An acid test for better diagnosis

Well begun is half done. An accurate diagnosis on time often proves to be the lifeline for patients suffering from serious diseases. Nucleic Acid Test (NAT) makes all this possible, Sushmi Dey finds out.

India, being a tropical country, is prone to several infections and outbreaks of diseases. According to recent United Nations report on HIV, India has the highest number of people living with AIDS (5.7 million) in the world and the overall Indian HIV cases account for two-thirds of Asia's total. This calls for an increasing need for fast and specific diagnosis and confirmation of diseases in order to control potential epidemics. All this and more has proved to be the contributing factor in the need for adopting and implementing Nucleic Acid Test (NAT) in India. NAT has also proved successful in ensuring blood safety to prevent transmitted infections.

NAT has been adopted by most of the developed nations of the world. The test is in use in USA, Canada, Brazil, Mexico, Australia, New Zealand, Japan, most of Europe, South Africa, Thailand, South Korea, Singapore, Hong Kong, Indonesia, and Malaysia. However, unlike most other countries NAT is not mandatory in India. "There is lack of awareness about blood safety among decision making bodies in India. The government also poses challenges by limiting the cost that can be charged for testing blood. Their role should be to enhance the minimum standards and not try to prevent better practices," opines Dr R N Makroo, Director, Department of Transfusion Medicines, Indraprastha Apollo Hospital.

Why is NAT better?

NAT is a method of testing blood that is more sensitive than conventional tests that require the presence of antibodies to trigger a positive test result. While an infection occurs, NAT is used to detect the low levels of viral genetic material present in the body. However, it happens before the body begins producing antibodies in response to a virus, giving the ability to detect a disease at an earlier stage.

According to Naveen Kulkarni, CEO, Polyclone Bioservices, NAT developed from a molecular biology technique called Polymerase Chain Reaction (PCR). PCR technique is based on amplifying a template DNA (sample) using a set of small DNA sequences called primers. The design of the primers and additional components like fluorescence, modifications etc will define the sensitivity and specificity of the PCR. "In NAT, the viral particles are lysed and then the viral nucleic acids are captured. The captured nucleic acids are then amplified to create billions of copies and specially prepared chemiluminescent probes get attached to the amplified products (amplicons). The chemiluminescent signal is measured by a luminometer and is reported as relative light units and shows the presence or absence of a particular viral nucleic acid," explains Sumit Bagaria, President, Hemogenomics. The presence or absence of the "product" determines the presence or absence of the infection whereas, the amount of amplification determines the seriousness of the infection. According to Kulkarni, such determinations help doctors to adjust the dosage of the medication depending on the extent of viral presence.

Across all diseases

Due to its ability of early and accurate diagnosis of diseases, NAT has been used in diagnostics for many years now. NAT plays a major role in diagnostics, detection of infectious diseases like HIV, Hepatitis C, STDs, tuberculosis and even in emerging tests for GBS, MRSA and respiratory viral infections. "NAT helps in the identification of microbacterial, fungal and bacterial pathogens. This helps in the timely treatment of these diseases," says Bagaria. In case of tuberculosis, the PCR methodology has been so designed that it not only detects the Tubercle bacilli but also determines the type of tuberculosis.

NAT is also useful in cancer and genetic testing as well as in NAT based blood donor screening. "The cancer sub-segment includes HPV testing as well as PCR, FISH and other technologies used for a variety of cancer types. Genetic testing includes, but is not limited to, CFTR, HLA, thrombophilia, chromosomal disorders and pharmacogenetic testing," tells Kulkarni. "Molecular techniques are being harnessed for the detection of organisms in a wide range of circumstances. Global trade has introduced new pests of crops and plants; simultaneously, there is universal concern from disease outbreaks such as BSE, foot and mouth and the dreaded bird-flu virus. Molecular diagnostic applications for the detection of food borne bacterial pathogens and genetically modified organisms in food are also becoming increasingly important," opines Dr Vijay Sharma, Technical Director, Stempeutics. According to Sharma, the PCR is also instrumental in determining whether a baby born to a HIV positive mother is infected with the virus or not.

NAT vs conventional tests

NAT is a direct test where it can detect the specific viral nucleic acid (RNA/DNA) for the identification of pathogenic organisms. According to Kulkarni, nucleic acid assays are also instrumental in monitoring a single cancer cell, microorganism, or as few as 50 copies of DNA/RNA (viral load). Genotyping allows the detection of minor variations in base sequences observed within an infectious agent. In comparison to viral cultures or patient response, molecular assays provide a more rapid turnaround time so that therapy can be initiated more quickly. "In terms of sensitivity and specificity, NAT has a clear advantage over conventional immunoassay or serotyping, as it is not dependent on the operators' personal judgment, and these inherent advantages are drivers for their increasing adoption worldwide," says Kulkarni.

According to Dr Satyajit Patra and Dr Satyamoorthy, Director MAHE Lifesciences Center, conventional diagnostic methods such as cell culture, antibody testing, and even symptom diagnosis consume considerable time as compared to NAT. While NAT significantly reduces the window period or the time between donor exposure to the virus and the appearance of detectable amount of antibodies, the conventional methodologies are incapable of addressing this primarily because these methodologies detect the antibodies formed by the human immune system against the virus.

NAT combines the advantages of direct detection of the organism with sensitivity, several orders of magnitude higher than that of traditional methods. By decreasing the window period, it allows for earlier detection of the infection. "The window period could be as short as two weeks to a few months. The PCR technology is used for early detection of the disease. This technique is not only highly specific but also highly sensitive and can pick up early infections," said Sharma. However, experts maintain that conventional serology testing is a good technology and the time taken in NAT is not as short as ELISA. "NAT should be used as an additional test to serology and together both the tests can make the blood supply safer than never before," suggests Makroo.

Advantages galore

Apart form being a specific, sensitive and effective method for several applications in the molecular diagnostic area, including clinical, industrial, veterinary and research applications; NAT also has advantages in terms of throughput, cost and time. NAT screening has a very high throughput. Automated NAT systems are also available now. "Semi-automated modular systems are more cost-effective in the Indian scenario and have minimum manual steps. In a semi-automated NAT system one person can test about 180 samples in less than seven hours with one set of instruments. The automated systems can do up to 1,000 tests per day," informs Bagaria. Conventional diagnostic methods such as cell culture, antibody testing, and even symptomatic diagnosis consume considerable time as compared to NAT.

Interestingly, NAT also offers multiplex assays where all the viruses (HIV, HBV and HCV) are simultaneously tested. Another important advantage of NAT is the Individual Donor Testing (IDT). According to Bagaria, pool testing often reduces the sensitivity of a particular test. IDT NAT has the advantage of highest possible sensitivity, minimum manpower, less capital cost and high throughput for releasing blood and blood products.

The flip side

"There are no drawbacks of NAT because all the work comes under the NAT includes in-vitro assay like collection of blood, tissue from patients and processing it inside a lab," say Patra and Satyamoorthy. However, Sharma believes that every technology has its drawbacks. "The high sensitivity and specificity of the test which are advantages can be turned into a disadvantage in untrained hands," opines Sharma. Cross-contamination of samples can yield disastrous results. To prevent any side-effects or mistakes in diagnosis the infrastructure set up including use of certified material and controls, training and awareness about NAT is essential.

Tthough the initial adoption of NAT has been slow in India, with increasing awareness the technology is now becoming a preferred diagnostic tool. The HIV boom has also helped in growing the market for NAT. "When we initially started NAT for tuberculosis detection the number of samples would range from four to five per month but with in a few years the samples touched approximately 100 per day in pioneer laboratories. The demand is ever increasing and as the number of infectious agents increase the NAT is now not only restricted to infectious diseases but also human genetic disorders, HLA typing for bone marrow transplants," says Sharma.

"The overall diagnostic market is estimated to be over Rs 1,000 crore and growing at the rate of 20 percent annually. Of this NAT is expected to start with about 20 percent and is expected to grow to atleast Rs 500 crore in the next three years," opines Kulkarni.

sushmi.dey@expressindia.com