Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

The overall goal of this procedure is to demonstrate the use of a lateral flow device for the rapid detection of aspergillus diagnostic antigen in human serum and BAL fluids. This is accomplished by first applying the serum or BAL sample to the release port of the lateral flow device. The second step is to allow the solution to migrate along the membrane.

The next step is to determine whether the test is valid by examining the internal control line. The final step is to interpret the test result by examining the test line. Ultimately, the lateral flow device is used to detect the presence of aspergillus diagnostic antigen in serum or BAL fluids.

The aspergillus LFD can be used as an adjunct test for the diagnosis of invasive pulmonary aspergillosis. The main advantage of this technique over existing methods like the aspergillus, galacto, Manan enzyme immunoassay, panf, fundal, beta glucan assay and aspergillus PCR test, is that it is a rapid point of care diagnostic test requiring minimal laboratory facilities and training. Though the LFD has been developed for the detection of aspergillus species, lateral flow technology can also be applied to the detection of other infectious pathogens such as cetosporium, fusarium, and riser species.

By incorporating monoclonal antibodies specific to these organisms, visual demonstration of this method is critical. Since interpretation of LFD results requires familiarization with the assay format To collect serum from untreated blood samples allow blood to clot at four degrees Celsius serum and bronchoalveolar lavage, or BAL fluids should be stored as aliquots at minus 20 degrees Celsius prior to use storage as aliquots limits the potential for degradation of the target antigen by repeated free storing of samples during repeat testing to prepare serum and BAL samples for testing, thaw samples at room temperature, and then keep on ice mix thoroughly by vortexing and then centrifuge for one minute at 14, 000 RPM For routine testing of human samples, dilute the serum one to one with tissue culture medium alternatively, and to dissociate the target antigen from serum immune complexes. Dilute serum one to two with PBS containing 4%EDTA heat for three minutes in a boiling water bath and centrifuge for five minutes of 14, 000.RPM.

Human BAL fluids do not require pre-treatments. A BAL sample has been thawed vortex and centrifuge is a neat sample that can be applied directly to the lateral flow device. For testing, the lateral flow devices or LFD are stored at room temperature or 23 degrees Celsius at which they're stable for 12 months.

To begin the assay, remove the devices from their pouches and place on a level surface. Using a sterile pipette tip, apply 100 microliters of pretreated serum to the release port of the device within seconds. The fluid should migrate by capillary action along the nitrous cellulose membrane in the observation window.

In the same manner, apply 100 microliters of a neat BAL sample to the release port of the device. Allow the assay to run for 15 minutes at room temperature, at which time the results of the test should be recorded. The test should not be left longer than 15 minutes before recording the results.

As this may bias result interpretation, a week reaction will not be enhanced by extending the incubation period. The LFD consists of an internal control line indicated by the letter C on the plastic housing and a test line indicated by the letter T.The control line should always appear irrespective of aspergillus antigen in the serum or BAL sample. This shows that the assay has run correctly if the aspergillus antigen is present in the serum or BAL sample.

The test line will also appear within 15 minutes of sample application. The intensities of the test, Lyme reactions are proportional to the concentrations of the target antigen in the serum and VAL samples. The most challenging aspect of this procedure is the interpretation of test results.

The LFD results should be read at 15 minutes and the test result compared to the representative example shown here, Shown in this figure are representative examples of weak, positive, moderate positive, and strong positive LFD results with BAL and serum samples. Any positive test line in the serum sample regardless of intensity would indicate invasive pary aspergillosis or IPA disease due to the presence of circulating aspergillus antigen in the bloodstream. Positive BAL reactions regardless of intensity would indicate germination of spores and development of potentially pathogenic hyphy in the lungs.

In the absence of aspergillus antigen, no test line will appear and the result is recorded as a negative. This table shows the results of LFD tests using BAL fluids from acute myeloid leukemia or a ML patients diagnosed according to the diagnostic criteria of the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Study Group. Included in this table are the corresponding clinical and psychological data, as well as results of an ASPERIA specific PCR test for each patient.

Under the 2002 E-O-R-T-C-M-S-G guidelines, patients 12, 13 and 16 were diagnosed with possible IPA on the basis of host factors and clinical criteria or GM positivity. According to the revised 2008 E-O-R-T-C-M-S-G guidelines, host factors and GM positivity alone or host factors and clinical criteria alone would not indicate possible invasive fungal infection unless accompanied by supporting evidence from clinical data and mycology respectively. Patient six was diagnosed with probable IPA under both 2002 and 2008 guidelines because of host factors major and minor clinical features and GM positivity.

Finally, note that while neither the LFD assay nor the PCR assay is currently included in E-O-R-T-C-M-S-G guidelines in the BAL samples of patients six and 12, there is strong agreement between the two assays and the commercial gala manin test indicating the presence of aspergillus antigen and nucleic acid Following this procedure. Other methods like the Pella Glactomannan enzyme amino assay, panf, fungal beta-glucan test or aspergillus PCR tests can be performed in parallel to confirm invasive fungal disease. Don't forget that working with human blood serum and BAL fluids can be extremely hazardous and operators of the device should always be vaccinated for diseases such as hepatitis B and tuberculosis.I.