by Jimmy LOH Jin Phang
e live in uncertain times. Bioterrorism represents a real threat whose ominous shadow overhangs the globe. The dreaded severe acute respiratory syndrome, commonly known as SARS, has quickly become a modern scourge that transcends all boundaries. Thus it is in the interest of every nation that researchers develop effective methods for detection and identification of infectious pathogens to preserve national safety and security.
Scientists have been increasing the use of molecular methods over the past ten years to improve the sensitivity and speed of diagnostics. Although the routine use of molecular methods remains limited to the detection of pathogens that are difficult to culture in vitro, real-time methods, commercial kits, quantification, and automation will increase potential applications.
The use of molecular methods, such as the polymerase chain reaction (PCR) process which amplifies DNA, has proven very sensitive and specific for detection and identification of pathogens in clinical specimens. In addition, these methods are now widely used for epidemiological fingerprinting of isolates that are of public health importance. Sequence-based identification and molecular strain typing will also assist in disease management and control.
The Defence Medical Research Institute (DMRI) of Singapore's Defence Science and Technology Agency (DSTA) has for the past three years been developing a real-time PCR diagnostics program to detect and identify infectious pathogens (Figures 1 and 2) as part of the nation's preparedness against bioterrorism. In particular, its Biomedical Sciences Laboratory (BSL) has been developing diagnostic assays that involve selecting gene targets unique to the bacterium or virus in question, designing PCR primers against the gene target, testing the PCR assay against reference strains of the bacterium or virus, and using clinical samples for validation.
With molecular diagnostics, crude specimens can be processed in a relatively short time. The process essentially involves extraction of nucleic acids (DNA/RNA) from the crude sample, followed by PCR analysis to detect the pathogen in question. Conventional PCR methods require at least two hours, followed by gelelectrophoresis analysis, which could take three to four hours. With real-time PCR on a capillary thermocycler (Figure 3), the whole process can be completed in less than 40 minutes. Therefore, detection and identification of causative pathogens, which used to take three to four days, now takes less than one day.
As a result of the project, DMRI's BSL has developed the capability to carry out a rapid response to bioterrorism as well as outbreaks of public health infectious diseases. The ability to respond quickly is crucial to identification and containment of the disease. Below are a few examples of actual applications of molecular diagnostics developed by BSL in outbreak investigations.
Gastroenteritis Outbreak Investigations
Gastroenteritis (GE) outbreaks occur fairly commonly, especially in urbanised countries where large concentrations of people consume food and water from common sources. Although investigations using conventional methods such as culturing have become routine, the investigators' success rate in finding the causative pathogen is low because routine culture methods traditionally involve testing for a small number of common pathogens. Specialised culture methods for rare, exotic or emerging pathogens usually suffer limitations and thus easily get overlooked.
Other problems include the possibility of difficult-to-culture causative pathogens owing to a slow growth rate or to fastidious growth requirements. After suspected pathogens have been successfully grown from sample specimens, investigators carry out identification using routine biochemical assays. By the time they complete the whole process, more than two days may have passed.
Research staff members at DMRI have developed real-time PCR diagnostics for several common GE causative pathogens, while carrying out diagnostic testing for the Singapore Armed Forces (SAF) during GE outbreak investigations. In one such outbreak, Salmonella enteritidis DNA was detected in stool samples of soldiers during an investigation. The time taken from the receipt of samples to the confirmation of results via real-time PCR was less than three hours. Final confirmation of the test results by culture methods took three days.
In a separate outbreak, large amounts of Clostridium perfringens DNA were detected in soldiers' stool samples. Results obtained in less than four hours' time were conveyed to the SAF, which was still conducting the investigation. Following this lead, the epidemiological team re-examined the freezers of the canteen. One of the freezers was found to be malfunctioning, and the food stored there was being held at sub-optimal temperatures.
Bacterial Meningitis Investigations
Bacterial meningitis, a severe infection affecting the central nervous system, can be deadly. Current laboratory diagnostic methods culturing blood to identify bacterial meningitis take 36 hours or more. In addition, the mounting practice of starting anti-microbial therapy prior to clinical-sample collection has hindered culture-method confirmation of bacterial meningitis pathogens.
Using real-time PCR, DMRI researchers have also been successful in detecting Neisseria meningitidis in blood and cerebrospinal fluid of patients suspected of suffering from bacterial meningitis within three hours after receipt of the specimen. PCR tests were also done to eliminate other possible causative pathogens such as Haemophilus influenzae and Streptococcus pneumoniae. Quick and accurate diagnosis in these cases allowed more specific treatment and enabled physicians to concentrate on the problem at hand as opposed to treatment of an unknown infection.
Investigations into viral diseases prove just as difficult as, if not more difficult than, those into bacterial diseases because of the need for specialised tissue-culture facilities and equipment. The SARS outbreak needs no introduction, and the pooling of resources among 11 worldwide laboratories to develop diagnostic assays highlights the work required. DMRI research staff support the Singapore General Hospital Pathology Department in this respect and work towards development and validation of a molecular diagnostic test. Development of such tests for formerly unknown viruses usually entails broad-spectrum PCR using generic primers followed by sequencing to identify the viruses.
Since the Sept 11 and anthrax letter incidents, bioterrorism has increasingly become a major concern of the world. To deal with the threat of bioterrorism, staff at DSTA's DMRI have developed a full range of real-time PCR diagnostic assays for the detection and identification of biothreatening agents such as anthrax, plague, and tularaemia. DMRI, in its new building next to the National University of Singapore's Clinical Research Centre, will also set up a P3 laboratory to further develop diagnostics for Biosafety Level 3 pathogens such as Bacillus anthracis and Yersinia pestis. This laboratory will also provide the safety and security for diagnostic testing of such samples when required.
For more information contact May Ann Lee at firstname.lastname@example.org