Diagnosing Malaria Sooner
Our third effort at developing a simple analytical diagnostic for malaria looks promising. It could help prevent the spread of the most devastating disease on the planet.
Over three billion people are at risk of malaria. In 2012, according to World Health Organization estimates, 207 million people were diagnosed with malaria and 600,000 people died from the disease; the main casualties were children under five and pregnant women.
There is an urgent need for diagnostics to detect the early stages of the parasite; they must be highly sensitive, cost effective, simple to use, and rugged enough to be transported to remote areas in tropical jungle communities. Current diagnostic tools include optical microscopy, which has a sensitivity of around 40 parasites/µl but which requires an experienced microscopist; monoclonal antibody-based rapid diagnostic tests (RDTs), which are easy to perform but do not quantify parasitemia (parasite load) and take about 20 minutes per test; and polymerase chain reaction (PCR) assays – the current gold standard – which have excellent sensitivity (one parasite/µl) but require expensive technology and reagents – and results take up to two hours to generate.
For a diagnostic technique to be effective it must be able to detect both the immature asexual (ring) stage of the parasite (the only stage that is present in the peripheral circulation in new infections) and the mature sexual stage, which appears later and is the only stage capable of transmission to mosquitoes.
We initially investigated the potential of Raman microspectroscopy in combination with multivariate data analysis (1). The technology showed potential for detecting hemozoin, a by-product of the catabolization of hemoglobin and also known as malaria pigment, but it took several hours to produce results, which is not acceptable.
To accelerate the analysis, we next detected hemozoin in a whole drop of blood. Using an ultrasonic acoustic levitation device we could probe the droplet with a Raman microscope with a right angle lens. An acoustic levitation device consists of a piezo electric transducer and a reflective sound plate, which together generate a standing wave with very stable ultrasonic nodes. A droplet of blood can be placed in one of the central nodes and levitated in air, which has the advantage of concentrating the droplet though evaporation and reducing the attenuation of Raman laser light, as there is no container. This enabled us to record high quality spectra and detect later stage ring-form parasites. However, it was not conducive to routine analysis as the droplets can become unstable after time and explode, and it did not detect the early stage rings found in peripheral blood. But it did demonstrate the ability to investigate a large population of cells with a spectroscopic modality.
Building on this, we identified a unique fatty acid signature for each stage of the parasite’s life-cycle at the single-cell level using the FTIR microscope on the infrared beamline at the Australian Synchrotron (2). Since a synchrotron clearly cannot be used as a routine clinical tool, we turned to total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This detected the earliest ring forms of the parasite and the gametocytes by analyzing specific fatty acids associated with the parasite membranes rather than relying on hemozoin. ATR-FTIR in combination with partial least squares (PLS) regression analysis enables parastemia detection down to 0.00001 percent in laboratory-spiked red blood cell samples – an improvement upon the PCR assay. Moreover, the technique is portable and rugged, so it can be placed in a bag and transported to remote jungle communities. It quantifies parastemia and does not require highly trained technicians. Sample preparation involves blood centrifugation, removal of the plasma and white blood cells, and the addition of methanol. A 20 µl aliquot of packed red blood cells is placed onto the ATR-FTIR window and the spectrum recorded in about 20 seconds. The spectrum is run through the PLS algorithm and the diagnosis, including degree of parastemia is determined in seconds (3).
The ability to detect very low levels of parastemia is crucial. People with low levels of malaria parasites often show none of the classic fever symptoms but infect more vulnerable members of their communities via mosquito bites. We will soon conduct a pilot study in Thailand to test the efficacy of the ATR-FTIR approach with clinical patients in remote communities.
- B. R. Wood et al., “Resonance Raman Microscopy in Combination with Partial Dark-Field Microscopy Lights Up a New Path in Malaria Diagnostics”, Analyst. 134, 1119-1125 (2009).
- G. T. Webster et al., “Discriminating the Intraerythrocytic Lifecycle Stages of the Malaria Parasite Using Synchrotron FT-IR Microspectroscopy and an Artificial Neural Network”, Anal. Chem. 81, 2516-2524 (2009).
- A. Khoshmanesh et al., “Detection and Quantification of Early-Stage Malaria Parasites in Laboratory Infected Erythrocytes by Attenuated Total Reflectance Infrared Spectroscopy and Multivariate Analysis”, Anal. Chem., 86 (9), 4379–4386 (2014).
Bayden Wood is an Australian Research Council Future Fellow and a founding member of the Centre for Biospectroscopy at Monash University. He completed his PhD in 1999 at Monash University and undertook his first post-doctoral position at Hunter College, City University of New York. He was awarded a Humboldt Fellowship in 2008 and undertook research at the Institute of Analytical Sciences in Dortmund with Volker Deckert and also at the Robert Koch Institute in Berlin with Dieter Naumann. “My primary research interests are the development and application of FTIR and Raman based systems for the analysis of cells and tissues with particular relevance to disease diagnosis and monitoring.”
