The use of molecular diagnostics has accelerated in recent years, and what was once a technique reserved for specialized centers is now in regular use in most microbiology laboratories. Indeed, integrated molecular diagnostics have now become a feature of even basic laboratories, targeting diseases such as tuberculosis, human immunodeficiency virus, methicillin-resistant Staphylococcus aureus, including the ability to diagnose drug resistance. The major use remains in the virology world, where molecular diagnostics have been able to replace lengthy culturing methods enabling safe and quick diagnosis. The full rollout of molecular tests in other fields has been slower to take hold, but with rapid advances in sequencing and the relative in expense of new technologies it will not be long before these techniques are widespread.

Multiplex polymerase chain reaction (PCR) has become very popular for diagnosing infections of intestinal tract and respiratory tract. Although most laboratories still use conventional cultures, new systems have been introduced to automate the entire laboratory processes with digital imaging technology. Efficiency, limited personnel, and financial resources have motivated laboratories to implement these systems. Point-of-care-testing (POCT), with the advances in technology and connectivity, has become a prominent laboratory service. It can significantly improve TAT by shortening pre- and post-analytical times. POCT requires an inter-disciplinary effort to ensure that quality results are obtained and its cost-effectiveness can be realized under controlled conditions.

Global market trends. The global molecular diagnostics market is anticipated to grow at a CAGR of 11.1 percent during 2013-19 to reach USD 8.7 billion. The rising geriatric population and the increasing prevalence of chronic diseases is fueling the growth all over the world. The next-generation sequencing market is emerging as the fastest growing technological segment of the global molecular diagnostics industry, reporting rapid growth. The brisk turnaround time of the next-generation sequencing technique has helped it gain momentum. The cost reduction of sequencing has also added to the growth of this market segment. In addition to this, the PCR market segment, which had once dominated the global molecular diagnostics market, is projected to lose ground on account of the regular advancement in high yield sequencing technologies.

Automated analyzers making way to diagnostic laboratories. With the arrival of the automated molecular diagnostic analyzers, the molecular laboratory underwent rapid growth and expansion. Automated analyzers provided a number of benefits - the ability to process a large number of samples simultaneously; the elimination of human error to increase accuracy and reliability of results; the reduction in labor costs to enable the performance of complex genetic testing. Automated liquid handlers can be integrated onto a singular automated platform that can automate most of the manual preparation steps and thermal cycling. By adding a data analysis platform to the back end, the system is able to achieve a high degree of semi-automation for a completely manual lab testing process. The automated liquid handlers, with their open architecture, help to automate the entire test process, no matter what the test process is, as long as it can be split into logical steps.

The evolution of the molecular analyzer has led to the advent of two additional types of analyzers. The first is the sample-to-result analyzer, which can take any test from the primary sample tube without the need for any operator manipulation of the sample. Essentially, this directly evolved from the previous iteration of automated analyzers by adding on to a primary sample handling system. While it may seem like a relatively simple matter to improve, the benefit of this is nonetheless extremely significant since it removes a key complication in infectious agent testing. The second new development is the emergence of the high-throughput systems. These new-age analyzers can provide an output of several thousand sample results in the same amount of time. In most cases these analyzers are not as automated as any of their ancestors, but their ability to run at rapid speeds compensates for their higher manual requirement.

Biomarkers invading the field of diagnostics. Biomarker detection research is pursuing clinically significant performance of diagnostic technologies through two main paths. On one side, there is the need for ultrasensitive detection of few biomolecules per specimen for early disease diagnosis and surveillance. On the other hand, state-of-the-art analytical technologies may have already reached appropriate sensitivity, but have too slow TAT to warrant sufficient clinical outcomes to benefit the patient. Therefore, diagnostic devices with TAT compatibility are a promising transformative area of research.The exponential increase in the use of molecular biomarkers as diagnostic, prognostic, and predictive aids in the management of cancer patients highlights the increasing importance of molecular biology in oncology.

Compared to available analytical methodologies, which provide sensitivity with costly, dedicated and sophisticated machinery in centralized labs, new devices are designed to be used at the proximity of the patient, whether this means at the patient's home, the doctor's office, the emergency room or in small decentralized, capillary spread laboratories. Ideally, such new devices would also provide less invasive sampling method and no requirement for sample pretreatment and specialized skills, like the analysis of a blood drop from a finger-prick or saliva from an oral swab. In such a scenario, the target analyte concentration in the biological fluids may fall in a non-analytically challenging range. However, sensitivity issues may still arise as a consequence of the miniaturization and handling of small amount of patient's specimen containing few analyte molecules.

Sample-to-use and digital PCR. Polymerase chain reaction (PCR) is improving patient care in the molecular laboratory through accuracy and timely results. The goal of molecular diagnostics is to create a sample-in answer-out system that is easy to use, fast, and cost-effective in order to drive faster time to result. To make PCR more relevant and enable it to be more widely used, the emphasis has been on automating the workflow from the sample to answer. Every step - sample introduction, lysis, nucleic acid purification, PCR amplification, read-out of results - is being integrated into a solution that provides actionable results in less time in order to have a positive impact on patient care. Currently, the approach is toward simple, walk-away sample loading. This has been accomplished with swabs for respiratory and sexually transmitted infections by expressing the sample into an elution, vortexing (if required), and introduction into a machine. Blood culture tests are also available for gram-negative and gram-positive bacteria and yeast. Fecal samples can be introduced into a system to test for bacteria, parasites, and viruses.

Second Opinion
Advances in MDx Instruments

In the last few years, molecular diagnostics (MDx) became an integral part of laboratory medicine. Moledular diagnostics are most specific PCR-based tests used for diagnosis and therapies of various diseases. These teste involve sophisticated instruments such as PCR, real-time PCR, automated DNA sequencer, microarrays, and next generation sequencer (NGS). Thermal cycler or PCR machine is the most commonly used machine to amplify segments of DNAs by the PCR. Modern PCR machines are equipped with multiple blocks and also have a gradient function to create different temperatures in different parts of the block. PCR machines are marketed by several international branded companies as Perkin Elmer, Bio-Rad, Life Technologies, Roche Diagnostics, and Applied Biosystem.

For further advancement in PCR technology, there is an introduction of real-time PCR (qPCR), which is very useful machine for quantitative analysis of various diseases such as infectious diseases, cancers, and genetic disorders. Real-time PCR machines are introduced by several international companies such as Roche Diagnostics, Applied Biosystem, Stategene, and Qiagen.

Next useful machine in molecular diagnostics is DNA sequencer, which plays an important role in evaluating disease causing mutations. These machines are marketed by international companies such as Applied Biosystem and Siemens. This machine is extensively used in completion of human genome project. Microarray platform has also been used in multiplex analysis and diagnosis of various hereditary and genetic diseases. In recent years, there has been a fast development and wide applications of NGS technology to get complete genomic sequence information for decoding life mysteries, NGS systems are marketed by their brand names - SOLiD/Ion Torrent PGM by Life Sciences, Genome Analyzer/HiSeq 2000/MiSeq by Illumina, and GS FLX Titanium/GS by Roche Diagnostics.

Overall, there is a great advancement in molecular diagnostics instruments for diagnosis and therapies of several complicated diseases.

Dr Pravin D Potdar
Head-Department of Molecular Medicine & Biology,
Sir Ganga Ram Hospital, New Delhi

Second Opinion
Newborn Screening - Role of Genetics

The newborn health challenge faced by India is bigger than that experienced by any other country. India contributes to 17.5 percent of the world's population and nearly 20 percent of the total live births. Its contribution to the global burden of newborn deaths is higher when compared to that of maternal and under-5 deaths. 16 percent of global maternal deaths, and 21 percent of under-5 deaths occur in India. A staggering 27 percent of newborn deaths occur in India.

Preventable newborn deaths account for 44 percent of all deaths among children under-5 globally. Incidences of inborn errors of metabolism (IEM) have been found in 74 of every 1000 babies screened in India.

Newborn screening programs. Recent advances in biotechnology have been utilized to craft newborn screening programs. Carried out via tandem mass spectrometry, these tests are aimed at early detection of serious disorders in asymptomatic newborns (for example, serious IEMs like amino acid disorders, fatty acid disorders, sugar metabolism disorders, and peroxisomal disorders) which facilitate elimination or reduction of associated mortality, morbidity and disabilities. These tests are often offered in the hospitals themselves, right after birth, before discharge of the mother and infant, for convenience and early identification of health issues, which could be addressed by timely interventions, such as dietary restrictions and enzyme/cofactor replacement therapy.

Conventionally, this kind of screening is aimed at diagnosis of disorders that are believed to be a serious health problem, and required immediate medical intervention in order to prevent serious and permanent illness, and for which treatment is available. With the increased understanding of capabilities that genomics has lent to healthcare, this screening can be extended to identify the underlying genetic basis of such diseases. Although the emphasis on common complex cases is understandable, more might be gained by focusing on rare diseases. A large number of individuals carry rare variants that might provide preventive advantages if knowledge about their genetic risk are available. Genomic firms can play a pivotal role by ensuring availability of newborn genetic screening tests to the medical community.

Director-Corporate Affairs,
Medgenome Labs Private Limited, Bangalore

Traditional PCR is an end-point analysis that is semi-quantitative as the amplified product is detected by agarose gel electrophoresis. Real-time PCR (qPCR) requires normalization to controls (either to a reference curve or to a standard curve), allowing only relative quantification. Furthermore, variations in amplification efficiency may affect qPCR results. Digital PCR (dPCR), on the other hand, has enabled precise and highly sensitive quantification of nucleic acids. dPCR builds on traditional PCR amplification and fluorescent-probe-based detection methods to provide highly sensitive absolute quantification of nucleic acids without the need for standard curves. dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. Digital PCR is a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. This technique is particularly useful for low abundance targets, targets in complex backgrounds, allelic variants, and for monitoring of subtle changes in target levels.

Future Outlook. Technologies will continue to evolve, allowing faster, more sensitive, and less expensive methods for pathogen surveillance and discovery. Although multiplex PCR is relatively mature, microarray technology is still in its infancy; near-term modifications already in development include microfluidic sample processing and direct measurement of conductance changes associated with hybridization. In chronic diseases the most substantive advances are likely to come not from technical improvements, but from investments in prospective serial sample collections and an appreciation that many diseases reflect intersections of genes and environment in a temporal context. Rapid development in genetic based molecular diagnostics has made wide application of tools, from basic research to detection of abnormalities in human health, possible. Soon, portable, hand-held, inexpensive POCT devices, equivalent to currently offered full-size systems will become available to aid in the detection of mutations or in the identification of infectious agents. The advent of analyzers with novel technologies may soon address the challenges faced while implementing genetic-based molecular diagnostics at a clinical stage. As these new trends develop, they reflect the increased rate of adoption of molecular medicine in the clinical lab and signal the impending transformation of healthcare, which may soon emerge to push the molecular diagnostics laboratory into yet another stage of evolution.

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