instance, anti-EGFE inhibitor treatment prescribed for colorectal cancer is only successful if the patient carries a particular genetic profile, which does not have mutations in the KRAS gene. Companion diagnostics testing performed on colorectal cancer not only helps in providing good treatment but also results in best use of scarce healthcare resources..

Biomarkers turning into companion diagnostics. Biomarkers are the biological characteristics, which undergo molecular, anatomic, physiologic and chemical changes during drug development research which results in turning biomarkers into companion diagnostics. These biomarkers could be used as basis for creating routine diagnostics test after they get approval to be used as in-vitro diagnostics tests. Large number of pharmaceutical and biotechnology companies are working hard on integrating this co-development concept but this phenomenon has been considered as rare until now. In current scenario, researchers are considering it quite challenging to find clinically predictive biomarkers early in a drug development program, majorly due to fact that they can only be determined on the basis of patient's responses to the drugs. The continuous efforts by the companies to use biomarkers for companion diagnostics are expected to make it successful.

Incorrect or inaccurate diagnosis of any disease is life-threatening for patients. Reliability of the relevant test is most important, especially when a disease has already developed inside the body. Molecular diagnostics testing in this context is considered as far superior to conventional method of diagnosis. The accurate diagnosis makes treatment more effective improving the outcome and ultimately reducing the total healthcare costs. The speed and accuracy of these tests and efficacy associated with the results coupled with the continuous technological advancements occurring in molecular diagnostics arena will assist the molecular diagnostics in achieving an exultant position in the healthcare diagnostics sector.

Industry Speak

MIC Test - An Important Diagnostics Tool

Grieston Fonseca

Product Management-Microbiology,

Transasia Bio-Medicals Limited

Irrational use of antibiotics has led to an incremental rise in drug resistance in India. The focus has always been on tackling resistance to TB drugs and antibiotics for other Gram negative bacteria. Lately, other Gram positive bacteria are also developing resistance, demanding immediate attention.

Microbiological diagnostics testing plays a crucial role in identifying infections and advising appropriate antibiotic treatment.

Importance of MIC Test. The minimum inhibitory concentration (MIC) test is important in diagnostics laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. Clinicians use MIC scores to select and determine the type and effective dose of antibiotic for patients with specific infections.

The interpretation of MICs requires an understanding of the mode of action of different antimicrobial classes and PK-PD studies. The available guidelines, especially CLSI & EUCAST, help in interpretation of MICs.

Method. Agar diffusion tests are often used as a qualitative method to determine whether a bacterium is resistant, intermediately resistant, or susceptible. However, the agar diffusion method can be used for determination of MIC values provided the necessary reference curves for conversion of inhibition zones into MIC values are available.

Broth dilution is another method of choice. In this method, identical doses of bacteria are cultured in wells of liquid media containing progressively lower drug concentrations. The minimum inhibitory concentration of the antibiotic is between the concentrations of the last well showing no bacteria growth and the next lower dose, which allowed bacterial growth.

Standardized commercial kits available for MIC testing in broth micro-dilution provide reliable results that can be adapted well by hospitals and reference laboratories for routine or specific use. The diagnostics importance of MIC test and its interpretation by the clinician has been widely discussed in recent times by the microbiological fraternity.

Most laboratories prefer MIC testing in broth dilution method. However, this method requires the use of a full panel format. ERBA Lachema, Czech Republic (a subsidiary of the ERBA-Transasia Group) has recently introduced single strips for individual antibiotics. The Mikrolatest MIC are single strips available in broth dilution format and are the first of its kind in India. Economically priced, these strips can be effectively used to confirm resistance patterns, in labs with or without automation.

The Mikrolatest MIC is exclusively available in India through Transasia Bio-Medicals Limited- India's leading in-vitro diagnostics company.

Second Opinion

Ushering in a New Era

Dr Meenakshi Sharma

Consultant Pathologist,

Manipal Hospital, Jaipur

Diagnostic tests are the foundation of a successful healthcare system providing critical information needed to make right medical decision. Molecular diagnostics is a dynamic and transformative area of medicine which detects and measures the presence of genetic material or protein associated with a specific health condition or disease.

Human body processes are driven by the interaction of genes and the proteins they produce that carry out specific functions. Molecular diagnostics helps assess an individual's health at this molecular level, uncovering the underlying mechanism of disease and enabling clinicians to tailor care at an individual level - facilitating the practice of personalized medicine.

The most commonly used techniques employed to detect and quantify specific DNA, RNA, and proteins are - polymerase chain reaction, in situ hybridization (ISH or FISH), chips and microarrays, mass spectrometry, and sequencing.

Clinical role. Molecular diagnostics are now becoming an essential tool in management of genetic diseases, infectious diseases, and oncology including risk assessment, screening, diagnosis, staging and prognosis-Mamma print, therapy selection-Her 2 in breast cancers, recurrence and therapeutic monitoring, and HIV viral load, BCR-ABL mutation in CML.

Scope. The overall global market for diagnostics was valued at USD 45.6 billion in 2012 of which USD 5 billion (11 percent) is contributed by molecular diagnostics. Globally, this is estimated to grow over 12 percent annually through 2017. The revenue currently is dominated by infectious disease testing (50-60 percent) with about another third attributable to genetic testing. But it has tremendous scope in pharmaceutical industry research, for discovering the next breakthrough targeted therapies.

Future. New technologies are emerging making it possible to gather more information to more fully characterize the disease state, the best treatment alternatives, and to rapidly identify a specific pathogen. Approaches that may address this need include a multiplex approach and gene sequencing that can detect multiple defects in a single assay.

Second Opinion

DNA Sequencing - The How and Why

Dr Shelly Mahajan

Visiting Scholar, Department of Pathology,

Penn Medicine, University of Pennsylvania, USA

DNA sequencing methods are used in pathology practice for diagnosing diseases by detecting genetic alterations. These techniques play an increasingly important role in diagnosing and prognosticating diseases. They also play an important role in predicting and monitoring patients' response to therapy. Human cells contain DNA (the blueprint that cells use to store & transmit information) that contains two strands of organic molecules called nucleotides, which encode genetic information. These nucleotides are arranged in specific sequences and make about 40,000 genes divided across 46 chromosomes. In turn, genes produce proteins depending on this sequence. This entire set-up of nucleotides, genes, and chromosomes constitutes a person's genome inherited from his/her parents.

The current process of DNA sequencing starts by breaking the bonds between its two strands by either heating to about 95 °C or placing the DNA in an alkaline solution. Then a DNA probe is used to identify the relevant portions (target sequences) of the genome. The probe is also made of DNA, but of a sequence that is complimentary to the target sequence, enabling the probe to bind to its target. This process is called hybridization. This forms the basis of most genetic tests to analyze specific portions of human genome. NextGen Sequencing (NGS) makes this identification easier and more comprehensive as it uses software packages that use bioinformatic alignment of sequences as opposed to traditional methods.

DNA sequencing has made it possible to identify and locate the gene sequences, and to compare and contrast genes between individuals. This helps identify diseases by distortion in amount and functions of genes. This may help in diagnosing cancer, customizing, predicting, and monitoring response to therapy, predicting relapse of disease, and monitoring tumor burden during therapy. Apart from oncology these molecular diagnostic techniques are also used in diagnosis of infectious diseases, inherited diseases, and identity tests.

Second Opinion

Rapid Molecular Diagnosis of DR-TB

Dr B Rayvathy

Assistant Professor, Department of Microbiology,

Dr. A. L. Mudaliar Post Graduate Institute of Basic Medical Sciences (ALM PG IBMS), Chennai

Tuberculosis, especially the drug resistant tuberculosis (DR-TB), has now become a major health problem worldwide with mortality ranging from 1.6 million to 2.2 million per year. In India, the incidence of MDR-TB has been found to be 1-3 percent in newly diagnosed cases and 12 percent in re-treatment cases. Rapid diagnosis of MDR-TB is essential for prompt initiation of effective second-line therapy to improve treatment outcome and limit transmission of this obstinate disease. Line Probe Assay (LPA) and GeneXpert are two promising molecular methods endorsed by WHO. The advantage of using molecular methods is that there is no need for growth of the organism and DST results can be determined in days rather than weeks.

LPA. This DNA strip test allows simultaneous molecular identification of tuberculosis and mutations causing resistance to rifampicin and isoniazid. This technology can diagnose MDR-TB directly from smear-positive sputum samples, providing results in just five hours which is an enormous improvement on the 1 to 2 months needed for conventional DST for 1st line drugs. The procedure is to extract the DNA, subject it for PCR amplification using biotinylated primers and hybridizing the biotinyalted PCR products with specific oligonucleotide probes immobilized on a strip. Captured labeled hybrids are then detected by colorimetric method using Biotin Avidin Alkaline phosphatase system resulting in formation of bands, which are compared with standard patterns.

GeneXpert. The next important method which has gained much importance in the recent times is GeneXpert which is a cartridge-based, automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to rifampicin (RIF) by nucleic acid amplification technique (NAAT). It is a hemi-nested real-time PCR of MTB-specific region of rpoB gene, which is then probed with molecular beacons for mutations. It is very simple to perform and is not prone to cross-contamination. It requires minimal biosafety facilities and training of man power. It can also be used to diagnose sputum negative and extra-pulmonary tuberculosis cases.

Though both these tests have proven to have good sensitivity and specificity they still have disadvantages like inability to detect heteroresistance and over-diagnosis of silent mutations. So in general, rapid molecular methods cannot replace conventional DST, but rather serve as a rapid screening method and/or supplement to conventional culture and DST.

Second Opinion

Innovations in Imaging

Dr Shah Hina J

Junior Consultant,

Department of Nuclear Medicine and PET/CT,

Jaslok Hospital and Research Centre, Mumbai

Radioactivity when used in a constructive manner can be useful in multiple ways in disease management - in diagnosis and therapy.

Radionuclides are tagged with different pharmaceuticals based on organ of interest to form a radiopaharmaceutical which is then used in diagnosing disease at molecular levels and thus help in making correct treatment decisions. These procedures are performed in dedicated nuclear medicine departments. Nuclear medicine is slowly growing and is now available in tier-II and tier-III cities. Molecular imaging using radionuclides consists mainly of two imaging modalities - single photon emission computed tomography (SPECT) and positron emission tomography (PET) scans. SPECT scans are used in diagnosing benign and malignant disease of various organs like brain, thyroid, parathyroid, kidneys, hepatobiliary, and gastrointestinal tract. PET scans are used in three main disease areas - oncology, neurology, and cardiology. Both these scans require specific radiotracers and imaging cameras.

Fluorodeoxyglucose which is mainstay of PET imaging exploits the increased glucose requirement by tumor/infective and inflammatory cells and non-invasively map the diseased areas in the body. There are now specific tracers available which look at specific receptors in the body and provide a non-invasive tool in diagnosis and also provide treatment options in some cases. If positive, one can also be provided with the treatment option again with the help of a radionuclide by replacing the diagnosing tracer with more lethal isotope (177-Lutetium DOTATATE or 90-Yttrium DOTATATE) which will reduce the tumor burden and cause improvement in quality of life in patient. The therapy with these tracers can be provided at a very low cost due to local availability of these radioisotopes by BARC.

The imaging equipment are also now made in India. Though this is in its initial stages, there is tremendous growth ahead and this will make the supply easier and faster.

The future for molecular imaging is exciting. With the advent and availability of new tracers and probes like PSMA for prostate cancer, exendin-4 for insulinoma and many more, this field is having tremendous growth. Similarly, there are parallel developments in the field of technology in the form of availability of new imaging machines like PET-MRI which will take fusion imaging to next level and new software algorithms which will help reduce radiation burden and provide accuracy. Lots of research is going in multiple areas to exploit the use of different radionuclides in management of array of diseases and when this research will see the light of day and be available in clinical practice, it will not only help physicians in disease management but also lead to overall improvement in patient care.


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