by Lay Leng TAN
ince a few years ago, the dreaded H5N1 avian flu has been affecting and killing people coming into contact with infected poultry, causing great concern in Asia. As the disease spreads through migratory birds to farm-bred poultry, it is critical to identify and localise animals carrying the virus before the deadly virus spreads to humans. The present determination of the disease usually relies on clinical symptoms, with high likelihood of wrong or missed diagnosis. Furthermore the available test takes more than one week to know the results, by which time the affected bird might have passed on the infection to other birds or even humans.
If the bug is caught early, the relevant authorities can take measures to reduce the chances of spread by culling infected birds, disposing carcasses properly, as well as cleaning and disinfecting affected areas. Doctors can use appropriate antibodies and antiviral therapy on affected patients; the prompt treatment will also reduce the possibility of antigenic shift where the virus can mutate into another strain.
Veredus Laboratories, a young Singapore medical diagnostics research company focusing on diagnostic assays for tropical infectious diseases, has come up with one of the world's first validated commercial avian flu diagnostic kit that drastically cuts the time required to accurately detect the H5N1 virus from 7 days to as short as 2 days. Although laboratories in China and Australia have developed such DNA kits, this Singapore product represents one of the first to be available in the market.
The kit uses patented nucleic acid diagnostic primers developed by the Genome Institute of Singapore. A primer is a short strand of DNA/ RNA whose presence is required for the formation of a longer chain of DNA/RNA. It helps initiate the replication of the viral genetic material such that minute levels can be magnified and observed. The molecular diagnostics is so sensitive that it can pick up 5 femtogram ( 10-15g) or as few as 1 ,000 copies of viral RNA.
The VereAVF-H5N1 kit has been tested on avian samples in Vietnam and Malaysia with an accuracy of 99%, without cross reactivity to other pathogens. It was able to detect all H5N1 positive samples, and is specific to this particular strain of deadly virus out of the 15 known subtypes. Validation tests on human are now ongoing in Thailand.
Veredus also launched patented test kits for malaria and dengue based on breakthrough technologies from the National University of Singapore (NUS) and Institute of Molecular and Cell Biology. These mosquito-borne diseases affect millions of people in the tropics, with malaria killing some 3 million people worldwide every year.
The present "gold standard" for testing the malarial Plasmodium parasite is by examining stained blood smear on a glass slide under the microscope. It detects about 50 parasites per microlitre, which means patients with low numbers may be overlooked. The process is time-consuming, laborious and requires skill, especially to differentiate between species of Plasmodium.
The Vereplas and Vereplas fv kits use polymerase chain reaction (PCR)-based assays - a technique where tiny amounts of DNA are amplified - to distinguish Plasmodium falciparum and P. vivax under 3 hours. Using DNA amplification of specific target gene in the parasite with species-specific primers, the kit can confirm the type of parasite causing the infection. The simple but robust procedure just needs a drop of blood, even from 6-month-old dried human blood spot, to pick up as few as 4 parasites per microlitre. The researchers have tried the kit successfully on more than 3,000 samples from countries such as India, Pakistan, Laos, Singapore, Indonesia, and Colombia.
The malaria kit is ideal for mass screening of immigrants and travellers to prevent the spread of non-localised disease, screening in blood banks to ensure the integrity of blood reserve, in epidemiological studies to check for malaria in animals or vectors, and for treatment management to determine the effectiveness of various malaria treatment.
Dengue, a disease seeing a record rise in the region, particularly in Singapore, is caused by one of four related RNA virus of Flavivirus. Current diagnosis resorts to signs which are general and inaccurate. Haemagglutination Inhibition Assay and ELISA/ Dengue IgG/IgM Rapid Test Kits being used now produce inconsistent results, and are unable to pinpoint which virus is the culprit among the Flavivirus family.
The RT-PCR approach in the Vereden kit is a highly sensitive technique capable of detecting as few as 1,000 copies of dengue viral RNA; it can ascertain the virus within 3-5 days of viraemia (presence of virus in the blood) compared to the usual 8 days by immunodiagnostics methods. The technique can also be configured to differentiate the four dengue viruses.
The kit has been deployed in Singapore by the Ministry of the Environment and the National University Hospital has used it for more than 3 years as a routine diagnostic test for dengue fever. NUS researcher Vincent Chow who invented the test has successfully identified dengue virus as the cause of the large outbreak of haemorrhagic fever in Karachi, Pakistan in 1994. The same technique correctly recognised the West Nile virus responsible for the 1999 outbreak of encephalitis in New York. Epidemiological tests can be performed in both human and mosquitoes.
For dengue and avian flu, the viral RNA has to be isolated first; 5 microlitres of sample are required for each test compared to the drop of blood for malaria testing.
The three kits allow multiple screening of 96 samples in under 4 hours, and can be adapted for high-throughput screening of up to 1,536 samples at a time. The training time takes only 15-30 minutes. Currently, the kits are done with gel-based PCR as it is more widely used and much cheaper. The PCR tests can also be run on any standard thermocycler available in the market or in a real-time reader to cut down the process to 2 hours.
Rosemary Tan, chief executive officer of Veredus, reveals that the kit for dengue was developed over 10 years; learning from the experience, the avian flu product was ready within a year. She says that the kits will be marketed to hospitals, diagnostic centres, and laboratories. Recommended shelf life of the products is 6 months, although this can be prolonged to a year under optimum conditions.
Tan reveals that in the pipeline are a1-step test that will detect both malaria or dengue in one go; kits for encephalitis, SARS, yellow fever, Japanese encephalitis, and chicken pox; as well as cancer markers.
Click here to download the full issue for USD 6.50