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Development of a rapid sensitive microarray (microchip) assay, for detection of infectious agents in patient samples

The rapid advancement of DNA microarrays, lead to a revolution in the analysis of cellular gene expression in neoplastic and normal cells (ref 1-10). This technology, if applied to identification of infectious agents, empowers us with the ability to carry out rapid identification of infectious agents present in various patient samples. Using microchip based technology it will be possible to identify several hundred infectious agents simultaneously in one single assay.
Introduction

With the large number of tests available for the detection of infectious agents affecting humans, it is often not recognized that most of these tests are indirect. For infections caused by agents such as Hepatitis C virus (HCV), Hepatitis B virus (HBV), Human Immunodeficiency virus (HIV) or other agents both viral or bacterial origin, commonly used tests determine the presence of specific antibodies produced by the patient's immune system in response to infectious agent. These tests are useful for blood screening and in a limited extent as diagnostic tools, but, because they offer only an indirect measure of infection, they do not tell the clinicians whether the infection is past or current, or if there is a response to the therapy. An antibody based test can also miss a recent infection, because generally it may take several days to weeks for the immune system to mount an antibody response to the infectious agent. This can be especially dangerous if the infectious agent rapidly spreads in the patient or if the blood from an apparently healthy but recently infected person (HIV and HCV) is used for blood transfusion.

New generation of tests has been developed in the past, which are based on measuring the presence of the infectious agent within the patient sample directly. These tests detect the presence of nucleic acids (the genetic material) of the infections agents in the blood or other samples from the patient. Two major methods have been developed and accepted for detection of infectious agents in patient samples. The most common is the Polymerase Chain Reaction (PCR) which can detect 100 copies or more of an infectious agent in a single sample. PCR uses an enzymatic reaction to amplify specific nucleic acid sequences from the infectious agent if they are present in the sample. There are several problems with this method. PCR uses specific nucleic acid sequences (Primers) from an already known sequence of the infectious agent. Therefore, if the infectious agent has not been sequenced, PCR can not be used. Similarly, if the infectious agent mutates very rapidly, the Primers may not recognize the infectious agent and a false negative test will result. This is a major problem with detection of HIV which is causes AIDS, because HIV undergo mutations very rapidly, specially in response to drug treatment. Since the PCR uses an enzymatic reaction, the enzyme can be inhibited by impurities in the patient sample, giving false negative results. In addition the short sequence Primers are only specific for an infectious agent at certain temperatures, which make this test dependent on very strict conditions. The specificity of the primers makes it also difficult to detect more than one agent simultaneously in one PCR reaction. Multiplex PCR reactions exist, but generally they are not quantitative and can only detect two or maximum three agents concurrently. To avoid some of the problems with the PCR it is necessary to add a second method after the PCR step for analysis of the result. This second step is a hybridization method where the amplified product is detected by hybridization to an infectious agent specific probe. The problems with specificity and sensitivity in the PCR method make this test available only at specific laboratories with extensive experience in this assay. This means that the samples must be sent to an outside laboratory and detection of an infectious agent in a patient's sample may take between 24-48 hours and generally the test results are only reported after 3-7 days following the assay.

The second method is based on direct hybridization of the infectious agent's nucleic acid to a synthetic nucleic acid probe. The hybridized infectious agent is then detected by amplifications using non-enzymatic methods. The major drawback of this method is that it is less sensitive (minimum 1,000 copies most be present in a sample) and requires a larger amount of the patient's sample (minimum 1 ml blood compared with 0.1 ml for PCR). The method is also time and work consuming and can only done in specialized laboratories. It takes 36 hours to finish the assay and like PCR it can only detect one single infectious agent. However, because it is based on hybridization, it is not affected by minor mutation in the nucleic acid sequences.

Microchip assay
There is a need for a sensitive and rapid assay, which is easy to perform, can be used in laboratories with very little experience or no experience in molecular biological methods. The 'first generation' diagnostic microarray developed in my laboratory can detect thirteen infectious agents simultaneously, and can be expanded do detect several hundreds. This type of assay once automated could be performed in Hospitals and at offices of Physicians. This method addresses the problems associated with both earlier methods, and reduces the detection time to less than 30 minutes. Because it is based on a rapid hybridization and no enzymatic amplification is used, it is not affected by impurities in the sample. At the present it can detect 500 copies or more of an infectious agent per sample. It can be adapted to any type of sample such as blood, stool or tissues. The size of the chip is less than one cm2 and the active site is less than 1 mm2.

The chip has been tested for detection and quantitation of 13 infectious agent (11 viruses and 2 bacteria) and as control a human gene. Recently, it has been extended to twenty infectious agents. Analysis of the chip allows us to quantitate the amount of infectious agent present in the sample and correlate to the patient's symptoms. The hybridization data from the patient samples were compared to the data from traditional quantitative PCR where the same patient samples were analyzed. Click here to see a quantitative summary of the microchip hybridization and QA-PCR data for some of the infectious agents.

Future Studies

The overall goal of the laboratory to apply the diagnostic microchip assay for use in clinical diagnosis. We expect to increase the number of probes present on the chip to cover most of the commonly tested infectious agents in patients. If this assay can be developed for commercialization it would allow the diagnosis of the infectious agents on the same day when the patient gets sick and would allow early treatment before complications or worsening occurred. It would also allow one to differentiate between diseases where the symptoms are very similar and only multiple testing may reveal the causative agent. Transplant patients, AIDS patients or others can be followed for opportunistic infections so they could be treated immediately, before requiring more extensive and expensive treatments. Red Cross and other blood banks can use it for rapid screening of blood from healthy blood donors. It could reduce the slight but still real risk of transferring infectious agents by transfusion from donors with recent viral infections. In a preliminary estimate the microchip-based diagnosis would be very much cheaper than other single diagnostic method and could identify up to several hundred agents simultaneously.