Validation of multiplex real-time PCR tests for intestinal parasites
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Introduction: Parasitic infections are one of the most common human infections around the world, especially in refugees. The current standard of diagnosis for parasitic infections is microscopy of stool specimens. However microscopy has several limitations, some of which include time, inexperience and subjectivity in diagnosis. To improve the specificity, sensitivity and turn-around time of the diagnosis method, multiplex real-time PCR (RT-PCR) diagnosis has recently been applied in this field. Multiplex RT-PCR can simultaneously amplify and detect the presence of multiple parasites in one sample in one run. Methods: The study population was comprised of 32 refugees arriving in Texas between the years of 2010 and 2012 that were infected with Giardia lamblia, Dientamoeba fragilis, and/or Strongyloides stercoralis. The probe/primer mixes to identify these parasites using multiplex RT-PCR were first validated in uniplex assays and then tested for effectiveness in multiplex assays. Results: The results for the uniplex assay suggest that the G. lamblia (100% agreement between uniplex and microscopy) and D. fragilis (82.4% agreement) probe/primer mixes were validated, but the S. stercoralis (0% agreement) probe/primer mix was not. A specific type of multiplex, known as a duplex, was conducted to identify the parasites G. lamblia and D. fragilis. In the multiplex assay, G. lamblia had a 100% agreement while D. fragilis had an 85.7% agreement. In the case of co-infections, there was a 100% agreement to identify the presence G. lamblia and a 66.7% agreement to identify D. fragilis. There was a 0% false positive rate for both uniplex and multiplex assays for all parasites. Conclusion: It was concluded that the multiplex RT-PCR assay to diagnose these two parasites was successful and the results agreed with microscopic diagnoses.