Malaria (Plasmodium) continues to remain one of the most devastating infectious diseases on the planet. While new initiatives aim to eradicate the disease, one important aspect of these projects is the necessity for improving diagnosis and surveillance methods. Despite inroads in molecular approaches, the gold standard for malaria diagnosis, epidemiology, and clinical trial efficacy evaluation continues to be microscopy, in particular the Giemsa-stained thick blood smears.
“Microscopy suffers from limitations such as sensitivity, with the average microscopist able to detect about 100 parasites per microliter of blood, but the threshold for fever and clinical disease is less than 10 parasites per microliter for nonimmune patients,” says MAJ Edwin Kamau, Ph.D., chief of the department of molecular diagnostics, Malaria Vaccine Branch at Walter Reed Army Institute of Research.
“As we move into an era working to control and eliminate malaria, it is critical that we develop a more sensitive and higher throughput means for detecting smaller amounts of the parasite useful for even subclinical infections. The malaria vaccine and drug development program at the Walter Reed Army Institute of Research is seeking better means of detection, particularly for those subclinically infected. To do this, we sought to develop a highly sensitive genus-specific RT-qPCR assay to detect Plasmodium.
“A number of such assays have been developed that can measure infection more than 1,000 times more sensitively than microscopy or even antigen detection tests. However, one challenge is that most qPCRs target the DNA of the multicopy 18S rRNA genes, which have enough genetic variation to be problematic. It is therefore important that qPCR target amplification of conserved regions of 18S rRNA genes.”
Dr. Kamau and colleagues developed a genus-specific reverse transcriptase RT-qPCR assay to detect Plasmodium. “The assay detects both rRNA and DNA and takes advantage of the high copy numbers of rRNA in the genome of the parasite. It can detect 0.002 parasites per microliter of blood, one of the lowest levels detected so far.”
According to Dr. Kamau, introduction of the reverse transcriptase step was key. “We were able to improve the assay by approximately 10-fold by adding this step. We verified our hypothesis that RT-qPCR was more reliable than microscopy at lower parasite densities, but not at higher densities. Thus another important finding was that dilution ranges of the sample are important.”
Before molecular diagnostic approaches replace microscopy, there are a number of remaining challenges to overcome including the need for stringent standardization of the methodology.